DEVICES, SYSTEMS AND METHODS FOR ANALYSIS OF NUCLEIC ACIDS

CROSS-REFERENCE

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/368,768 filed July 18, 2022; U.S. Provisional Application Serial No. 63/371,331 filed August 12, 2022; U.S. Provisional Application Serial No. 63/376,259 filed September 19, 2022; U.S. Provisional Application Serial No. 63/376,995 filed September 23, 2022; U.S. Provisional Application Serial No. 63/381,526 filed October 28, 2022; and U.S. Provisional Application Serial No. 63/480,266 filed January 17, 2023, each of which is incorporated herein in its entirety for all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Contract No. N66001-21-C- 4048 awarded by the Department of Defense, Defense Advanced Research Projects Agency (DARPA). The US government has certain rights in the invention.

BACKGROUND

[0003] Detection of ailments, especially at the early stages of disease or infection, can provide guidance on treatment or intervention to reduce the progression or transmission of said ailments. Such ailments can be detected at the point of need by devices capable of running diagnostic assays. Various biological species associated with an organism, disease state, phenotype, or genotype can be detected by these devices. Challenges in deploying such devices include developing methods to immobilize diagnostic assay components on surfaces without compromising the performance of the assay, as well as performing amplification of samples without significant additional instrumentation.

SUMMARY

[0004] In certain aspects, described herein are embodiments of a device, system and method for target detection, comprising: providing a sample comprising at least one sequence of interest; separating the sample into a plurality of sub-samples; receiving the plurality of sub-samples in a detection chamber and contacting the plurality of sub-samples with at least one probe, wherein said at least one probe is connected to a surface of said detection chamber by a linkage, wherein said at least one probe may comprise a programmable nuclease, a guide nucleic acid, a reporter or a combination thereof; and using a plurality of sensors to determine a presence of said at least one sequence of interest by detecting a signal produced upon cleavage of said reporter by said programmable nuclease.

[0005] Described herein are various embodiments of a device comprising: a sample interface configured to receive a sample that may comprise one or more target sequences of interest; one or more channels to separate said sample into partitioned samples, wherein said one or more channels are in fluid communication with said sample interface and a reaction chamber comprising a surface, wherein at least one probe may comprise a programmable nuclease, a guide nucleic acid, a reporter or a combination thereof, wherein said at least one probe is connected to said surface by a linkage; and a plurality of sensors for determining a presence of said one or more target sequences of interest by detecting a signal emitted upon cleavage of said reporter by said programmable nuclease.

[0006] In one aspect, described herein is a microfluidic device comprising: a sample interface configured to receive a sample; a first actuator configured to provide positive pressure to an upstream portion of the device proximate to the sample interface; a second actuator configured to provide negative pressure to a downstream portion of the device distal to the sample interface; a heating channel in fluid communication with the sample interface and first actuator, wherein the heating channel comprises a first portion in a first plane and a second portion in a second plane parallel to the first plane; a first heating element disposed between and in thermal contact with the first and second portions of the heating channel; a reagent mixing chamber fluidically connected downstream of the heating channel by a first valve; a plurality of reaction chambers in fluid communication with the second actuator; a channel multiplexing unit fluidically connected downstream of the mixing chamber by a second valve, wherein the channel multiplexing unit comprises a main channel and a plurality of side channels, wherein each of the side channels is in fluid communication with one inlet of a plurality of inlets positioned along the main channel, and wherein each of the side channels comprises an outlet in fluid communication with one of the plurality of reaction chambers; a first valve actuator configured to actuate the first valve; and a second valve actuator configured to actuate the second valve.

[0007] In some embodiments, (i) each of the first valve and second valve is thermally actuated, and (ii) each valve actuator comprises a heating element in thermal contact with the respective valve. In some embodiments, each of the first and second valves comprises a valve inlet channel and a valve outlet channel; and wherein a cross-sectional area of the valve inlet channel is less than a cross-sectional area of the corresponding valve outlet channel. In some embodiments, each of the valves is filled with a material configured to change between liquid and solid phases when heated by the heating element of the corresponding valve actuator. In some embodiments, the first heating element is regulated by a thermistor. In some embodiments, the heating channel has a serpentine configuration. In some embodiments, the main channel is serially divided into a plurality of subchannel portions and a tolerance channel, wherein the tolerance channel is positioned at a distal end of the main channel. In some embodiments, each of the subchannel portions and tolerance channel are configured to subsequently reduce in cross-sectional area from a most proximal subchannel to the tolerance channel. In some embodiments, a volume of the sample is (a) greater than a combined volume of the plurality of side channels and the plurality of reaction chambers, and (b) less than the combined volume the plurality of side channels, the plurality of reaction chambers, and the tolerance channel. In some embodiments, each of the plurality of inlets comprises a porous frit. In some embodiments, each of the plurality of reaction chambers is connected to the downstream portion by each of a plurality of hydrophobic venting membranes. In some embodiments, a shape of the first heating element corresponds to a shape of the heating channel such that a desired heating profile is achieved within or along the heating channel. In some embodiments, the first heating element is configured to trace in alignment with the heating channel. In some embodiments, the first heating element occupies a surface area that is larger than a surface area occupied by the heating channel. In some embodiments, a shape of the first heating element: (a) corresponds to a shape of the heating channel, and (b) occupies a surface area that is larger than a surface area occupied by the heating channel. In some embodiments, the microfluidic device further comprises a selfsealing vent in fluid communication with a distal end of the second portion, wherein the vent is configured to seal when wetted. In some embodiments, the first actuator and second actuator are operably connected such that actuation of the first actuator triggers actuation of the second actuator.

[0008] In some embodiments, the microfluidic device further comprises one or more reaction heating elements in thermal contact with the reaction chambers; optionally wherein the one or more reaction heating elements are regulated by a thermistor. In some embodiments, the first actuator is operably connected to trigger a timing mechanism that controls the heating elements and valve actuators. In some embodiments, the microfluidic device further comprises a power supply in electrical contact with each of the heating elements. In some embodiments, the power supply comprises a battery (e.g., an alkaline or paper battery). In some embodiments the power supply comprises a plurality of batteries. In some embodiments, the microfluidic device further comprises a computing unit in electrical communication with the power supply and each of the heating elements. In some embodiments, the computing unit is programmed to control heating of each of the plurality of heating elements to control fluid flow through each of the first and second valves. In some embodiments, the computing unit comprises a plurality of status LEDs. [0009] In one aspect, described herein is a microfluidic device comprising: a sample interface configured to receive a sample; a first actuator configured to provide positive pressure to an upstream portion of the device proximate to the sample interface; a second actuator configured to provide negative pressure to a downstream portion of the device distal to the sample interface; a heating channel in fluid communication with the sample interface and first actuator; a first heating element disposed in thermal contact with the heating channel; a reagent input comprising a reagent interface and a reagent channel; a reagent mixing unit fluidically connected downstream of an intersection by a first valve, wherein the intersection is fluidically connected (i) downstream of the heating channel by a second valve, and (ii) downstream of the reagent channel by a third valve; a plurality of reaction chambers; a channel multiplexing unit fluidically connected downstream of the mixing unit by a fourth valve, wherein the channel multiplexing unit comprises a plurality of side channels, wherein each of the plurality of side channels is in fluid communication with a plurality of subchannels, wherein each of the plurality of subchannels comprises an outlet in fluid communication with one of the plurality of reaction chambers; a plurality of detection chambers, wherein each of the plurality of detection chambers is (i) fluidically connected downstream of a corresponding reaction chamber by a further valve, and (ii) in fluid communication with the second actuator; a first valve actuator configured to actuate the first valve; a second valve actuator configured to actuate the second valve; a third valve actuator configured to actuate the third valve; a fourth valve actuator configured to actuate the fourth valve; and one or more further valve actuators configured to actuate the further valves. [0010] In some embodiments, (i) each of the valves is thermally actuated, and (ii) each valve actuator comprises a heating element in thermal contact with the respective valve. In some embodiments, the microfluidic device further comprises a first self-sealing vent in fluid communication with the heating channel, and a second self-sealing vent in fluid communication with the mixing channel, wherein the first self-sealing vent and second self-sealing vent are each configured to seal when wetted. In some embodiments, the second actuator is a sliding unit. In some embodiments, the microfluidic device further comprises at least one flow restrictor positioned between the reagent interface and the reagent channel. In some embodiments, each of the valves comprises a valve inlet channel and a valve outlet channel; and wherein a cross- sectional area of the valve inlet channel is less than a cross-sectional area of the corresponding valve outlet channel. In some embodiments, each of the valves is filled with a material configured to change between liquid and solid phases when heated by the heating element of the corresponding valve actuator. In some embodiments, the first heating element is regulated by a thermistor. In some embodiments, the heating channel has a serpentine configuration.

[0011] In some embodiments, (i) the heating channel comprises a first portion in a first plane and a second portion in a second plane parallel to the first plane, and (ii) the first heating element is disposed between and in thermal contact with the first and second portions of the heating channel. In some embodiments, the first actuator and second actuator are operably connected such that actuation of the first actuator triggers actuation of the second actuator. In some embodiments, the microfluidic device further comprises one or more reaction heating elements in thermal contact with the reaction chambers; optionally wherein the one or more reaction heating elements are regulated by a thermistor. In some embodiments, the microfluidic device further comprises a power supply in electrical contact with each of the heating elements. In some embodiments, the power supply comprises a battery (e.g., an alkaline or paper battery). In some embodiments the power supply comprises a plurality of batteries. In some embodiments, the microfluidic device further comprises a computing unit in electrical communication with the power supply and each of the heating elements. In some embodiments, the computing unit is programmed to control heating of each of the plurality of heating elements to control fluid flow through each of the first, second, third, fourth, and further valves. In some embodiments, the computing unit comprises a plurality of status LEDs.

[0012] In one aspect, described herein is a system comprising (a) a microfluidic device described herein, and (b) a signal detection device.

[0013] In some embodiments, each of the reaction chambers comprises a programmable nuclease, a guide nucleic acid, and a reporter, wherein: (a) the guide nucleic acid comprises a sequence configured to bind to a target nucleic acid; (b) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (c) cleavage of the reporter is effective to produce a detectable product; and (d) at least two of the reaction chambers comprise different guide nucleic acids.

[0014] In one aspect, described herein is a method of using a microfluidic device disclosed herein, the method comprising: (a) applying a sample to the sample interface, wherein said applying forms a sample fluid; (b) actuating flow of the sample fluid through the heating channel to each of the reaction chambers, (c) reacting the sample fluid with the programmable nuclease, the guide nucleic acid, and the reporter, and (d) detecting a detectable signal when a target nucleic acid is present in the sample.

[0015] In one aspect, the present disclosure provides systems comprising a reaction chamber comprising a composition described herein, such as with regard to any of the various aspects described herein. In some embodiments, the reaction chamber is a chamber within a device disclosed herein.

[0016] In one aspect, the present disclosure provides a composition for detecting a target nucleic acid, the composition comprising a programmable nuclease, a guide nucleic acid, and a reporter. In some embodiments, (a) the guide nucleic acid comprises a sequence selected from a table described herein and is configured to bind to the target nucleic acid; (b) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; and (c) cleavage of the reporter is effective to produce a detectable product. In some embodiments, the reporter is coupled to the guide nucleic acid. In some embodiments, the composition comprises a plurality of different guide nucleic acids, wherein the plurality of different guide nucleic acids are collectively configured to bind different target sequences within the target nucleic acid. In some embodiments, the composition comprises a plurality or all of guide nucleic acids represented by (a) SEQ ID Nos 987-1010, (b) SEQ ID Nos 1011-1037, (c) SEQ ID Nos 1038-1048, (d) SEQ ID Nos 1049-1061, (e) SEQ ID Nos 1062-1096, (f) SEQ ID Nos 1097-1123, (g) SEQ ID Nos 1124- 1158, (h) SEQ ID Nos 1159-1170, or (i) SEQ ID Nos 1171-1175.

[0017] In one aspect, the present disclosure provides a composition for detecting a target nucleic acid, the composition comprising a programmable nuclease, a guide nucleic acid, a first reporter, an enzyme, and a second reporter. In some embodiments, (a) the guide nucleic acid comprises a sequence selected from a table as described herein and is configured to bind to the target nucleic acid; (b) the programmable nuclease is effective to cleave the first reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (c) the cleavage of the first reporter is effective to separate a first nucleic acid section from a second nucleic acid section thereof; (d) the first nucleic acid section is effective to activate the enzyme; and (e) the activated enzyme is effective to cleave the second reporter to produce a detectable product comprising a detection moiety. In some embodiments, the enzyme is an endonuclease and the second reporter comprises a polynucleotide substrate of the enzyme. In some embodiments, the endonuclease is a NucC endonuclease. In some embodiments, the first nucleic acid section comprises adenosine residues. In some embodiments, the adenosine residues comprise cyclic adenylate (cA3). In some embodiments, the second nucleic acid section comprises RNA residues (such as a plurality of uracil residues). In some embodiments, the second nucleic acid section comprises DNA residues (such as a plurality of thymine residues). In some embodiments, (a) the second reporter comprises a fluorescent label and a quencher, and (b) cleavage of the second reporter by the activated enzyme is effective to separate the fluorescent label from the quencher.

[0018] In one aspect, the present disclosure provides a method of detecting a target nucleic acid in a sample. In some embodiments, the method comprises: (a) contacting the sample with a composition described herein; (b) cleaving the first reporter with the programmable nuclease in response to formation of the complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid, thereby releasing the first nucleic acid section; (c) activating the enzyme with the first nucleic acid section; (d) cleaving the second reporter with the activated enzyme, thereby producing the detectable product comprising the detection moiety; and (e) detecting the detection moiety, thereby detecting the presence of the target nucleic acid in the sample. In some embodiments, (a) the second reporter comprises a polynucleotide substrate of the enzyme, and (b) the enzyme is a NucC. In some embodiments, step (d) is performed at a temperature of at least 40 °C.

[0019] In one aspect, the present disclosure provides a method of detecting a target nucleic acid in a sample. In some embodiments, the method comprises: (a) contacting the sample with a composition described herein; (b) cleaving the reporter with the programmable nuclease in response to formation of the complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid, thereby producing a detectable product; and (c) detecting the detectable product, thereby detecting the presence of the target nucleic acid in the sample.

[0020] In one aspect, the present disclosure provides a device for detecting a target nucleic acid, the device comprising: a sample interface configured to receive a sample; a reaction chamber in fluid communication with the sample interface; and a composition described herein.

[0021] In one aspect, the present disclosure provides a composition for detecting a target nucleic acid in a reaction chamber, the composition comprising a programmable nuclease, a guide nucleic acid, a forward primer, a reverse primer, a polymerase, a nicking endonuclease, and a reporter. In some embodiments, (a) the guide nucleic acid comprises a sequence selected from a table described herein and is configured to bind to the target nucleic acid; (b) the forward primer comprises (i) a 5’ portion comprising a first hairpin, and (ii) a 3’ portion that is configured to bind the target nucleic acid at a first overlapping region with respect to the guide nucleic acid; (c) the reverse primer comprises (i) a 5’ portion comprising a second hairpin, and (ii) a 3’ portion that is configured to bind a complement of the target nucleic acid at a second overlapping region with respect to the guide nucleic acid; (d) the first and second hairpins are cleavage substrates for the nicking endonuclease; (e) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and (i) the target nucleic acid or (ii) an amplicon of the target nucleic acid; and (f) the cleavage of the reporter is effective to produce a detectable product comprising a detection moiety. In some embodiments, (a) the sequence of the target nucleic acid to which the 3’ portion of the first primer is configured to bind defines a first sequence of the target nucleic acid; (b) the sequence of the 3’ portion of the reverse primer defines a second sequence of the target nucleic acid; and (c) the first sequence and second sequence are separated by about 5 to about 10 nucleotides along the target nucleic acid. In some embodiments, the 3’ portions of the forward primer and reverse primer are about 16 to about 20 nucleotides in length. In some embodiments, overlap between the 3’ portion of the reverse primer and the sequence to which the guide nucleic acid is configured to bind overlap by 1 to 5 nucleotides, 2 to 5 nucleotides, or 3 nucleotides. In some embodiments, the first hairpin and/or the second hairpin are 10 to 20 nucleotides in length, 16 to 20 nucleotides in length, or 16 nucleotides in length. In some embodiments, the programmable nuclease is a Cas protein (such as a Casl2 protein or a Casl4 protein).

[0022] In one aspect, the present disclosure provides a method of detecting a target nucleic acid in a sample. In some embodiments, the method comprises: (a) contacting the sample with a composition described herein; (b) performing nicking enzyme amplification reaction (NEAR) reaction to amplify the target nucleic acid; (c) forming a complex comprising the programmable nuclease, the guide nucleic acid, and (i) the target nucleic acid, or (ii) an amplicon of the target nucleic acid; (d) cleaving the reporter with the programmable nuclease activated by formation of the complex, thereby producing the detectable cleavage product; and (e) detecting the detection moiety, thereby detecting the presence of the target nucleic acid in the sample.

[0023] In one aspect, the present disclosure provides a microfluidic device including: a sample interface configured to receive a sample; an actuator configured to provide positive pressure to an upstream portion of the device proximate to the sample interface; a heating channel in fluid communication with the sample interface and first actuator; a channel heating element in thermal contact with the heating channel; a reagent mixing chamber fluidically connected downstream of the heating channel by a first valve, wherein the first valve is a thermally actuated valve in thermal contact with a first valve heating element; a plurality of reaction chambers fluidically connected downstream of the reagent mixing chamber by one or more second valves, wherein each of the one or more second valves is a thermally actuated valve in thermal contact with a respective second valve heating element; and a power supply in electrical contact with each of the heating elements, wherein the power supply is configured to sequentially power the channel heating element, the first valve heating element, and the respective second valve heating element upon activation of the power supply.

[0024] In some embodiments, the microfluidic device further includes a computing unit in electrical communication with the power supply and each of the plurality of heating elements, wherein the computing unit is programmed to control timing and duration of heating of each of the heating elements. In some embodiments, the computing unit is programmed to trigger one or more timing mechanisms that control the heating elements.

[0025] In one aspect, the present disclosure provides a method of detecting a target nucleic acid in a sample, the method comprising: (a) contacting the sample with a programmable nuclease, a guide nucleic acid, and a reporter at a first location of a device, wherein: (i) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and (1) the target nucleic acid or (2) an amplicon of the target nucleic acid; and (ii) the cleavage of the reporter is effective to produce a detectable product comprising a detection moiety; (b) flowing the detectable product to a second location of the device, wherein uncleaved reporter molecules are retained in the first location; and (c) detecting the detection moiety at the second location, thereby detecting the presence of the target nucleic acid in the sample.

[0026] In some embodiments, the uncleaved reporter molecules are retained by (i) attachment to a surface at the first location, or (2) a filter that restricts passage of uncleaved reporter and allows passage of the detectable product. In some embodiments, the detection moiety comprises a molecule that is captured at a surface at the second location, and (ii) the detecting comprises detection of a change in electrical signal at the surface at the second location resulting from the capture of the detection moiety. In some embodiments, the uncleaved reporter molecules are immobilized to the surface at the first location, and individual uncleaved reporter molecules comprise a surface linker, an RNA portion that is cleavable by the programmable nuclease, and a DNA portion that is released as part of the detectable product upon cleavage of the RNA portion, wherein the detecting comprises detecting a change in electrical signal resulting from hybridization of the DNA portion to a surface-bound probe oligonucleotide. In some embodiments, the uncleaved reporter molecules are immobilized to the surface at the first location, and individual uncleaved reporter molecules comprise a surface linker, an RNA portion that is cleavable by the programmable nuclease, and a gold nanoparticle (AuNP) that is released as part of the detectable product upon cleavage of the RNA portion, wherein the detecting comprises detecting a change in electrical signal resulting from the AuNP binding to a sensor surface at the second location. In some embodiments, the uncleaved reporter molecules are immobilized to the surface at the first location, and individual uncleaved reporter molecules comprise a surface linker, an RNA portion that is cleavable by the programmable nuclease, and a hairpin DNA portion that is released as part of the detectable product upon cleavage of the RNA portion, wherein the hairpin DNA portion comprises a first binding moiety, and wherein the detecting comprises detecting a change in electrical signal resulting from capture of the DNA hairpin and first binding moiety by a second binding moiety at a sensor surface at the second location. In some embodiments, the uncleaved reporter molecules are immobilized to the surface at the first location, and individual uncleaved reporter molecules comprise a surface linker, an RNA portion that is cleavable by the programmable nuclease, and a platinum nanoparticle (PtNP) that is released as part of the detectable product upon cleavage of the RNA portion, wherein the detecting comprises detecting a change in pH resulting from hydrogen peroxide reacting with the PtNP at the second location. In some embodiments, the uncleaved reporter molecules are free in a solution at the first location, and individual uncleaved reporter molecules comprise a binding moiety restricted by the filter, an RNA portion that is cleavable by the programmable nuclease, and a DNA portion that is released as part of the detectable product upon cleavage of the RNA portion, wherein the detecting comprises detecting a change in electrical signal resulting from hybridization of the DNA portion to a surface-bound probe oligonucleotide. In some embodiments, the uncleaved reporter molecules are free in a solution at the first location, and individual uncleaved reporter molecules comprise a binding moiety restricted by the filter, an RNA portion that is cleavable by the programmable nuclease, and a gold nanoparticle (AuNP) that is released as part of the detectable product upon cleavage of the RNA portion, wherein the detecting comprises detecting a change in electrical signal resulting from the AuNP binding to a sensor surface at the second location.

[0027] In one aspect, the present disclosure provides a method of detecting a target nucleic acid in a sample, the method comprising: (a) contacting the sample with a programmable nuclease, a guide nucleic acid, and a reporter at a first location of a device, wherein: (i) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and (1) the target nucleic acid or (2) an amplicon of the target nucleic acid; (ii) the cleavage of the reporter is effective to produce a detectable product comprising a detection moiety; and (iii) individual uncleaved reporter molecules comprise a binding moiety, an RNA portion that is cleavable by the programmable nuclease, and a gold nanoparticle (AuNP) that is released as part of the detectable product upon cleavage of the RNA portion; (b) flowing the detectable product and uncleaved reporter molecules to a second location of the device, wherein the uncleaved reporter molecules are captured to surface at the second location; and (c) detecting presence of the detection moiety at the second location, thereby detecting the presence of the target nucleic acid in the sample, wherein the detecting comprises detecting a change in electrical signal resulting from reduced AuNP binding to a sensor surface at the second location relative to a negative control.

INCORPORATION BY REFERENCE

[0028] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The novel features of the invention are set forth with particularity in the appended claims.

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0030] FIGS. 1A-1C illustrate front views and a cross-sectional view of a microfluidic device for programmable nuclease-based detection, in accordance with some embodiments. FIG. 1A shows an exploded view of the device, for user input and operation. FIG. IB shows a perspective view of the device. FIG. 1C shows a cross-sectional view along line Y of FIG. 1A. [0031] FIG. 2 illustrates a rear view of the exemplary microfluidic device of FIGS. 1A-1C for programmable nuclease-based detection.

[0032] FIGS. 3A-3G illustrate component views of portions of the exemplary microfluidic device of FIGS. 1A-1C. FIGS. 3A-3C illustrate detailed views of a front, back, and top side of an upstream portion, respectively, and FIGS. 3D-3F illustrate detailed views of a front, back, and top side of a downstream portion, respectively. FIG. 3G shows front and back views of a serpentine heating channel, with an upstream portion on one side, and a downstream portion on the other side, in which the upstream and downstream portions sandwich a heating element therebetween.

[0033] FIGS. 4A-4C show an embodiment of the valves compatible for use in the exemplary microfluidic device of FIGS. 1A-1C. FIG. 4A illustrates a detailed view of an embodiment of the plurality of valves. FIGS. 4B-4C illustrate perspective views of heaters used as valve actuators.

[0034] FIGS. 5A-5D illustrate front views of a microfluidic device for programmable nuclease- based detection, in accordance with some embodiments. FIG. 5A shows a perspective view of the device, for user input and operation. FIG. 5B shows a perspective view, FIG. 5C shows a perspective view wherein a top surface of each of an upstream portion cover and a downstream portion cover are transparent, and FIG. 5D shows a top view of the device without upstream portion cover.

[0035] FIG. 6 shows a process flow chart for a programmable nuclease-based detection device, whereby a sample comprising one or more target sequences is collected and prepared before the one or more target sequences are detected. Sample preparation includes compartmentalized thermocycling.

[0036] FIGS. 7A-7B show top down and cross-section views of a programmable nuclease-based detection device, as described herein.

[0037] FIGS. 8A-8B illustrate a cross sectional view of a programmable nuclease-based detection device comprising a plurality of thermocycling compartments with movable mechanisms, as described herein.

[0038] FIGS. 9A-9B illustrate a programmable nuclease probe comprising a programmable nuclease and a guide nucleic acid complexed with the programmable nuclease before and after a complementary binding event, as described herein.

[0039] FIGS. 10A-10B show a programmable nuclease probe before and after a complementary binding event and the generation of a signal indicating a presence of a target sequence or target nucleic acid, as described herein.

[0040] FIGS. 11A-11C illustrates immobilization strategies for CRISPR-Cas diagnostic assay components, as described herein

[0041] FIG. 12 illustrates an embodiment where immobilization strategies are combined to enable CRISPR diagnostic readouts, as described herein.

[0042] FIG. 13 presents an overview of sRCA.

[0043] FIG. 14 presents an overview of RCA positive feedback for Casl3.

[0044] FIG. 15 presents an overview of CasPin.

[0045] FIG. 16 presents potential hairpin structures for CasPin.

[0046] FIG. 17 presents results for an initial design using two hairpins.

[0047] FIGS. 18A-18B illustrate an embodiment of a multiplex lateral flow assay strip, as described herein.

[0048] FIG. 19 illustrates an embodiment of a workflow with multiplex “HotPot” as described herein.

[0049] FIG. 20 illustrates an embodiment for HRP paper-based detection, as described herein.

[0050] FIG. 21 illustrates an embodiment for an HRP -based multiplex lateral flow assay, as described herein.

[0051] FIG. 22 illustrates an embodiment for Multiplexed Casl3 immobilization approach to an HRP-based multiplex lateral flow assay, as described herein.

[0052] FIGS. 23A-23B illustrate the use of multiple Cas-complex probes guide pooling enhanced signal detection to a lateral flow assay, as described herein.

[0053] FIG. 24 shows a schematic of an exemplary workflow for a multiplexed programmable nuclease assay. The illustrated reactions may include detection in the respective reaction chamber, or optionally moved to another location for separate detection reactions.

[0054] FIG. 25 shows an exemplary hydrogel comprising immobilized reporters co-polymerized therein.

[0055] FIGS. 26A and 26B show exemplary multiplexing strategies for hydrogel immobilized DETECTR systems. [0056] FIGS. 27A-27B show an exemplary positive feedback system for signal amplification. [0057] FIG. 28 shows an exemplary workflow for DETECTR-based HotPot reactions.

[0058] FIG. 29 shows a schematic of the NEAR reaction. A forward and reverse primer consisting of a nicking enzyme stabilization site and recognition region able amplify a target region of interest into a single-stranded DNA molecule. A guide RNA will bind a region complementary to the amplified ssDNA, allowing further detection by a DETECTR system. [0059] FIGS. 30A-30B show a signal amplification strategy involving the release of a tethered enzyme by a programmable nuclease. Presence of a target nucleic acid may activate the programmable nuclease, which may cleave a nucleic acid tether that immobilizes the enzyme on a surface. Cleaving the nucleic acid tether may release the enzyme into solution, which may be contacted with an enzyme substrate to create a detectable and amplified signal.

[0060] FIGS. 31A-31C show a signal amplification strategy involving the release of a programmable nuclease-enzyme fusion protein. Presence of a target nucleic acid may activate a programmable nuclease portion of the programmable nuclease-enzyme fusion protein, wherein the activated programmable nuclease may cleave a nucleic acid tether that immobilizes the programmable nuclease-enzyme fusion protein on a surface. Cleaving the nucleic acid tether may release the programmable nuclease-enzyme fusion protein into solution, wherein an enzyme portion of the programmable nuclease-enzyme fusion protein may be contacted with an enzyme substrate to create a detectable and amplified signal. In some cases, contacting may comprise contacting the solution comprising the released programmable nuclease-enzyme fusion protein with another solution comprising the enzyme substrate.

[0061] FIG. 32 shows a signal generation and amplification strategy involving the binding of two programmable nuclease-split enzyme fusion proteins with a target nucleic acid, wherein the two programmable nuclease-split enzyme fusion proteins each comprise an inactivated programmable nuclease portion and a split enzyme subunit portion. The programmable nuclease portion of each programmable nuclease-split enzyme fusion protein may be configured to bind to a different portion of a target nucleic acid. When the inactivated programmable nuclease portion of each programmable nuclease-split enzyme fusion protein binds to the target nucleic acid, the split enzyme subunit portion of each programmable nuclease-split enzyme fusion protein may dock to form an active enzyme. The active enzyme may be contacted with an enzyme substrate to create a detectable and amplified signal.

[0062] FIGS. 33A-33E show various exemplary reporters for use in the signal amplification strategy shown in FIGS. 30A-30B. Each reporter comprises at least one of the following: a detection moiety comprising a horseradish peroxidase (HRP) enzyme for signal enhancement, a nucleic acid-based linker-section, and a functionality for immobilization to a surface. In some embodiments, a carbon spacer may be positioned between the functionality and the nucleic acid and/or detection moiety. In FIG. 33A (e.g., repl61), the reporter comprises an enzyme (e.g., an HRP enzyme), a nucleic acid-based linker (e.g., a single-stranded nucleic acid comprising 20 thymines (T20)), and a biotin functionality. In FIG. 33B (e.g., repl94), the reporter comprises an enzyme (e.g., an HRP enzyme), a nucleic acid-based linker (e.g., a single-stranded nucleic acid comprising 12 thymines (T12) each on either side of a cleavable section comprising 5 uracils), a carbon spacer, and a biotin functionality. In FIG. 33C (e.g., repl88), the reporter comprises an enzyme (e.g., an HRP enzyme), a nucleic acid-based linker (e.g., a single-stranded nucleic acid comprising 20 thymines (T20)), and an acrydite functionality. In FIG. 33D (e.g., repl90), the reporter comprises an enzyme (e.g., an HRP enzyme), a nucleic acid-based linker (e.g., a singlestranded nucleic acid comprising 20 thymines), a carbon spacer, and an amine functionality. In FIG. 33E (e.g., repl97), the reporter comprises an enzyme (e.g., a poly-HRP enzyme), a first carbon spacer, a nucleic acid-based linker (e.g., a single-stranded nucleic acid comprising 20 thymines (T20)), a second carbon spacer, and an amine functionality. In FIG. 33E (e.g., repl97), a poly-HRP detection moiety (e.g., streptavidin (SA) molecule functionalized with multiple HRP groups) is conjugated to the nucleic acid linker via a biotin and the first carbon spacer. In at least some instances, the incorporation of multiple enzymes on one reporter may provide an assay signal boost upon a single cleavage event of the reporter induced by the activated programmable nuclease. In some embodiments, a linker comprises at least one linker segment, the functionality for immobilization to a surface, or a combination thereof. In some embodiments, the linker is referred to as a tether.

[0063] FIG. 34 shows a schematic of a reporter immobilized to a surface of a carboxylic acid magnetic bead (MB) via an amide bond. The depicted reporter comprises a poly-HRP reporter and may be substantially similar to the reporter shown in FIG. 33E.

[0064] FIGS. 35A-35F show a schematic (FIG. 35A) and results (FIGS. 35B-35F) of a programmable nuclease-based detection assay utilizing NHS-activated resin as the solid support for a reporter comprising an HRP signal enhancement enzyme (rep 190).

[0065] FIG. 36 shows a schematic of a signal amplification strategy involving downstream activation of an endonuclease following activation of a programmable nuclease in response to a target nucleic acid. Presence of the target nucleic acid may activate the programmable nuclease, which may cleave the first nucleic acid section of the first reporter from the second nucleic acid section of the first reporter, thereby freeing the first nucleic acid section to bind to and activate the endonuclease. The activated endonuclease may then cleave the second reporter and release a detection moiety that may be detected as described herein.

[0066] FIG. 37 shows illustrative structures for first reporters comprising first and second nucleic acid sections in accordance with some embodiments, such as the strategy illustrated in FIG. 36

[0067] FIG. 38 shows a schematic of a signal amplification strategy involving reporters comprising an enzyme conjugated to a linker, in which the linker comprises a functionality for immobilization to a support. Presence of a target nucleic acid may activate the programmable nuclease, which may cleave a nucleic acid tether to separate the enzyme from the functionality for immobilization. The solution is then contacted with beads that bind the functionality for immobilization. Enzymes cleaved from the nucleic acid tether remain free, whereas in the absence of cleavage, enzymes become complexed with the beads. The solution is separated from the beads and tested for enzyme activity in the presence of enzyme substrate to create a detectable and amplified signal indicative of the presence of the target nucleic acid.

[0068] FIG. 39 shows a schematic representation of a process for concentrating enzymes released from a reporter (e.g., HRP) in an assay.

[0069] FIG. 40 shows a schematic representation of a surface-immobilized positive-feedback system.

[0070] FIG. 41 shows a schematic representation of a surface-immobilized positive-feedback system wherein the Cas#l is initially in the solution and Cas#2 is initially immobilized. In a subsequent step, Cas #1 forms an activated complex upon binding to a first target nucleic acid and cleaves the ssDNA tether linking Cas#2 to the surface. The released Cas#2 is activated upon complexing with a second target nucleic acid (immobilized target #2), and the activated Cas#2 is effective to release additional immobilized Cas#2 and to cleave the reporter to produce a detectable product.

[0071] FIG. 42 shows a schematic representation of tethered guide-reporter chimera hybrid molecules with HRP wherein the tethered guide-reporter hybrid molecule is anchored on a surface with a carbon spacer connecting the programmable nuclease to the surface, and then a subsequent carbon spacer connecting the programmable nuclease to the reporter DNA, followed by a final carbon spacer linking the target DNA to HRP.

[0072] FIG. 43 illustrates a front view of a microfluidic device for programmable nuclease- based detection, in accordance with some embodiments, with a heating channel occupying a surface area smaller than that of a heater trace.

[0073] FIGS. 44A-44C illustrate front views of microfluidic devices for programmable nuclease-based detection, in accordance with some embodiments. FIG. 44A shows a front view of a bent serpentine heater trace without the heating channel. FIG. 44B shows a front view of the bent serpentine heater trace with a serpentine heating channel. FIG. 44C shows a front view of the bent serpentine heater trace with a spiral heating channel. [0074] FIG. 45 illustrates a back view of a microfluidic device for programmable nuclease- based detection using lateral flow assay (LFA) strips, in accordance with some embodiments. [0075] FIG. 46 illustrates a front view of a microfluidic device for programmable nuclease- based detection using a paper battery or capacitor as a power supply, in accordance with some embodiments.

[0076] FIG. 47 illustrates a microfluidic device for programmable nuclease-based detection using a lateral flow assay (LFA) strip, in accordance with some embodiments.

[0077] FIGS. 48A-48B illustrate schematics for programmable nuclease-based detection in which signal generation is based on binding of a nucleic acid to a sensor-bound probe oligonucleotide, in accordance with some embodiments. FIG. 48A shows a target nucleic acid bound to a surface, and FIG. 48B shows a target nucleic acid in solution.

[0078] FIGS. 49A-49C illustrate schematics for programmable nuclease-based detection in which signal generation is based on binding of a nanoparticle to a sensor, in accordance with some embodiments. FIG. 49A shows a nanoparticle bound to a surface, FIG. 49B shows a nanoparticle in solution, and FIG. 49C shows a nanoparticle bound to an attachment moiety. [0079] FIG. 50 illustrates a schematic for programmable nuclease-based detection in which signal generation is based on binding of a hairpin nucleic acid with a biotin moiety to a streptavidin-coated sensor, in accordance with some embodiments.

[0080] FIG. 51 illustrates a schematic for programmable nuclease-based detection in which signal generation is based on reaction of a platinum nanoparticle released from a cleaved reporter to produce a detectable change in pH, in accordance with some embodiments.

DETAILED DESCRIPTION

[0081] The present disclosure provides devices, systems, and methods for nucleic acid target detection. The systems and methods of the present disclosure can be implemented using the devices that are configured for programmable nuclease-based detection. In some embodiments, the devices can be configured for single reaction detection. In some embodiments, the devices can be disposable devices. The devices disclosed herein can be particularly well suited for carrying out highly efficient, rapid, and accurate reactions for detecting whether a target is present in a sample. The target can comprise a target sequence or target nucleic acid. As used herein, a target can be referred to interchangeably as a target nucleic acid. Further, a target can be referred to as a target amplicon or a target nucleic acid amplicon if such target undergoes amplification (e.g., through a thermocycling process as described elsewhere herein). The target nucleic acid can be a portion of a nucleic acid of interest, e.g., a target nucleic acid from any plant, animal, virus, or microbe of interest. The devices provided herein can be used to perform rapid tests in a single integrated system.

[0082] The target nucleic acid can be a nucleic acid or a portion of a nucleic acid from a pathogen, virus, bacterium, fungi, protozoa, worm, or other agent(s) or organism(s) responsible for and/or related to a disease or condition in living organisms (e.g., humans, animals, plants, crops, and the like). The target nucleic acid can be a nucleic acid, or a portion thereof. The target nucleic acid can be a portion of a nucleic acid from a gene expressed in a cancer or genetic disorder in the sample. The target nucleic acid can be a portion of an RNA or DNA from any organism in the sample. In some embodiments, one or more programmable nucleases as disclosed herein can be activated to initiate trans cleavage activity of a reporter (also referred to herein as a reporter molecule). A programmable nuclease as disclosed herein can, in some cases, bind to a target sequence or target nucleic acid to initiate trans cleavage of a reporter. The programmable nuclease can be referred to as an RNA-activated programmable RNA nuclease. In some instances, the programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of an RNA reporter. Such a programmable nuclease can be referred to herein as a DNA-activated programmable RNA nuclease. In some cases, a programmable nuclease as described herein can be activated by a target RNA or a target DNA. For example, a programmable nuclease, e.g., a Cas enzyme, can be activated by a target RNA nucleic acid or a target DNA nucleic acid to cleave RNA reporters. In some embodiments, the Cas enzyme can bind to a target ssDNA which initiates trans cleavage of RNA reporters. In some instances, a programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of a DNA reporter, and this programmable nuclease can be referred to as a DNA-activated programmable DNA nuclease.

[0083] The nucleic acids described and referred to herein can comprise a plurality of base pairs. A base pair can be a biological unit comprising two nucleobases bound to each other by hydrogen bonds. Nucleobases can comprise adenine, guanine, cytosine, thymine, and/or uracil. In some cases, the nucleic acids described and referred to herein can comprise different base pairs. In some cases, the nucleic acids described and referred to herein can comprise one or more modified base pairs. The one or more modified base pairs can be produced when one or more base pairs undergo a chemical modification leading to new bases. The one or more modified base pairs can be, for example, Hypoxanthine, Inosine, Xanthine, Xanthosine, 7-Methylguanine, 7- Methylguanosine, 5,6-Dihydrouracil, Dihydrouridine, 5-Methylcytosine, 5-Methylcytidine, 5- hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxylcytosine (5caC).

[0084] The programmable nuclease can become activated after binding of a guide nucleic acid that is complexed with the programmable nuclease with a target nucleic acid, and the activated programmable nuclease can cleave the target nucleic acid, which can result in a trans cleavage activity. Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the target nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released or separated from the reporter and can directly or indirectly generate a detectable signal. The reporter and/or the detection moiety can be immobilized on a support medium. Often the detection moiety is at least one of a fluorophore, a dye, a polypeptide, or a nucleic acid. Sometimes the detection moiety binds to a capture molecule on the support medium to be immobilized. The detectable signal can be visualized on the support medium to assess the presence or concentration of one or more target nucleic acids associated with an ailment, such as a disease, cancer, or genetic disorder.

[0085] The systems and methods of the present disclosure can be implemented using a device that is compatible with any type of programmable nuclease that is human-engineered or naturally occurring. The programmable nuclease can comprise a nuclease that is capable of being activated when complexed with a guide nucleic acid and a target nucleic acid segment or a portion thereof. A programmable nuclease can become activated when complexed with a guide nucleic acid and a target sequence of a target gene of interest. The programmable nuclease can be activated upon binding of a guide nucleic acid to a target nucleic acid and can exhibit or enable trans cleavage activity once activated. In any instances or embodiments where a CRISPR-based programmable nuclease is described or used, it is recognized herein that any other type of programmable nuclease can be used in addition to or in substitution of such CRISPR-based programmable nuclease.

[0086] The systems and methods of the present disclosure can be implemented using a device that is compatible with a plurality of programmable nucleases. The device can comprise a plurality of programmable nuclease probes comprising the plurality of programmable nucleases and one or more corresponding guide nucleic acids. The plurality of programmable nuclease probes can be the same. Alternatively, the plurality of programmable nuclease probes can be different. For example, the plurality of programmable nuclease probes can comprise different programmable nucleases and/or different guide nucleic acids associated with the programmable nucleases.

Microfluidic Device for Nucleic Acid Target Detection

[0087] Disclosed herein are microfluidic devices and uses thereof, e.g., use for detection of target nucleic acids. Devices described herein can be used for a programmable nuclease-based detection (e.g., DETECTR) assay. In some embodiments, the devices are compatible with multiplex lateral flow detection. In some embodiments, the devices are configured to perform one or more of the reactions described herein (e.g., amplification, detection, etc.) in separate chambers. In at least some instances, isolating portions of a liquid sample for detection of different target nucleic acids may facilitate multiplexing (e.g., by air gaps separating the liquid contents of various chambers during a reaction). In some embodiments, the devices described herein can be used in combination with enzyme-based methods for signal amplification of a binding event between one or more programmable nuclease probes and one or more target nucleic acids. In some embodiments, signal detection is performed on the device (e.g., in a reaction chamber, or in a detection chamber connected to the reaction chamber). In some embodiments, the device is configured to allow removal of the contents of a reaction chamber to perform a signal detection step. Methods for signal detection compatible with the devices are also disclosed herein.

[0088] In one aspect, described herein is a microfluidic device comprising: a sample interface configured to receive a sample; a first actuator configured to provide positive pressure to an upstream portion of the device proximate to the sample interface; a second actuator configured to provide negative pressure to a downstream portion of the device distal to the sample interface; a heating channel in fluid communication with the sample interface and first actuator, wherein the heating channel comprises a first portion in a first plane and a second portion in a second plane parallel to the first plane; a first heating element disposed between and in thermal contact with the first and second portions of the heating channel; a reagent mixing chamber fluidically connected downstream of the heating channel by a first valve; a plurality of reaction chambers in fluid communication with the second actuator; a channel multiplexing unit fluidically connected downstream of the mixing chamber by a second valve, wherein the channel multiplexing unit comprises a main channel and a plurality of side channels, wherein each of the side channels is in fluid communication with one inlet of a plurality of inlets positioned along the main channel, and wherein each of the side channels comprises an outlet in fluid communication with one of the plurality of reaction chambers; a first valve actuator configured to actuate the first valve; and a second valve actuator configured to actuate the second valve.

[0089] FIGS. 1A-1B illustrate front perspective views of a microfluidic device 100 for programmable nuclease-based detection, in accordance with some embodiments. FIG. 1A shows an exploded view of the device 100, for user operation. In some embodiments, the device 100 includes a first actuator 102 for providing positive pressure to the upstream portion 112 of the device proximate to a sample interface at sample input 101, wherein sample input 101 may comprise a well, a chamber, a channel, an inlet, or any other appropriate orifice for a sample downstream of an input chamber 107, and a second actuator 103 for providing negative pressure to a downstream portion 113 of the device 100 distal to the sample interface. In some embodiments, input chamber 107 can be configured to enable insertion of various types of actuators, such as a plunger, a spring-actuated plunger, or a spring mechanism, used for moving the sample into sample input 101 and the channels therebeyond. The actuators cause actuation of the sample in the form of liquid, herein sample liquid, to flow through the system and react with various reagents, which include but may not be limited to a programmable nuclease, a guide nucleic acid, and a reporter. A target nucleic acid is detected in the form of a detectable signal as a result of the reaction between the sample liquid, or a portion thereof, and the programmable nuclease-based reagents, as described herein. In some embodiments, the first actuator 102 includes an input plunger snap lock 104. In some embodiments, second actuator 103 is adjacent to a vacuum chamber 124 for removing air and/or pressure from the device 100 for providing the negative pressure to the liquid sample. In some embodiments, at least one of first actuator 102 and second actuator 103 is a sliding unit. In some embodiments, the upstream portion 112 is in a first plane and the downstream portion 113 of the device 100 is in a second plane parallel to the first plane. In some embodiments, a volume of the sample fluid, or sample volume, is about 200 pL to about 500 pL, about 250 pL to about 400 pL, about 275 pL to about 350 pL, or about 300 pL to about 320 pL. In some embodiments, the sample fluid, or sample volume, is about 310 pL. [0090] In some embodiments, device 100 includes status LEDs 105a-c for indicating device status such as on, if there is an error, if the device 100 is in use or complete, etc. In some embodiments, device 100 includes at least one temperature probe 106 for measuring and/or controlling the temperature of device 100 at different locations. In some embodiments, device 100 includes a programming and diagnostics connector 109 for programming a computing unit operably connected to the device 100. In some embodiments, device 100 includes a power supply 108. In some embodiments, power supply 108 comprises a battery or a plurality of batteries. The power supply 108 may comprise an alkaline battery(s), a paper-based battery(s) as shown in FIG. 46, or a coin battery, a capacitor, or other similarly portable power source. For example, the power supply 108 may comprise two AA batteries as shown in FIG. 1A. Alternatively, or in combination, the power supply 108 may comprise one or more paper batteries, e.g., one paper battery per heating element and/or light source within the device 100, as shown in FIG. 46. The one or more batteries may be used to power any number of features on the device including, but not limited to, one or more heat sources or heating elements, one or more light sources, one or more detectors or sensors, one or more computing units, one or more valves and/or valve actuators, one or more feedback elements (e.g., thermistors, etc.), or the like. [0091] FIG. IB shows a perspective view of the assembled device 100, and FIG. 1C shows a cross-sectional view along line Y of FIG. 1A. In some embodiments, device 100 comprises a heating channel comprising a first portion 114a and a second portion 114b (e.g., as shown in at least FIGS. 3A and 3D), wherein the heating channel is in fluid communication with the sample interface and first actuator 102. In some embodiments, first portion 114a is in the first plane and second portion 114b is in the second plane parallel to the first plane, as shown in FIG. 1C. In some embodiments, device 100 comprises a first heating element 116 in thermal contact with both the first portion 114a and second portion 114b of the heating channel. In some embodiments, the first heating element 116 is shaped according to a shape the first portion 114a and the second portion 114b of the heating channel to facilitate thermal contact therewith (e.g., serpentine, spiral, and/or a combination of both configurations, etc.). In some embodiments, first heating element 116 comprises two heater traces layered onto opposite sides of a printed circuit board (PCB) 110 and connected through a via 118, wherein the first heating element 116 is disposed between and in thermal contact with the first portion 114a and second portion 114b of the heating channel. In some embodiments, first portion 114a and second portion 114b are fluidically connected by a bridging hole 115 that crosses PCB 110. In some embodiments, first heating element 116 is controllable by the computing unit. In some embodiments, first heating element 116 comprises two or more heating elements that are not connected through a via, and are individually controllable by the computing unit. In some embodiments, at least one of first portion 114a and second portion 114b of the heating channel has a serpentine configuration or a spiral configuration. In some embodiments, first heating element 116 is configured to trace in alignment with at least one of the first portion 114a and second portion 114b of the heating channel.

[0092] Traditional heating elements on PCBs positioned for transferring heat to heating channels are often inefficient in heat utilization due to heat loss through the PCB side, or backside, of the heating elements. Therefore, the positioning of each of the first portion 114a and second portion 114b of the heating channel provides a method of increased heat efficiency, as heat that would have ordinarily dissipated from the PCB is now captured by or in thermal communication with an opposite portion, either first portion 114a or second portion 114b of the heating channel, thereby minimizing heat loss. In some embodiments, first heating element 116 is regulated by a thermistor. In some embodiments, fluid (e.g., a sample liquid) is retained in the heating channel until the sample has been heated to a target temperature or held at a target temperature for a desired period of time. Retention in the heating channel may be facilitated by a valve 122a. In some embodiments, the liquid within the heating channel contains lysis reagents in addition to the sample liquid, and is retained in the heating channel for a sufficient length of time to lyse one or more components of the sample (e.g., cells or viral particles).

[0093] FIG. 2 illustrates a rear view of the exemplary microfluidic device 100 for programmable nuclease-based detection. In some embodiments, device 100 optionally comprises vacuum plunger alignment features 117 that align second actuator 103 with PCB 110.

[0094] Microfluidic device 100 also optionally comprises a channel multiplexing unit 125. FIGS. 3A-3C illustrate detailed views of a front, back, and top side of upstream portion 112, respectively, and FIGS. 3D-3F illustrate detailed views of a front, back, and top side of downstream portion 113, respectively. FIG. 3G shows front and back views of an exemplary heating channel having a serpentine configuration, with an upstream portion 112 on one side, a downstream portion 113 on the other side, in which the upstream and downstream portions sandwich a first heating element 116 therebetween. In some embodiments, device 100 comprises a reagent mixing chamber 121 fluidically connected downstream of the heating channel by a first valve 122a (e.g., as shown in at least FIG. 3D). In some embodiments, the reagent mixing chamber 121 comprises one or more reagents for sample processing, such as for inactivating one or more lysis reagents active in a previous lysis step. In some embodiments, said previous lysis step can be performed using lysis reagents. Non-limiting examples of lysis reagents, and reagents for the inactivation thereof are provided herein. In some embodiments, lysis reagents are included in sample input 101, or are added through a separate input chamber that is fluidly connected thereto. In some embodiments, fluid is retained in the mixing chamber 121 for a desired period of time to facilitate adequate mixing, which retention may be facilitated by a valve 122b. In some embodiments, a channel multiplexing unit 125 is fluidically connected downstream of the reagent mixing chamber 121 by a mixing channel 123 and a second valve 122b. In some embodiments, each of the first and second valves 122a-122b are actuated by a first valve actuator 140a and second valve actuator 140b, respectively (e.g., as shown in at least FIG. 3G). In some embodiments, a plurality of reaction chambers 126a-126f are each fluidically connected to the channel multiplexing unit 125, wherein the reaction chambers 126 are further in fluid communication with the second actuator 103 such that the second actuator 103 is configured to pull, via negative pressure, the reaction fluid from the reagent mixing chamber 121 to each of the reaction chambers 126a-126f (e.g., as shown in at least FIG. 3F). In some embodiments, reaction chambers 126a-126f can comprise any combination of various reagents prior to the addition of a sample, including a programmable nuclease, a guide nucleic acid, a reporter, or a combination thereof. In some embodiments, the reaction chambers 126a-126f are preloaded with guide nucleic acids, and the programmable nuclease and/or reporters are either preloaded along with the guide nucleic acids or preloaded to another location of the device, such as the mixing chamber 121. In some embodiments, mixing chamber 121 can comprise any combination of said reagents. In some embodiments, the mixing chamber 121 can comprise any combination of said reagents and reaction chambers 126a-126f can include a remainder of said reagents. As reaction fluid enters reaction chambers 126a-126f, each fluid is available for detection of a detectable signal when a target nucleic acid is present in the sample, as described herein. In some embodiments, each of the plurality of reaction chambers 126a-126f is layered between the PCB 110 and each of a plurality of hydrophobic venting membranes, wherein each of the hydrophobic venting membranes is positioned between a corresponding reaction chamber 126 and vacuum chamber 124. In some embodiments, downstream portion 113 further comprises a self-sealing vent 137 in fluid communication with a distal end of second portion 114b, wherein the vent 137 is configured to seal when wetted with sample liquid. In some embodiments, each of the reaction chambers 126a-126f is in thermal contact with at least one of a plurality of heating elements to facilitate further reaction processes of the reaction fluid (such as a detection reaction in the reaction chambers 126a-126f). The heating elements may be configured to surround the reaction chambers 126a-126f in order to provide integrated, sustained, even heating during isothermal amplification and/or programmable nuclease-based detection reactions. In some embodiments, each of the plurality of heating elements is regulated by a thermistor.

[0095] In some embodiments, first heating element 116 comprises any shape relative to at least one of first portion 114a and second portion 114b of the heating channel that achieves a desired heating profile within or along the heating channel. In some embodiments, first heating element 116 is shaped to directly correspond to a shape of at least one of the first portion 114a and second portion 114b of the heating channel. In some embodiments, first heating element 116 occupies a greater surface area of the device relative to at least one of first portion 114a and second portion 114b of the heating channel. In some embodiments, the shape of the first heating element 116 both corresponds to a shape of, and occupies a greater surface area of the device relative to, at least one of first portion 114a and second portion 114b of the heating channel. FIG. 43 illustrates a front view of a microfluidic device for programmable nuclease-based detection, in accordance with some embodiments, with a heating channel occupying a surface area smaller than that occupied by a heater trace of a first heating element 116, and in which both have a serpentine shape. In some embodiments, this allows a fluidic pathway of the heating channel to avoid potential interfacial areas with lower temperatures, improving heating uniformity of at least one of first portion 114a and second portion 114b.

