CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Patent Application No.

63/217,229, filed June 30, 2021, which is incorporated by reference herein in its entirety for all purposes.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

[0002] The contents of the text file submitted electronically herewith are incorporated herein by reference in its entirety: A computer readable format copy of the Sequence Listing (filename: MABI_016_01WO_SeqList_ST25.txt, date created: June 28, 2022, file size: -678,278 bytes).

STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH

[0003] 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 U.S. government has certain rights in the invention.

BACKGROUND

[0004] The quantification of target biological molecules can provide precise and accurate diagnosis of ailments, especially at the early stages of disease or infection. Additionally, it can provide guidance on treatment or intervention to reduce the progression or transmission of ailments. Challenges in deploying such devices include developing methods to quantify target nucleic acids quickly and accurately, with minimal footprint, without compromising the performance of the assay. Existing technologies often employ cumbersome procedures, vary in sensitivity and specificity, and require substantial instrumentation which can be bulky, costly, and/or require special training to perform involved quantification methods.

SUMMARY

[0005] Devices, systems, and methods for nucleic acid quantification which provide fast, accurate, easy to understand results are desired. Not necessarily all such aspects or advantages are achieved by any particular embodiment. Thus, various embodiments may be carried out in a manner that achieves or optimizes one advantage or group of advantages taught herein without necessarily achieving other aspects or advantages as may also be taught or suggested herein.

[0006] The present disclosure generally relates to nucleic acid quantification and more particularly relates to programmable nuclease-based nucleic acid quantification.

[0007] Described herein are various devices comprising: an array comprising a plurality of surface locations, wherein a first surface location of the plurality of surface locations comprises at least one of a first programmable nuclease, a first reporter, or a first guide nucleic acid immobilized thereto, wherein a second surface location of the plurality of surface locations comprises at least one of a second programmable nuclease, a second reporter, or a second guide nucleic acid immobilized thereto, and wherein the first surface location is spatially distinct from the second surface location. In some embodiments, the first guide nucleic acid and the first reporter are immobilized to the first surface location, wherein the first programmable nuclease is complexed with the first guide nucleic acid, and wherein the first reporter is configured to be cleaved when the first programmable nuclease is activated upon binding of the first guide nucleic acid to a first target nucleic acid. In some embodiments, the second guide nucleic acid and the second reporter are immobilized to the second surface location, wherein the second programmable nuclease is complexed with the second guide nucleic acid, and wherein the second reporter is configured to be cleaved when the second programmable nuclease is activated upon binding of the second guide nucleic acid to a second target nucleic acid. In some embodiments, the plurality of surface locations comprises a plurality of spots, a plurality of particles, or a plurality of microwells, and wherein each surface location of the plurality of surface locations comprises at least one programmable nuclease, at least one reporter, and at least one guide nucleic acid. In some embodiments, the plurality of particles comprises a plurality of beads. In some embodiments, the array comprises a plurality of spots, a plurality of particles, or a plurality of microwells, and wherein the first surface location and the second surface location are located in a same spot of the plurality of spots, on a same particle of the plurality of particles, or in a same microwell of the plurality of microwells, respectively. In some embodiments, at least one of the first programmable nuclease, the first reporter, or the first guide nucleic acid are immobilized within a matrix disposed on the first surface. In some embodiments, the matrix comprises a hydrogel. In some embodiments, at least one of the first programmable nuclease, the first reporter, or the first guide nucleic acid are immobilized with a covalent linkage. In some embodiments, the covalent linkage comprises a bifunctional silane. In some embodiments, the first reporter and the second reporter are different types of reporters. In some embodiments, the first reporter and the second reporter are a same type of reporter. In some embodiments, the first reporter or the second reporter comprises a detection moiety or a quenching moiety configured to be released upon cleavage of the first reporter or the second reporter, respectively. In some embodiments, the detection moiety comprises a fluorophore, a particle, an affinity molecule, an enzyme, or an enzyme substrate. In some embodiments, the particle comprises a quantum dot. In some embodiments, the particle is configured for encoded particle multiplexing. In some embodiments, the particle comprises one or more lanthanide elements. In some embodiments, the reporter comprises a fluorophore and a quencher, wherein the quenching moiety comprises the quencher, and wherein release of the quenching moiety results in fluorescence of the fluorophore. In some embodiments, the reporter comprises a fluorophore and a quencher, wherein the detection moiety comprises the fluorophore, and wherein release of the detection moiety results in fluorescence of the fluorophore. In some embodiments, the first programmable nuclease and the second programmable nuclease are different types of programmable nuclease. In some embodiments, the first programmable nuclease and the second programmable nuclease are the same type of programmable nuclease. In some embodiments, the first guide nucleic acid and the second guide nucleic acids are configured to bind to the same target nucleic acid sequence. In some embodiments, the first guide nucleic acid and the second guide nucleic acids are configured to bind to different target nucleic acid sequences.

[0008] Described herein are various devices comprising: an inlet; and a first fluidic channel fluidly coupled to the inlet, wherein the first fluidic channel comprises at least one of a first programmable nuclease, a first reporter, or a first guide nucleic acid immobilized to a first surface thereof, wherein the first fluidic channel comprises at least one of a second programmable nuclease, a second reporter, or a second guide nucleic acid immobilized to a second surface thereof, and wherein the first surface is spatially distinct from the second surface. In some embodiments, the first guide nucleic acid and the first reporter are immobilized to the first surface, wherein the first programmable nuclease is complexed with the first guide nucleic acid, and wherein the first reporter is configured to be cleaved when the first programmable nuclease is activated upon binding of the first guide nucleic acid to a first target nucleic acid. In some embodiments, the second guide nucleic acid and the second reporter are immobilized to the second surface, wherein the second programmable nuclease is complexed with the second guide nucleic acid, and wherein the second reporter is configured to be cleaved when the second programmable nuclease is activated upon binding of the second guide nucleic acid to a second target nucleic acid. In some embodiments, the first surface and the second surface are adjacent sections of the first fluidic channel. In some embodiments, the first surface and the second surface are parallel stripes in a same section of the first fluidic channel. In some embodiments, at least one of the first programmable nuclease, the first reporter, or the first guide nucleic acid are immobilized within a matrix disposed on the first surface. In some embodiments, the matrix comprises a hydrogel. In some embodiments, at least one of the first programmable nuclease, the first reporter, or the first guide nucleic acid are immobilized with a covalent linkage. In some embodiments, the covalent linkage comprises a bifunctional silane. In some embodiments, the first reporter is immobilized to the first surface, wherein the first programmable nuclease or the first guide nucleic acid is coupled to a detection moiety of the first reporter, wherein the first programmable nuclease is complexed with the first guide nucleic acid, wherein the first reporter is configured to be cleaved when the first programmable nuclease is activated upon binding of the first guide nucleic acid to a first target nucleic acid, and wherein cleavage of the first reporter releases the detection moiety or quenching moiety and the first programmable nuclease from the surface of the first fluidic channel. In some embodiments, a second fluidic channel is downstream of the first fluidic channel and coupled to a detector, wherein the detector is configured to detect the detection moiety as the first programmable nuclease flows through the second fluidic channel after being released by cleavage of the first reporter. In some embodiments, the first fluidic channel comprises a branching channel structure having a plurality of subchannels and configured to digitize or compartmentalize a sample into a plurality of compartments. In some embodiments, the plurality of compartments comprises a plurality of microwells, a plurality of nanowells, or a plurality of droplets. In some embodiments, the first reporter and the second reporter are different types of reporters. In some embodiments, the first reporter and the second reporter are the same type of reporter. In some embodiments, the first reporter or the second reporter comprises a detection moiety or a quenching moiety configured to be released upon cleavage of the first reporter or the second reporter, respectively. In some embodiments, the detection moiety comprises a fluorophore, a particle, an affinity molecule, an enzyme, or an enzyme substrate. In some embodiments, the particle comprises a quantum dot. In some embodiments, the particle is configured for encoded particle multiplexing. In some embodiments, the particle comprises one or more lanthanide elements. In some embodiments, the reporter comprises a fluorophore and a quencher, wherein the quenching moiety comprises the quencher, and wherein release of the quenching moiety results in fluorescence of the fluorophore. In some embodiments, the reporter comprises a fluorophore and a quencher, wherein the detection moiety comprises the fluorophore, and wherein release of the detection moiety results in fluorescence of the fluorophore. In some embodiments, the first programmable nuclease and the second programmable nuclease are different types of programmable nuclease. In some embodiments, the first programmable nuclease and the second programmable nuclease are the same type of programmable nuclease. In some embodiments, the first guide nucleic acid and the second guide nucleic acids are configured to bind to the same target nucleic acid sequence. In some embodiments, the first guide nucleic acid and the second guide nucleic acids are configured to bind to different target nucleic acid sequences.

[0009] Described herein are various methods comprising: providing a surface comprising a plurality of programmable nuclease complexes and a plurality of reporters immobilized thereon, wherein each programmable nuclease complex of the plurality of programmable nuclease complexes comprises a programmable nuclease and a guide nucleic acid; applying a sample to the surface, wherein the sample comprises a plurality of nucleic acids, wherein an amount of programmable nuclease complexes in the plurality of programmable nuclease complexes is greater than an amount of nucleic acids in the plurality of nucleic acids; and quantifying cleavage of the plurality of reporters, wherein at least a first reporter of the plurality of reporters is configured to be cleaved upon binding of a first guide nucleic acid of the plurality of programmable nuclease complexes to a first target nucleic acid of the plurality of nucleic acids, and wherein cleavage of the first reporter indicates a presence of the first target nucleic acid in the sample. In some embodiments, the surface is a surface of a spotted microarray, an array of particles, or an array of microwells, and wherein the surface comprises a plurality of locations, each of plurality of locations comprises at least one programmable nuclease complex of the plurality of programmable nuclease complexes and at least one reporter of the plurality of reporters immobilized on the surface. In some embodiments, the plurality of particles comprises a plurality of beads. In some embodiments, a first location of the plurality of locations comprises the first programmable nuclease complex and the first reporter. In some embodiments, the surface is a surface of a fluidic channel and wherein applying the sample comprises flowing the sample over the surface. In some embodiments, cleavage of the first reporter induces a change in the surface at a location corresponding to a location of the first reporter, and wherein quantifying cleavage comprises detecting the change. In some embodiments, the change comprises a change in conductivity, a change in fluorescence intensity, a change in fluorescence wavelength, a change in absorbance, a change in luminescence, or an amperometric change. In some embodiments, the first programmable nuclease or the first guide nucleic acid is coupled to a detection moiety of the first reporter, wherein cleavage of the first reporter releases the detection moiety and the first programmable nuclease from the surface of the first fluidic channel and wherein quantifying cleavage comprises detection moiety as the first programmable nuclease flows past a detector after being released by cleavage of the first reporter. In some embodiments, a ratio of the amount of nucleic acids to the amount of programmable nuclease complexes is about 1 :2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10. In some embodiments, a ratio of the amount of nucleic acids to the amount of programmable nuclease complexes is about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1 :70, about 1 :80, about 1 :90, or about 1 : 100. In some embodiments, the sample is not amplified prior to cleavage of the plurality of reporters.

INCORPORATION BY REFERENCE

[0010] 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

[0011] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings of which:

[0012] FIGS. 1A-1C illustrate exemplary immobilization strategies for programmable nuclease-based diagnostic assay components, in accordance with embodiments.

[0013] FIG. 2 illustrates an exemplary immobilization strategy combination which enables programmable nuclease-based diagnostic readouts, in accordance with embodiments. [0014] FIG. 3A illustrates an exemplary microfluidic structure for sample fractionation, in accordance with embodiments.

[0015] FIG. 3B shows an exemplary flow channel where sample solutions are both continuously fractionated and continuously reacted with programmable nuclease-based diagnostic assay components within a branching channel structure such as the structure of FIG. 3 A, in accordance with embodiments.

[0016] FIG. 3C shows another exemplary flow channel where sample solutions are both continuously fractionated and continuously reacted with programmable nuclease-based diagnostic assay components within a branching channel structure such as the structure of FIG. 3 A, in accordance with embodiments.

[0017] FIG. 3D shows another exemplary flow channel where sample solutions are both continuously fractionated and continuously reacted with programmable nuclease-based diagnostic assay components within a branching channel structure such as the structure of FIG. 3 A, in accordance with embodiments.

[0018] FIG. 4A shows an exemplary detection array, in accordance with embodiments.

[0019] FIG. 4B shows an exemplary detection channel, in accordance with embodiments.

DETAILED DESCRIPTION

Definitions

[0020] Unless otherwise indicated, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise indicated or obvious from context, the following terms have the following meanings:

[0021] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0022] Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

[0023] As used herein, the term “including”, as well as other forms, such as “includes” and “included,” is not limiting.

[0024] As used herein, the term “comprise” and its grammatical equivalents specifies the presence of stated features, integers, steps, operations, elements, and/or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0025] As used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers +/- 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range. [0026] 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. 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):l l-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 l):387-95). For the purposes of calculating identity to the sequence, extensions, such as tags, are not included.

[0027] The terms, “amplification” and “amplifying,” as used herein, refer to a process by which a unit is copied to increase said unit relative to its surroundings. For example, 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, or a signal can be increased, for example by modifying the signal-to-noise ratio. [0028] 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) 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.

[0029] The term, “clustered regularly interspaced short palindromic repeats

(CRISPR),” as used herein refers to a segment of DNA found in the genomes of certain prokaryotic organisms, including some bacteria and archaea, that includes repeated short sequences of nucleotides interspersed at regular intervals between unique sequences of nucleotides derived from the DNA of a pathogen (e.g., virus) that had previously infected the organism and that functions to protect the organism against future infections by the same pathogen.

[0030] The term, “CRISPR RNA (crRNA),” as used herein refers to a nucleic acid comprising a first sequence, often referred to as a “spacer sequence,” that hybridizes to a target sequence of a target nucleic acid, and a second sequence that either a) hybridizes to a portion of a tracrRNA or b) is capable of being non-covalently bound by a nuclease. In some embodiments, the crRNA is covalently linked to an additional nucleic acid (e.g., a tracrRNA), wherein the additional nucleic acid interacts with the nuclease.

[0031] The term, “detectable signal,” as used herein refers to a signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical, and other detection methods described herein or known in the art.

[0032] 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.

[0033] The term, “detection moiety,” as used herein refers to a molecule that can release a signal that can be detected using optical, fluorescent, chemiluminescent, electrochemical, calorimetric and other detection methods known in the art.

[0034] The term, “genetic disorder,” as used herein refers to a disease, disorder, condition, or syndrome caused by one or more mutations in the DNA of an organism. Mutations can be due to several different cellular mechanisms, including, but not limited to, an error in DNA replication, recombination, or repair, or due to environmental factors. A genetic disease comprises, in some embodiments, a single gene disorder, a chromosome disorder, or a multifactorial disorder.

[0035] A “syndrome”, as used herein, refers to a group of symptoms which, taken together, characterize a condition.

[0036] 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 a nuclease. 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.

[0037] The term, “linked amino acids," as used herein refers to at least two amino acids linked by an amide bond.

[0038] The terms, “linkage” or “linker,” as used herein refers to a bond or molecule that links a first polypeptide or polynucleotide to a second polypeptide or polynucleotide or to a surface. A “peptide linker” comprises at least two amino acids linked by an amide bond. [0039] The terms, “non-naturally occurring” and “engineered,” as used herein are used interchangeably and indicate the involvement of the hand of man. 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 non-limiting example, a composition may include a nuclease and a guide nucleic acid that do not naturally occur together. Conversely, and as a non-limiting further clarifying example, a nuclease or guide nucleic acid that is “natural,” “naturally-occurring,” or “found in nature” includes a nuclease and a guide nucleic acid from a cell or organism that have not been genetically modified by the hand of man.

[0040] The term, “nuclease activity,” as used herein refers to the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bonds between the nucleotide subunits of nucleic acids; the term “endonuclease activity” refers to the enzymatic activity of an enzyme which allows the enzyme to cleave the phosphodiester bond within a polynucleotide chain. An enzyme with nuclease activity may be referred to as a “nuclease.”

[0041] The term, “protospacer adjacent motif (PAM),” as used herein refers to a nucleotide sequence found in a target nucleic acid that directs a nuclease to detect and modify the target nucleic acid at a specific location. A PAM sequence may be required for a complex having a nuclease and a guide nucleic acid to hybridize to and modify the target nucleic acid. However, a given nuclease may not require a PAM sequence being present in a target nucleic acid for the nuclease to modify the target nucleic acid. [0042] The term, “quenching moiety,” as used herein refers to a molecule that can quench, or significantly lessen, a signal that was detectable using optical, fluorescent, chemiluminescent, electrochemical, calorimetric and other detection methods known in the art. [0043] The term, “reporter,” as used herein refers to a molecule that can provide a detectable signal upon cleavage thereof by a nuclease.

[0044] 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 non-limiting example, samples may be modified or manipulated with purification techniques, heat, nucleic acid amplification, salts, and/or buffers.

[0045] The term, “subject,” as used herein can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a member of the animal kingdom. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In some instances, the subject is not necessarily diagnosed or suspected of being at high risk for the disease. As used herein, the terms “individual,” “subject,” and “patient” are used interchangeably.

[0046] The term, “target nucleic acid,” as used herein refers to a nucleic acid that is selected as the nucleic acid for detection, 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).

[0047] 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 an equal length portion of a guide nucleic acid. Hybridization of the guide nucleic acid to the target sequence may bring a nuclease into contact with the target nucleic acid.

[0048] The term, “trans cleavage,” is used herein in reference to cleavage (hydrolysis of a phosphodiester bond) of one or more nucleic acids by a nuclease that is complexed with a guide nucleic acid and a target nucleic acid. The one or more nucleic acids may include the target nucleic acid as well as non-target nucleic acids.

[0049] The term, “trans-activating RNA (tracrRNA),” as used herein refers to a nucleic acid that comprises a first sequence that is capable of being non-covalently bound by a nuclease. TracrRNAs may comprise a second sequence that hybridizes to a portion of a crRNA, which may be referred to as a repeat hybridization sequence. In some embodiments, tracrRNAs are covalently linked to a crRNA.

[0050] The terms, “treatment” or “treating,” as used herein are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subj ect at risk of developing a particular disease, or to a subj ect reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

Systems, Devices, or Methods for CRISPR/Cas-based Nucleic Acid Quantification [0051] In the following detailed description, reference is made to the accompanying figures, which form a part hereof. In the figures, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, figures, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the scope of the subject matter presented herein. It will be readily understood that aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein. [0052] Although certain embodiments and examples are disclosed below, inventive subject matter extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses, and to modifications and equivalents thereof. Thus, the scope of the claims appended hereto is not limited by any of the particular embodiments described below. For example, in any method or process disclosed herein, the acts or portions of the method or process may be performed in any suitable sequence and are not necessarily limited to any particular disclosed sequence. Various operations may be described as multiple discrete operations in turn, in a manner that may be helpful to understanding certain embodiments, however, the order of the description should not be construed to imply that these operations are order dependent. Additionally, structures, systems, and/or devices described herein may be embodied as integrated components or as separate components.

[0053] For the purposes of comparing various embodiments, certain aspects and advantages of these embodiments are described. Not necessarily all such aspects or advantages are achieved by any particular embodiment. Thus, for example, various embodiments may be carried out in a manner that achieves or optimizes one advantage or group of advantages taught herein without necessarily achieving other aspects or advantages as may also be taught or suggested herein.

[0054] The present disclosure is described in relation to systems, devices, or methods for CRISPR/Cas-based nucleic acid quantification. However, one of ordinary skill in the art will appreciate that this is not intended to be limiting and the devices and methods disclosed herein may be used with other programmable nucleases and/or other nucleic acid detection systems and methods.

[0055] Described herein are devices, systems, kits, and methods for quantifying an amount of a target nucleic acid in a sample. Such quantification can be used in a rapid test (e.g., a lab test or point-of-care test) for detection of a target nucleic acid of interest. For example, disclosed herein are particular microfluidic devices and compositions (e.g., programmable nucleases, guide nucleic acids, reporters, or any combination thereof) for use in said devices that are particularly well-suited for carrying out a highly efficient, rapid, and accurate reactions for detecting whether a target nucleic acid is present in a sample and in what amount. In many embodiments, the rapid tests can be performed in a single system. The target nucleic acid may be a portion of a nucleic acid of interest, e.g., a target nucleic acid from any plant, animal, virus, or microbe of interest. [0056] In some embodiments, the device for quantifying nucleic acids comprises an array comprising a plurality of surface locations. In certain embodiments, a surface location of the plurality of surface locations comprises at least one of a programmable nuclease, a reporter, or a guide nucleic acid immobilized thereto. In certain embodiments, the plurality of surface locations is spatially distinct from each other. In certain embodiments, a first surface location of the plurality of surface locations comprises at least one of a first programmable nuclease, a first reporter, or a first guide nucleic acid immobilized thereto. In certain embodiments, a second surface location of the plurality of surface locations comprises at least one of a second programmable nuclease, a second reporter, or a second guide nucleic acid immobilized thereto. In certain embodiments, the first surface location is spatially distinct from the second surface location.

