Cross-Reference to Related Application

This application claims the benefit of priority to U.S. Provisional Application No. 63/367,988, filed July 8, 2022, the disclosure of which is incorporated by reference herein.

Incorporation by Reference of Sequence Listing

A Sequence Listing is provided herewith as XML file “2349574.xml” created on July 6, 2023 and having a size of 101,264 bytes. The content of the XML file is incorporated by reference herein in its entirety.

Background

Detection of respiratory infections, including SARS-CoV-2 and influenza A and B, is critical for targeting locations and populations that need medical assistance. For example, the estimated U.S. influenza illnesses in the 2019-2020 season was approximately 38 million people. In that same influenza season, approximately 400,000 people were hospitalized and approximately 22,000 died from the disease.

Current respiratory virus diagnostic assays include RT-qPCR nucleic-acid based tests (NATs) that require lab-based equipment and personnel or rapid influenza diagnostics (RIDTs) that detect viral antigens. These assays are not quantitative or multiplexed with other relevant respiratory viruses. These assays are also not appropriate for use by inexperienced or untrained personnel, such as for at home use.

A rapid, easy-to-use detection assay for viral RNA from respiratory body fluid samples is needed for identifying respiratory infections.

Summary

Described herein are methods, compositions, and devices for detecting and quantifying target viral RNA, such as Influenza A and B, that are faster and more readily deployable in the field than currently available methods and devices. In addition, the methods, compositions, and devices can readily detect and distinguish between strains and variants of the target viral RNA. Current rapid influenza diagnostic tests (RIDTs) are immunoassays that can identify the presence of influenza A and B viral nucleoprotein antigens in respiratory specimens and display the result in a qualitative way (positive vs. negative). However, RIDTs are known to have limited sensitivity to detect influenza in respiratory specimens compared to time-consuming RT-PCR or viral culture methods. Negative RIDT has the potential for false negative results, especially during peak influenza activity in a community.

The methods described herein can include: (a) incubating a sample suspected of containing Influenza A or B RNA or virus with one or more Cast 3 protein, at least one CRISPR guide RNA (crRNA), and at least one reporter RNA for a period of time sufficient to form at least one RNA cleavage product; and (b) detecting reporter RNA cleavage product(s) with a detector. Such methods are useful for detecting whether the sample contains one or more copies of Influenza A or B viral RNA. The methods are also useful for detecting the absence of infection with the virus carrying the target viral RNA. Moreover, the methods and compositions described herein can also readily identify whether a variant or mutant strain of virus carrying the target viral RNA is present in a sample, and what is the variant or mutation.

The methods described herein are useful for diagnosing Influenza infections in a variety of complex biological samples. For example, the samples can include human saliva, sputum, mucus, nasopharyngeal materials, blood, serum, plasma, urine, aspirate, biopsy tissue, or a combination thereof.

Description of the Figures

Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale. Instead, emphasis is placed on clearly illustrating the principles of the present disclosure. Furthermore, components can be shown as transparent in certain views for clarity of illustration only and not to indicate that the illustrated component is necessarily transparent.

FIG. 1A-1B illustrates use of CRISPR-Casl3 and CRISPR guide RNAs (crRNAs) to detect target RNA. FIG. 1A is a schematic diagram illustrating CRISPR-Casl3 detection of target viral RNA using a CRISPR-Casl3 protein that binds CRISPR guide RNAs (crRNA) to form a ribonucleoprotein (RNP) complex. The crRNA targets or guides the CRISPR-Casl3 protein to target viral RNA sequences (e.g., Influenza RNA), where the Casl3 protein is activated to cleave RNA, including the reporter RNA. FIG. IB is a similar schematic diagram further illustrating a Casl3a:crRNA ribonucleoprotein (RNP) complex binding of target viral RNA, resulting in activation of the Cast 3a nuclease (denoted by scissors). Upon target recognition and RNP activation, Cast 3a indiscriminately cleaves a quenched-fluorophore RNA reporter, allowing for fluorescence detection as a proxy for Cast 3a activation and the presence of target RNA.

FIG. 2 is a schematic diagram illustrating methods for detection of the SARS-CoV-2 RNA genome and fluorescent detection of reporter RNA. CRISPR guide RNAs (crRNA) that can target or bind to SARS-CoV-2 RNA are used. As illustrated, in a first step the CRISPR-Casl3 protein binds CRISPR guide RNAs (crRNA) to form a ribonucleoprotein (RNP) complex. The RNP complex is inactive but, when mixed with the sample to be tested, binding of the RNP complex to the SARS-CoV-2 RNA in the sample activates the Casl3 protein to cut RNA, including reporter RNA molecules added to the assay mixture. Cleavage of the reporter RNA leads to fluorescence, which can be detected by a fluorescence detector.