[0096] In some embodiments, first heating element 116 is a heater trace with a serpentine configuration that is bent to form right angles near the ends of the serpentine turns, forming a bent serpentine path, as shown in FIGS. 44A-44C. FIG. 44A shows a front view of the bent serpentine heater trace without a heating channel. FIG. 44B shows a front view of the bent serpentine heater trace with a first portion 114a of a heating channel with a serpentine configuration. FIG. 44C shows a front view of a device utilizing a first heating element 116 comprising a bent serpentine heater trace, wherein a first portion 114a of a heating channel comprises a spiral configuration. In some embodiments, the long axis of the heater trace along an outside perimeter is perpendicular to the long axis of the heater trace along a central portion thereof. It should be noted that the heating element 116 is not limited to any particular size or shape, and may be selected to obtain a desired heating profile within at least one of first portion 114a and second portion 114b of the heating channel.

[0097] In some embodiments, channel multiplexing unit 125 comprises a main channel 131 and a plurality of side channels 132a-132f, wherein each of the side channels 132a-132f is in fluid communication with one inlet of a plurality of inlets 133a-133f positioned along the main channel 131. In some embodiments, each of the side channels 132a-132f comprises an outlet 134a-134f, wherein each outlet is in fluid communication with a corresponding one of the plurality of reaction chambers 126a-126f.

[0098] In some embodiments, the device comprises one or more flow-restriction elements 130a- 130f at one or more points along the main channel 131. In some embodiments, flow restriction elements 130a-130f are arranged such that the last reaction chamber along the main channel 131 is the last to fill. For example, one or more flow restriction elements 130a-130f may be placed along the main channel 131 between an inlet 133a-133f to the second to last reaction chamber and the last reaction chamber 126f. Examples of flow restriction elements 130a-130f include changes (e.g., decreases) in cross-sectional area from an upstream portion of the main channel 131 to a downstream portion of the main channel 131, and/or porous frits at downstream reaction chamber inlets 133a-133f having smaller pore sizes than frits at upstream chamber inlets. In some embodiments, main channel 131 is serially divided into a plurality of subchannel portions 135a-135e and a tolerance channel 136. In some embodiments, each of the plurality of inlets 133a-133f comprises a porous frit for restricting flow.

[0099] In some embodiments, the main channel 131 is designed to ensure each of reaction chambers 126a-126f finishes in order from first to last such that the last chamber (e.g., for containing a control reaction), in this case 126f, is the last to finish filling with sample liquid. This way, if there is a shortage of sample liquid, the control chamber 126f will reflect the shortage. Even though chambers may finish filling in a desired order, two or more chambers may be filling in parallel at a given point in time. In some embodiments, filling order is implemented by designing each of the subchannel portions 135a-135e and tolerance channel 136 to subsequently reduce in cross-sectional area from a most proximal channel, e.g., subchannel 135a, to a most distal channel, e.g., tolerance channel 136. This change in cross-sectional area can be achieved by changing a corresponding width and/or depth of each of subchannels 135a- 135e and tolerance channel 136, thereby subsequently increasing in a flow resistance and therefore prioritizing filling most proximal of reaction chambers 126a-126f with sample liquid before most distal of reaction chambers 126a-126f.

[0100] In some embodiments, tolerance channel 136 is configured to accommodate variation of the sample volume by a desired volume (e.g., +/- 5 pL, 10 pL, 15 pL, 20 pL, 25 pL, 30 pL, 40 pL, 50 pL, or more) through the system while ensuring precise volumes enter each of the reaction chambers 126a-126f. In some embodiments, the variation accommodated by the tolerance channel is +/- 10 pL. Preferably, tolerance channel 136 is positioned at a distal end of the main channel 131, while subchannel portions 135a-135e are positioned proximal to tolerance channel 136. In some embodiments, tolerance channel 136 has a volume of 20 pL, and at a target sample volume of 310 pL the sample liquid is configured to fill halfway through tolerance channel 136, whereas a lower volumes tolerance channel 136 has less fluid and at higher volumes has more fluid. Preferably, the sample volume is greater than a combined volume of the plurality of side channels 132a-132f and the plurality of reaction chambers 126a-126f, and (b) less than the combined volume the plurality of 132a-132f, the plurality of reaction chambers 126a-126f, and the tolerance channel 136. In some embodiments, these volume ranges are effective to ensure that when each of the reaction chambers 126a-126f are filled, air gaps in the main channel 131 form to separate the inlets to each of the reaction chambers 126a-126f, thereby avoiding diffusion of reagents from one chamber to another.

[0101] In some embodiments, the device includes at least one lateral flow assay (LFA) strip 150, such as is shown in FIG. 45. Each reaction chamber 126a-126f may be configured to interface with a detection region comprising an LFA strip 150. In some embodiments, the detection region may be in fluid communication with the reaction chamber. In some embodiments, the detection region may be contacted to the reaction chamber 126a-126f after amplification and the programmable nuclease-based reactions have been performed in the reaction chamber 126a- 126f. In some embodiments, the LFA strips 150 may be configured to be inserted into the reaction chambers 126a-126f. In some embodiments, the LFA strips 150 may be configured to be inserted into the reaction chambers 126a-126f at the same time or at different times.

[0102] In some embodiments, one or more fluid channels directs a detection product of the programmable nuclease-based reactions from each reaction chamber 126a-126f to a different LFA strip 150. In some embodiments, the detection product can be directed by additional valves 122 as needed. In some embodiments, detection products from different reaction chambers 126a- 126f can be directed to the same LFA strip 150 using spatial multiplexing wherein multiple different target nucleic acids are detected at the same time, but the detection reactions for different targets carried out on LFA strip 150 are spatially separated. In some embodiments, detection products from multiple reaction chambers 126a-126f can be directed to multiple LFA strips 150 for detection (e.g., with detection products from different reaction chambers being directed to different LFA strips 150).

[0103] In some embodiments, a plurality of valves 122 are configured to restrict flow in a first direction through the one or more channels towards the sample interface. The plurality of valves 122 can be configured to selectively permit flow in a second direction through the one or more channels towards the reaction chamber 126a-126f. A first valve and a second valve 122a-122b of the plurality of valves 122 can be configured to physically, fluidically, and/or thermally isolate a first portion of the sample from a second portion of the sample when the first valve and the second valve 122a-122b are in a closed state. Nonlimiting examples of valves 122 include phase-change valves, wax valves, capillary valves, electrostatic valves, check valves, sliding valves, rotary valves, pneumatic valves, vacuum valves, pinch valves, and burst valves. One or more valves can control a flow or a movement of the sample through the channel. The one or more valves can comprise a check valve that is configured to restrict a movement of the sample or the one or more droplets such that the sample or the one or more droplets do not travel backwards towards an inlet portion of the channel. The plurality of valves 122 may all be the same type of valve (e.g., a thermally actuated valve). Alternatively, the plurality of valves 122 can comprise a mixture of valve types. A valve may be controlled by a valve actuator, the selection of which can depend on the type of valve. For example, a thermally actuated valve may be under control of a heating element.

[0104] FIG. 4A illustrates a detailed view of an embodiment of the plurality of valves 122. In some embodiments, first and second valves 122a-122b of the plurality of valves 122 are thermally actuated, such that each of the first and second valve actuators 140a- 140b comprises at least one heating element 141 in thermal contact with a respective valve 122. In this way, each of the plurality of valves can control when the sample liquid progresses from one section of the device 100 to another (e.g., from the heating channel to the mixing chamber 121, and from the mixing chamber 121 to the reaction chambers 126a-126f).

[0105] In some embodiments, each of the first and second valves 122a-122b comprises an upstream channel 144, a valve inlet channel 142 and a valve outlet channel 143. In some embodiments, a cross-sectional area of the valve inlet channel 142 is less than a cross-sectional area of the corresponding valve outlet channel 143. In some embodiments, each valve 122 comprises an upper section 145, a middle section 146, and a lower section 147, wherein each section 145-147 is in fluid communication with each other section. In some embodiments, lower section 147 is relatively flat, such that a height of lower section 147 is equivalent to a height of the valve inlet channel 142. In some embodiments, the upper section 145, middle section 146, and lower section 147 of each of the plurality of valves 122 is filled with a material 148 configured to change between liquid and solid phases when heating by the heating elements 141 of the corresponding valve actuator 140.

[0106] In some embodiments, the material 148 is wax. In some embodiments, the wax can be natural or synthetic. Preferably, the material 148 changes phase between 50-57°C. When no heat is applied from each heating element 141, material 148 maintains a solid phase, restricting the sample liquid from flowing through each valve 122. As heat is applied from each heating element 141, the material 148 in the lower section 147 such that the material 148 melts, allowing sample liquid to flow through the valve 122 and solidify as it enters the valve outlet channel 143. Pressure in at least the valve inlet channel 142 and the valve outlet channel 143 force the material 148, once melted, out of the way to allow the flow. Due to the increase in cross- sectional area from the valve inlet channel 142 to the valve outlet channel 143, the material 148 solidifies in a manner such that a risk of clogging of the valve outlet channel 143 is reduced, thereby allowing sample liquid to continuing flowing through the valve outlet channel 143. This mechanism provides an efficient method for advancing sample liquid to subsequent regions in the channel when desired. Further, the design of valve 122 for phase transition materials such as wax promotes a decrease in manufacturing time for high volume production of the microfluidic device 100, as the material 148 need only be filled from a top side, e.g., upper section 145, of the valve 122. Upper section 145, as illustrated in FIG. 4A, allows for the material 148 to subsequently dispense into the middle section 146 and lower section 147, the latter of which restricts the sample liquid or reaction fluid in a closed state, and upon melting, e.g., changing phase, opens to allow the respective fluid to flow through the valve. A fill amount of the material 148 is not critical as long as there is some fill above the lower section 147 to stop fluid from coming out of the valve through upper section 145. Upper section 145, middle section 146, and/or lower section 147 can be any of a variety of three-dimensional geometric shapes.

[0107] FIGS. 4B-4C illustrate perspective views of valve actuators 140 (e.g., heaters, in the case of thermally actuated valves). Nonlimiting examples of the additional valves 122 can comprise, for example, a phase-change valve, a wax valve, a capillary valve, an electrostatic valve, a check valve, a sliding valve, a rotary valve, a pneumatic valve, a vacuum valve, a pinch valve, or a burst valve. In some embodiments, the at least one valve actuator comprises a plunger, a spring- actuated plunger, or a spring mechanism. In some embodiments, at least one of the plurality of heating elements 141 is in thermal contact with at least one resistor 1411 on an opposite side 1102 of the PCB 110. This thermal contact can be achieved by vias 1412, connecting heating elements 141 on a valve side 1101 of the PCB 110 proximal to at least one of the plurality of valves 122 to heating elements 141 on the opposite side 1102 of the PCB 110. As the at least one resistor 1411 heats up when triggered by the computing unit, vias 1412 allow heat to transfer from heating elements 141 on opposite side 1102 to heating elements 141 on valve side 1101,

- l- generating heat from heating elements 141 on valve side 1101 and thereby inducing phase change, or melting, material 148 in the corresponding valve 122.

[0108] FIGS. 5A-5C illustrate front views of a further microfluidic device 300 for programmable nuclease-based detection, in accordance with some embodiments. Microfluidic device 300 is similar to microfluidic device 100, though with differences in how various elements are oriented with respect to each other and how actuation is achieved. FIG. 5A shows a perspective view of the device 300, for user input and operation. In some embodiments, the device 300 includes an upstream portion cover 318 that covers an upstream portion 312 of a heating channel. In some embodiments, device 300 comprises a downstream portion cover 319. In some embodiments, the device 300 includes a first actuator 302 for providing positive pressure to the upstream portion 312 of the device proximate to a sample interface at sample input well 301 and a second actuator 303 for providing negative pressure to a downstream portion 313 of a heating channel of the device 300 distal to the sample interface, wherein in this embodiment, the first actuator 302 and the second actuator 303 are each oriented perpendicular to the upstream portion 312 and the downstream portion 313. In some embodiments, the actuators cause actuation of the sample in the form of fluid, herein sample liquid, to flow through the system and react with various reagents, such as reagents introduced through a reagent input 304 for inactivating one or more lysis reagents active in a previous lysis step, or one or more of a programmable nuclease, a guide nucleic acid, and a reporter in one or more reaction chambers 330a-330f. A target nucleic acid is detected in the form of a detectable signal as a result of a detection reaction between the sample liquid, or a portion thereof, and the programmable nuclease-based reagents, as described herein. In some embodiments, first actuator 302 includes a reagent input chamber 304. In some embodiments, input well 301 can contain the lysis reagents. In other embodiments, the lysis reagents can be included in a separate input chamber that is fluidically connected to the input well 304. In some embodiments, second actuator 303 is adjacent to a vacuum chamber 324 for removing air or pressure from the device 300 for providing the negative pressure to the fluid sample. In some embodiments, at least one of first actuator 302 and second actuator 303 is a translatable unit. In some embodiments, the translatable unit can be a sliding unit.

[0109] FIG. 5B shows a perspective view of the device 300, FIG. 5C shows a perspective view of the device 300 wherein a top surface of each of upstream portion cover 318 and downstream portion cover 319 are transparent, and FIG. 5D shows a top view of the device 300 without upstream portion cover 318. In some embodiments, device 300 comprises a heating channel 314, wherein the heating channel is in fluid communication with the sample interface and first actuator 302. In some embodiments, device 300 comprises a first heating element 316, wherein the first heating element 316 comprises a heater trace layered onto a PCB 310, wherein the first heating element 316 is disposed between and in thermal contact with the heating channel 314. In some embodiments, first heating element 316 is substantially similar to first heating element 116 as shown in FIG. 1A. In some embodiments, first heating element 316 is controllable by the computing unit. In some embodiments, heating channel 314 has a serpentine configuration. In some embodiments, first heating element 316 is configured to trace in alignment with heating channel 314. In some embodiments, first heating element 316 is regulated by a thermistor. In some embodiments, the heating channel passes through two planes with the heating trace located therebetween an in thermal contact with both, as in the device 100 described above. In some embodiments, fluid is retained in the heating channel until the sample has been heated to a target temperature or held at a target temperature for a desired period of time. Retention in the heating channel may be facilitated by a valve 323. In some embodiments, the fluid contains lysis reagents, and is retained in the heating channel for a sufficient length of time to lyse components of the sample (e.g., cells or viral particles).

[0110] In some embodiments, reagent input 304 comprises a reagent interface 305 and a reagent channel 306. In some embodiments, the reagent input 304 comprises various reagents for sample processing, such as for inactivating one or more lysis reagents active in a previous lysis step, such that the sample liquid mixes with the reagents to form a reaction fluid at a downstream location. In some embodiments, device 300 comprises a reagent mixing unit 320 connected downstream of an intersection 321 by a first valve 322 configured to restrict or completely inhibit a flow of the sample liquid through one or more sections of the channel. In some embodiments, the intersection 321 is fluidically connected downstream, in parallel, of the heating channel 314 by a second valve 323, and of the reagent channel 306 by a third valve 324. In some embodiments, reagent channel 306 further comprises additional flow restrictors 307 configured to restrict a movement of the sample liquid such that the sample liquid does not travel backwards towards an inlet portion of the reagent channel 306.

[OHl] The sample liquid can be circulated through the reagent mixing unit 320 to produce a reaction fluid using a bulk circulation mechanism that is configured to stir the remixed sample around. The one or more sample liquid flow paths can be used to target delivery of at least a portion of the mixed fluid to one or more detection regions associated with the one or more programmable nucleases. The fluid can be circulated through the reagent mixing unit 320 along one or more desired fluid flow paths with aid of a piezoelectric device.

[0112] In some embodiments, a channel multiplexing unit 326 is fluidically connected downstream of the reagent mixing unit 320 by at least one of a fourth valve 325 and a first selfsealing vent 327 that seals upon being wetted to prevent backward travel of fluid. In some embodiments, each of the first, second, third and fourth valves 322-325 is actuated by a first, second, third and fourth valve actuator, respectively, similarly to valves and actuators described for device 100.

[0113] In some embodiments, a plurality of reaction chambers 330a-330f are each fluidically connected downstream of the channel multiplexing unit 326. In some embodiments, the channel multiplexing unit 326 comprises a plurality of side channels 331 that each subsequently splits into a plurality of subchannels 332, wherein each of the plurality of subchannels comprises an outlet in fluid communication with one of the plurality of reaction chambers 330a-330f. In some embodiments, a plurality of detection chambers 333a-333f are fluidically connected downstream of a corresponding reaction chamber 330. In some embodiments, the plurality of detection chambers 333a-333f are fluidically connected downstream of a corresponding reaction chamber 330 via at least one of a plurality of additional valves 335 similar to valves 322-325. In some embodiments, each of the additional valves 335 is actuated by at least one of an additional valve actuator, similar to valves and actuators of device 100. In some embodiment, each of the plurality of detection chambers 333a-333f is further in fluid communication with the second actuator 303 such that the second actuator 303 is configured to pull, via negative pressure, the reaction fluid from the detection chambers 333a-333f. As reaction fluid enters detection chambers 333a-333f, each fluid is available for detection of a detectable signal when a target nucleic acid is present in the sample, as described herein. In some embodiments, each of a plurality of additional flow restrictors 307 is positioned between a corresponding detection chamber 333 and vacuum chamber 334, wherein each of the flow restrictors 307 is configured to seal when wetted with reaction fluid. In some embodiments, each of the reaction chambers 330a- 330f is in thermal contact with at least one of a plurality of heating elements to facilitate further reaction processes of the reaction fluid. In some embodiments, each of the plurality of heating elements is regulated by a thermistor. In some embodiments, each of the plurality of heating elements is controllable by the computing unit.

[0114] In some embodiments, each of the additional valves 335 is configured to selectively permit flow in a second direction through the one or more channels. In some embodiments, each of the additional valves 335 is configured to physically, fluidically, or thermally isolate a first portion of the sample from a second portion of the sample when at least one of the additional valves 335 is in a closed state. Nonlimiting examples of valves 335 include phase-change valves, wax valves, capillary valves, electrostatic valves, check valves, sliding valves, rotary valves, pneumatic valves, vacuum valves, pinch valves, and burst valves. One or more valves can control a flow or a movement of the sample through the channel. The one or more valves can comprise a check valve that is configured to restrict a movement of the sample or the one or more droplets such that the sample or the one or more droplets do not travel backwards towards an inlet portion of the channel. In some embodiments, each of the additional valves 335 is equivalent to any of valves 122. The additional valves 335 may all be the same type of valve (e.g., a thermally actuated valve). Alternatively, the additional valves 335 can comprise a mixture of valve types. A valve may be controlled by a valve actuator, the selection of which can depend on the type of valve. In some embodiments, one or more of the various valves are thermally actuated valves, and the respective one or more actuators comprise heaters, such as described with respect to device 100.

[0115] In some embodiments, the power supply 108 may comprise one or more paper batteries or capacitor power supply 108a as shown in FIG. 46. For example, the device 100 may comprise one paper battery per heating element and/or light source within the device 100, a single paper battery to power all heaters in the device, or a plurality of paper batteries each connected to one or more heaters. The one or more paper batteries 108a may be used to power any number of features on the device including, but not limited to, one or more heat sources or heating elements, one or more light sources, one or more detectors or sensors, one or more computing units, one or more valves and/or valve actuators, one or more feedback elements (e.g., thermistors, etc.), or the like.

[0116] In some embodiments, the battery 108a utilizes a water-based redox reaction. In some embodiments, the water-based redox reaction is initiated when the first actuator 102 primes device 100 with positive and negative pressure. In some embodiments, the priming punctures a capsule or otherwise opens an aqueous fluid-containing chamber. In some embodiments, this puncturing releases fluid that wets or causes wetting of the paper battery or the capacitor power supply 108a, providing power to at least one of a plurality of heating elements 116 to open valves and heat reaction chambers/channels as described herein. In some embodiments, the power provided by the power supply 108 or 108a is used to (a) thermally actuate valves in system 100, such as valves 122, and/or (b) stimulate heating elements in thermal contact with reaction chambers, such as reaction chambers 126a-126f. In some embodiments, activating the power supply (e.g., wetting a paper battery in response to priming by the first actuator 102) initiates a sequence in which flow through the device 100, and temperature and residence times within certain regions (e.g., the heating channel and reaction chambers) is regulated by heat from the various heating elements as powered by the power supply. In some embodiments, a computing unit is connected to the power supply, and is programmed to control the timing of activation of the various heaters of the device 100, which in turn regulates the timing a flow of fluid within the device 100.

[0117] FIG. 47 illustrates a further exemplary device 4800 for a programmable nuclease-based detection using an LFA strip, in accordance with some embodiments. In some embodiments, a sample (e.g., a sample solution) is tested for the presence of at least a first and second target nucleic acid sequences. In some embodiments, the sample is introduced into a sample input 4801 (e.g., a well, a chamber, a channel, or an inlet) at a proximal location of a device 4800. In some embodiments, the device 4800 comprises a lyophilization reservoir 4802 in fluidic communication with sample input 4801 such that the sample is configured to flow from the sample input 4801 into the lyophilization reservoir 4802 when pressure is applied to sample input 4801. In some embodiments, sample input 4801 and lyophilization reservoir 4802 are fluidically separated by a valve 4810. In some embodiments, valve 4810 is a thermally actuated valve as described herein.

[0118] In some embodiments, the lyophilization reservoir 4802 comprises one or more sample preparation reagents. In some embodiments, the sample preparation reagents may be dried reagents. Dried reagents may comprise solids and/or semi-solids. In some embodiments, the sample preparation reagents may comprise lyophilized reagents (e.g., amplification reagents, programmable nucleases, buffers, excipients, etc.). In some embodiments, lyophilized reagents may be in the form of lyophilized beads, spheres, and/or particulates. In some embodiments, the lyophilized bead, sphere, and/or particulate may comprise either single or multiple compounds. In some embodiments, the lyophilized bead, sphere, and/or particulate may have diameters between about 0.5 millimeters to about 5 millimeters in diameter.

[0119] In some embodiments, the device 4800 comprises a reaction chamber 4803 in fluidic communication with lyophilization reservoir 4802, wherein reaction chamber 4803 comprises one or more locations 4803(l-N), each of the one or more locations comprising a different guide nucleic acid immobilized to a surface of the reaction chamber 4803. In some embodiments, each guide nucleic acid comprises an sgRNA. The reaction chamber 4803 may be heated or thermocycled in order to facilitate programmable nuclease-based reporter cleavage, sample preparation (e.g., cell lysis, etc.), and/or nucleic acid amplification as described herein.

[0120] In some embodiments, the guide nucleic acids form part of programmable nuclease probes immobilized to a surface. In some embodiments, a guide nucleic acid is designed to specifically bind to a target nucleic acid in the sample. In some embodiments, each different guide nucleic acid is complimentary to a different target nucleic acid sequence that may or may not be present in the sample. In some embodiments, in addition to the programmable nuclease probes containing guide nucleic acids, each location is functionalized with one or more reporter probes having distinct functional groups, which are different from the functional groups of reporters at other locations. In some embodiments, the reporter probes are in close enough proximity to be cleaved by the programmable nuclease probes upon activation. In some embodiments, binding between a particular guide nucleic acid and the corresponding target nucleic acid is effective to activate the programmable nuclease probe, which in turn cleaves a reporter, with reporter cleavage releasing a detectable product. In some embodiments, the reporter is functionalized with a label.

[0121] In some embodiments, the device 4800 includes at least one lateral flow assay (LFA) strip 4804. The mode of detection on the LFA strip will depend on detectable product selected in the design of the reporters. Non-limiting examples of multiplex detection using lateral flow strips are described herein, such as in connection with FIGS. 18A-22, which may form part of a device as illustrated in FIG. 47. In some embodiments, the LFA strip 4804 contains a detection region comprising one or more detection spots 4804(l-N), where each detection spot contains a different capture moiety (e.g., a complementary oligo for capture of a nucleic acid detectable product, or an antibody for capture of antigen detectable product) to achieve multiplexing capabilities. In some embodiments, each capture moiety specifically binds to a particular detectable product of one of the reporters. In some embodiments, reaction chamber 4803 and LFA strip 4804 are fluidically separated by a valve 4811. In some embodiments, valve 4811 is a thermally actuated valve as described herein.

[0122] In some embodiments, the solution containing cleaved reporters is contacted to a sample pad 4805 of the lateral flow assay strip 4804 along with chase buffer. In some embodiments, the sample pad 4805 has one or more flowing capture probes (e.g., anti-biotin-AuNP) disposed thereon. In some embodiments, the sample solution containing the detectable product of the cleaved reporters, along with the chase buffer, flow across the sample pad 4805, where the detectable products are bound to conjugates (e.g., anti-biotin-gold nanoparticles). In some embodiments, the solution containing the cleaved reporters is contacted to the sample pad 4805 being drawn from a chamber in fluid connection with the sample pad 4805, such as by the application of upstream pressure, downstream vacuum, or both. In some embodiments, the solution containing the detectable products is contacted to the sample pad 4805 by being drawn from a chamber in which the assay resulting in the cleaved reporter solution occurs. In some embodiments, the solution is drawn into and out of the sample pad 4805 by capillary action, or wicking. In some embodiments, the capillary action or wicking is caused by the liquid being drawn into an absorption pad. In some embodiments, the capillary action or wicking is caused by the liquid being drawn into an absorption pad, not requiring electrical power. In some cases, the solution is drawn into or out of the sample pad 4805 by a pressure gradient. In some embodiments, the conjugates bound to detectable products will selectively bind to the one or more detection spot 4804(l-N) containing a corresponding capture moiety, thus indicating the presence of the target nucleic acid sequence in the sample. The location and/or signal type of the detection spot 4804(l-N) indicates the detectable product being detected, which in turn indicates the reporter that was cleaved, thereby indicating which programmable nuclease probe was activated and which corresponding target nucleic acid(s) is in the sample. In this manner, for some embodiments, parallel detection of two or more target nucleic acid sequences present in a multiplexed sample is enabled.

[0123] In some embodiments, the LFA strip 4804 may comprise a control spot or line 4806. In some embodiments, the control line 4806 may comprise anti-IgG that is complimentary to all flowing capture probes. In some embodiments, when a flowing capture probe does not bind to a detectable product, the flowing capture probe will be captured by the anti-IgG on the control line, ensuring the user that the device is working properly even if no signal is read from the test line. In some embodiments, the control line 4806 comprises a capture moiety for a detectable product released from cleavage of a control reporter following activation of a control programmable nuclease probe for a sequence expected to be in any sample, or added to the sample.

[0124] In some embodiments, device 4800 includes one or more power source such as a battery 4807. In some embodiments, the one or more battery may be used to power valve 4810 and valve 4811. Nonlimiting examples of valves 4810 and 4811 include phase-change valves, wax valves, capillary valves, electrostatic valves, check valves, sliding valves, rotary valves, pneumatic valves, vacuum valves, pinch valves, and burst valves. The power source may comprise any of the power sources described herein.

Method of Use of Microfluidic Device for Nucleic Acid Target Detection

[0125] Disclosed herein is a method of microfluidic devices described herein (e.g., device 100 or 300) for nucleic acid target detection. In some embodiments, the method comprises applying a sample to the sample interface. In some embodiments, said applying forms a sample liquid. In some embodiments, the method can comprise sample collection. The method can further comprise sample preparation. In some embodiments, the method may comprise using a physical filter to filter one or more particles from the sample that do not comprise the at least one analyte of interest (e.g., a target nucleic acid). In some embodiments, the method may comprise lysing the sample before detecting the analyte. In some embodiments, the method may comprise performing enzyme (e.g., PK or savinase) inactivation on the sample. In some embodiments, the method may comprise performing heat inactivation on the sample. In some embodiments, the method may comprise performing nucleic acid purification on the sample. In some embodiments, the method may comprise contacting a plurality of sub-samples with a plurality of programmable nuclease probes comprising different guide RNAs. In some instances, the sample is diluted with a buffer or a fluid or concentrated prior to application to the detection system. In some embodiments, the sample can be provided manually to the detection device of the present disclosure. For example, a swab sample can be dipped into a solution and the sample/solution can be pipetted into the device. In other embodiments, the sample can be provided via an automated syringe. The automated syringe can be configured to control a flow rate at which the sample is provided to the detection device. The automated syringe can be configured to control a volume of the sample that is provided to the detection device over a predetermined period. In some embodiments, the sample can be provided directly to the detection device of the present disclosure. For example, a swab sample can be inserted into a sample chamber on the detection device. The sample can be prepared before one or more targets are detected within the sample. The sample preparation steps described herein can process a crude sample to generate a pure or purer sample. Sample preparation may comprise one or more physical or chemical processes, including, for example, nucleic acid purification, lysis, binding, washing, and/or eluting. In certain instances, sample preparation can comprise the following steps, including sample collection, nucleic acid purification, heat inactivation, enzyme inactivation, and/or base/acid lysis.

[0126] In some embodiments, the method further comprises actuating flow of the sample liquid through the heating channel to each of the reaction chambers. In some embodiments, the method of actuating comprises actuating using a plunger, a spring-actuated plunger, or a spring mechanism. In some embodiments, the actuation is manual. In some embodiments, actuation is configured to move the sample from the sample interface to the heating region via manual actuation of the first actuator. In some embodiments, the device is configured to be operated manually without electrical power. In some embodiments, actuation is achieved using a pneumatic pump, a sliding device, a rotary device, and/or a lateral flow device.

[0127] In some embodiments, the method further comprises reacting the sample liquid with the programmable nuclease, the guide nucleic acid, and the reporter. In some embodiments, the reagents described herein may include a composition for improving detection signal strength, detection reaction time, detection reaction efficiency, stability, solubility, or the like. In some embodiments, the reaction may generate a colorimetric signal, a fluorescent signal, an electrochemical signal, a chemiluminescent signal, or another type of signal. In some embodiments, the reaction may induce color-change in substances.

[0128] In some embodiments, the method further comprises detecting a detectable signal when a target nucleic acid is present in the sample. The method can further comprise using a programmable nuclease-based detection module to detect one or more targets (e.g., target sequences or target nucleic acids) in the sample. In some cases, the sample can be divided into a plurality of aliquots or subsamples to facilitate sample preparation and to enhance the detection capabilities of the devices of the present disclosure. In some cases, the sample is not divided into subsamples. In some embodiments, the detectable signal is a colorimetric signal, a fluorescent signal, an electrochemical signal, a chemiluminescent signal, or another type of signal. In some embodiments, the detectable signal may be a color-change in substances. In some embodiments, detection is achieved using a sensor or detector. In some embodiments, detection is achieved either directly or indirectly. Additional illustrative embodiments for detecting a target nucleic acid using devices described herein are provided herein.

Devices for Amplifying Signals using Positive Feedback Systems

[0129] Any of the devices described herein may be configured for amplifying a detection signal using a positive feedback system. In some embodiments, a device may comprise one or more compartments configured to: (a) bind a first nuclease (15101) with a first guide nucleic acid (15102) and a first target nucleic acid (15103) to generate a first complex; (b) release one or more second guide nucleic acids (15104) each comprising a nucleic acid tether (15106) by cleaving the nucleic acid tether(s) (15106) with the first complex; (c) bind the second guide nucleic acids (15104) each with a second nuclease (15101) and a second target nucleic acid (15108) to generate a plurality of second complexes; (d) cleave a plurality of reporters with the first and second complexes as described herein. Additional second complexes may be formed by further cleavage by the first and second complexes as described herein.

[0130] In some embodiments, a device may comprise one or more compartments comprising: (a) a first set of nucleases (15101) each comprising a first guide nucleic acid (15402), wherein each nuclease in the first set of nucleases is configured to bind with a first target nucleic acid (15103) and then cleave a plurality of nucleic acids comprising a first sequence; (b) a plurality of secondary target-specific guide nucleic acids (15104)) each comprising a nucleic acid tether (15106) comprising the first sequence; (c) a second set of nucleases (15101) each configured to bind with the secondary target-specific guide nucleic acid (15104) and a second target nucleic acid (15108) and then cleave a plurality of nucleic acids; (d) a plurality of second target nucleic acids (15108).

[0131] In some embodiments, one or more compartments may be in fluid communication, optical communication, thermal communication, or any combination thereof with one another. In some embodiments, one or more compartments may be arranged in a sequence. In some embodiments, one or more compartments may be arranged in parallel. In some embodiments, one or more compartments may be arranged in sequence, parallel, or both. In some embodiments, one or more compartments may comprise a well. In some embodiments, one or more compartments may comprise a flow strip. In some embodiments, one or more compartments may comprise a heating element.

[0132] In some embodiments, the device may be a handheld device. In some embodiments, the device may be point-of-need device. In some embodiments, the device may comprise any one of the device configurations described in this disclosure. In some embodiments, the device may comprise one or more parts of any one of the device configurations described herein.

Programmable Nucleases

[0133] Disclosed herein are programmable nucleases and uses thereof, e.g., detection and editing of target nucleic acids. In some cases, a programmable nuclease is capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment. A programmable nuclease can be capable of being activated when complexed with a guide nucleic acid and the target sequence. The programmable nuclease can be activated upon binding of the guide nucleic acid to its target nucleic acid and can non-specifically degrade a non-target nucleic acid in its environment. The programmable nuclease has trans cleavage activity once activated. A programmable nuclease can be a Cas protein (also referred to, interchangeably, as a Cas nuclease or Cas effector protein). A guide nucleic acid (e.g., crRNA) and Cas protein can form a CRISPR enzyme (also referred to herein as a programmable nuclease complex or probe).

[0134] In some embodiments, one or more programmable nucleases as disclosed herein can be activated to initiate trans cleavage activity of a reporter (also referred to herein as a reporter molecule). A programmable nuclease as disclosed herein can, in some cases, bind to a target sequence or target nucleic acid to initiate trans cleavage of a reporter. The programmable nuclease can be referred to as an RNA-activated programmable RNA nuclease. In some instances, the programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of an RNA reporter. Such a programmable nuclease can be referred to herein as a DNA-activated programmable RNA nuclease. In some cases, a programmable nuclease as described herein can be activated by a target RNA or a target DNA. For example, a programmable nuclease, e.g., a Cas enzyme, can be activated by a target RNA nucleic acid or a target DNA nucleic acid to cleave RNA reporters. In some embodiments, the programmable nuclease can bind to a target ssDNA which initiates trans cleavage of RNA reporters. In some instances, a programmable nuclease as disclosed herein can bind to a target DNA to initiate trans cleavage of a DNA reporter, and this programmable nuclease can be referred to as a DNA- activated programmable DNA nuclease.

[0135] As used herein, a programmable nuclease generally refers to any enzyme that can cleave nucleic acid. The programmable nuclease can be any enzyme that can be or has been designed, modified, or engineered by human contribution so that the enzyme targets or cleaves the nucleic acid in a sequence-specific manner. Programmable nucleases can include, for example, zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and/or RNA- guided nucleases such as the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) nucleases or Cpfl. Programmable nucleases can also include, for example, PfAgo and/or NgAgo.

[0136] ZFNs can cut genetic material in a sequence- specific matter and can be designed, or programmed, to target specific viral targets. A ZFN is composed of two domains: a DNA- binding zinc-finger protein linked to the Fokl nuclease domain. The DNA-binding zinc-finger protein is fused with the non-specific Fokl cleave domain to create ZFNs. The protein will typically dimerize for activity. Two ZFN monomers form an active nuclease; each monomer binds to adjacent half- sites on the target. The sequence specificity of ZFNs is determined by ZFPs. Each zinc-finger recognizes a 3-bp DNA sequence, and 3-6 zinc-fingers are used to generate a single ZFN subunit that binds to DNA sequences of 9-18 bp. The DNA-binding specificities of zinc-fingers is altered by mutagenesis. New ZFPs are programmed by modular assembly of pre-characterized zinc fingers.

[0137] Transcription activator-like effector nucleases (TALENs) can cut genetic material in a sequence-specific matter and can be designed, or programmed, to target specific viral targets. TALENs contain the Fokl nuclease domain at their carboxyl termini and a class of DNA binding domains known as transcription activator- like effectors (TALEs). TALENs are composed of tandem arrays of 33-35 amino acid repeats, each of which recognizes a single base-pair in the major groove of target viral DNA. The nucleotide specificity of a domain comes from the two amino acids at positions 12 and 13 where Asn-Asn, Asn-Ile, His-Asp and Asn-Gly recognize guanine, adenine, cytosine and thymine, respectively. That pattern allows one to program TALENs to target various nucleic acids.

[0138] The programmable nuclease can comprise any type of engineered enzyme. Alternatively, the programmable nuclease can comprise CRISPR enzymes derived from naturally occurring bacteria or phage. A programmable nuclease can be a Cas effector protein (also referred to, interchangeably, as a Cas nuclease). A guide nucleic acid (e.g., a crRNA) and Cas effector protein can form a CRISPR enzyme. The programmable nuclease can be a CRISPR-Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated) nucleoprotein complex with trans cleavage activity, which can be activated by binding of a guide nucleic acid with a target nucleic acid. The programmable nuclease can comprise one or more amino acid modifications. The programmable nuclease can be a nuclease derived from a CRISPR-Cas system. The programmable nuclease can be a nuclease derived from recombineering. In some embodiments, the programmable nuclease further comprises a Cas enzyme. In some embodiments, the Cas enzyme is selected from the group consisting of Casl2, Casl3, Casl4, Casl4a, Casl4al, and CasPhi.

[0139] In some cases, the programmable nuclease is Casl3. Sometimes the Casl3 is Casl3a, Casl3b, Casl3c, Casl3d, Casl3e, or Casl3f. In some cases, the programmable nuclease is Mad7 or Mad2. In some cases, the programmable nuclease is Casl2. Sometimes the Casl2 is Casl2a, Cas 12b, Cas 12c, Cas 12d, Casl2e, Casl2f, Cas 12g, Casl2h, Casl2i, Casl2j, or Cas 12k. In some cases, the Cas 12 can be a Cas 12 variant having the sequence of SEQ ID NO: 17 (or a sequence with at least 60%, 70%, 80%, 90%, or 95% identity thereto). In some cases, the programmable nuclease is Csml, Cas9, C2c4, C2c8, C2c5, C2cl0, C2c9, or CasZ. Sometimes, the Csml is also called smCmsl, miCmsl, obCmsl, or suCmsl. Sometimes Casl3a is also called C2c2.

Sometimes CasZ is also called Casl4a, Casl4b, Casl4c, Casl4d, Casl4e, Casl4f, Casl4g, or Casl4h. Sometimes, the programmable nuclease is a type V CRISPR-Cas system. In some cases, the programmable nuclease is a type VI CRISPR-Cas system. Sometimes the programmable nuclease is a type III CRISPR-Cas system. In some embodiments, the programmable nuclease can be an engineered nuclease that is not from a naturally occurring CRISPR-Cas system. In some cases, the programmable nuclease is from at least one of Leptotrichia shahii (Lsh), Listeria seeligeri (Lse), Leptotrichia buccalis (Lbu), Leptotrichia wadeu (Lwa), Rhodobacter capsulatus (Rea), Herbinix hemicellulosilytica (Hhe), Paludibacter propionicigenes (Ppr), Lachnospiraceae bacterium (Lba), [Eubacterium] rectale (Ere), Listeria newyorkensis (Lny), Clostridium aminophilum (Cam), Prevotella sp. (Psm), Capnocytophaga canimorsus (Cea Lachnospiraceae bacterium (Lba), Bergeyella zoohelcum (Bzo), Prevotella intermedia (Pin), Prevotella buccae (Pbu), Alistipes sp. (Asp), Riemerella anatipestifer (Ran), Prevotella aurantiaca (Pau), Prevotella saccharolytica (Psa), Prevotella intermedia (Pin2), Capnocytophaga canimorsus (Cea), Porphyromonas gulae (Pgu), Prevotella sp. (Psp), Porphyromonas gingivalis (Pig), Prevotella intermedia (Pin3), Enterococcus italicus (Ei), Lactobacillus salivarius (Ls), or Thermus thermophilus (Tt). Sometimes the Casl3 is at least one of LbuCasl3a, LwaCasl3a, LbaCasl3a, HheCasl3a, PprCasl3a, EreCasl3a, CamCasl3a, or LshCasl3a. The trans cleavage activity of the CRISPR enzyme can be activated when the crRNA is complexed with the target nucleic acid. The trans cleavage activity of the CRISPR enzyme can be activated when the guide nucleic acid comprising a tracrRNA and crRNA are complexed with the target nucleic acid. The target nucleic acid can be RNA or DNA.

[0140] Disclosed herein are programmable nucleases and uses thereof, e.g., detection and editing of target nucleic acids. In some instances, programmable nucleases comprise a Type V CRISPR/Cas protein. In some instances, Type V CRISPR/Cas proteins comprise nucleic acid cleavage activity. In some instances, Type V CRISPR/Cas proteins cleave or nick singlestranded nucleic acids, double, stranded nucleic acids, or a combination thereof. In some cases, Type V CRISPR/Cas proteins cleave single-stranded nucleic acids. In some cases, Type V CRISPR/Cas proteins cleave double-stranded nucleic acids. In some cases, Type V CRISPR/Cas proteins nick double-stranded nucleic acids. Typically, guide nucleic acids of Type V CRISPR/Cas proteins hybridize to ssDNA or dsDNA. However, the trans cleavage activity of Type V CRISPR/Cas protein is typically directed towards ssDNA. In some cases, the Type V CRISPR/Cas protein comprises a catalytically inactive nuclease domain. A catalytically inactive domain of a Type V CRISPR/Cas protein may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 mutations relative to a wild type nuclease domain of the Type V CRISPR/Cas protein. Said mutations may be present within a cleaving or active site of the nuclease. [0141] In some instances, the Type V Cas protein is a Cas protein. A Cas protein can function as an endonuclease that catalyzes cleavage at a specific sequence in a target nucleic acid. A programmable Cas nuclease may have a single active site in a RuvC domain that is capable of catalyzing pre-crRNA processing and nicking or cleaving of nucleic acids. This compact catalytic site may render the programmable Cas nuclease especially advantageous for genome engineering and new functionalities for genome manipulation.

[0142] In some instances, the programmable nuclease is a Type VI Cas protein. In some embodiments, the Type VI Cas protein is a programmable Casl3 nuclease. The general architecture of a Cas 13 protein includes an N-terminal domain and two HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domains separated by two helical domains. The HEPN domains each comprise aR-X4-H motif. Shared features across Cas 13 proteins include that upon binding of the crRNA of the guide nucleic acid to a target nucleic acid, the protein undergoes a conformational change to bring together the HEPN domains and form a catalytically active RNase. Thus, two activatable HEPN domains are characteristic of a programmable Casl3 nuclease of the present disclosure. However, programmable Cas 13 nucleases also consistent with the present disclosure include Casl3 nucleases comprising mutations in the HEPN domain that enhance the Cas 13 proteins cleavage efficiency or mutations that catalytically inactivate the HEPN domains. Programmable Cas 13 nucleases consistent with the present disclosure also Casl3 nucleases comprising catalytic components. In some instances, the Cas effector is a Cas 13 effector. In some instances, the Casl3 effector is a Casl3a, a Casl3b, a Cas 13c, a Cas 13d, or a Cas 13e effector protein.

[0143] In some embodiments, the programmable nuclease comprises a Casl2 protein, wherein the Cas 12 enzyme binds and cleaves double stranded DNA and single stranded DNA. In some embodiments, programmable nuclease comprises a Casl3 protein, wherein the Casl3 enzyme binds and cleaves single stranded RNA. In some embodiments, programmable nuclease comprises a Casl4 protein, wherein the Casl4 enzyme binds and cleaves both double stranded DNA and single stranded DNA.

[0144] Table 1 provides illustrative amino acid sequences of programmable nucleases having trans-cleavage activity. In some instances, programmable nucleases described herein comprise an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 98%, at least 99%, or 100% identical to any one of SEQ ID Nos: 1-71. The programmable nuclease may consist of an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one or SEQ ID Nos: 1-71. The programmable nuclease may comprise at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500 consecutive amino acids of any one of SEQ ID NOs: 1-71.

TABLE 1 - Example Protein Sequences

[0145] Other Exemplary protein sequences are described in the following applications: PCT/US2021/033271, PCT/US2021/035031, PCT/US2022/028865, PCT/US2022/034110, and PCT/US2022/034596, all of which are herein incorporated by reference in their entireties.

[0146] In some instances, the Type V CRISPR/Cas protein has been modified (also referred to as an engineered protein). For example, a Type V CRISPR/Cas protein disclosed herein or a variant thereof may comprise a nuclear localization signal (NLS). In some cases, the NLS may comprise a sequence of KRPAATKKAGQAKKKKEF . Type V CRISPR/Cas proteins may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the Type V CRISPR/Cas protein is codon optimized for a human cell.

[0147] In some cases, the effector proteins comprise a RuvC domain (e.g., a partial RuvC domain). In some instances, the RuvC domain may be defined by a single, contiguous sequence, or a set of partial RuvC domains that are not contiguous with respect to the primary amino acid sequence of the protein. An effector protein of the present disclosure may include multiple partial RuvC domains, which may combine to generate a RuvC domain with substrate binding or catalytic activity. For example, an effector protein may include three partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains) that are not contiguous with respect to the primary amino acid sequence of the effector protein, but form a RuvC domain once the protein is produced and folds. In some cases, effector proteins comprise a recognition domain with a binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex. In some instances, the effector protein does not comprise a zinc finger domain. In some instances, the effector protein does not comprise an HNH domain.

[0148] Effector proteins disclosed herein may function as an endonuclease that catalyzes cleavage at a specific position (e.g., at a specific nucleotide within a nucleic acid sequence) in a target nucleic acid. The target nucleic acid may be single stranded RNA (ssRNA), double stranded DNA (dsDNA) or single-stranded DNA (ssDNA). In some instances, the target nucleic acid is single-stranded DNA. In some instances, the target nucleic acid is single-stranded RNA. The effector proteins may provide cis cleavage activity, trans cleavage activity, nickase activity, or a combination thereof. Cis cleavage activity is cleavage of a target nucleic acid that is hybridized to a guide nucleic acid e.g., a dual gRNA or a sgRNA), wherein cleavage occurs within or directly adjacent to the region of the target nucleic acid that is hybridized to guide nucleic acid. Trans cleavage activity (also referred to as transcollateral cleavage) is cleavage of ssDNA or ssRNA that is near, but not hybridized to the guide nucleic acid. Trans cleavage activity is triggered by the hybridization of guide nucleic acid to the target nucleic acid. Nickase activity is a selective cleavage of one strand of a dsDNA.

[0149] Effector proteins of the present disclosure, dimers thereof, and multimeric complexes thereof may cleave or nick a target nucleic acid within or near a protospacer adjacent motif (PAM) sequence of the target nucleic acid. In some instances, cleavage occurs within 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides of a 5’ or 3’ terminus of a PAM sequence. A target nucleic acid may comprise a PAM sequence adjacent to a sequence that is complementary to a guide nucleic acid spacer region.

[0150] In some instances, the Type V CRISPR/Cas protein has been modified (also referred to as an engineered protein). For example, a Type V CRISPR/Cas protein disclosed herein or a variant thereof may comprise a nuclear localization signal (NLS). Type V CRISPR/Cas proteins may be codon optimized for expression in a specific cell, for example, a bacterial cell, a plant cell, a eukaryotic cell, an animal cell, a mammalian cell, or a human cell. In some embodiments, the Type V CRISPR/Cas protein is codon optimized for a human cell.

[0151] Several programmable nucleases are consistent with the methods and devices of the present disclosure. For example, Cas proteins are programmable nucleases used in the methods and systems disclosed herein. Cas proteins can include any of the known Classes and Types of CRISPR/Cas enzymes. Programmable nucleases disclosed herein include Class 1 Cas proteins, such as the Type I, Type IV, or Type III Cas proteins. Programmable nucleases disclosed herein also include the Class 2 Cas proteins, such as the Type II, Type V, and Type VI Cas proteins. Programmable nucleases included in the devices disclosed herein and methods of use thereof include a Type V or Type VI Cas proteins.

[0152] In some instances, the programmable nuclease is a Type V Cas protein. In general, a Type V Cas effector protein comprises a RuvC domain, but lacks an HNH domain. In most instances, the RuvC domain of the Type V Cas effector protein comprises three patrial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains). In some instances, the three RuvC subdomains are located within the C-terminal half of the Type V Cas effector protein. In some instances, none of the RuvC subdomains are located at the N terminus of the protein. In some instances, the RuvC subdomains are contiguous. In some instances, the RuvC subdomains are not contiguous with respect to the primary amino acid sequence of the Type V Cas protein, but form a RuvC domain once the protein is produced and folds. In some instances, there are zero to about 50 amino acids between the first and second RuvC subdomains. In some instances, there are zero to about 50 amino acids between the second and third RuvC subdomains. In some instances, the Cas effector is a Casl4 effector. In some instances, the Casl4 effector is a Casl4a, Casl4al, Casl4b, Casl4c, Casl4d, Casl4e, Casl4f, Casl4g, Casl4h, or Casl4u effector. In some instances, the Cas effector is a CasPhi effector. In some

-n- instances, the Cas effector is a Cast 2 effector. In some instances, the Cast 2 effector is a Cast 2a, Cast 2b, Cas 12c, Cast 2d, Casl2e, or Casl2j effector.

[0153] In some instances, the Type V CRISPR/Cas protein comprises a Casl4 protein. Casl4 proteins may comprise a bilobed structure with distinct amino-terminal and carboxy-terminal domains. The amino- and carboxy-terminal domains may be connected by a flexible linker. The flexible linker may affect the relative conformations of the amino- and carboxyl-terminal domains. The flexible linker may be short, for example less than 10 amino acids, less than 8 amino acids, less than 6 amino acids, less than 5 amino acids, or less than 4 amino acids in length. The flexible linker may be sufficiently long to enable different conformations of the amino- and carboxy-terminal domains among two Cas 14 proteins of a Cas 14 dimer complex (e.g., the relative orientations of the amino- and carboxy-terminal domains differ between two Casl4 proteins of a Casl4 homodimer complex). The linker domain may comprise a mutation which affects the relative conformations of the amino- and carboxyl-terminal domains. The linker may comprise a mutation which affects Casl4 dimerization. For example, a linker mutation may enhance the stability of a Cas 14 dimer.

[0154] In some instances, the amino-terminal domain of a Casl4 protein comprises a wedge domain, a recognition domain, a zinc finger domain, or any combination thereof. The wedge domain may comprise a multi-strand P-barrel structure. A multi-strand P-barrel structure may comprise an oligonucleotide/oligosaccharide-binding fold that is structurally comparable to those of some Casl2 proteins. The recognition domain and the zinc finger domain may each (individually or collectively) be inserted between P-barrel strands of the wedge domain. The recognition domain may comprise a 4-a-helix structure, structurally comparable but shorter than those found in some Casl2 proteins. The recognition domain may comprise a binding affinity for a guide nucleic acid or for a guide nucleic acid-target nucleic acid heteroduplex. In some cases, a REC lobe may comprise a binding affinity for a PAM sequence in the target nucleic acid. The amino-terminal may comprise a wedge domain, a recognition domain, and a zinc finger domain. The carboxy-terminal may comprise a RuvC domain, a zinc finger domain, or any combination thereof. The carboxy-terminal may comprise one RuvC and one zinc finger domain.

[0155] Casl4 proteins may comprise a RuvC domain or a partial RuvC domain. The RuvC domain may be defined by a single, contiguous sequence, or a set of partial RuvC domains that are not contiguous with respect to the primary amino acid sequence of the Cas 14 protein. In some instances, a partial RuvC domain does not have any substrate binding activity or catalytic activity on its own. A Cas 14 protein of the present disclosure may include multiple partial RuvC domains, which may combine to generate a RuvC domain with substrate binding or catalytic activity. For example, a Casl4 may include 3 partial RuvC domains (RuvC-I, RuvC-II, and RuvC-III, also referred to herein as subdomains) that are not contiguous with respect to the primary amino acid sequence of the Casl4 protein, but form a RuvC domain once the protein is produced and folds. A Casl4 protein may comprise a linker loop connecting a carboxy terminal domain of the Cast 4 protein with the amino terminal domain of the Cas 14 protein, and wherein the carboxy terminal domain comprises one or more RuvC domains and the amino terminal domain comprises a recognition domain.

[0156] Cas 14 proteins may comprise a zinc finger domain. In some instances, a carboxy terminal domain of a Casl4 protein comprises a zinc finger domain. In some instances, an amino terminal domain of a Casl4 protein comprises a zinc finger domain. In some instances, the amino terminal domain comprises a wedge domain (e.g., a multi-P-barrel wedge structure), a zinc finger domain, or any combination thereof. In some cases, the carboxy terminal domain comprises the RuvC domains and a zinc finger domain, and the amino terminal domain comprises a recognition domain, a wedge domain, and a zinc finger domain.