[0057] In some embodiments, the device for quantifying nucleic acids comprises an inlet and one or more fluidic channels fluidly coupled to the inlet. In certain embodiments, each of the fluidic channels comprises a plurality of surface locations. In certain embodiments, the plurality of surface locations are adjacent sections of a fluidic channel. In certain embodiments, the plurality of surface locations are parallel stripes in a same section of a fluidic channel. In certain embodiments, each of the fluidic channel comprises at least one of a programmable nuclease, a reporter, or a guide nucleic acid immobilized to a surface thereof. In certain embodiments, the plurality of surface locations is spatially distinct from each other. In certain embodiments, the device for quantifying nucleic acids comprises an inlet and a first fluidic channel fluidly coupled to the inlet. In certain embodiments, the first fluidic channel comprises at least one of a first programmable nuclease, a first reporter, or a first guide nucleic acid immobilized to a first surface thereof. In certain embodiments, the first fluidic channel comprises at least one of a second programmable nuclease, a second reporter, or a second guide nucleic acid immobilized to a second surface thereof. In certain embodiments, the first surface is spatially distinct from the second surface.

[0058] In some embodiments, the method for quantifying nucleic acids comprises: (i) providing a surface comprising a plurality of programmable nuclease complexes and a plurality of reporters immobilized thereon, wherein each programmable nuclease complex of the plurality of programmable nuclease complexes comprises a programmable nuclease and a guide nucleic acid; (ii) applying a sample to the surface, wherein the sample comprises a plurality of nucleic acids, wherein an amount of programmable nuclease complexes in the plurality of programmable nuclease complexes is greater than an amount of nucleic acids in the plurality of nucleic acids; and (iii) quantifying cleavage of the plurality of reporters, wherein at least a first reporter of the plurality of reporters is configured to be cleaved upon binding of a first guide nucleic acid of the plurality of programmable nuclease complexes to a first target nucleic acid of the plurality of nucleic acids, and wherein cleavage of the first reporter indicates a presence of the first target nucleic acid in the sample. In certain embodiments, the surface is a surface of a fluidic channel and wherein applying the sample comprises flowing the sample over the surface. In certain embodiments, cleavage of the first reporter induces a change in the surface at a location corresponding to a location of the first reporter, and wherein quantifying cleavage comprises detecting the change.

[0059] In some embodiments, the plurality of surface locations comprises a plurality of spots, a plurality of particles, or a plurality of microwells. In certain embodiments, the plurality of particles comprises a plurality of beads. In certain embodiments, different surface locations are located in a same spot of the plurality of spots, on a same particle of the plurality of particles, or in a same microwell of the plurality of microwells, respectively.

[0060] In some embodiments, a known quantity of a plurality of programmable nucleases is bound to a known quantity of beads that have reporters attached thereto and is mixed with a plurality of nucleic acids. In some embodiments, fractionation is done before the plurality of nucleic acids are added, and in some embodiments, the fractionation occurs afterwards. In some embodiments, a detector detects a signal indicative of cleavage of the reporter. In some embodiments, the plurality of nucleic acids is less than the known quantity of programmable nucleases, and the cleaved products can be used to quantify an absolute value as to the quantity of the plurality of nucleic acids. In some embodiments, the cleaved products can be compared to one or more standards having known values in order to quantify a relative value as to the quantity of the plurality of nucleic acids.

[0061] Any of the devices, systems, or kits described herein may comprise one or more reagents for programmable nuclease-based detection of a target nucleic acid. Any of the devices, systems, or kits described herein may be configured to monitor and/or visualize a target nucleic acid in a sample. Exemplary devices may include a particular microfluidic device (e.g., a pneumatic valve device, a sliding valve device, a rotating valve device, or the like), a lateral flow device, or a sample preparation device. In some embodiments, any of the devices, systems, or kits described herein may be configured to perform one or more method steps, such as some or all of the steps of sample preparation, programmable nuclease reaction incubation, and/or signal detection or readout. [0062] Any of the devices, systems, kits, or methods described herein may be used to detect and/or quantify an amount of a target nucleic acid. The target nucleic acid may be from any organism, including, but not limited to, a bacterium, a virus, a parasite, a protozoon, a fungus, a mammal, a plant, and an insect. In some embodiments, a target nucleic acid may be at least a portion of a nucleic acid from a virus, a bacterium, or other pathogenic agent responsible for a disease. In some embodiments, a target nucleic acid may be at least a portion of a nucleic acid from a gene expressed in a cancerous cell or responsible for a genetic disorder. In some embodiments, a target nucleic acid may be at least a portion of a nucleic acid from a gene expressed in response by a host (e.g., a growth factor, cytokine, etc.) in response to an agent responsible for disease (e.g., a cancer cell, virus, etc.). In some embodiments, a target nucleic acid may contain a mutation (e.g., single strand polymorphism, point mutation, insertion, or deletion), be contained in an amplicon, or be uniquely identifiable from the surrounding nucleic acids (e.g, contain a unique sequence of nucleotides). The target nucleic acid may be at least a portion of a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) from any organism present in the sample.

[0063] The detection of the target nucleic acid in the sample may indicate the presence of the disease in the sample and may provide information to enable a user to take appropriate action, such as to reduce the transmission of the disease to individuals in the disease-affected environment or near the disease-carrying individual. Detection of the target nucleic acid in the sample may indicate the presence of a disease-causing or disease-related mutation, such as a single nucleotide polymorphism (SNP) that provides antibiotic resistance to a disease-causing bacteria. The detection of the target nucleic acid in the sample may indicate the presence of cancer or genetic disorder in the subject and may provide information to a user which may be useful for informing treatment and/or slowing progression of the cancer or genetic disorder in a subject. The detection of the target nucleic acid may be facilitated by a programmable nuclease as described herein.

[0064] Any of the devices, systems, kits, or methods described herein may be used to detect and/or quantify an amount of a target nucleic acid using one or more programmable nucleases.

Programmable Nucleases

[0065] 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.

[0066] 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 (also referred to herein as programmable nuclease complexes) 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. [0067] 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. Programmable nucleases can also include, for example, PfAgo and/or NgAgo.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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 instances, the Cas effector is a Cas 12 effector. In some instances, the Cas 12 effector is a Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, or Casl2j effector.

[0072] In some instances, the Type V 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.

[0073] 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 b-barrel structure. A multi-strand b-barrel structure may comprise an oligonucleotide/oligosaccharide-binding fold that is structurally comparable to those of some Cas 12 proteins. The recognition domain and the zinc finger domain may each (individually or collectively) be inserted between b-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.

[0074] 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 Casl4 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 Casl4 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.

[0075] Casl4 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. [0076] Casl4 proteins may be relatively small compared to many other Cas proteins, making them suitable for nucleic acid detection or gene editing. For instance, a Cas 14 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.

[0077] In some instances, a Casl4 protein may function as an endonuclease that catalyzes cleavage at a specific position within a target nucleic acid. In some instances, a Cas 14 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.

[0078] In some instances, the Type V Cas protein is a CasO protein. A CasO protein can function as an endonuclease that catalyzes cleavage at a specific sequence in a target nucleic acid. A programmable CasO 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 CasO nuclease especially advantageous for genome engineering and new functionalities for genome manipulation. [0079] In some instances, the programmable nuclease is a Type VI Cas protein. In some embodiments, the Type VI Cas protein is a programmable Cas 13 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 Cas 13 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.

[0080] 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, Cas 13a of the present disclosure can be activated by a target RNA to initiate trans cleavage activity of the Cas 13a for the cleavage of an RNA reporter and can be activated by a target DNA to initiate trans cleavage activity of the Casl3a for trans cleavage of an RNA reporter. An RNA reporter can be an RNA-based reporter. In some embodiments, the Casl3a recognizes and detects ssDNA to initiate transcleavage of RNA reporters. Multiple Casl3a 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 Casl3 nuclease, such as Casl3a) 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 Casl3a. 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 Casl3 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 Casl3a, 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.

[0081] 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.

[0082] In some embodiments, the devices, systems, kits, or methods described herein comprise two programmable nucleases. In certain embodiments, a first programmable nuclease and thae second programmable nuclease are different types of programmable nuclease. In certain embodiments, the first programmable nuclease and the second programmable nuclease are a same type of programmable nuclease. Several programmable nucleases are consistent with the methods and devices of the present disclosure. Programmable nucleases disclosed herein can include Class 1 CRISPR/Cas proteins, such as a Type I, Type IV, or Type III CRISPR/Cas proteins. Programmable nucleases disclosed herein also may include the Class 2 CRISPR/Cas proteins, such as the Type II, Type V, and Type VI CRISPR/Cas proteins.

[0083] In some embodiments, the Type V CRISPR/Cas proteins can be a programmable Type V CRISPR/Cas protein (e.g., Casl2, Casl4, Case] , etc.). In some embodiments the programmable nuclease may lack an HNH domain. In some embodiments the programmable nuclease may comprise a RuvC domain (e.g., comprised of three RuvC subdomains). A programmable nuclease of the present disclosure can cleave a nucleic acid via a single catalytic RuvC domain. The RuvC domain can be within a nuclease, or “NUC” lobe of the protein. The programmable nuclease can further comprise a recognition, or “REC” lobe. The REC and NUC lobes can be connected by a bridge helix (e.g., Casl2). The programmable nuclease can include a protospacer adjacent motif (PAM) recognition domain. In some embodiments the nuclease can comprise two domains for PAM recognition, (e.g., termed the PAM interacting (PI) domain and the wedge (WED) domain).

[0084] In some embodiments, the Type V CRISPR/Cas programmable nuclease can be a Casl2 protein. Some non-limited examples of programmable nucleases can include a Casl2a protein, a Casl2b protein, a Casl2c protein, a Casl2d protein, or a Casl2e protein. [0085] In some embodiments, the programmable nuclease can be a Casl3 protein. In some embodiments, the programmable nuclease can be a Casl3a, Casl3b, Casl3c, Casl3d, or Casl3e protein. In some cases, the programmable nuclease can be Mad7 or Mad2. In some cases, the programmable nuclease can be a Csml, Cas9, C2c4, C2c8, C2c5, C2cl0, C2c9, or CasZ protein. In some cases, the programmable nuclease can be a smCmsl, miCmsl, obCmsl, or suCmsl protien. In some cases, the programmable nuclease can be a C2c2 protein. In some cases, the programmable nuclease can be a CasZ protein. In some cases, the programmable nuclease can be a Casl4a, Casl4b, Casl4c, Casl4d, Casl4e, Casl4f, Casl4g, Casl4h, or Casl4u protein.

[0086] Sometimes, the programmable nuclease can be a type V CRISPR-Cas system.

In some cases, the programmable nuclease can be a type VI CRISPR-Cas system. Sometimes the programmable nuclease can be a type III CRISPR-Cas system. Sometimes the programmable nuclease can be an engineered nuclease that is not from a naturally occurring CRISPR-Cas system. In some cases, the programmable nuclease can be from a bacteria. In some cases, the programmable nuclease can be from a bacteriophage. In some cases, the programmable nuclease can be human engineered. In some embodiments, the programmable nuclease may be recombineered. In some embodiments, the programmable nuclease may be derived 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 (Cca, 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 (Cca), 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 may be at least one of LbuCasl3a, LwaCasl3a, LbaCasl3a, HheCasl3a, PprCasl3a, EreCasl3a, CamCasl3a, orLshCasl3a.

[0087] In some embodiments, the programmable nuclease comprises a Casl2 protein, wherein the Casl2 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. [0088] 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: Amino Acid Sequences of Exemplary Programmable Nucleases

[0089] 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.

[0090] 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.

Engineered Proteins

[0091] In some instances, effector proteins disclosed herein are engineered proteins.

Engineered proteins are not identical to a naturally-occurring protein. Engineered proteins may provide enhanced nuclease or nickase activity as compared to a naturally occurring nuclease or nickase. An engineered protein may comprise a modified form of a wild type counterpart protein.

[0092] In some instances, effector proteins comprise at least one amino acid change e.g. , deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the effector protein relative to the wild type counterpart. For example, a nuclease domain (e.g, RuvC domain) of an effector protein may be deleted or mutated relative to a wild type counterpart effector protein so that it is no longer functional or comprises reduced nuclease activity. The effector protein 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 a guide nucleic acid to activate or repress transcription of a target nucleic acid sequence. In some instances, the enzymatically inactive protein is fused with a protein comprising recombinase activity.

[0093] In some instances, effector proteins comprise at least one amino acid change

( e.g ., deletion, insertion, or substitution) that increases the nucleic acid-cleaving activity of the effector protein relative to the wild type counterpart. The effector protein may provide at least about 20%, at least about 30%, at least about 40%, at least about 50% at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% more nucleic acid cleaving activity relative to that of the wild-type counterpart. The effector protein may provide at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold or at least about 10 fold more nucleic acid-cleaving activity relative to that of the wild-type counterpart.

Fusion Proteins

[0094] In some instances, an effector protein is a fusion protein, wherein the fusion protein comprises a Cas effector protein and a fusion partner protein. A fusion partner protein is also simply referred to herein as a fusion partner. The fusion partner may comprise a protein or a functional domain thereof. Non-limiting examples of fusion partners include cell surface receptor proteins, intracellular signaling proteins, transcription factors, or functional domains thereof. The fusion partner may comprise a signaling peptide, e.g., a nuclear localization signal (NLS).

[0095] In some instances, the fusion partner modulates transcription (e.g, inhibits transcription, increases transcription) of a target nucleic acid. In some instances, the fusion partner is a protein (or a domain from a protein) that inhibits transcription of a target nucleic acid, also referred to as a transcriptional repressor. Transcriptional repressors may inhibit transcription via recruitment of transcription inhibitor proteins, modification of target DNA such as methylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, or a combination thereof. In some instances, the fusion partner is a protein (or a domain from a protein) that increases transcription of a target nucleic acid, also referred to as a transcription activator. Transcriptional activators may promote transcription via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, or a combination thereof. [0096] In some instances, the fusion protein is a base editor. In general, a base editor comprises a deaminase. In some instances, a fusion protein that comprises a deaminase and a Cas effector protein changes a nucleobase to a different nucleobase, e.g ., cytosine to thymine or guanine to adenine.

[0097] In some instances, fusion partners provide enzymatic activity that modifies a target nucleic acid. Such enzymatic activities include, but are not limited to, histone acetyltransferase activity, histone deacetylase activity, nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, kinase activity, phosphatase activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity, and glycosylase activity. In some instances, the fusion partner comprises an RNA splicing factor.

Multimeric Complexes

[0098] In some instances, an effector protein may form a multimeric complex with another protein. In general, a multimeric complex comprises multiple programmable nucleases that non-covalently interact with one another. A multimeric complex may comprise enhanced activity relative to the activity of any one of its programmable nucleases alone. For example, a multimeric complex comprising two programmable nucleases may comprise greater nucleic acid binding affinity, cis-cleavage activity, and/or transcollateral cleavage activity than that of either of the programmable nucleases provided in monomeric form. A multimeric complex may have an affinity for a target region of a target nucleic acid and is capable of catalytic activity (e.g., cleaving, nicking or modifying the nucleic acid) at or near the target region. Multimeric complexes may be activated when complexed with a guide nucleic acid. Multimeric complexes may be activated when complexed with a guide nucleic acid and a target nucleic acid. In some instances, the multimeric complex cleaves the target nucleic acid. In some instances, the multimeric complex nicks the target nucleic acid.

[0099] In some instances, the multimeric complex is a dimer comprising two programmable nucleases of identical amino acid sequences. In some instances, the multimeric complex comprises a first programmable nuclease and a second programmable nuclease, wherein the amino acid sequence of the first programmable nuclease is at least 90%, at least 92%, at least 94%, at least 96%, at least 98% identical, or at least 99% identical to the amino acid sequence of the second programmable nuclease. In some instances, the multimeric complex is a heterodimeric complex comprising at least two programmable nucleases of different amino acid sequences. In some instances, the multimeric complex is a heterodimeric complex comprising a first programmable nuclease and a second programmable nuclease, wherein the amino acid sequence of the first programmable nuclease is less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% identical to the amino acid sequence of the second programmable nuclease.

[00100] In some instances, a multimeric complex comprises at least two programmable nucleases. In some instances, a multimeric complex comprises more than two programmable nucleases. In some instances, multimeric complexes comprise at least one Type V CRISPR/Cas protein, or a fusion protein thereof. In some instances, a multimeric complex comprises two, three or four Casl4 proteins.

Thermostable Programmable Nucleases

[00101] Described herein are various embodiments of thermostable programmable nucleases. In some embodiments, a programmable nuclease is referred to as a programmable nuclease. A programmable nuclease may be thermostable. In some instances, known programmable nucleases (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 a programmable nuclease 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, at least 10-fold, 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. In some instances, the trans cleavage activity of a programmable nuclease 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, at least 10- fold, 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. In some instances, the trans cleavage activity of a programmable nuclease 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, at least 10-fold, 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. In some instances, the trans cleavage activity of a programmable nuclease 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, at least 10-fold, 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. In some instances, the trans cleavage activity of a programmable nuclease 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, at least 10-fold, 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. In some instances, the trans cleavage activity of a programmable nuclease 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, at least 10-fold, 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. In some instances, the trans cleavage activity of a programmable nuclease in a trans cleavage assay at 70 °C, 75 °C. 80 °C, or more may be at least 50, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, 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 , at least 10-fold , 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.

Engineered Guide Nucleic Acids

[00102] Any of the devices, systems, kits, or methods described herein may comprise one or more guide nucleic acids. Guide nucleic acids and portions thereof may be found in or identified from a CRISPR array present in the genome of a host organism. A guide nucleic acid may comprise a nucleic acid that binds to a programmable nuclease to form a programmable nuclease complex. At least a portion of the guide nucleic acid may be complimentary to a target sequence of a target nucleic acid. Guide nucleic acids, when complexed with a nuclease, may bring the nuclease into proximity of a target nucleic acid. Sufficient conditions for hybridization of a guide nucleic acid to a target nucleic acid and/or for binding of a guide nucleic acid to a nuclease include in vivo physiological conditions of a desired cell type or in vitro conditions sufficient for assaying catalytic activity of a protein, polypeptide or peptide described herein, such as the nuclease activity of a nuclease. In some embodiments, the guide nucleic acid may comprise a crispr RNA (crRNA), a short-complementarity untranslated RNA (scoutRNA), a trans-activating crispr RNA (tracrRNA), a single guide RNA (sgRNA) (e.g., a crRNA linked to a tracrRNA), or a combination thereof. In some embodiments, the crRNA and the tracrRNA are covalently linked. In some embodiments, the crRNA and tracrRNA are linked by a phosphodiester bond. In some instances, the crRNA and tracrRNA are linked by one or more linked nucleotides. A crRNA may be the product of processing of a longer precursor CRISPR RNA (pre-crRNA) transcribed from the CRISPR array by cleavage of the pre-crRNA within each direct repeat sequence to afford shorter, mature crRNAs. A crRNA may be generated by a variety of mechanisms, including the use of dedicated endonucleases (e.g., Cas6 or Cas5d in Type I and III systems), coupling of a host endonuclease (e.g, RNase III) with tracrRNA (Type II systems), or a ribonuclease activity endogenous to the nuclease itself (e.g, Casl2a, from Type V systems). A crRNA may also be specifically generated outside of processing of a pre-crRNA and individually contacted to a nuclease in vivo or in vitro. A tracrRNA may be separate from, but form a complex with, a guide nucleic acid and a nuclease. The tracrRNA may be attached ( e.g ., covalently) by an artificial linker to a guide nucleic acid. A tracrRNA may include a nucleotide sequence that hybridizes with a portion of a guide nucleic acid. A tracrRNA may also form a secondary structure (e.g., one or more hairpin loops) that facilitates the binding of a nuclease to a guide nucleic acid and/or modification activity of a nuclease on a target nucleic acid. A tracrRNA may include a repeat hybridization region and a hairpin region. The repeat hybridization region may hybridize to all or part of the repeat sequence of a guide nucleic acid. The repeat hybridization region may be positioned 3’ of the hairpin region. The hairpin region may include a first sequence, a second sequence that is reverse complementary to the first sequence, and a stem-loop linking the first sequence and the second sequence. In some embodiments, the guide nucleic acid may comprise deoxyribonucleotides, ribonucleotides, chemically modified nucleosides, or a combination thereof (e.g, RNA with a thymine base). Guide nucleic acids may be referred to herein as a guide RNA (gRNA). However, a guide RNA is not limited to ribonucleotides, but may comprise deoxyribonucleotides and other chemically modified nucleotides. The guide nucleic acid may be engineered to contain a chemical or biochemical modification. The guide nucleic acid may be chemically synthesized or recombinantly produced. The sequence of the engineered guide nucleic acid, or a portion thereof, may be different from the sequence of a naturally occurring nucleic acid.

[00103] Provided herein are compositions comprising one or more engineered guide nucleic acids. A guide nucleic acid can comprise a sequence that is reverse complementary to the sequence of a target nucleic acid. Guide nucleic acids are often referred to as a “guide RNA.” However, a guide nucleic acid may comprise deoxyribonucleotides. The term “guide RNA,” as well as crRNA and tracrRNA, includes guide nucleic acids comprising DNA bases, RNA bases, and modified nucleobases. In general, a guide nucleic acid is a nucleic acid molecule that binds to an effector protein (e.g, a Cas effector protein), thereby forming a ribonucleoprotein complex (RNP). In some instances, the engineered guide RNA imparts activity or sequence selectivity to the effector protein. In general, the engineered guide nucleic acid comprises a CRISPR RNA (crRNA) that is at least partially complementary to a target nucleic acid. In some instances, the engineered guide nucleic acid comprises a trans-activating crRNA (tracrRNA), at least a portion of which interacts with the effector protein. The tracrRNA may hybridize to a portion of the guide RNA that does not hybridize to the target nucleic acid. In some instances, the crRNA and tracrRNA are provided as a single guide nucleic acid, also referred to as a single guide RNA (sgRNA). In some instances, a crRNA and tracrRNA function as two separate, unlinked molecules.

[00104] In some instances, the length of the crRNA is not greater than about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleosides. In some instances, the length of the crRNA is about 30 to about 120 linked nucleosides. In some instances, the length of a crRNA is about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 90, about 50 to about 80, about 50 to about 70, or about 50 to about 60 linked nucleosides. In some instances, the length of a crRNA is about 40, about 45, about 50, about 55, about 60, about 65, about 70 or about 75 linked nucleosides.

[00105] In general, crRNAs comprise a spacer region that hybridizes to a target sequence of a target nucleic acid, and a repeat region that interacts with the effector protein. The repeat region may also be referred to as a “protein-binding segment.” Typically, the repeat region is adjacent to the spacer region. For example, a guide RNA that interacts with the effector protein comprises a repeat region that is 5’ of the spacer region. The spacer region of the guide RNA may comprise complementarity with ( e.g ., hybridize to) a target sequence of a target nucleic acid. In some cases, the spacer region is 15-28 linked nucleosides in length. In some cases, the spacer region is 15-26, 15-24, 15-22, 15-20, 15-18, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 17-26, 17-24, 17-22, 17-20, 17-18, 18-26, 18-24, or 18-22 linked nucleosides in length. In some cases, the spacer region is 18-24 linked nucleosides in length. In some cases, the spacer region is at least 15 linked nucleosides in length. In some cases, the spacer region is at least 16, 18, 20, or 22 linked nucleosides in length. In some cases, the spacer region comprises 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 some cases, the spacer region is at least 17 linked nucleosides in length. In some cases, the spacer region is at least 18 linked nucleosides in length. In some cases, the spacer region is at least 20 linked nucleosides in length. In some cases, the spacer region is at least 80%, at least 85%, at least 90%, at least 95% or 100% complementary to a target sequence of the target nucleic acid. In some cases, the spacer region is 100% complementary to the target sequence of the target nucleic acid. In some cases, the spacer region comprises at least 15 contiguous nucleobases that are complementary to the target nucleic acid. Pooling Guide Nucleic Acids

[00106] In some embodiments, compositions, systems, or methods provided herein may comprise a pool of guide nucleic acids. In some instances, the pool of guide nucleic acids may be tiled against a target nucleic acid, e.g., the genomic locus of interest or uses thereof. In some instances, a guide nucleic acid may be selected from a group of guide nucleic acids that have been tiled against a nucleic acid sequence of a genomic locus of interest. The genomic locus of interest may belong to a viral genome, a bacterial genome, or a mammalian genome. Non limiting examples of viral genomes are an HPV genome, an HIV genome, an influenza genome, or a coronavirus genome. These guide nucleic acids may be pooled for detecting a target nucleic acid in a single assay. Pooling of guide nucleic acids may ensure broad spectrum identification, or broad coverage, of a target species within a single reaction. This may be particularly helpful in diseases or indications, like sepsis, that may be caused by multiple organisms. This may also be useful in quantification when multiple programmable nucleases complexed with guide nucleic acids tiled against different sequences of the same target nucleic acid are pooled at a single detection location, thereby increasing the chances of binding of one or more of the programmable nuclease complexes with the target nucleic acid and improving detection efficiency. The pool of guide nucleic acids may enhance the detection of a target nucleic acid using systems of methods described herein relative to detection with a single guide nucleic acid. The pool of guide nucleic acids may ensure broad coverage of the target nucleic acid within a single reaction using the methods described herein. In some instances, the pool of guide nucleic acids may be collectively complementary to 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 97%, or 100% of the target nucleic acid. In some instances, the guide nucleic acids of the pool bind to a same target nucleic acid sequence. In some instances, at least a portion of the guide nucleic acids of the pool may overlap in sequence. In some instances, at least a portion of the guide nucleic acids of the pool may not overlap in sequence. In some cases, the pool of guide nucleic acids may comprise at least 2, at least 3, at least 4, at least 5, or at least 6 guide nucleic acids targeting different sequences of a target nucleic acid. In some cases, the pool of guide nucleic acids may comprise at least 2, at least 3, at least 4, at least 5, or at least 6 guide nucleic acids targeting different target nucleic acids.

[00107] The programmable nuclease can become activated after binding of a guide nucleic acid with a target nucleic acid. The activated programmable nuclease can cleave the target nucleic acid or remain bound to the target nucleic acid once activated. Activation of the programmable nuclease may activate the trans-cleavage activity of the programmable nuclease. Trans-cleavage activity can result in non-specific cleavage of nearby single-stranded nucleic acids by the activated programmable nuclease, such as trans-cleavage of one or more reporters comprising a detection moiety as described herein. Once the reporter is cleaved by the activated programmable nuclease, the detection moiety can be released or separated from the remainder of the reporter in order to generate a detectable signal. In some embodiments, the reporter may be immobilized on a support medium. Often, the detection moiety may be at least one of a fluorophore, a dye, a polypeptide, or a nucleic acid. In some embodiments, the released detection moiety may bind to a capture molecule immobilized on the support medium. In some embodiments, the detectable signal can be visualized on the support medium to assess the presence or level (e.g., to quantify an amount) of the target nucleic acid associated with an ailment, such as a disease, cancer, or genetic disorder. In some embodiments, the detectable signal can be measured in solution. The programmable nuclease can be a CRISPR-Cas (clustered regularly interspaced short palindromic repeats - CRISPR associated) nucleoprotein complex having trans-cleavage activity, which can be activated by binding of a guide nucleic acid with a target nucleic acid as described herein.

[00108] A ratio of the amount of target nucleic acids to the amount of programmable nuclease complexes may be about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100, or more. In some embodiments, the sample comprising the target nucleic acids are not amplified prior to cleavage of the plurality of reporters.

Reporters

[00109] In some instances, systems disclosed herein comprise a reporter. By way of non limiting and illustrative example, a reporter may comprise a single stranded nucleic acid and a detection moiety (e.g., a labeled single stranded RNA reporter), wherein the nucleic acid is capable of being cleaved by a programmable nuclease (e.g., a Type V CRISPR/Cas protein as disclosed herein) or a multimeric complex thereof, releasing the detection moiety, and, generating a detectable signal. As used herein, "reporter" is used interchangeably with "reporter nucleic acid" or "reporter molecule". The programmable nucleases disclosed herein, activated upon hybridization of a guide RNA to a target nucleic acid, may cleave the reporter. Cleaving the "reporter" may be referred to herein as cleaving the "reporter nucleic acid," the "reporter molecule," or the "nucleic acid of the reporter." Reporters may comprise RNA. Reporters may comprise DNA. Reporters may be double-stranded. Reporters may be single-stranded.

[00110] In some cases, the reporter comprises a detection moiety. In some instances, the reporter comprises a cleavage site, wherein the detection moiety is located at a first site on the reporter, wherein the first site is separated from the remainder of reporter upon cleavage at the cleavage site. In some cases, the detection moiety is 3' to the cleavage site. In some cases, the detection moiety is 5' to the cleavage site. Sometimes the detection moiety is at the 3' terminus of the nucleic acid of a reporter. In some cases, the detection moiety is at the 5' terminus of the nucleic acid of a reporter.

[00111] In some embodiments, the reporter may comprise a nucleic acid and a detection moiety. In some embodiments, a reporter is connected to a surface by a linkage. In some embodiments, a reporter may comprise at least one of a nucleic acid, a chemical functionality, a detection moiety, a quenching moiety, or a combination thereof. In some embodiments, a reporter is configured for the detection moiety to remain immobilized to the surface and the quenching moiety to be released into solution upon cleavage of the reporter. In some embodiments, a reporter is configured for the quenching moiety to remain immobilized to the surface and for the detection moiety to be released into solution, upon cleavage of the reporter. Often the detection moiety is at least one of a label, a polypeptide, a dendrimer, or a nucleic acid, or a combination thereof. In some embodiments, the reporter contains a label. In some embodiments, the label may be FITC, DIG, TAMRA, Cy5, AF594, or Cy3. In some embodiments, the label may comprise a dye, a nanoparticle configured to produce a signal. In some embodiments, the dye may be a fluorescent dye. In some embodiments, the at least one chemical functionality may comprise biotin. In some embodiments, the at least one chemical functionality may be configured to be captured by a capture probe. In some embodiments, the at least one chemical functionality may comprise biotin and the capture probe may comprise anti-biotin, streptavidin, avidin or other molecule configured to bind with biotin. In some embodiments, the dye is the chemical functionality. In some embodiments, a capture probe may comprise a molecule that is complementary to the chemical functionality. 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. In some embodiments, the detection moiety can be the chemical functionality. [00112] In some instances, reporters comprise a detection moiety capable of generating a signal. A signal may be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some cases, the reporter comprises a detection moiety. Suitable detectable labels and/or moieties that may provide a signal include, but are not limited to, an enzyme, a radioisotope, a member of a specific binding pair, a fluorophore, a fluorescent protein, a quantum dot, and the like.

[00113] In some cases, the reporter comprises a detection moiety and a quenching moiety. In some instances, the reporter comprises a cleavage site, wherein the detection moiety is located at a first site on the reporter and the quenching moiety is located at a second site on the reporter, wherein the first site and the second site are separated by 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 nucleic acid of a reporter. Sometimes the detection moiety is at the 3' terminus of the nucleic acid of a reporter. In some cases, the detection moiety is at the 5' terminus of the nucleic acid of a reporter. In some cases, the quenching moiety is at the 3' terminus of the nucleic acid of a reporter.

[00114] In some instances, reporters may comprise a protein capable of generating a signal. A signal may be a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric. In some cases, the reporter may comprise a detection moiety. Suitable detectable labels and/or moieties that may provide a signal may include, but are not limited to, an enzyme, a radioisotope, a member of a specific binding pair; a fluorophore; a fluorescent protein; a quantum dot; and the like. In some instances, reporters may comprise a fluorophore, a quencher, or a combination thereof. In some instances, reporters may comprise a fluorophore. In some instances, reporters may comprise a quencher. The devices, systems, kits, or methods described herein may comprise one or more reporters. In some embodiments, the devices, systems, kits, or methods described herein comprise two reporters. In certain embodiments, the first reporter and the second reporter are different types of reporters. In certain embodiments, the first reporter and the second reporter are a same type of reporter.

[00115] In some cases, the reporter may comprise a detection moiety and a quenching moiety. In some instances, the reporter may comprise a cleavage site. In some instances, the detection moiety or the quenching moiety is configured to be released upon cleavage of the reporter. In some instances, the release results in signals from the detection moiety (e.g., fluorescence of the fluorophore). The detection moiety may be located at a first site on the reporter and the quenching moiety may be located at a second site on the reporter. The first site and the second site may be separated by the cleavage site. Sometimes the quenching moiety may be a fluorescence quenching moiety. In some cases, the quenching moiety may be 5’ to the cleavage site and the detection moiety may be 3’ to the cleavage site. In some cases, the detection moiety may be 5’ to the cleavage site and the quenching moiety may be 3’ to the cleavage site. Sometimes the quenching moiety may be at the 5’ terminus of the nucleic acid of a reporter. Sometimes the detection moiety may be at the 3’ terminus of the nucleic acid of a reporter. In some cases, the detection moiety may be at the 5’ terminus of the nucleic acid of a reporter. In some cases, the quenching moiety may be at the 3’ terminus of the nucleic acid of a reporter.

[00116] The detection moiety can be a fluorophore, a particle, an affinity molecule, an enzyme, or an enzyme substrate. In certain embodiments, the particle comprises a quantum dot. In certain embodiments, the particle is configured for encoded particle multiplexing. In certain embodiments, the particle comprises one or more lanthanide elements.

[00117] Suitable fluorescent proteins may include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t- HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFPl, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Suitable enzymes include, but are not limited to, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, b-glucuronidase, invertase, Xanthine Oxidase, firefly luciferase, and glucose oxidase (GO).

[00118] In some instances, the detection moiety may comprise an invertase. The substrate of the invertase may be sucrose. A DNS reagent may be included in the system to produce a colorimetric change when the invertase converts sucrose to glucose. In some cases, the reporter nucleic acid and invertase may be conjugated using a heterobifunctional linker via sulfo-SMCC chemistry. [00119] Suitable fluorophores may provide a detectable fluorescence signal in the same range as 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). Non-limiting examples of fluorophores are fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester). The fluorophore may be an infrared fluorophore. The fluorophore may emit fluorescence in the range of 500 nm and 720 nm. In some cases, the fluorophore may emit fluorescence at a wavelength of 700 nm or higher. In other cases, the fluorophore may emit fluorescence at about 665 nm. In some cases, the fluorophore may emit fluorescence in the range of 500 nm to 520 nm, 500 nm to 540 nm, 500 nm to 590 nm, 590 nm to 600 nm, 600 nm to 610 nm, 610 nm to 620 nm, 620 nm to 630 nm, 630 nm to 640 nm, 640 nm to 650 nm, 650 nm to 660 nm, 660 nm to 670 nm, 670 nm to 680 nm, 690 nm to 690 nm, 690 nm to 700 nm, 700 nm to 710 nm, 710 nm to 720 nm, or 720 nm to 730 nm. In some cases, the fluorophore emits fluorescence in the range 450 nm to 750 nm, 500 nm to 650 nm, or 550 to 650 nm.

[00120] Systems described herein may comprise a quenching moiety. A quenching moiety may be chosen based on its ability to quench the detection moiety. A quenching moiety may be a non-fluore scent fluorescence quencher. A quenching moiety may quench a detection moiety that emits fluorescence in the range of 500 nm and 720 nm. A quenching moiety may quench a detection moiety that emits fluorescence in the range of 500 nm and 720 nm. In some cases, the quenching moiety may quench a detection moiety that emits fluorescence at a wavelength of 700 nm or higher. In other cases, the quenching moiety may quench a detection moiety that emits fluorescence at about 660 nm or about 670 nm. In some cases, the quenching moiety may quench a detection moiety that emits fluorescence in the range of 500 to 520, 500 to 540, 500 to 590, 590 to 600, 600 to 610, 610 to 620, 620 to 630, 630 to 640, 640 to 650, 650 to 660, 660 to 670, 670 to 680, 690 to 690, 690 to 700, 700 to 710, 710 to 720, or 720 to 730 nm. In some cases, the quenching moiety may quench a detection moiety that emits fluorescence in the range 450 nm to 750 nm, 500 nm to 650 nm, or 550 to 650 nm. A quenching moiety may quench fluorescein amidite, 6-Fluorescein, IRDye 700, TYE 665, Alex Fluor 594, or ATTO TM 633 (NHS Ester). A quenching moiety may be Iowa Black RQ, Iowa Black FQ or IRDye QC-1 Quencher. A quenching moiety may quench fluorescein amidite, 6-Fluorescein (Integrated DNA Technologies), IRDye 700 (Integrated DNA Technologies), TYE 665 (Integrated DNA Technologies), Alex Fluor 594 (Integrated DNA Technologies), or ATTO TM 633 (NHS Ester) (Integrated DNA Technologies). A quenching moiety may be Iowa Black RQ (Integrated DNA Technologies), Iowa Black FQ (Integrated DNA Technologies) or IRDye QC-1 Quencher (LiCor). Any of the quenching moieties described herein may be from any commercially available source, may be an alternative with a similar function, a generic, or a non-tradename of the quenching moieties listed.

[00121] The generation of the detectable signal from the release of the detection moiety or the quenching moiety may indicate that cleavage by the programmable nuclease has occurred and that the sample contains a copy of the target nucleic acid. In some cases, the detection moiety may comprise a fluorescent dye. Sometimes the detection moiety may comprise a fluorescence resonance energy transfer (FRET) pair. In some cases, the detection moiety may comprise an infrared (IR) dye. In some cases, the detection moiety may comprise an ultraviolet (UV) dye. Alternatively, or in combination, the detection moiety may comprise a protein. Sometimes the detection moiety may comprise a biotin. Sometimes the detection moiety may comprise at least one of avidin or streptavidin. In some instances, the detection moiety may comprise a polysaccharide, a polymer, or a nanoparticle. In some instances, the detection moiety may comprise a gold nanoparticle or a latex nanoparticle. In some instances, the detection moiety may comprise an encoded particle, e.g., a lanthanide series encoded particle. In some instances, the detection moiety may comprise a quantum dot.

[00122] A detection moiety may be any moiety capable of generating a calorimetric, potentiometric, amperometric, optical (e.g., fluorescent, colorimetric, etc.), or piezo-electric signal. In some embodiments, the reporter may comprise a protein-nucleic acid that is capable of generating 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 nucleic acids of a reporter. Sometimes, a calorimetric signal is heat absorbed after cleavage of the nucleic acids of a reporter. A potentiometric signal, for example, is electrical potential produced after cleavage of the nucleic acids of a reporter. An amperometric signal may be movement of electrons produced after the cleavage of nucleic acid of a reporter. Often, the signal is an optical signal, such as a colorimetric signal or a fluorescence signal (e.g., a change in fluorescence intensity or a change in fluorescence wavelength). An optical signal is, for example, a light output produced after the cleavage of the nucleic acids of a reporter. Sometimes, an optical signal is a change in light absorbance or luminescence between before and after the cleavage of nucleic acids of a reporter. Often, a piezo-electric signal is a change in mass between before and after the cleavage of the nucleic acid of a reporter.

[00123] The detectable signal may be a colorimetric signal or a signal visible by eye. In some instances, the detectable signal may be fluorescent, electrical, chemical, electrochemical, or magnetic. In some cases, a first detection signal may be generated by binding of the detection moiety to the capture molecule in a detection region, location, or channel, where the first detection signal may indicate that the sample contained a first target nucleic acid. Sometimes systems and devices described herein may be capable of detecting more than one type of target nucleic acid, wherein the system comprises more than one type of guide nucleic acid and more than one type of reporter nucleic acid. In some cases, the detectable signal may be generated directly by the cleavage event. Alternatively, or in combination, the detectable signal may be generated indirectly by the cleavage event. Sometimes the detectable signal may not be a fluorescent signal. In some instances, the detectable signal may be a colorimetric or color-based signal. In some cases, the detected target nucleic acid may be identified based on its spatial location within the fluidic channel, array, or the like. In some cases, a second detectable signal may be generated in a spatially distinct location than the first generated signal.

[00124] In some embodiments, programmable nucleases disclosed herein may be activated to initiate trans cleavage activity of an RNA reporter by RNA or DNA target nucleic acids. As used herein, “trans cleavage” is used interchangeably with “collateral cleavage.” A programmable nuclease as disclosed herein may, in some cases, bind to a target RNA to initiate trans cleavage of an RNA reporter, and this programmable nuclease can be referred to as an RNA-activated programmable RNA nuclease. In some instances, a programmable nuclease as disclosed herein may bind to a target DNA to initiate trans cleavage of an RNA reporter, and this programmable nuclease can be referred to as a DNA-activated programmable RNA nuclease. In some cases, a programmable nuclease as described herein may be capable of being activated by a target RNA or a target DNA. For example, a Casl3, such as Casl3a, may be activated by a target RNA nucleic acid or a target DNA nucleic acid to transcollaterally cleave RNA reporter molecules. In some embodiments, the Casl3 may bind to a target ssDNA to initiate trans cleavage of one or more RNA reporters. In some instances, a programmable nuclease may 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. Trans cleavage may occur near, but not within or directly adjacent to, the region of the target nucleic acid that is hybridized to the guide nucleic acid. [00125] A reporter may be attached to a solid support. The solid support, for example, is a surface. A surface can be an electrode. Sometimes the solid support is a bead. Often the bead is a magnetic bead. Upon cleavage, the detection moiety is liberated from the solid support and interacts with other mixtures. For example, the detection moiety is an enzyme, and upon cleavage of the nucleic acid of the enzyme-nucleic acid, the enzyme flows through a chamber into a mixture comprising the substrate. When the enzyme meets the enzyme substrate, a reaction occurs, such as a colorimetric reaction, which is then detected. As another example, the detection moiety is an enzyme substrate, and upon cleavage of the nucleic acid of the enzyme substrate-nucleic acid, the enzyme substrate flows through a chamber into a mixture comprising the enzyme. When the enzyme substrate meets the enzyme, a reaction occurs, such as a calorimetric reaction, which is then detected.

[00126] In some embodiments, the reporter comprises a nucleic acid conjugated to an affinity molecule which is in turn conjugated to the fluorophore (e.g., nucleic acid - affinity molecule - fluorophore) or the nucleic acid conjugated to the fluorophore which is in turn conjugated to the affinity molecule (e.g., nucleic acid - fluorophore - affinity molecule). In some embodiments, a linker conjugates the nucleic acid to the affinity molecule. In some embodiments, a linker conjugates the affinity molecule to the fluorophore. In some embodiments, a linker conjugates the nucleic acid to the fluorophore. A linker can be any suitable linker known in the art. In some embodiments, the nucleic acid of the reporter can be directly conjugated to the affinity molecule and the affinity molecule can be directly conjugated to the fluorophore or the nucleic acid can be directly conjugated to the fluorophore and the fluorophore can be directly conjugated to the affinity molecule. In this context, “directly conjugated” indicates that no intervening molecules, polypeptides, proteins, or other moieties are present between the two moieties directly conjugated to each other. For example, if a reporter comprises a nucleic acid directly conjugated to an affinity molecule and an affinity molecule directly conjugated to a fluorophore - no intervening moiety is present between the nucleic acid and the affinity molecule and no intervening moiety is present between the affinity molecule and the fluorophore. The affinity molecule can be biotin, avidin, streptavidin, or any similar molecule.