[0157] Cas 14 proteins may be relatively small compared to many other Cas proteins, making them suitable for nucleic acid detection or gene editing. For instance, a Casl4 protein may be less likely to adsorb to a surface or another biological species due to its small size. The smaller nature of these proteins also allows for them to be more easily packaged as a reagent in a system or assay, and delivered with higher efficiency as compared to other larger Cas proteins. In some cases, a Casl4 protein is 400 to 800 amino acid residues long, 400 to 600 amino acid residues long, 440 to 580 amino acid residues long, 460 to 560 amino acid residues long, 460 to 540 amino acid residues long, 460 to 500 amino acid residues long, 400 to 500 amino acid residues long, or 500 to 600 amino acid residues long. In some cases, a Casl4 protein is less than about 550 amino acid residues long. In some cases, a Casl4 protein is less than about 500 amino acid residues long.

[0158] In some instances, a Cas 14 protein may function as an endonuclease that catalyzes cleavage at a specific position within a target nucleic acid. In some instances, a Casl4 protein is capable of catalyzing non-sequence-specific cleavage of a single stranded nucleic acid. In some cases, a Casl4 protein is activated to perform trans cleavage activity after binding of a guide nucleic acid with a target nucleic acid. This trans cleavage activity is also referred to as “collateral” or “transcollateral” cleavage. Trans cleavage activity may be non-specific cleavage of nearby single-stranded nucleic acid by the activated programmable nuclease, such as trans cleavage of reporters with a detection moiety.

[0159] In some embodiments, the Type V CRISPR/Cas enzyme is a programmable Casl2 nuclease. Type V CRISPR/Cas enzymes (e.g., Cas 12 or Cas 14) lack an HNH domain. A Cas 12 nuclease of the present disclosure cleaves a nucleic acid via a single catalytic RuvC domain. The RuvC domain is within a nuclease, or “NUC” lobe of the protein, and the Casl2 nucleases further comprise a recognition, or “REC” lobe. The REC and NUC lobes are connected by a bridge helix and the Cast 2 proteins additionally include two domains for PAM recognition termed the PAM interacting (PI) domain and the wedge (WED) domain. In some instances, a programmable Cast 2 nuclease can be a Cast 2a protein, a Cast 2b protein, Cast 2c protein, Cast 2d protein, or a Casl2e protein.

[0160] In some embodiments, a programmable nuclease as disclosed herein is an RNA-activated programmable RNA nuclease. In some embodiments, a programmable nuclease as disclosed herein is a DNA-activated programmable RNA nuclease. In some embodiments, a programmable nuclease is capable of being activated by a target RNA to initiate trans cleavage of an RNA reporter and is capable of being activated by a target DNA to initiate trans cleavage of an RNA reporter, such as a Type VI CRISPR/Cas enzyme (e.g., a Casl3 nuclease). For example, Casl3a of the present disclosure can be activated by a target RNA to initiate trans cleavage activity of the Cast 3a for the cleavage of an RNA reporter and can be activated by a target DNA to initiate trans cleavage activity of the Cast 3a for trans cleavage of an RNA reporter. An RNA reporter can be an RNA-based reporter. In some embodiments, the Cast 3a recognizes and detects ssDNA to initiate transcleavage of RNA reporters. Multiple Cast 3a isolates can recognize, be activated by, and detect target DNA, including ssDNA, upon hybridization of a guide nucleic acid with the target DNA. For example, Lbu-Casl3a and Lwa-Casl3a can both be activated to transcollaterally cleave RNA reporters by target DNA. Thus, Type VI CRISPR/Cas enzyme (e.g., a Cast 3 nuclease, such as Cast 3 a) can be DNA-activated programmable RNA nucleases, and therefore can be used to detect a target DNA using the methods as described herein. DNA- activated programmable RNA nuclease detection of ssDNA can be robust at multiple pH values. For example, target ssDNA detection by Casl3 can exhibit consistent cleavage across a wide range of pH conditions, such as from a pH of 6.8 to a pH of 8.2. In contrast, target RNA detection by Casl3 can exhibit high cleavage activity of pH values from 7.9 to 8.2. In some embodiments, a DNA-activated programmable RNA nuclease that also is capable of being an RNA-activated programmable RNA nuclease, can have DNA targeting preferences that are distinct from its RNA targeting preferences. For example, the optimal ssDNA targets for Casl3a have different properties than optimal RNA targets for Cast 3 a. As one example, gRNA performance on ssDNA can not necessarily correlate with the performance of the same gRNAs on RNA. As another example, gRNAs can perform at a high level regardless of target nucleotide identity at a 3’ position on a target RNA sequence. In some embodiments, gRNAs can perform at a high level in the absence of a G at a 3’ position on a target ssDNA sequence. Furthermore, target DNA detected by Cast 3 disclosed herein can be directly taken from organisms or can be indirectly generated by nucleic acid amplification methods, such as PCR and LAMP or any amplification method described herein. Key steps for the sensitive detection of a target DNA, such as a target ssDNA, by a DNA-activated programmable RNA nuclease, such as Cast 3 a, can include: (1) production or isolation of DNA to concentrations above about 0.1 nM per reaction for in vitro diagnostics, (2) selection of a target sequence with the appropriate sequence features to enable DNA detection as these features are distinct from those required for RNA detection, and (3) buffer composition that enhances DNA detection.

Engineered programmable nuclease probes

[0161] Disclosed herein are non-naturally occurring compositions and systems comprising at least one of an engineered Cas protein and an engineered guide nucleic acid, which may simply be referred to herein as a Cas protein and a guide nucleic acid, respectively. In general, an engineered Cas protein and an engineered guide nucleic acid refer to a Cas protein and a guide nucleic acid, respectively, that are not found in nature. In some instances, systems and compositions comprise at least one non-naturally occurring component. For example, compositions and systems may comprise a guide nucleic acid, wherein the sequence of the guide nucleic acid is different or modified from that of a naturally-occurring guide nucleic acid. In some instances, compositions and systems comprise at least two components that do not naturally occur together. For example, compositions and systems may comprise a guide nucleic acid comprising a repeat region and a spacer region which do not naturally occur together. Also, by way of example, composition and systems may comprise a guide nucleic acid and a Cas protein that do not naturally occur together. Conversely, and for clarity, a Cas protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes Cas proteins and guide nucleic acids from cells or organisms that have not been genetically modified by a human or machine.

[0162] In some instances, the guide nucleic acid may comprise a non-natural nucleobase sequence. In some instances, the non-natural sequence is a nucleobase sequence that is not found in nature. The non-natural sequence may comprise a portion of a naturally occurring sequence, wherein the portion of the naturally occurring sequence is not present in nature absent the remainder of the naturally-occurring sequence. In some instances, the guide nucleic acid may comprise two naturally occurring sequences arranged in an order or proximity that is not observed in nature. In some instances, compositions and systems comprise a ribonucleotide complex comprising a CRISPR/Cas effector protein and a guide nucleic acid that do not occur together in nature. Engineered guide nucleic acids may comprise a first sequence and a second sequence that do not occur naturally together. For example, an engineered guide nucleic acid may comprise a sequence of a naturally occurring repeat region and a spacer region that is complementary to a naturally occurring eukaryotic sequence. The engineered guide nucleic acid may comprise a sequence of a repeat region that occurs naturally in an organism and a spacer region that does not occur naturally in that organism. An engineered guide nucleic acid may comprise a first sequence that occurs in a first organism and a second sequence that occurs in a second organism, wherein the first organism and the second organism are different. The guide nucleic acid may comprise a third sequence disposed at a 3’ or 5’ end of the guide nucleic acid, or between the first and second sequences of the guide nucleic acid. For example, an engineered guide nucleic acid may comprise a naturally occurring crRNA and tracrRNA coupled by a linker sequence.

[0163] In some instances, compositions and systems described herein comprise an engineered Cas protein that is similar to a naturally occurring Cas protein. The engineered Cas protein may lack a portion of the naturally occurring Cas protein. The Cas protein may comprise a mutation relative to the naturally-occurring Cas protein, wherein the mutation is not found in nature. The Cas protein may also comprise at least one additional amino acid relative to the naturally- occurring Cas protein. For example, the Cas protein may comprise an addition of a nuclear localization signal relative to the natural occurring Cas protein. In certain embodiments, the nucleotide sequence encoding the Cas protein is codon optimized (e.g., for expression in a eukaryotic cell) relative to the naturally occurring sequence.

[0164] An engineered protein may comprise a modified form of a wildtype counterpart protein. The modified form of the wildtype counterpart may comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the programmable nuclease. For example, a nuclease domain (e.g., RuvC domain) of a Type V CRISPR/Cas protein may be deleted or mutated so that it is no longer functional or comprises reduced nuclease activity. The modified form of the programmable nuclease may have less than 90 %, less than 80 %, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity of the wild-type counterpart. Engineered proteins may have no substantial nucleic acid-cleaving activity. Engineered proteins may be enzymatically inactive or “dead,” that is it may bind to a nucleic acid but not cleave it. An enzymatically inactive protein may comprise an enzymatically inactive domain (e.g., inactive nuclease domain). Enzymatically inactive may refer to an activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% activity compared to the wild-type counterpart. A dead protein may associate with an engineered guide nucleic acid to activate or repress transcription of a target nucleic acid sequence. In some embodiments, the enzymatically inactive protein is fused with a protein comprising recombinase activity.

[0165] An engineered protein may comprise a modified form of a wildtype counterpart protein. The modified form of the wildtype counterpart may comprise an amino acid change (e.g., deletion, insertion, or substitution) that increase the nucleic acid-cleaving activity of the programmable nuclease. For example, a nuclease domain (e.g., RuvC domain) of a Type V CRISPR/Cas protein may be mutated so that it is exhibits improved functionality or comprises increased nuclease activity. The modified form of the programmable nuclease may have more than 190 %, more than 180 %, more than 170%, more than 160%, more than 150%, more than 140%, more than 130%, more than 120%, more than 110%, more than 105%, or more than 101% of the nucleic acid-cleaving activity of the wild-type counterpart. In some embodiments, compositions, systems, devices, kits, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more amino acid alterations relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more alterations comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least sixteen, at least twenty, or more amino acid alterations relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more alterations comprises one to twenty, one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, or sixteen to twenty amino acid alterations relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more alterations comprises one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid alterations relative to any one of the sequences recited in TABLE 1. In some embodiments, the effector protein comprising one or more amino acid alterations is a variant of an effector protein described herein. It is understood that any reference to an effector protein herein also refers to an effector protein variant as described herein. In some embodiments, the one or more amino acid alterations comprises conservative substitutions, non-conservative substitutions, conservative deletions, non-conservative deletions, or combinations thereof. In some embodiments, an effector protein or a nucleic acid encoding the effector protein comprises 1 amino acid alteration, 2 amino acid alterations, 3 amino acid alterations, 4 amino acid alterations, 5 amino acid alterations, 6 amino acid alterations, 7 amino acid alterations, 8 amino acid alterations, 9 amino acid alterations, 10 amino acid alterations or more relative to any one of the sequences recited in TABLE 1.

[0166] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more substitutions relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more substitutions comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least sixteen, at least twenty, or more substitutions relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more substitutions comprises one to twenty, one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, or sixteen to twenty substitutions relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more substitutions comprise one, two, three, four, five, six, seven, eight, nine, ten or more substitutions relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more substitutions comprise one or more conservative substitutions, one or more non-conservative substitutions, or combinations thereof.

[0167] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more conservative substitutions relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more conservative substitutions comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least sixteen, at least twenty, or more conservative substitutions relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more conservative substitutions comprises one to twenty, one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, or sixteen to twenty conservative substitutions relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more conservative substitutions comprise one, two, three, four, five, six, seven, eight, nine, ten or more conservative substitutions relative to any one of the sequences recited in TABLE 1.

[0168] In some embodiments, compositions, systems, and methods described herein comprise an effector protein, or a nucleic acid encoding the effector protein, wherein the effector protein comprises one or more non-conservative substitutions relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more non-conservative substitutions comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least twelve, at least sixteen, at least twenty, or more non-conservative substitutions relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more non-conservative substitutions comprises one to twenty, one to sixteen, one to twelve, one to eight, one to four, four to twenty, four to sixteen, four to twelve, four to eight, eight to twenty, eight to sixteen, eight to twelve, twelve to twenty, twelve to sixteen, or sixteen to twenty non-conservative substitutions relative to any one of the sequences recited in TABLE 1. In some embodiments, the one or more non-conservative substitutions comprise one, two, three, four, five, six, seven, eight, nine, ten or more non-conservative substitutions relative to any one of the sequences recited in TABLE 1.

[0169] In some instances, compositions and systems provided herein comprise a multi-vector system encoding a Cas protein and a guide nucleic acid described herein, wherein the guide nucleic acid and the Cas protein are encoded by the same or different vectors. In some embodiments, the engineered guide and the engineered Cas protein are encoded by different vectors of the system.

Programmable nuclease fusion proteins

[0170] Described herein are various embodiments of programmable nuclease fusion proteins. In some embodiments, a programmable nuclease may be fused with a heterologous polypeptide to form a programmable nuclease fusion protein. In some instances, “fusion partner” can refer to a polypeptide that is fused to another polypeptide. In some embodiments, the heterologous polypeptide may be an enzyme, wherein the enzyme is configured to react an enzyme substrate to generate a detectable signal. In some embodiments, the heterologous polypeptide may provide a tag (i.e., the heterologous polypeptide is a detectable label) for ease of tracking and/or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, and the like; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).

[0171] "Heterologous," as used herein, can refer to a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively. In some cases, a heterologous polypeptide may comprise a sequence different from a sequence of a given protein. For example, a sequence of an HRP enzyme may be considered heterologous to a sequence of a programmable nuclease when the sequence of the HRP enzyme is different from the sequence of the programmable nuclease. In some embodiments, a programmable nuclease (e.g., a Casl2 protein) can be fused to an active domain from a non-programmable nuclease protein (e.g., an HRP), and the sequence of the active domain may be considered to be a heterologous polypeptide (i.e., it is heterologous to the programmable nuclease).

[0172] In some instances, a programmable nuclease is a fusion protein, wherein the fusion protein comprises a protein comprising the amino acid sequence of any one of SEQ ID NOs: 1- 71. In some instances, the fusion protein comprises a programmable nuclease and a fusion partner protein. [0173] A fusion partner protein is also simply referred to herein as a fusion partner. In some cases, the fusion partner promotes the formation of a multimeric complex of the programmable nuclease. In some cases, the fusion partner is an additional programmable nuclease. In some cases, the multimeric complex comprising the programmable nuclease and the additional programmable nuclease binds a guide nucleic acid. The programmable nucleases of the multimeric complex may bind the guide nucleic acid in an asymmetric fashion. In some cases, one programmable nuclease of the multimeric complex interacts more strongly with the guide nucleic acid than the additional programmable nuclease of the multimeric complex. In some cases, a programmable nuclease interacts more strongly with a target nucleic acid when it is complexed with the guide nucleic acid relative to when the programmable nuclease or the multimeric complex is not complexed with the guide nucleic acid.

[0174] In some instances, fusion partners include, but are not limited to, a protein that directly and/or indirectly provides for increased or decreased transcription and/or translation of a target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription and/or translation regulator, a translation-regulating protein, etc.). In some instances, fusion partners that increase or decrease transcription include a transcription activator domain or a transcription repressor domain, respectively.

[0175] In some cases, a terminus of the programmable nuclease is linked to a terminus of the fusion partner through an amide bond. In some cases, a programmable nuclease is coupled to a fusion partner via a linker protein. In some cases, a programmable nuclease is coupled to a fusion partner via a linker protein. The linker protein may have any of a variety of amino acid sequences. A linker protein may comprise a region of rigidity (e.g., beta sheet, alpha helix), a region of flexibility, or any combination thereof. In some instances, the linker comprises small amino acids, such as glycine and alanine, that impart high degrees of flexibility. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any desired element may include linkers that are all or partially flexible, such that the linker may include a flexible linker as well as one or more portions that confer less flexible structure. Suitable linkers include proteins of 4 linked amino acids to 40 linked amino acids in length, or between 4 linked amino acids and 25 linked amino acids in length. These linkers may be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or may be encoded by a nucleic acid sequence encoding a fusion protein (e.g., an programmable nuclease coupled to a fusion partner). Examples of linker proteins include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, GSGGSn, GGSGGSn, and GGGSn, where n is an integer of at least one), glycine-alanine polymers, and alanine-serine polymers. Exemplary linkers may comprise amino acid sequences including, but not limited to, GGSG, GGSGG, GSGSG, GSGGG, GGGSG, and GSSSG.

Thermostable programmable nuclease

[0176] Described herein are various embodiments of thermostable programmable nucleases. In some embodiments, a programmable nuclease is referred to as an effector protein. An effector protein may be thermostable. In some instances, known effector proteins (e.g., Casl2 nucleases) are relatively thermo-sensitive and only exhibit activity (e.g., cis and/or trans cleavage) sufficient to produce a detectable signal in a diagnostic assay at temperatures less than 40° C, and optimally at about 37° C. A thermostable protein may have enzymatic activity, stability, or folding comparable to those at 37 °C. In some instances, the trans cleavage activity (e.g., the maximum trans cleavage rate as measured by fluorescent signal generation) of an effector protein in a trans cleavage assay at 40 °C may be at least 50% of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 40 °C may be at least 1- fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 40 °C may be at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.

[0177] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 45 °C may be at least 11 -fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.

[0178] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 50 °C may be at least 11 -fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.

[0179] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 55 °C may be at least 11 -fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.

[0180] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 60 °C may be at least 11 -fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.

[0181] In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 50 % of that at 37 °C (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 1-fold of that at 37 °C (e.g., at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold of that at 37 °C). In some instances, the trans cleavage activity of an effector protein in a trans cleavage assay at 65 °C may be at least 11 -fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that at 37 °C.

[0182] In some instances, the trans cleavage activity may be measured against a negative control in a trans cleavage assay. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 37 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 37 °C may be at least 11 -fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 40 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 40 °C may be at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 45 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 45 °C may be at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 50 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 50 °C may be at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 55 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 55 °C may be at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 60 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 60 °C may be at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 65 °C may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10- fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 65 °C may be at least 11- fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 70 °C, 75 °C, 80 °C, or more may be at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 100 %, at least 1-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold of that against a negative control nucleic acid. In some instances, the trans cleavage activity of an effector protein against a nucleic acid in a trans cleavage assay at 70 °C, 75 °C, 80 °C, or more may be at least 11 -fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold or more of that against a negative control nucleic acid. [0183] The reporters described herein can be RNA reporters. The RNA reporters can comprise at least one ribonucleic acid and a detectable moiety. In some embodiments, a programmable nuclease probe or a CRISPR probe comprising a Cas enzyme can recognize and detect ssDNA and, further, can specifically trans-cleave RNA reporters. The detection of the target nucleic acid in the sample can indicate the presence of the disease (or disease-causing agent) in the sample and can provide information for taking action to reduce the transmission of the disease to individuals in the disease-affected environment or near the disease-carrying individual.

[0184] Cleavage of a reporter (e.g., a protein-nucleic acid) can produce a signal. The signal can indicate a presence of the target nucleic acid in the sample, and an absence of the signal can indicate an absence of the target nucleic acid in the sample. In some cases, cleavage of the protein-nucleic acid can produce a calorimetric signal, a potentiometric signal, an amperometric signal, an optical signal, or a piezo-electric signal. Various devices and/or sensors can be used to detect these different types of signals, which indicate whether a target nucleic acid is present in the sample. The sensors usable to detect such signals can include, for example, optical sensors (e.g., imaging devices for detecting fluorescence or optical signals with various wavelengths and frequencies), electric potential sensors, surface plasmon resonance (SPR) sensors, interferometric sensors, or any other type of sensor suitable for detecting calorimetric signals, potentiometric signals, amperometric signals, optical signals, or piezo-electric signals.

[0185] In one aspect, the present disclosure provides a method for non-fluorescence-based target detection, such as those illustrated in schematics shown in FIGS. 48A-50. FIGS. 48A-48B illustrate schematics for programmable nuclease-based detection in which signal generation is based on binding of a nucleic acid to a sensor-bound probe oligonucleotide in accordance with some embodiments. In some embodiments, as illustrated in FIG. 48A, a reporter is immobilized to a solid surface in a reaction chamber. In some embodiments, the reporter may comprise a surface linker, an RNA portion that is cleavable by an activated programmable nuclease, and a DNA portion that is released as part of a detectable product upon cleavage of the RNA portion. In some embodiments, the linker is bound to the solid surface using an attachment moiety. In some embodiments, the DNA portion of the reporter comprises single-stranded DNA (ssDNA). Upon cleavage, the DNA portion is released from the surface as part of the detectable product into solution. Solution containing released detectable products comprising DNA portions may flow (e.g., within a device) or be transferred (e.g., by pipetting) to a reaction chamber having a sensor region with a sensor that forms part of or is in contact with a surface of the reaction chamber. In some embodiments, the sensor region comprises an electrode. In some embodiments, the sensor region comprises a surface-bound probe oligonucleotide. In some embodiments, the surface-bound probe oligonucleotide comprises a surface linker, wherein the surface linker is bound to the surface of the sensor region by an attachment moiety. The surface- bound probe oligonucleotide is complementary to the released DNA portion from the reporter, and is capable of forming a double-stranded complex therewith. An amount of binding between the target nucleic acid and probe oligonucleotide results in a corresponding change in sensor signal (e.g., a change in resistance at an electrical sensor). In some embodiments, the corresponding sensor signal is at least one of a potentiometric signal and an amperometric signal. For example, binding between the target nucleic acid (or an amplicon thereof) and the probe oligonucleotide may ultimately result in a decrease in capacitance detected at the sensor.

[0186] In some embodiments, as illustrated in FIG. 48B, the reporter is cleaved in solution. In some embodiments, the reporter comprises an RNA portion that is cleavable by an activated programmable nuclease, and a DNA portion that is released as part of a detectable product upon cleavage of the RNA portion. In some embodiments, the RNA portion comprises a binding moiety. In some embodiments, the binding moiety is biotin. In some embodiments, the DNA portion of the reporter comprises single-stranded DNA (ssDNA). Upon cleavage, the DNA portion is released from the reporter as part of the detectable product. The solution is then flowed through a filter that restricts passage of uncleaved reporter and allows passage of the detectable product. In some embodiments, the filter comprises beads (e.g., beads with a capture moiety that binds the binding moiety of the RNA portion). The solution containing released DNA portions may flow (e.g., within a device) or be transferred (e.g., by pipetting) to a reaction chamber having a sensor region with a sensor that forms part of or is in contact with a surface of the reaction chamber. At this point, detection is similar to that described for FIG. 48A, such as by use of sensor coupled to a surface-bound probe oligonucleotide complementary to a released DNA portion, the binding of which may change an electrical property at the sensor which can be detected as a change in a sensor signal.

[0187] FIGS. 49A-49C illustrate additional nucleic acid detection methods based on binding of a detectable product to a sensor surface, in which the detectable product is released from a cleaved reporter and said release indicates the presence of a target nucleic acid in a sample. In some embodiments, as illustrated in FIG. 49A, a reporter is immobilized to a solid surface in a reaction chamber. This method is similar to the method as illustrated in FIG. 48A, except the reporter comprises a AuNP bound to the RNA portion (instead of the DNA portion), forming an AuNP-reporter conjugate. Upon cleavage of the RNA portion by an activated programmable nuclease, the AuNP is released as part of the detectable product and flows or is transferred from the reaction chamber to the sensor region, where the AuNP binds to the sensor surface resulting in a corresponding sensor signal.

[0188] In some embodiments, as illustrated in FIG. 49B, the reporter is in solution and comprises a linker, an RNA portion that is cleavable by an activated programmable nuclease, and a AuNP forming the AuNP-reporter conjugate. In some embodiments, the linker comprises a binding moiety. In some embodiments, the binding moiety is biotin. Similar to the method illustrated in FIG. 48B, cleavage of the reporter releases the detectable product (comprising the AuNP in this illustration). The solution is then flowed through a filter that restricts passage of uncleaved reporter and allows passage of the detectable product. In some embodiments, the filter comprises beads (e.g., beads with a capture moiety that binds the binding moiety of the RNA portion). Solution containing released AuNP may flow (e.g., within a device) or be transferred (e.g., by pipetting) to a reaction chamber having a sensor region with a sensor that forms part of or is in contact with a surface of the reaction chamber. AuNP binding produces a detectable signal that is detected at the sensor (e.g., a change in an electrical property of the surface at the sensor).

[0189] In some embodiments, as illustrated in FIG. 49C, it is a decrease in binding at the sensor that is detected as indicative of a cleavage event. For example, unlike the example shown in FIG. 49B, upon cleavage due to nuclease activity, the uncleaved AuNP-reporter conjugates and the cleaved AuNP-reporter conjugates both flow and/or are transferred to the sensor region without passing through a filter. In some embodiments, the sensor surface of the sensor region comprises a coating, wherein the coating comprises a functional moiety for immobilization (e.g., streptavidin coated surface for capture of targets comprising a biotin functionality). When the linker of the AuNP-reporter conjugate comprises a biotin moiety, the uncleaved AuNP-reporter conjugates that have flowed into the sensor region bind to the streptavidin-coated surface, resulting in a corresponding sensor signal. In some embodiments, more nuclease activity results in fewer uncleaved AuNP-reporter conjugates binding to the streptavidin coated surface, thus resulting in a detectable change in sensor signal relative to the uncleaved condition. In some embodiments, the signal is a change in an electrical property of the surface at the sensor.

[0190] FIG. 50 illustrates a schematic for programmable nuclease-based detection in which signal generation is based on binding of detectable product to a sensor surface, in which the detectable product is released from a cleaved reporter and indicating the presence of a target nucleic acid in a sample. In some embodiments, the reporter comprises a linker, an RNA portion that is cleavable by an activated programmable nuclease, and a DNA portion with self- complementary regions that fold to form a hairpin structure. In some embodiments, the DNA hairpin is bound to the RNA portion on a first end, and to a binding moiety on a second end. In some embodiments, the binding moiety is biotin. Upon cleavage of the RNA portion, DNA hairpin is released from the reporter as part of the detectable product into solution. The solution containing released DNA hairpins may flow (e.g., within a device) or be transferred (e.g., by pipetting) to a reaction chamber having a sensor region with a sensor that forms part of or is in contact with a surface of the reaction chamber. In some embodiments, the sensor region comprises an electrode. In some embodiments, the sensor region comprises a functional moiety for capture and immobilization of the detectable product (e.g., streptavidin). An amount of binding of DNA hairpin to the surface results in a corresponding change in sensor signal (e.g., a change in resistance or capacitance at an electrical sensor). In some embodiments, the corresponding sensor signal is at least one of a potentiometric signal and an amperometric signal. For example, binding between the target nucleic acid (or an amplicon thereof) and the probe oligonucleotide may ultimately result in a decrease in capacitance detected at the sensor.

[0191] FIG. 51 illustrates an additional method for non-fluorescence-based target nucleic acid detection. In some embodiments, the reporter is immobilized to a solid surface in a reaction chamber. In some embodiments, the reporter may comprise a surface linker, an RNA portion that is cleavable by an activated programmable nuclease, and a platinum nanoparticle (PtNP), forming a Pt-NP reporter conjugate. In some embodiments, the linker is bound to the solid surface using an attachment moiety. Upon cleavage of the RNA portion by an activated programmable nuclease, the PtNP is released as part of a detectable product and flows or is transferred from the reaction chamber to the sensor region. The sensor region may comprise hydrogen peroxide (H2O2) and a pH sensor. As the PtNP enters the sensor region, the H2O2 reacts with the PtNP to form H2O and O2, resulting in a change in pH. This pH change is measured by the pH sensor and produces a corresponding sensor signal indicative of the nuclease activity and presence of the target nucleic acid in the sample. For example, cleavage of the reporter in the presence of the target nucleic acid (or an amplicon thereof) may ultimately result in an increase in pH detected at the sensor.

[0192] In an aspect, the present disclosure provides a method for target detection. The method can comprise sample collection. The method can further comprise sample preparation. The method can further comprise detection of one or more target nucleic acids in the collected and prepared sample.

[0193] In another aspect, the present disclosure provides a nucleic acid detection device for target detection. The nucleic acid detection device can be configured for multiplexed target detection. The nucleic acid detection device can be used to collect one or more samples, prepare or process the one or more samples for detection, and optionally divide the one or more samples into a plurality of droplets, aliquots, or subsamples for amplification of one or more target sequences or target nucleic acids. The target sequences may comprise, for example, a biological sequence. The biological sequence can comprise a nucleic acid sequence or an amino acid sequence. In some embodiments, the target sequences are associated with an organism of interest, a disease of interest, a disease state of interest, a phenotype of interest, a genotype of interest, or a gene of interest.

[0194] The nucleic acid detection device can be configured to amplify target nucleic acids contained within the plurality of droplets, aliquots, or subsamples. The nucleic acid detection device can be configured to amplify the target sequences or target nucleic acids contained within the plurality of droplets by individually processing each of the plurality of droplets (e.g., by using a thermocycling process or any other suitable amplification process as described in greater detail below). In some cases, the plurality of droplets can undergo separate thermocycling processes. In some cases, the thermocycling processes can occur simultaneously. In other cases, the thermocycling processes can occur at different times for each droplet.

[0195] The nucleic acid detection device can be further configured to remix the droplets, aliquots, or subsamples after the target nucleic acids in each of the droplets undergo amplification. The nucleic acid detection device can be configured to provide the remixed sample comprising the droplets, aliquots, or subsamples to a detection chamber of the device. The detection chamber can be configured to direct the remixed droplets, aliquots, or subsamples to a plurality of programmable nuclease probes. The detection chamber can be configured to direct the remixed droplets, aliquots, or subsamples along one or more fluid flow paths such that the remixed droplets, aliquots, or subsamples are positioned adjacent to and/or in contact with the one or more programmable nuclease probes. In some cases, the detection chamber can be configured to recirculate or recycle the remixed droplets, aliquots, or subsamples such that the remixed droplets, aliquots, or subsamples are repeatedly placed in contact with one or more programmable nuclease probes over a predetermined period of time.

[0196] The nucleic acid detection device can comprise one or more sensors. The one or more sensors of the nucleic acid detection device can be configured to detect one or more signals that are generated after one or more programmable nucleases of the one or more programmable nuclease probes become activated due to a binding of a guide nucleic acid of the programmable nuclease probes with a target nucleic acid present in the sample. As described elsewhere herein, the activated programmable nuclease can cleave the target nucleic acid, which can result in a trans cleavage activity. Trans cleavage activity can be a non-specific cleavage of nearby singlestranded nucleic acids by the activated programmable nuclease, such as trans cleavage of target nucleic acids with a detection moiety. Once the target nucleic acids are cleaved by the activated programmable nucleases, the detection moiety can be released or separated from the reporter, thereby generating one or more detectable signals. The one or more sensors of the nucleic acid detection device can be configured to register and/or process the one or more detectable signals to confirm a presence and/or an absence of a particular target (e.g., a target nucleic acid).

[0197] The one or more programmable nuclease probes of the nucleic acid detection device can be configured for multiplexed detection. In some cases, each programmable nuclease probe can be configured to detect a particular target. In other cases, each programmable nuclease probe can be configured to detect a plurality of targets. In some cases, a first programmable nuclease probe can be configured to detect a first target or a first set of targets, and a second programmable nuclease probe can be configured to detect a second target or a second set of targets. In other cases, a first programmable nuclease probe can be configured to detect a first set of targets, and a second programmable nuclease probe can be configured to detect a second set of targets. The programmable nuclease probes of the present disclosure can be used to detect a plurality of different target sequences or target nucleic acids. In any of the embodiments described herein, the sample provided to the nucleic acid detection device can comprise a plurality of target sequences or target nucleic acids. In any of the embodiments described herein, the sample provided to the nucleic acid detection device can comprise multiple classes of target sequences or target nucleic acids. Each class of target sequences or class of target nucleic acids can comprise a plurality of target sequences or target nucleic acids associated with a particular organism, disease state, phenotype, or genotype present within the sample. In some cases, each programmable nuclease probe can be used to detect a particular class of target sequences or a particular class of target nucleic acids associated with a particular organism, disease state, phenotype, or genotype present within the sample. In some cases, two or more programmable nuclease probes can be used to detect different classes of target sequences or different classes of target nucleic acids. In such cases, the two or more programmable nuclease probes can comprise different sets or classes of guide nucleic acids complexed to the programmable nucleases of the probes.

[0198] In any of the embodiments described herein, the nucleic acid detection device can comprise a single integrated system that is configured to perform sample collection, sample processing, droplet generation, droplet processing (e.g., amplification of target nucleic acids in droplets), droplet remixing, and/or circulation of the remixed droplets within a detection chamber so that at least a portion of the remixed droplets is placed in contact with one or more programmable nuclease probes coupled to the detection chamber. The nucleic acid detection devices of the present disclosure can be disposable devices configured to perform one or more rapid single reaction or multi -reach on tests to detect a presence and/or an absence of one or more target sequences or target nucleic acids.

[0199] The systems and methods of the present disclosure can be used to detect one or more target sequences or nucleic acids in one or more samples. The one or more samples can comprise one or more target sequences or nucleic acids for detection of an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry and are compatible with the reagents and support mediums as described herein. Generally, a sample can be taken from any place where a nucleic acid can be found. Samples can be taken from an individual/human, a non-human animal, or a crop, or an environmental sample can be obtained to test for presence of a disease, virus, pathogen, cancer, genetic disorder, or any mutation or pathogen of interest. A biological sample can be blood, serum, plasma, lung fluid, exhaled breath condensate, saliva, spit, urine, stool, feces, mucus, lymph fluid, peritoneal , cerebrospinal fluid, amniotic fluid, breast milk, gastric secretions, bodily discharges, secretions from ulcers, pus, nasal secretions, sputum, pharyngeal exudates, urethral secretions/mucus, vaginal secretions/mucus, anal secretion/mucus, semen, tears, an exudate, an effusion, tissue fluid, interstitial fluid (e.g., tumor interstitial fluid), cyst fluid, tissue, or, in some instances, any combination thereof. A sample can be an aspirate of a bodily fluid from an animal (e.g., human, animals, livestock, pet, etc.) or plant. A tissue sample can be from any tissue that can be infected or affected by a pathogen (e.g., a wart, lung tissue, skin tissue, and the like). A tissue sample (e.g., from animals, plants, or humans) can be dissociated or liquified prior to application to detection system of the present disclosure. A sample can be from a plant (e.g., a crop, a hydroponically grown crop or plant, and/or house plant). Plant samples can include extracellular fluid, from tissue (e.g., root, leaves, stem, trunk etc.). A sample can be taken from the environment immediately surrounding a plant, such as hydroponic fluid/ water, or soil. A sample from an environment can be from soil, air, or water. In some instances, the environmental sample is taken as a swab from a surface of interest or taken directly from the surface of interest. In some instances, the raw sample is applied to the detection system. In some instances, the sample is diluted with a buffer or a fluid or concentrated prior to application to the detection system. In some cases, the sample is contained in no more than about 200 nanoliters (nL). In some cases, the sample is contained in about 200 nL. In some cases, the sample is contained in a volume that is greater than about 200 nL and less than about 20 microliters (pL). In some cases, the sample is contained in no more than 20 pl. In some cases, the sample is contained in no more than 1, 5, 10, 15, 20, 25, 30, 35 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400, 500 pl, or any of value from 1 pl to 500 pl. In some cases, the sample is contained in from 1 pL to 500 pL, from 10 pL to 500 pL, from 50 pL to 500 pL, from 100 pL to 500 pL, from 200 pL to 500 pL, from 300 pL to 500 pL, from 400 pL to 500 pL, from 1 pL to 200 pL, from 10 pL to 200 pL, from 50 pL to 200 pL, from 100 pL to 200 pL, from 1 pL to 100 pL, from 10 pL to 100 pL, from 50 pL to 100 pL, from 1 pL to 50 pL, from 10 pL to 50 pL, from 1 pL to 20 pL, from 10 pL to 20 pL, or from 1 pL to 10 pL. Sometimes, the sample is contained in more than 500 pl.

[0200] In some instances, the sample is taken from a single-cell eukaryotic organism; a plant or a plant cell; an algal cell; a fungal cell; an animal or an animal cell, tissue, or organ; a cell, tissue, or organ from an invertebrate animal; a cell, tissue, fluid, or organ from a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; a cell, tissue, fluid, or organ from a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine. In some instances, the sample is taken from nematodes, protozoans, helminths, or malarial parasites. In some cases, the sample may comprise nucleic acids from a cell lysate from a eukaryotic cell, a mammalian cell, a human cell, a prokaryotic cell, or a plant cell. In some cases, the sample may comprise nucleic acids expressed from a cell.

[0201] The sample used for disease testing can comprise at least one target sequence that can bind to a guide nucleic acid of the reagents described herein. In some cases, the target sequence is a portion of a nucleic acid. A nucleic acid can be from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA. A nucleic acid can be from 5 to 100, 5 to 90, 5 to 80, 5 to 70, 5 to 60, 5 to 50, 5 to 40, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides in length. A nucleic acid can be from 10 to 90, from 20 to 80, from 30 to 70, or from 40 to 60 nucleotides in length. A nucleic acid sequence can be from 10 to 95, from 20 to 95, from 30 to 95, from 40 to 95, from 50 to 95, from 60 to 95, from 10 to 75, from 20 to 75, from 30 to 75, from 40 to 75, from 50 to 75, from 5 to 50, from 15 to 50, from 25 to 50, from 35 to 50, or from 45 to 50 nucleotides in length. A nucleic acid can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,

24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides in length. The target nucleic acid can be reverse complementary to a guide nucleic acid. In some cases, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,

25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides of a guide nucleic acid can be reverse complementary to a target nucleic acid.

[0202] In some cases, the target sequence is a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease in the sample. The target sequence, in some cases, is a portion of a nucleic acid from a sexually transmitted infection or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from an upper respiratory tract infection, a lower respiratory tract infection, or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from a hospital acquired infection or a contagious disease, in the sample. The target sequence, in some cases, is a portion of a nucleic acid from sepsis, in the sample. These diseases can include but are not limited to respiratory viruses (e.g., SARS-CoV-2 (i.e., a virus that causes COVID-19), SARS, MERS, influenza, Adenovirus, Coronavirus HKU1, Coronavirus NL63, Coronavirus 229E, Coronavirus OC43, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Human Metapneumovirus (hMPV), Human Rhinovirus/Enterovirus, Influenza A, Influenza A/Hl, Influenza A/H3, Influenza A/Hl-2009, Influenza B, Influenza C, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Respiratory Syncytial Virus) and respiratory bacteria (e.g., Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Mycoplasma pneumoniae). Other viruses include human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis. Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites. Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms. Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis. Examples of pathogens such as parasitic/protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii. Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, and Candida albicans. Pathogenic viruses include but are not limited to: respiratory viruses (e.g., adenoviruses, parainfluenza viruses, severe acute respiratory syndrome (SARS), coronavirus, MERS), gastrointestinal viruses (e.g., noroviruses, rotaviruses, some adenoviruses, astroviruses), exanthematous viruses (e.g., the virus that causes measles, the virus that causes rubella, the virus that causes chickenpox/shingles, the virus that causes roseola, the virus that causes smallpox, the virus that causes fifth disease, chikungunya virus infection); hepatic viral diseases (e.g., hepatitis A, B, C, D, E); cutaneous viral diseases (e.g., warts (including genital, anal), herpes (including oral, genital, anal), molluscum contagiosum); hemmorhagic viral diseases (e.g., Ebola, Lassa fever, dengue fever, yellow fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever); neurologic viruses (e.g., polio, viral meningitis, viral encephalitis, rabies), sexually transmitted viruses (e.g., HIV, HPV, and the like), immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like. Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Klebsiella pneumoniae, Acinetobacter baumannii, Bacillus anthracis, Bortadella pertussis, Burkholderia cepacia, Corynebacterium diphtheriae, Coxiella burnetii, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella longbeachae, Legionella pneumophila, Leptospira interrogans, Moraxella catarrhalis, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria elongate, Neisseria gonorrhoeae, Parechovirus, Pneumococcus, Pneumocystis jirovecii, Cryptococcus neoformans, Histoplasma capsulatum, Haemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus (RSV), M. genitalium, T. Vaginalis, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, Reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium, M. pneumoniae, Enterobacter cloacae, Kiebsiella aerogenes, Proteus vulgaris, Serratia macesens, Enterococcus faecalis, Enterococcus faecium, Streptococcus inter mdius, Streptococcus pneumoniae, and Streptococcus pyogenes. Often the target nucleic acid may comprise a sequence from a virus or a bacterium or other agents responsible for a disease that can be found in the sample. In some cases, the target nucleic acid is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus in at least one of: human immunodeficiency virus (HIV), human papillomavirus (HPV), chlamydia, gonorrhea, syphilis, trichomoniasis, sexually transmitted infection, malaria, Dengue fever, Ebola, chikungunya, and leishmaniasis. Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, and Schistosoma parasites. Helminths include roundworms, heartworms, and phytophagous nematodes, flukes, Acanthocephala, and tapeworms. Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis. Examples of pathogens such as parasitic/protozoan pathogens include, but are not limited to: Plasmodium falciparum, P. vivax, Trypanosoma cruzi and Toxoplasma gondii. Fungal pathogens include, but are not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. Pathogenic viruses include but are not limited to immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis Virus C; Hepatitis Virus A; Hepatitis Virus B; papillomavirus; and the like. Pathogens include, e.g., HIV virus, Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Legionella pneumophila, Streptococcus pyogenes, Streptococcus salivarius, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus (RSV), M. genitalium, T. vaginalis, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, Reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and AL pneumoniae. In some cases, the target sequence is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus of bacterium or other agents responsible for a disease in the sample comprising a mutation that confers resistance to a treatment, such as a single nucleotide mutation that confers resistance to antibiotic treatment. [0203] In some embodiments, the Coronavirus HKU1 sequence is a target of an assay. In some embodiments, the Coronavirus NL63 sequence is a target of an assay. In some embodiments, the Coronavirus 229E sequence is a target of an assay. In some embodiments, the Coronavirus OC43 sequence is a target of an assay. In some embodiments, the SARS-CoV-1 sequence is a target of an assay. In some embodiments, the MERS sequence is a target of an assay. In some embodiments, the SARS-CoV-2 sequence is a target of an assay. In some embodiments, the Respiratory Syncytial Virus A sequence is a target of an assay. In some embodiments, the Respiratory Syncytial Virus B sequence is a target of an assay. In some embodiments, the Influenza A sequence is a target of an assay. In some embodiments, the Influenza B sequence is a target of an assay. In some embodiments, the Human Metapneumovirus sequence is a target of an assay. In some embodiments, the Human Rhinovirus sequence is a target of an assay. In some embodiments, the Human Enterovirus sequence is a target of an assay. In some embodiments, the Parainfluenza Virus 1 sequence is a target of an assay. In some embodiments, the Parainfluenza Virus 2 sequence is a target of an assay. In some embodiments, the Parainfluenza Virus 3 sequence is a target of an assay. In some embodiments, the Parainfluenza Virus 4 sequence is a target of an assay. In some embodiments, the Alphacoronavirus genus sequence is a target of an assay. In some embodiments, the Betacoronavirus genus sequence is a target of an assay. In some embodiments, the Sarbecovirus subgenus sequence is a target of an assay. In some embodiments, the SARS- related virus species sequence is a target of an assay. In some embodiments, the Gammacoronavirus Genus sequence is a target of an assay. In some embodiments, the Deltacoronavirus Genus sequence is a target of an assay. In some embodiments, the Influenza B - Victoria VI sequence is a target of an assay. In some embodiments, the Influenza B - Yamagata Y1 sequence is a target of an assay. In some embodiments, the Influenza A Hl sequence is a target of an assay. In some embodiments, the Influenza A H2 sequence is a target of an assay. In some embodiments, the Influenza A H3 sequence is a target of an assay. In some embodiments, the Influenza A H4 sequence is a target of an assay. In some embodiments, the Influenza A H5 sequence is a target of an assay. In some embodiments, the Influenza A H6 sequence is a target of an assay. In some embodiments, the Influenza A H7 sequence is a target of an assay. In some embodiments, the Influenza A H8 sequence is a target of an assay. In some embodiments, the Influenza A H9 sequence is a target of an assay. In some embodiments, the Influenza A H10 sequence is a target of an assay. In some embodiments, the Influenza A Hl 1 sequence is a target of an assay. In some embodiments, the Influenza A H12 sequence is a target of an assay. In some embodiments, the Influenza A Hl 3 sequence is a target of an assay. In some embodiments, the Influenza A H14 sequence is a target of an assay. In some embodiments, the Influenza A H15 sequence is a target of an assay. In some embodiments, the Influenza A H16 sequence is a target of an assay. In some embodiments, the Influenza A N1 sequence is a target of an assay. In some embodiments, the Influenza A N2 sequence is a target of an assay. In some embodiments, the Influenza A N3 sequence is a target of an assay. In some embodiments, the Influenza A N4 sequence is a target of an assay. In some embodiments, the Influenza A N5 sequence is a target of an assay. In some embodiments, the Influenza A N6 sequence is a target of an assay. In some embodiments, the Influenza A N7 sequence is a target of an assay. In some embodiments, the Influenza A N8 sequence is a target of an assay. In some embodiments, the Influenza A N9 sequence is a target of an assay. In some embodiments, the Influenza A N10 sequence is a target of an assay. In some embodiments, the Influenza A N11 sequence is a target of an assay. In some embodiments, the Influenza A/Hl-2009 sequence is a target of an assay. In some embodiments, the Human endogenous control 18S rRNA sequence is a target of an assay. In some embodiments, the Human endogenous control GAPDH sequence is a target of an assay. In some embodiments, the Human endogenous control HPRT1 sequence is a target of an assay. In some embodiments, the Human endogenous control GUSB sequence is a target of an assay. In some embodiments, the Human endogenous control RNASe P sequence is a target of an assay. In some embodiments, the Influenza A oseltamivir resistance sequence is a target of an assay. In some embodiments, the Human Bocavirus sequence is a target of an assay. In some embodiments, the SARS-CoV-2 85A sequence is a target of an assay. In some embodiments, the SARS-CoV-2 T1001I sequence is a target of an assay. In some embodiments, the SARS- CoV-2 3675-3677A sequence is a target of an assay. In some embodiments, the SARS-CoV-2 P4715L sequence is a target of an assay. In some embodiments, the SARS-CoV-2 S5360L sequence is a target of an assay. In some embodiments, the SARS-CoV-2 69-70A sequence is a target of an assay. In some embodiments, the SARS-CoV-2 Tyrl44fs sequence is a target of an assay. In some embodiments, the SARS-CoV-2 242-244A sequence is a target of an assay. In some embodiments, the SARS-CoV-2 Y453F sequence is a target of an assay. In some embodiments, the SARS-CoV-2 S477N sequence is a target of an assay. In some embodiments, the SARS-CoV-2 E848K sequence is a target of an assay. In some embodiments, the SARS- CoV-2 N501 Y sequence is a target of an assay. In some embodiments, the SARS-CoV-2 D614G sequence is a target of an assay. In some embodiments, the SARS-CoV-2 P681R sequence is a target of an assay. In some embodiments, the SARS-CoV-2 P681H sequence is a target of an assay. In some embodiments, the SARS-CoV-2 L21F sequence is a target of an assay. In some embodiments, the SARS-CoV-2 Q27Stop sequence is a target of an assay. In some embodiments, the SARS-CoV-2 Mlfs sequence is a target of an assay. In some embodiments, the SARS-CoV-2 R203fs sequence is a target of an assay. In some embodiments, the Human adenovirus - pan assay sequence is a target of an assay. In some embodiments, the Bordetella parapertussis sequence is a target of an assay. In some embodiments, the Bordetella pertussis sequence is a target of an assay. In some embodiments, the Chlamydophila pneumoniae sequence is a target of an assay. In some embodiments, the Mycoplasma pneumoniae sequence is a target of an assay. In some embodiments, the Legionella pneumophila sequence is a target of an assay. In some embodiments, the Bordetella bronchoseptica sequence is a target of an assay. In some embodiments, the Bordetella holmesii sequence is a target of an assay. In some embodiments, the Human adenovirus Type A sequence is a target of an assay. In some embodiments, the Human adenovirus Type B sequence is a target of an assay. In some embodiments, the Human adenovirus Type C sequence is a target of an assay. In some embodiments, the Human adenovirus Type D sequence is a target of an assay. In some embodiments, the Human adenovirus Type E sequence is a target of an assay. In some embodiments, the Human adenovirus Type F sequence is a target of an assay. In some embodiments, the Human adenovirus Type G sequence is a target of an assay. In some embodiments, the MERS-CoV sequence is a target of an assay. In some embodiments, the human metapneumovirus sequence is a target of an assay. In some embodiments, the human parainfluenza 1 sequence is a target of an assay. In some embodiments, the human parainfluenza 2 sequence is a target of an assay. In some embodiments, the human parainfluenza 4 sequence is a target of an assay. In some embodiments, the hCoV-OC43 sequence is a target of an assay. In some embodiments, the human parainfluenza 3 sequence is a target of an assay. In some embodiments, the RSV-A sequence is a target of an assay. In some embodiments, the RSV-B sequence is a target of an assay. In some embodiments, the hCoV-229E sequence is a target of an assay. In some embodiments, the hCoV-HKUl sequence is a target of an assay. In some embodiments, the hCoV-NL63 sequence is a target of an assay. In some embodiments, the Gammacoronavirus sequence is a target of an assay. In some embodiments, the Deltacoronavirus sequence is a target of an assay. In some embodiments, the Alphacoronavirus sequence is a target of an assay. In some embodiments, the Rhinovirus C sequence is a target of an assay. In some embodiments, the Betacoronavirus sequence is a target of an assay. In some embodiments, the Influenza A sequence is a target of an assay. In some embodiments, the Influenza B sequence is a target of an assay. In some embodiments, the SARS-CoV-2 sequence is a target of an assay. In some embodiments, the SARS-CoV-1 sequence is a target of an assay. In some embodiments, the Sarbecovirus subgenus sequence is a target of an assay. In some embodiments, the SARS- related viruses sequence is a target of an assay. In some embodiments, the MS2 sequence is a target of an assay.