[00127] In some cases, the reporter comprises a substrate-nucleic acid. The substrate may be sequestered from its cognate enzyme when present as in the substrate-nucleic acid, but then is released from the nucleic acid upon cleavage, wherein the released substrate can contact the cognate enzyme to produce a detectable signal. Often, the substrate is sucrose and the cognate enzyme is invertase, and a DNS reagent can be used to monitor invertase activity. [00128] A reporter may be a hybrid nucleic acid reporter. A hybrid nucleic acid reporter comprises a nucleic acid with at least one deoxyribonucleotide and at least one ribonucleotide. In some embodiments, the nucleic acid of the hybrid nucleic acid reporter can be of any length and can have any mixture of DNAs and RNAs. For example, in some cases, longer stretches of DNA can be interrupted by a few ribonucleotides. Alternatively, longer stretches of RNA can be interrupted by a few deoxyribonucleotides. Alternatively, every other base in the nucleic acid may alternate between ribonucleotides and deoxyribonucleotides. A major advantage of the hybrid nucleic acid reporter is increased stability as compared to a pure RNA nucleic acid reporter. For example, a hybrid nucleic acid reporter can be more stable in solution, lyophilized, or vitrified as compared to a pure DNA or pure RNA reporter.

[00129] The reporter can be lyophilized or vitrified. 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 is immobilized on beads, such as magnetic beads, in a chamber of a device as disclosed herein where they can be held in position by a magnet placed below the chamber.

[00130] In some cases, the reporter is a single-stranded nucleic acid comprising deoxyribonucleotides. In some cases, the reporter nucleic acid is a single-stranded nucleic acid sequence comprising ribonucleotides. The nucleic acid of a reporter may be a single-stranded nucleic acid sequence comprising at least one ribonucleotide. In some cases, the nucleic acid of a reporter 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 nucleic acid of a reporter comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 ribonucleotide residues at an internal position. In some cases, the nucleic acid of a reporter comprises 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 reporter 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 nucleic acid of a reporter has only ribonucleotide residues. In some cases, the nucleic acid of a reporter has only deoxyribonucleotide residues. In some cases, the nucleic acid comprises nucleotides resistant to cleavage by the programmable nuclease described herein. In some cases, the nucleic acid of a reporter comprises synthetic nucleotides. In some cases, the nucleic acid of a reporter comprises at least one ribonucleotide residue and at least one non-ribonucleotide residue.

[00131] In some cases, the nucleic acid of a reporter comprises at least one uracil ribonucleotide. In some cases, the nucleic acid of a reporter comprises at least two uracil ribonucleotides. Sometimes the nucleic acid of a reporter has only uracil ribonucleotides. In some cases, the nucleic acid of a reporter comprises at least one adenine ribonucleotide. In some cases, the nucleic acid of a reporter comprises at least two adenine ribonucleotide. In some cases, the nucleic acid of a reporter has only adenine ribonucleotides. In some cases, the nucleic acid of a reporter comprises at least one cytosine ribonucleotide. In some cases, the nucleic acid of a reporter comprises at least two cytosine ribonucleotide. In some cases, the nucleic acid of a reporter comprises at least one guanine ribonucleotide. In some cases, the nucleic acid of a reporter comprises at least two guanine ribonucleotide. In some instances, a nucleic acid of a reporter comprises a single unmodified ribonucleotide. In some instances, a nucleic acid of a reporter comprises only unmodified ribonucleotides. In some instances, a nucleic acid of a reporter comprises only unmodified deoxyribonucleotides.

[00132] In some cases, the nucleic acid of a reporter is 5 to 20, 5 to 15, 5 to 10, 7 to 20, 7 to 15, or 7 to 10 nucleotides in length. In some cases, the nucleic acid of a reporter is 3 to 20,

4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 13 to 20, 15 to 20, 3 to 15, 4 to 15,

5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 3 to 8, 4 to 8, 5 to 8, 6 to 8, or 7 to 8, nucleotides in length. In some cases, the nucleic acid of a reporter is 5 to 12 nucleotides in length. In some cases, the reporter nucleic acid is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotides in length. In some cases, the reporter 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 reporter can be 5, 8, or 10 nucleotides in length. For cleavage by a programmable nuclease comprising Casl2, a reporter can be 10 nucleotides in length.

[00133] In some cases, systems comprise a plurality of reporters. The plurality of reporters may comprise a plurality of signals. In some cases, systems comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 30, at least 40, or at least 50 reporters. In some cases, there are 2 to 50, 3 to 40, 4 to 30, 5 to 20, or 6 to 10 different reporters. [00134] In some instances, systems comprise a Type V CRISPR/Cas protein and a reporter nucleic acid configured to undergo transcollateral cleavage by the Type V CRISPR/Cas protein. Transcollateral cleavage of the reporter may generate a signal from the reporter or alter a signal from the reporter. In some cases, the signal is an optical signal, such as a fluorescence signal or absorbance band. Transcollateral cleavage of the reporter may alter the wavelength, intensity, or polarization of the optical signal. For example, the reporter may comprise a fluorophore and a quencher, such that transcollateral cleavage of the reporter separates the fluorophore and the quencher thereby increasing a fluorescence signal from the fluorophore. In some embodiments described herein is a method of assaying for a target nucleic acid in a sample comprising contacting the target nucleic acid with a programmable nuclease, a non-naturally occurring guide nucleic acid that hybridizes to a segment of the target nucleic acid, and a reporter nucleic acid, and assaying for a change in a signal, wherein the change in the signal is produced by cleavage of the reporter nucleic acid.

[00135] In the presence of a large amount of non-target nucleic acids, an activity of a programmable nuclease (e.g., a Type V CRISPR/Cas protein as disclosed herein) may be inhibited. If total nucleic acids are present in large amounts, they may outcompete reporters for the programmable nucleases. In some instances, systems comprise an excess of reporter(s), such that when the system is operated and a solution of the system comprising the reporter is combined with a sample comprising a target nucleic acid, the concentration of the reporter in the combined solution-sample is greater than the concentration of the target nucleic acid. In some instances, the sample comprises amplified target nucleic acid. In some instances, the sample comprises an unamplified target nucleic acid. In some instances, the concentration of the reporter is greater than the concentration of target nucleic acids and non-target nucleic acids. The non-target nucleic acids may be from the original sample, either lysed or unlysed. The non-target nucleic acids may comprise byproducts of amplification. In some instances, systems comprise a reporter wherein the concentration of the reporter in a solution 1.5 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, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 16 fold, at least 17 fold, at least 18 fold, at least 19 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold excess of total nucleic acids. 1.5 fold to 100 fold, 2 fold to 10 fold, 10 fold to 20 fold, 20 fold to 30 fold, 30 fold to 40 fold, 40 fold to 50 fold, 50 fold to 60 fold, 60 fold to 70 fold, 70 fold to 80 fold, 80 fold to 90 fold, 90 fold to 100 fold, 1.5 fold to 10 fold, 1.5 fold to 20 fold, 10 fold to 40 fold, 20 fold to 60 fold, or 10 fold to 80 fold excess of total nucleic acids.

Modified Nucleic Acids

[00136] In some cases, a reporter and/or guide nucleic acid can comprise one or more modifications, e.g., a base modification, a backbone modification, a sugar modification, etc., to provide the nucleic acid with a new or enhanced feature (e.g., improved stability).

[00137] Examples of suitable modifications include modified nucleic acid backbones and non-natural internucleoside linkages. Nucleic acids having modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphor amidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Suitable oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts (such as, for example, potassium or sodium), mixed salts and free acid forms are also included. Also suitable are nucleic acids having morpholino backbone structures. Suitable modified polynucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and Chh component parts. [00138] Other suitable modifications include nucleic acid mimetics. The term "mimetic" as it is applied to polynucleotides is intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the intemucleotide linkage are replaced with non- furanose groups, replacement of only the furanose ring is also referred to in the art as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid, a polynucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA, the sugar-backbone of a polynucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Another such mimetic is a morpholino-based polynucleotide based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A further class of nucleic acid mimetic is referred to as a cyclohexenyl nucleic acid (CeNA). The furanose ring normally present in a DNA/RNA molecule is replaced with a cyclohexenyl ring. Another modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C,4'- C-oxymethylene linkage thereby forming a bicyclic sugar moiety.

[00139] The nucleic acids described herein can include one or more substituted sugar moieties. Suitable polynucleotides comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.l to CIO alkyl or C2 to CIO alkenyl and alkynyl. Particularly suitable are 0((CH2)n0)mCH3, 0(CH2)n0CH3, 0(CH ₂ )nNH2, 0(CH ₂ )nCH3, 0(CH ₂ )n0NH ₂ , and 0(CH2)n0N((CH ₂ )nCH3)2, where n and m are from 1 to about 10. Other suitable polynucleotides comprise a sugar substituent group selected from: Ci to CIO lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O- alkaryl or O-aralkyl, SH, SCH ₃ , OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. [00140] Other suitable sugar substituent groups include methoxy (-O-CH3), aminopropoxy (— OCH2 CH ₂ CH2NH2), allyl (-CH ₂ -CH=CH ₂ ), -O-allyl (-O-CH2— CH=CH ₂ ) and fluoro (F). 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position. A suitable 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[00141] The nucleic acids described herein may include nucleobase modifications or substitutions. A labeled detector ssDNA (and/or a guide RNA) may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5-trifluoromethyl and other 5- substituted uracil s and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7- deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido(5,4- b)(l,4)benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin- 2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H- pyrimido(5,4-(b) (l,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2- one), pyridoindole cytidine (Hpyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one). Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2- pyridone.

[00142] 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).

Target Nucleic Acids

[00143] Disclosed herein are compositions, systems and methods for detecting a target nucleic acid. 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 programmable nuclease-based detection reagents (e.g., programmable nuclease, guide nucleic acid, and/or reporter). In some embodiments, the target nucleic acid is a double stranded nucleic acid. In some embodiments, the double stranded nucleic acid is DNA. The target nucleic acid may be an RNA. 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 instances, the target nucleic acid is complementary DNA (cDNA) synthesized from a single-stranded RNA template in a reaction catalyzed by a reverse transcriptase. In some cases, the target nucleic acid is single-stranded RNA (ssRNA) or mRNA. In some cases, the target nucleic acid is from a virus, a parasite, or a bacterium described herein.

[00144] In some cases, the target nucleic acid comprises 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. In some cases, the target nucleic acid comprises 10 to 90, 20 to 80, 30 to 70, or 40 to 60 nucleotides in length. In some instances, the target 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. In some cases, the target nucleic acid comprises 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. In some instances, the target nucleic acid comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 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.

[00145] A programmable nuclease-guide nucleic acid complex may comprise high selectivity for a target sequence. In some cases, a ribonucleoprotein may comprise a selectivity of at least 200:1, 100:1, 50:1, 20:1, 10:1, or 5:1 for a target nucleic acid over a single nucleotide variant of the target nucleic acid. In some cases, a ribonucleoprotein may comprise a selectivity of at least 5 : 1 for a target nucleic acid over a single nucleotide variant of the target nucleic acid. Leveraging programmable nuclease selectivity, some methods described herein may 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 comprises 1 to 10,000, 100 to 8000, 400 to 6000, 500 to 5000, 1000 to 4000, or 2000 to 3000 target nucleic acids. In some cases, the method detects target nucleic acid present at least at one copy per 10 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 10 ¹⁰ non-target nucleic acids.

[00146] Often, the target nucleic acid may be from 0.05% to 20% of total nucleic acids in the sample. Sometimes, the target nucleic acid is 0.1% to 10% of the total nucleic acids in the sample. The target nucleic acid, in some cases, is 0.1% to 5% of the total nucleic acids in the sample. The target nucleic acid may also be 0.1% to 1% of the total nucleic acids in the sample. The target nucleic acid may be DNA or RNA. The target nucleic acid may be any amount less than 100% of the total nucleic acids in the sample. The target nucleic acid may be 100% of the total nucleic acids in the sample.

[00147] The target nucleic acid may be 0.05% to 20% of total nucleic acids in the sample. Sometimes, the target nucleic acid is 0.1% to 10% of the total nucleic acids in the sample. The target nucleic acid, in some cases, is 0.1% to 5% of the total nucleic acids in the sample. Often, a sample comprises the segment of the target nucleic acid and at least one nucleic acid comprising less than 100% sequence identity to the segment of the target nucleic acid but no less than 50% sequence identity to the segment of the target nucleic acid. For example, the segment of the target nucleic acid comprises a mutation as compared to at least one nucleic acid comprising less than 100% sequence identity to the segment of the target nucleic acid but no less than 50% sequence identity to the segment of the target nucleic acid. Often, the segment of the target nucleic acid comprises a single nucleotide mutation as compared to at least one nucleic acid comprising less than 100% sequence identity to the segment of the target nucleic acid but no less than 50% sequence identity to the segment of the target nucleic acid.

[00148] A target nucleic acid may be an amplified nucleic acid of interest. The nucleic acid of interest may be any nucleic acid disclosed herein or from any sample as disclosed herein. The nucleic acid of interest may be an RNA that is reverse transcribed before amplification. The nucleic acid of interest may be amplified then the amplicons may be transcribed into RNA.

[00149] In some instances, compositions described herein exhibit indiscriminate trans cleavage of ssRNA, enabling their use for detection of RNA in samples. In some cases, target ssRNA are generated from many nucleic acid templates (RNA) in order to achieve cleavage of the FQ reporter in a programmable nuclease-based assay. Certain programmable nucleases may be activated by ssRNA, upon which they may exhibit trans-cleavage of ssRNA and may, thereby, be used to cleave ssRNA FQ reporter molecules in a programmable nuclease-based assay. These programmable nucleases may target ssRNA present in the sample, or generated and/or amplified from any number of nucleic acid templates (RNA). Described herein are reagents comprising a single stranded reporter nucleic acid comprising a detection moiety, wherein the reporter nucleic acid (e.g., the ssDNA-FQ reporter described above) is capable of being cleaved by the programmable nuclease, upon generation and amplification of ssRNA from a nucleic acid template using the methods disclosed herein, thereby generating a first detectable signal.

[00150] In some instances, target nucleic acids comprise at least one nucleic acid comprising at least 50% sequence identity to the target nucleic acid or a portion thereof. Sometimes, the at least one nucleic acid comprises an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an equal length portion of the target nucleic acid. Sometimes, the at least one nucleic acid comprises an amino acid sequence that is 100% identical to an equal length portion of the target nucleic acid. Sometimes, the amino acid sequence of the at least one nucleic acid is at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the target nucleic acid. Sometimes, the target nucleic acid comprises an amino acid sequence that is less than 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an equal length portion of the at least one nucleic acid.

[00151] In some embodiments, samples comprise a target nucleic acid at a concentration of less than 1 nM, less than 2 nM, less than 3 nM, less than 4 nM, less than 5 nM, less than 6 nM, less than 7 nM, less than 8 nM, less than 9 nM, less than 10 nM, less than 20 nM, less than 30 nM, less than 40 nM, less than 50 nM, less than 60 nM, less than 70 nM, less than 80 nM, less than 90 nM, less than 100 nM, less than 200 nM, less than 300 nM, less than 400 nM, less than 500 nM, less than 600 nM, less than 700 nM, less than 800 nM, less than 900 nM, less than 1 mM, less than 2 pM, less than 3 pM, less than 4 pM, less than 5 pM, less than 6 pM, less than 7 pM, less than 8 pM, less than 9 pM, less than 10 pM, less than 100 pM, or less than 1 mM. In some embodiments, the sample comprises a target nucleic acid sequence at a concentration of 1 nM to 2 nM, 2 nM to 3 nM, 3 nM to 4 nM, 4 nM to 5 nM, 5 nM to 6 nM, 6 nM to 7 nM, 7 nM to 8 nM, 8 nM to 9 nM, 9 nM to 10 nM, 10 nM to 20 nM, 20 nM to 30 nM, 30 nM to 40 nM, 40 nM to 50 nM, 50 nM to 60 nM, 60 nM to 70 nM, 70 nM to 80 nM, 80 nM to 90 nM, 90 nM to 100 nM, 100 nM to 200 nM, 200 nM to 300 nM, 300 nM to 400 nM, 400 nM to 500 nM, 500 nM to 600 nM, 600 nM to 700 nM, 700 nM to 800 nM, 800 nM to 900 nM, 900 nM to 1 pM, 1 pM to 2 pM, 2 pM to 3 pM, 3 pM to 4 pM, 4 pM to 5 pM, 5 pM to 6 pM, 6 pM to 7 pM, 7 pM to 8 pM, 8 pM to 9 pM, 9 pM to 10 pM, 10 pM to 100 pM, 100 pM to 1 mM, 1 nM to 10 nM, 1 nM to 100 nM, 1 nM to 1 pM, 1 nM to 10 pM, 1 nM to 100 pM, 1 nM to 1 mM, 10 nM to 100 nM, 10 nM to 1 pM, 10 nM to 10 pM, 10 nM to 100 pM, 10 nM to 1 mM, 100 nM to 1 pM, 100 nM to 10 pM, 100 nM to 100 pM, 100 nM to 1 mM, 1 pM to 10 pM, 1 pM to 100 pM, 1 pM to 1 mM, 10 pM to 100 pM, 10 pM to 1 mM, or 100 pM to 1 mM. In some embodiments, the sample comprises a target nucleic acid at a concentration of 20 nM to 200 pM, 50 nM to 100 pM, 200 nM to 50 pM, 500 nM to 20 pM, or 2 pM to 10 pM. In some embodiments, the target nucleic acid is not present in the sample. [00152] In some embodiments, samples comprise fewer than 10 copies, fewer than 100 copies, fewer than 1000 copies, fewer than 10,000 copies, fewer than 100,000 copies, or fewer than 1,000,000 copies of a target nucleic acid sequence. In some embodiments, the sample comprises 10 copies to 100 copies, 100 copies to 1000 copies, 1000 copies to 10,000 copies, 10,000 copies to 100,000 copies, 100,000 copies to 1,000,000 copies, 10 copies to 1000 copies, 10 copies to 10,000 copies, 10 copies to 100,000 copies, 10 copies to 1,000,000 copies, 100 copies to 10,000 copies, 100 copies to 100,000 copies, 100 copies to 1,000,000 copies, 1,000 copies to 100,000 copies, or 1,000 copies to 1,000,000 copies of a target nucleic acid sequence. In some embodiments, the sample comprises 10 copies to 500,000 copies, 200 copies to 200,000 copies, 500 copies to 100,000 copies, 1000 copies to 50,000 copies, 2000 copies to 20,000 copies, 3000 copies to 10,000 copies, or 4000 copies to 8000 copies. In some embodiments, the target nucleic acid is not present in the sample.

[00153] A number of target nucleic acid populations are consistent with the methods and compositions disclosed herein. Some methods described herein may 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 3 to 50, 5 to 40, or 10 to 25 target nucleic acid populations. In some cases, the method detects 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 10 ¹⁰ non-target nucleic acids. The target nucleic acid populations may be present at different concentrations or amounts in the sample.

[00154] In some embodiments, target nucleic acids may activate a programmable nuclease to initiate sequence-independent cleavage of a nucleic acid-based reporter (e.g., a reporter comprising an RNA sequence, or a reporter comprising DNA and RNA). For example, a programmable nuclease of the present disclosure is activated by a target nucleic acid to cleave reporters having an RNA (also referred to herein as an "RNA reporter"). Alternatively, a programmable nuclease of the present disclosure is activated by a target RNA to cleave reporters having an RNA (also referred to herein as a "RNA reporter"). The RNA reporter may comprise a single-stranded RNA labeled with a detection moiety or may be any RNA reporter as disclosed herein.

[00155] In some embodiments, the target nucleic acid as described in the methods herein does not initially comprise a PAM sequence. However, any target nucleic acid of interest may be generated using the methods described herein to comprise a PAM sequence, and thus be a PAM target nucleic acid. A PAM target nucleic acid, as used herein, refers to a target nucleic acid that has been amplified to insert a PAM sequence that is recognized by a CRISPR/Cas system.

[00156] In some embodiments, the target nucleic acid is in a cell. In some embodiments, the cell is a single-cell eukaryotic organism; a plant cell an algal cell; a fungal cell; an animal cell; a cell an invertebrate animal; a cell a vertebrate animal such as fish, amphibian, reptile, bird, and mammal; or a cell a mammal such as a human, a non-human primate, an ungulate, a feline, a bovine, an ovine, and a caprine. In preferred embodiments, the cell is a eukaryotic cell. In preferred embodiments, the cell is a mammalian cell, a human cell, or a plant cell.