[0204] In some embodiments, the assay is directed to one or more target sequences. In some embodiments, a target sequence is a portion of an antimicrobial resistance (AMR) gene, such as CTX-M-1, CTX-M-2, CTX-M-25, CTX-M-8, CTX-M-9, or IMP. In some embodiments, a target sequence is a Mycobacterium tuberculosis sequence, such as a portion of IS 1081 or IS6110. In some embodiments, a target sequence is an orthopox virus sequence. In some embodiments, a target sequence is a pseudorabies virus sequence. In some embodiments, a target sequence is a Staphylococcus aureus sequence, such as a portion of gyrA or gyrB, or a portion of a S. aureus thermonuclease. In some embodiments, a target sequence is a Stenotrophomonas maltophilia sequence, such as a sequence of S. maltophilia alpha, S. maltophilia beta, or S. maltophilia gamma. In some embodiments, a target sequence is a Bordetalla sp. sequence, such as a sequence of Bordetella bronchoseplica. Bordetella holmesii. Bordetella parapertussis, or Bordetella pertussis. In some embodiments, a target sequence is a Chlamydophila pneumoniae sequence. In some embodiments, a target sequence is a Human adenovirus sequence, such as a sequence of human adenovirus Type A, Type B, Type C, Type D, Type E, Type F, or Type G. In some embodiments, a target sequence is a human bocavirus sequence. In some embodiments, a target sequence is a Legionella pneumophila sequence. In some embodiments, a target sequence is Mycoplasma pneumoniae sequence. In some embodiments, a target sequence is an Acinetobacter spp. (e.g., A. pitii, A. baumannii, or A. nosocomialis) sequence, such as a portion of gyrB or a 16S-23S ribosomal RNA intergenic spacer sequence. In some embodiments, a target sequence is a Proteus spp. (e.g., P. mirabilis, P. vulgaris, P. penneri, or P. hauseri) sequence, such as a portion of rpoD or 16S. In some embodiments, a target sequence is an Enterobacter spp. (e.g., E. nimipressuralis, E. cloacae, E. asburiae, E. hormaechei, E. kobei, E. ludwigii, or E. mori) sequence, such as a portion of dnaJ, purG, or 16S. In some embodiments, a target sequence is a. Bacillus anthracis sequence, such as a portion of pagA or capB. In some embodiments, a target sequence is a Brucella spp. sequence, such as a portion of 23 S, bcsp31, or omp2a. In some embodiments, a target sequence is a Coxiella burnetiid sequence, such as a portion of coml or IS110. In some embodiments, a target sequence is aFrancisella tularensis sequence, such as a portion of 16S. In some embodiments, a target sequence is a Rickettsia spp. sequence, such as a portion of 16S, 23S, or 782-17K genus common antigen. In some embodiments, a target sequence is a Yersinia pestis sequence, such as a portion of pMTl, pCDl, or pPCPl. In some embodiments, a target sequence is a A. calcoaceticus sequence, such as a portion of gyrB. In some embodiments, a target sequence is a Francisella tularensis sequence, such as a portion of tul4 or fopA. In some embodiments, a target sequence is a Nocardia spp. sequence, such as a portion of 16S, hsp65, gyrB, secAl, or sodA. In some embodiments, a target sequence is a Cryptococcus spp. sequence, such as a portion of 18S, URA5, ITS, 28S, or CTX1. In some embodiments, a target sequence is an Actinomyces spp. sequence, such as a portion of 16S. In some embodiments, a target sequence is a Streptococcus spp. sequence, such as a portion of 16S, tuf, sodA, or rpoB. In some embodiments, a target sequence is an rRNA sequence, such as a portion of 28S rRNA or 18S rRNA. In some embodiments, a target sequence is a coronavirus sequence, such as a sequence of an alphacoronavirus, betacoronavirus, deltacoronavirus, or gammacoronavirus. In some embodiments, a target sequence is a human coronavirus (hCoV) sequence, such as a sequence of hCoV-229E, hCoV-HKUl, hCoV-NL63, hCoV-OC43. In some embodiments, a target sequence is a MERS-CoV sequence. In some embodiments, the sequence is a mammarenavirus sequence, such as a sequence of a Argentinian mammarenavirus (Junin arenavirus), Lassa mammarenavirus, Lujo mammarenavirus (e.g., an L segment or S segment thereof), or Machupo mammarenavirus. In some embodiments, a target sequence is a human metapneumovirus sequence. In some embodiments, a target sequence is a human parainfluenza sequence, such as a sequence of human parainfluenza 1, human parainfluenza 2, human parainfluenza 3, or human parainfluenza 4. In some embodiments, a target sequence is an influenza A virus sequence, such as a sequence of influenza A Hl, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl 1, H12, H13, H14, H15, H16, Nl, N2, N3, N4, N5, N6, N7, N8, or N9. In some embodiments, a target sequence is an influenza B sequence, such as a sequence of influenza B- Victoria VI or influenza B-Yamagata Yl. In some embodiments, a target sequence is a bacteriophage MS2 sequence. In some embodiments, a target sequence is a rhinovirus C sequence. In some embodiments, a target sequence is a respiratory syncytial virus (RSV) sequence, such as a sequence of RSV-A or RSV-B. In some embodiments, a target sequence is a Sarbecovirus sequence. In some embodiments, a target sequence is a severe acute respiratory syndrome coronavirus (SARS-CoV) sequence, such as a sequence of SARS-CoV-1 or SARS- CoV-2. In some embodiments, a target sequence is a portion of a SARS-COV-2 S gene, such as a sequence comprising 144/145 wild-type (WT), deletion (del) 144/145 (alpha variant), 156/157 WT, dell56/157 (delta variant), 241/243 WT, del241/243 (beta variant), 69/70 WT, del69/70 (alpha variant), A570 WT, A570D (alpha variant), A701 WT, A701 V (beta variant), DI 118 WT, DI 118H (alpha variant), D215 WT, D215G (beta variant), D614 WT, D614G (beta variant), D80 WT, D80A (beta variant), E484 WT, E484K (gamma variant), P681 WT, P681H (alpha variant), P681R (delta variant), S982 WT, S982A (alpha variant), T19 WT, T19R (delta variant), T716 WT, T716F (gamma variant). In some embodiments, a target sequence is a SARS-related virus sequence. In some embodiments, a target sequence is a portion of a gene selected from 16S, 18S, 23S, 28S, ACTB, ATP5ME, ATP5MF, ATP5MG, ATP5PB, BCSP31, CAPB, CHMP2A, Clorf43, COMI, CTX1, DNAJ, EMC7, FOP A, GPI, GAPDH, GUSB, GYRB, HRPT1, HSP65, NDUFB3, NDUFB4, NDUFB8, OMP2A, PAGA, PRDX1, PSMB2, PSMB4, PURG, RAB7A, REEP5, RNaseP, RPL13, RPL19, RPL27A, RPL30, RPL31, RPL32, RPL37A, RPOB, RPOD, RPS10, RPS27, RPS29, RPS6, SECA1, SNRPD3, SODA, TUF, TUL4, URA5, VCP, VPS29, and YWHAG.

[0205] In some embodiments, the one or more targets may be at a concentration of 1 copy/reaction, at least about 2 copies/reaction, at least about 3 copies/reaction, at least about 4 copies/reaction, at least about 5 copies/reaction, at least about 6 copies/reaction, at least about 7 copies/reaction, at least about 8 copies/reaction, at least about 9 copies/reaction, at least about 10 copies/reaction, at least about 20 copies/reaction, at least about 30 copies/reaction, at least about 40 copies/reaction, at least about 50 copies/reaction, at least about 60 copies/reaction, at least about 70 copies/reaction, at least about 80 copies/reaction, at least about 90 copies/reaction, at least about 100 copies/reaction, at least about 200 copies/reaction, at least about 300 copies/reaction, at least about 400 copies/reaction, at least about 500 copies/reaction, at least about 600 copies/reaction, at least about 700 copies/reaction, at least about 800 copies/reaction, at least about 900 copies/reaction, at least about 1000 copies/reaction, at least about 2000 copies/reaction, at least about 3000 copies/reaction, at least about 4000 copies/reaction, at least about 5000 copies/reaction, at least about 6000 copies/reaction, at least about 7000 copies/reaction, at least about 8000 copies/reaction, at least about 9000 copies/reaction, at least about 10000 copies/reaction, at least about 20000 copies/reaction, at least about 30000 copies/reaction, at least about 40000 copies/reaction, at least about 50000 copies/reaction, at least about 60000 copies/reaction, at least about 70000 copies/reaction, at least about 80000 copies/reaction, at least about 90000 copies/reaction, or at least about 100000 copies/reaction. [0206] The sample used for cancer testing or cancer risk testing can comprise at least one target sequence or target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene with a mutation associated with cancer, from a gene whose overexpression is associated with cancer, a tumor suppressor gene, an oncogene, a checkpoint inhibitor gene, a gene associated with cellular growth, a gene associated with cellular metabolism, or a gene associated with cell cycle. Sometimes, the target nucleic acid encodes for a cancer biomarker, such as a prostate cancer biomarker or non-small cell lung cancer. In some cases, the assay can be used to detect “hotspots” in target nucleic acids that can be predictive of cancer, such as lung cancer, cervical cancer, in some cases, the cancer can be a cancer that is caused by a virus. Some non-limiting examples of viruses that cause cancers in humans include Epstein-Barr virus (e.g., Burkitt’s lymphoma, Hodgkin’s Disease, and nasopharyngeal carcinoma); papillomavirus (e.g., cervical carcinoma, anal carcinoma, oropharyngeal carcinoma, penile carcinoma); hepatitis B and C viruses (e.g., hepatocellular carcinoma); human adult T-cell leukemia virus type 1 (HTLV- 1) (e.g., T-cell leukemia); and Merkel cell polyomavirus (e.g., Merkel cell carcinoma). One skilled in the art will recognize that viruses can cause or contribute to other types of cancers. In some cases, the target nucleic acid is a portion of a nucleic acid that is associated with a blood fever. In some cases, the target nucleic acid segment is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a locus of at least one of: ALK, APC, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CASR, CDC73, CDH1, CDK4, CDKN1B, CDKN1C, CDKN2A, CEBPA, CHEK2, CTNNA1, DICER1, DIS3L2, EGFR, EPCAM, FH, FLCN, GATA2, GPC3, GREM1, HOXB13, HRAS, KIT, MAX, MEN1, MET, MITF, MLH1, MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NF2, NTHL1, PALB2, PDGFRA, PHOX2B, PMS2, POLDI, POLE, POTI, PRKAR1A, PTCHI, PTEN, RAD50, RAD51C, RAD51D, RBI, RECQL4, RET, RUNX1, SDHA, SDHAF2, SDHB, SDHC, SDHD, SMAD4, SMARCA4, SMARCB1, SMARCE1, STK11, SUFU, TERC, TERT, TMEM127, TP53, TSC1, TSC2, VHL, WRN, and WT1.

[0207] The sample used for genetic disorder testing can comprise at least one target sequence or target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. In some embodiments, the genetic disorder is hemophilia, sickle cell anemia, P-thalassemia, Duchene muscular dystrophy, severe combined immunodeficiency, or cystic fibrosis. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene with a mutation associated with a genetic disorder, from a gene whose overexpression is associated with a genetic disorder, from a gene associated with abnormal cellular growth resulting in a genetic disorder, or from a gene associated with abnormal cellular metabolism resulting in a genetic disorder. In some cases, the target nucleic acid segment is a portion of a nucleic acid from a genomic locus, a transcribed mRNA, or a reverse transcribed cDNA from a locus of at least one of: CFTR, FMRI, SMN1, ABCB11, ABCC8, ABCD1, ACAD9, ACADM, ACADVL, ACAT1, ACOX1, ACSF3, ADA, ADAMTS2, ADGRG1, AGA, AGL, AGPS, AGXT, AIRE, ALDH3A2, ALDOB, ALG6, ALMS1, ALPL, AMT, AQP2, ARG1, ARSA, ARSB, ASL, ASNS, ASP A, ASS1, ATM, ATP6V1B1, ATP7A, ATP7B, ATRX, BBS1, BBS10, BBS12, BBS2, BCKDHA, BCKDHB, BCS1L, BLM, BSND, CAPN3, CBS, CDH23, CEP290, CERKL, CHM, CHRNE, CIITA, CLN3, CLN5, CLN6, CLN8, CLRN1, CNGB3, COL27A1, COL4A3, COL4A4, COL4A5, COL7A1, CPS1, CPT1A, CPT2, CRB1, CTNS, CTSK, CYBA, CYBB, CYP11B1, CYP11B2, CYP17A1, CYP19A1, CYP27A1, DBT, DCLRE1C, DHCR7, DHDDS, DLD, DMD, DNAH5, DNAI1, DNAI2, DYSF, EDA, EIF2B5, EMD, ERCC6, ERCC8, ESCO2, ETFA, ETFDH, ETHE1, EVC, EVC2, EYS, F9, FAH, FAM161A, FANCA, FANCC, FANCG, FH, FKRP, FKTN, G6PC, GAA, GALC, GALK1, GALT, GAMT, GBA, GBE1, GCDH, GFM1, GJB1, GJB2, GLA, GLB1, GLDC, GLE1, GNE, GNPTAB, GNPTG, GNS, GRHPR, HADHA, HAX1, HBA1„ HBA2, HBB, HEXA, HEXB, HGSNAT, HLCS, HMGCL, HOGA1, HPS1, HPS3, HSD17B4, HSD3B2, HYAL1, HYLS1, IDS, IDUA, IKBKAP, IL2RG, IVD, KCNJ11, LAMA2, LAMA3, LAMB3, LAMC2, LCA5, LDLR, LDLRAP1, LHX3, LIFR, LIPA, LOXHD1, LPL, LRPPRC, MAN2B1, MCOLN1, MED 17, MESP2, MFSD8, MKS1, MLC1, MMAA, MMAB, MMACHC, MMADHC, MPI, MPL, MPV17, MTHFR, MTM1, MTRR, MTTP, MUT, MY07A, NAGLU, NAGS, NBN, NDRG1, NDUFAF5, NDUFS6, NEB, NPC1, NPC2, NPHS1, NPHS2, NR2E3, NTRK1, OAT, OP A3, OTC, PAH, PC, PCCA, PCCB, PCDH15, PDHA1, PDHB, PEX1, PEX10, PEX12, PEX2, PEX6, PEX7, PFKM, PHGDH, PKHD1, PMM2, POMGNT1, PPT1, PROP1, PRPS1, PSAP, PTS, PUS1, PYGM, RAB23, RAG2, RAPSN, RARS2, RDH12, RMRP, RPE65, RPGRIP1L, RSI, RTEL1, SACS, SAMHD1, SEPSECS, SGCA, SGCB, SGCG, SGSH, SLC12A3, SLC12A6, SLC17A5, SLC22A5, SLC25A13, SLC25A15, SLC26A2, SLC26A4, SLC35A3, SLC37A4, SLC39A4, SLC4A11, SLC6A8, SLC7A7, SMARCAL1, SMPD1, STAR, SUMF1, TAT, TCIRG1, TECPR2, TFR2, TGM1, TH, TMEM216, TPP1, TRMU, TSFM, TTP A, TYMP, USH1C, USH2A, VPS 13 A, VPS13B, VPS45, VRK1, VSX2, WNT10A, XPA, XPC, and ZFYVE26.

[0208] The sample used for phenotyping testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene associated with a phenotypic trait.

[0209] The sample used for genotyping testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene associated with a genotype.

[0210] The sample used for ancestral testing can comprise at least one target nucleic acid segment that can bind to a guide nucleic acid of the reagents described herein. The target nucleic acid segment, in some cases, is a portion of a nucleic acid from a gene associated with a geographic region of origin or ethnic group.

[0211] The sample can be used for identifying a disease status. For example, a sample is any sample described herein, and is obtained from a subject for use in identifying a disease status of a subject. The disease can be a cancer or genetic disorder. Sometimes, a method may comprise obtaining a serum sample from a subject; and identifying a disease status of the subject. Often, the disease status is prostate disease status. In any of the embodiments described herein, the device can be configured for asymptomatic, pre-symptomatic, and/or symptomatic diagnostic applications, irrespective of immunity. In any of the embodiments described herein, the device can be configured to perform one or more serological assays on a sample (e.g., a sample comprising blood).

[0212] In some embodiments, the sample can be used to identify a mutation in a target nucleic acid of a plant or of a bacteria, virus, or microbe associated with a plant or soil. The devices and methods of the present disclosure can be used to identify a mutation of a target nucleic acid that affects the expression of a gene. A mutation that affects the expression of gene can be a mutation of a target nucleic acid within the gene, a mutation of a target nucleic acid comprising RNA associated with the expression of a gene, or a target nucleic acid comprising a mutation of a nucleic acid associated with regulation of expression of a gene, such as an RNA or a promoter, enhancer, or repressor of the gene. Often, the mutation is a single nucleotide mutation

[0213] In some instances, the target nucleic acid is a single stranded nucleic acid. Alternatively, or in combination, the target nucleic acid is a double stranded nucleic acid and is prepared into single stranded nucleic acids before or upon contacting the reagents. The target nucleic acid can be a RNA, DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples. The target nucleic acids include but are not limited to mRNA, rRNA, tRNA, non- coding RNA, long non-coding RNA, and microRNA (miRNA). In some cases, the target nucleic acid is mRNA. In some cases, the target nucleic acid is from a virus, a parasite, or a bacterium described herein. In some cases, the target nucleic acid is transcribed from a gene as described herein.

[0214] A number of target nucleic acids are consistent with the systems and methods disclosed herein. Some methods described herein can detect a target nucleic acid present in the sample in various concentrations or amounts as a target nucleic acid population. In some cases, the sample has at least 2 target nucleic acids. In some cases, the sample has at least 3, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 target nucleic acids. In some cases, the sample has from 1 to 10,000, from 100 to 8000, from 400 to 6000, from 500 to 5000, from 1000 to 4000, or from 2000 to 3000 target nucleic acids. In some cases, the sample has from 100 to 9500, from 100 to 9000, from 100 to 8500, from 100 to 8000, from 100 to 7500, from 100 to 7000, from 100 to 6500, from 100 to

6000, from 100 to 5500, from 100 to 5000, from 250 to 9500, from 250 to 9000, from 250 to

8500, from 250 to 8000, from 250 to 7500, from 250 to 7000, from 250 to 6500, from 250 to

6000, from 250 to 5500, from 250 to 5000, from 2500 to 9500, from 2500 to 9000, from 2500 to

8500, from 2500 to 8000, from 2500 to 7500, from 2500 to 7000, from 2500 to 6500, from 2500 to 6000, from 2500 to 5500, or from 2500 to 5000 target nucleic acids. In some cases, the method detects target nucleic acid present at least at one copy per 10 ¹ non-target nucleic acids, 10 ² non-target nucleic acids, 10 ³ non-target nucleic acids, 10 ⁴ non-target nucleic acids, 10 ⁵ nontarget nucleic acids, 10 ⁶ non-target nucleic acids, 10 ⁷ non-target nucleic acids, 10 ⁸ non-target nucleic acids, 10 ⁹ non-target nucleic acids, or 10 ¹⁰ non-target nucleic acids.

[0215] A number of target nucleic acid populations are consistent with the systems and methods disclosed herein. Some methods described herein can be implemented to detect two or more target nucleic acid populations present in the sample in various concentrations or amounts. In some cases, the sample has at least 2 target nucleic acid populations. In some cases, the sample has at least 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 target nucleic acid populations. In some cases, the sample has from 3 to 50, from 5 to 40, or from 10 to 25 target nucleic acid populations. In some cases, the sample has from 2 to 50, from 5 to 50, from 10 to 50, from 2 to 25, from 3 to 25, from 4 to 25, from 5 to 25, from 10 to 25, from 2 to 20, from 3 to 20, from 4 to 20, from 5 to 20, from 10 to 20, from 2 to 10, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, or from 9 to 10 target nucleic acid populations. In some cases, the methods of the present disclosure can be implemented to detect target nucleic acid populations that are present at least at one copy per 10 ¹ non-target nucleic acids, 10 ² non-target nucleic acids, 10 ³ non-target nucleic acids, 10 ⁴ non-target nucleic acids, 10 ⁵ non-target nucleic acids, 10 ⁶ non-target nucleic acids, 10 ⁷ non-target nucleic acids, 10 ⁸ non-target nucleic acids, 10 ⁹ non-target nucleic acids, or IO ¹⁰ non-target nucleic acids. The target nucleic acid populations can be present at different concentrations or amounts in the sample.

[0216] FIG. 6 illustrates an exemplary method for programmable nuclease-based detection. The method can comprise collecting a sample. The sample can comprise any type of sample as described herein. The method can comprise preparing the sample. Sample preparation can comprise one or more sample preparation steps. The one or more sample preparation steps can be performed in any suitable order. The one or more sample preparation steps can comprise physical filtration of non-target materials using a macro filter. The one or more sample preparation steps can comprise nucleic acid purification. The one or more sample preparation steps can comprise lysis. The one or more sample preparation steps can comprise heat inactivation. The one or more sample preparation steps can comprise enzyme (e.g., PK) inactivation. The one or more sample preparation steps can comprise adding one or more enzymes or reagents to prepare the sample for target detection.

[0217] The method can comprise generating one or more droplets, aliquots, or subsamples from the sample. The one or more droplets, aliquots, or subsamples can correspond to a volumetric portion of the sample. The sample can be divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more droplets, aliquots, or subsamples. In some embodiments, the sample is not divided into subsamples.

[0218] The method can comprise amplifying one or more targets within each droplet, aliquot, or subsample. Amplification of the one or more targets within each droplet can be performed in parallel and/or simultaneously for each droplet. Dividing the sample into a plurality of droplets can enhance a speed and/or an efficiency of the amplification process (e.g., a thermocycling process) since the droplets comprise a smaller volume of material than the bulk sample introduced. Amplifying the one or more targets within each individual droplet can also permit effective amplification of various target nucleic acids that cannot be amplified as efficiently in a bulk sample containing the various target nucleic acids if the bulk sample were to undergo a singular amplification process. In some embodiments, amplification is performed on the bulk sample without first dividing the sample into subsamples.

[0219] The method can further comprise using a CRISPR-based or programmable nuclease- based detection module to detect one or more targets (e.g., target sequences or target nucleic acids) in the sample. In some cases, the sample can be divided into a plurality of droplets, aliquots, or subsamples to facilitate sample preparation and to enhance the detection capabilities of the devices of the present disclosure. In some cases, the sample is not divided into subsamples.

-I l l- [0220] In some embodiments, the sample can be provided manually to the nucleic acid detection device of the present disclosure. For example, a swab sample can be dipped into a solution and the sample/solution can be pipetted into the device. In other embodiments, the sample can be provided via an automated syringe. The automated syringe can be configured to control a flow rate at which the sample is provided to the nucleic acid detection device. The automated syringe can be configured to control a volume of the sample that is provided to the nucleic acid detection device over a predetermined period.

[0221] In some embodiments, the sample can be provided directly to the nucleic acid detection device of the present disclosure. For example, a swab sample can be inserted into a sample chamber on the nucleic acid detection device.

[0222] The sample can be prepared before one or more targets are detected within the sample. The sample preparation steps described herein can process a crude sample to generate a pure or purer sample. Sample preparation can one or more physical or chemical processes, including, for example, nucleic acid purification, lysis, binding, washing, and/or eluting. In certain instances, sample preparation can comprise the following steps, in any order, including sample collection, nucleic acid purification, heat inactivation, enzyme inactivation, and/or base/acid lysis.

[0223] In some embodiments, nucleic acid purification can be performed on the sample. Purification can comprise disrupting a biological matrix of a cell to release nucleic acids, denaturing structural proteins associated with the nucleic acids (nucleoproteins), inactivating nucleases that can degrade the isolated product (RNase and/or DNase), and/or removing contaminants (e.g., proteins, carbohydrates, lipids, biological or environmental elements, unwanted nucleic acids, and/or other cellular debris).

[0224] In some embodiments, lysis of a collected sample can be performed. Lysis can be performed using a protease (e.g., a Proteinase K or PK enzyme). Exemplary proteases include serine proteases (e.g., Proteinase K, Savinase®, trypsin, Protamex®, etc.), metalloproteinases (e.g., MMP-3, etc.), cysteine proteases (e.g., cathepsin B, papin, etc.), threonine proteases, aspartic proteases (e.g., renin, pepsin, cathepsin D, etc.), glutamic proteases, asparagine peptide lyases, or the like. In some cases, a solution of reagents can be used to lyse the cells in the sample and release the nucleic acids so that they are accessible to the programmable nuclease. Active ingredients of the solution can be chaotropic agents, detergents, salts, and can be of high osmolality, ionic strength, and pH. Chaotropic agents or chaotropes are substances that disrupt the three-dimensional structure in macromolecules such as proteins, DNA, or RNA. One example protocol may comprise a 4 M guanidinium isothiocyanate, 25 mM sodium citrate.2H20, 0.5% (w/v) sodium lauryl sarcosinate, and 0.1 M P-mercaptoethanol), but numerous commercial buffers for different cellular targets can also be used. Alkaline buffers can also be used for cells with hard shells, particularly for environmental samples. Detergents such as sodium dodecyl sulphate (SDS) and cetyl trimethylammonium bromide (CTAB) can also be implemented to chemical lysis buffers. Cell lysis can also be performed by physical, mechanical, thermal or enzymatic means, in addition to chemically-induced cell lysis mentioned previously. In some cases, depending on the type of sample, nanoscale barbs, nanowires, acoustic generators, integrated lasers, integrated heaters, and/or microcapillary probes can be used to perform lysis. [0225] In certain instances, heat inactivation can be performed on the sample. In some embodiments, a processed/lysed sample can undergo heat inactivation to inactivate, in the lysed sample, the proteins used during lysing (e.g., a PK enzyme or a lysing reagent) and/or other residual proteins in the sample (e.g., RNases, DNases, viral proteins, etc.). In some cases, a heating element integrated into the nucleic acid detection device can be used for heatinactivation. The heating element can be powered by a battery or another source of thermal or electric energy that is integrated with the nucleic acid detection device.

[0226] In certain instances, enzyme inactivation can be performed on the sample. In some embodiments, a processed/lysed sample can undergo enzyme inactivation to inhibit or inactivate, in the lysed sample, the proteins used during lysing (e.g., a PK enzyme or a lysing reagent) and/or other residual proteins in the sample (e.g., RNases, DNases, etc.). In some cases, a solution of reagents can be used to inactivate one or more enzymes present in the sample. Enzyme inactivation can occur before, during, or after lysis, when lysis is performed. For example, an RNase inhibitor may be included as a lysis reagent to inhibit native RNases within the sample (which might otherwise impair target and/or reporter detection downstream). Exemplary RNase inhibitors include RNAse Inhibitor, Murine (NEB), Rnaseln Plus (Promega), Protector Rnase Inhibitor (Roche), Superasein (Ambion), RiboLock (Thermo), Ribosafe (Bioline), or the like. Alternatively, or in combination, when a protease is used for sample lysis, a protease inhibitor can be applied to the lysed sample to inactivate the protease prior to contacting the sample nucleic acids to the programmable nuclease. Additional application of heat may not be required to inhibit the protease (e.g., proteinase K) sufficiently to prevent additional activity of the protease (which could potentially impair programmable nuclease activity downstream, in some embodiments). Exemplary protease inhibitors include AEBSF, antipain, aprotinin, bestatin, chymostatin, EDTA, leupeptin, pepstatin A, phosphoramidon, PMSF, soybean trypsin inhibitor, TPCK, or the like. In some instances, enzyme inactivation may occur before, during, after, or instead of heat inactivation.

[0227] In some cases, a target nucleic acid within the sample can undergo amplification before binding to a guide nucleic acid, for example a crRNA of a CRISPR enzyme. The target nucleic acid within a purified sample can be amplified. In some instances, amplification can be accomplished using loop mediated amplification (LAMP), isothermal recombinase polymerase amplification (RPA), and/or polymerase chain reaction (PCR). In some instances, digital droplet amplification can used. Such nucleic acid amplification of the sample can improve at least one of a sensitivity, specificity, or accuracy of the detection of the target RNA. The reagents for nucleic acid amplification can comprise a recombinase, an oligonucleotide primer, a single-stranded DNA binding (SSB) protein, and a polymerase. The nucleic acid amplification can be transcription mediated amplification (TMA). Nucleic acid amplification can be helicase dependent amplification (HD A) or circular helicase dependent amplification (cHDA). In additional cases, nucleic acid amplification is strand displacement amplification (SDA). The nucleic acid amplification can be recombinase polymerase amplification (RPA). The nucleic acid amplification can be at least one of loop mediated amplification (LAMP) or the exponential amplification reaction (EXPAR). Nucleic acid amplification is, in some cases, by rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence-based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA). The nucleic acid amplification can be performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes. Sometimes, the nucleic acid amplification is performed for from 1 to 60, from 5 to 55, from 10 to 50, from 15 to 45, from 20 to 40, or from 25 to 35 minutes. Sometimes, the nucleic acid amplification is performed for from 5 to 60, from 10 to 60, from 15 to 60, from 30 to 60, from 45 to 60, from 1 to 45, from 5 to 45, from 10 to 45, from 30 to 45, from 1 to 30, from 5 to 30, from 10 to 30, from 15 to 30, from 1 to 15, from 5 to 15, or from 10 to 15 minutes.

[0228] In some embodiments, amplification can comprise thermocycling of the sample. Thermocycling can be carried out for one or more droplets of the sample in parallel and/or independently in separate locations. This can be accomplished by methods such as (1) by holding droplets stationary in locations where a heating element is in close proximity to the droplet on one of the droplet sides and a heat sink element is in close proximity to the other side of the droplet, or (2) flowing the droplet through zones in a fluid channel where heat flows across it from a heating source to a heat sink. In some cases, one or more resistive heating elements can be used to perform thermocycling. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 20-45°C. The nucleic acid amplification reaction can be performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. The nucleic acid amplification reaction can be performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. In some cases, the nucleic acid amplification reaction is performed at a temperature of from 20°C to 45°C, from 25°C to 40°C, from 30°C to 40°C, or from 35°C to 40°C. In some cases, the nucleic acid amplification reaction is performed at a temperature of from 45°C to 65°C, from 50°C to 65°C, from 55°C to 65°C, or from 60°C to 65°C. In some cases, the nucleic acid amplification reaction can be performed at a temperature that ranges from about 20 °C to 45 °C, from 25 °C to 45 °C, from 30 °C to 45 °C, from 35 °C to 45 °C, from 40 °C to 45 °C, from 20 °C to 37 °C, from 25 °C to 37 °C, from 30 °C to 37 °C, from 35 °C to 37 °C, from 20 °C to 30 °C, from 25 °C to 30 °C, from 20 °C to 25 °C, or from about 22 °C to 25 °C. In some cases, the nucleic acid amplification reaction can be performed at a temperature that ranges from about 40 °C to 65 °C, from 45 °C to 65 °C, from 50 °C to 65 °C, from 55 °C to 65 °C, from 60 °C to 65 °C, from 40 °C to 60 °C, from 45 °C to 60 °C, from 50 °C to 60 °C, from 55 °C to 60 °C, from 40 °C to 55 °C, from 45 °C to 55 °C, from 50 °C to 55 °C, from 40 °C to 50 °C, or from about 45 °C to 50 °C.

[0229] Additionally, target nucleic acid can optionally be amplified before binding to the guide nucleic acid (e.g., crRNA) of the programmable nuclease (e.g., CRISPR enzyme). This amplification can be PCR amplification or isothermal amplification. This nucleic acid amplification of the sample can improve at least one of sensitivity, specificity, or accuracy of the detection the target RNA. The reagents for nucleic acid amplification can comprise a recombinase, a oligonucleotide primer, a single-stranded DNA binding (SSB) protein, and a polymerase. The nucleic acid amplification can be transcription mediated amplification (TMA). Nucleic acid amplification can be helicase dependent amplification (HD A) or circular helicase dependent amplification (cHDA). In additional cases, nucleic acid amplification is strand displacement amplification (SDA). The nucleic acid amplification can be recombinase polymerase amplification (RPA). The nucleic acid amplification can be at least one of loop mediated amplification (LAMP) or the exponential amplification reaction (EXPAR). Nucleic acid amplification is, in some cases, by rolling circle amplification (RCA), ligase chain reaction (LCR), simple method amplifying RNA targets (SMART), single primer isothermal amplification (SPIA), multiple displacement amplification (MDA), nucleic acid sequence based amplification (NASBA), hinge-initiated primer-dependent amplification of nucleic acids (HIP), nicking enzyme amplification reaction (NEAR), or improved multiple displacement amplification (IMDA). The nucleic acid amplification can be performed for no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 60 minutes.

Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 20- 45°C. Sometimes, the nucleic acid amplification reaction is performed at a temperature of around 45-65 °C. The nucleic acid amplification reaction can be performed at a temperature no greater than 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C. The nucleic acid amplification reaction can be performed at a temperature of at least 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.

[0230] FIG. 8A illustrates an example of a channel through which a continuous flow of a sample can travel to undergo a thermocycling procedure. The device can comprise one or more movable mechanisms that are integrated into the device. The one or more movable mechanisms can be powered using a battery that is integrated with the device. The one or more movable mechanisms can be configured to stop and start the continuous flow of the sample through the channel at one or more predetermined time intervals. The one or more movable mechanisms can be configured to chop or divide the continuous flow of the sample into a plurality of smaller volumes, which can be referred to herein as “droplets.” The one or more movable mechanisms can have an open configuration and a closed configuration. The open configuration can permit a continuous flow of the sample through one or more sections of the channel, as seen in FIG. 8A. The closed configuration can restrict or completely inhibit a flow of the sample through one or more sections of the channel, as seen in FIG. 8B. The closed configuration can permit a physical and/or thermal separation of one or more volumes or portions of the sample flowing through the channel. When the movable mechanisms are in the closed position, the movable mechanisms can provide a physical barrier between different volumes or portions of the sample flowing through the channel. The droplet volume can range from 0.01 to 1 microliter, 1 to 5 microliters and 5 to 50 microliters. The different volumes or partitions can correspond to the droplets described elsewhere herein. The movable mechanisms can switch from the open position to the closed position, or from the closed position to the open position, depending on an operation of a syringe that is providing the sample or another flow regulator that is controlling a flow of the sample through the channel. The movable mechanisms can be powered using a battery that is integrated with the device.

[0231] In some cases, the movable mechanism can comprise a plurality of valves. The plurality of valves can comprise, for example, a check valve. In some cases, the movable mechanism can comprise a plunger or a bristle. The plunger or bristle can have an open configuration and a closed configuration. As described above, the open configuration can permit a continuous flow of the sample through one or more sections of the channel, and the closed configuration can restrict or completely inhibit a flow of the sample through one or more sections of the channel. The closed configuration can permit a physical and/or thermal separation of one or more volumes or portions of the sample flowing through the channel. When in the open configuration, the plunger or the bristle can be positioned flush to a bottom of the channel so that the sample can flow through the channel. When in the closed configuration, the plunger or the bristle can be configured to extend from a bottom portion of the channel to a top portion of the channel so that the sample flow is restricted and the sample is divided into a plurality of different droplets, or partitions, that are physically and/or thermally isolated from each other. In some cases, the movable mechanism can comprise any physical object (e.g., a plate) that can be configured to restrict flow through the channel at one or more sections of the channel. In some cases, the movable mechanism can comprise a hinge or spring mechanism to move the movable mechanism between an open configuration and a closed configuration.

[0232] The movable mechanisms can be used to generate one or more droplets, aliquots, or sub samples. Each of the one or more droplets, aliquots, or subsamples generated using the movable mechanism can be physically and/or thermally isolated within a plurality of different portions within the channel. The droplets, aliquots, or subsamples can be physically constrained within different portions within the channel. The droplets, aliquots, or subsamples can be constrained between a first movable mechanism that is in a closed position and a second movable mechanism that is in a closed position. The first movable mechanism can be located at a first distance from an inlet of the channel, and the second movable mechanism can be located at a second distance from the inlet of the channel. The channel can be part of a closed system through which the sample can flow. In some cases, when the sample flow through an inlet of the channel is stopped (e.g., a plunger of a syringe containing the sample is pulled back), the one or more movable mechanisms can be placed in a closed configuration, thereby separating the sample already within the channel into a plurality of thermally and physically isolated droplets. Generating droplets, aliquots, or subsamples can simplify the solution, reduce a complexity of the solution, and enhance an accessibility of targets for amplification.

[0233] The one or more droplets, aliquots, or subsamples generated using the movable mechanism can undergo an amplification step or a thermocycling step as described elsewhere herein. In some cases, the one or more droplets generated using the movable mechanisms can come into contact with separate heating units and heat sinks while constrained between two movable mechanisms. Different sections of the channel can comprise a plurality of heating units and heat sinks configured to perform thermocycling for different droplets. Individual thermocycling of the droplets, aliquots, or subsamples can permit more efficient thermocycling of smaller volumes of fluid, and can require less energy usage (e.g., from a battery). One or more valves can control a flow or a movement of the sample through the channel. The one or more valves can comprise a check valve that is configured to restrict a movement of the sample or the one or more droplets such that the sample or the one or more droplets do not travel backwards towards an inlet portion of the channel. The one or more valves can control when the sample or the droplets come into thermal contact with the heating unit and/or the heat sink. The timing of such thermal contact can correspond to a timing of one or more thermocycling steps. In some cases, a first droplet of the sample can be in thermal contact with a first heating unit and a first heat sink, a second droplet of the sample can be in thermal contact with a second heating unit and a second heat sink, and so on.

[0234] As described above, the devices of the present disclosure can be configured to perform droplet digitization or droplet generation. Droplet digitization or generation can comprise splitting a volume of the sample into multiple droplets, aliquots, or subsamples. The sample can have a volume that ranges from about 10 microliters to about 500 microliters. The plurality of droplets, aliquots, or subsamples can have a volume that ranges from about 0.01 microliters to about 100 microliters. The plurality of droplets, aliquots, or subsamples can have a same or substantially similar volume. In some cases, the plurality of droplets, aliquots, or subsamples can have different volumes. In some cases, the droplets, aliquots, or subsamples can be generated using a physical filter or the one or more movable mechanisms described above. In some cases, each droplet of the sample can undergo one or more sample preparation steps (e.g., nucleic acid purification, lysis, heat inactivation, enzyme inactivation, amplification, etc.) independently and/or in parallel while the droplets are physically constrained or thermally isolated between two movable mechanisms.

[0235] After amplification, the sample can be remixed. The sample can be circulated through the detection chamber using a bulk circulation mechanism that is configured to stir the remixed sample around such that the remixed sample comes into contact with one or more programmable nuclease probes. In some cases, the sample can be provided on a portion of a surface of the detection chamber that is proximal to one or more programmable nuclease probes. In some cases, the detection chamber can be configured to direct the sample along one or more fluid flow paths that position the remixed sample adjacent and/or proximal to one or more programmable nuclease probes. The one or more fluid flow paths can be used to target delivery of at least a portion of the remixed droplets to one or more detection regions associated with the one or more programmable nuclease probes. The remixed droplets can be circulated through the detection chamber along one or more desired fluid flow paths with aid of a piezoelectric device.

[0236] In some embodiments, electrowetting can be used by the device for sample transport. In some cases, the device can be configured for electrowetting-on-dielectric (EWOD) applications. The devices of the present disclosure can comprise an array of independently addressable electrodes integrated into the device.

[0237] Described herein are various embodiments of a device for programmable nuclease-based (e.g., CRISPR-based) assays. FIG. 7 A illustrates a top-down view of an exemplary device for CRISPR-based detection. The device can comprise a sample interface (200) that is configured to receive a sample. The sample can undergo one or more processing steps as described elsewhere herein. Any of the devices described herein may comprise a physical filter (201) to filter one or more particles from the sample that do not comprise the one or more targets (e.g., a gene of interest). In some embodiments, the device may comprise thermocycling components (202). In some cases, the sample can be divided into a plurality of digitized droplets. The plurality of digitized droplets can be provided in a plurality of different chambers of the device. The plurality of digitized droplets can undergo separate processing steps (e.g., thermocycling). In some embodiments, the independent digital reactions can be conducted in parallel with no cross-talk therebetween. In other embodiments, the independent digital reactions can be conducted in parallel with a minimal level or amount of cross-talk. The plurality of droplets can be mixed together after the separate processing steps upon completion of the processing steps. A plurality of programmable nuclease probes comprising one or more programmable nucleases (e.g., Cas enzymes) can be operatively coupled to the detection chamber (203) to detect one or more targets in the sample or the plurality of digitized droplets that are mixed together in the detection chamber. One or more detectors (204) may be configured to detect a signal from the detection chamber as described herein. A side profile of an embodiment of an exemplary programmable nuclease-based device is shown in FIG. 7B. In some embodiments, the device may comprise one or more thermocycling compartments (205) as seen in sideview. In some embodiments, the device may comprise a detection chamber (206). In some embodiments, the device may comprise one or more detectors (207). In some embodiments, the device may comprise a battery (208) or plurality of batteries as described herein. In some embodiments, the device may comprise telemedicine components.

[0238] FIGS. 9A, 9B, 10A, and 10B illustrate an exemplary programmable nuclease probe that can be used in a compatible manner with the devices of the present disclosure. The programmable nuclease probe can comprise a guide nucleic acid complexed with a programmable nuclease. The programmable nuclease can comprise any type of programmable nuclease as described herein. In some cases, the programmable nuclease probe may comprise a guide nucleic acid complexed with a CRISPR enzyme. For example, FIG. 9A shows unbound target amplicons in the circulation chamber prior to binding to a guide RNA, which in turn is contacted to a programmable nuclease (e.g., a CRISPR enzyme). The guide RNA-CRISPR enzyme complex also includes a reporter. The programmable nuclease probe (e.g., a CRISPR probe) is immobilized to an immobilization matrix, where the interior side of the immobilization matrix is exposed to the inside wall of the circulation chamber. The guide nucleic acid or guide RNA is exposed to the target amplicons inside the circulation chamber. The reporter is in proximity to the “exterior” side of the immobilization matrix, where the exterior side of the immobilization matrix be in proximity to a detection region. FIG. 9B illustrates a programmable nuclease probe (e.g., a CRISPR probe) after binding with a complementary target amplicon. The binding event triggers a trans-cut that releases the reporter into a detectable region or changes the reporter. Detection mechanisms can involve interferometry, surface plasmon resonance, electrochemical detection such as potentiometry, or other detection mechanisms.

[0239] In certain instances, as seen in FIGS. 10A and 10B, the reporter of the programmable nuclease probe can initiate a signal amplification reaction with another molecular species after the complementary binding induced trans-cutting. Such species can be a reactive solid or gel matrix, or other reactive entity to generate an amplified signal during detection. The signal amplification reaction can be physical or chemical in nature. In certain instances, as seen in FIGS. 10A and 10B, after a complementary binding induced trans-cut, the released reporter, X, can initiate an interaction and/or a reaction with another entity, Y to produce an amplified or modified signal. Such entities can comprise a molecular species, a solid, a gel, or other entities. The signal amplification interaction can be a physical or chemical reaction. In some embodiments, the interaction involves free-radical, anionic, cationic or coordination polymerization reactions. In other embodiments the cut reporter can trigger aggregation, or agglutination, of molecules, cells, or nanoparticles. In some instances, the cut reporter can interact with a semiconductor material. In some embodiments, the chemical or physical change caused by the interaction is detected by optical detection means such as interferometry, surface plasmon resonance, reflectivity or other. In other embodiments, the chemical or physical change caused by the interaction is detected by potentiometric, amperometric, field effect transistor, or other electronic means

[0240] The programmable nuclease probe can comprise a programmable nuclease and/or a guide nucleic acid. The guide nucleic acid can bind to a target nucleic acid, as described in greater detail below. In some case, to minimize off-target binding (which can slow down detection or inhibit accurate detection), the device can be configured to generate an electro-potential gradient or to provide heat energy to one or more regions proximal to the programmable nuclease probe, to enhance targeting.

[0241] In some embodiments, one or more guide nucleic acids can be used to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample. The guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease as described herein. The guide nucleic acid can bind to the single stranded target nucleic acid comprising a portion of a nucleic acid from a bacterium or other agents responsible for a disease as described herein and further comprising a mutation, such as a single nucleotide polymorphism (SNP), which can confer resistance to a treatment, such as antibiotic treatment. The guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a cancer gene or gene associated with a genetic disorder as described herein. The guide nucleic acid is complementary to the target nucleic acid. Often the guide nucleic acid binds specifically to the target nucleic acid. The target nucleic acid can be a RNA, DNA, or synthetic nucleic acids. A guide nucleic acid can comprise a sequence that is reverse complementary to the sequence of a target nucleic acid. A guide nucleic acid can be a crRNA. Sometimes, a guide nucleic acid may comprise a crRNA and tracrRNA. The guide nucleic acid can bind specifically to the target nucleic acid. In some cases, the guide nucleic acid is not naturally occurring and made by artificial combination of otherwise separate segments of sequence. Often, the artificial combination is performed by chemical synthesis, by genetic engineering techniques, or by the artificial manipulation of isolated segments of nucleic acids. The target nucleic acid can be designed and made to provide desired functions. In some cases, the targeting region of a guide nucleic acid is 20 nucleotides in length. The targeting region of the guide nucleic acid can have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some instances, the targeting region of the guide nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the targeting region of a guide nucleic acid has a length from exactly or about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some cases, the targeting region of a guide nucleic acid has a length of from about 10 nt to about 60 nt, from about 20 nt to about 50 nt, or from about 30 nt to about 40 nt. In some cases, the targeting region of a guide nucleic acid has a length of from 15 nt to 55 nt, from 25 nt to 55 nt, from 35 nt to 55 nt, from 45 nt to 55 nt, from 15 nt to 45 nt, from 25 nt to 45 nt, from 35 nt to 45 nt, from 15 nt to 35 nt, from 25 nt to 35 nt, or from 15 nt to 25 nt. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to bind specifically or be specifically hybridizable. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid, in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid. The guide nucleic acid, in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid. [0242] The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of an infection or genomic locus of interest. The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of HPV 16 or HPV 18. Often, guide nucleic acids that are tiled against the nucleic acid of a strain of an infection or genomic locus of interest can be pooled for use in a method described herein. Often, these guide nucleic acids are pooled for detecting a target nucleic acid in a single assay. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can enhance the detection of the target nucleic using the methods described herein. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can ensure broad coverage of the target nucleic acid within a single reaction using the methods described herein. The tiling, for example, is sequential along the target nucleic acid. Sometimes, the tiling is overlapping along the target nucleic acid. In some instances, the tiling may comprise gaps between the tiled guide nucleic acids along the target nucleic acid. In some instances, the tiling of the guide nucleic acids is non-sequential. Often, a method for detecting a target nucleic acid may comprise contacting a target nucleic acid to a pool of guide nucleic acids and a programmable nuclease, wherein a guide nucleic acid of the pool of guide nucleic acids has a sequence selected from a group of tiled guide nucleic acid that is reverse complementary to a sequence of a target nucleic acid; and assaying for a signal produce by cleavage of at least some detector nucleic acids of a population of detector nucleic acids. Pooling of guide nucleic acids can ensure broad spectrum identification, or broad coverage, of a target species within a single reaction. This can be particularly helpful in diseases or indications, like sepsis, that can be caused by multiple organisms.

[0243] In some embodiments, programmable nucleases can be used to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample. A programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid and can have trans cleavage activity. Trans cleavage activity can be non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the detector nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released from the detector nucleic acid and can generate a signal. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. Often, the signal is present prior to detector nucleic acid cleavage and changes upon detector nucleic acid cleavage. Sometimes, the signal is absent prior to detector nucleic acid cleavage and is present upon detector nucleic acid cleavage. The detectable signal can be immobilized on a support medium for detection. The programmable nuclease can be a CRISPR-Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated) nucleoprotein complex with trans cleavage activity, which can be activated by binding of a guide nucleic acid with a target nucleic acid. The CRISPR-Cas nucleoprotein complex can comprise a Cas protein (also referred to as a Cas nuclease) complexed with a guide nucleic acid, which can also be referred to as CRISPR enzyme. A guide nucleic acid can be a CRISPR RNA (crRNA). Sometimes, a guide nucleic acid may comprise a crRNA and a transactivating crRNA (tracrRNA).

[0244] The programmable nuclease system used to detect modified target nucleic acids can comprise CRISPR RNAs (crRNAs), trans-activating crRNAs (tracrRNAs), Cas proteins, and detector nucleic acids.

[0245] Described herein are reagents comprising a programmable nuclease capable of being activated when complexed with the guide nucleic acid and the target nucleic acid segment or portion. A programmable nuclease can be capable of being activated when complexed with a guide nucleic acid and the target sequence. The programmable nuclease can be activated upon binding of the guide nucleic acid to its target nucleic acid and degrades non-specifically nucleic acid in its environment. The programmable nuclease has trans cleavage activity once activated. A programmable nuclease can be a Cas protein (also referred to, interchangeably, as a Cas nuclease). A crRNA and Cas protein can form a CRISPR enzyme.

[0246] Several programmable nucleases are consistent with the methods and devices of the present disclosure. For example, CRISPR/Cas enzymes are programmable nucleases used in the methods and systems disclosed herein. CRISPR/Cas enzymes can include any of the known Classes and Types of CRISPR/Cas enzymes. Programmable nucleases disclosed herein include Class 1 CRISPR/Cas enzymes, such as the Type I, Type IV, or Type III CRISPR/Cas enzymes. Programmable nucleases disclosed herein also include the Class 2 CRISPR/Cas enzymes, such as the Type II, Type V, and Type VI CRISPR/Cas enzymes. Preferable programmable nucleases included in the several devices disclosed herein (e.g., a microfluidic device such as a pneumatic valve device or a sliding valve device or a lateral flow assay) and methods of use thereof include a Type V or Type VI CRISPR/Cas enzyme. Non-limiting examples of programmable nucleases are described herein.

[0247] The detection of a target DNA by a DNA-activated programmable RNA nuclease can be connected to a variety of readouts including fluorescence, lateral flow, electrochemistry, or any other readouts described herein. Multiplexing of programmable DNA nuclease, such as a Type V CRISPR-Cas protein, with a DNA-activated programmable RNA nuclease, such as a Type VI protein, with a DNA reporter and an RNA reporter, can enable multiplexed detection of target ssDNAs or a combination of a target dsDNA and a target ssDNA, respectively. Multiplexing of different RNA-activated programmable RNA nucleases that have distinct RNA reporter cleavage preferences can enable additional multiplexing. Methods for the generation of ssDNA for DNA- activated programmable RNA nuclease-based diagnostics can include (1) asymmetric PCR, (2) asymmetric isothermal amplification, such as RPA, LAMP, SDA, etc. (3) NEAR for the production of short ssDNA molecules, and (4) conversion of RNA targets into ssDNA by a reverse transcriptase followed by RNase H digestion. Thus, DNA-activated programmable RNA nuclease detection of target DNA is compatible with the various systems, kits, compositions, reagents, and methods disclosed herein. For example, target ssDNA detection by Casl3a can be employed in a nucleic acid detection device as disclosed herein.

[0248] In any of the embodiments described herein, the programmable nuclease can comprise a programmable nuclease capable of being activated when complexed with a guide nucleic acid and target nucleic acid. The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid, in which the activated programmable nuclease can cleave the target nucleic acid, which can initiate trans cleavage activity. In some cases, the trans cut or trans cleavage can cut and/or release a reporter. In other cases, the trans cut or trans cleavage can produce an analog of a target, which can be directly detected. Trans cleavage activity can be non-specific cleavage of nearby nucleic acids by the activated programmable nuclease, such as trans cleavage of detector nucleic acids with a detection moiety. Once the detector nucleic acid is cleaved by the activated programmable nuclease, the detection moiety can be released from the detector nucleic acid and can generate a signal. For example, the detection moiety can correspond to the element, or moiety, (X) shown in FIG. 9A, 9B, 10A, and 10B. The signal can be immobilized on a support medium for detection. The signal can be visualized to assess whether a target nucleic acid is present or absent.

[0249] Reporters, which can be referred to interchangeably as reporters or detector nucleic acids, can be used in conjunction with the compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, etc.) to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample. The reporter can be suspended in solution or immobilized on a surface. For example, the reporter can be immobilized on the surface of a chamber in a device as disclosed herein. In some cases, the reporter can be immobilized on beads, such as magnetic beads, in a chamber of a device as disclosed herein where they are held in position by a magnet placed below the chamber. The reporter can be capable of being cleaved by the activated programmable nuclease, thereby generating a detectable signal. The detectable signal can correspond to a release of one or more elements (X) as illustrated in FIGS. 10A and 10B. The release of the one or more elements (X) can initiate a reaction with another element (Y) when the element (Y) is in the presence of the element (X). The reaction between the element (Y) and the element (X) can initiate a chemical chain reaction in a solid phase material. Such a chemical chain reaction can produce one or more physical or chemical changes. In some cases, the physical or chemical changes can be optically detected. In some embodiments, one or more cascade amplification reactions can occur to further amplify the signal before sensing or detection. There can be a single point of attachment between the reporter and the element (X). Cutting the single point of attachment can release a macro molecule (X), which can undergo a series of reactions based on the macro molecule (X) itself. In any of the embodiments described herein, the reporter can comprise a single stranded detector nucleic acid comprising a detection moiety.

[0250] As used herein, a detector nucleic acid is used interchangeably with reporter or reporter. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising deoxyribonucleotides. In other cases, the detector nucleic acid is a single-stranded nucleic acid comprising ribonucleotides. The detector nucleic acid can be a single-stranded nucleic acid comprising at least one deoxyribonucleotide and at least one ribonucleotide. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some cases, the detector nucleic acid may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position. In some cases, the detector nucleic acid may comprise from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position. In some cases, the detector nucleic acid may comprise from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 8, from 3 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 5, from 3 to 5, or from 4 to 5 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between non-ribonucleotide residues. In some cases, the detector nucleic acid has only ribonucleotide residues. In some cases, the detector nucleic acid has only deoxyribonucleotide residues. In some cases, the detector nucleic acid may comprise nucleotides resistant to cleavage by the programmable nuclease described herein. In some cases, the detector nucleic acid may comprise synthetic nucleotides. In some cases, the detector nucleic acid may comprise at least one ribonucleotide residue and at least one non-ribonucleotide residue. In some cases, the detector nucleic acid is 5-20, 5-15, 5-10, 7-20, 7-15, or 7-10 nucleotides in length. In some cases, the detector nucleic acid is from 3 to 20, from 4 to 20, from 5 to 20, from 6 to 20, from 7 to 20, from 8 to 20, from 9 to 20, from 10 to 20, from 15 to 20, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, from 10 to 15, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, or from 7 to 8 nucleotides in length. In some cases, the detector nucleic acid may comprise at least one uracil ribonucleotide. In some cases, the detector nucleic acid may comprise at least two uracil ribonucleotides. Sometimes the detector nucleic acid has only uracil ribonucleotides. In some cases, the detector nucleic acid may comprise at least one adenine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two adenine ribonucleotides. In some cases, the detector nucleic acid has only adenine ribonucleotides. In some cases, the detector nucleic acid may comprise at least one cytosine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two cytosine ribonucleotides. In some cases, the detector nucleic acid may comprise at least one guanine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two guanine ribonucleotides. A detector nucleic acid can comprise only unmodified ribonucleotides, only unmodified deoxyribonucleotides, or a combination thereof. In some cases, the detector nucleic acid is from 5 tol2 nucleotides in length. In some cases, the detector nucleic acid is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the detector nucleic acid is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. For cleavage by a programmable nuclease comprising Casl3, a detector nucleic acid can be 5, 8, or 10 nucleotides in length. For cleavage by a programmable nuclease comprising Casl2, a detector nucleic acid can be 10 nucleotides in length.