[00157] In some embodiments, the target nucleic acid sequence comprises a nucleic acid sequence of a virus, a bacterium, or other pathogen responsible for a disease in a plant (e.g., a crop). Methods and compositions of the disclosure may be used to treat or detect a disease in a plant. For example, the methods of the disclosure may be used to target a viral nucleic acid sequence in a plant. A programmable nuclease of the disclosure (e.g., Casl4) may cleave the viral nucleic acid. In some embodiments, the target nucleic acid sequence comprises a nucleic acid sequence of a virus or a bacterium or other agents (e.g., any pathogen) responsible for a disease in the plant (e.g., a crop). In some embodiments, the target nucleic acid comprises RNA. The target nucleic acid, in some cases, is a portion of a nucleic acid from a virus or a bacterium or other agents responsible for a disease in the plant (e.g., a crop). In some cases, the target nucleic acid is a portion of a nucleic acid from a genomic locus, or any NA amplicon, such as a reverse transcribed mRNA or a cDNA from a gene locus, a transcribed mRNA, or a reverse transcribed cDNA from a gene locus in at a virus or a bacterium or other agents (e.g., any pathogen) responsible for a disease in the plant (e.g., a crop). A virus infecting the plant may be an RNA virus. A virus infecting the plant may be a DNA virus. Non-limiting examples of viruses that may be targeted with the disclosure include Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Cauliflower mosaic virus (CaMV) (RT virus), Plum pox virus (PPV), Brome mosaic virus (BMV) and Potato virus X (PVX).

Mutations

[00158] In some instances, target nucleic acids comprise a mutation. In some instances, a sequence comprising a mutation may be modified to a wildtype sequence with a composition, system or method described herein. In some instances, a sequence comprising a mutation may be detected with a composition, system or method described herein. The mutation may be a mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. Non-limiting examples of mutations are insertion-deletion (indel), single nucleotide polymorphism (SNP), and frameshift mutations. In some instances, guide nucleic acids described herein hybridize to a region of the target nucleic acid comprising the mutation. The mutation may be located in a non-coding region or a coding region of a gene.

[00159] In some instances, target nucleic acids comprise a mutation, wherein the mutation is a SNP. The single nucleotide mutation or SNP may be associated with a phenotype of the sample or a phenotype of the organism from which the sample was taken. The SNP, in some cases, is associated with altered phenotype from wild type phenotype. The SNP may be a synonymous substitution or a nonsynonymous substitution. The nonsynonymous substitution may be a missense substitution or a nonsense point mutation. The synonymous substitution may be a silent substitution. The mutation may be a deletion of one or more nucleotides. Often, the single nucleotide mutation, SNP, or deletion is associated with a disease such as cancer or a genetic disorder. The mutation, such as a single nucleotide mutation, a SNP, or a deletion, may be encoded in the sequence of a target nucleic acid from the germline of an organism or may be encoded in a target nucleic acid from a diseased cell, such as a maycer cell.

[00160] In some instances, target nucleic acids comprise a mutation, wherein the mutation is a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. The mutation may be a deletion of about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 nucleotides. The mutation may be a deletion of 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, 95 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900, 900 to 1000, 1 to 50, 1 to 100, 25 to 50, 25 to 100, 50 to 100, 100 to 500, 100 to 1000, or 500 to 1000 nucleotides.

Samples

[00161] 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 mΐ. 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 mΐ, or any of value from 1 mΐ to 500 mΐ. 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 pi. [00162] 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.

[00163] 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.

[00164] 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.

[00165] 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.

[00166] 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).

[00167] 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- CoV-1, MERS-CoV, influenza, Adenovirus, Coronavirus HKU1, Coronavirus NL63, Coronavirus 229E, Coronavirus OC43, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Human Metapneumovirus (hMPV), Human Rhinovirus (HRVs A, B, C), Human Enterovirus, Influenza A, Influenza A/HI, Influenza A/H2, Influenza A/H3, Influenza A/H4, Influenza A/H5, Influenza A/H6, Influenza A/H7, Influenza A/H8, Influenza A/H9, Influenza A/H10, Influenza A/Hl l, Influenza A/H12, Influenza A/H13, Influenza A/H14, Influenza A/H15, Influenza A/H16, Influenza A/H 1-2009, Influenza A/Nl, Influenza A/N2, Influenza A/N3, Influenza A/N4, Influenza A/N5, Influenza A/N6, Influenza A/N7, Influenza A/N8, Influenza A/N9, Influenza A/N10, Influenza A/Nl l, oseltamivir-resistant Influenza A, Influenza B, Influenza B - Victoria VI, Influenza B - Yamagata Yl, Influenza C, Parainfluenza Virus 1, Parainfluenza Virus 2, Parainfluenza Virus 3, Parainfluenza Virus 4, Respiratory Syncytial Virus A, Respiratory Syncytial Virus B) and respiratory bacteria (e.g., Bordetella parapertussis , Bordetella pertussis , Bordetella bronchiseptica , Bordetella holmesii , 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, Bordetella 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 intermdius, 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), Alphacoronavirus, Betacoronavirus, Sarbecovirus, SARS-related virus, Gammacoronavirus, Deltacoronavirus, M genitalium , T vaginalis, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, human adenovirus (type A, B, C, D, E, F, G), 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, Human Bocavirus, 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. or ale, M. arginini, Acholeplasma laidlawii, M. salivarium and M pneumoniae. SARS-CoV-2 Variants include Coronavirus HKU1, Coronavirus NL63, Coronavirus 229E, Coronavirus OC43, SARS-CoV-2 85D, SARS-CoV-2 T1001I, SARS- CoV-2 3675-3677D, SARS-CoV-2 P4715L, SARS-CoV-2 S5360L, SARS-CoV-2 69-70D, SARS-CoV-2 Tyrl44fs, SARS-CoV-2 242-244D, SARS-CoV-2 Y453F, SARS-CoV-2 S477N, SARS-CoV-2 E848K, SARS-CoV-2 N501Y, SARS-CoV-2 D614G, SARS-CoV-2 P681R, SARS-CoV-2 P681H, SARS-CoV-2 L21F, SARS-CoV-2 Q27Stop, SARS-CoV-2 Mlfs, and SARS-CoV-2 R203fs. 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.

[00168] In some instances, the target sequence is a portion of a nucleic acid from a subject having cancer. The cancer may be a solid cancer (tumor). The cancer may be a blood cell cancer, including leukemias and lymphomas. Non-limiting types of cancer that could be treated with such methods and compositions include colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, bladder cancer, cancer of the kidney or ureter, lung cancer, cancer of the small intestine, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, brain cancer ( e.g ., glioblastoma), cancer of the head or neck, melanoma, uterine cancer, ovarian cancer, breast cancer, testicular cancer, cervical cancer, stomach cancer, Hodgkin's Disease, non-Hodgkin's lymphoma, thyroid cancer. The cancer may be a leukemia, such as, by way of non-limiting example, acute myeloid (or myelogenous) leukemia (AML), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), and chronic lymphocytic leukemia (CLL).

[00169] In some instances, the target sequence is a portion of a nucleic acid from a cancer cell. A cancer cell may be a cell harboring one or more mutations that results in unchecked proliferation of the cancer cell. Such mutations are known in the art. Non-limiting examples of antigens are ADRB3, AKAP-4,ALK, Androgen receptor, B7H3, BCMA, BORIS, BST2, CAIX, CD 179a, CD 123, CD171, CD 19, CD20, CD22, CD24, CD30, CD300LF, CD33, CD38, CD44v6, CD72, CD79a, CD79b, CD97, CEA, CLDN6, CLEC12A, CLL-1, CS-1, CXORF61, CYP1B1, Cyclin B 1, E7, EGFR, EGFRvIII, ELF2M, EMR2, EPCAM, ERBB2 (Her2/neu), ERG (TMPRSS2 ETS fusion gene), ETV6-AML, EphA2, Ephrin B2, FAP, FCAR, FCRL5, FLT3, Folate receptor alpha, Folate receptor beta, Fos-related antigen 1, Fucosyl GM1, GD2, GD3, GM3, GPC3, GPR20, GPRC5D, GloboH, HAVCR1, HMWMAA, HPV E6, IGF- I receptor, IL-13Ra2, IL-1 IRa, KIT, L AGE-1 a, LAIR1, LCK, LILRA2, LMP2, LY6K, LY75, LewisY, MAD-CT-1, MAD-CT-2, MAGE Al, MAGE-A1, ML-IAP, MUC1, MYCN, Mel an A/M ARTl , Mesothelin, NA17, NCAM, NY-BR-1, NY-ESO-1, OR51E2, OY- TES 1, PANX3, PAP, PAX3, PAX5, PCTA-l/Galectin 8, PDGFR-beta, PLAC1, PRSS21, PSCA, PSMA, Polysialic acid, Prostase, RAGE-1, ROR1, RU1, RU2, Ras mutant, RhoC, SART3, S SEA-4, SSX2, TAG72, TARP, TEM1/CD248, TEM7R, TGS5, TRP-2, TSHR, Tie 2, Tn Ag, UPK2, VEGFR2, WT1, XAGE1, and IGLL1.

[00170] In some cases, the target sequence is a portion of a nucleic acid from a control gene in a sample. In some embodiments, the control gene is an endogenous control. The endogenous control may include human 18S rRNA, human GAPDH, human HPRT1, human GUSB, human RNase P, MS2 bacteriophage, or any other control sequence of interest within the sample.

[00171] 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 mΐ. 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 mΐ, or any of value from 1 mΐ to 500 mΐ. 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 pi.

[00172] 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.

[00173] 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.

[00174] 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 HI 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 HI 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 HI 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 HI 5 sequence is a target of an assay. In some embodiments, the Influenza A HI 6 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 85D 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 3675-3677D 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 69-70D 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-2 242-244D 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 N501Y 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.

[00175] 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 hronchoseptica , 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 a 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 & 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 a Francisella tularensis sequence, such as a portion of 16S. In some embodiments, a target sequence is & Rickettsia spp. sequence, such as a portion of 16S, 23 S, 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 Francisella tularensis sequence, such as a portion of tul4 or fopA. 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 HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 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, del 156/157 (delta variant), 241/243 WT, del241/243 (beta variant), 69/70 WT, del69/70 (alpha variant), A570 WT, A570D (alpha variant), A701 WT, A701V (beta variant), Dl l 18 WT, D1118H (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, 23S, ACTB, ATP5ME, ATP5MF, ATP5MG, ATP5PB, BCSP31, CAPB, CHMP2A, Clorf43, COM1, DNAJ, EMC7, FOP A, GPI, GAPDH, GUSB, GYRB, HRPTl, NDUFB3, NDUFB4, NDUFB8, OMP2A, PAGA, PRDX1, PSMB2, PSMB4, PURG, RAB7A, REEP5, RNaseP, RPL13, RPL19, RPL27A, RPL30, RPL31, RPL32, RPL37A, RPOD, RPS10, RPS27, RPS29, RPS6, SNRPD3, TUL4, VCP, VPS29, and YWHAG.

[00176] 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, BAPl, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CASR, CDC73, CDH1, CDK4, CDKN1B, CDKN1C, CDKN2A, CEBPA, CHEK2, CTNNA1, DICERl, DIS3L2, EGFR, EPCAM, FH, FLCN, GATA2, GPC3, GREMl, HOXB13, HRAS, KIT, MAX, MENl, MET, MITF, MLHl, MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NF2, NTHL1, PALB2, PDGFRA, PHOX2B, PMS2, POLD1, POLE, POT1, PRKARIA, PTCH1, PTEN, RAD50, RAD51C, RAD51D, RBI, RECQL4, RET, RUNX1, SDHA, SDHAF2, SDHB, SDHC, SDHD, SMAD4, SMARCA4, SMARCBl, SMARCEl, STK11, SUFU, TERC, TERT, TMEM127, TP53, TSC1, TSC2, VHL, WRN, and WT1.

[00177] 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, b -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: AAVS1, ABCA4, ABCB11, ABCC8, ABCD1, ACAD9, AC ADM, ACADVL, ACAT1, ACOX1, ACSF3, ADA, ADAMTS2, ADGRGl, AGA, AGL, AGPS, AGXT, AHI1, AIRE, ALDH3A2, ALDOB, ALG6, ALK, ALKBH5, ALMSl, ALPL, AMRC9, AMT, ANGPTL3, APC, Apo(a), APOCIII, AROEe4, APOLl, APP, AQP2, AR, ARFRPl, ARGl, ARL13B, ARL6, ARSA, ARSB, ASL, ASNS, ASP A, ASS1, ATM, ATP6V1B1, ATP7A, ATP7B, ATRX, ATXN1, ATXN10, ATXN2, ATXN3, ATXN7, ATXN80S, AXIN1, AXIN2, B2M, BACE-1, BAKl, BAPl, BARDl, BAX2, BBS1, BBS10, BBS12, BBS2, BCKDHA, BCKDHB, BCL2L2, BCS1L, BEST1, Betaglobin gene, BLM, BMPR1A, BRAFV600E, BRCA1, BRCA2, BRIP1, BSND, C282Y, C9orf72, CA4, CACNA1A, CAPN3, CASR, CBS, CC2D2A, CCR5, CDC73, CDH1, CDH23, CDK11, CDK4, CDKN1B, CDKN1C, CDKN2A, CEBPA, CEP290, CERKL, CFTR, CHCHD10, CHEK2, CHM, CHRNE, CIITA, CLN3, CLN5, CLN6, CLN8, CLRN1, CLTA, CNBP, CNGB1, CNGB3, COL1A1, COL1A2, COL27A1, COL4A3, COL4A4, COL4A5, COL7A1, CPS1, CPT1A, CPT2, CRB1, CRX, CTNNA1, CTNNB1, CTNND2, CTNS, CTSK, CYBA, CYBB, CYPl lBl, CYP11B2, CYP17A1, CYP19A1, CYP27A1, DBT, DCLREIC, DERL2, DFNA36, DFNB31, DGAT2, DHCR7, DHDDS, DICERl, DIS3L2, DLD, DMD, DMPK, DNAH5, DNAI1, DNAI2, DNM2, DNMT1, DYSF, EDA, EDN3, EDNRB, EGFR, EIF2B5, EMC2, EMC3, EMD, EMX1, EPCAM, ERCC6, ERCC8, ESC02, ETFA, ETFDH, ETHE1, EVC, EVC2, EYS, F5, F9, FactorB, FactorXI, FAH, FAM161A, FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, FANCN, FANCP, FANCS, FBN1, FGF14, FGFR2, FGFR3, FH, FHL1, FKRP, FKTN, FLCN, FMRl, FOXP3, FSCN2, FUS, FUT8, FVIII, FXII, FXN, G6PC, GAA, GALC, GALKl, GALT, GAMT, GATA2, GBA, GBE1, GCDH, GCGR, GDNF, GFAP, GFM1, GHR, GJB1, GJB2, GLA, GLB1, GLDC, GLE1, GNE, GNPTAB, GNPTG, GNS, GPC3, GPR98, GREMl, GRHPR, GRIN2B, H2AX, HADHA, HAX1, HBA1, HBA2, HBB, HEXA, HEXB, HGSNAT, HLCS, HMGCL, HOGA1, HOXB13, HPRPF3, HPRT1, HPS1, HPS3, HRAS, HSD17B4, HSD3B2, HTT, HYALl, HYLSl, IDS, IDUA, IFITM5, IKBKAP, IL2RG, IMPDH1, INPP5E, IRF4, ITPR1, IVD, JAG1, KCNC3, KCND3, KCNJ11, KLHL7, KRAS, LAMA2, LAMA3, LAMB3, LAMC2, LCA5, LDLR, LDLRAPl, LHX3, LIFR, LIP A, LMNA, LOXHD1, LPL, LRAT, LRP6, LRPPRC, LRRK2, MAN2B1, MAPT, MAX, MCOLN1, MECP2, MED 17, MEFV, MENl, MERTK, MESP2, MET, METexl4, MFN2, MFSD8, MITF, MKS1, MLC1, MLHl, MLH3, MMAA, MMAB, MMACHC, MMADHC, MMD, MPI, MPL, MPV17, MSH2, MSH3, MSH6, MTHFR, MTM1, MTRR, MTTP, MUT, MUTYH, MY07A, NAGLU, NAGS, NBN, NDRG1, NDUFAF5, NDUFS6, NEB, NF1, NF2, NOTCH2, NPCl, NPC2, NPHPl, NPHSl, NPHS2, NR2E3, NTHL1, NTRK, NTRK1, OAT, OCT4, OFD1, OP A3, OTC, PAH, PALB2, PAQR8, PAX3, PC, PCCA, PCCB, PCDH15, PCSK9, PD1, PDCD1, PDE6B, PDGFRA, PDHA1, PDFffl, PEX1, PEX10, PEX12, PEX13, PEX14, PEX16, PEX19, PEX2, PEX26, PEX3, PEX5, PEX6, PEX7, PFKM, PHGDH, PHOX2B, PKD1, PKD2, PKITD1, PKK, PLEKHG4, PMM2, PMP22, PMS1, PMS2, PNPLA3, POLD1, POLE, POMGNT1, POT1, POU5F1, PPM1A, PPP2R2B, PPT1, PRCD, PRKAR1A, PRKCG, PRNP, PROM1, PROP1, PRPF31, PRPF8, PRPH2, PRPS1, PSAP, PSD95, PSEN1, PSEN2, PTCH1, PTEN, PTS, PUS1, PYGM, RAB23, RAD50, RAD51C, RAD51D, RAG2, RAPSN, RARS2, RBI, RDH12, RECQL4, RET, RHO, RICTOR, RMRP, ROS1, RP1, RP2, RPE65, RPGR, RPGRIPIL, RPL32P3, RSI, RTEL1, RUNX1, SACS, SAMHD1, SCN1A, SCN2A, SDHA, SDHAF2, SDFffl, SDHC, SDITD, SEL1L, SEPSECS, SERPING1, SGCA, SGCB, SGCG, SGSH, SIRT1, SLC12A3, SLC12A6, SLC17A5, SLC22A5, SLC25A13, SLC25A15, SLC26A2, SLC26A4, SLC35A3, SLC37A4, SLC39A4, SLC4A11, SLC6A8, SLC7A7, SMAD4, SMARCA4, SMARCALl, SMARCBl, SMARCEl, SMN1, SMPD1, SNAI2, SNCA, SNRNP200, SOD1, SOX10, SPARA7, SPTBN2, STAR, STAT3, STK11, SUFU, SUMF1, SYNE1, SYNE2, SYS1, TARDBP, TAT, TBK1, TBP, TCIRG1, TCTN3, TECPR2, TERC, TERT, TFR2, TGFBR2, TGM1, TH, TLE3, TMEM127, TMEM138, TMEM216, TMEM43, TMEM67, TMPRSS6, TOPI, TOPORS, TP53, TPP1, TRAC, TRMU, TSFM, T SPAN 14, TTBK2, TTC8, TTPA, TTR, TULP1, TYMP, UBE2G2, UBE2J1, UBE3A, USH1C, USH1G, USH2A, VEGF, VHL, VPS13A, VPS13B, VPS35, VPS45, VRK1, VSX2, VWF, WDR19, WNT10A, WS2B, WS2C, XPA, XPC, XPF, YAPl, ZFYVE26, and ZNF423. [00178] 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.

[00179] 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.

[00180] 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.

[00181] 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).

[00182] 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.

[00183] Further description of detecting a target nucleic acid in the foregoing genes can be found in more detail in Kim et al., “ Enhancement of target specificity of CRISPR-Casl2a by using a chimeric DNA-RNA guide ”, Nucleic Acids Res. 2020 Sep 4;48(15): 8601 -8616; Wang et al., “ Specificity profiling of CRISPR system reveals greatly enhanced off-target gene editingf , Scientific Reports volume 10, Article number: 2269 (2020); Tuladhar et al., “ CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation” , Nature Communications volume 10, Article number: 4056 (2019); Dong et al., “ Genome-Wide Off-Target Analysis in CRISPR-Cas9 Modified Mice and Their Offspring G3, Volume 9, Issue 11, 1 November 2019, Pages 3645-3651; Winter et al., “ Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus a-hemolysin-mediated toxicity’ Scientific Reports volume 6, Article number: 24242 (2016); and Ma et al., “4 CRISPR-Based Screen Identifies Genes Essential for West-Nile-Virus-Induced Cell Death” , Cell Rep. 2015 Jul 28;12(4):673-83, which are hereby incorporated by reference in their entirety.

[00184] Any of the devices, systems, kits, or methods described herein may be used to quantify one or more target nucleic acid populations within a sample. Quantification of the target nucleic acids in the sample may be accomplished by immobilizing a plurality of programmable nucleases, a plurality of guide nucleic acids, and/or a plurality of reporters to a surface. Various methods may be used to immobilize one or more programmable nuclease- based detection reagents to the surface at a desired location (e.g., at a detection spot, in a detection well, on a detection particle, etc.). Any of the devices described herein may comprise one or more immobilized detection reagents (e.g., programmable nuclease, guide nucleic acid, and/or reporter). In some embodiments, one or more detection reagents may be modified with biotin. In some embodiments, these biotinylated components may be immobilized on surfaces coated with streptavidin.

[00185] FIGS. 1A-1C show exemplary immobilization strategies for programmable nuclease-based diagnostic assay components which may be utilized for quantification of a target nucleic acid population in a sample as described herein. In some embodiments, as shown in FIG. 1A, one or more chemical modifications may be made to amino acid residues in the programmable nuclease in order to enable its attachment to and immobilization on a surface at a desired surface location. The surface may be any of the surfaces described herein (e.g., a detection array, a channel wall, a detection particle, a detection well, etc.). Alternatively, or in combination, as shown in FIG. IB, a guide nucleic acid may be immobilized at the desired surface location by adding various chemical modifications at the 5’ or 3’ end of the guide nucleic acid that are compatible with the selected surface chemistry. Alternatively, or in combination, as shown in FIG. 1C, a reporter may be immobilized at the desired surface location using similar chemical modifications as the guide nucleic acids.

[00186] In some embodiments, the programmable nuclease, the guide nucleic acid, and/or the reporter are immobilized to a surface. In some embodiments, the programmable nuclease, the guide nucleic acid, and/or the reporter may be immobilized to a surface by a linkage or linker. In some embodiments, the linkage may comprise 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 may comprise non-specific absorption. In some embodiments, the programmable nuclease may be immobilized to the surface by the linkage, wherein the linkage may be between the programmable nuclease and the surface. In some embodiments, the reporter may be immobilized to the device surface by the linkage, wherein the linkage may be between the reporter and the surface. In some embodiments, the guide nucleic acid may be immobilized to the surface by the linkage, wherein the linkage may be between the 5’ end of the guide nucleic acid and the surface. In some embodiments, the guide nucleic acid may be immobilized to the surface by the linkage, wherein the linkage may be between the 3’ end of the guide nucleic acid and the surface.

[00187] In some embodiments, one or more detection reagents (e.g., one or more of the programmable nuclease, the guide nucleic acid, and/or the reporter) may be immobilized in discrete detection locations of a surface by a covalent linkage. In some embodiments, the covalent linkage comprises a bifunctional silane.

[00188] In some embodiments, the one or more detection reagents can be immobilized in discrete detection locations using NHS-amine chemistry. For example, a primary amine- modified guide nucleic acid and a primary amine-modified reporter may be conjugated to an NHS-coated surface of the detection region.

[00189] In some embodiments, the one or more detection reagents may be immobilized using streptavidin-biotin chemistry. For example, a biotinylated reporter and a biotinylated guide nucleic acid may be immobilized to a streptavidin-coated surface of the detection region. [00190] In some embodiments, the one or more detection reagents may be immobilized using maleimide-thiol chemistry. For example, a thiol-modified guide nucleic acid and a thiol- modified reporter may be conjugated to a maleimide-coated surface of the detection region. [00191] In many embodiments, the programmable nuclease and/or guide nucleic acid may be immobilized in close enough proximity to an immobilized reporter in order to enable cleavage of the reporter when a target nucleic acid binds to the guide nucleic acid to activate the programmable nuclease as described herein. Cleavage of the reporter may release at least a portion of the reporter (e.g., a portion comprising a detection moiety) from the surface. Release of at least a portion of the reporter may result in a detectable signal at the surface location or at a downstream location as described herein.

[00192] In some embodiments, one or more detection reagents may be coupled to one or more other detection reagents in order to immobilize the detection reagents on the surface. For example, the programmable nuclease and/or guide nucleic acid may be coupled directly or indirectly (e.g., via a linker) to an immobilized reporter. Activation of the programmable nuclease upon binding of the guide nucleic acid to a target nucleic acid may result in cleavage of the reporter and release of the programmable nuclease complex and a portion of the reporter (e.g., a portion comprising a detection moiety) from the surface. Release of at least a portion of the reporter may result in a detectable signal at the surface location or at a downstream location as described herein. [00193] In some embodiments, the one or more detection reagents can be immobilized within a matrix disposed on the surface. In some embodiments, the matrix may comprise a hydrogel.

[00194] FIG. 2 depicts a surface 200 comprising an immobilized programmable nuclease-guide nucleic acid complex 208, 202 and a plurality of reporters 204, with one reporter having been cleaved 214 by an activated programmable nuclease 208. In some embodiments, one or more detection reagents may be immobilized on a surface 200. For example, a guide nucleic acid 202 and one or more reporters 204 may be immobilized on the surface 200 as shown. In some embodiments, one or more detection reagents may be immobilized on a surface 200 of the detection region via a linker or linkage 206 as described herein. The programmable nuclease 208 may be complexed with a guide nucleic acid 202 complementary to a specific target nucleic acid sequence as described herein. When a target nucleic acid is present in the sample, the programmable nuclease 208 may be activated by binding of the guide nucleic acid 202 (which is complexed thereto) to the target nucleic acid. Activation of the programmable nuclease 208 may enable trans-cleavage of the reporter 204 as described herein. In some embodiments, the reporter 204 may comprise a detection moiety as described herein. In some embodiments, the reporter 204 may comprise a detection moiety (e.g., a fluorophore 210) and a quenching moiety (e.g., a quencher 212) configured to generate a detectable signal when separated from one another. In some embodiments, trans-cleavage of the reporter 204 by the activated programmable nuclease 208 may release the detection moiety (or a quenching moiety, depending on the signal), thereby generating a signal indicative of the presence or absence of the target nucleic acid in the sample as described herein. The reporter 204 may comprise any of the reporters described herein and may comprise any detection moiety described herein (and/or other moieties or molecules described herein which facilitate signal detection). The surface 200 may be any of the surfaces described herein (e.g., a detection array, a detection spot, a channel wall, a detection particle, a detection well, etc.).

[00195] As shown in FIG. 2, cleavage of the reporter 204 may release a cleaved portion 214 comprising the quencher 212 into solution, thereby allowing the fluorophore 210 which was quenched when the reporter 204 was intact to fluoresce at the surface location. The presence of fluorescence at the surface location may therefore indicate the presence of the target nucleic acid. In another example, the reporter may not comprise a quencher and cleavage of the reporter may instead release a fluorophore into solution, thereby reducing fluorescence at the surface location. The absence of fluorescence at the surface location may therefore indicate the presence of the target nucleic acid. In some embodiments, cleavage of the reporter may result in a non-fluorescent signal as described herein. For example, cleavage of the reporter can produce a calorimetric signal, a potentiometric signal, an amperometric signal, a colorimetric signal, or a piezo-electric signal as described herein.

[00196] FIG. 3A illustrates an exemplary microfluidic structure 300 for continuous sample fractionation. The microfluidic structure may comprise an inlet 301 fluidly coupled to a first fluidic channel 310. In some embodiments, the first fluidic channel 310 may comprise a branching channel structure having a plurality of subchannels 311 and configured to digitize or compartmentalize a sample into a plurality of compartments. In some embodiments, the plurality of compartments may comprise a plurality of microwells, a plurality of nanowells, a plurality of particles, or a plurality of droplets. In some embodiments, digitization or compartmentalization of the sample nucleic acids may distribute the sample into the plurality of compartments such that each compartment has at most one nucleic acid therein. In some embodiments, each compartment may be spatially separated from every other compartment so as to enable precise detection and quantification of target nucleic acid binding events. In some embodiments, the compartments may comprise individual droplets or particles flowing in a fluid channel. In some embodiments, each compartment may be localized to a specific location within the device. For example, each compartment may be localized to a specific location on a surface of the first fluidic channel, to a specific microwell or nanowell, or to a specific location on a surface of an array. In some embodiments, the nucleic acids of the sample may not be amplified prior to compartmentalization.

[00197] In some embodiments, the first fluidic channel may be coupled to a second fluidic channel downstream of the first fluidic channel which may be configured to facilitate detection of a detection moiety (e.g., as shown in FIG. 4B) by a detector as described herein. [00198] A sample solution containing a plurality of nucleic acids may be introduced into the first fluidic channel 310 via an inlet 301. In some embodiments, positive or negative pressure may be used to transfer the sample from the inlet into the diverging channel system 300. In some embodiments, capillary action may drive the sample solution through the branching channel system 300.

[00199] In some embodiments, the plurality of nucleic acids may be contacted to a surface comprising one or more programmable nuclease complexes and reporters immobilized thereto prior to, during, or after introduction into the first fluidic channel 310 via inlet 301. For example, the plurality of nucleic acids may be contacted to a plurality of particles comprising programmable nuclease complexes and reporters immobilized thereto prior to introduction into the first fluidic channel 310 via inlet 301 as described herein. In some embodiments, the plurality of nucleic acids may be contacted to a channel wall comprising programmable nuclease complexes and reporters immobilized thereto during or after introduction into the first fluidic channel 310 via inlet 301 as described herein.

[00200] In some embodiments, the branching channel system 300 may be composed of a large diameter channel (or subchannel) adjacent the inlet that reduces in size at each subsequent downstream branch thereof. In some embodiments, the branching channel system 300 may be composed of channels (or subchannels) of the same diameter. In some embodiments, channel branches (or subchannels) may be coupled to one another at a branching juncture. In some embodiments, the branching juncture may comprise a fluid reservoir configured to facilitate pressure balancing between the channel branches coupled thereto and/or across the various branches of the first fluidic channel in order to encourage even loading into the channel branches (or subchannels).

[00201] FIG. 3B shows an exemplary flow channel 310 where sample solutions are both continuously fractionated and continuously reacted with programmable nuclease-based diagnostic assay components within a branching channel structure such as the structure of FIG. 3A. A sample solution containing first target nucleic acids 306a may be introduced into the branching channel system as described herein. In some embodiments, the sample may be continuously fractionated and continuously detected along the entire length of the branching channel system. For example, the first target nucleic acids 306a may be flown over a first surface 302a of the first fluidic channel 310 and continuously exposed to a first plurality of programmable nuclease complexes 304a immobilized on the first surface 302a of the channel 310. In some embodiments, the first plurality of programmable nuclease complexes 304a may be adjacent a first plurality of reporters comprising first detection moieties 313a (e.g., as shown in FIG. 2). Cleavage of the first reporter may release the first detection moiety 313a and induce a change in the surface 302a at a location corresponding to a location of the first reporter. In some embodiments, the first detection moiety 313a may comprise a fluorophore as described herein. In some embodiments, the first detection moiety 313a may comprise a quencher as described herein. The change in the surface 302a may be detected by a detector. The change in the surface 302a may be detected by a detector at the surface location where the first reporter was cleaved. [00202] In some embodiments, the sample may further comprise second target nucleic acids 306b. The second target nucleic acids 306b may be flown over a second surface 302b of the first fluidic channel 310 and continuously exposed to a second plurality of programmable nuclease complexes 304b immobilized on the second surface 302b of the channel. In some embodiments, the second plurality of programmable nuclease complexes 304b may be adjacent to a second plurality of reporters comprising second detection moieties 313b (e.g., as shown in FIG. 2). Cleavage of the second reporter may release the second detection moiety 313b and induce a change in the surface 302b at a location corresponding to a location of the second reporter. In some embodiments, the second detection moiety 313b may comprise a fluorophore as described herein. In some embodiments, the second detection moiety 313b may comprise a quencher as described herein. The change in the surface 302b may be detected by a detector at the surface location where the second reporter was cleaved.

[00203] In some embodiments, the change in the first and/or second surface 302a, 302b may comprise a change in conductivity, a change in fluorescence intensity, a change in fluorescence wavelength, a change in absorbance, a change in luminescence, or an amperometric change as described herein.

[00204] In some embodiments, the first programmable nuclease and the second programmable nuclease may be different types of programmable nuclease. In some embodiments, the first programmable nuclease and the second programmable nuclease may be the same type of programmable nuclease.

[00205] In some embodiments, the first guide nucleic acid and the second guide nucleic acids may be configured to bind to a same target nucleic acid sequence. In some embodiments, the first guide nucleic acid and the second guide nucleic acids may be configured to bind to different target nucleic acid sequences. In some embodiments, the different target nucleic acid sequences may be different portions of a same target nucleic acid (e.g., for guide pooling). In some embodiments, the different target nucleic acid sequences may be from different target nucleic acids.

[00206] In some embodiments, the first and second surfaces may be spatially distinct from one another. In some embodiments, the first surface and the second surface may be adjacent sections of the first fluidic channel (e.g., similar to regions 307 and 308 of FIG. 3C). In some embodiments, the first surface and the second surface may be parallel stripes within a same section of the first fluidic channel. In some embodiments, the first fluidic channel may comprise a plurality of spatially distinct surfaces (e.g., parallel stripes comprising programmable nuclease-based detection reagents) in order to enable target multiplexing within the channel.

[00207] In some embodiments, the surface may comprise more of each type of programmable nuclease complexes than nucleic acids in the sample so as to ensure that every nucleic acid has a chance to interact with a programmable nuclease (and thus, every target nucleic acid has a chance to bind to a programmable nuclease complementary thereto). A ratio of the amount of nucleic acids to the amount of programmable nuclease complexes may be about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1 :90, or about 1 : 100, or more.

[00208] In some embodiments, one or more of the assay components (e.g., first programmable nuclease complex and/or first reporter) may be embedded in a hydrogel matrix disposed on the first surface. In some embodiments, one or more of the assay components (e.g., second programmable nuclease complex and/or second reporter) may be embedded in a hydrogel matrix disposed on the second surface. In some embodiments, one or more of the assay components (e.g., first programmable nuclease complex and/or first reporter) may be directly immobilized to the first surface and no hydrogel matrix may be present. In some embodiments, one or more of the assay components (e.g., second programmable nuclease complex and/or second reporter) may be directly immobilized to the second surface and no hydrogel matrix may be present.

[00209] FIG. 3C shows another exemplary flow channel where sample solutions are both continuously fractionated and continuously reacted with programmable nuclease-based diagnostic assay components within a branching channel structure such as the structure of FIG. 3A. A sample solution containing target nucleic acids 306 may be introduced into the branching channel system 300 through inlet 301 as described herein. In some embodiments, the sample may be continuously fractionated and continuously detected along the entire length of the branching channel system. For example, the target nucleic acids 306 may be flown over a surface 302 of the first fluidic channel 310 and continuously exposed to a plurality of programmable nuclease complexes 304 immobilized on the surface 302 of the channel. In some embodiments, the plurality of programmable nuclease complexes 304 may be coupled to a plurality of reporters comprising detection moieties 313. Cleavage of the reporter may release the activated programmable nuclease complex 314 and detection moiety coupled thereto. In some embodiments, the detection moiety may comprise an affinity molecule as described herein. The affinity molecule may be complementary to an affinity molecule on a detectable particle 305 disposed on the surface of the first fluidic channel 310. The released programmable nuclease complex coupled to the detection moiety may be configured to bind to the particle 305 as it flows through the channel to form a detectable complex 309 which may be detected by a downstream detector or at a downstream location as described herein.

[00210] In some embodiments, the surface may comprise more programmable nuclease complexes 304 than nucleic acids in the sample so as to ensure that every nucleic acid has a chance to interact with a programmable nuclease (and thus, every target nucleic acid has a chance to bind to a programmable nuclease complementary thereto). A ratio of the amount of nucleic acids to the amount of programmable nuclease complexes may be about 1 :2, about 1 :3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100, or more.

[00211] In some embodiments, one or more of the assay components (e.g., programmable nuclease complex 304 and/or particle 305) may be embedded in a hydrogel matrix 303 that lines the walls 302 of the fluid channel 310. In some embodiments, one or more of the assay components (e.g., programmable nuclease complex 304 and/or particle 305) may be directly immobilized to the channel wall 302 and no hydrogel matrix 303 may be present. [00212] In some embodiments, a first region 307 of the channel may contain a programmable nuclease complex 304 and a second region 308 of the channel may contain the detectable particle 305 configured to bind to the detection moiety. The first region 307 may be upstream of the second region 308.

[00213] In some embodiments, the channel 310 may comprise a plurality of adjacent regions comprising complementary programmable nuclease complexes 304 and detectable particles 305, respectively. In some embodiments, one or more of the adjacent region pairs may be configured to detect the same target nucleic acid. In some embodiments, one or more of the adjacent region pairs may be configured to detect different target nucleic acids. In some embodiments, the adjacent region pairs may be spatially encoded (e.g., with different detection moieties, different programmable nucleases, different guide nucleic acids, different reporter types, etc.) for target multiplexing and/or spatial reconstruction.

[00214] In some embodiments, the detectable particle 305 may comprise a quantum dot. In some embodiments, the detectable particle may be configured for encoded particle multiplexing. In some embodiments, the detectable particle may comprise one or more lanthanide elements.

[00215] In some embodiments, multiple assay components can be mixed together in one region.

[00216] In some embodiments, the assay components may be separately lyophilized in order to be coated to different regions (such as different subchannels, sections, or stripes) of the branching channel system.

[00217] FIG. 3D shows another exemplary flow channel 310 where sample solutions are both continuously fractionated and continuously reacted with programmable nuclease- based diagnostic assay components within a branching channel structure such as the structure of FIG. 3A. A sample solution containing first target nucleic acids 306a may be introduced into the branching channel system 300 as described herein. In some embodiments, the sample may be continuously fractionated and continuously detected along the entire length of the branching channel system 300. For example, the first target nucleic acids 306a may be flown over a first surface 302a of the first fluidic channel 310 and continuously exposed to a first plurality of programmable nuclease complexes 304a immobilized on the first surface 302a of the channel. In some embodiments, the first plurality of programmable nuclease complexes 304a may be coupled to a first plurality of reporters comprising first detection moieties 313a. Cleavage of the first reporter may release a first activated programmable nuclease complex 314a comprising the first programmable nuclease complex 304a and first detection moiety 313a coupled thereto. In some embodiments, the first detection moiety 313a may comprise a fluorophore as described herein. The first released programmable nuclease complex 314a coupled to the first detection moiety 313a may form a first detectable complex 309a that can be flown through the channel and detected by a downstream detector or at a downstream location as described herein.

[00218] In some embodiments, the sample may further comprise second target nucleic acids 306b. The second target nucleic acids 306b may be flown over a second surface 302b of the first fluidic channel and continuously exposed to a second plurality of programmable nuclease complexes 304b immobilized on the second surface 302b of the channel. In some embodiments, the second plurality of programmable nuclease complexes 304b may be coupled to a second plurality of reporters comprising second detection moieties 313b. Cleavage of the second reporter may release a second activated programmable nuclease complex 314b comprising the second programmable nuclease complex 304b and second detection moiety 313b coupled thereto. In some embodiments, the second detection moiety 313b may comprise a fluorophore as described herein. The second released programmable nuclease complex 314b coupled to the second detection moiety 313b may form a second detectable complex 309b that can be flown through the channel and detected by a downstream detector or at a downstream location as described herein.

[00219] In some embodiments, the first programmable nuclease and the second programmable nuclease may be different types of programmable nuclease. In some embodiments, the first programmable nuclease and the second programmable nuclease may be the same type of programmable nuclease.

[00220] In some embodiments, the first guide nucleic acid and the second guide nucleic acids may be configured to bind to a same target nucleic acid sequence. In some embodiments, the first guide nucleic acid and the second guide nucleic acids may be configured to bind to different target nucleic acid sequences. In some embodiments, the different target nucleic acid sequences may be different portions of a same target nucleic acid (e.g., for guide pooling). In some embodiments, the different target nucleic acid sequences may be from different target nucleic acids.

[00221] In some embodiments, the first and second surfaces may be spatially distinct from one another. In some embodiments, the first surface and the second surface may be adjacent sections of the first fluidic channel (e.g., similar to regions 307 and 308 of FIG. 3C). In some embodiments, the first surface and the second surface may be parallel stripes within a same section of the first fluidic channel. In some embodiments, the first fluidic channel may comprise a plurality of spatially distinct surfaces (e.g., parallel stripes comprising programmable nuclease-based detection reagents) in order to enable target multiplexing within the channel.

[00222] In some embodiments, the surface may comprise more of each type of programmable nuclease complexes than nucleic acids in the sample so as to ensure that every nucleic acid has a chance to interact with a programmable nuclease (and thus, every target nucleic acid has a chance to bind to a programmable nuclease complementary thereto). A ratio of the amount of nucleic acids to the amount of programmable nuclease complexes may be about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1 :90, or about 1 : 100, or more. [00223] In some embodiments, one or more of the assay components (e.g., first programmable nuclease complex and/or first reporter) may be embedded in a hydrogel matrix disposed on the first surface. In some embodiments, one or more of the assay components (e.g., second programmable nuclease complex and/or second reporter) may be embedded in a hydrogel matrix disposed on the second surface. In some embodiments, one or more of the assay components (e.g., first programmable nuclease complex and/or first reporter) may be directly immobilized to the first surface and no hydrogel matrix may be present. In some embodiments, one or more of the assay components (e.g., second programmable nuclease complex and/or second reporter) may be directly immobilized to the second surface and no hydrogel matrix may be present.

[00224] FIG. 4A shows an exemplary detection array 400. The array 400 may comprise a plurality of surface locations. In some embodiments, the detection array 400 may be downstream of a fluidic channel configured to digitize, fractionate, or compartmentalize a sample. For example, the detection array 400 may be downstream of a branched fluidic channel structure (e.g., fluidic channel 310 of FIG. 3A). In some embodiments, the detection array 400 may be configured to receive an unfractionated sample. The detection array 400 may be coupled to a detector (e.g., a confocal microscope). The detector may be configured to detect a detection moiety released by cleavage of a reporter as described herein.

[00225] In some embodiments, the plurality of surface locations may comprise at least a first surface location 401 and a second surface location 402. The first surface location may comprise at least one of a first programmable nuclease, a first reporter, and/or a first guide nucleic acid immobilized thereto. The second surface location may comprise at least one of a second programmable nuclease, a second reporter, and/or a second guide nucleic acid immobilized thereto. The first surface location and the second surface location may be spatially distinct from one another.