[0251] The single stranded detector nucleic acid can comprise a detection moiety capable of generating a first detectable signal. Sometimes the detector nucleic acid may comprise a protein capable of generating a signal. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some cases, a detection moiety is on one side of the cleavage site. Optionally, a quenching moiety is on the other side of the cleavage site. Sometimes the quenching moiety is a fluorescence quenching moiety. In some cases, the quenching moiety is 5’ to the cleavage site and the detection moiety is 3’ to the cleavage site. In some cases, the detection moiety is 5’ to the cleavage site and the quenching moiety is 3’ to the cleavage site. Sometimes the quenching moiety is at the 5’ terminus of the detector nucleic acid. Sometimes the detection moiety is at the 3’ terminus of the detector nucleic acid. In some cases, the detection moiety is at the 5’ terminus of the detector nucleic acid. In some cases, the quenching moiety is at the 3’ terminus of the detector nucleic acid. In some cases, the single-stranded detector nucleic acid is at least one population of the single-stranded nucleic acid capable of generating a first detectable signal. In some cases, the single-stranded detector nucleic acid is a population of the single stranded nucleic acid capable of generating a first detectable signal. Optionally, there are more than one population of single-stranded detector nucleic acid. In some cases, there are 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, or greater than 50, or any number spanned by the range of this list of different populations of single-stranded detector nucleic acids capable of generating a detectable signal. In some cases, there are from 2 to 50, from 3 to 40, from 4 to 30, from 5 to 20, or from 6 to 10 different populations of single-stranded detector nucleic acids capable of generating a detectable signal. In some cases there are from 2 to 50, from 5 to 50, from 10 to 50, from 15 to 50, from 20 to 50, from 25 to 50, from 30 to 50, from 35 to 50, from 40 to 50, from 2 to 40, from 5 to 40, from 10 to 40, from 15 to 40, from 20 to 40, from 25 to 40, from 30 to 40, from 35 to 40, from 2 to 30, from 5 to 30, from 10 to 30, from 15 to 30, from 20 to 30, from 25 to 30, from 2 to 20, from 5 to 20, from 10 to 20, from 15 to 20, from 2 to 10, or from 5 to 10 different populations of singlestranded detector nucleic acids capable of generating a detectable signal.

[0252] In some embodiments, target nucleic acid amplicons are detected by immobilized programmable nuclease probes, such as, for example, CRISPR CAS guide RNA probes (referred to as CRISPR probe). Upon a complementary binding event between a target nucleic acid amplicon and a programmable nuclease probe (e.g., an immobilized CRISPR CAS / guide RNA complex) a cutting event will occur that release a reporter that is then detected by a sensor.

[0253] As described above, the single stranded detector nucleic acid can comprise a detection moiety capable of generating a first detectable signal. Sometimes the detector nucleic acid may comprise a protein capable of generating a signal. A signal can be a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. The generation of the detectable signal from the release of the detection moiety indicates that cleavage by the programmable nuclease has occurred and that the sample contains the target nucleic acid. A detection moiety can be any moiety capable of generating a colorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. A detector nucleic acid, sometimes, is protein-nucleic acid that can generate a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal upon cleavage of the nucleic acid. Often a calorimetric signal is heat produced after cleavage of the detector nucleic acids. Sometimes, a calorimetric signal is heat absorbed after cleavage of the detector nucleic acids. A potentiometric signal, for example, is electrical potential produced after cleavage of the detector nucleic acids. An amperometric signal can be movement of electrons produced after the cleavage of detector nucleic acid. Often, the signal is an optical signal, such as a colorimetric signal or a fluorescence signal. An optical signal is, for example, a light output produced after the cleavage of the detector nucleic acids. Sometimes, an optical signal is a change in light absorbance between before and after the cleavage of detector nucleic acids. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the detector nucleic acid.

[0254] Detecting the presence or absence of a target nucleic acid of interest can involve measuring a signal emitted from a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease. The signal can be measured using one or more sensors integrated with the device or operatively coupled to the device. Thus, the detecting steps disclosed herein can involve measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or, measuring a signal indicating that the target nucleic acid is absent in a sample. In some embodiments, a signal is generated upon cleavage of the detector nucleic acid by the programmable nuclease. In other embodiments, the signal changes upon cleavage of the detector nucleic acid by the programmable nuclease. In other embodiments, a signal can be present in the absence of detector nucleic acid cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease. For example, a signal can be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.

[0255] In some cases, the signal can comprise a colorimetric signal or a signal visible by eye. In some instances, the signal is fluorescent, electrical, chemical, electrochemical, or magnetic. A signal can be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some cases, the detectable signal is a colorimetric signal or a signal visible by eye. In some instances, the detectable signal is fluorescent, electrical, chemical, electrochemical, or magnetic. In some cases, the first detection signal is generated by binding of the detection moiety to the capture molecule in the detection region, where the first detection signal indicates that the sample contained the target nucleic acid. Sometimes the system can detect more than one type of target nucleic acid, wherein the system may comprise more than one type of guide nucleic acid and more than one type of detector nucleic acid. In some cases, the detectable signal is generated directly by the cleavage event. Alternatively, or in combination, the detectable signal is generated indirectly by the signal event. Sometimes the detectable signal is not a fluorescent signal. In some instances, the detectable signal is a colorimetric or color-based signal. In some cases, the detected target nucleic acid is identified based on its spatial location on the detection region of the support medium.

[0256] In some cases, the one or more detectable signals generated after cleavage can produce an index of refraction change or one or more electrochemical changes. In some cases, real-time detection of the Cas reaction can be achieved using fluorescence, electrochemical detection, and/or electrochemiluminescence.

[0257] In some cases, the detectable signals can be detected and analyzed in various ways. For example, the detectable signals can be detected using an imaging device. The imaging device can a digital camera, such a digital camera on a mobile device. The mobile device can have a software program or a mobile application that can capture fluorescence, ultraviolet (UV), infrared (IR), or visible wavelength signals. Any suitable detection or measurement device can be used to detect and/or analyze the colorimetric, fluorescence, amperometric, potentiometric, or electrochemical signals described herein. In some embodiments, the colorimetric, fluorescence, amperometric, potentiometric, or another electrochemical sign can be detected using a measurement device connected to a detection chamber of the device (e.g., a fluorescence measurement device, a spectrophotometer, and/or an oscilloscope).

[0258] In certain aspects of this disclosure, multiplexing refers to parallel sensing of multiple target nucleic acid sequences in one sample by multiple probes.

[0259] The devices of the present disclosure can be used to implement for detection of one or more target nucleic acids within the sample. The devices of the present disclosure can comprise one or multiple pumps, valves, reservoirs, and chambers for sample preparation, optional amplification of a target nucleic acid within the sample, mixing with a programmable nuclease, and detection of a detectable signal arising from cleavage of detector nucleic acids by a programmable nuclease.

[0260] Methods consistent with the present disclosure include a multiplexing method of assaying for a plurality of target nucleic acids in a sample. A multiplexing method may comprise contacting the sample to a complex comprising a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease that exhibits sequence independent cleavage upon forming a complex comprising the segment of the guide nucleic acid binding to the segment of the target nucleic acid; and assaying for a signal indicating cleavage of at least some reporters (e.g., protein-nucleic acids) of a population of reporter moleucles (e.g., protein-nucleic acids), wherein the signal indicates a presence of the target nucleic acid in the sample and wherein absence of the signal indicates an absence of the target nucleic acid in the sample.

[0261] Multiplexing can comprise spatial multiplexing wherein multiple different target nucleic acids are detected at the same time, but the reactions are spatially separated. In some cases, the multiple target nucleic acids are detected using the same programmable nuclease, but different guide nucleic acids. The multiple target nucleic acids sometimes are detected using the different programmable nucleases. In the case wherein multiple target nucleic acids are detected using the different programmable nucleases, the method involves using a first programmable nuclease, which upon activation (e.g., after binding of a first guide nucleic acid to a first target), cleaves a nucleic acid of a first reporter and using a second programmable nuclease, which upon activation (e.g., after binding of a second guide nucleic acid to a second target), cleaves a nucleic acid of a second reporter.

[0262] Sometimes, multiplexing can be single reaction multiplexing wherein multiple different target acids are detected in a single reaction volume. Often, at least two different programmable nucleases are used in single reaction multiplexing. For example, multiplexing can be enabled by immobilization of multiple categories of detector nucleic acids within a fluidic system, to enable detection of multiple target nucleic acids within a single fluidic system. Multiplexing allows for detection of multiple target nucleic acids in one kit or system. In some cases, the multiple target nucleic acids comprise different target nucleic acids to a virus, a bacterium, or a pathogen responsible for one disease. In some cases, the multiple target nucleic acids comprise different target nucleic acids associated with a cancer or genetic disorder. Multiplexing for one disease, cancer, or genetic disorder increases at least one of sensitivity, specificity, or accuracy of the assay to detect the presence of the disease in the sample. In some cases, the multiple target nucleic acids comprise target nucleic acids directed to different viruses, bacteria, or pathogens responsible for more than one disease. In some cases, multiplexing allows for discrimination between multiple target nucleic acids, such as target nucleic acids that comprise different genotypes of the same bacteria or pathogen responsible for a disease, for example, for a wildtype genotype of a bacteria or pathogen and for genotype of a bacteria or pathogen comprising a mutation, such as a single nucleotide polymorphism (SNP) that can confer resistance to a treatment, such as antibiotic treatment. Multiplexing, thus, allows for multiplexed detection of multiple genomic alleles. For example, multiplexing may comprise method of assaying comprising a single assay for a microorganism species using a first programmable nuclease and an antibiotic resistance pattern in a microorganism using a second programmable nuclease. Sometimes, multiplexing allows for discrimination between multiple target nucleic acids of different HPV strains, for example, HP VI 6 and HP VI 8. In some cases, the multiple target nucleic acids comprise target nucleic acids directed to different cancers or genetic disorders. Often, multiplexing allows for discrimination between multiple target nucleic acids, such as target nucleic acids that comprise different genotypes, for example, for a wild-type genotype and for SNP genotype. Multiplexing for multiple diseases, cancers, or genetic disorders provides the capability to test a panel of diseases from a single sample. For example, multiplexing for multiple diseases can be valuable in a broad panel testing of a new patient or in epidemiological surveys. Often multiplexing is used for identifying bacterial pathogens in sepsis or other diseases associated with multiple pathogens.

[0263] Furthermore, signals from multiplexing can be quantified. For example, a method of quantification for a disease panel may comprise assaying for a plurality of unique target nucleic acids in a plurality of aliquots from a sample, assaying for a control nucleic acid control in a second aliquot of the sample, and quantifying a plurality of signals of the plurality of unique target nucleic acids by measuring signals produced by cleavage of detector nucleic acids compared to the signal produced in the second aliquot. In this context, a unique target nucleic acid refers to the sequence of a nucleic acid that has an at least one nucleotide difference from the sequences of the other nucleic acids in the plurality. Multiple copies of each target nucleic acid can be present. For example, a unique target nucleic population can comprise multiple copies of the unique target nucleic acid. Often the plurality of unique target nucleic acids is from a plurality of bacterial pathogens in the sample.

[0264] In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 2 different target nucleic acids in a single reaction. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 3 different target nucleic acids in a single reaction. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 4 different target nucleic acids in a single reaction. In some instances, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 5 different target nucleic acids in a single reaction. In some cases, the multiplexed devices, systems, fluidic devices, kits, and methods detect at least 6, 7, 8, 9, or 10 different target nucleic acids in a single reaction. In some instances, the multiplexed kits detect at least 2 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 3 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 4 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 5 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect at least 6, 7, 8, 9, or 10 different target nucleic acids in a single kit. In some instances, the multiplexed kits detect from 2 to 10, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 9, from 3 to 9, from 4 to 9, from 5 to 9, from 6 to 9, from 7 to 9, from 8 to 9, from 2 to 8, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 7, from 3 to 7, from 4 to 7, from 5 to 7, from 6 to 7, from 2 to 6, from 3 to 6, from 4 to 6, from 5 to 6, from 2 to 5, from 3 to 5, from 4 to 5, from 2 to 4, from 3 to 4, or from 2 to 3 different target nucleic acids in a single kit. Multiplexing can be carried out in a single-pot or “one-pot” reaction, where reverse transcription, amplification, in vitro transcription, or any combination thereof, and detection are carried out in a single volume. Multiplexing can be carried out in a “two-pot reaction”, where reverse transcription, amplification, in vitro transcription, or any combination thereof, are carried out in a first volume and detection is carried out in a second volume.

[0265] In some cases, multiplexing can comprise detecting multiple targets with a single probe. Alternatively, multiplexing can comprise detecting multiple targets with multiple probes. The multiple probes can be configured to detect a presence of a particular sequence, target nucleic acid, or a plurality of different target sequences or nucleic acids.

[0266] The devices of the present disclosure can be manufactured from a variety of different materials. Exemplary materials that can be used include plastic polymers, such as polymethacrylate (PMMA), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene (PE), high-density polyethylene (HDPE), polypropylene (PP); glass; and silicon. Features of the device (e.g., chambers, channels, etc.) can be manufactured by various processes. For example, the features can be (1) embossed using injection molding, (2) micro-milled or micro-engraved using computer numerical control (CNC) micromachining or non-contact laser drilling (by means of a CO2 laser source); (3) generated using additive manufacturing, and/or (4) generated using one or more photolithographic or stereolithographic methods.

[0267] In some embodiments, any of the devices of the present disclosure can comprise a sample interface configured to receive a sample that may comprise at least one gene of interest. The device can further comprise a channel in fluid communication with the sample interface and a detection chamber. In some cases, the channel may comprise one or more movable mechanisms to separate the sample into a plurality of droplets. As used herein, a droplet can refer to a volumetric portion of the sample, a partitioned sub-sample of the sample, and/or an aliquot of the sample. In some cases, the detection chamber is configured to receive and contact the plurality of droplets with at least one programmable nuclease probe disposed on a surface of said detection chamber. The at least one programmable nuclease probe can comprise a guide nucleic acid complexed with a programmable nuclease. In some cases, the programmable nuclease probe may comprise a CRISPR/Cas enzyme. In some cases, the guide nucleic acid may comprise a guide RNA. In some embodiments, the device may comprise a plurality of programmable nuclease probes comprising different guide RNAs.

[0268] The device can further comprise a plurality of sensors that determine a presence of said at least one gene of interest by detecting a signal produced upon cleavage of a target nucleic acid region in said at least one gene of interest by said at least one programmable nuclease probe. The cleavage of the target nucleic acid region can occur after a complementary binding of said target nucleic acid region to said guide nucleic acid of said at least one programmable nuclease probe. [0269] As described elsewhere herein, the one or more movable mechanisms can comprise one or more valves configured to restrict flow through one or more sections of the channel. The one or more movable mechanisms can comprise a plunger or a bristle that is configured to restrict flow through one or more sections of the channel. The one or more movable mechanisms can be operatively coupled to a power supply that is integrated with or insertable into the device. The power supply can comprise a battery or a plurality of batteries as described herein.

[0270] In some cases, any of the devices described herein may comprise a physical filter to filter one or more particles from the sample that do not comprise the one or more targets (e.g., a gene of interest). In some cases, the device may comprise a sample preparation chamber. The sample preparation chamber can comprise a lysing agent. The sample preparation chamber can comprise a heating unit configured for heat inactivation. The sample preparation chamber can comprise one or more reagents for nucleic acid purification.

[0271] In some cases, the channel between the sample interface and the detection chamber may comprise a plurality of heating elements and a plurality of heat sinks for amplifying the at least one gene of interest or a portion thereof. The plurality of heating elements and the plurality of heat sinks can be configured to perform one or more thermocycling operations on the sample or at least a portion of the sample (e.g., the plurality of droplets).

[0272] As described elsewhere herein, the signal produced upon cleavage of a target nucleic acid can be associated with a physical, chemical, or electrochemical change or reaction. The signal can comprise an optical signal, a fluorescent or colorimetric signal, a potentiometric or amperometric signal, and/or a piezo-electric signal. In some cases, the signal is associated with a change in an index of refraction of a solid or gel volume in which the at least one programmable nuclease probe is disposed.

[0273] In some embodiments, the device may comprise a sample interface configured to receive a sample that may comprise one or more genomic targets of interest. In some cases, the one or more genomic targets of interest comprise a sequence of nucleic acids comprising the nucleic acid.

[0274] The device can further comprise one or more channels comprising one or more movable mechanisms to separate the sample into partitioned samples. The one or more channels can be in fluid communication with the sample interface and a reaction chamber that is configured to receive and contact the partitioned samples with an enzyme, reagent, or programmable detection agent that is configured to cleave a nucleic acid of said one or more genomic targets of interest. [0275] The device can further comprise a plurality of sensors for determining a presence of the one or more genomic targets of interest by detecting one or more reporters released by said cleavage of said nucleic acid. The programmable detection agent can be a CRISPR/Cas enzyme. In some cases, the reporter may comprise a nucleic acid and a detection moiety. In some cases, the reporter may comprise at least one ribonucleotide or at least one deoxyribonucleotide. In some cases, the reporter may comprise a DNA nucleic acid or an RNA nucleic acid. The reported molecule can be immobilized on a surface of the detection chamber (i.e., a movement of the reporter can be physically or chemically constrained).

[0276] In some cases, the one or more movable mechanisms comprise a plurality of valves configured to restrict flow in a first direction through the one or more channels towards the sample interface. The plurality of valves can be configured to selectively permit flow in a second direction through the one or more channels towards the reaction chamber. A first valve and a second valve of the plurality of valves can be configured to physically, fluidically, or thermally isolate a first portion of the sample from a second portion of the sample when the first valve and the second valve are in a closed state.

[0277] The one or more channels can comprise a plurality of heating elements and a plurality of heat sinks to perform thermocycling on the partitioned samples. A first heating element of the plurality of heating elements and a first heat sink of the plurality of heat sinks can be positioned between a first movable mechanism and a second movable mechanism of the one or more movable mechanisms.

[0278] In any of the embodiments described herein, the device can further comprise a telemedicine unit configured to provide one or more detection results to a computing unit that is remote from the device. In some embodiments, the telemedicine unit provides one or more detection results to a computing unit that is remote to the device through a cloud-based connection. In some embodiments, the telemedicine unit is HIPAA compliant. In some embodiments, the telemedicine unit transmits encrypted data. The computing unit can comprise a mobile device or a computer. The one or more detection results can indicate a presence or an absence of a target nucleic acid of interest in the sample.

[0279] In another aspect, the present disclosure provides a method for target detection. The method can comprise contacting a sample with the device of any of the preceding claims and detecting a presence or an absence of one or more genes of interest in said sample. In some cases, the method can comprise generating one or more detection results indicating the presence or the absence of the one or more genes of interest in the sample. In some cases, the method can comprise transmitting the one or more detection results to a remote computing unit. The remote computing unit can comprise, for example, a mobile device.

[0280] In another aspect, the present disclosure provides a method for target detection. The method can comprise providing a sample comprising at least one gene of interest. The method can comprise separating the sample into a plurality of sub-samples using one or more movable mechanisms described herein. The method can comprise receiving the plurality of sub-samples in a detection chamber and contacting the plurality of sub-samples with at least one programmable nuclease probe disposed on a surface of said detection chamber. The at least one programmable nuclease probe can comprise a guide nucleic acid complexed with a programmable nuclease. In some cases, the method can comprise contacting the plurality of subsamples with a plurality of programmable nuclease probes comprising different guide RNAs. The method can comprise using a plurality of sensors to determine a presence or an absence of said at least one gene of interest by detecting a signal produced upon cleavage of a target nucleic acid region in said at least one gene of interest by said at least one programmable nuclease probe. [0281] In some cases, the method can further comprise amplifying the at least one gene of interest after separating the sample into a plurality of sub-samples. In some cases, the method can further comprise amplifying the at least one gene of interest before mixing the plurality of sub-samples in the detection chamber. Amplifying the at least one gene of interest can comprise using a plurality of heating elements and a plurality of heat sinks to perform thermocycling on the plurality of sub-samples.

[0282] In some cases, the method can comprise using a physical filter to filter one or more particles from the sample that do not comprise the one or more target genes of interest. In some cases, the method can comprise lysing the sample before detecting the one or more target genes of interest. In some cases, the method can comprise performing heat inactivation on the sample. In some cases, the method can comprise performing enzyme (e.g., PK) inactivation on the sample. In some cases, the method can comprise performing nucleic acid purification on the sample.

[0283] In some cases, the nucleic acid detection devices described herein can be configured to implement process control procedures to ensure that the sample preparation, target amplification, and target detection processes are performed accurately and as intended.

Electrochemical DETECTR reaction

[0284] The present disclosure provides various devices, systems, fluidic devices, and kits for rapid tests, which may quickly assess whether a target nucleic acid is present in a sample by using a programmable nuclease that can interact with functionalized surfaces of the fluidic systems to generate a detectable signal. For example, disclosed herein are particular microfluidic devices, lateral flow devices, sample preparation devices, and compositions (e.g., programmable nucleases, guide RNAs, reagents for in vitro transcription, amplification, reverse transcription, and reporters, or any combination thereof) for use in said devices that are particularly well suited to carry out a highly efficient, rapid, and accurate reactions for detecting the presence of a target nucleic acid (e.g., a DETECTR reaction). The systems and programmable nucleases disclosed herein can be used as a companion diagnostic with any of the diseases disclosed herein (e.g., RSV, sepsis, flu, COVID-19), or can be used in reagent kits, point-of- care diagnostics, or over- the-counter diagnostics. The systems may be used as a point of care diagnostic or as a lab test for detection of a target nucleic acid and, thereby, detection of a condition, for example, in a subject from which the sample was taken. The systems may be used in various sites or locations, such as in laboratories, in hospitals, in physician offices/laboratories (POLs), in clinics, at remotes sites, or at home. Sometimes, the present disclosure provides various devices, systems, fluidic devices, and kits for consumer genetic use or for over-the-counter use.

Guide nucleic acids

[0285] Guide nucleic acids are compatible for use in the devices described herein (e.g., pneumatic valve devices, sliding valve devices, rotating valve devices, and lateral flow devices) and may be used in conjunction with compositions disclosed herein (e.g., programmable nucleases, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, and reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample (e.g., DETECTR reactions). The guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease as described herein. The guide nucleic acid can bind to the single stranded target nucleic acid comprising a portion of a nucleic acid from a bacterium or other agents responsible for a disease as described herein and further comprising a mutation, such as a single nucleotide polymorphism (SNP), which can confer resistance to a treatment, such as antibiotic treatment. The guide nucleic acid binds to the single stranded target nucleic acid comprising a portion of a nucleic acid from a cancer gene or gene associated with a genetic disorder as described herein. The guide nucleic acid is complementary to the target nucleic acid. Often the guide nucleic acid binds specifically to the target nucleic acid. The target nucleic acid may be a RNA, DNA, or synthetic nucleic acids. A guide nucleic acid can comprise a sequence that is reverse complementary to the sequence of a target nucleic acid. A guide nucleic acid can be a crRNA. Sometimes, a guide nucleic acid may comprise a crRNA and tracrRNA. The guide nucleic acid can bind specifically to the target nucleic acid. In some cases, the guide nucleic acid is not naturally occurring and made by artificial combination of otherwise separate segments of sequence. Often, the artificial combination is performed by chemical synthesis, by genetic engineering techniques, or by the artificial manipulation of isolated segments of nucleic acids. The target nucleic acid can be designed and made to provide desired functions. In some cases, the targeting region of a guide nucleic acid is 20 nucleotides in length. The targeting region of the guide nucleic acid may have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some instances, the targeting region ofthe guide nucleic acid is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the targeting region of a guide nucleic acid has a length from exactly or about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt. In some cases, the targeting region of a guide nucleic acid has a length of from about 10 nt to about 60 nt, from about 20 nt to about 50 nt, or from about 30 nt to about 40 nt. In some cases, the targeting region of a guide nucleic acid has a length of from 15 nt to 55 nt, from 25 nt to 55 nt, from 35 nt to 55 nt, from 45 nt to 55 nt, from 15 nt to 45 nt, from 25 nt to 45 nt, from 35 nt to 45 nt, from 15 nt to 35 nt, from 25 nt to 35 nt, or from 15 nt to 25 nt. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable or bind specifically. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid, in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a modification variable region in the target nucleic acid. The guide nucleic acid can have a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid. The guide nucleic acid, in some cases, has a sequence comprising at least one uracil in a region from nucleic acid residue 5 to 9, 10 to 14, or 15 to 20 that is reverse complementary to a methylation variable region in the target nucleic acid.

[0286] The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of an infection or genomic locus of interest. The guide nucleic acid can be selected from a group of guide nucleic acids that have been tiled against the nucleic acid of a strain of HPV 16 or HPV 18. Often, guide nucleic acids that are tiled against the nucleic acid of a strain of an infection or genomic locus of interest can be pooled for use in a method described herein. Often, these guide nucleic acids are pooled for detecting a target nucleic acid in a single assay. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can enhance the detection of the target nucleic using the methods described herein. The pooling of guide nucleic acids that are tiled against a single target nucleic acid can ensure broad coverage of the target nucleic acid within a single reaction using the methods described herein. The tiling, for example, is sequential along the target nucleic acid. Sometimes, the tiling is overlapping along the target nucleic acid. In some instances, the tiling may comprise gaps between the tiled guide nucleic acids along the target nucleic acid. In some instances, the tiling of the guide nucleic acids is non-sequential. Often, a method for detecting a target nucleic acid may comprise contacting a target nucleic acid to a pool of guide nucleic acids and a programmable nuclease, wherein a guide nucleic acid of the pool of guide nucleic acids has a sequence selected from a group of tiled guide nucleic acid that is reverse complementary to a sequence of a target nucleic acid; and assaying for a signal produce by cleavage of at least some detector nucleic acids of a population of detector nucleic acids. Pooling of guide nucleic acids can ensure broad spectrum identification, or broad coverage, of a target species within a single reaction. This can be particularly helpful in diseases or indications, like sepsis, that may be caused by multiple organisms. Exemplary guide nucleic acids are listed in Table 2. In Table 2, references to a “Casl2 Variant” refer to a Casl2 variant having the sequence of SEQ ID NO: 17, and references to “Casl3 Variant” refer to a Casl3 variant having the sequence of SEQ ID NO: 21.

TABLE 2. Exemplary guide nucleic acids

[0287] In some embodiments, a composition (e.g., a composition in a detection chamber for a detection reaction) comprises a plurality of guide nucleic acids selected from the sequences of Table 2. In some embodiments, the plurality of guide nucleic acids are directed to a plurality of different target sequences (e.g., different target sequences from one or more SARS-CoV2, one or more hCOV, one or more hMPV, one or more enterovirus and/or rhinovirus, one or more influenza A virus, one or more influenza B virus, one or more human parainfluenza virus, one or more RSV, or one or more 18S). In some embodiments, the plurality of different guide nucleic acids are collectively configured to bind different target sequences within the target nucleic acid. In some embodiments, the plurality of different guide nucleic acids are collectively configured in the same reaction vessel. In some embodiments, the composition includes a plurality or all of guide nucleic acids represented by SEQ ID NOs 987-1010. In some embodiments, the composition includes a plurality or all of guide nucleic acids represented by SEQ ID NOs 1011- 1037. In some embodiments, the composition includes a plurality or all of guide nucleic acids represented by SEQ ID NOs 1038-1048. In some embodiments, the composition includes a plurality or all of guide nucleic acids represented by SEQ ID NOs 1049-1061. In some embodiments, the composition includes a plurality or all of guide nucleic acids represented by SEQ ID NOs 1062-1096. In some embodiments, the composition includes a plurality or all of guide nucleic acids represented by SEQ ID NOs 1097-1123. In some embodiments, the composition includes a plurality or all of guide nucleic acids represented by SEQ ID NOs 1124- 1158. In some embodiments, the composition includes a plurality or all of guide nucleic acids represented by SEQ ID NOs 1159-1170. In some embodiments, the composition includes a plurality or all of guide nucleic acids represented by SEQ ID NOs 1171-1175.

Reporter

[0288] Reporters, which can be referred to interchangeably reporters, or detector nucleic acids, described herein are compatible for use in the devices described herein (e.g., pneumatic valve devices, sliding valve devices, rotating valve devices, and lateral flow devices) and may be used in conjunction with compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample (e.g., DETECTR reactions). Described herein is a reporter comprising a single stranded detector nucleic acid comprising a detection moiety, wherein the reporter is capable of being cleaved by the activated programmable nuclease, thereby generating a first detectable signal. As used herein, a detector nucleic acid is used interchangeably with reporter or reporter. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising deoxyribonucleotides. In other cases, the detector nucleic acid is a single-stranded nucleic acid comprising ribonucleotides. The detector nucleic acid can be a single-stranded nucleic acid comprising at least one deoxyribonucleotide and at least one ribonucleotide. In some cases, the detector nucleic acid is a single-stranded nucleic acid comprising at least one ribonucleotide residue at an internal position that functions as a cleavage site. In some cases, the detector nucleic acid may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 ribonucleotide residues at an internal position. In some cases, the detector nucleic acid may comprise from 2 to 10, from 3 to 9, from 4 to 8, or from 5 to 7 ribonucleotide residues at an internal position. In some cases, the detector nucleic acid may comprise from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 2 to 8, from 3 to 8, from 5 to 8, from 6 to 8, from 7 to 8, from 2 to 5, from 3 to 5, or from 4 to 5 ribonucleotide residues at an internal position. Sometimes the ribonucleotide residues are continuous. Alternatively, the ribonucleotide residues are interspersed in between nonribonucleotide residues. In some cases, the detector nucleic acid has only ribonucleotide residues. In some cases, the detector nucleic acid has only deoxyribonucleotide residues. In some cases, the detector nucleic acid may comprise nucleotides resistant to cleavage by the programmable nuclease described herein. In some cases, the detector nucleic acid may comprise synthetic nucleotides. In some cases, the detector nucleic acid may comprise at least one ribonucleotide residue and at least one non-ribonucleotide residue. In some cases, the detector nucleic acid is 5-20, 5-15, 5-10, 7-20, 7-15, or 7-10 nucleotides in length. In some cases, the detector nucleic acid is from 3 to 20, from 4 to 20, from 5 to 20, from 6 to 20, from 7 to 20, from 8 to 20, from 9 to 20, from 10 to 20, from 15 to 20, from 3 to 15, from 4 to 15, from 5 to 15, from 6 to 15, from 7 to 15, from 8 to 15, from 9 to 15, from 10 to 15, from 3 to 10, from 4 to 10, from 5 to 10, from 6 to 10, from 7 to 10, from 8 to 10, from 9 to 10, from 3 to 8, from 4 to 8, from 5 to 8, from 6 to 8, or from 7 to 8 nucleotides in length. In some cases, the detector nucleic acid may comprise at least one uracil ribonucleotide. In some cases, the detector nucleic acid may comprise at least two uracil ribonucleotides. Sometimes the detector nucleic acid has only uracil ribonucleotides. In some cases, the detector nucleic acid may comprise at least one adenine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two adenine ribonucleotide. In some cases, the detector nucleic acid has only adenine ribonucleotides. In some cases, the detector nucleic acid may comprise at least one cytosine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two cytosine ribonucleotide. In some cases, the detector nucleic acid may comprise at least one guanine ribonucleotide. In some cases, the detector nucleic acid may comprise at least two guanine ribonucleotide. A detector nucleic acid can comprise only unmodified ribonucleotides, only unmodified deoxyribonucleotides, or a combination thereof. In some cases, the detector nucleic acid is from 5 to 12 nucleotides in length. In some cases, the detector nucleic acid is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some cases, the detector nucleic acid is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. For cleavage by a programmable nuclease comprising Casl3, a detector nucleic acid can be 5, 8, or 10 nucleotides in length. For cleavage by a programmable nuclease comprising Casl2, a detector nucleic acid can be 10 nucleotides in length.

Signals

[0289] The devices, systems, fluidic devices, kits, and methods for detecting the presence of a target nucleic acid in a sample described herein may comprise a generation of a signal indicative of the presence or absence of the target nucleic acid in the sample. The generation of a signal indicative of the presence or absence of the target nucleic acid in the sample as described herein is compatible with the methods and devices described herein (e.g., pneumatic valve devices, sliding valve devices, rotating valve devices, and lateral flow devices) and may result from the use of compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample (e.g., DETECTR reactions). As disclosed herein, in some embodiments, detecting the presence or absence of a target nucleic acid of interest involves measuring a signal emitted from a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease. Alternatively, or in combination, in some embodiments, detecting the presence or absence of a target nucleic acid of interest involves measuring a signal emitted from a conjugate bound to a detection moiety present in a reporter, after cleavage of the reporter by an activated programmable nuclease. The conjugates may comprise a nanoparticle, a gold nanoparticle, a latex nanoparticle, a quantum dot, a chemiluminescent nanoparticle, a carbon nanoparticle, a selenium nanoparticle, a fluorescent nanoparticle, a liposome, or a dendrimer. The surface of the conjugate may be coated by a conjugate binding molecule that binds to the detection moiety or another affinity molecule of the cleaved detector molecule as described herein. Thus, the detecting steps disclosed herein involve indirectly (e.g., via a reporter) measuring the presence of a target nucleic acid, quantifying how much of the target nucleic acid is present, or, measuring a signal indicating that the target nucleic acid is absent in a sample. In some embodiments, a signal is generated upon cleavage of the detector nucleic acid by the programmable nuclease. In other embodiments, the signal changes upon cleavage of the detector nucleic acid by the programmable nuclease. In other embodiments, a signal may be present in the absence of detector nucleic acid cleavage and disappear upon cleavage of the target nucleic acid by the programmable nuclease. For example, a signal may be produced in a microfluidic device or lateral flow device after contacting a sample with a composition comprising a programmable nuclease.

Buffers

[0290] The reagents described herein can also include buffers, which are compatible with the devices, systems, fluidic devices, kits, and methods disclosed herein. The buffers described herein are compatible for use in the devices described herein (e.g., pneumatic valve devices, sliding valve devices, rotating valve devices, and lateral flow devices) and may be used in conjunction with compositions disclosed herein (e.g., programmable nucleases, guide nucleic acids, reagents for in vitro transcription, reagents for amplification, reagents for reverse transcription, reporters, or any combination thereof) to carry out highly efficient, rapid, and accurate reactions for detecting whether the target nucleic acid is in the sample (e.g., DETECTR reactions). These buffers are compatible with the other reagents, samples, and support mediums as described herein for detection of an ailment, such as a disease, cancer, or genetic disorder, or genetic information, such as for phenotyping, genotyping, or determining ancestry. The methods described herein can also include the use of buffers, which are compatible with the methods disclosed herein. For example, a buffer may comprise HEPES, MES, TCEP, EGTA, Tween 20, KC1, MgCl ² , glycerol, or any combination thereof. In some instances, a buffer may comprise Tris-HCl pH 8.8, VLB, EGTA, CH3COOH, TCEP, IsoAmp®, (NH4)2SO4, KC1, MgSO4, Tween20, KO Ac, MgOAc, BSA, TCEP, or any combination thereof. In some instances the buffer may comprise from 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10,5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM HEPES pH 6.8. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM KC1. In other instances the buffer may comprise 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM MgCl ² . The buffer can comprise 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30% glycerol. The buffer can comprise from 0% to 30%, from 5% to 30%, from 10% to 30%, from 15% to 30%, from 20% to 30%, from 25% to 30%, from 0% to 25%, from 2% to 25%, from 5% to 25%, from 10% to 25%, from 15% to 25%, from 20% to 25%, from 0% to 20%, from 5% to 20%, from 10% to 20%, from 15% to 20%, from 0% to 15%, from 5% to 15%, from 10% to 15%, from 0% to 10%, from 5% to 10%, or from 0% to 5% glycerol. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM Tris-HCl pH 8.8. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM KO Ac. The buffer can comprise to 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10, 5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 5 to 150, 5 to 200, 5 to 250, 5 to 300, 5 to 400, 5 to 500, 25 to 50, 25 to 75, 25 to 100, 50 to 100, 50 150, 50 to 200, 50 to 250, 50 to 300, 100 to 200, 100 to 250, 100 to 300, or 150 to 250 mM MgOAc. In some instances the buffer may comprise from 0 to 100, 0 to 75, 0 to 50, 0 to 25, 0 to 20, 0 to 10, 0 to 5, 5 to 10,5 to 15, 5 to 20, 5 to 25, to 30, 5 to 40, 5 to 50, 5 to 75, 5 to 100, 10 to 20, 10 to 30, 10 to 40, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 4, 15 to 50, 20 to 25, 20 to 30, 20 to 40, or 20 to 50 mM EGTA. The buffer can comprise from 0% to 30%, from 5% to 30%, from 10% to 30%, from 15% to 30%, from 20% to 30%, from 25% to 30%, from 0% to 25%, from 2% to 25%, from 5% to 25%, from 10% to 25%, from 15% to 25%, from 20% to 25%, from 0% to 20%, from 5% to 20%, from 10% to 20%, from 15% to 20%, from 0% to 15%, from 5% to 15%, from 10% to 15%, from 0% to 10%, from 5% to 10%, or from 0% to 5% Tween 20.

Compositions Comprising One or More Additives for Improving Assay Signals

[0291] In some embodiments, the reagents described herein may include a composition for improving detection signal strength, detection reaction time, detection reaction efficiency, stability, solubility, or the like. In some embodiments, the composition may comprise one or more additives. The one or more additives may, for example, comprise amino acids or derivatives thereof, chaotrpes, chelators, cyclodextrins, inhibitors, ionic liquids, linkers, metals, non detergent sulfobetaines, organic acids, osmolytes, peptides, polyamides, polymers, polyols, polyols and salts, salts, or combinations thereof. In some embodiments, the one or more additives may, for example, comprise one or more of trichloroacetic acid, L-Arginine, L- Glutamic acid, glycine, L-Proline, L-Histidine, beta(|3)-Alanine, L-Serine, L-Arginine ethyl ester dihydrochloride, L-Argininamide dihydrochloride, 6-Aminohexanoic acid, Gly-gly peptide, Gly- gly-gly peptide, tryptone, betaine monohydrate, D-(+)-Trehalose dihydrate, Xylitol, D-Sorbitol, sucrose, hydroxyectoine, Trimethylamine N-oxide dihydrate, methyl alpha(a)-D- gluocopyranoside, triethylene glycol, spermine tetrahydrochloride, spermidine, 5-aminovaleric acid, glutaric acid, adipic acid, ethylenediamine dihydrochloride, guanidine hydrochloride, urea, N-methylurea, N-ethylurea, N-methylformamide, hypotauring, TCEP hydrochloride, GSH (L- Glutathione reduced), GSSG (L-Glutathione oxidized), benzaminidine hydrochloride, ethylenediaminetetraacetic acid disodium salt dihydrate, magnesium chloride hexahydrate, calcium chloride dihydrate, cadmium chloride hydrate, cobalt(II) chloride hexahydrate, Non Detergent Sulfobetaine 195 (NDSB-195), NDSB-201, NDSB-211, NDSB-221, NDSB-256, taurine, acetamide, oxalic acid dihydrate, sodium malonate pH 7.0, succinic acid pH 7.0, tacsimate pH 7.0, tetraethylammonium bromide, cholin acetate, l-Ethyl-3-methylimidazolium acetate, l-Butyl-3-methylimidazolium chloride, ethylammonium nitrate, ammonium sulfate, ammonium chloride, magnesium sulfate hydrate, potassium thiocynate, gadolinium(III) chloride hexahydrate, cesium chloride, 4-aminobutyric acid (GABA), lithium nitrate, DL-malic acid pH 7.0, lithium citrate tribasic tetrahydrate, ammonium acetate, sodium benzenesulfonate, sodium p- toluenesulfonate, sodium chloride, potassium chloride, sodium phosphate monobasic monohydrate, potassium phosphate dibasic, sodium sulfate decahydrate, lithium chloride, sodium bromide, glycerol, ethylene glycol, polyethylene glycol 200 (PEG-200), PEG 3350, PEG 8000, PEG monomethyl ether 550, PEG monomethyl ether 750, PEG monomethyl ether 1900, formamide, polypropylene glycol P 400, pentaerythritol ethoxylate, 1,2-Propanediol, polyvinylpyrrolidone K 15, 6-O-a-Maltosyl-|3-cyclodextrin, (2-Hydroxypropyl)-|3-cyclodextrin, a-cyclodextrin, P-cyclodextrin, Methyl-|3-cyclodextrin, or any combination thereof.

[0292] In some embodiments, the reagents described herein may include a composition for increasing the signal strength of any of the assays described herein. In some embodiments, the composition comprises water and an additive. In some embodiments, the additive may comprise trehalose, xylitol, D-sorbitol, sucrose, and trimethylamine N-oxide dihydrate, or any combination thereof. In some embodiments, the composition further comprises one or more targets, one or more enzymes, one or more reporters, one or more substrates, or any combination thereof as described herein.

[0293] In some embodiments, the reagents described herein may include a composition for reducing the time it takes for the signal of any of the assays described herein to saturate. In some embodiments, the composition comprises an additive. In some embodiments, the additive may comprise betaine monohydrate, acetamide, GABA, L-proline, beta-alanine, 6-aminohexanonic acid, urea, methylurea, ethylurea, hypotaurine, NDSB-256, ammonium acetate, or any combination thereof. In some embodiments, the composition further comprises one or more targets, one or more enzymes, one or more reporters, one or more substrates, or any combination thereof as described herein.

[0294] In some embodiments, the one or more enzymes may comprise a nuclease. In some embodiments, the one or more enzymes may comprise a programmable nuclease. In some embodiments, the one or more enzymes may comprise a Casl2 enzyme. In some embodiments, the one or more enzymes may comprise a Casl4 enzyme. In some embodiments, the one or more enzymes may comprise a CasPhi enzyme. In some embodiments, the one or more enzymes may comprise a Cast 3 enzyme. In some embodiments, the one or more enzymes may comprise HRP. In some embodiments, the one or more enzymes may comprise any one or combination of enzymes presented in this disclosure.

[0295] In some embodiments, the one or more reporters may comprise a reporter free in solution. In some embodiments, the one or more reporters may comprise a reporter immobilized on a surface of a support. In some embodiments, the one or more reporters may comprise biotin. In some embodiments, the one or more reporters may comprise a fluorescent moiety. In some embodiments, the one or more reporters may comprise a nucleic acid tether. In some embodiments, the one or more reporters may comprise a linker. In some embodiments, the one or more reporters may comprise any one or combination of reporters presented in this disclosure. [0296] In some embodiments, the one or more supports may comprise a glassy substance. In some embodiments, the one or more substrates may comprise a polymeric substance. In some embodiments, the one or more substrates may comprise a hydrogel. In some embodiments, the one or more substrates may comprise any one or combination of substrates presented in this disclosure.

DETECTR assay immobilization

[0297] CRISPR diagnostic reactions are generally performed in solution where the Cas protein- RNA complexes can freely bind target nucleic acids and reporters. However, reactions where all components are in solution limit the designs of CRISPR diagnostic assays, especially in microfluidic devices. A system where various components of the CRISPR diagnostic reaction are immobilized on a surface enables designs where multiple readouts can be accomplished within a single reaction chamber.

[0298] Described herein are various methods to immobilize CRISPR diagnostic reaction components to the surface of a reaction chamber or other surface (e.g., a surface of a bead). Any of the devices described herein may comprise one or more immobilized detection reagent components (e.g., programmable nuclease, guide nucleic acid, and/or reporter), or component capable of being immobilized at some point during a particular assay (e.g., through the inclusion of a functionality for immobilization to a surface, such as the surface of a bead). In some embodiments, the surface is a bead, which may be present at the beginning of a reaction, or added later to capture one or more components of a reaction (e.g., unreacted reporters comprising an enzyme, a linker, and a functionality for immobilization). The term “bead” is not limited to any particular size or shape. Beads may be uniform or non-uniform, spherical or non- spherical, regular or irregular. In embodiments, beads are magnetic or paramagnetic. The surface of the beads may comprise a reaction partner for the functional moiety for immobilization (e.g., streptavidin coated beads for capture of targets comprising a biotin functionality). In some embodiments, the beads are microparticles or nanoparticles. A variety of beads for use with nucleic acid based assays are commercially available, a non-limiting example of which includes DYNABEADS in various formats. In certain instances, methods include immobilization of programmable nucleases (e.g., Cas proteins or Cas enzymes), reporters, guide nucleic acids (e.g., gRNAs), or a combination of two or all of these. In some embodiments, various CRISPR diagnostic reaction components are modified with biotin. In some embodiments, these biotinylated CRISPR diagnostic reaction components are tested for immobilization on surfaces coated with streptavidin. In some embodiments, the biotin-streptavidin interaction is used as a model system for other immobilization chemistries.

[0299] Table 3 presents gRNA and reporter immobilization sequences

Table 3: gRNA and reporter immobilization sequences

[0300] FIGS. 11A-11C illustrate three exemplary immobilization strategies for CRISPR-Cas diagnostic assay components. In some embodiments, as seen in FIG. 11 A, chemical modifications of amino acid residues in the Cas protein enable attachment to a surface. In some embodiments, as seen in FIG. 11B, gRNAs are immobilized by adding various chemical modifications at the 5’ or 3’ end of the gRNA that are compatible with a selected surface chemistry. In some embodiments, as seen in FIG. 11C fluorescence-quenching (FQ), or other reporter chemistries, are attached to surfaces using similar chemical modifications as gRNAs. In some embodiments, these attached reporters are activated by a Cas protein, which leads to either activated molecules that remain attached to the surface or activated molecules that are released into solution.

[0301] For some embodiments, described herein, FIG. 12 provides an illustrative example of immobilization strategies for use with methods and compositions described herein where the RNP complex is immobilized by a gRNA and cleaves surrounding FQ reporters that are also immobilized to a surface. Here, the quencher is released into solution, leaving a localized fluorescent signal.

[0302] In some embodiments, the programmable nuclease, guide nucleic acid, or the reporter are immobilized to a device surface by a linkage or linker. In some embodiments, the linkage comprises a covalent bond, a non-covalent bond, an electrostatic bond, a bond between streptavidin and biotin, an amide bond, or any combination thereof. In some embodiments, the linkage comprises non-specific absorption. In some embodiments, the programmable nuclease is immobilized to the device surface by the linkage, wherein the linkage is between the programmable nuclease and the surface. In some embodiments, the reporter is immobilized to the device surface by the linkage, wherein the linkage is between the reporter and the surface. In some embodiments, the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 5’ end of the guide nucleic acid and the surface. In some embodiments, the guide nucleic acid is immobilized to the surface by the linkage, wherein the linkage is between the 3’ end of the guide nucleic acid and the surface.

Sample Preparation and Lyophilization

[0303] Described herein are various methods of sample preparation and reagent storage. Any of the devices described herein may comprise one or more sample preparation reagents Any of the devices described herein may comprise sample preparation reagents as dried reagents. Dried reagents may comprise solids and/or semi-solids. In certain instances, dried reagents may comprise lyophilized reagents. Any of the devices described herein may comprise one or more lyophilized reagents (e.g., amplification reagents, programmable nucleases, buffers, excipients, etc.). In certain instances, methods include sample lysis, concentration, and/or filtration. In certain instances, methods include reconstitution of one or more lyophilized reagents. In some embodiments, lyophilized reagents may be in the form of lyophilized beads, spheres, and/or particulates. In some embodiments, the lyophilized bead, sphere, and/or particulate may comprise either single or multiple compounds. In some embodiments, the lyophilized bead, sphere, and/or particulate may be adjusted to various moisture levels or hygroscopy. In some embodiments, the lyophilized bead, sphere, and/or particulate may comprise assay internal standards. In some embodiments, the lyophilized bead, sphere, and/or particulate may have diameters between about 0.5 millimeters to about 5 millimeters in diameter.

[0304] Described herein are various embodiments of the DETECTR reaction involving optimization of sample preparation and lyophilization. Such embodiments allow for adapting the buffer for binding a substrate to perform a concentration step. In some embodiments, experiments may be performed to evaluate the lysis (sample is evaluated directly in the assay) and binding (the sample is eluted from magnetic beads) characteristics of buffers with different components. In such embodiments, the input sample is the same concentration as the eluted sample.

One-pot sRCA: Rolling circle amplification with Casl4a DETECTR in single reaction volume

[0305] Described herein are various methods of sample amplification and detection in a single reaction volume. Any of the devices described herein may be configured to perform amplification and detection in a same well, chamber, channel, or volume in the device. In certain instances, methods include simultaneous amplification and detection in the same volume. In certain instances, methods include sequential amplification and detection in the same volume. In some embodiments, sample amplification may comprise RCA.

[0306] FIG. 13 presents an overview of sRCA. In this system, a target nucleic acid is added to a system that contains components for a one-pot RCA + Cas protein reaction. The RCA portion of the system is composed of a dumbbell-shaped DNA template, a primer, and a DNA polymerase. For some embodiments, the DETECTR portion of the reaction is composed of a Cas protein, such as Cas 12 or Cas 14, a crRNA that targets the amplicon generated by RCA, but not the dumbbell-shaped DNA template, and a FQ ssDNA reporter. A target nucleic acid (e.g., viral RNA), which is capable of binding to the dumbbell DNA template, is added to the system. The target nucleic acid base pairs with the DNA template. The more extensive base pairing between the target and the DNA template causes the internal base pairing of the dumbbell to be disrupted, which opens up a binding site for the primer. The DNA polymerase can then use this primer to begin RCA. In some embodiments, As RCA proceeds, the amplicon is generated which contains the target site for the Cas protein. The Cas protein recognizes this site through base pairing with the crRNA and initiates trans-cleavage of the FQ ssDNA reporter. In some embodiments, the system contains fewer components than other one-pot approaches and does not require a RT enzyme.

CasPin: Casl3 positive-feedback loop leveraging Casl3 ssDNA targeting

[0307] Described herein are various methods of signal amplification. Any of the devices described herein may be configured to perform signal amplification after the reporter has been cleaved by the programmable nuclease. Signal amplification may improve detection of rare targets in a complex sample. In certain instances, methods include leveraging ssDNA targeting of the programmable nuclease (e.g., Casl3) to create a positive feedback loop upon biding of the programmable nuclease to the target nucleic acid to cleave additional reporters and amplify the signal generated by the presence of the target nucleic acid.

[0308] FIG. 15 presents an overview of CasPin. In some embodiments, the CasPin system uses two populations of Casl3. One is programmed with a crRNA that targets an RNA of interest, such as a viral genome. The other population is programmed with a crRNA that is optimal for ssDNA detection. In some embodiments, the systems also contains a hairpin-shaped oligo that is composed of both DNA and RNA. Finally, in some embodiments, there is a FQ RNA reporter that is used to readout the result of the assay. When Casl3 detects the RNA of interest, it can either cleave the FQ RNA reporter or the RNA on the hairpin oligo. When the RNA on the hairpin oligo is cleaved, it dissociates from the DNA revealing a ssDNA target site that can be recognized by the other population of Cast 3 RNPs. This initiates a positive feedback loop where Cast 3 recognizes the ssDNA target and cleaves more hairpin molecules, which increases the overall amount of target in the system, and leads to further activation of the system. As this process continues more and more FQ RNA reporter is cleaved, which is the ultimate readout of the assay.

[0309] FIG. 16 presents potential structures of hairpins for CasPin. In some embodiments, the target ssDNA sequence is indicated by the purple rectangle. RNA loop structures could occur on either side of the target strand (either 5’, 3’ or both). The strand that is complementary to the target site could be DNA or RNA. The strand that is complementary to the target site could also be a perfect match to the target site, be shorter than the target site, or contain mismatches to help destabilize or promote trans-cleavage by Casl3.

[0310] In some embodiments, two hairpins are used on either end of the target site. FIG. 17 presents results for such an embodiment and indicates capability for blocking Cast 3 from recognizing the ssDNA target site. In some embodiments, CasPin oligos have varying lengths of hairpin stems. In some embodiments, CasPin oligo do not have stem structures. In some embodiments, CasPin oligos contain another DNA sequence. Such embodiments were evaluated and found to not be recognized by the crRNA. Both raw oligos from the manufacture and those that had been denatured and refolded at 25C in a Cast 3 DETECTR reaction were tested. The results of this experiment demonstrated that Casl3 was able to recognize the target site regardless of the stem length. In some embodiments, longer stem length oligos block Cast 3 recognition without RNA cleavage to release the structure.

One-pot DETECTR on handheld microfluidic device

[0311] Described herein are various devices and methods for running one-pot DETECTR assays on a handheld device. Any of the devices described herein may be configured to perform a one- pot DETECTR assay.

Multiplexed DETECTR assay-based lateral flow assay

[0312] Described herein are various methods of multiplexing detection. Any of the devices described herein may be configured for multiplexing (e.g., detecting multiple target nucleic acids). In certain instances, multiplexed detection may utilize one or more lateral flow assay strips.

[0313] Described here are various devices and methods for a DETECTR™ assay based multiplex lateral flow assay strip as illustrated in FIG. 18. In some embodiments, reporters (10701) are immobilized to a surface (10700) of a solid support. In some embodiments, programmable nuclease (e.g., Cas-complex) probes (10707) are immobilized to a surface (10700). In some embodiments, programmable nuclease probes (10707) comprise guide nucleic acid such as a single guide RNA (sgRNA) (10708). In some embodiments, a programmable nuclease probe (10707) may comprise a sgRNA (10708) that is designed to be a compliment for a target nucleic acid of a sample. In some embodiments, programmable nuclease probes (10707) and reporters (10701) are both immobilized to a surface (10700). In some embodiments, programmable nuclease probes (10707) and reporters (10701) are both immobilized to a surface (10700) in close enough proximity that the reporter (10701) can be cleaved by the programmable nuclease of the programmable nuclease probe (10707). In some embodiments, programmable nuclease probes (10707) and reporters (10701) are both immobilized to a surface (10700) in close enough proximity that the reporter (10701) can be cleaved (10709) by the programmable nuclease of the programmable nuclease probe (10707) upon binding of a target nucleic acid to an sgRNA (10708) of the programmable nuclease probe (10707) when target nucleic acid and sgRNA (10708) are compliments. In such an embodiment, this indicates the presence of and is a “hit” for the target. In some embodiments, binding of a target nucleic acid that is complimentary to a sgRNA (10708) of the programmable nuclease probe (10707) results in the programmable nuclease of the programmable nuclease probe (10707) initiating cleavage of nucleic acids within a close enough proximity of the programmable nuclease. In some embodiments, the surface (10700) is in the bottom of a well. In some embodiments, a collection of a first programmable nuclease probe (10707) and a first reporter (10701) are immobilized to a surface at one location of the surface (10700).