[00226] In some embodiments, the first guide nucleic acid and the first reporter may be immobilized to the first surface location. The first programmable nuclease may be complexed with the first guide nucleic acid as described herein. The first reporter may be configured to be cleaved when the first programmable nuclease is activated upon binding of the first guide nucleic acid to a first target nucleic acid as described herein. In some embodiments, a plurality of first reporters may be immobilized adjacent the first programmable nuclease complex (e.g., as shown in FIG. 2). In some embodiments, the first reporter may comprise a first detection moiety as described herein. Cleavage of the first reporter may release the first detection moiety and induce a change in the surface at a location corresponding to a location of the first reporter. In some embodiments, the first detection moiety may comprise a fluorophore as described herein. In some embodiments, the first detection moiety may comprise a quencher as described herein. The change in the surface may be detected by a detector at the surface location where the first reporter was cleaved.

[00227] In some embodiments, the second guide nucleic acid and the second reporter may be immobilized to the second surface location. The second programmable nuclease may be complexed with the second guide nucleic acid as described herein. The second reporter may be configured to be cleaved when the second programmable nuclease is activated upon binding of the second guide nucleic acid to a second target nucleic acid as described herein. In some embodiments, a plurality of second reporters may be immobilized adjacent the second programmable nuclease complex (e.g., as shown in FIG. 2). In some embodiments, the second reporter may comprise a second detection moiety as described herein. Cleavage of the second reporter may release the second detection moiety and induce a change in the surface at a location corresponding to a location of the second reporter. In some embodiments, the second detection moiety may comprise a fluorophore as described herein. In some embodiments, the second detection moiety may comprise a quencher as described herein. The change in the surface may be detected by a detector at the surface location where the second reporter was cleaved.

[00228] In some embodiments, the change in the first and/or second surface location may comprise a change in conductivity, a change in fluorescence intensity, a change in fluorescence wavelength, a change in absorbance, a change in luminescence, or an amperometric change as described herein.

[00229] In some embodiments, the first programmable nuclease and the second programmable nuclease may be different types of programmable nuclease. In some embodiments, the first programmable nuclease and the second programmable nuclease may be the same type of programmable nuclease.

[00230] In some embodiments, the first guide nucleic acid and the second guide nucleic acids may be configured to bind to a same target nucleic acid sequence. In some embodiments, the first guide nucleic acid and the second guide nucleic acids may be configured to bind to different target nucleic acid sequences. In some embodiments, the different target nucleic acid sequences may be different portions of a same target nucleic acid (e.g., for guide pooling). In some embodiments, the different target nucleic acid sequences may be from different target nucleic acids.

[00231] In some embodiments, the number of surface locations may be more than the number of nucleic acids in a sample applied thereto so as to ensure that every nucleic acid has a chance to interact with a programmable nuclease (and thus, every target nucleic acid has a chance to bind to a programmable nuclease complementary thereto). Each surface location may comprise at least one programmable nuclease. A ratio of the amount of nucleic acids to the number of surface locations may be about 1 :2, about 1 :3, about 1 :4, about 1:5, about 1 :6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100, or more.

[00232] In some embodiments, the array may comprise more of each type of programmable nuclease complexes than nucleic acids in the sample so as to ensure that every nucleic acid has a chance to interact with a programmable nuclease (and thus, every target nucleic acid has a chance to bind to a programmable nuclease complementary thereto). A ratio of the amount of nucleic acids to the amount of programmable nuclease complexes may be about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1 :90, or about 1 : 100, or more.

[00233] In some embodiments, the array 400 may comprise a plurality of spots disposed on a surface of a microarray and each surface location may comprise a spot. In some embodiments, the array 400 may comprise a plurality of particles disposed on a surface of a microarray and each surface location may comprise a surface of a particle. In some embodiments, the plurality of particles comprises a plurality of beads. In some embodiments, the sample may be flown over the microarray. In some embodiments, the sample may be statically applied to the microarray.

[00234] In some embodiments, the array 400 may comprise a plurality of microwells or nanowells and each surface location of the array 400 may correspond to a microwell or nanowell, respectively.

[00235] In some embodiments, the array 400 may comprise a plurality of spots disposed within a microwell or nanowell. Each of the plurality of spots may be configured to detect a different target nucleic acid and produce a different detectable signal (e.g., different wavelength fluorescent signals). The array 400 may be configured to receive a digitized or compartmentalized portion of a sample (e.g., from a branching channel structure as shown in FIG. 3A) comprising at most one nucleic acid therein. Each of a plurality of microwells or nanowells may comprise an identical array 400 disposed therein. The plurality of microwells or nanowells may outnumber the amount of nucleic acids in the sample such that after compartmentalization of the sample, each microwell or nanowell has at most one nucleic acid therein. If the nucleic acid is a target nucleic acid complementary to one of the programmable nuclease complexes within the array 400, a detectable signal may be produced at the detection location within the nanowell or microwell.

[00236] The target nucleic acid(s) may be quantified by counting the number (and type) of detection moieties detected by the detector.

[00237] In some embodiments, target nucleic acids may be quantitatively detected using a dual system of immobilized probes composed of an immobilized programmable nuclease complex comprising a Cas protein and a guide nucleic acid that selectively binds to a target nucleic acid as shown in FIG. 2. In such an embodiment, adjacent to the programmable nuclease complex may be one or more reporters. The one or more reporters may comprise a fluorophore and a quencher as described herein. When the target nucleic acid selectively binds to a guide nucleic acid, the Cas protein may be activated to cleave all cleavable regions of nucleic acids around it. In some embodiments, the reporter may comprise a cleavable region located between the fluorophore and the quencher. In such an embodiment, upon cleavage, the fluorophore and quencher may be separated, thereby stopping the quenching mechanism and causing the fluorophore to emit fluorescence and thus produce a signal for the presence of a target.

[00238] In some embodiments, the reporter and programmable nuclease complex may be coupled to one another as described herein such that only the reporter need be immobilized to the surface.

[00239] In some embodiments, a plurality of reporters may be located adjacent a single programmable nuclease in order to amplify the signal produced by activation of the programmable nuclease upon binding of the target nucleic acid thereto. For example, one, two, three, four, five, six, seven, eight, nine, ten, or more reporters may be located within a distance allowing for cleavage by the programmable nuclease to occur.

[00240] In some embodiments, the array 400 may be configured for multiplexed detection of a plurality of different target nucleic acids. In some embodiments, a first programmable nuclease complex may be immobilized at a first known location 401 on the array 400. In some embodiments, the sample containing multiple target nucleic acid sequences may be introduced to the surface of the array and allowed to react in a static fashion. In some embodiments, the sample may be continuously moving over the surface of the array. In some embodiments, the sample solution may be washed away after 5 minutes incubation on the array. In some embodiments, the sample solution may be washed away after 10 minutes incubation on the array. In some embodiments, the signal from the array may be detected by scanning confocal microscopy. In some embodiments, emission of fluorescence at a surface location may signify a hit. In some embodiments, loss of emission of fluorescence at a surface location may signify a hit.

[00241] In some embodiments, the sample solution may comprise fluidically isolated volumes corresponding to each surface location.

[00242] FIG. 4B shows an exemplary detection channel 405. The detection channel 405 may be downstream of a fluidic channel configured to digitize, fractionate, or compartmentalize a sample. For example, the detection channel may be downstream of a branched fluidic channel structure (e.g., fluidic channel 310 of FIG. 3A). The detection channel 405 may be coupled to a detector 403. The detector 403 may be configured to detect a detection moiety released by cleavage of a reporter as described herein. For example, a programmable nuclease complex coupled to a detection moiety 408 (which may be generated as described in FIG. 3C or FIG. 3D or which may be introduced directly into the fluidic channel 310 for fractionation) may be detected in a flow cytometric fashion as seen in FIG. 4B. in some embodiments, the programmable nuclease-detection moiety complex 408 may be substantially similar to any of the detectable complexes (e.g., complex 309 or 314) described herein. A detector 403 may focus light 404 in a region of the channel to detect the programmable nuclease-detection moiety complex 408 within the channel 405. In some embodiments, one or more crowding agents 407 may be introduced along the detection channel 405 in order to further isolate the programmable nuclease-detection moiety complexes 408 from each other for more sensitive detection. In some embodiments, quantitative detection in flow systems may be carried out in conjunction with branching channel systems. In some embodiments, there is no movement of sample solution 406 during detection. In some embodiments, the sample solution 406 may be flown through the channel past the detector 403 during detection. The target nucleic acid(s) may be quantified by counting the number (and type) of detection moieties or detectable complexes detected by the detector 403. In some embodiments, backscatter interferometry may be used to detect the detection moieties or detectable complexes. For example, the refractive index of a particle such as a bead with uncleaved reporter bound thereto (i.e., a bead which has not encountered a target nucleic acid) may be distinguishable from the refractive index of a particle such as a bead with cleaved reporter bound thereto (i.e., a bead which has encountered a target nucleic acid) by a detector configured to perform backscatter interferometery. In some embodiments, when the detectable complex comprises a particle such as a bead, the particle may comprise a first label and the detectable complex may comprise a second label different from the first label. For example, the particle may comprise a first quantum dot that fluoresces at a first wavelength and the second label may comprise a second quantum dot that fluoresces at a second wavelength when exposed to a predetermined wavelength of excitation light. Detectable complexes comprising activated programmable nuclease complex(es) immobilized on beads can be distinguished from particles that have not encountered a target nucleic acid based on the fluorescent signature of the different labels. It will be understood by one of ordinary skill in the art based on the teachings herein that various permutations of labels may be used in order to generate any number of detectable complexes, which may be of particular value for their ability to be distinguished from one another when multiplexing as described herein.

Multiplexing

[00243] The systems, devices, and methods described herein can be multiplexed in a number of ways. Multiplexing may include assaying for two or more target nucleic acids in a sample. Multiplexing can be spatial multiplexing wherein multiple different target nucleic acids are detected from the same sample at the same time, but the reactions are spatially separated. Often, 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. Sometimes, multiplexing can be single reaction multiplexing wherein multiple different target nucleic acids are detected in a single reaction volume (e.g., within a flow channel as shown in FIG. 4B, with different programmable nucleases and/or different reporters generating different, distinguishable signals in the presence of the different target nucleic acids). 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 reporters within a device (e.g., on a surface of an array, channel, particle, etc. as described herein), to enable detection of multiple target nucleic acids. 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. In some cases, the multiple target nucleic acids comprise different target nucleic acids associated with at least a first disease and a second disease. Multiplexing for one disease can increase 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 indicative of the presence and/or severity of a disease, such as target nucleic acids that comprise a mutation, such as one or more single nucleotide polymorphism (SNP), associated with a particular cancer and/or cancer subtype. In some cases, the multiple target nucleic acids comprise target nucleic acids indicative of the host response to a disease or pathogen, such as target nucleic acids that indicate changes in the patient’s immune response, angiogenesis, cell stress or death, growth factor production, or the like. In some cases, the multiple target nucleic acids comprise target nucleic acids indicative of a host or pathogen response to treatment, which may help in determining thresholds for therapy and/or enable therapeutic adjustments. 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 wild-type 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. For example, multiplexing methods may comprise 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 influenza strains, for example, influenza A and influenza B. 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 a mutant (e.g., SNP) genotype. Multiplexing for multiple viral infections can provide 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. [00244] Furthermore, signals from multiplexing can be quantified. For example, a method of quantification for a disease panel comprises assaying for a plurality of unique target nucleic acids in a plurality of aliquots (e.g., fractions or compartments) from a sample, assaying for a control nucleic acid control in another 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 reporters compared to the signal produced in the second aliquot. Often the plurality of unique target nucleic acids is from a plurality of viruses in the sample. Sometimes the quantification of a signal of the plurality correlates with a concentration of a unique target nucleic acid of the plurality for the unique target nucleic acid of the plurality that produced the signal of the plurality. The disease panel can be for any disease.

[00245] In some cases, the combination of a guide nucleic acid, a programmable nuclease, and a reporter configured to detect one target nucleic acid is provided in its own reagent chamber or its own support medium (e.g., particle or channel wall). In this case, multiple reagent chambers or support mediums are provided, where each reagent chamber is designed to detect one target nucleic acid. In some cases, multiple different target nucleic acids may be detected in the same chamber or support medium.

[00246] In some instances, the multiplexed devices and methods detect at least 2 different target nucleic acids in a single reaction. In some instances, the multiplexed devices and methods detect at least 3 different target nucleic acids in a single reaction. In some instances, the multiplexed devices and methods detect at least 4 different target nucleic acids in a single reaction. In some instances, the multiplexed devices and methods detect at least 5 different target nucleic acids in a single reaction. In some cases, the multiplexed devices and methods detect at least 6, 7, 8, 9, or 10 different target nucleic acids in a single reaction.

NUMBERED EMBODIMENTS

[00247] Notwithstanding the appended claims, the disclosure sets forth the following numbered embodiments:

[00248] 1. A method for quantifying nucleic acids, the method comprising: providing a plurality of particles comprising a plurality of programmable nuclease complexes, wherein each particle of the plurality of particles comprises one or more programmable nuclease complex of the plurality of programmable nuclease complexes and a plurality of reporters immobilized thereon, wherein each programmable nuclease complex of the plurality of programmable nuclease complexes comprises a programmable nuclease and a guide nucleic acid; applying a sample to the plurality of particles, wherein the sample comprises a plurality of nucleic acids, wherein an amount of programmable nuclease complexes in the plurality of programmable nuclease complexes is greater than an amount of nucleic acids in the plurality of nucleic acids; and quantifying cleavage of the plurality of reporters, wherein at least a first reporter of the plurality of reporters is configured to be cleaved upon binding of a first guide nucleic acid of the one or more of programmable nuclease complex of a first particle to a first target nucleic acid of the plurality of nucleic acids, and wherein cleavage of the first reporter indicates a presence of the first target nucleic acid in the sample.

[00249] 2. The method of embodiment 1, further comprising, after applying the sample to the plurality of particles, fractionating the plurality of particles into a plurality of compartments.

[00250] 3. The method of embodiment 2, wherein the plurality of particles comprises a plurality of beads.

[00251] 4. The method of embodiment 2, wherein each compartment of the plurality of compartments comprises at most one particle of the plurality of particles.

[00252] 5. The method of embodiment 2, wherein each compartment of the plurality of compartments comprises at least one particle of the plurality of particles.

[00253] 6. The method of embodiment 1, wherein cleavage of the first reporter induces a change in a surface of the first particle, and wherein quantifying cleavage comprises detecting the change.

[00254] 7. The method of embodiment 6, wherein the change comprises a change in conductivity, a change in fluorescence intensity, a change in fluorescence wavelength, a change in absorbance, a change in luminescence, or an amperometric change.

[00255] 8. The method of embodiment 6, wherein the first programmable nuclease or the first guide nucleic acid is coupled to a detection moiety or a quenching moiety of the first reporter, wherein cleavage of the first reporter releases the moiety and leads to a change in the intensity of a signal from the first particle which is indicative of the change in the surface after release of the moiety and detecting said signal, and optionally wherein detecting the signal occurs as the first particle flows through a channel past a detector.

[00256] 9. The method of embodiment 1, wherein a ratio of the amount of nucleic acids to the amount of programmable nuclease complexes is about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10. [00257] 10. The method of embodiment 1, wherein a ratio of the amount of nucleic acids to the amount of programmable nuclease complexes is about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100.

[00258] 11. The method of embodiment 1, wherein the sample is not amplified prior to cleavage of the plurality of reporters.

[00259] 12. The method of embodiment 1, wherein a ratio of the amount of nucleic acids to the amount of particles is about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10.

[00260] 13. The method of embodiment 1 , wherein a ratio of the amount of nucleic acids to the amount of particles is about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100.

[00261] 14. The method of embodiment 1, wherein cleavage of the plurality of reporters comprises comparing a signal generated by the first particle to a reference signal.

[00262] 15. The method of embodiment 1, further comprising quantifying the number of particles in the plurality of particles having cleaved reporters following application of the sample to the plurality of particles, wherein each particle of the plurality of particles comprises at least one reporter of the plurality of reporters configured to be cleaved upon binding of a guide nucleic acid of the one or more of programmable nuclease complex of the particle to a target nucleic acid of the plurality of nucleic acids.

[00263] 16. The method of embodiment 15, wherein each particle generates a first signal prior to cleavage of the at least one reporter and a second signal after cleavage of the at least one reporter.

[00264] 17. The method of embodiment 16, wherein quantifying the number of particles comprises detecting the number of particles generating the second signal.

[00265] 18. A device for quantifying nucleic acids, the device comprising: an array comprising a plurality of surface locations, wherein a first surface location of the plurality of surface locations comprises at least one of a first programmable nuclease, a first reporter, or a first guide nucleic acid immobilized thereto, wherein a second surface location of the plurality of surface locations comprises at least one of a second programmable nuclease, a second reporter, or a second guide nucleic acid immobilized thereto, and wherein the first surface location is spatially distinct from the second surface location. [00266] 19. The device of embodiment 18, wherein the first guide nucleic acid and the first reporter are immobilized to the first surface location, wherein the first programmable nuclease is complexed with the first guide nucleic acid, and wherein the first reporter is configured to be cleaved when the first programmable nuclease is activated upon binding of the first guide nucleic acid to a first target nucleic acid.

[00267] 20. The device of embodiment 18, wherein the second guide nucleic acid and the second reporter are immobilized to the second surface location, wherein the second programmable nuclease is complexed with the second guide nucleic acid, and wherein the second reporter is configured to be cleaved when the second programmable nuclease is activated upon binding of the second guide nucleic acid to a second target nucleic acid. [00268] 21. The device of embodiment 18, wherein the plurality of surface locations comprises a plurality of spots, a plurality of particles, or a plurality of microwells, and wherein each surface location of the plurality of surface locations comprises at least one programmable nuclease, at least one reporter, and at least one guide nucleic acid.

[00269] 22. The device of embodiment 21, wherein the plurality of particles comprises a plurality of beads.

[00270] 23. The device of embodiment 18, wherein the array comprises a plurality of spots, a plurality of particles, or a plurality of microwells, and wherein the first surface location and the second surface location are located in a same spot of the plurality of spots, on a same particle of the plurality of particles, or in a same microwell of the plurality of microwells, respectively.

[00271] 24. The device of embodiment 18, wherein at least one of the first programmable nuclease, the first reporter, or the first guide nucleic acid are immobilized within a matrix disposed on the first surface.

[00272] 25. The device of embodiment 24, wherein the matrix comprises a hydrogel.

[00273] 26. The device of embodiment 18, wherein at least one of the first programmable nuclease, the first reporter, or the first guide nucleic acid are immobilized with a covalent linkage.

[00274] 27. The device of embodiment 26, wherein the covalent linkage comprises a bifunctional silane.

[00275] 28. The device of embodiment 1, wherein the first reporter and the second reporter are different types of reporters. [00276] 29. The device of embodiment 18, wherein the first reporter and the second reporter are a same type of reporter.

[00277] 30. The device of embodiment 18, wherein the first reporter or the second reporter comprises a detection moiety or a quenching moiety configured to be released upon cleavage of the first reporter or the second reporter, respectively.

[00278] 31. The device of embodiment 30, wherein the detection moiety comprises a fluorophore, a particle, an affinity molecule, an enzyme, or an enzyme substrate.

[00279] 32. The device of embodiment 31, wherein the particle comprises a quantum dot.

[00280] 33. The device of embodiment 31, wherein the particle is configured for encoded particle multiplexing.

[00281] 34. The device of embodiment 31, wherein the particle comprises one or more lanthanide elements.

[00282] 35. The device of embodiment 30, wherein the reporter comprises a fluorophore and a quencher, wherein the quenching moiety comprises the quencher, and wherein release of the quenching moiety results in fluorescence of the fluorophore.

[00283] 36. The device of embodiment 30, wherein the reporter comprises a fluorophore and a quencher, wherein the detection moiety comprises the fluorophore, and wherein release of the detection moiety results in fluorescence of the fluorophore.

[00284] 37. The device of embodiment 18, wherein the first programmable nuclease and the second programmable nuclease are different types of programmable nuclease.

[00285] 38. The device of embodiment 18, wherein the first programmable nuclease and the second programmable nuclease are a same type of programmable nuclease.

[00286] 39. The device of embodiment 18, wherein the first guide nucleic acid and the second guide nucleic acids are configured to bind to a same target nucleic acid sequence. [00287] 40. The device of embodiment 18, wherein the first guide nucleic acid and the second guide nucleic acids are configured to bind to different target nucleic acid sequences. [00288] 41. A device for quantifying nucleic acids, the device comprising: an inlet; and a first fluidic channel fluidly coupled to the inlet, wherein the first fluidic channel comprises at least one of a first programmable nuclease, a first reporter, or a first guide nucleic acid immobilized to a first surface thereof, wherein the first fluidic channel comprises at least one of a second programmable nuclease, a second reporter, or a second guide nucleic acid immobilized to a second surface thereof, and wherein the first surface is spatially distinct from the second surface.

[00289] 42. The device of embodiment 41, wherein the first guide nucleic acid and the first reporter are immobilized to the first surface, wherein the first programmable nuclease is complexed with the first guide nucleic acid, and wherein the first reporter is configured to be cleaved when the first programmable nuclease is activated upon binding of the first guide nucleic acid to a first target nucleic acid.