[0314] In some embodiments, as illustrated in FIG. 18A, the reporter (10701) may comprise a surface linker (10702), a nucleic acid (10703), a second linker (10706), a detection moiety (e.g., a label) (10704), and an affinity molecule (e.g., a binding moiety) (10705). In some embodiments, the binding moiety (10705) is biotin. In some embodiments, there is more than one copy of the same reporter (10701) immobilized to the surface.

[0315] In some embodiments, lateral flow assay strips (10710) are used to detect cleaved reporters (10709). In some embodiments, cleaved reporters (10709) are contacted to the sample pad (10711) of the lateral flow assay strip (10710). In some embodiments, the cleaved reporters (10709) bind to conjugate particles present in the sample pad. In some embodiments, the conjugate particles are gold nanoparticles. In some embodiments, the gold nanoparticles are functionalized with anti-biotin. In some embodiments, the anti-biotin functionalized gold nanoparticles bind to the cleaved reporter which contains one or more biotins in the binding moiety (10705).

[0316] In some embodiments, the reporter contains a second linker. In some embodiments, the second linker links one or more binding moieties to the nucleic acid. In some embodiments, the second linker links one or more labels to the nucleic acid. In some embodiments, the second linker links both one or more binding moieties and one or more labels to the nucleic acid of the reporter. In some embodiments, the reporter is a dendrimer or trebler molecule.

[0317] In some embodiments, the reporter contains a label. In some embodiments, label is FITC, DIG, TAMRA, Cy5, AF594, Cy3, or any appropriate label for a lateral flow assay.

[0318] In some embodiments, the reporter may comprise chemical functional group for binding. In some embodiments, the chemical functional group is biotin. In some embodiments, the chemical functional group is complimentary to a capture probe on the flowing capture probe (e.g., conjugate particle or capture molecule). In some embodiments, the flowing capture probe is a gold nanoparticle functionalized with anti-biotin. In some embodiments, the flow capture probe is located in the sample pad. In some embodiments, the flowing capture probe is located in a conjugate pad in contact with the sample pad, wherein both lateral flow assay strip may comprise both the sample pad and conjugate pad, further wherein both the sample pad and the conjugate pad are in fluid communication with the detection region.

[0319] In some embodiments, the lateral flow assay strip (10710) contains a detection region (10712). In some embodiments, the detection region (10712) may comprise one or more detection spots. In some embodiments, the detection spots contain a stationary capture probe (e.g., capture molecule). In some embodiments, the stationary capture probe may comprise one or more capture antibodies. In some embodiments, the capture antibodies are anti-FITC, anti- DIG, anti-TAMRA, anti-Cy5, anti-AF594, or any other appropriate capture antibody capable of binding the detection moiety or conjugate.

[0320] In some embodiments, the flowing capture probe comprising FITC is captured by a stationary capture probe comprising anti-FITC antibody. In some embodiments, the flowing capture probe comprising TAMRA is captured by a stationary capture probe comprising anti- TAMRA antibody. In some embodiments, the flowing capture probe comprising DIG is captured by the stationary capture probe comprising anti-DIG antibody. In some embodiments, the flowing capture probe comprising Cy5 is captured by the stationary capture probe comprising anti-Cy5 antibody. In some embodiments, the flowing capture probe comprising AF574 is captured by the stationary capture probe comprising anti-AF594 antibody.

[0321] In some embodiments, the lateral flow assay strip (10710) may comprise a control line (10714). In some embodiments, the control line (10714) may comprise anti-IgG that is complimentary to all flowing capture probes. In some embodiments, when a flowing capture probe does not bind to a reporter the flowing capture probe will be captured by the anti-IgG on the control line, ensuring the user that the device is working properly even no signal is read from the test line.

[0322] In some embodiments, the lateral flow assay strip (10710) may comprise a sample pad. In some embodiments, the flowing capture probe may comprise anti-biotin. In some embodiments, the flowing capture probe may comprise HRP. In some embodiments, the flowing capture probe may comprise HRP-anti-biotin. In some embodiments, the flowing capture probe is HRP-anti- biotin DAB/TMB.

[0323] In some embodiments, as shown in FIG. 45, the lateral flow assay strips may be incorporated into microfluidic devices for parallel readout.

[0324] Described here are various devices and methods for a DETECTR™ assay based multiplex lateral flow assay strip as illustrated in FIG. 19. FIG. 19 depicts a non-limiting exemplary workflow for a DETECTR™ assay read out on a lateral flow assay strip. In some embodiments, a sample (10801) contains one or more target nucleic acid sequences. In some embodiments, a sample (10801) (e.g., a sample solution) contains at least first and second target nucleic acid sequences. In some embodiments, the sample (10801) is introduced into a well (10802) (e.g., D1-D5) where at one or more locations there are different guide nucleic acids such as sgRNAs immobilized to the surface of the well. In some embodiments, the sgRNAs are part of a programmable nuclease probe immobilized to a surface. In some embodiments, a sgRNA is designed to specifically bind to a target nucleic acid in the sample. In some embodiments, there are different sgRNAs corresponding to different locations (e.g., locations D1-D5) on the surface of the well, where each different sgRNA is complimentary for a different target nucleic acid sequence that may or may not be present in the sample. In some embodiments, in addition to the programmable nuclease probes containing sgRNAs, each location is functionalized with one or more reporter probes having distinct functional groups. In some embodiments, the reporter probes are in close enough proximity to be cleaved by the programmable nuclease probes. In some embodiments, binding between a particular sgRNA and the target nucleic acid to which the sgRNA is designed to specifically bind allows for a section of one or more reporters are cleaved from a corresponding nucleic acid and released into the sample solution. In some embodiments, the reporter is functionalized with a label. In some embodiments, the lateral flow assay strip contains a detection region comprising detection spots (e.g., 10803, 10804), where each detection spot contains a different type of capture antibody. In some embodiments, each capture antibody type specifically binds to a particular label type of a reporter. In some embodiments, a first detection spot (10803) contains the capture antibody anti-FITC. In some embodiments, location D5 on the surface of the well (10802) contains a first immobilized programmable nuclease probe including the sgRNA specific to the first target nucleic acid sequence. In some embodiments, D5 additionally contains the immobilized first reporter (10806), which is labeled with FITC. In some embodiments, upon binding of the first target nucleic acid sequence to the programmable nuclease probe causes the cleavable nucleic acid of the first reporter (10806) to be cleaved and released into solution. Alternatively, or in combination, in some embodiments, a second detection spot (10804) contains the capture antibody anti -DIG. In some embodiments, a second location D4 contains the immobilized programmable nuclease probe including the sgRNA specific to the second target nucleic acid sequence. In some embodiments, D4 additionally contains the immobilized second reporter (10805), which is labeled with DIG. Therefore, in some embodiments, upon binding of the second target nucleic acid sequence, the immobilized second reporter (10805) is cleaved and released into solution. In some embodiments, the solution containing cleaved first and second reporters (10805) and (10806) is contacted to the sample pad of the lateral flow assay strip along with chase buffer. In some embodiments, the sample pad has one or more flowing capture probes (e.g., anti-biotin-AuNP) disposed thereon. In some embodiments, the sample solution containing the cleaved first and second reporters, along with the chase buffer, flow across the sample pad, where the reporters are bound to conjugates (e.g., anti-biotin -gold nanoparticles). In some embodiments, the solution containing the cleaved reporters is contacted to the sample pad by manually pipetting. In some embodiments, the solution containing the cleaved reporters is contacted to the sample pad being drawn from a chamber in fluid connection with the sample pad. In some embodiments, the solution containing the cleaved reporters is contacted to the sample pad by being drawn from a chamber in which the assay resulting in the cleaved reporter solution occurs. In some embodiments, the reporters are cleaved in the sample pad. In some embodiments, the reporters are cleaved in the sample pad by a DETECTR™ assay. In some embodiments, the solution is drawn into and out of the sample pad by capillary action, or wicking. In some embodiments, the capillary action or wicking is caused by the liquid being drawn into an absorption pad. In some embodiments, the capillary action or wicking is caused by the liquid being drawn into an absorption pad, not requiring electrical power. In some cases, the solution is drawn into or out of the sample pad by a pressure gradient. In some embodiments, the gold nanoparticle-reporter conjugates having reporter (10806) labeled with FITC will selectively bind to the first detection spot (10803) containing the capture antibody anti-FITC, thus indicating the presence of the first target nucleic acid sequence in the sample. In some embodiments, the AuNP-reporter conjugates having mostly reporter (10805) labeled with DIG will selectively bind to the second detection spot (10804) containing the capture antibody anti -DIG, thus indicating the presence of the second target nucleic acid sequence in the sample. In this manner, for some embodiments, parallel detection of two or more target nucleic acid sequences present in a multiplexed sample is enabled.

[0325] Described herein are various embodiments of lateral flow-based detection as illustrated in FIG. 20. In some embodiments, horse radish peroxidase (HRP) (10901) is used to enhance detection in lateral flow based DETECTR™ assays. In some embodiments, a sample containing a target(s) nucleic acid sequence is exposed to a surface (10900) upon which programmable nuclease probes and reporter probes are immobilized on the surface. In some embodiments, the reporter probes contain HRP molecules. In some embodiments, upon cleavage of the reporter by the programmable nuclease following a specific binding event between the target and the guide RNA, the cleaved portion of the reporter is released into the sample solution (10906). In some embodiments, the sample solution is then exposed to a lateral flow assay strip (10902) comprising or adjacent to a sample pad containing sodium percarbonate (10904), which generates H2O2 when exposed to an aqueous solution. In some embodiments, the rehydration of the sodium percarbonate to form H2O2 occurs when the sample is wicked through the region. In some embodiments, the substrate contains DAB, TMB, or any other sufficient substrate. In some embodiments, the “spot” changes from blue to red, indicating the presence of HRP, and in turn a “hit” for the target nucleic acid sequence. In some embodiments, the readout is accomplished in solution, upon a color change of the sample solution (10908).

[0326] Described here are various methods and devices utilizing HRP-enhanced multiplexed DETECTR™ assays utilizing lateral flow assay strips for readout. In some embodiments, an HRP-signal enhanced multiplexed lateral flow assay as illustrated in FIG. 21. In some embodiments, the immobilized surface (11000) of a support medium and detection on the lateral flow assay strip (11010) are carried out as described in FIGS. 18A-16 with the exception that signal enhancement is not carried out by gold nanoparticles scattering light. Instead, in some embodiments, the anti -biotin labeled AuNP are supplanted by HRP-anti -biotin DAB/TMB. In some embodiments, the HRP is activated by sodium percarbonate present in the lateral flow assay strip which is rehydrated by the reaction and or chase buffer. In some embodiments, HRP allows for strong enough signal so as not to require sample amplification such as PCR.

[0327] Described herein are various embodiments for multiplexed target nucleic acid detection utilizing Casl3 RNA cleaving specificity over DNA, HRP-signal enhancement, and capture oligo probe specificity. In some embodiments, as shown in FIG. 22, the sample (11100) contains different target nucleic acids. In some embodiments, the sample (11100) is then contacted to the surface of the well (11101) that is functionalized at one or more locations (e.g., five locations, D1-D5). In some embodiments, there are one or more locations. In some embodiments, Cast 3 enzyme is present in the programmable nuclease probe. In some embodiments, Casl3 cleaves RNA but not DNA, enabling the use of a reporter (11102) that contains nucleic acid sequences with both DNA and RNA strands. In some embodiments, upon binding of the target nucleic acid to the sgRNA, the RNA of the reporter is cleaved by the Cast 3 enzyme and a fragment containing a portion of the RNA, the complete DNA sequence, and a FITC label is released into solution. In some embodiments, this action is repeated in parallel at each location, or spot with different reporters. In some embodiments, this action is repeated in parallel at locations DI through D5 for five different target nucleic acids, producing five distinct reporter fragments. In some embodiments, the solution is then contacted to the sample pad of the lateral flow assay strip, where the sample pad contains HRP-anti-FITC. In some embodiments, the FITC-labeled reporter fragment then binds to the HRP-anti-FITC, forming a complex (11103) and is carried downstream across the detection region, binding specifically to the detection spot containing a capture oligo that has been designed to be the compliment for the oligo in the complex (11103).

Guide RNA pooling for signal enhancement

[0328] In some embodiments, one or more programmable nuclease probes (11300-11302) are used for guide pooling to achieve enhanced signal detection in lateral flow assays as shown in FIG. 23A. In some embodiments, a first programmable nuclease probe (11300) may comprise a first sgRNA that is complimentary for a first segment of a target nucleic acid. In some embodiments, a second programmable nuclease probe (11301) may comprise a second sgRNA that is complimentary for a second segment of a same target nucleic acid. In some embodiments, a third programmable nuclease probes (11302) may comprise a third sgRNA that is complimentary for a third segment of the same target nucleic acid. In some embodiments, the first programmable nuclease probe, the second programmable nuclease probe, and the third programmable nuclease probe are all located close enough to allow for sufficient cleaving of a reporter that is labeled to indicate the presence of the target nucleic acid. FIG. 23B shows a typical lateral flow assay strip comprising a sample pad (11303), a test line (11304), and a control line (11305).

DETECTR based multiplexed detection

[0329] FIG. 24 shows a schematic of an exemplary workflow for a multiplexed programmable nuclease assay. Any of the devices described herein may be configured to perform one or more of the reactions described herein (e.g., amplification, detection, etc.) in separate chambers. In at least some instances, fluidly isolating reactions for different target nucleic acids may facilitate multiplexing.

[0330] In some embodiments, each reaction chamber may comprise one or more guide nucleic acids (e.g., sgRNAs). In some embodiments, the one or more guide nucleic acids may be immobilized to a surface (e.g., a glass bead or hydrogel disposed within a reaction chamber). In some embodiments, the guide nucleic acids are part of a programmable nuclease (e.g., Cas- complex) probe immobilized to a surface. In some embodiments, a guide nucleic acid is designed to specifically bind to a target nucleic acid in the sample. In some embodiments, there are different guide nucleic acids corresponding to different reaction chambers, where each different guide nucleic acid is complimentary for a different target nucleic acid sequence that may or may not be present in the sample. In some embodiments, in addition to the programmable nuclease probes containing guide nucleic acids, each reaction chamber may contain or be functionalized with one or more reporters having distinct functional groups as described herein. In some embodiments, the reporters may be in close enough proximity to be cleaved by the programmable nuclease probes. In some embodiments, reporters are cleaved and portion thereof (e.g., a detection moiety) is released into the solution upon binding between a particular guide nucleic acid and the target nucleic acid to which the guide nucleic acid is designed to specifically bind. In some embodiments, reporters are functionalized with a detection moiety (e.g., a label). [0331] In some embodiments, chemical heating may be used. In some embodiments, chemical heating may be used to supply energy to initiate and run reactions. In some embodiments, chemical heating may be used to supply energy to initiate and run programmable nuclease assay reactions. In some embodiments, chemical heating may be used to heat reaction or heating zones. In some embodiments, chemical heating may be used to heat regions, chambers, volumes, zones, surfaces, or areas of a device.

[0332] In some embodiments, the device may comprise one or more lateral flow assay strips in a detection region disposed downstream of the reaction chamber. In some embodiments, the device may comprise one or more lateral flow assay strips in a detection region which may be brought into fluid communication with the reaction chamber. Each lateral flow assay strip contains one or more detection regions or spots, where each detection region or spot contains a different type of capture antibody. In some embodiments, each lateral flow assay strip contains a different type of capture antibody. In some embodiments, each capture antibody type specifically binds to a particular label type of a reporter. In some embodiments, a first lateral flow assay strip contains the capture antibody anti-FITC. In some embodiments, a first DETECTR region or surface location (e.g., within a first reaction chamber) contains the immobilized programmable nuclease (e.g., Cas-complex) including the guide nucleic acid (e.g., sgRNA) specific to the first target nucleic acid sequence. In some embodiments, the first DETECTR region or surface location additionally contains a first immobilized reporter which is labeled with a first detection moiety (e.g., FITC). In some embodiments, upon binding of the first target nucleic acid sequence, the first immobilized reporter is cleaved and released into solution. In some embodiments, the first detection moiety is released into solution and the remainder of the first reporter remains immobilized on the surface. Alternatively, or in combination, in some embodiments, a second lateral flow assay strip contains the capture antibody anti-DIG. In some embodiments, a second DETECTR region or surface location (e.g., within a second reaction chamber) contains the immobilized programmable nuclease (e.g., Cas complex) including the guide nucleic acid (e.g., sgRNA) specific to the second target nucleic acid sequence. In some embodiments, the second DETECTR region or surface location additionally contains a second immobilized reporter which is labeled with a second detection moiety (e.g., DIG). Therefore, in some embodiments, upon binding of the second target nucleic acid sequence, the second immobilized reporter is cleaved and released into solution. In some embodiments, the second detection moiety is released into solution and the remainder of the second reporter remains immobilized on the surface. FIG. 45 presents an exemplary microfluidic device that implements the LFA strips for parallel readout.

[0333] In some embodiments, the solutions containing the first or second cleaved reporters are transferred from their respective reaction chambers to a first lateral flow assay strip and a second lateral flow assay strip, respectively. In some embodiments, a chase buffer or diluent is introduced into a diluent input and negative pressure is applied to the negative pressure port to contact the solutions containing the first or second cleaved reporters to their respective lateral flow assay strips. The reporters may be bound to conjugate molecules e.g., anti-biotin-AuNPs. In some embodiments, the AuNP-reporter conjugates having the first reporter labeled with the first detection moiety (e.g., FITC) will selectively bind to a first detection region or spot containing the first capture antibody (e.g., anti-FITC) on the first lateral flow assay strip, thus indicating the presence of the first target nucleic acid sequence in the sample. In some embodiments, the AuNP-reporter conjugates having mostly the second reporter labeled with the second detection moiety (e.g., DIG) will selectively bind to a second detection region or spot containing the second capture antibody (e.g., anti-DIG) on the second lateral flow assay strip, thus indicating the presence of the second target nucleic acid sequence in the sample. In this manner, for some embodiments, parallel detection of two or more target nucleic acid sequences present in a multiplexed sample is enabled.

[0334] In some embodiments, the device may comprise more than one lateral flow assay strip. For example, the device may comprise two, three, four, five, six, seven, eight, nine, ten, or more lateral flow assay strips. In some embodiments, each reaction chamber may be interfaced with a lateral flow assay strip for multiplexing. In some embodiments, one or more lateral flow assay strips are configured to detect a control sequence instead of or in addition to a target sequence. For example, a device comprising six lateral flow assay strips may comprise five lateral flow assay strips configured to detect one or more target sequences (e.g., five different target sequences) and one lateral flow assay strip configured to detect a control sequence.

[0335] In some embodiments, the device may comprise one or more detection chambers or volumes in a detection region disposed downstream of the reaction chamber. In some embodiments, the device may comprise one or more detection chambers or volumes in a detection region which may be brought into fluid communication with the reaction chamber. In some embodiments, at least a portion of the reporter (e.g., a cleavage product thereof) may be transferred from a reaction chamber to one or more detection chambers. For example, when the reporter comprises an HRP detection moiety immobilized on a surface of the reaction chamber by a cleavage nucleic acid, the presence of the target nucleic acid results in cleavage of the reporter and release of the HRP detection moiety into solution as described herein. The released HRP-containing cleavage product may be transferred from the reaction chamber to a detection chamber containing an HRP substrate (e.g., TMB) and a detectable signal may be generated when HRP acts on its substrate in the detection chamber as described herein.

Enzyme-Linked Signal Amplification Strategies

[0336] Disclosed herein are systems, compositions, and methods for amplifying a signal from a target nucleic acid using an enzyme. In some embodiments, enzyme-based assays may allow for signal amplification of a binding event between one or more programmable nuclease probes and one or more target nucleic acids.

[0337] Disclosed herein are some strategies that use enzymes with programmable nucleases (e.g., DETECTR based assays) to amplify detection signals indicative of the presence of one or more target nucleic acids in a sample.

[0338] FIGS. 30A-30B show an enzyme-based signal generation and amplification strategy that involves a reporter (19208), wherein the reporter comprises a nucleic acid tether and an enzyme (19204). Activation of a programmable nuclease (19206) upon binding with its guide nucleic acid (19209) and a target nucleic acid (19205), may enable the programmable nuclease to carry out trans-cleavage of the nucleic acid tether of the reporter. The trans-cleavage of the nucleic acid tether may release the enzyme into solution. Release of the enzyme may create a detectable signal, for example, by the enzyme reacting with an enzyme substrate (19207) in a solution to induce a color change in the solution, thereby indicating the presence of the target nucleic acid in the sample.

[0339] FIG. 30A shows two exemplary embodiments of a reporter (19208) comprising an enzyme (19204) and a nucleic acid tether, wherein the nucleic acid tether immobilizes the reporter to a surface (19200). In some embodiments, the reporter may comprise an enzyme and a nucleic acid tether, e.g., a ssDNA tether (19202) or a RNA tether (19203). In some embodiments, the nucleic acid tether may comprise a mixture of DNA and RNA.

[0340] FIG. 30B shows an exemplary signal generation strategy utilizing a reporter (19208) comprising an enzyme (19204) and a nucleic acid tether (19210), wherein the nucleic acid tether immobilizes the reporter to a surface (19200). The first panel from the left shows a programmable nuclease complex, comprising a programmable nuclease (19206) with a guide nucleic acid (19209), and a target nucleic acid (19205) prior to encountering the reporter immobilized to the surface. Upon binding of a target nucleic acid to the programmable nuclease complex, the programmable nuclease may be activated and may cleave the nucleic acid tether of the reporter, thereby releasing the enzyme into solution. In some embodiments, an enzyme substrate (19207) may be added to the solution after the enzyme is cleaved off the reporter, as shown in the last panel from the left. In some embodiments, the solution (or supernatant) containing the released enzyme can be separated and added to a solution comprising an enzyme substrate (19207) upon which the enzyme (19204) can act to produce a detectable signal in the solution. FIG. 38 illustrates an alternative method in which the reporters comprise the linker and functionality for immobilization, but are free in solution at the time of exposure to the programmable nuclease. In the presence of a target nucleic acid, an activated programmable nuclease complex cleaves the reporter, separating the enzyme from the functionality. The reaction is then contacted with beads reactive with the functionality, leaving only released enzyme (if present) in the solution. The solution is then separated from the beads, and exposed to a substrate of the enzyme. If enzyme is present (resulting from cleavage of the reporter in response to presence of the target nucleic acid), that enzyme reacts with its substrate to produce a detectable signal. Without wishing to be bound by theory, reporters that are unbound in solution may be more accessible to activated programmable nuclease, while inclusion of the functionality for immobilization still provides the benefits of surface-bound reagents in later steps. Where reporters are surface-immobilized at the beginning of the reaction, longer linkers may be used to increase accessibility by the activated programmable nuclease.

[0341] In some embodiments, the method may comprise (a) providing a solution comprising a target nucleic acid, a programmable nuclease complex configured to bind to the target nucleic acid, and a reporter comprising an enzyme immobilized on a surface, and (b) adding an enzyme substrate configured to be acted on by the enzyme to induce color change of the solution.

[0342] In some embodiments, the method may comprise (a) providing a solution comprising a programmable nuclease complex configured to bind to a target nucleic acid and a reporter comprising an enzyme immobilized on a surface, and (b) adding the target nucleic acid and an enzyme substrate configured to be acted on by the enzyme to induce color change of the solution.

[0343] In some embodiments, the method may comprise (a) providing a solution comprising a target nucleic acid and a reporter comprising an enzyme immobilized on a surface, and (b) adding a programmable nuclease complex configured to bind to the target nucleic acid and an enzyme substrate configured to be acted on by the enzyme to induce color change of the solution.

[0344] In some embodiments, the method may comprise (a) providing a solution comprising a target nucleic acid and a programmable nuclease complex configured to bind to the target nucleic acid, and (b) adding a reporter comprising an enzyme immobilized on a surface and an enzyme substrate configured to be acted on by the enzyme to induce color change of the solution.

[0345] In some embodiments, the method may comprise (a) providing a solution comprising a reporter comprising an enzyme immobilized on a surface, and (b) adding a target nucleic acid, a programmable nuclease complex configured to bind to the target nucleic acid, and an enzyme substrate configured to be acted on by the enzyme to induce color change of the solution.

[0346] In some embodiments, the method may comprise (a) providing a solution comprising a target nucleic acid, a programmable nuclease complex configured to bind to the target nucleic acid, and an enzyme substrate configured to be acted on by an enzyme to induce color change of the solution, and (b) adding reporter comprising the enzyme immobilized on one or more surfaces.

[0347] In some embodiments, the method may comprise (a) providing a solution comprising programmable nuclease complex configured to bind to a target nucleic acid and an enzyme substrate configured to be acted on by an enzyme to induce color change of the solution, and (b) adding the target nucleic acid and a reporter comprising the enzyme immobilized on one or more surfaces.

[0348] In some embodiments, the method may comprise (a) providing a solution comprising a target nucleic acid and an enzyme substrate configured to be acted on by an enzyme to induce color change of the solution, and (b) adding a programmable nuclease complex configured to bind to the target nucleic acid and a reporter comprising the enzyme immobilized on one or more surfaces.

[0349] In some embodiments, the method may comprise (a) providing a solution comprising substrates configured to be acted on by an enzyme to induce color change of the solution, and (b) adding a target nucleic acid, a programmable nuclease complex configured to bind to the target nucleic acid, and a reporter comprising the enzyme immobilized on one or more surfaces.

[0350] In some embodiments, the reporter may comprise any of the reporters disclosed herein. In some embodiments, the programmable nuclease may comprise any one of the programmable nucleases disclosed herein. In some embodiments, the nucleic acid tether may comprise any of the nucleic acid tethers disclosed herein.

[0351] The nucleic acid tether of the reporter may be attached to, bound to, coupled to, or otherwise immobilized on the surface using any of the immobilization chemistries or linkages described herein. In some embodiments, the immobilization chemistry or linkage may be between: streptavidin and biotin; an amine group and N-hydroxysuccinimide (NHS), an amine group and an epoxy group, or a malemide group and a thiol group.

[0352] In some embodiments, the surface may be any surface disclosed herein. In some embodiments the surface may be of a support as described herein. In some embodiments, the support may comprise glass, PDMS, a hydrogel, plastic, magnetic beads, agarose beads, nitrocellulose, or any other support whose surface can be modified to bind nucleic acids. In some embodiments, the surface of the support, or of a reaction chamber, comprises streptavidin, biotin, an amine group, a carboxyl group, an epoxy group, an NHS group, a malemide group, or a thiol group. In some embodiments, the target nucleic acid may be any target nucleic acid disclosed herein. In some embodiments, the target nucleic acid may be a single-stranded DNA, a singlestranded RNA, a double-stranded DNA, a double-stranded RNA, or a double-stranded DNA/RNA hybrid.

[0353] In some embodiments, the guide nucleic acid may be any guide nucleic acid disclosed herein. In some embodiments, the guide nucleic acid may be a single-stranded DNA or a singlestranded RNA. In some embodiments, the nucleic acid tethering the reporter may comprise any of the reporter nucleic acids described herein.

[0354] In some embodiments, the enzyme may be HRP, AP, beta-galactosidase, acetylcholinesterase, catalase, catacolase, tyronase, nitrocefelin, alkaline phosphatase, or invertase. In some embodiments, the enzyme may be any one of the enzymes described herein. [0355] In some embodiments, the enzyme may bind with an enzyme substrate and produce a detectable signal. In some embodiments, the enzyme substrate may be 3, 3', 5,5'- tetramethylbenzidine (TMB), 2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]- diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD), p-Nitrophenyl Phosphate (PNPP), o-nitrophenyl-P-D-galactopyranoside (ONPG), 3,3 ’-diaminobenzidine (DAB), p-hydroxyphenylacetic acid, 3-(p-hydroxyphenyl)-propionic acid, homovanillic acid, or o-aminophenol. In some embodiments, the enzyme substrate may be a commercial enzyme substrate including SuperSignal ELISA Pico, SuperSignal Elisa Femto, CDP-Star Substrate, CSPD Substrate, DynaLight Substrate with RapidGlow Enhancer, QuantaBlu, QuantaRed, or Amplex.

[0356] In some embodiments, the enzyme may generate a colorimetric signal, a fluorescent signal, an electrochemical signal, a chemiluminescent signal, or another type of signal. In some embodiments, the enzyme may induce color-change in substances.

[0357] FIGS. 31A-31C show a signal generation and amplification strategy involving the release of a programmable nuclease-enzyme fusion protein comprising a programmable nuclease and an enzyme. Presence of a target nucleic acid may activate the programmable nuclease of the programmable nuclease-enzyme fusion protein, wherein the activated programmable nuclease may cleave a nucleic acid tether that immobilizes the programmable nuclease-enzyme fusion protein on a surface. Cleaving the nucleic acid tether may release the programmable nuclease- enzyme fusion protein into solution, wherein the enzyme of the programmable nuclease-enzyme fusion protein may be contacted with an enzyme substrate to create a detectable and amplified signal.

[0358] FIG. 31A shows two exemplary embodiments of a programmable nuclease-enzyme fusion protein comprising (i) an enzyme portion and (ii) a programmable nuclease portion complexed with a guide nucleic acid. In some embodiments, the programmable nuclease-enzyme fusion protein may be complexed with a guide nucleic acid (19309) tethered or immobilized to a surface (19303) as described herein. In some embodiments, the guide nucleic acid may be immobilized to the surface with a ssDNA tether (19304). In some embodiments, the guide nucleic acid may be immobilized to the surface with an RNA tether (19305). [0359] In some embodiments, the programmable nuclease of the programmable nuclease- enzyme fusion protein may be Cast 2, Cast 3, Cast 4, or CasPhi. In some embodiments, the programmable nuclease of the programmable nuclease-enzyme fusion protein may be any programmable nuclease disclosed herein.

[0360] In some embodiments, the enzyme of the programmable nuclease-enzyme fusion protein may be HRP, AP, beta-galactosidase, acetylcholinesterase, or catalase. In some embodiments, the enzyme of the programmable nuclease-enzyme fusion protein may be any enzyme disclosed herein.

[0361] In some embodiments, the programmable nuclease may be fused by a peptide linker to the enzyme. In some embodiments, the C-terminus of the programmable nuclease portion may be fused by a peptide linker to the N-terminus of the enzyme. In some embodiments, the N- terminus of the programmable nuclease portion may be fused by a peptide linker to the C- terminus of the enzyme. In some embodiments, the peptide linker may be XTEN.

[0362] FIG. 31B shows an example procedure using a programmable nuclease-enzyme fusion protein comprising (i) an enzyme and (ii) a programmable nuclease complexed with a guide nucleic acid. In some embodiments, a target nucleic acid (19306) may be added to a solution comprising the programmable nuclease-enzyme fusion protein bound to an immobilized guide nucleic acid. The target nucleic acid may bind with the programmable nuclease-enzyme fusion protein on the surface of a detection location (19308) and activate the trans-cleavage activity of the programmable nuclease of the programmable nuclease-enzyme fusion protein. The activated programmable nuclease may then cleave the nucleic acid tether (e.g., a ssDNA tether or a RNA tether) and release the programmable nuclease-enzyme fusion protein into solution (in a manner analogous to release of a detection moiety from a reporter as described herein). The solution (or the supernatant) comprising the released programmable nuclease-enzyme fusion protein can be added to a second solution comprising an enzyme substrate (19307) which can be acted upon by the enzyme of the programmable nuclease-enzyme fusion protein to produce a detectable signal as described herein (e.g., to induce a color change of the second solution) as shown in FIG. 31C. [0363] In some embodiments, the method may comprise (a) providing a solution comprising an enzyme substrate and a programmable nuclease-enzyme fusion protein immobilized on a surface, wherein the programmable nuclease-enzyme fusion protein comprises a programmable nuclease and an enzyme, wherein the programmable nuclease is configured to bind to a target nucleic acid, and (b) adding the target nucleic acid.

[0364] In some embodiments, the method may comprise (a) providing a solution comprising an enzyme substrate and a target nucleic acid, and (b) adding a programmable nuclease-enzyme fusion protein immobilized on a surface, wherein the programmable nuclease-enzyme fusion protein comprises a programmable nuclease and an enzyme, wherein the programmable nuclease is configured to bind to a target nucleic acid.

[0365] In some embodiments, the programmable nuclease-enzyme fusion protein may comprise any one of the programmable nucleases disclosed herein. In some embodiments, the target nucleic acid may be any one of the target nucleic acids disclosed herein. In some embodiments, the guide nucleic acid may comprise any one of the guide nucleic acids disclosed herein. In some embodiments, the nucleic acid tethering the guide nucleic acid to the surface may comprise any of the reporter nucleic acids described herein. In some embodiments, the programmable nuclease-enzyme fusion protein may comprise any of the enzymes described herein.

[0366] FIG. 32 shows an exemplary signal generation strategy utilizing a split-enzyme based assay strategy. A first programmable nuclease-split enzyme fusion protein (19402) may comprise a first catalytically inactive programmable nuclease (19403), e.g., a first dead Cas (dCas) protein, fused to a first subunit (19404) of a split enzyme. A second programmable nuclease-split enzyme fusion protein (19406) may comprise a second catalytically inactive programmable nuclease (19407), e.g., a second dead Cas (dCas) protein, fused to a second subunit (19408) of the split enzyme. The first catalytically inactive programmable nuclease (19403) may be complexed to a first guide nucleic acid (19405) configured to bind to a first portion or sequence (19410) of a target nucleic acid. The second catalytically inactive programmable nuclease (19407) may be complexed to a second guide nucleic acid (19409) configured to bind to a second portion or sequence (19411) of a target nucleic acid. The first portion (19410) and the second portion (19411) of the target nucleic acid may be sufficiently close to bring the first subunit (19404) and the second subunit (19408) of the split enzyme together to form a functional enzyme (19414). Once activated, the functional enzyme (19414) may act upon an enzyme substrate (19413) to generate a detectable signal as described herein. [0367] In some embodiments, a catalytically inactive programmable nuclease may have no substantial nucleic acid-cleaving activity. In some embodiments, a catalytically inactive programmable nuclease may be catalytically inactive or “dead,” that is, they may bind to a target nucleic acid but not cleave the target nucleic acid. A catalytically inactive or dead programmable nuclease may comprise a catalytically inactive domain (e.g., inactive nuclease domain). In some instances, a catalytically inactive domain may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 mutations relative to an active domain of a catalytically active programmable nuclease. In some instances, the mutations may be present within a cleaving or active site of the programmable nuclease. In some instances, a catalytically inactive or dead programmable nuclease is fused to a fusion partner. In some instances, catalytically inactive may refer to an activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% activity compared to a wild-type counterpart of the programmable nuclease.

[0368] In an exemplary embodiment, a target nucleic acid (19401) may bind with a programmable nuclease-split enzyme fusion protein comprising a dCas protein, an “S” subunit of a split HRP protein, and a first guide nucleic acid. In some embodiments, the dCas protein is not capable of cis or trans cleavage. The target nucleic acid (19402) may also bind with another programmable nuclease-split enzyme fusion protein (19406) comprising a dCas protein (19407), an “L” subunit of a split HRP protein (19408), and a second guide nucleic acid (19409). The first guide nucleic acid (19402) may be configured to target a first portion (19410) of the target nucleic sequence. The second guide nucleic acid (19409) may be configured to target a second portion (19411) of the target nucleic acid sequence, wherein the second portion is sufficiently close to the first portion such that the “S” subunit of the HRP (19404) and the “L” subunit of the HRP (19408) can dock together to form an activated HRP protein (19412). The activated HRP protein (19412) then may act on an enzyme substrate (19413) in solution to induce a color change.

[0369] In some embodiments, the programmable nuclease-split enzyme fusion protein may comprise any one of the programmable nucleases disclosed herein. In some embodiments, the target nucleic acid may be any one of the target nucleic acids disclosed herein. In some embodiments, the guide nucleic acids may comprise any of the guide nucleic acids disclosed herein. In some embodiments, the programmable nuclease-split enzyme fusion protein may comprise any of the enzymes described herein in split form.

[0370] FIG. 36 shows a schematic of a signal amplification strategy involving downstream activation of an endonuclease (e.g., NucC, Csm6, etc.) following activation of a programmable nuclease (e.g., any of the programmable nucleases described herein) in response to a target nucleic acid. The programmable nuclease may be configured to cleave a first reporter comprising a first nucleic acid section and a second nucleic acid section. The first nucleic acid section may act as an activator for the endonuclease when separated from the second nucleic acid section. The second nucleic acid section may act as a blocker nucleic acid and prevent the first nucleic acid from binding to and/or activating the endonuclease. Presence of the target nucleic acid may activate the programmable nuclease, which may cleave the first nucleic acid section from the second nucleic acid section, thereby freeing the first nucleic acid section to bind to and activate the endonuclease. The activated endonuclease may then cleave the second reporter and release a detection moiety where may be detected as described herein.

[0371] In one aspect, the present disclosure provides a composition for detecting a target nucleic acid, in which the composition comprises a programmable nuclease, a guide nucleic acid, a first reporter, an enzyme, and a second reporter. In some embodiments, (a) the guide nucleic acid is configured to bind to the target nucleic acid; (b) the programmable nuclease is effective to cleave the first reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (c) the cleavage of the first reporter is effective to separate a first nucleic acid section from a second nucleic acid section thereof; (d) the first nucleic acid section is effective to activate the enzyme; and (e) the activated enzyme is effective to cleave the second reporter to produce a detectable product comprising a detection moiety. In some embodiments, the programmable nuclease is a Cas protein or fragment thereof. In some embodiments, the programmable nuclease is a Casl3. In some embodiments, the programmable nuclease is a Casl3a. In some embodiments, the programmable nuclease is a Casl3c. In some embodiments, the programmable nuclease is thermostable at elevated temperatures. In some embodiments, the enzyme is an endonuclease and the second reporter comprises a polynucleotide substrate of the enzyme. In some embodiments, the endonuclease is a NucC endonuclease. In some embodiments, the first nucleic acid section comprises adenosine residues (such as cyclic adenylate) effective to activate the NucC. In some embodiments, the second nucleic acid section comprises RNA residues, optionally wherein the RNA residues comprise a plurality of uracil residues (e.g., at least 4, 5, 6, 7, 8, 9 or 10 RNA residues, such as uracils). In some embodiments, the second nucleic acid section comprises DNA residues, optionally wherein the DNA residues comprise a plurality of thymine residues (e.g., at least 4, 5, 6, 7, 8, 9 or 10 DNA residues, such as thymines). In some embodiments, (a) the second reporter comprises a fluorescent label and a quencher, and (b) cleavage of the second reporter by the activated enzyme is effective to separate the fluorescent label from the quencher.

[0372] In some embodiments, such as when the endonuclease is NucC, the first reporter may comprise a first nucleic acid section comprising a plurality of adenosine residues and a second nucleic acid section comprising a plurality of uracil residues. In some embodiments, one or more of the plurality of adenosine residues may be modified (e.g., fluoro-adenosine, etc.) as described herein. When the target nucleic acid is a target RNA, the programmable nuclease may comprise a Cas 13 nuclease which, when activated by the target RNA, may preferentially cleave the uracil residues off of the first reporter and release the first nucleic acid section comprising the adenosine residues. NucC is naturally activated by cyclic adenylate (cA3) and cleaves doublestranded DNA, thus in an exemplary embodiment the plurality of adenosine residues may be at least two or three adenosine residues and the second reporter may comprise a detection moiety (e.g., a fluorophore) and a nucleic acid comprising double-stranded DNA. In some embodiments, the second reporter may comprise a detection moiety (e.g., a fluorophore) and a quencher moiety. Cleavage of the second reporter may separate the detection moiety from the quencher moiety and generate a detectable signal as described herein.

[0373] In some embodiments, such as when the endonuclease is NucC, the composition for detecting the target nucleic acid and the target nucleic acid are reacted and analyzed at room temperature. In some embodiments, the composition for detecting the target nucleic acid and the target nucleic acid are reacted and analyzed at an elevated temperature. In some embodiments, the elevated temperature is a temperature selected from: 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, or 65 °C.

[0374] In various aspects, provided herein are methods for detecting a target nucleic acid in a sample, the methods comprising contacting the sample with a composition, device, or system described herein. In an embodiment, the method comprises (a) contacting the sample with composition described herein; (b) cleaving the first reporter with the programmable nuclease in response to formation of the complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid, thereby releasing the first nucleic acid section; (c) activating the enzyme with the first nucleic acid section; (d) cleaving the second reporter with the activated enzyme, thereby producing the detectable product comprising the detection moiety; and; and (e) detecting the detection moiety, thereby detecting the presence of the target nucleic acid in the sample.

Enzyme-based reporters for DETECTR signal enhancement

[0375] Described herein are various compositions, devices, and methods for detecting a target nucleic acid, utilizing a DETECTR assay as described herein. In some embodiments, the DETECTR assay utilizes reporters comprising one or more detection moieties. One or more of the detection moieties may comprise an enzyme configured for assay signal enhancement as described herein. In some embodiments, the enzyme is horseradish peroxidase (HRP). In some embodiments, enzyme-modified reporters may be immobilized to a surface and configured to release the enzyme upon cleavage of a nucleic acid of the reporter by an activated programmable nuclease-guide complex bound to a target nucleic acid as described herein. In some embodiments, one or more enzymes are conjugated to a linker, the comprises one or more nucleic acid sections, and the linker comprises a functionality for immobilization to a support (but is not immobilized to the support at the time of cleavage by an activated programmable nuclease). In some embodiments, the reporters may comprise two or more enzymes configured for signal enhancement. In some embodiments, a reporter comprising two or more enzymes configured for signal enhancement is referred to as a poly-enzyme reporter. In some embodiments, each of the two or more enzymes configured for signal enhancement are HRP. In some embodiments, wherein the two or more enzymes configured for signal enhancement are each HRP, the reporter is referred to as a poly-HRP reporter.

[0376] Described herein are various devices for detecting a target nucleic acid, comprising: a sample interface configured to receive a sample comprising the target nucleic acid; a first reaction chamber in fluid communication with the sample interface, wherein the first reaction chamber comprises a plurality of reporters, each reporter of the plurality comprising two or more enzymes; and a surface of the first reaction chamber, wherein each reporter of the plurality of reporters is immobilized to the surface of the first reaction chamber via a nucleic acid (which may comprise or be a part of a linker or tether as described herein), and a second reaction chamber in fluid communication with the first reaction chamber, wherein the second reaction chamber comprises an enzyme substrate, and wherein cleaving the nucleic acid of the reporter by a programmable nuclease complexed with a guide nucleic acid, the guide nucleic acid being configured to bind to the target nucleic acid, may release the two or more enzymes into solution, and wherein each enzyme of the two or more enzymes may be contacted with the enzyme substrate in the second reaction chamber to generate a detectable and amplified signal indicative of a presence or absence of the target nucleic acid in the sample. In some embodiments, each enzyme of the two or more enzymes comprises HRP. In some embodiments, the reporter comprises a detection moiety comprising a streptavidin conjugated to the two or more enzymes. In some embodiments, the nucleic acid of the reporter may be immobilized to the surface via a functionality (e.g., a biotin, an acrydite, an amine, or the like) as described herein. In some embodiments, the surface of the first reaction chamber may befunctionalized with a corresponding functionality (e.g., a hydrogel, carboxyl group, NHS groups, or like) as described herein. In some embodiments, the tether comprises a segment(s) of cleavable DNA, cleavable RNA, or a combination thereof. In some embodiments, the tether comprises a segment(s) of non- cleavable DNA, non-cleavable RNA, or a combination thereof. In some embodiments, the enzyme comprises HRP and the enzyme substrate comprises 3, 3', 5, 5'-tetramethylbenzidine (TMB). In some embodiments, the surface of the first reaction chamber comprises the inner wall of the first reaction chamber. In some embodiments, the surface of the first reaction chamber comprises a surface of a bead, wherein the bead is contained within the first reaction chamber. In some embodiments, the bead is a magnetic bead.

[0377] Described herein are various devices and methods for detecting a target nucleic acid, utilizing a DETECTR assay as described herein. In some embodiments, the DETECTR assay utilizes reporters comprising a detection moiety comprising an enzyme configured for assay signal enhancement as described herein. Non-limiting examples of enzymes for assay signal enhancement include horseradish peroxidase (HRP), alkaline phosphatase, beta-galactosidase, catacholase, invertase, glucose, beta-lactamase, luciferase, and complexes comprising two or more thereof (e.g., two or more of the same enzyme, or two or more different enzymes). In some embodiments, the enzyme is HRP. In some embodiments, the enzyme is a poly-HRP. In some embodiments, enzyme-modified reporters may be immobilized to a surface and configured to release the enzyme upon cleavage of a nucleic acid of the reporter by an activated programmable nuclease-guide complex bound to a target nucleic acid as described herein. In some embodiments, the reporters comprise two or more enzymes configured for signal enhancement. In some embodiments, the incorporation of the two or more enzymes configured for signal enhancement may provide for assay signal enhancement upon a single cleavage event of the reporter induced by the activated programmable nuclease. In some embodiments, the two or more enzymes comprise a group, wherein the group further comprises a protein. In some embodiments, the two or more enzymes comprise a group, wherein the group further comprises a protein, the protein comprising streptavidin (SA). In some embodiments, the group comprises two or more HRP. In some embodiments, the group comprising two or more enzymes is attached to the nucleic acid of the reporter via a Biotin-Streptavidin interaction (e.g., as shown in FIG. 33E).

[0378] In some embodiments, the reporter comprises a linker (also referred to herein as a tether). In some embodiments, the linker comprises one or more linker-sections. In some embodiments, the one or more linker sections comprises a cleavable section comprising one or more nucleic acids which may be cleaved by an activated programmable nuclease as described herein. In some embodiments, the one or more linker sections comprises cleavable and non-cleavable section, the non-cleavable section comprising one or more nucleic acids, carbon spacers, or other molecules which may not be cleaved by an activated programmable nuclease. For example, FIG. 33B shows an exemplary reporter comprising a linker comprising a carbon spacer, a first linker section comprising a first plurality of thymines (T12), a second linker section comprising a plurality of uracils (UUUUU), and a third linker section comprising a second plurality of thymines (T12). The carbon spacer may be uncleavable by the programmable nuclease. When the programmable nuclease is a DNA-targeting programmable nuclease, the first and second pluralities of thymines may be cleavable linker sections and the plurality of uracils may be a non-cleavable linker section. When the programmable nuclease is an RNA-targeting programmable nuclease, the first and second pluralities of thymines may be non-cleavable linker sections and the plurality of uracils may be a cleavable linker section.

[0379] In some embodiments, the linker comprises one or more nucleic acid sections, and the only nucleic acids in the linker consist of DNA. In some embodiments, the linker comprises one or more nucleic acid sections, and the only nucleic acids in the linker consist of RNA. In some embodiments, the linker comprises one or more sections of DNA and one or more sections of RNA (e.g., one section of each, one section of RNA between two sections of DNA, one section of DNA between two sections of RNA, or greater numbers of each). In linkers comprising DNA and RNA, the DNA and RNA may be linked (e.g., covalently linked) directly to one another, or separated by one or more intervening molecules or linkages to which both the DNA and RNA are linked (e.g., covalently linked). The type, length, and arrangement of the one or more nucleic acid sections can be selected based on the programmable nuclease(s) to be used. In some embodiments, one or more DNA sections comprise one or more poly-T sections consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or more contiguous thymine nucleotides. In some embodiments, the poly-T section consists of 12 contiguous thymine nucleotides (T12). In some embodiments, the poly-T section consists of 20 contiguous thymine nucleotides (T20). In a linker comprising two or more poly-T sections, the poly-T sections may be of the same or different lengths (e.g., two of T12, a T12 and a T20, etc.). In some embodiments, one or more RNA sections comprise one or more poly-U sections consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or more contiguous uracil nucleotides. In some embodiments, the poly-U section consists of 5 contiguous uracil nucleotides (UUUUU). In a linker comprising two or more poly-U sections, the poly-U sections may be of the same or different lengths (e.g., two of UUUUU, a UUUUU and a U8, etc.) In some embodiments, the RNA section is about or less than about 20, 15, 10, or 5 nucleotides in length. In some embodiments, a linker includes only one RNA section of fewer than 20 nucleotides (e.g., about 5 nucleotides) and is adjusted to a desired length by the inclusion of one or more DNA sections (e.g., at one end or at each end). In some embodiments, the desired length adjustment is the length of 24 DNA nucleotides (e.g., as in two T12 sections), 40 nucleotides (e.g., as in two T20 sections), a length intermediate to these (e.g., a T12 and a T20), or another length as specified herein. In some embodiments, the length is adjusted to a combined length of at least about 20, 30, 40, 50, 75, 100, or 200 nucleotides. In some embodiments, the length is adjusted to a combined length of about 10 to 200 nucleotides, 20 to 100 nucleotides, or 25 to 75 nucleotides. In some embodiments, the length is adjusted to a combined length of at least about 20, 30, 40, or 50 nucleotides. In some embodiments, the length is adjusted to a combined length of at least about 30 nucleotides. In some embodiments, the one or more nucleic acid sections are single-stranded.

[0380] In some embodiments, the one or more linker sections comprise at least one hydrocarbon chain. In some embodiments, the one or more hydrocarbon chains comprise a linear chain of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 75 or more carbons. In some embodiments, a hydrocarbon chain comprises one or more units (e.g., 2, 3, 4, 5, or more units) of an 18-carbon chain. In some embodiments the hydrocarbon chain is a chain of 4 carbons (C4H8). In some embodiments the hydrocarbon chain is a chain of 8 carbons (CsHie). In some embodiments, the hydrocarbon chain is a chain of at least 36 carbons. In some embodiments, the hydrocarbon chain is a chain of at least 54-carbon chain. In a linker comprising two or more hydrocarbon chains, the hydrocarbon chains can be chains of the same or different lengths (e.g., two chains of 4 carbons, a chain of 4 carbons and a chain of 8 carbons, a chain of 36 carbons and a chain of 54 carbons, etc.). In some embodiments of a linker comprising two hydrocarbon chains separated by one or more nucleic acid sections, the two hydrocarbon chains have a combine length of about or at least about 30, 50, 75, 100, or more carbons. In some embodiments, the combined chain length is at least 75 carbons. In some embodiments, a hydrocarbon chain is located at one or more of (i) between a nucleic acid section of a linker and a solid support to which the linker is attached, (ii) between a nucleic acid section of a linker and an enzyme for signal enhancement (e.g., an HRP or poly- HRP), or (iii) between two nucleic acid sections of the same or different type. In some embodiments, the linker comprises a single nucleic acid section (e.g., an RNA section) with a hydrocarbon chain at both ends, one joining the nucleic acid section to the support at one end, and the other joining the nucleic acid section to the enzyme at the other end. In some embodiments, a linker includes only one RNA section of fewer than 20 nucleotides (e.g., about 5 nucleotides) and is adjusted to a desired length by the inclusion of one or more hydrocarbon chains (e.g., at one end or at each end). In some embodiments one or more hydrocarbon chains and one or more DNA sections are used to adjust the linker length.

[0381] In some embodiments, the reporter may comprise a functionality for binding to a solid support as described herein. In some embodiments, the reporter may comprise a functionality for binding to a surface of a solid support as described herein. In some embodiments, the surface may comprise a surface of a chamber, the surface of a particle, a bead, a polymer matrix, or other scaffold as described herein. In some embodiments, the functionality may comprise a biotin, an acrydite, an amine, or the like, or a combination thereof, as described herein. FIGS. 33A-33E show various non-limiting examples of reporters, each comprising at least one enzyme for signal enhancement of the DETECTR assay. FIG. 33A shows a reporter, repl61, comprising an HRP enzyme for signal enhancement, a T20 linker segment, and a biotin for immobilization to a support, as described herein. FIG. 33B shows a reporter, rep 194, comprising an HRP enzyme for signal enhancement, a T12 linker segment, a UUUUU linker segment, a second T12 linker segment and a biotin functionality for immobilization to a support. FIG. 33C shows a reporter, rep 188, comprising an HRP enzyme for signal enhancement, a T20 linker segment and an acrydite functionality for immobilization to a support. FIG. 33D shows a reporter, rep 190, comprising an HRP enzyme for signal enhancement, a T20 linker segment, a carbon spacer, and an amine functionality for immobilization to a support. FIG. 33E shows a reporter, rep 197, comprising an SA Poly-HRP for signal enhancement, a first carbon spacer, a T20 linker segment, a second carbon spacer, and an amine functionality for immobilization to a support.

[0382] Table 4 describes reporters as shown in FIG. 33.

[0383] FIG. 34 shows a non-limiting example of a reporter comprising a streptavidin-based poly-HRP detection moiety coupled to a carboxyl-functionalized magnetic bead. The multiple HRP units released per trans-cleavage event is designed to increase signal intensity indicative of activation of the programmable nuclease. In general, the term “poly-HRP” is used to refer to a plurality of HRP enzymes complexed to one another by interactions with one or more complexing molecules. Typically, the complexing molecule is streptavidin. Illustrations of poly- HRP, such as in the figures, are simplified for illustrative purposes. The term “poly-HRP” does not require any particular stoichiometry or arrangement of molecules within the complex. A variety of suitable poly-HRP complexes are commercially available.

[0384] In some embodiments, the reporter may be immobilized onto a solid support as described herein. In some embodiments, the reporter may be a poly-enzyme reporter. In some embodiments, one or more of the poly-enzymes of the reporter may be HRP. In some embodiments, the poly-HRP reporter may be immobilized to a bead functionalized with a functionality such as streptavidin. A variety of suitable beads are available, non-limiting examples of which include DYNABEADS MYONE carboxylic acid magnetic beads (1pm diameter), 1 m PIERCE streptavidin magnetic beads, 4.5pm DYNABEADS epoxy magnetic beads, 2.7pm DYNABEADS epoxy magnetic beads, and 1pm PIERCE NHS-activated magnetic beads. In some embodiments, during reporter immobilization onto beads functionalized with streptavidin, the reporter may be present at a concentration of about 0.1 nM to about 1,000 nM, for example within a range of about 0.1 nM to about 10 nM. In some embodiments, during reporter immobilization, the reporter is present at a concentration of about 0.1 nM to about 1 nM, about 0.1 nM to about 10 nM, about 0.1 nM to about 100 nM, about 0.1 nM to about 250 nM, about 0.1 nM to about 500 nM, about 0.1 nM to about 1,000 nM, about 1 nM to about 10 nM, about 1 nM to about 100 nM, about 1 nM to about 250 nM, about 1 nM to about 500 nM, about 1 nM to about 1,000 nM, about 10 nM to about 100 nM, about 10 nM to about 250 nM, about 10 nM to about 500 nM, about 10 nM to about 1,000 nM, about 100 nM to about 250 nM, about 100 nM to about 500 nM, about 100 nM to about 1,000 nM, about 250 nM to about 500 nM, about 250 nM to about 1,000 nM, or about 500 nM to about 1,000 nM. In some embodiments, during reporter immobilization, the reporter is present at a concentration of about 0.1 nM, about 1 nM, about 10 nM, about 100 nM, about 250 nM, about 500 nM, or about 1,000 nM. In some embodiments, during reporter immobilization, the reporter is present at a concentration of at least about 0.1 nM, about 1 nM, about 10 nM, about 100 nM, about 250 nM, or about 500 nM. In some embodiments, during reporter immobilization, the reporter is present at a concentration of at most about 1 nM, about 10 nM, about 100 nM, about 250 nM, about 500 nM, or about 1,000 nM.

[0385] In some embodiments, reporters configured for HRP-reporter-based programmable nuclease-based detection (e.g., DETECTR) assays may be immobilized onto a solid support as described herein. In some embodiments, one or more HRP-conjugated reporters may be immobilized onto a solid support comprising a resin. In some embodiments, one or more HRP- conjugated reporters may be immobilized onto a solid support comprising an activated resin. In some embodiments, one or more HRP-conjugated reporters may be immobilized onto a solid support comprising a resin activated with N-Hydroxysuccinimide (NHS) functionalities. In some embodiments, one or more HRP-conjugated reporters are immobilized onto a solid support comprising a resin activated with NHS functionalities, wherein the reporters comprise corresponding amine functionalities as described herein. In some embodiments, one or more HRP-conjugated reporters are immobilized onto a solid support comprising a resin activated with NHS functionalities, wherein the reporters comprise a protein, for example an enzyme such as HRP. In some embodiments, one or more reporters are configured for HRP-based DETECTR assays and may be immobilized onto one or more solid support comprising a resin activated with NHS functionalities, wherein the reporters comprise an HRP and an amine functionality connected by a linker as described herein. In some embodiments, the resin may comprise agarose.

[0386] FIGS. 35A-35F show a schematic and results for a programmable nuclease-based detection assay utilizing NHS-activated resin as a solid support for an HRP-based reporter for signal enhancement (rep 190). FIG. 35A shows a schematic describing the immobilization of an HRP-reporter and an HRP-based detection assay. In FIG. 35A a reporter, 20001, comprising HRP and an amine functionality is immobilized onto the NHS activated resin packed into a 0.2 mL column 20002. The column may then be washed and exposed to programmable-nuclease based detection (e.g., DETECTR) reagents 20004 comprising a programmable nuclease (e.g., SEQ ID NO: 34) complexed with a guide nucleic acid, as described herein. In the last step of FIG. 35A, the eluate, which contains cleaved reporter fragments comprising HRP 20005 when the target is present, may be mixed with a substrate (e.g., TMB) for generation of a detectable signal (e.g., a color change which can be detected by absorbance measurements) as described herein.

[0387] FIG. 35B shows assay results in the form of absorbance (O.D. at 650 nm) versus time, for the assay eluate comprising a substrate and the cleaved HRP -based reporter, rep 190, as described herein. FIGS. 35C-35E show measurements of the eluates from a first wash, a second wash and a third wash, respectively, of the resin packed column. In FIG. 35B, the two top traces show results for the assay carried out with 1 nM target, and the two bottom traces show results for the NFW condition. In FIG. 35C, the NFW condition is represented by the two middle traces. In FIGS. 35D-35E, traces for the two conditions overlapped. Additional details concerning the illustrative assays are described in the examples below.

[0388] In some embodiments, reporters configured for poly enzyme reporter-based DETECTR assays may be immobilized onto solid-supports. In some embodiments, reporters configured for poly HRP reporter-based DETECTR assays may be immobilized onto solid-supports comprising beads. In some embodiments, the beads are functionalized with streptavidin or NHS. In some embodiments, the poly HRP -reporters are immobilized onto the beads at a concentration of about 0.01 nM to about 0.5 pM, such as about 0.1 nM to about 250 nM, about 0.5 nM to about 100 nM, or about 1 nM to about 50 nM. In some embodiments, poly-HRP reporter molecules are immobilized at a concentration of about 10 nM. In some embodiments, mono-HRP reporter molecules are immobilized at a concentration of about 1 nM to about 50 pM, about 10 nM to about 25 pM, about 50 nM to about 10 pM, or about 100 nM to about 5 pM. In some embodiments, mono-HRP reporter molecules are immobilized at a concentration of about 1 pM. In some embodiments, HRP -DETECTR reagents are added to the beads functionalized with reporters, wherein the % solid content (e.g., concentration) of the beads is about 1 to about 50 mg/mL (e.g., about 5 to about 25, or about 10 mg/mL). In some embodiments, the reporter- functionalized beads are used at a concentration of about 10 mg/mL.

[0389] In various aspects, the present disclosure provides methods of using the compositions, devices, and systems disclosed herein to detect a target nucleic acid. In some embodiments, the method comprises (a) contacting the sample with a composition disclosed herein to produce a reaction fluid, wherein (i) the linker is conjugated to the support, (ii) the composition further comprises a guide nucleic acid configured to bind to the target nucleic acid, and (iii) the programmable nuclease cleaves the linker in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (b) separating the reaction fluid from the support; (c) contacting the separated reaction fluid with a substrate of the one or more enzymes, wherein presence of the one or more enzymes in the fluid produces a detectable signal from the enzyme substrate; and (d) detecting the detectable signal. In some embodiments, the method comprises (a) contacting the sample with a composition disclosed herein to produce a reaction fluid, wherein (i) the composition further comprises a programmable nuclease and a guide nucleic acid, (ii) the guide nucleic acid is configured to bind to the target nucleic acid, and (iii) the programmable nuclease cleaves the linker in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (b) contacting the reaction fluid with a plurality of beads, wherein each bead reacts with a plurality of the functionalities, thereby immobilizing the functionalities to the beads; (c) separating the reaction fluid from the beads; (d) contacting the separated reaction fluid with a substrate of the one or more enzymes, wherein presence of the one or more enzymes in the fluid produces a detectable signal from the enzyme substrate; and (e) detecting the detectable signal. [0390] In some embodiments, the method further comprises concentrating the one or more enzymes prior to contacting the one or more enzymes with the substrate, wherein the concentrating comprises separating the one or more enzymes from at least a portion of the separated reaction fluid. For example, the enzyme may be captured to a support, such as a polymer matrix. The enzyme may be retained on the polymer matrix having a volume smaller than the reaction fluid applied to the matrix, thereby concentrating the enzyme. The enzyme may be reacted with a substrate while complexed to the matrix, or may be eluted in a fluid for subsequent reaction with the substrate. In embodiments where the enzyme is reacted to the substrate while complexed to the matrix, the substrate solutions comprising the substrate may be added serially (e.g., 2, 3, 4, 5, or more times), which may then be combined to detect enzymatic activity. In some embodiments, the enzyme comprises a horseradish peroxidase (HRP). [0391] In some cases, the reporters are immobilized to the surface by attachment to the non- naturally occurring guide nucleic acid, a linkage immobilizing the guide nucleic acid to a surface, or both. In some instances, at least one of the reporters is immobilized to the surface by attachment to an end of the non-naturally occurring guide nucleic acid that is distal to the linkage. For example, at least one of the reporters can be immobilized by attaching to the 3’ end of a spacer region of the guide nucleic acid. In some cases, at least one of the reporters is immobilized to the surface by attachment to the linkage. For example, at least one of the reporters can be immobilized by attaching to a tether. In some embodiments, a plurality of reporters (e.g., at least 2, 3, 4, 5, 10, 15, or more) are attached to the linkage. In some embodiments, reporters are attached to both the linkage and an end of the guide nucleic acid. In some cases, a reporter comprises a detection moiety. In some cases, a reporter comprises an enzyme, such as HRP, as shown in FIG. 42. Molecules comprising a guide nucleic acid joined to a reporter are also referred to herein as “gREPs”.

Methods of Making Polymer Matrices with Immobilized Reporters

[0392] FIG. 25 shows an exemplary polymer immobilization matrix (14901) comprising a plurality of immobilized DETECTR reaction components. The DETECTR reaction components may comprise one or more reporters, one or more programmable nucleases, and/or one or more guide nucleic acids. In some embodiments, the polymer matrix may comprise a hydrogel. In the exemplary embodiment shown in FIG. 25, a plurality of reporters (14902) may be immobilized within a hydrogel (14901) matrix. In some embodiments, methods of immobilizing a reporter (14902) and/or other DETECTR reaction component may comprise (a) providing a polymerizable composition comprising: (i) a plurality of oligomers, (ii) a plurality of polymerizable (e.g., functionalized) oligomers, (iii) a set of polymerizable (e.g., functionalized) reporters (and/or other DETECTR reaction components), and (iv) a set of polymerization initiators; and (b) initiating the polymerization reaction by providing an initiation stimulus. [0393] Co-polymerization of the reporter into the hydrogel may result in a higher density of reporter/unit volume or reporter/unit area than other immobilization methods utilizing surface immobilization (e.g., onto beads). Co-polymerization of the reporter into the hydrogel may result in less undesired release of the reporter (e.g., during an assay, a measurement, or on the shelf), and thus may cause less background signal, than other immobilization strategies (e.g., conjugation to a pre-formed hydrogel, bead, etc.). In at least some instances this may be due to better incorporation of reporters into the hydrogel as a co-polymer and fewer “free” reporter molecules retained on the hydrogel via non-covalent interactions or non-specific binding interactions.

[0394] In some embodiments, the plurality of oligomers and the plurality of polymerizable oligomers may comprise an irregular or non-uniform mixture. The irregularity of the mixture of polymerizable oligomers and unfunctionalized oligomers may allow pores to form within the hydrogel (i.e., the unfunctionalized oligomers may act as a porogen). For example, the irregular mixture of oligomers may result in phase separation during polymerization that allows for the generation of pores of sufficient size for programmable nucleases to diffuse into the hydrogel and access internal reporter molecules. The relative percentages and/or molecular weights of the oligomers may be varied to vary the pore size of the hydrogel. For example, pore size may be tailored to increase the diffusion coefficient of the programmable nucleases.

[0395] In some embodiments, the functional groups attached to the reporters may be selected to preferentially incorporate the reporters into the hydrogel matrix via covalent binding at the functional group versus other locations along the nucleic acid of the reporter. In some embodiments, the functional groups attached to the reporters may be selected to favorably transfer free radicals from the functionalized ends of polymerizable oligomers to the functional group on the end of the reporter (e.g., 5’ end), thereby forming a covalent bond and immobilizing the reporter rather than destroying other parts of the reporter molecules.

[0396] In some embodiments, the polymerizable composition may further comprise one or more polymerizable nucleic acids. In some embodiments, the polymerizable nucleic acids may comprise guide nucleic acids (e.g., guide nucleic acids 15003a, 15003b, or 15003c shown in FIGS. 26A-26B). In some embodiments, the polymerizable nucleic acids may comprise linker or tether nucleic acids. In some embodiments, the polymerizable nucleic acids may be configured to bind to a programmable nuclease (e.g., programmable nuclease 15004a, 15004b, or 15004c shown in FIGS. 26A-26B). In some embodiments, the programmable nuclease may be immobilized in the polymer matrix.

[0397] In some embodiments, the oligomers may form a polymer matrix comprising a hydrogel. In some embodiments, the oligomers may comprise poly(ethylene glycol) (PEG), poly(siloxane), poly(hydroxyethyl acrylate, poly(acrylic acid), poly(vinyl alcohol), poly(butyl acrylate), poly(2- ethylhexyl acrylate), poly(methyl acrylate), poly(ethyl acrylate), poly(acrylonitrile), poly(methyl methacrylate), poly(acrylamide), poly(TMPTA methacrylate), chitosan, alginate, or the like, or any combination thereof. One of ordinary skill in the art will recognize that the oligomers may comprise any oligomer or mix of oligomers capable of forming a hydrogel.

[0398] In some embodiments, the oligomers may comprise polar monomers, nonpolar monomers, protic monomers, aprotic monomers, solvophobic monomers, or solvophillic monomers, or any combination thereof.

[0399] In some embodiments, the oligomers may comprise a linear topology, branched topology, star topology, dendritic topology, hyperbranched topology, bottlebrush topology, ring topology, catenated topology, or any combination thereof. In some embodiments, the oligomers may comprise 3-armed topology, 4-armed topology, 5-armed topology, 6-armed topology, 7-armed topology, 8-armed topology, 9-armed topology, or 10-armed topology.

[0400] In some embodiments, the oligomers may comprise a homopolymer, a copolymer, a random copolymer, a block copolymer, an alternative copolymer, a copolymer with regular repeating units, or any combination thereof.

[0401] In some embodiments, the oligomers may comprise 1 type of monomer, 2 types of monomers, 3 types of monomers, 4 types of monomers, 5 types of monomers, 6 types of monomers, 7 types of monomers, 8 types of monomers, 9 types of monomers, or 10 types of monomers. [0402] The polymerizable oligomers may comprise any of the oligomers described herein. In some embodiments, the polymerizable oligomers may comprise one or more functional groups. In some embodiments, the functional group may comprise an acrylate group, N- hydroxysuccinimide ester group, thiol group, carboxyl group, azide group, alkyne group, an alkene group, or any combination thereof. One of ordinary skill in the art will recognize that a variety of functional groups may be used to functionalize oligomers into polymerizable oligomers depending on the desired properties of the polymerizable oligomers.

[0403] In some embodiments, the polymerizable oligomers may form a polymer matrix comprising a hydrogel. In some embodiments, the polymerizable oligomers may comprise PEG, poly(siloxane), poly(hydroxyethyl acrylate, poly(acrylic acid), poly(vinyl alcohol), or any combination thereof. One of ordinary skill in the art will recognize that the set of polymerizable oligomers may comprise any polymer capable of forming a hydrogel.

[0404] In some embodiments, the set of polymerizable oligomers comprises polar monomers, nonpolar monomers, protic monomers, aprotic monomers, solvophobic monomers, or solvophillic monomers.

[0405] In some embodiments, the set of polymerizable oligomers comprises a linear topology, branched topology, star topology, dendritic topology, hyperbranched topology, bottlebrush topology, ring topology, catenated topology, or any combination thereof. In some embodiments, the set of polymerizable oligomers comprises 3-armed topology, 4-armed topology, 5-armed topology, 6-armed topology, 7-armed topology, 8-armed topology, 9-armed topology, or 10- armed topology.

[0406] In some embodiments, the set of polymerizable oligomers comprises a homopolymer, a copolymer, a random copolymer, a block copolymer, an alternative copolymer, a copolymer with regular repeating units, or any combination thereof.

[0407] In some embodiments, the set of polymerizable oligomers comprises 1 type of monomer, 2 types of monomers, 3 types of monomers, 4 types of monomers, 5 types of monomers, 6 types of monomers, 7 types of monomers, 8 types of monomers, 9 types of monomers, or 10 types of monomers.

[0408] In some embodiments, the polymerizable composition may comprise a mix of unfunctionalized or unmodified oligomers and polymerizable oligomers as described herein. In some embodiments, the unfunctionalized or unmodified oligomers may act as porogens to generate pores within the polymer matrix.

[0409] The polymerizable reporters may comprise any of the reporters described herein. In some embodiments, the set of polymerizable reporters may comprise one or more functional groups. In some embodiments, the functional group may comprise a single stranded nucleic acid, a double stranded nucleic acid, an acrydite group, a 5’ thiol modifier, a 3’ thiol modifier, an amine group, a I-Linker™ group, methacryl group, or any combination thereof. One of ordinary skill in the art will recognize that a variety of functional groups may be used with the set of polymerizable reporters depending on the desired properties of the polymerizable reporters.

[0410] In some embodiments, the set of initiators may comprise one or more photoinitiators or thermal initiators. In some embodiments, the set of initiators may comprise cationic initiators, anionic initiators, or radical initiators.

[0411] In some embodiments, the initiation stimulus is UV light. In some embodiments, the initiation stimulus is UV light through a photomask. In some embodiments, the initiation stimulus is heat.

[0412] In some embodiments, the hydrogel may comprise a circular cross-sectional shape, a rectangular cross-sectional shape, a star cross-sectional shape, a dollop shape, an amorphous shape, or any shape of interest, or any combination thereof (e.g., as shown in FIGS. 26A-26B). [0413] In some embodiments, a mask may be used to shape the initiation stimulus deposition on the polymerizable components (e.g., oligomers, etc.) and thereby shape the resulting polymer matrix. In some embodiments, the mask may comprise a circular shape, a rectangular shape, a star shape, a dollop shape, an amorphous shape, or any shape of interest, or any combination thereof.

Hydrogel Compositions with Immobilized Reporters

[0414] FIG. 25 and FIGS. 26A-26B show examples of hydrogels comprising immobilized reporters. In some aspects, provided herein are compositions comprising a hydrogel (14901) comprising (a) a network of covalently bound oligomers (14903) and (b) immobilized reporters (14902) covalently bound to said network (14903).

[0415] FIG. 25 shows an exemplary hydrogel (14901) comprising a plurality of reporters (14902) co-polymerized with a plurality of oligomers (modified and unmodified) to form a network or matrix (14903). FIGS. 26A-26B show exemplary multiplexing schemes utilizing hydrogel-immobilized reporters which may be implemented in any of the devices or methods described herein. Multiplexing could be distinguished through spatial multiplexing by knowing the location of hydrogels functionalized with each guide nucleic acid and/or through shape, by using different shapes of hydrogel for each guide nucleic acid.

[0416] In some embodiments, the composition may comprise a hydrogel (15001) comprising (a) a polymer network comprising covalently bound oligomers co-polymerized with reporters (15002) to covalently bind and immobilize the reporters to said network, and (b) immobilized programmable nuclease complexes covalently bound to said network (e.g., via co-polymerization or after reporter-immobilized polymer formation), wherein said programmable nuclease complexes may comprise a programmable nuclease (15004) and a guide nucleic acid (15003). In some embodiments, the guide nucleic acid (15003) and/or the programmable nuclease (15004) may be immobilized to or in the hydrogel as described herein (e.g., during or after formation of the hydrogel).

[0417] In some embodiments, the network of covalently bound oligomers may comprise a network formed by polymerizing one or more PEG species. In some embodiments, the network of covalently bound oligomers may comprise a network formed by polymerizing PEG comprising acrylate functional groups. In some embodiments, the acrylate functional groups may be PEG end groups. In some embodiments, the network may be formed by polymerizing PEG comprising acrylate functional groups with unmodified PEG. The molecular weight of the acrylate-modified PEG (e.g., PEG-diacrylate) and the unmodified PEG may be the same or different.

[0418] In some embodiments, the network of covalently bound oligomers may comprise a network formed from polymerizing one or more PEG species, wherein each PEG species may comprise a linear topology, branched topology, star topology, dendritic topology, hyperbranched topology, bottlebrush topology, ring topology, catenated topology, or any combination thereof. In some embodiments, the network of covalently bound oligomers may comprise a network formed from polymerizing one or more PEG species comprising a 3-armed topology, a 4-armed topology, a 5-armed topology, a 6-armed topology, a 7-armed topology, a 8-armed topology, a 9- armed topology, or a 10-armed topology.

[0419] In some embodiments, the immobilized reporter may comprise a reporter molecule covalently bound to a linker molecule, wherein the linker molecule is covalently bound to the hydrogel (e.g., via co-polymerization with the oligomers as described herein). In some embodiments, the linker molecule may comprise a single stranded nucleic acid, a double stranded nucleic acid, an acrydite group, a 5’ thiol modifier, a 3’ thiol modifier, an amine group, a I-Linker™ group, or any combination thereof. One of ordinary skill in the art will recognize that a variety of linker molecules may be used.

[0420] In some cases, the immobilized guide nucleic acid may comprise a guide nucleic acid covalently bound to a linker molecule, wherein the linker molecule is covalently bound to the hydrogel. In some embodiments, the linker molecule may comprise a single stranded nucleic acid, a double stranded nucleic acid, an acrydite group, a 5’ thiol modifier, a 3’ thiol modifier, an amine group, a I-Linker™ group, or any combination thereof. One of ordinary skill in the art will recognize that a variety of linker molecules may be used.

[0421] In some cases, the immobilized programmable nuclease may comprise a programmable nuclease covalently bound to a linker molecule, wherein the linker molecule is covalently bound to the hydrogel. In some embodiments, the linker molecule may comprise a single stranded nucleic acid, a double stranded nucleic acid, an acrydite group, a 5’ thiol modifier, a 3’ thiol modifier, an amine group, a I-Linker™ group, or any combination thereof. One of ordinary skill in the art will recognize that a variety of linker molecules may be used.

Methods of Using Hydrogels with Immobilized Reporters

[0422] Any of the methods described herein may utilize hydrogels (14901) with immobilized reporters (14902) for target detection assays. In some embodiments, the hydrogel (14901) comprises (a) a network of covalently bound oligomers (14903) and (b) immobilized reporters (14902) covalently bound to said network (14903) as shown in FIG. 25. A solution comprising target nucleic acid molecules and programmable nuclease complexes may be applied to the hydrogel (e.g., by pipetting or flowing over the hydrogel). The immobilized reporters (14902) may comprise a nucleic acid with a sequence cleavable by the programmable complex when the programmable nuclease complex is activated by binding of its associated guide nucleic acid to a target nucleic acid molecule as described herein. When activated, the programmable nuclease complex may trans-cleave the cleavable nucleic acid of the reporter molecule and generates a detectable signal as described herein. For example, the reporter may comprise a detection moiety which may be release upon cleavage of the reporter as described herein. The detection moiety may comprise FAM-biotin which may be captured by one or more capture molecules coupled to a surface of a support (e.g., a lateral flow assay strip) at a detection location as described herein. Detection of the detectable signal generated at the detection location by the detection moiety may indicate the presence of the target nucleic acid in the sample as described herein.

[0423] Any of the multiplexing methods described herein may utilize hydrogels (15001a, 15001b, 15001c, etc.) with immobilized reporters (15002) for multiplexed target detection assays. In some embodiments, each hydrogel (15001a, 150001b, 15001c, etc.) may comprise (a) a polymer network of covalently bound oligomers co-polymerized with reporters (15002) to covalently bind and immobilize the reporters to said network, and (b) one or more immobilized programmable nuclease complexes covalently bound to said network as shown in FIGS. 26A- 26B. Each of the programmable nuclease complexes may comprise a programmable nuclease (15004a, 15004b, 15004c, etc.) and a guide nucleic acid (15003a, 15003b, 15003c, etc.). In some embodiments, the guide nucleic acid (15003) and/or the programmable nuclease (15004) may be immobilized to or in the hydrogel as described herein (e.g., during or after formation of the hydrogel). In some embodiments, multiplexing for a plurality of different targets may be facilitated by providing a plurality different and/or spatially separated hydrogels comprising a plurality of different DETECTR reaction components. In some embodiments, each hydrogel may comprise a different programmable nuclease as described herein. Alternatively, or in combination, each hydrogel may comprise a different guide nucleic acid configured to bind to a different target nucleic acid sequence as described herein. Alternatively, or in combination, each hydrogel may comprise a different reporter as described herein. Alternatively, or in combination, each hydrogel may comprise a different shape and be deposited on a surface of a support at different detection locations. For example, as shown in FIGS. 26A-26B, a first hydrogel (15001a) may comprise a first programmable nuclease (15004a), a first guide nucleic acid (15003a) configured to bind a first target nucleic acid, and a first reporter (15002). A second hydrogel (15001b) may comprise a second programmable nuclease (15004b), a second guide nucleic acid (15003b) configured to bind a second target nucleic acid, and a second reporter (15002). A third hydrogel (15001c) may comprise a third programmable nuclease (15004c), a third guide nucleic acid (15003c) configured to bind a third target nucleic acid, and a third reporter (15002). The programmable nucleases (15004a, 15004b, 15004c) may be the same programmable nuclease or different programmable nuclease. The guide nucleic acids (15003a, 15003b, 15003c) may be different guide nucleic acids configured to recognize different target nucleic acids. The reporters (15002) may be the same reporter or different reporters. A solution comprising one or more target nucleic acid molecules may be applied to the hydrogels (15001a, 15002b, 15003c), e.g., by pipetting or flowing over the hydrogels. The immobilized reporters (15002) may comprise a nucleic acid with a sequence cleavable by the programmable nuclease complexes (15004a, 15004b, 15004c) when the programmable nuclease complexes are activated by binding of their respective guide nucleic acids (15003a, 15003b, 15003c) to their respective target nucleic acid molecules as described herein. When activated, the programmable nuclease complexes may trans-cleave the cleavable nucleic acid of the reporter molecule and generates a detectable signal at the detection location as described herein. For example, the reporter may comprise a detection moiety which may be release upon cleavage of the reporter as described herein. The detection moiety may comprise FAM-biotin as shown in FIG. 26A which may be captured by one or more capture molecules coupled to a surface of a support (e.g., a lateral flow assay strip) at a detection location as described herein. Alternatively, the detection moiety may comprise a quencher moiety which may be released from the hydrogel upon cleavage of the reporter, thereby allowing a fluorescent moiety on the other end of the reporter to fluoresce at the detection location comprising the hydrogel as shown in FIG. 26B. Detection of the detectable signal generated at the detection locations by the detection moiety may indicate the presence of the target nucleic acid in the sample as described herein. Each hydrogel (15001a, 15001b, 15001c) may have a different shape and detection of a target nucleic acid may comprise detecting a particular fluorescent shape corresponding to the hydrogel shape at the detection location.

Devices Comprising Hydrogels with Immobilized Reporters

[0424] Any of the systems or devices described herein may comprise one or more hydrogels with immobilized reporters.

[0425] In some embodiments, the systems and devices described herein may comprise a plurality of hydrogels each comprising reporter molecules (e.g., in order to facilitate multiplexing and/or improve signal). In some embodiments, a first hydrogel may comprise a shape different from a shape of a second hydrogel. In some embodiments, the first hydrogel may comprise a plurality of first reporter molecules different from a plurality of second reporter molecules of the second hydrogel. In some embodiments, the reporters are the same in the first and second hydrogels. In some embodiments, the first hydrogel may comprise a circular shape, a square shape, a star shape, or any other shape distinguishable from a shape of the second hydrogel. In some embodiments, the plurality of first reporter molecules may each comprise a sequence cleavable by a programmable nuclease complex comprising a first programmable nuclease and a first guide nucleic acid. In some embodiments, the plurality of second reporter molecules may each comprise a sequence not cleavable by the first programmable nuclease complex.

[0426] Any of the systems or devices described herein may comprise a plurality of hydrogels each comprising reporter molecules. For example, a first hydrogel may comprise a plurality of first reporter molecules different from a plurality of second reporter molecules of a second hydrogel. In some embodiments, the plurality of first reporter molecules may each comprise a first fluorescent moiety, wherein the first fluorescent moiety is different than second fluorescent moieties of in each of the plurality of second reporter molecules. In some embodiments, the plurality of first reporter molecules may each comprise a sequence cleavable by a first programmable nuclease complex comprising a first programmable nuclease and a first guide nucleic acid. In some embodiments, the plurality of second reporter molecules may each comprise a sequence cleavable by a second programmable nuclease complex comprising a second programmable nuclease and a second guide nucleic acid.

[0427] Any of the systems or devices described herein may comprise at least about 2 hydrogels, at least about 3 hydrogels, at least about 4 hydrogels, at least about 5 hydrogels, at least about 6 hydrogels, at least about 7 hydrogels, at least about 8 hydrogels, at least about 9 hydrogels, at least about 10 hydrogels, at least about 20 hydrogels, at least about 30 hydrogels, at least about 40 hydrogels, at least about 50 hydrogels, at least about 60 hydrogels, at least about 70 hydrogels, at least about 80 hydrogels, at least about 90 hydrogels, at least about 100 hydrogels, at least about 200 hydrogels, at least about 300 hydrogels, at least about 400 hydrogels, at least about 500 hydrogels, at least about 600 hydrogels, at least about 700 hydrogels, at least about 800 hydrogels, at least about 900 hydrogels, at least about 1000 hydrogels,

[0428] Any of the systems or devices described herein may comprise one or more compartments, chambers, channels, or locations comprising the one or more hydrogels. In some embodiments, two or more of the compartments may be in fluid communication, optical communication, thermal communication, or any combination thereof with one another. In some embodiments, two or more compartments may be arranged in a sequence. In some embodiments, two or more compartments may be arranged in parallel. In some embodiments, two or more compartments may be arranged in sequence, parallel, or both. In some embodiments, one or more compartments may comprise a well. In some embodiments, one or more compartments may comprise a flow strip. In some embodiments, one or more compartments may comprise a heating element.

[0429] In some embodiments, the device may be a handheld device. In some embodiments, the device may be point-of-need device. In some embodiments, the device may comprise any one of the device configurations described herein. In some embodiments, the device may comprise one or more parts of any one of the device configurations described herein.

Amplifying Signals using Positive Feedback Systems

[0430] Any of the methods described herein may comprise amplifying a detection signal using a positive feedback system. FIGS. 27A-27B illustrates an exemplary positive feedback system for signal amplification. In some embodiments, a method for signal amplification may comprise binding a first nuclease, e.g., a first programmable nuclease (15101a) bound to a first guide nucleic acid (15102) with a first target nucleic acid (15103) to generate a first activated programmable nuclease complex (15101b), as shown in FIG. 27A. The first target nucleic acid (15103) may be present in a sample. Activation of the first programmable nuclease may result in release of one or more secondary target-specific guide nucleic acids (15104) from a first location (15105). The secondary target-specific guide nucleic acids (15104) may each comprise a nucleic acid tether (15106) capable of being cleaved by the first activated programmable nuclease complex. The secondary target-specific guide nucleic acids may be released by trans-cleaving the nucleic acid tethers (15106) via the first activated programmable nuclease complex. The secondary target-specific guide nucleic acids (15104) may then bind to an uncomplexed second programmable nuclease (15101a) present at the first location (15105), as shown in FIG. 27B. The second programmable nuclease (15101a) may then bind a second target nucleic acid (15108) at a second location (15109) to generate a second activated programmable nuclease complex (15101b). The second activated programmable nuclease complex (15101b) may then cleave the second target nucleic acid (15108) or remain immobilized at the second location (15109). One or more reporters may be present (e.g., free-floating, immobilized to a surface of a support at a third location, etc.) which may be cleaved by the first and second activated programmable nuclease complexes as described herein. Alternatively, or in combination, one or more of the secondary target-specific guide nucleic acid (15104), the tether (15106), and/or the second target nucleic acid (15108) may comprise a detection moiety which may provide a detectable signal upon cleavage of the nucleic acid species to which it is bound. In some embodiments, the detection moiety comprises a quencher-fluorophore pair as described herein.

[0431] A single first target nucleic acid can lead to the release of a plurality of secondary targetspecific guide nucleic acids and the generation of the plurality of second activated programmable nuclease complexes as described herein. Then each second activated programmable nuclease complex can lead to the generation of another plurality of secondary target-specific guide nucleic acids and another plurality of second activated programmable nuclease complexes. And so on. The second activated programmable nuclease complexes can generate additional signal beyond that of the first activated programmable nuclease complex alone as described herein. Therefore, the detection of the single first target nucleic acid can activate a positive feedback loop for amplifying its signal.

[0432] In some aspects, provided herein are compositions for amplifying a detection signal using a positive feedback system. In some embodiments, a composition may comprise: (a) a first set of programmable nucleases (15101) each comprising a first guide nucleic acid (15102), wherein each programmable nuclease in the first set of programmable nucleases is configured to bind with a first target nucleic acid (15103) and then trans-cleave a plurality of nucleic acids comprising a first sequence; (b) a plurality of secondary target-specific guide nucleic acids (15104) each comprising a nucleic acid tether (15106) comprising the first sequence; (c) a second set of programmable nucleases (15101) each configured to bind with a secondary targetspecific guide nucleic acid which is configured to bind with a second target nucleic acid and then cleave a plurality of nucleic acids comprising a second sequence; a plurality of second target nucleic acids (15108) each comprising the second sequence.

[0433] In some embodiments, the first programmable nuclease (15101) may be free in solution, as illustrated in FIGS. 27A-27B. In some embodiments, the first programmable nuclease (15401) may be immobilized to a substrate (e.g., 15105, 15109, etc.). In some embodiments, the second programmable nuclease (15101) may be free in solution. In some embodiments, the second programmable nuclease (15101) may be immobilized to a substrate (e.g., 15105, 15109, etc.). [0434] In some embodiments, one or more components of the composition may be immobilized on a substrate (e.g., 15105, 15109).

[0435] In some embodiments, the substrate (15105, 15109) may comprise a hydrogel, as illustrated in FIG. 27. In some embodiments, a first hydrogel (15105) may comprises a secondary target-specific guide nucleic acid (15104) immobilized by a single-stranded nucleic acid (15106). In some embodiments, a second hydrogel (15109) may comprise an immobilized second target nucleic acid (15108). In some embodiments, the substrate (15105, 15109) may comprise a reporter-incorporated hydrogel as described herein. In some embodiments, the substrate (15105, 15109) may be in the form of a bead. In some embodiments, the substrate (15105, 15109) may be a glassy material. In some embodiments, the substrate (15105, 15109) may be a polymeric material.

[0436] In some embodiments, the secondary target-specific guide nucleic acids (15104) may be immobilized to a substrate (15105, 15109). In some embodiments, the secondary target-specific guide nucleic acids (15104) may be immobilized with a single stranded nucleic acid tether (15106). In some embodiments, the secondary target-specific guide nucleic acids (15104) may be free in solution. In some embodiments, the secondary target-specific guide nucleic acids (15104) may comprise reporters. In some embodiments, the secondary target-specific guide nucleic acids (15104) may comprise detection moieties (15111).

[0437] In some embodiments, the second target nucleic acids (15108) may be immobilized on a substrate (15105, 15109). In some embodiments, the second target nucleic acids (15108) may be free in solution. In some embodiments, the second target nucleic acids (15108) may comprise reporters. In some embodiments, the second target nucleic acids (15108) may comprise detection moieties (15111).

[0438] In some embodiments, the compositions for amplifying a detection signal using a positive feedback system can be described step-wise as shown in FIG. 40 which shows a schematic representation of a surface-immobilized positive-feedback system wherein the Cas#l is initially in the solution and Cas#2 is initially immobilized. In a subsequent step, Cas #1 forms an activated complex upon binding to a first target nucleic acid and cleaves the ssDNA tether linking Cas#2 to the surface. The released Cas#2 is activated upon complexing with a second target nucleic acid (immobilized target #2), and the activated Cas#2 is effective to release additional immobilized Cas#2 and to cleave the reporter to produce a detectable product. In some embodiments, Cas#l and Cas#2 are the same. In some embodiments, Cas#l and Cas#2 are different. In some embodiments, target #1 is different for each of a plurality of reactions (e.g., in a plurality of wells), and target #2 is (a) different from each target #1, and (b) the same as each target #2 in the plurality of reactions. [0439] The programmable nucleases may comprise any of the programmable nucleases described herein. In some embodiments, the nuclease is an endonuclease. In some embodiments, the nuclease is a Cas9 enzyme. In some embodiments, the nuclease is a mutant Cas9 enzyme. In some embodiments, the nuclease is an engineered Cas9 enzyme. In some embodiments, the nuclease is a Casl2 enzyme. In some embodiments, the nuclease is a Casl3 enzyme. In some embodiments, the nuclease is a Casl4 enzyme. In some embodiments, the nuclease is a CasPhi enzyme.

[0440] In some embodiments, the first and second programmable nucleases are the same. In some embodiments, the first and second programmable nucleases are different. In some embodiments, the programmable nucleases carry out cis cleavage. In some embodiments, the programmable nucleases carry out trans cleavage.

[0441] In some embodiments, cleaving by a programmable nuclease activates a reporter. In some embodiments, cleaving by a programmable nuclease activates (e.g., releases, unquenches, etc.) a detection moiety. In some embodiments, cleaving a nucleic acid tether activates a reporter. In some embodiments, cleaving a second target nucleic acid activates a reporter.

Nicking Enzyme Amplification Reaction (NEAR)

[0442] In some embodiments, a target nucleic acid may be amplified using a nicking enzyme amplification reaction (NEAR). NEAR may be used to amplify a region of a nucleic acid comprising a target nucleic acid. NEAR may comprise a forward primer and a reverse primer that at least partially anneals to complementary strands of a target nucleic acid 3’ of the region to be amplified. The forward primer and the reverse primer may comprise a stabilizing region that is not complementary to the target sequence. The forward primer and the reverse primer may comprise recognition regions that may be nicked by a nicking enzyme. A polymerase may polymerize a nucleic acid 5’ to 3’ from the 3’ end of the forward primer or the 3’ end of the reverse primer, using the strand to which the primer is annealed as a template, resulting in a double stranded nucleic acid product or amplicon. The newly synthesized strand may comprise a restriction site and may then serve as a template. The resulting double stranded nucleic acid amplicon may comprise nicking sites in both strands. A nicking enzyme may nick a single strand of the double stranded product or amplicon. The polymerase may polymerize a nucleic acid 5’ to 3’ from the 3’ end of the nucleic acid amplicon 5’ of the nick. The process may be repeated, thereby amplifying the target nucleic acid.

[0443] As described herein, a target nucleic acid may be detected using a DNA-activated programmable RNA nuclease (e.g., a Casl3), a DNA-activated programmable DNA nuclease (e.g., a Casl2), or an RNA-activated programmable RNA nuclease (e.g., a Casl3) and other reagents disclosed herein (e.g., RNA components). The target nucleic acid may be detected using DETECTR, as described herein. The target nucleic acid may be an RNA, reverse transcribed RNA, DNA, DNA amplicon, amplified DNA, synthetic nucleic acids, or nucleic acids found in biological or environmental samples. In some embodiments, the target nucleic acid is amplified prior to or concurrent with detection. In some embodiments, the target nucleic acid is reverse transcribed prior to amplification. The target nucleic acid may be amplified via NEAR of a target nucleic acid sequence. In some embodiments, the nucleic acid is amplified using NEAR coupled with reverse transcription (RT-NEAR). The NEAR amplification may be performed independently, or the NEAR amplification may be coupled to DETECTR for detection of the target nucleic acid. The RT-NEAR amplification may be performed independently, or the RT- NEAR amplification may be coupled to DETECTR for detection of the target nucleic acid. The DETECTR reaction may be performed using any method consistent with the methods disclosed herein.

[0444] NEAR may be performed as an isothermal reaction, for example NEAR may be performed at from about 37 °C to about 42 °C. In some embodiments, NEAR may be performed at about room temperature. In some embodiments, a nucleic acid sample may be heated prior to isothermal amplification. In some embodiments, the nucleic acid sample heated prior to isothermal amplification may comprise one or more primers. The nucleic acid sample may be heated to about 95 °C prior to isothermal amplification. The nucleic acid sample may be heated to a temperature sufficient to dissociate two strands of a double stranded nucleic acid sequence. [0445] NEAR may amplify a target nucleic acid to detectable levels within about 30 seconds, 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes.

[0446] NEAR Amplification and Detection Reaction Mixtures

[0447] NEAR reaction components may comprise a polymerase, a nicking enzyme, dNTPs, and one or more nucleic acid primers. In some embodiments, the reaction may further comprise a reverse transcriptase as described herein. In some embodiments, the polymerase may be an exo- Klenow polymerase. The nicking enzyme may be capable of nicking a single strand of a double stranded nucleic acid sequence. In some embodiments, the nicking enzyme may be capable of nicking an unthiolated strand of a double stranded nucleic acid sequence comprising a thiolated strand and an unthiolated strand. In some embodiments, the nicking enzyme may be capable of nicking a single strand comprising an unthiolated region of a double stranded nucleic acid comprising at one or more thiolated regions and one or more unthiolated regions. In some embodiments, the nicking enzyme is a restriction enzyme capable of nicking a single strand of a double stranded nucleic acid sequence. In some embodiments, the nicking enzyme is a modified restriction enzyme. The nicking enzyme may be a strand-limited restriction enzyme. The restriction enzyme may be Hindi. In some embodiments, the restriction enzyme may be Alul, BamHI, EcoP15I, EcoRI, EcoRII, EcoRV, Haelll, Hgal, Hindll, Hindlll, HinFI, Kpnl, Notl, PstI, PvuII, Sad, Sall, Sau3, Seal, Smal, Spel, SphI, Stul, TaqI, or Xbal, or the like. The nicking enzyme may be Nt.BspQI, Nt.CvPII, Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nb.BbvCI, Nb.BsmI, Nb.BssSI, Nt.BsmAI, Nb.BpulOI, Nt.Bpul 101, Nb.Mval269I, or I-Hmul, or the like. The one or more nudeic acid primers may comprise two primers. For example, the one or more nucleic acid primers may comprise a first primer (e.g., an SI primer) and a second primer (e.g., an S2 primer). The target nucleic acid may be single stranded DNA or double stranded DNA. In some embodiments, a target nucleic acid comprising RNA may be reverse transcribed into DNA using a reverse transcriptase prior to NEAR amplification. A reverse transcription reaction may comprise primers, dNTPs, and a reverse transcriptase. In some embodiments, the reverse transcription reaction and the NEAR amplification reaction may be performed in the same reaction. A combined RT-NEAR reaction may comprise NEAR primers, reverse transcription primers, dNTPs, a reverse transcriptase, a polymerase, and dNTPs. In some embodiment, the NEAR primers may comprise the reverse transcription primers.

[0448] A DETECTR reaction to detect the target nucleic acid sequence may comprise a guide nucleic acid comprising a segment that is reverse complementary to a segment of the target nucleic acid and a programmable nuclease. The programmable nuclease when activated, as described elsewhere herein, exhibits sequence-independent cleavage of a reporter (e.g., a nucleic acid comprising a moiety that becomes detectable upon cleavage of the nucleic acid by the programmable nuclease). The programmable nuclease is activated upon the guide nucleic acid hybridizing to the target nucleic acid. A combined NEAR DETECTR reaction may comprise a polymerase, a restriction enzyme, dNTPs, one or more nucleic acid primers, a guide nucleic acid, a programmable nuclease, and a substrate nucleic acid. A combined RT-NEAR DETECTR reaction may comprise reverse transcription primers, a reverse transcriptase, a polymerase, a restriction enzyme, dNTPs, one or more nucleic acid primers, a guide nucleic acid, a programmable nuclease, and a substrate nucleic acid. In some embodiment, the primers may comprise the reverse transcription primers. NEAR and DETECTR can be carried out in the same sample volume. NEAR and DETECTR can be carried out concurrently in separate sample volumes or in the same sample volume. RT-NEAR and DETECTR can be carried out in the same sample volume. RT-NEAR and DETECTR can be carried out concurrently in separate sample volumes or in the same sample volume. A NEAR reaction may be multiplexed to amplify a plurality of target nucleic acid sequences in a single reaction. [0449] NEAR Primers and Guide Nucleic Acids

[0450] A number of NEAR primers and NEAR primer design methods are consistent with the methods compositions, reagents, enzymes, and kits disclosed herein. NEAR may comprise a set of primers. In some embodiments, NEAR may be an RT-NEAR reaction, a NEAR DETECTR reaction, or an RT-NEAR DETECTR reaction. The set of NEAR primers may comprise two primers, a first primer and a second primer. In some embodiments, a first primer may comprise a sequence of the first region at the 3’ end of the first primer. The sequence of the first region may be from about 16 nucleic acids to about 25 nucleic acids long, or about 20 nucleic acids long. The 3’ end of the first primer may hybridize to the first complementary region of the target. The first complementary region may be 3’ of the target nucleic acid. The first complementary region may be 3’ of a sequence reverse complementary to the target nucleic acid. The first primer may further comprise a cut site 5’ of the sequence of the first region that may be recognized and cleaved by a nicking enzyme. The 3’ end of the first primer may further comprise a recognition site for a nicking enzyme. In some embodiments, the 3’ end of the first primer may further comprise a nicking enzyme stabilization region. The sequence of the second region may be from about 30 nucleic acids to about 38 nucleic acids long. The 3’ end of the second primer may hybridize to the second complementary region. The second complementary region may be 3’ of a sequence reverse complementary to the target nucleic acid. The second complementary region may be 3’ of the target nucleic acid. The second complementary region may be 3’ of a sequence reverse complementary to the target nucleic acid. The second primer may further comprise a cut site 5’ of the sequence of the second region that may be recognized and cleaved by a nicking enzyme. The 3’ end of the second primer may further comprise a recognition site for a nicking enzyme. In some embodiments, the 3’ end of the second primer may further comprise a nicking enzyme stabilization region.

[0451] The NEAR primers are designed depending on the site of the optimal guide RNA placement, which may or may not be determined by an available PAM sequence. When performing a NEAR-DETECTR reaction, single-stranded DNA is produced by the designed primers. Because the DETECTR reaction will detection single stranded DNA species, the amplification reaction can be biased to produce more of the particular strand than another. This can be done through changing of the ratio of the forward and reverse primer concentrations. In some embodiments, the concentration of forward primer can be 5 times, 4 times, 3 times, 2 times, or equal to the concentration of reverse primer. In some embodiments, the concentration of reverse primer can be 5 times, 4 times, 3 times, 2 times, or equal to the concentration of forward primer.

[0452] In some embodiments, the first region, the second region, or both may be about 8 nucleic acids, about 10 nucleic acids, about 12 nucleic acids, about 14 nucleic acids, about 16 nucleic acids, about 18 nucleic acids, about 20 nucleic acids, about 22 nucleic acids, about 24 nucleic acids, about 26 nucleic acids, about 28 nucleic acids, about 30 nucleic acids, about 32 nucleic acids, about 34 nucleic acids, about 36 nucleic acids, about 38 nucleic acids, about 40 nucleic acids, about 42 nucleic acids, about 44 nucleic acids, about 46 nucleic acids, about 48 nucleic acids, or about 50 nucleic acids long.

[0453] In some embodiments, the first region, the second region, or both may be from about 8 to about 12, from about 8 to about 16, from about 8 to about 20, from about 8 to about 24, from about 8 to about 28, from about 8 to about 30, from about 8 to about 32, from about 8 to about 34, from about 8 to about 36, from about 8 to about 38, from about 8 to about 40, from about 8 to about 42, from about 8 to about 44, from about 8 to about 48, or from about 8 to about 50 nucleic acids long.

[0454] In some embodiments, the first region, the second region, or both may comprise a GC content of about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In some embodiments, the first region, the second region, or both may comprise a GC content of from about 1% to about 95%, from about 5% to about 90%, from about 10% to about 80%, from about 15% to about 70%, from about 20% to about 60%, from about 25% to about 50%, or from about 30% to about 40%

[0455] In some embodiments, the first region, the second region, or both may have a melting temperature of about 38 °C, about 40 °C, about 42 °C, about 44 °C, about 46 °C, about 48 °C, about 50 °C, about 52 °C, about 54 °C, about 56 °C, about 58 °C, about 60 °C, about 62 °C, about 64 °C, about 66 °C, about 68 °C, about 70 °C, about 72 °C, about 74 °C, about 76 °C, about 78 °C, about 80 °C, about 82 °C, about 84 °C, about 86 °C, about 88 °C, about 90 °C, or about 92 °C. In some embodiments, the first region, the second region, or both may have a melting temperature of from about 35 °C to about 40 °C, from about 35 °C to about 45 °C, from about 35 °C to about 50 °C, from about 35 °C to about 55 °C, from about 35 °C to about 60 °C, from about 35 °C to about 65 °C, from about 35 °C to about 70 °C, from about 35 °C to about 75 °C, from about 35 °C to about 80 °C, or from about 35 °C to about 85 °C.

[0456] A set of NEAR primers may be designed for use in combination with a DETECTR reaction. The amplified nucleic acid sequence may comprise a sequence that hybridizes to a guide RNA. The amplified nucleic acid sequence may comprise a target nucleic acid. The guide RNA may hybridize to the target nucleic acid. The amplified nucleic acid sequence may comprise corresponding to a guide RNA. The amplified nucleic acid sequence may comprise a sequence reverse complementary to the target nucleic acid. All or part of the guide RNA may be reverse complementary to all or part of the target nucleic acid. The amplified nucleic acid sequence may comprise a protospacer adjacent motif (PAM) positioned next to or near the target sequence. The PAM may be 3’ of the target nucleic acid. In some embodiments, a portion of a sequence that hybridizes the guide RNA may be located between the first region and the second complementary region. The portion of a sequence that hybridizes the guide RNA located between the first region and the second complementary region may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more of the sequence that hybridizes guide

RNA. In some embodiments, the 5’ end of the sequence that hybridizes the guide RNA is 3’ of the 3’ end of the first region and 3’ end of the sequence that hybridizes the guide RNA is 5’ of the 5’ end of the second complementary region. In some embodiments, a portion of a sequence that hybridizes the guide RNA may be located between the second region and the first complementary region. The portion of a sequence that hybridizes the guide RNA located between the second region and the first complementary region may comprise at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more of the sequence that hybridizes the guide RNA. In some embodiments, the 5’ end of the sequence that hybridizes the guide RNA is 3’ of the 3’ end of the second region and 3’ end of the sequence that hybridizes the guide RNA is 5’ of the 5’ end of the first complementary region.

[0457] In some embodiments, a sequence that hybridizes the guide RNA may overlap the first region, the first complementary region, the second region, or the second complementary region by no more than no more than about 5%, no more than about 10%, no more than about 15%, no more than about 20%, no more than about 25%, no more than about 30%, no more than about 35%, no more than about 40%, no more than about 45%, no more than about 50%, no more than about 55%, no more than about 60%, no more than about 65%, no more than about 70%, no more than about 75%, no more than about 80%, no more than about 85%, no more than about 90%, or no more than about 95%. In some embodiments, the sequence that hybridizes the guide RNA does not overlap the first region, the first complementary region, the second region, or the second complementary region. In some embodiments, the guide RNA does not hybridize to the first primer or the second primer.

[0458] In some embodiments, a NEAR primer set may be designed using a commercially available primer design software. A NEAR primer set may be designed for use in combination with a DETECR reaction, a reverse transcription reaction, or both. One or more methods of designing a set of NEAR primers may be readily apparent to one skilled in the art and may be employed in any of the compositions, kits and methods described herein.

TABLE 5 - Exemplary NEAR Primers Amplification and Detection of a Gene of Interest

[0459] A DETECTR reaction may be used to detect the presence of a specific target gene in the same. The DETECTR reaction may produce a detectable signal, as described elsewhere herein, in the presence of a target nucleic acid sequence comprising a target gene. The DETECTR reaction may not produce a signal in the absence of the target nucleic acid or in the presence of a nucleic acid sequence that does not comprise the specific SNP allele or comprises a different SNP allele. In some embodiments, a DETECTR reaction may comprise a guide RNA reverse complementary to a portion of a target nucleic acid sequence comprising a specific SNP allele. The guide RNA and the target nucleic acid comprising the specific SNP allele may bind to and activate a programmable nuclease, thereby producing a detectable signal as described elsewhere herein. The guide RNA and a nucleic acid sequence that does not comprise the specific SNP allele may not bind to or activate the programmable nuclease and may not produce a detectable signal. In some embodiments, a target nucleic acid sequence that may or may not comprise a specific SNP allele may be amplified using, for example, a LAMP amplification reaction, an RPA amplification reaction, an SDA amplification reaction, a NEAR amplification reaction, or any other amplification method. In some embodiments, the amplification reaction may be combined with a reverse transcription reaction, a DETECTR reaction, or both. For example, the amplification reaction may be an RT-NEAR reaction, a NEAR DETECTR reaction, or an RT- NEAR DETECTR reaction. In some embodiments, the target nucleic acid sequence can comprise a SNP. In some embodiments, the target nucleic acid sequence can comprise a sequence indicative of a human disease.