[00290] 43. The device of embodiment 41, wherein the second guide nucleic acid and the second reporter are immobilized to the second surface, wherein the second programmable nuclease is complexed with the second guide nucleic acid, and wherein the second reporter is configured to be cleaved when the second programmable nuclease is activated upon binding of the second guide nucleic acid to a second target nucleic acid.

[00291] 44. The device of embodiment 41, wherein the first surface and the second surface are adjacent sections of the first fluidic channel.

[00292] 45. The device of embodiment 41, wherein the first surface and the second surface are parallel stripes in a same section of the first fluidic channel.

[00293] 46. The device of embodiment 41, wherein at least one of the first programmable nuclease, the first reporter, or the first guide nucleic acid are immobilized within a matrix disposed on the first surface.

[00294] 47. The device of embodiment 46, wherein the matrix comprises a hydrogel.

[00295] 48. The device of embodiment 41, wherein at least one of the first programmable nuclease, the first reporter, or the first guide nucleic acid are immobilized with a covalent linkage.

[00296] 49. The device of embodiment 48, wherein the covalent linkage comprises a bifunctional silane.

[00297] 50. The device of embodiment 41, wherein the first reporter is immobilized to the first surface, wherein the first programmable nuclease or the first guide nucleic acid is coupled to a detection moiety of the first reporter, wherein the first programmable nuclease is complexed with the first guide nucleic acid, wherein the first reporter is configured to be cleaved when the first programmable nuclease is activated upon binding of the first guide nucleic acid to a first target nucleic acid, and wherein cleavage of the first reporter releases the detection moiety and the first programmable nuclease from the surface of the first fluidic channel.

[00298] 51. The device of embodiment 50, further comprising a second fluidic channel downstream of the first fluidic channel and coupled to a detector, wherein the detector is configured to detect the detection moiety as the first programmable nuclease flows through the second fluidic channel after being released by cleavage of the first reporter.

[00299] 52. The device of embodiment 41, wherein the first fluidic channel comprises a branching channel structure having a plurality of subchannels and configured to digitize or compartmentalize a sample into a plurality of compartments.

[00300] 53. The device of embodiment 52, wherein the plurality of compartments comprises a plurality of microwells, a plurality of nanowells, or a plurality of droplets.

[00301] 54. The device of embodiment 41, wherein the first reporter and the second reporter are different types of reporters.

[00302] 55. The device of embodiment 41, wherein the first reporter and the second reporter are a same type of reporter.

[00303] 56. The device of embodiment 41, wherein the first reporter or the second reporter comprises a detection moiety or a quenching moiety configured to be released upon cleavage of the first reporter or the second reporter, respectively.

[00304] 57. The device of embodiment 56, wherein the detection moiety comprises a fluorophore, a particle, an affinity molecule, an enzyme, or an enzyme substrate.

[00305] 58. The device of embodiment 57, wherein the particle comprises a quantum dot.

[00306] 59. The device of embodiment 57, wherein the particle is configured for encoded particle multiplexing.

[00307] 60. The device of embodiment 59, wherein the particle comprises one or more lanthanide elements.

[00308] 61. The device of embodiment 58, wherein the reporter comprises a fluorophore and a quencher, wherein the quenching moiety comprises the quencher, and wherein release of the quenching moiety results in fluorescence of the fluorophore.

[00309] 62. The device of embodiment 58, wherein the reporter comprises a fluorophore and a quencher, wherein the detection moiety comprises the fluorophore, and wherein release of the detection moiety results in fluorescence of the fluorophore. [00310] 63. The device of embodiment 41, wherein the first programmable nuclease and the second programmable nuclease are different types of programmable nuclease.

[00311] 64. The device of embodiment 41, wherein the first programmable nuclease and the second programmable nuclease are a same type of programmable nuclease.

[00312] 65. The device of embodiment 41, wherein the first guide nucleic acid and the second guide nucleic acids are configured to bind to a same target nucleic acid sequence. [00313] 66. The device of embodiment 41, wherein the first guide nucleic acid and the second guide nucleic acids are configured to bind to different target nucleic acid sequences. [00314] 67. A method for quantifying nucleic acids, the method comprising: providing a surface comprising a plurality of programmable nuclease complexes and a plurality of reporters immobilized thereon, wherein each programmable nuclease complex of the plurality of programmable nuclease complexes comprises a programmable nuclease and a guide nucleic acid; applying a sample to the surface, wherein the sample comprises a plurality of nucleic acids, wherein an amount of programmable nuclease complexes in the plurality of programmable nuclease complexes is greater than an amount of nucleic acids in the plurality of nucleic acids; and quantifying cleavage of the plurality of reporters, wherein at least a first reporter of the plurality of reporters is configured to be cleaved upon binding of a first guide nucleic acid of the plurality of programmable nuclease complexes to a first target nucleic acid of the plurality of nucleic acids, and wherein cleavage of the first reporter indicates a presence of the first target nucleic acid in the sample.

[00315] 68. The method of embodiment 67, wherein the surface is a surface of a spotted microarray, an array of particles, or an array of microwells, and wherein the surface comprises a plurality of locations, each of plurality of locations comprises at least one programmable nuclease complex of the plurality of programmable nuclease complexes and at least one reporter of the plurality of reporters immobilized on the surface.

[00316] 69. The method of embodiment 68, wherein the plurality of particles comprises a plurality of beads.

[00317] 70. The method of embodiment 68, wherein a first location of the plurality of locations comprises the first programmable nuclease complex and the first reporter. [00318] 71. The method of embodiment 67, wherein the surface is a surface of a fluidic channel and wherein applying the sample comprises flowing the sample over the surface.

[00319] 72. The method of embodiment 71, wherein cleavage of the first reporter induces a change in the surface at a location corresponding to a location of the first reporter, and wherein quantifying cleavage comprises detecting the change.

[00320] 73. The method of embodiment 72, wherein the change comprises a change in conductivity, a change in fluorescence intensity, a change in fluorescence wavelength, a change in absorbance, a change in luminescence, or an amperometric change.

[00321] 74. The method of embodiment 71, wherein the first programmable nuclease or the first guide nucleic acid is coupled to a detection moiety of the first reporter, wherein cleavage of the first reporter releases the detection moiety and the first programmable nuclease from the surface of the first fluidic channel and wherein quantifying cleavage comprises detection moiety as the first programmable nuclease flows past a detector after being released by cleavage of the first reporter.

[00322] 75. The method of embodiment 67, wherein a ratio of the amount of nucleic acids to the amount of programmable nuclease complexes is about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10.

[00323] 76. The method of embodiment 67, wherein a ratio of the amount of nucleic acids to the amount of programmable nuclease complexes is about 1:10, about 1 :20, about 1 :30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100.

[00324] 77. The method of embodiment 67, wherein the sample is not amplified prior to cleavage of the plurality of reporters.

[00325] 78. A system or kit comprising a first plurality of particles, wherein each particle of the first plurality of particles comprises one or more first programmable nuclease complex and a first plurality of reporters immobilized thereon, wherein the one or more first programmable nuclease complex comprises a first programmable nuclease and a first guide nucleic acid, wherein a first portion of the first guide nucleic acid is configured to recognize a first target nucleic acid sequence and a second portion of the first guide nucleic acid is configured to bind to the first programmable nuclease to form the first programmable nuclease complex, and optionally wherein each reporter of the first plurality of reporters comprises a detection moiety and/or a quencher moiety. [00326] 79. The system or kit of embodiment 78, further comprising a second plurality of particles, wherein each particle of the second plurality of particles comprises one or more second programmable nuclease complex and a second plurality of reporters immobilized thereon, wherein the one or more second programmable nuclease complex comprises a second programmable nuclease and a second guide nucleic acid, wherein a first portion of the second guide nucleic acid is configured to recognize a second target nucleic acid sequence and a second portion of the second guide nucleic acid is configured to bind to the second programmable nuclease to form the second programmable nuclease complex, and optionally wherein each reporter of the second plurality of reporters comprises a detection moiety and/or a quencher moiety.

[00327] 80. The system or kit of embodiment 79, wherein: i) the first programmable nuclease and the second programmable nuclease are a same type of programmable nuclease; ii) the first programmable nuclease and the second programmable nuclease are a different type of programmable nuclease; iii) the first reporter and the second reporter are a same type of reporter; iv) the first reporter and the second reporter are a different type of reporter; v) the first target nucleic acid sequence and the second target nucleic acid sequence are different portions of a same target nucleic acid; and/or vi) the first target nucleic acid sequence and the second target nucleic acid sequence are from different target nucleic acids.

[00328] 81. The system or kit of any one of embodiments 78, 79, or 80, wherein the first plurality of particles generates a first signal prior to cleavage of the first reporter and a second signal after cleavage of the first reporter.

[00329] 82. The system of kit of any one of embodiments 79, 80, or 81, wherein the second plurality of particles generates a third signal prior to cleavage of the second reporter and a fourth signal after cleavage of the second reporter.

[00330] 83. The system or kit of embodiment 82, wherein the first signal and third signal are the same.

[00331] 84. The system or kit of embodiment 82, wherein the first signal and the third signal are different. [00332] 85. The system or kit of any one of embodiments 82-84, wherein the second signal and the fourth signal are the same.

[00333] 86. The system or kit of any one of embodiments 82-84, wherein the second signal and the fourth signal are different.

EXAMPLES

[00334] 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: Continuous sample fractionation and reaction with CRISPR-Cas diagnostic assay components

[00335] This example describes a branching channel system for both continuous fractionation and continuous reaction of a sample solution containing target nucleic acids with CRISPR-Cas diagnostic assay components as illustrated in FIGS. 3A-3D. Here, a liquid sample containing target nucleic acids (306) is introduced into the branching channel system (300) on the left-hand side inlet. In this example, pressure is applied to the sample which is then directed into the diverging channel system. The branching structure is intended to draw the sample out into fractions of liquid. While the solution sample is being continuously fractionated, the target nucleic acids (306) are continuously exposed to a series of programmable nuclease-based reagents (304 and 305) embedded in a hydrogel matrix (303). The hydrogel matrix (303) lines the walls of the fluid channel (302), as can be seen in the expanded view of a section of the branching channel system (301). In this example, different regions (307) and (308) of the channel have different assay components (304) and (305), respectively, which allows for a series of assay steps to occur as the sample proceeds through the channel system, thus forming a Target-Cas-Reporter complex that is detected in a manner similar to flow cytometry. Additionally, in this example, crowding agents (407) are introduced along the channel in order to further isolate the target-probe complexes from each other for more efficient quantitative detection. At this point target-Cas-reporter complexes are detected in a flow cytometric fashion as seen in FIG. 4B. Example 2: Quantitative detection of target nucleic acids using programmable nuclease- based probes in a microarray format

[00336] In this example, target nucleic acids are quantitatively detected using a dual system of immobilized probes composed of an immobilized programmable nuclease probe complex (201) comprising a Cas protein (208) and a guide RNA (202) that selectively binds to a target nucleic acid (306), as seen in FIG. 2. Adjacent to the programmable nuclease probe (201) are one or more reporter probes (204) comprising a fluorophore (210) and a quencher (212). When the target nucleic acid (306) selectively binds to a guide nucleic acid (202), the Cas protein (208) is then triggered to cleave all cleavable regions of DNA around it. In this case those cleavable regions are located between the fluorophore (210) and the quencher (212). Therefore, upon cleavage, the fluorophore (210) and quencher (212) separate, which stops the quenching mechanism and causes the fluorophore to emit fluorescence and thus emit a signal for the presence of a target. In this example, there are 4 reporter probes (204) in close enough proximity to be cleaved by the Cas Protein. Therefore, this dual system of immobilized probes has a 1:4 ratio of target to number of fluorophores, allowing for signal amplification. Further, the dual probe system allows for sample multiplexing when used in a microarray format. Here, specific probe types are immobilized as spots (402) in known locations in a microarray format (400) as seen in FIG. 4A. In this example, the multiplexed sample containing multiple target nucleic acid sequences is introduced to the surface of the microarray and allowed to react in a static fashion. After the requisite time, the sample solution is washed away, and the array is read out by fluorescence microscopy. Spots that emit fluorescence signify a hit for a target nucleic acid known to correspond to the guide nucleic acid at that location.

Example 3: Quantitative detection of target nucleic acids using programmable nuclease- based probes in a flow-based format

[00337] In this example, target nucleic acids are quantitatively detected using a dual system of immobilized probes composed of an immobilized programmable nuclease probe complex (201) comprising a Cas protein (208) and, a guide RNA (202) that selectively binds to a target nucleic acid (306), as seen in FIG. 2. Adjacent to the programmable nuclease probe (201) are one or more reporter probes (204) comprising a fluorophore (210) and a quencher (212). The programmable nuclease probe complex (201) and reporter(s) (204) are immobilized on a particle, such as a bead. When the target nucleic acid (306) selectively binds to a guide nucleic acid (202), the Cas protein (208) is then triggered to cleave all cleavable regions of DNA around it. In this case those cleavable regions are located between the fluorophore (210) and the quencher (212). Therefore, upon cleavage, the fluorophore (210) and quencher (212) separate, which stops the quenching mechanism and causes the fluorophore to emit fluorescence and thus emit a signal for the presence of a target. In this example, there are 4 reporter probes (204) in close enough proximity to be cleaved by the Cas Protein. Each particle comprises a single Cas Protein. Therefore, this dual system of immobilized probes has a 1:4 ratio of target to number of fluorophores, allowing for signal amplification upon detection of a target. A sample suspected of comprising a target nucleic acid is mixed with a plurality of particles, each particle comprising a Cas protein and a plurality of reporters immobilized thereto. The plurality of particles is in excess of the amount of nucleic acids in the sample (e.g., about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100, or more) so as to ensure that every nucleic acid has a chance to interact with a programmable nuclease (and thus, every target nucleic acid has a chance to bind to a programmable nuclease probe complementary thereto). Upon binding of the programmable nuclease probe to a target nucleic acid, the programmable nuclease probe cleaves the reporters and releases the quenchers therefrom to generate a detectable complex comprising the cleaved reporters and activated programmable nuclease complex bound to the particle. The plurality of particles may optionally be washed (e.g., to remove the released quenchers and/or other contaminants), concentrated, or otherwise processed before being introduced into the branching channel system (300) on the left-hand side inlet shown in FIG. 3A. In this example, pressure is applied to the sample which is then directed into the diverging channel system. The branching structure is intended to draw the sample out into fractions of liquid. As the plurality of particles flows through the branching structure, the solution is continuously fractionated until they reach a detection channel at a single particle density for flow cytometric-like detection as shown in FIG. 4B.

Example 4: Quantitative multiplexed detection of target nucleic acids using programmable nuclease-based probes in a flow-based format

[00338] In this example, two different target nucleic acids are quantitatively detected using a first and second plurality of particles. A first system of immobilized probes composed of an immobilized first programmable nuclease probe complex (201) comprising a first Cas protein (208) and, a first guide RNA (202) that selectively binds to a first target nucleic acid (306), as seen in FIG. 2. Adjacent to the first programmable nuclease probe (201) are one or more first reporter probes (204) comprising a first fluorophore (210) and a first quencher (212). The first programmable nuclease probe complex (201) and first reporter(s) (204) are immobilized on a first particle, such as a bead. When the first target nucleic acid (306) selectively binds to the first guide nucleic acid (202), the first Cas protein (208) is then triggered to cleave all cleavable regions of DNA around it. In this case those cleavable regions are located between the first fluorophore (210) and the first quencher (212). Therefore, upon cleavage, the first fluorophore (210) and first quencher (212) separate, which stops the quenching mechanism and causes the first fluorophore to emit fluorescence and thus emit a first signal for the presence of a first target. In this example, there are 4 first reporter probes (204) in close enough proximity to be cleaved by the first Cas Protein. Each first particle comprises at least one first Cas Protein. A second system of immobilized probes composed of an immobilized second programmable nuclease probe complex (201) comprising a second Cas protein (208) and, a second guide RNA (202) that selectively binds to a second target nucleic acid (306), as seen in FIG. 2. Adjacent to the second programmable nuclease probe (201) are one or more second reporter probes (204) comprising a second fluorophore (210) and a second quencher (212). The second programmable nuclease probe complex (201) and second reporter(s) (204) are immobilized on a second particle, such as a bead. When the second target nucleic acid (306) selectively binds to the second guide nucleic acid (202), the second Cas protein (208) is then triggered to cleave all cleavable regions of DNA around it. In this case those cleavable regions are located between the second fluorophore (210) and the second quencher (212). Therefore, upon cleavage, the second fluorophore (210) and second quencher (212) separate, which stops the quenching mechanism and causes the second fluorophore to emit fluorescence and thus emit a second signal for the presence of a second target. In this example, there are 4 second reporter probes (204) in close enough proximity to be cleaved by the second Cas Protein. Each second particle comprises at least one second Cas Protein. A sample suspected of comprising a first target nucleic acid and/or a second target nucleic acid is mixed with a plurality of first particles and a plurality of second particles, each particle comprising a first or second Cas protein and a plurality of first or second reporters immobilized thereto, respectively. The plurality of first particles is in excess of the the amount of nucleic acids in the sample (e.g., about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100, or more) so as to ensure that every nucleic acid has a chance to interact with a first programmable nuclease (and thus, every first target nucleic acid has a chance to bind to a first programmable nuclease probe complementary thereto). The plurality of second particles is in excess of the the amount of nucleic acids in the sample (e.g., about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100, or more) so as to ensure that every nucleic acid has a chance to interact with a second programmable nuclease (and thus, every second target nucleic acid has a chance to bind to a second programmable nuclease probe complementary thereto). Upon binding of the first programmable nuclease probe to a first target nucleic acid, the first programmable nuclease probe cleaves the first reporters and releases the first quenchers therefrom to generate a first detectable complex comprising the cleaved first reporters and activated first programmable nuclease complex bound to the first particle. Upon binding of the second programmable nuclease probe to a second target nucleic acid, the second programmable nuclease probe cleaves the second reporters and releases the second quenchers therefrom to generate a second detectable complex comprising the cleaved second reporters and activated second programmable nuclease complex bound to the second particle. The plurality of first and second particles may optionally be washed (e.g., to remove the released quenchers and/or other contaminants), concentrated, or otherwise processed before being introduced into the branching channel system (300) on the left-hand side inlet shown in FIG. 3A. In this example, pressure is applied to the sample which is then directed into the diverging channel system. The branching structure is intended to draw the sample out into fractions of liquid. As the plurality of first and second particles flows through the branching structure, the solution is continuously fractionated until they reach a detection channel at a single particle density for flow cytometric like detection as shown in FIG. 4B. Detection of a first signal from the first detectable complex indicates the presence of the first target nucleic acid. Detection of a second signal from the second detectable complex indicates the second target nucleic acid. The number of instances of detection of the first signal and/or second signal may be quantified in order to quantify the number of target nucleic acids as described herein.

[00339] In some examples, the first programmable nuclease and the second programmable nuclease are a different type of programmable nuclease, for example a Type V and a Type VI Cas effector protein, etc.. In some examples, the first programmable nuclease and the second programmable nuclease are a same type of programmable nuclease, for example two different Type V Cas enzymes (e.g., a Cas 12 and a Casl4, a Casl2a or a Casl2b, two different Casl2a, or two of the same effector proteins, etc.).

[00340] In some examples, the first reporter and the second reporter are a same type of reporter (e.g., two DNA-based reporters, two RNA-based reporters, two identical reporters, etc.). In some examples, the first reporter and the second reporter are a different type of reporter (e.g., one DNA-based reporter and one RNA-based reporter, two RNA-based reporters with different nucleic acid sequences and/or detection moieties, etc.).

[00341] In some examples, the first guide nucleic acid and the second guide nucleic acid may hybridize to different portions of the same target nucleic acid. For example, the first and second guide nucleic acid may recognize different target nucleic acid sequences in the same target nucleic acid (e.g., two different sections of the S-gene of SARS-CoV-2 which have different SNPs in order to enable variant differentiation). In some examples, the first guide nucleic acid and the second guide nucleic acid may hybridize to different target nucleic acids. For example, the first and second guide nucleic acid may recognize different target nucleic acids in the same sample (e.g., a first target nucleic acid from SARS-CoV-2 and a second target nucleic acid from Influenza A so as to differentiate between two different viruses, or a first target nucleic acid from a cancer and a second target nucleic acid from a healthy cell as a control in order to quantify a percentage of cancerous cells, or a first target nucleic acid from a virus and a second target nucleic acid from a control gene like RNase P in order to confirm that the sample was collected and processed correctly, etc.).

[00342] In some examples, the first plurality of particles generates a first signal (e.g., fluorescence at a first wavelength) prior to cleavage of the first reporter and a second signal (e.g., fluorescence at a second wavelength) after cleavage of the first reporter. In some examples, the second plurality of particles generates a first signal (e.g., fluorescence at a first wavelength) prior to cleavage of the second reporter and a second signal (e.g., fluorescence at a second wavelength) after cleavage of the second reporter. The signal(s) generated by the first and second plurality of particles before and after cleavage of the first and second reporters, respectively, may be distinguishable from one another so as to quantify how many of the first plurality of particles encountered the first target nucleic acid and how many of the second plurality of particles encountered the second target nucleic acid.

[00343] 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.