[0460] A DETECTR reaction, as described elsewhere herein, may produce a detectable signal specifically in the presence of a target nucleic acid sequence comprising a target gene. In addition to the DETECTR reaction, the target nucleic acid having the target gene may be concurrently, sequentially, concurrently together in a sample, or sequentially together in a sample be carried out alongside NEAR or RT- NEAR. For example, the reactions can comprise NEAR and DETECTR reactions, or RT- NEAR and DETECTR reactions. Performing a DETECTR reaction in combination with a NEAR reaction may result in an increased detectable signal as compared to the DETECTR reaction in the absence of the NEAR reaction. In some embodiments, the target nucleic acid sequence can comprise a SNP. In some embodiments, the target nucleic acid sequence can comprise a sequence indicative of a human disease.

[0461] In some embodiments, the detectable signal produced in the DETECTR reaction may be higher in the presence of a target nucleic acid comprising target nucleic acid than in the presence of a nucleic acid that does not comprise the target nucleic acid. In some embodiments, the DETECTR reaction may produce a detectable signal that is at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40- fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 300-fold, at last 400-fold, at least 500-fold, at least 1000-fold, at least 2000-fold, at least 3000-fold, at least 4000-fold, at least 5000-fold, at least 6000-fold, at least 7000-fold, at least 8000-fold, at least 9000-fold, at least lOOOO-fold, at least 50000-fold, at least lOOOOO-fold, at least 500000-fold, or at least lOOOOOO- fold greater in the presence of a target nucleic acid comprising a target nucleic acid than in the presence of a nucleic acid that does not comprise the target nucleic acid. In some embodiments, the DETECTR reaction may produce a detectable signal that is from 1-fold to 2-fold, from 2-fold to 3-fold, from 3-fold to 4-fold, from 4-fold to 5-fold, from 5-fold to 10-fold, from 10-fold to 20- fold, from 20-fold to 30-fold, from 30-fold to 40-fold, from 40-fold to 50-fold, from 50-fold to 100-fold, from 100-fold to 500-fold, from 500-fold to 1000-fold, from 1000-fold to 10,000-fold, from 10,000-fold to 100,000-fold, or from 100,000-fold to 1,000,000-fold greater in the presence of a target nucleic acid comprising a specific SNP allele than in the presence of a nucleic acid that does not comprise the specific SNP allele. In some embodiments, the target nucleic acid sequence can comprise a SNP. In some embodiments, the target nucleic acid sequence can comprise a sequence indicative of a human disease.

[0462] A DETECTR reaction may be used to detect the presence of a target nucleic acid associated with a disease or a condition in a nucleic acid sample. The DETECTR reaction may reach signal saturation within about 30 seconds, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 75 minutes, about 80 minutes, or about 85 minutes and be used to detect the presence of a target gene associated with an increased likelihood of developing a disease or a condition in a nucleic acid sample. The DETECTR reaction may be used to detect the presence of a target gene associated with a phenotype in a nucleic acid sample. For example, a DETECTR reaction may be used to detect target gene associated with a disease such as phenylketonuria (PKU), cystic fibrosis, sickle-cell anemia, albinism, Huntington's disease, myotonic dystrophy type 1, hypercholesterolemia, neurofibromatosis, polycystic kidney disease, hemophilia, muscular dystrophy, hypophosphatemic rickets, Rett's syndrome, or spermatogenic failure. A DETECTR reaction may be used to detect a SNP allele associated with an increased risk of cancer, for example bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, gallbladder cancer, stomach cancer, leukemia, liver cancer, lung cancer, oral cancer, esophageal cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, testicular cancer, thyroid cancer, neuroblastoma, or lymphoma. A DETECTR reaction may be used to detect a SNP allele associated with an increased risk of a disease, for example Alzheimer’s disease, Parkinson’s disease, amyloidosis, heterochromatosis, celiac disease, macular degeneration, or hypercholesterolemia. A DETECTR reaction may be used to detect a SNP allele associated with a phenotype, for example, eye color, hair color, height, skin color, race, alcohol flush reaction, caffeine consumption, deep sleep, genetic weight, lactose intolerance, muscle composition, saturated fat and weight, or sleep movement. A DETECTR reaction may also be used to detect the presence of a pathological organism. In some embodiments, the pathological organism is a prokaryote, eukaryote, or a protozoa. In some embodiments, the pathological organism is a virus, an opportunistic pathogen, a parasite, a bacterium, or any combination thereof. In some embodiments, the pathological organism is SARS-CoV-2 or Streptococcus pyogenes.

[0463] The terms “sample interface”, “sample input”, “input port”, “input”, “port” as used herein, generally refers to a portion of a device that is configured to receive a sample.

[0464] The terms “heating region”, “heated region”, “heat chambers”, “heat volumes”, “heat zones”, “heat surfaces”, “heat areas”, and the like, as used herein, generally refers to a portion of a device that is in thermal communication with a heating unit.

[0465] The terms “heater”, “heating unit”, “heating element”, “heat source”, and the like, as used herein, generally refers to an element that is configured to produce heat and is in thermal communication with a portion of a device.

[0466] The term “reagent mix”, “reagent master mix”, “reagents”, and the like, as used herein, generally refers to a formulation comprising one or more chemicals that partake in a reaction that the reagent mix is formulated for.

[0467] The term “non-cycled temperature profile,” as used herein, generally refers to a temperature profile that is cyclical or sinusoidal in that the temperature profile has an initial temperature, a target temperature, and a final temperature.

[0468] The term “capture probe”, “capture molecule”, and the like, as used herein, generally refers to a molecule that selectively binds to a target nucleic acid and only nonspecifically binds to other nucleic acids that can be washed away.

[0469] The term “collection tube,” as used herein, generally refers to a compartment that is used to collect a sample and deliver the sample to the sample interface of a device. In some embodiments, the collection tube may be portable. In some embodiments, the collection tube is a syringe.

[0470] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word “about” or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/- 0.1% of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), or +/- 10% of the stated value (or range of values). Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points.

[0471] As used herein, the terms “thermostable” and “thermostability” refer to the stability of a composition disclosed herein at one or more temperatures, such as an elevated operating temperature for a given reaction. Stability may be assessed by the ability of the composition to perform an activity, e.g., cleaving a target nucleic acid or reporter. Improving thermostability means improving the quantity or quality of the activity at one or more temperatures.

[0472] As used herein, the terms “percent identity,” “% identity,” and “% identical” refer to the extent to which two sequences (nucleotide or amino acid) have the same residue at the same positions in an alignment. For example, “an amino acid sequence is X% identical to SEQ ID NO: Y” refers to % identity of the amino acid sequence to SEQ ID NO: Y and is elaborated as X% of residues in the amino acid sequence are identical to the residues of sequence disclosed in SEQ ID NO: Y in an alignment between the two. Generally, computer programs may be employed for such calculations. Illustrative programs that compare and align pairs of sequences, include ALIGN (Myers and Miller, Comput Appl Biosci. 1988 Mar;4(l): 11-7), FASTA (Pearson and Lipman, Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444-8; Pearson, Methods Enzymol. 1990;183:63-98) and gapped BLAST (Altschul et al., Nucleic Acids Res. 1997 Sep l;25(17):3389-40), BLASTP, BLASTN, or GCG (Devereux et al., Nucleic Acids Res. 1984 Jan 11 ; 12(1 Pt 1):387-95). For the purposes of calculating identity to the sequence, extensions, such as tags, are not included.

[0473] As used herein, a “one-pot” reaction refers to a reaction in which more than one reaction occurs in a single volume alongside a programmable nuclease-based detection (e.g., DETECTR) assay. For example, in a one-pot assay, sample preparation, reverse transcription, amplification, in vitro transcription, or any combination thereof, and programmable nuclease-based detection (e.g., DETECTR) assays (optionally including signal amplification) are carried out in a single volume. In some embodiments, amplification and detection are carried out within a same volume or region of a device (e.g., within a detection region). Readout of the detection (e.g., DETECTR) assay may occur in the single volume or in a second volume. For example, the product of the one-pot DETECTR reaction (e.g., a cleaved detection moiety comprising an enzyme) may be transferred to another volume (e.g., a volume comprising an enzyme substrate) for signal generation and indirect detection of reporter cleavage by a sensor or detector (or by eye in the case of a colorimetric signal).

[0474] As used herein, “HotPot” refers to a one-pot reaction in which both amplification (e.g., RT-LAMP) and detection (e.g., DETECTR) reactions occur simultaneously. In many embodiments, a HotPot reaction may utilize a thermostable programmable nuclease which exhibits trans cleavage at elevated temperatures (e.g., greater than 37C).

[0475] The terms, “nucleic acid amplification” and “amplifying a nucleic acid,” as used herein, refer to a process by which a nucleic acid molecule is enzymatically copied to generate a plurality of nucleic acid molecules containing the same sequence as the original nucleic acid molecule or a distinguishable portion thereof.

[0476] The term, “complementary,” as used herein with reference to a nucleic acid refers to the characteristic of a polynucleotide having nucleotides that base pair with their Watson-Crick counterparts (C with G; or A with T/U) in a reference nucleic acid. For example, when every nucleotide in a polynucleotide forms a base pair with a reference nucleic acid, that polynucleotide is said to be 100% complementary to the reference nucleic acid. In a double stranded DNA or RNA sequence, the upper (sense) strand sequence is in general, understood as going in the direction from its 5'- to 3 '-end, and the complementary sequence is thus understood as the sequence of the lower (antisense) strand in the same direction as the upper strand.

Following the same logic, the reverse sequence is understood as the sequence of the upper strand in the direction from its 3'- to its 5 '-end, while the ‘reverse complement’ sequence or the ‘reverse complementary’ sequence is understood as the sequence of the lower strand in the direction of its 5'- to its 3 '-end. Each nucleotide in a double stranded DNA or RNA molecule that is paired with its Watson-Crick counterpart called its complementary nucleotide.

[0477] The term, “cleavage assay,” as used herein refers to an assay designed to visualize, quantitate or identify cleavage of a nucleic acid. In some cases, the cleavage activity may be cis- cleavage activity. In some cases, the cleavage activity may be trans-cleavage activity.

[0478] Assays which leverage the transcollateral cleavage properties of programmable nuclease enzymes (e.g., CRISPR-Cas enzymes) are often referred to herein as DNA endonuclease targeted CRISPR trans reporter (DETECTR) reactions. As used herein, detection of programmable nuclease-based reporter cleavage (directly or indirectly) to determine the presence of a target nucleic acid sequence may be referred to as “DETECTR”.

[0479] The term, “detectable signal,” as used herein refers to a signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical or other detection methods known in the art.

[0480] The term, “detecting a nucleic acid” and its grammatical equivalents, as used herein refers to detecting the presence or absence of the target nucleic acid in a sample that potentially contains the nucleic acid being detected.

[0481] The term, “effector protein,” as used herein refers to a protein, polypeptide, or peptide that non-covalently binds to a guide nucleic acid to form a complex that contacts a target nucleic acid, wherein at least a portion of the guide nucleic acid hybridizes to a target sequence of the target nucleic acid. In some embodiments, the complex comprises multiple effector proteins. In some embodiments, the effector protein modifies the target nucleic acid when the complex contacts the target nucleic acid. In some embodiments, the effector protein does not modify the target nucleic acid, but it is fused to a fusion partner protein that modifies the target nucleic acid. A non-limiting example of modifying a target nucleic acid is cleaving (hydrolysis) of a phosphodiester bond. Additional examples of modifying target nucleic acids are described herein and throughout. In some embodiments, the term, “effector protein” refers to a protein that is capable of modifying a nucleic acid molecule (e.g., by cleavage, deamination, recombination). Modifying the nucleic acid may modulate the expression of the nucleic acid molecule (e.g., increasing or decreasing the expression of a nucleic acid molecule). The effector protein may be a Cas protein (i.e., an effector protein of a CRISPR-Cas system).

[0482] The term, “guide nucleic acid,” as used herein refers to a nucleic acid comprising: a first nucleotide sequence that hybridizes to a target nucleic acid; and a second nucleotide sequence that is capable of being non-covalently bound by an effector protein. The first sequence may be referred to herein as a spacer sequence. The second sequence may be referred to herein as a repeat sequence. In some embodiments, the first sequence is located 5’ of the second nucleotide sequence. In some embodiments, the first sequence is located 3’ of the second nucleotide sequence.

[0483] The terms, “non-naturally occurring” and “engineered,” as used herein are used interchangeably and indicate the involvement of human intervention. The terms, when referring to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid, refer to a nucleic acid, nucleotide, protein, polypeptide, peptide or amino acid that is at least substantially free from at least one other feature with which it is naturally associated in nature and as found in nature, and/or contains a modification (e.g., chemical modification, nucleotide sequence, or amino acid sequence) that is not present in the naturally occurring nucleic acid, nucleotide, protein, polypeptide, peptide, or amino acid. The terms, when referring to a composition or system described herein, refer to a composition or system having at least one component that is not naturally associated with the other components of the composition or system. By way of a nonlimiting example, a composition may include an effector protein and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, an effector protein or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes an effector protein and a guide nucleic acid from a cell or organism that have not been genetically modified by human intervention.

[0484] The term, “protospacer adjacent motif (PAM),” as used herein refers to a nucleotide sequence found in a target nucleic acid that directs an effector protein to modify the target nucleic acid at a specific location. A PAM sequence may be required for a complex having an effector protein and a guide nucleic acid to hybridize to and modify the target nucleic acid. However, a given effector protein may not require a PAM sequence being present in a target nucleic acid for the effector protein to modify the target nucleic acid.

[0485] The terms, “reporter” and “reporter nucleic acid,” are used interchangeably herein to refer to a non-target nucleic acid molecule that can provide a detectable signal upon cleavage by an effector protein. Examples of detectable signals and detectable moieties that generate detectable signals are provided herein.

[0486] The term, “sample,” as used herein generally refers to something comprising a target nucleic acid. In some instances, the sample is a biological sample, such as a biological fluid or tissue sample. In some instances, the sample is an environmental sample. The sample may be a biological sample or environmental sample that is modified or manipulated. By way of nonlimiting example, samples may be modified or manipulated with purification techniques, heat, nucleic acid amplification, salts and buffers.

[0487] The term, “target nucleic acid,” as used herein refers to a nucleic acid that is selected as the nucleic acid for modification, binding, hybridization or any other activity of or interaction with a nucleic acid, protein, polypeptide, or peptide described herein. A target nucleic acid may comprise RNA, DNA, or a combination thereof. A target nucleic acid may be single-stranded (e.g., single-stranded RNA or single-stranded DNA) or double-stranded (e.g., double-stranded DNA).

[0488] The term, “target sequence,” as used herein when used in reference to a target nucleic acid refers to a sequence of nucleotides that hybridizes to a portion (preferably an equal length portion) of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring an effector protein into contact with the target nucleic acid.

[0489] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

ILLUSTRATIVE EMBODIMENTS

[0490] The present disclosure provides the following illustrative embodiments.

[0491] Embodiment 1. A microfluidic device comprising: a sample interface configured to receive a sample; a first actuator configured to provide positive pressure to an upstream portion of the device proximate to the sample interface; a second actuator configured to provide negative pressure to a downstream portion of the device distal to the sample interface; a heating channel in fluid communication with the sample interface and first actuator, wherein the heating channel comprises a first portion in a first plane and a second portion in a second plane parallel to the first plane; a first heating element disposed between and in thermal contact with the first and second portions of the heating channel; a reagent mixing chamber fluidically connected downstream of the heating channel by a first valve; a plurality of reaction chambers in fluid communication with the second actuator; a channel multiplexing unit fluidically connected downstream of the mixing chamber by a second valve, wherein the channel multiplexing unit comprises a main channel and a plurality of side channels, wherein each of the side channels is in fluid communication with one inlet of a plurality of inlets positioned along the main channel, and wherein each of the side channels comprises an outlet in fluid communication with one of the plurality of reaction chambers; a first valve actuator configured to actuate the first valve; and a second valve actuator configured to actuate the second valve.

[0492] Embodiment 2. The microfluidic device of Embodiment 1, wherein (i) each of the first valve and second valve is thermally actuated, and (ii) each valve actuator comprises a heating element in thermal contact with the respective valve.

[0493] Embodiment 3. The microfluidic device of Embodiment 2, wherein each of the first and second valves comprises a valve inlet channel and a valve outlet channel; and wherein a cross- sectional area of the valve inlet channel is less than a cross-sectional area of the corresponding valve outlet channel. [0494] Embodiment 4. The microfluidic device of Embodiment 2 or 3, wherein each of the valves is filled with a material configured to change between liquid and solid phases when heated by the heating element of the corresponding valve actuator.

[0495] Embodiment 5. The microfluidic device of any one of Embodiments 1-4, wherein the first heating element is regulated by a thermistor.

[0496] Embodiment 6. The microfluidic device of any one of Embodiments 1-5, wherein the heating channel has a serpentine configuration.

[0497] Embodiment 7. The microfluidic device of any one of Embodiments 1-6, wherein the main channel is serially divided into a plurality of subchannel portions and a tolerance channel, wherein the tolerance channel is positioned at a distal end of the main channel.

[0498] Embodiment 8. The microfluidic device of Embodiment 7, wherein each of the subchannel portions and tolerance channel are configured to subsequently reduce in cross- sectional area from a most proximal subchannel to the tolerance channel.

[0499] Embodiment 9. The microfluidic device of Embodiment 7, wherein a volume of the sample is (a) greater than a combined volume of the plurality of side channels and the plurality of reaction chambers, and (b) less than the combined volume the plurality of side channels, the plurality of reaction chambers, and the tolerance channel.

[0500] Embodiment 10. The microfluidic device of Embodiment 7, wherein each of the plurality of inlets comprises a porous frit.

[0501] Embodiment 11. The microfluidic device of any one of Embodiments 1-10, wherein each of the plurality of reaction chambers is connected to the downstream portion by each of a plurality of hydrophobic venting membranes.

[0502] Embodiment 12. The microfluidic device of any one of Embodiments 1-11, wherein the first heating element is configured to trace in alignment with the heating channel.

[0503] Embodiment 13. The microfluidic device of any one of Embodiments 1-11, wherein the first heating element occupies a surface area that is larger than a surface area occupied by the heating channel.

[0504] Embodiment 14. The microfluidic device of any one of Embodiments 1-11, wherein a shape of the first heating element: (a) corresponds to a shape of the heating channel, and (b) occupies a surface area that is larger than a surface area occupied by the heating channel.

[0505] Embodiment 15. The microfluidic device of any one of Embodiments 1-14, further comprising a self-sealing vent in fluid communication with a distal end of the second portion, wherein the vent is configured to seal when wetted. [0506] Embodiment 16. The microfluidic device of any one of Embodiments 1-15, wherein the first actuator and second actuator are operably connected such that actuation of the first actuator triggers actuation of the second actuator.

[0507] Embodiment 17. The microfluidic device of any one of Embodiments 1-16, further comprising one or more reaction heating elements in thermal contact with the reaction chambers; optionally wherein the one or more reaction heating elements are regulated by a thermistor.

[0508] Embodiment 18. The microfluidic device of any one of Embodiments 1-17, wherein the first actuator is operably connected to trigger a timing mechanism that controls the heating elements and valve actuators.

[0509] Embodiment 19. The microfluidic device of any one of Embodiments 1-18, further comprising a power supply in electrical contact with each of the heating elements.

[0510] Embodiment 20. The microfluidic device of Embodiment 19, wherein the power supply comprises one or more batteries, optionally wherein the one or more batteries comprise an alkaline battery, a paper battery, a coin battery, or a capacitor, or any combination thereof. [0511] Embodiment 21. The microfluidic device of Embodiment 19, further comprising a computing unit in electrical communication with the power supply and each of the heating elements.

[0512] Embodiment 22. The microfluidic device of Embodiment 21, wherein the computing unit is programmed to control heating of each of the plurality of heating elements to control fluid flow through each of the first and second valves.

[0513] Embodiment 23. The microfluidic device of Embodiment 21 or 22, wherein the computing unit comprises a plurality of status LEDs.

[0514] Embodiment 24. A microfluidic device comprising: a sample interface configured to receive a sample; a first actuator configured to provide positive pressure to an upstream portion of the device proximate to the sample interface; a second actuator configured to provide negative pressure to a downstream portion of the device distal to the sample interface; a heating channel in fluid communication with the sample interface and first actuator; a first heating element disposed in thermal contact with the heating channel; a reagent input comprising a reagent interface and a reagent channel; a reagent mixing unit fluidically connected downstream of an intersection by a first valve, wherein the intersection is fluidically connected (i) downstream of the heating channel by a second valve, and (ii) downstream of the reagent channel by a third valve; a plurality of reaction chambers; a channel multiplexing unit fluidically connected downstream of the mixing unit by a fourth valve, wherein the channel multiplexing unit comprises a plurality of side channels, wherein each of the plurality of side channels is in fluid communication with a plurality of subchannels, wherein each of the plurality of subchannels comprises an outlet in fluid communication with one of the plurality of reaction chambers; a plurality of detection chambers, wherein each of the plurality of detection chambers is

(i) fluidically connected downstream of a corresponding reaction chamber by a further valve, and

(ii) in fluid communication with the second actuator; a first valve actuator configured to actuate the first valve; a second valve actuator configured to actuate the second valve; a third valve actuator configured to actuate the third valve; a fourth valve actuator configured to actuate the fourth valve; and one or more further valve actuators configured to actuate the further valves.

[0515] Embodiment 25. The microfluidic device of Embodiment 24, wherein (i) each of the valves is thermally actuated, and (ii) each valve actuator comprises a heating element in thermal contact with the respective valve.

[0516] Embodiment 26. The microfluidic device of Embodiment 24 or 25, further comprising a first self-sealing vent in fluid communication with the heating channel, and a second self-sealing vent in fluid communication with the mixing channel, wherein the first self-sealing vent and second self-sealing vent are each configured to seal when wetted.

[0517] Embodiment 27. The microfluidic device of any one of Embodiments 24-26, wherein the second actuator is a sliding unit.

[0518] Embodiment 28. The microfluidic device of any one of Embodiments 24-27, further comprising at least one flow restrictor positioned between the reagent interface and the reagent channel.

[0519] Embodiment 29. The microfluidic device of any one of Embodiments 25-28, wherein each of the valves comprises a valve inlet channel and a valve outlet channel; and wherein a cross-sectional area of the valve inlet channel is less than a cross-sectional area of the corresponding valve outlet channel.

[0520] Embodiment 30. The microfluidic device of any one of Embodiments 25-29, wherein each of the valves is filled with a material configured to change between liquid and solid phases when heated by the heating element of the corresponding valve actuator.

[0521] Embodiment 31. The microfluidic device of any one of Embodiments 24-30, wherein the first heating element is regulated by a thermistor. [0522] Embodiment 32. The microfluidic device of any one of Embodiments 24-31, wherein the heating channel has a serpentine configuration.

[0523] Embodiment 33. The microfluidic device of any one of Embodiments 24-32, wherein (i) the heating channel comprises a first portion in a first plane and a second portion in a second plane parallel to the first plane, and (ii) the first heating element is disposed between and in thermal contact with the first and second portions of the heating channel.

[0524] Embodiment 34. The microfluidic device of any one of Embodiments 24-33, wherein the first actuator and second actuator are operably connected such that actuation of the first actuator triggers actuation of the second actuator.

[0525] Embodiment 35. The microfluidic device of any one of Embodiments 24-34, further comprising one or more reaction heating elements in thermal contact with the reaction chambers; optionally wherein the one or more reaction heating elements are regulated by a thermistor.

[0526] Embodiment 36. The microfluidic device of any one of Embodiments 24-35, further comprising a power supply in electrical contact with each of the heating elements.

[0527] Embodiment 37. The microfluidic device of Embodiment 36, wherein the power supply comprises one or more batteries, optionally wherein the one or more batteries comprise an alkaline battery, a paper battery, a coin battery, or a capacitor, or any combination thereof .

[0528] Embodiment 38. The microfluidic device of Embodiment 36, further comprising a computing unit in electrical communication with the power supply and each of the heating elements.

[0529] Embodiment 39. The microfluidic device of Embodiment 38, wherein the computing unit is programmed to control heating of each of the plurality of heating elements to control fluid flow through each of the first, second, third, fourth, and further valves.

[0530] Embodiment 40. The microfluidic device of Embodiment 38 or 39, wherein the computing unit comprises a plurality of status LEDs.

[0531] Embodiment 41. A system comprising (a) the microfluidic device of any one of Embodiments 1-40, and (b) a signal detection device.

[0532] Embodiment 42. The microfluidic device of any one of Embodiments 1-40 or the system of Embodiment 41, wherein each of the reaction chambers comprises a programmable nuclease, a guide nucleic acid, and a reporter, wherein:

(a) the guide nucleic acid comprises a sequence configured to bind to a target nucleic acid;

(b) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid; (c) cleavage of the reporter is effective to produce a detectable product; and

(d) at least two of the reaction chambers comprise different guide nucleic acids.

[0533] Embodiment 43. A method of using the microfluidic device or system of Embodiment 42, the method comprising:

(a) applying a sample to the sample interface, wherein said applying forms a sample fluid;

(b) actuating flow of the sample fluid through the heating channel to each of the reaction chambers,

(c) reacting the sample fluid with the programmable nuclease, the guide nucleic acid, and the reporter, and

(d) detecting a detectable signal when a target nucleic acid is present in the sample. [0534] Embodiment 44. A composition for detecting a target nucleic acid, the composition comprising a programmable nuclease, a guide nucleic acid, and a reporter, wherein:

(a) the guide nucleic acid comprises a sequence selected from Table 2 and is configured to bind to the target nucleic acid;

(b) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid;

(c) cleavage of the reporter is effective to produce a detectable product.

[0535] Embodiment 45. The composition of Embodiment 44, wherein the reporter is coupled to the guide nucleic acid.

[0536] Embodiment 46. The composition of Embodiment 44 or 45, further comprising a plurality of different guide nucleic acids, wherein the plurality of different guide nucleic acids are collectively configured to bind different target sequences within the target nucleic acid.

[0537] Embodiment 47. The composition of any one of Embodiments 44-46, wherein the composition comprises a plurality or all of guide nucleic acids represented by (a) SEQ ID Nos 987-1010, (b) SEQ ID Nos 1011-1037, (c) SEQ ID Nos 1038-1048, (d) SEQ ID Nos 1049-1061,

(e) SEQ ID Nos 1062-1096, (f) SEQ ID Nos 1097-1123, (g) SEQ ID Nos 1124-1158, (h) SEQ ID Nos 1159-1170, or (i) SEQ ID Nos 1171-1175.

[0538] Embodiment 48. A composition for detecting a target nucleic acid, the composition comprising a programmable nuclease, a guide nucleic acid, a first reporter, an enzyme, and a second reporter, wherein:

(a) the guide nucleic acid comprises a sequence selected from Table 2 and is configured to bind to the target nucleic acid;

(b) the programmable nuclease is effective to cleave the first reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid;

(c) the cleavage of the first reporter is effective to separate a first nucleic acid section from a second nucleic acid section thereof;

(d) the first nucleic acid section is effective to activate the enzyme; and

(e) the activated enzyme is effective to cleave the second reporter to produce a detectable product comprising a detection moiety.

[0539] Embodiment 49. The composition of Embodiment 48, wherein the enzyme is an endonuclease and the second reporter comprises a polynucleotide substrate of the enzyme. [0540] Embodiment 50. The composition of Embodiment 49, wherein the endonuclease is a NucC endonuclease.

[0541] Embodiment 51. The composition of Embodiment 50, wherein the first nucleic acid section comprises adenosine residues.

[0542] Embodiment 52. The composition of Embodiment 51, wherein the adenosine residues comprise cyclic adenylate (cA3).

[0543] Embodiment 53. The composition of any one of Embodiments 48-52, wherein the second nucleic acid section comprises RNA residues, optionally wherein the RNA residues comprise a plurality of uracil residues.

[0544] Embodiment 54. The composition of any one of Embodiments 48-52, wherein the second nucleic acid section comprises DNA residues, optionally wherein the DNA residues comprise a plurality of thymine residues.

[0545] Embodiment 55. The composition of any one of Embodiments 48-52, wherein (a) the second reporter comprises a fluorescent label and a quencher, and (b) cleavage of the second reporter by the activated enzyme is effective to separate the fluorescent label from the quencher. [0546] Embodiment 56. A method of detecting a target nucleic acid in a sample, the method comprising:

(a) contacting the sample with the composition of any one of Embodiments 48-52;

(b) cleaving the first reporter with the programmable nuclease in response to formation of the complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid, thereby releasing the first nucleic acid section;

(c) activating the enzyme with the first nucleic acid section;

(d) cleaving the second reporter with the activated enzyme, thereby producing the detectable product comprising the detection moiety; and

(e) detecting the detection moiety, thereby detecting the presence of the target nucleic acid in the sample. [0547] Embodiment 57. The method of Embodiment 56, wherein (a) the second reporter comprises a polynucleotide substrate of the enzyme, and (b) the enzyme is a NucC.

[0548] Embodiment 58. The method of Embodiment 57, wherein step (d) is performed at a temperature of at least 40 °C.

[0549] Embodiment 59. A method of detecting a target nucleic acid in a sample, the method comprising:

(a) contacting the sample with the composition of any one of Embodiments 44-47;

(b) cleaving the reporter with the programmable nuclease in response to formation of the complex comprising the programmable nuclease, the guide nucleic acid, and the target nucleic acid, thereby producing a detectable product; and

(c) detecting the detectable product, thereby detecting the presence of the target nucleic acid in the sample.

[0550] Embodiment 60. A device for detecting a target nucleic acid, comprising:

[0551] a. a sample interface configured to receive a sample;

[0552] b. a reaction chamber in fluid communication with the sample interface; and

[0553] c. the composition of any one of Embodiments 44-55.

[0554] Embodiment 61. A composition for detecting a target nucleic acid in a reaction chamber, the composition comprising a programmable nuclease, a guide nucleic acid, a forward primer, a reverse primer, a polymerase, a nicking endonuclease, and a reporter, wherein:

(a) the guide nucleic acid comprises a sequence selected from Table 2 and is configured to bind to the target nucleic acid;

(b) the forward primer comprises (i) a 5’ portion comprising a first hairpin, and (ii) a 3’ portion that is configured to bind the target nucleic acid at a first overlapping region with respect to the guide nucleic acid;

(c) the reverse primer comprises (i) a 5’ portion comprising a second hairpin, and (ii) a 3’ portion that is configured to bind a complement of the target nucleic acid at a second overlapping region with respect to the guide nucleic acid;

(d) the first and second hairpins are cleavage substrates for the nicking endonuclease;

(e) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and (i) the target nucleic acid or (ii) an amplicon of the target nucleic acid; and

(f) the cleavage of the reporter is effective to produce a detectable product comprising a detection moiety.

[0555] Embodiment 62. The composition of Embodiment 61, wherein (a) the sequence of the target nucleic acid to which the 3’ portion of the first primer is configured to bind defines a first sequence of the target nucleic acid; (b) the sequence of the 3’ portion of the reverse primer defines a second sequence of the target nucleic acid; and (c) the first sequence and second sequence are separated by about 5 to about 10 nucleotides along the target nucleic acid.

[0556] Embodiment 63. The composition of Embodiment 61, wherein the 3’ portions of the forward primer and reverse primer are about 16 to about 20 nucleotides in length.

[0557] Embodiment 64. The composition of Embodiment 61, wherein overlap between the 3’ portion of the reverse primer and the sequence to which the guide nucleic acid is configured to bind overlap by 1 to 5 nucleotides, 2 to 5 nucleotides, or 3 nucleotides.

[0558] Embodiment 65. The composition of Embodiment 61, wherein the first hairpin and/or the second hairpin are 10 to 20 nucleotides in length, 16 to 20 nucleotides in length, or 16 nucleotides in length.

[0559] Embodiment 66. The composition of Embodiment 61, wherein the programmable nuclease is a Cas protein, optionally wherein the Cas protein is a Casl2 protein or a Casl4 protein.

[0560] Embodiment 67. A method of detecting a target nucleic acid in a sample, the method comprising:

(a) contacting the sample with the composition of any one of Embodiments 61-66;

(b) performing nicking enzyme amplification reaction (NEAR) reaction to amplify the target nucleic acid;

(c) forming a complex comprising the programmable nuclease, the guide nucleic acid, and (i) the target nucleic acid, or (ii) an amplicon of the target nucleic acid;

(d) cleaving the reporter with the programmable nuclease activated by formation of the complex, thereby producing the detectable cleavage product; and

(e) detecting the detection moiety, thereby detecting the presence of the target nucleic acid in the sample.

[0561] Embodiment 68. A microfluidic device comprising: a sample interface configured to receive a sample; an actuator configured to provide positive pressure to an upstream portion of the device proximate to the sample interface; a heating channel in fluid communication with the sample interface and first actuator; a channel heating element in thermal contact with the heating channel; a reagent mixing chamber fluidically connected downstream of the heating channel by a first valve, wherein the first valve is a thermally actuated valve in thermal contact with a first valve heating element; a plurality of reaction chambers fluidically connected downstream of the reagent mixing chamber by one or more second valves, wherein each of the one or more second valves is a thermally actuated valve in thermal contact with a respective second valve heating element; and a power supply in electrical contact with each of the heating elements, wherein the power supply is configured to sequentially power the channel heating element, the first valve heating element, and the respective second valve heating element upon activation of the power supply. [0562] Embodiment 69. The microfluidic device of Embodiment 68, further comprising a computing unit in electrical communication with the power supply and each of the plurality of heating elements, wherein the computing unit is programmed to control timing and duration of heating of each of the heating elements.

[0563] Embodiment 70. The microfluidic device of Embodiment 69, wherein the computing unit is programmed to trigger one or more timing mechanisms that control the heating elements.

[0564] Embodiment 71. A method of detecting a target nucleic acid in a sample, the method comprising:

(a) contacting the sample with a programmable nuclease, a guide nucleic acid, and a reporter at a first location of a device, wherein:

(i) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and (1) the target nucleic acid or (2) an amplicon of the target nucleic acid; and

(ii) the cleavage of the reporter is effective to produce a detectable product comprising a detection moiety;

(b) flowing the detectable product to a second location of the device, wherein uncleaved reporter molecules are retained in the first location; and

(c) detecting the detection moiety at the second location, thereby detecting the presence of the target nucleic acid in the sample.

[0565] Embodiment 72. The method of Embodiment 71, wherein the uncleaved reporter molecules are retained by (i) attachment to a surface at the first location, or (ii) a filter that restricts passage of the uncleaved reporter molecules and allows passage of the detectable product.

[0566] Embodiment 73. The method of Embodiment 71 or 72, wherein (i) the detection moiety comprises a molecule that is captured at a surface at the second location, and (ii) the detecting comprises detection of a change in electrical signal at the surface at the second location resulting from the capture of the detection moiety.

[0567] Embodiment 74. The method of Embodiment 72 or 73, wherein the uncleaved reporter molecules are immobilized to the surface at the first location, and further wherein individual uncleaved reporter molecules comprise: (a) a surface linker, an RNA portion that is cleavable by the programmable nuclease, and a DNA portion that is released as part of the detectable product upon cleavage of the RNA portion, wherein the detecting comprises detecting a change in electrical signal resulting from hybridization of the DNA portion to a surface-bound probe oligonucleotide;

(b) a surface linker, an RNA portion that is cleavable by the programmable nuclease, and a gold nanoparticle (AuNP) that is released as part of the detectable product upon cleavage of the RNA portion, wherein the detecting comprises detecting a change in electrical signal resulting from the AuNP binding to a sensor surface at the second location;

(c) a surface linker, an RNA portion that is cleavable by the programmable nuclease, and a hairpin DNA portion that is released as part of the detectable product upon cleavage of the RNA portion, wherein the hairpin DNA portion comprises a first binding moiety, and wherein the detecting comprises detecting a change in electrical signal resulting from capture of the DNA hairpin and first binding moiety by a second binding moiety at a sensor surface at the second location; or

(d) a surface linker, an RNA portion that is cleavable by the programmable nuclease, and a platinum nanoparticle (PtNP) that is released as part of the detectable product upon cleavage of the RNA portion, wherein the detecting comprises detecting a change in pH resulting from hydrogen peroxide reacting with the PtNP at the second location.

[0568] Embodiment 75. The method of Embodiment 72 or 73, wherein the uncleaved reporter molecules are free in a solution at the first location, and further wherein individual uncleaved reporter molecules comprise:

(a) a binding moiety restricted by the filter, an RNA portion that is cleavable by the programmable nuclease, and a DNA portion that is released as part of the detectable product upon cleavage of the RNA portion, wherein the detecting comprises detecting a change in electrical signal resulting from hybridization of the DNA portion to a surfacebound probe oligonucleotide; or

(b) a binding moiety restricted by the filter, an RNA portion that is cleavable by the programmable nuclease, and a gold nanoparticle (AuNP) that is released as part of the detectable product upon cleavage of the RNA portion, wherein the detecting comprises detecting a change in electrical signal resulting from the AuNP binding to a sensor surface at the second location.

[0569] Embodiment 76. A method of detecting a target nucleic acid in a sample, the method comprising:

(a) contacting the sample with a programmable nuclease, a guide nucleic acid, and a reporter at a first location of a device, wherein: (i) the programmable nuclease is effective to cleave the reporter in response to formation of a complex comprising the programmable nuclease, the guide nucleic acid, and (1) the target nucleic acid or (2) an amplicon of the target nucleic acid;

(ii) the cleavage of the reporter is effective to produce a detectable product comprising a detection moiety; and

(iii) individual uncleaved reporter molecules comprise a binding moiety, an RNA portion that is cleavable by the programmable nuclease, and a gold nanoparticle (AuNP) that is released as part of the detectable product upon cleavage of the RNA portion;

(b) flowing the detectable product and uncleaved reporter molecules to a second location of the device, wherein the uncleaved reporter molecules are captured to surface at the second location; and

(c) detecting presence of the detection moiety at the second location, thereby detecting the presence of the target nucleic acid in the sample, wherein the detecting comprises detecting a change in electrical signal resulting from reduced AuNP binding to a sensor surface at the second location relative to a negative control.

EXAMPLES

[0570] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1: DETECTR-based lateral flow assay strip

[0571] The purpose of this example is to demonstrate a DETECTR™-based multiplexed assay using a lateral flow assay (LFA) strip for parallel readout as illustrated in FIG. 18A-18B. To perform the DETECTR™ assay, a surface (10700) is immobilized with programmable nuclease probes (10707) and reporter probes (10701). In this example, the surface is the bottom of a well, separate from the LFA strip. The reporter probes (10701) contain a surface linker (10702), a cleavable nucleic acid sequence (10703), a label (10704) and binding moiety for the flowing capture probe (10705) of the strip. In this example the binding moiety is biotin. The label and binding moiety are attached to the nucleic acid (10703) by a second linker (10706), where in this example the linker is a dendrimer or trebler molecule. The programmable nuclease probe (10707) contains a surface linker and a programmable nuclease (e.g., Cas enzyme) that in turn contains an sgRNA (10708). The sgRNA contains a repeat unit (or hairpin) and a recognition sequence. The recognition sequence is the compliment for a target nucleic acid of the sample. Anti-biotin labeled gold nanoparticles are located in the sample pad (10711) of the LFA strip (10710) as shown in FIG. 18B. In this example, the first step is to contact the surface (10700) with a sample containing target nucleic acids. Upon binding of a target nucleic acid that is complimentary to the sgRNA (10708) of the immobilized programmable nuclease probe (10707), the reporters (10701) immobilized in near proximity, are cleaved by the programmable nuclease, releasing a cleaved section of the reporter (10709) into solution. The sample solution now containing cleaved reporters corresponding to target nucleic acids that were present in the sample, is then contacted to the sample pad (10711) of the LFA strip (10710). In this example, the sample pad has flowing capture probes (e.g., anti-biotin labeled gold nanoparticles) disposed thereon. Once the sample solution containing released sections of reporters is contacted with the sample pad, said sample solution then flows across the sample pad (for e.g., by being wicked). The cleaved reporters in the sample solution contact and bind to the anti-biotin gold nanoparticle flowing capture probes upon contact in solution. The complex of reporter and nanoparticle is then carried downstream with the rest of the liquid sample by capillary action through the detection region (10712) of the LFA strip. In this example, the detection region (10712) may comprise six detection spots (10713), where each detection spot (10713) contains a different capture antibody type that is specific for a reporter’s dye (10704). In this example, the dye (10704) is FITC and the stationary capture probe is an anti-FITC antibody functionalized to the detection spot (10713) allowing for the specific detection of the FITC labeled reporter among other reporters that are specific for other target nucleic acids. A control line (10714) is present, functionalized with anti-IgG so that all flowing capture probes, not bound to a FITC labeled reporter fragment, (e.g., detection moiety) are captured and detected. It should be noted that in this example, multiple labels and binding moieties were present via the dendritic linker of the detection moiety (10706) to amplify the signal. In this example, multiple detections spots (10713) are present, allowing for the possibility parallel detection of multiplexed samples, as explained in Example 2. FIG. 45 presents an additional exemplary microfluidic device that implements the LFA strips for parallel readout.

Example 2: Multiplexed DETECTR-based lateral flow assay strip

[0572] The purpose of this example is to demonstrate a lateral flow assay (LFA) strip workflow utilizing a multiplex “Hotpot” assay as illustrated in FIG. 19. In this example, a sample (10801) contains target nucleic acid sequences 1 and 2. The sample (10801) is contacted to the surface (10802) of a well where at each of five locations, DI through D5, there are different sgRNA’s immobilized to the surface. For example, each of the 5 different sgRNA’s are part of 5 different programmable nuclease probes (e.g., see FIG. 18) immobilized in the five different locations DI through D5, as depicted in FIG. 19. Additionally, each of the 5 different sgRNA’s are designed to specifically bind to different target nucleic acids in the sample, thus allowing for sample multiplexing. In addition to the immobilized programmable nuclease probes containing sgRNAs, each location, DI through D5, is functionalized with reporter probes having distinct functional groups. The reporter probes are in close enough proximity to be cleaved by the programmable nuclease probes. Therefore, as described in Example 1, reporters are cleaved and released into the solution upon binding between a sgRNA and the target nucleic acid that the sgRNA is designed to bind specifically to. In this example, D4 and D5 each contain reporters labeled with two different labels or capture antibody recognition elements. Once the sample has contacted with the wells (DI to D5), the respective cleaved sections of reporters are released into the sample solution (as described in Example 1). The sample solution with the released reporters are then contacted with a sample pad, wherein in this example, is situated on lateral flow assay. In this example each detection spot contains a different type of capture antibody, where each capture antibody type specifically binds to a particular label of a reporter. For this example, detection spot (10803) contains the capture antibody anti-FITC, whereas well D5 contains 1) the immobilized Cas-complex including the sgRNA specific to a first target nucleic acid sequence, and 2) the immobilized reporter (10806), which is labeled with FITC. Therefore, upon binding of the first target nucleic acid sequence with the corresponding sgRNA, the linkage between the corresponding immobilized reporter (10806) and corresponding nucleic acid (for example see ref. char. 10701 in FIG. 18) is cleaved, thereby releasing the reporter into solution. By contrast, for this example, detection spot (10804) contains the capture antibody anti -DIG, whereas well D4 contains 1) the immobilized Cas complex including the sgRNA specific to a second target nucleic acid sequence, and 2) the immobilized reporter (10805), which is labeled with DIG. Therefore, upon binding of the second target nucleic acid sequence with the corresponding sgRNA, the linkage between the corresponding immobilized reporter (10805) and corresponding nucleic acid (for example see ref. char. 10701 in FIG. 18) is cleaved, thereby releasing the reporter the solution. The solution now containing cleaved reporters (10805 and 10806) is then contacted to the sample pad of the LFA strip along with chase buffer, where the reporters bind with and pick up flowing capture probes (e.g., anti-biotin-AuNPs) that are disposed on the sample pad. The AuNP -reporter conjugates having reporter (10806) labeled with FITC will selectively bind to detection spot (10803) containing the capture antibody anti-FITC, thus indicating the presence of the first target nucleic acid sequence in the sample. The AuNP- reporter conjugates having reporter (10805) labeled with DIG will selectively bind to detection spot (10804) containing the capture antibody anti -DIG, thus indicating the presence of the second target nucleic acid sequence in the sample. In this manner, parallel detection of 2 or more target nucleic acid sequences present in a multiplexed sample is enabled. In some examples, the detection spots are spaced apart from each other in prescribed locations, such that detection of a reporter at a given detection spot will correlate with a specific target nucleic acid. FIG. 45 illustrates the incorporation of LFA strips into a microfluidic device for parallel readout.

Example 3: Alkaline battery-driven programmable nuclease-based detection device

[0573] The purpose of this example is to demonstrate an exemplary programmable nuclease- based assay workflow run in a handheld alkaline battery-powered fluidic device. FIGS. 1A-4C show an exemplary microfluidic device 100 including a first actuator 102 for providing positive pressure to the upstream portion 112 of the device proximate to a sample interface at sample input 101 and a second actuator 103 for providing negative pressure to a downstream portion 113 of the device 100 distal to the sample interface. A sample (e.g., a liquid sample or a swab comprising the sample) is inserted into the sample input 101 before actuation of the first actuator 102 by the user. The first actuator 102 and the second actuator 103 are coupled to one another such that actuation of the first actuator 102 by the user, to generate positive pressure to an upstream portion of the device proximate to the sample interface, also actuates the second actuator 103, to provide negative pressure to a downstream portion of the device distal to the sample interface. Actuation of the first actuator 102 also causes the second actuator 103 to turn on the power supply 108, comprising two AA batteries, which are coupled to the circuitry of the PCB 110 including heat element(s) 116, valve actuators 112a, 122b, 140a, 140b, LEDs 105a- 105c, reaction chamber heating elements, detection elements or sensors, and any other device elements requiring power as described herein. Actuation of the first actuator 102 also applies positive pressure to the sample input 101 and moves the samples from the input chamber 107 into the first and second portions of the heating channel 114a, 114b where the crude sample is lysed and or otherwise processed in advance of the programmable nuclease-based reaction. After a pre-determined amount of time, the circuitry triggers activation of the first valve actuator 140a and actuation (e.g., melting) of the first valve 122a, which enables the lysed sample (driven by the positive pressure generated by the first actuator 102) to enter the reagent mixing chamber 121 which contains reagents for inactivating the lysis reagents (e.g., protease inhibitors), programmable nucleases, and reporters. After a pre-determined amount of time, to enable sufficient mixing and/or reactions to occur, the second valve actuator 140b is activated and the second valve 122b is actuated (e.g., melted), which enables the processed sample (driven by the

-Tll- negative pressure generated by the second actuator 103) to enter the channel multiplexing unit 125 comprising reaction chambers 126a-126f. Each of the reaction chambers 126a-126f comprises a different guide nucleic acid to facilitate multiplexed analysis of the sample (e.g., for detecting SARS-CoV-2, Influenza A, Influenza B, RSV A/B, rhinovirus, and/or RNase P, etc. in a single sample). The reaction chambers 126a-126f are then heated by their corresponding reaction chamber heating element(s) to the programmable nuclease-based reaction temperature. Upon binding of the programmable nuclease-guide nucleic acid complex to its target nucleic acid within a reaction chamber, reporters therein are cleaved. In this exemplary embodiment, the reporters comprise a fluorescence-quencher reporter such that cleavage in the reporter results in an increase in fluorescence within the reaction chamber. A fluorescence detector (coupled to the reaction chamber or external to the device) is used to detect cleavage, thereby detecting the presence of the target nucleic acid within the reaction chamber.

Example 4: Paper battery-based power source testing

[0574] The purpose of this example is to demonstrate the performance of a paper-based battery, as shown in FIG. 46, as an alternative, more eco-friendly power source for programmable nuclease-based detection devices. Paper batteries can be activated by an aqueous (e.g., waterbased activation solution. In an exemplary embodiment, upon activation by contact with an aqueous activation solution (e.g., water), paper batteries are used to power a metal trace acting as a resistive heating element to heat 200 pL of water with continuous power output. In a first example, water is heated from 25°C to 37°C for 5 minutes and held constant with a single, continuous discharge. In a second example, the water is heated from 25°C to 95°C for 2 minutes with a single, continuous discharge similar to that of two AA batteries. In another example, the water is heated from 25°C to 95°C for 2 minutes, cooled to 25°C for 5 minutes, the heated to 56°C for 10 minutes and held constant with a single, continuous discharge. In another example, the water is heated from 25°C to 95°C in 2 minutes, cooled to 25°C, heated to 56°C for 10 minutes, cooled to 25°C, and heated to 95°C for 2 minutes, demonstrating the utility of the paper battery to heat samples rapidly and cycle through a variety of temperatures in a single, continuous discharge. Such heating could be utilized for isothermal and/or thermocycling techniques, as desired to run an amplification and/or programmable nuclease-based nucleic acid detection assay.

[0575] In another example, upon activation by contact with an aqueous activation solution (e.g., water), paper batteries are used to power four LEDs with 300 ohm resistors for 30 minutes with continuous power output. [0576] In another example, upon activation by contact with an aqueous activation solution (e.g., water), paper batteries are used to power 10 metal traces acting as resistive heating elements to heat 10 wax valves with continuous power output.

[0577] In another example, upon activation by contact with an aqueous activation solution (e.g., water), paper batteries are used to power a small motor pump for 1 minute (duty cycle: 20 seconds on, 2 seconds off) to pump water within a 500 |1L volume of water.

Example 5: Paper battery-driven programmable nuclease-based detection device

[0578] The purpose of this example is to demonstrate an exemplary programmable nuclease- based assay workflow run in a handheld paper battery-powered fluidic device. FIGS. 1A-4C show an exemplary microfluidic device 100 including a first actuator 102 for providing positive pressure to the upstream portion 112 of the device proximate to a sample interface at sample input 101 and a second actuator 103 for providing negative pressure to a downstream portion 113 of the device 100 distal to the sample interface. A sample (e.g., a liquid sample or a swab comprising the sample) is inserted into the sample input 101 before actuation of the first actuator 102 by the user. The first actuator 102 and the second actuator 103 are coupled to one another such that actuation of the first actuator 102 by the user, to generate positive pressure to an upstream portion of the device proximate to the sample interface, also actuates the second actuator 103, to provide negative pressure to a downstream portion of the device distal to the sample interface. Actuation of the first actuator 102 also causes the second actuator 103 to turn on the power supply 108, comprising a plurality of water-activated paper-based batteries, which are coupled to the circuitry of the PCB 110 including heat element(s) 116, valve actuators 112a, 122b, 140a, 140b, LEDs 105a-105c, reaction chamber heating elements, detection elements or sensors, and any other device elements requiring power as described herein. In an exemplary embodiment, turning on the paper-based batteries comprises opening a chamber comprising an aqueous activation solution and allowing the activation solution to contact the paper-based battery(s). Actuation of the first actuator 102 also applies positive pressure to the sample input 101 and moves the samples from the input chamber 107 into the first and second portions of the heating channel 114a, 114b where the crude sample is lysed and or otherwise processed in advance of the programmable nuclease-based reaction. After a pre-determined amount of time, the circuitry triggers activation of the first valve actuator 140a and actuation (e.g., melting) of the first valve 122a, which enables the lysed sample (driven by the positive pressure generated by the first actuator 102) to enter the reagent mixing chamber 121 which contains reagents for inactivating the lysis reagents (e.g., protease inhibitors), programmable nucleases, and reporters. In some embodiments, a second paper battery is turned on (e.g., by contacting it with an aqueous activation solution, as described herein) which controls the first valve actuator 140a. After a predetermined amount of time, to enable sufficient mixing and/or reactions to occur, the second valve actuator 140b is activated and the second valve 122b is actuated (e.g., melted), which enables the processed sample (driven by the negative pressure generated by the second actuator 103) to enter the channel multiplexing unit 125 comprising reaction chambers 126a-126f. In some embodiments, a third paper battery is turned on (e.g., by contacting it with an aqueous activation solution, as described herein) which controls the second valve actuator 140b. Each of the reaction chambers 126a-126f comprises a different guide nucleic acid to facilitate multiplexed analysis of the sample (e.g., for detecting SARS-CoV-2, Influenza A, Influenza B, RSV A/B, rhinovirus, and/or RNase P, etc. in a single sample). The reaction chambers 126a-126f are then heated by their corresponding reaction chamber heating element(s) to the programmable nuclease-based reaction temperature. In some embodiments, one or more additional paper batteries (e.g., one paper battery coupled to all of the reaction chamber heating elements) is turned on to provide power to the reaction chamber heating element(s). Upon binding of the programmable nuclease-guide nucleic acid complex to its target nucleic acid within a reaction chamber, reporters therein are cleaved. In this exemplary embodiment, the reporters comprise a fluorescence-quencher reporter such that cleavage in the reporter results in an increase in fluorescence within the reaction chamber. A fluorescence detector (coupled to the reaction chamber and a fourth paper battery, or external to the device entirely) is used to detect cleavage, thereby detecting the presence of the target nucleic acid within the reaction chamber. An illustration of a microfluidic device incorporating a paper battery is provided in FIG. 46.

[0579] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein can be employed. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.