FIG. 3 illustrates a point-of-care (POC) method for detecting influenza. As illustrated, a sample can be collected (e.g., a patient’s saliva, sputum, mucus, or nasopharyngeal sample), the cells and/or viruses in the sample can be lysed to release any viral RNA that may be present, and the RNA from the sample can be mixed with reporter RNAs and a CRISPR-Casl3 protein-crRNA ribonucleoprotein (RNP) complex. Background fluorescence from control reactions can be subtracted and the fluorescence of the sample can be detected. Detection can be by a fluorometer or other suitable device. Such point-of-care detection allows mobilization of medical support and medical personnel.

FIGS. 4A-C shows the detection of influenza strains with specific RNA guides. The Influenza A and Influenza B RNA guides were designed to detect H1N1 or H3N2 strains of Influenza A or Influenza B (FluB). The RNA guides were tested against H1N1 , H3N2, FluB target viral RNA, and ribonucleoprotein (RNP) background control with no target viral RNA. FIG. 4A lists signal slope results for different Influenza A and Influenza B RNA guides. As shown in FIG. 4A, the signals from each reaction were measured over two hours and the signal slopes were calculated. Slope ratios were calculated by dividing the slope of a guide RNA + target (i.e. RNP + target viral RNA) reaction by the slope of guide RNA + no target (i.e. RNP control only) reaction. FIG. 4B also lists signal slope results for different Influenza A and Influenza B RNA guides. The comparative slope ratio of the target viral RNA to the RNP control shown in FIG. 4B was obtained by dividing the signal slopes of H1N1, H3N2, or FluB RNA guides by the signal slopes of the RNP control. When the comparative ratio is high (e.g., greater than 1), the guide RNAs employed in the assay mixture detect H1N1, H3N2, or FluB target viral RNA strains more efficiently. But when the comparative ratio is low (e.g., less than 1), the guide RNAs employed in the assay mixture detect the target viral RNA similarly to the RNP control. FIG. 4C lists RNA guide names for H1N1 and H3N2 strains of Influenza A with slope ratios of more than three and the RNA guides for FluB with slope ratios of more than five.

FIGS. 5A-B show the validation and cross-reactivity of Influenza B (FluB) RNA guides against host RNA and nasal swabs. FIG. 5A shows the signal slopes of the RNA guides for FluB having a slope ratio of more than five, as shown in FIG. 4C, as tested against host RNA and nasal swabs. The signals from each reaction were measured over two hours and the signal slopes were calculated. FIG. 5B shows the comparative slope ratio between the target viral RNA and the RNP control as obtained by dividing the signal slopes of the RNA guides for FluB by the signal slopes of the RNP control. FluB RNA guides FluB crlO and FluB-crl3 were found to cross-react significantly with nasal swab material that was positive for FluB. FluB_crl2 and FluB-crl4 were found to not cross-react to the same extent with the nasal swab material that was positive for FluB.

FIGS. 6A-B illustrate improved specificity and/or signals obtained for combined guide RNAs FluB_crl2 and FluB-crl4 that were targeted to different viral RNAs. FIG. 6A graphically illustrates that signal slopes from each reaction of target viral RNA for H3N1, H1N1, FluB, or RNP alone with the RNA guides FluB_crl2 alone, FluB-crl4 alone, or FluB_crl2 and FluB-crl4 combined as measured over two hours. FIG. 6B graphically illustrates the comparative slope ratio between the target viral RNA and the RNP control as obtained by dividing the signal slopes of the target viral RNA by the signal slopes of the RNP control. Combining the RNA guides FluB_crl2 and FluB-crl4 improves detection of FluB target viral RNA more than use of these RNA guides separately. Detection of H3N1, H1N1, or RNP alone was not increased by combining the RNA guides FluB_crl2 and FluB-crl4.

FIGS. 7A-B show the validation and cross-reactivity of Influenza A (H1N1 and H3N2 strains) RNA guides against host RNA and nasal swabs. FIG. 7A graphically illustrates the signal slopes from each reaction that were measured over two hours. The RNA guides for Influenza A having a slope ratio of more than three, as shown in FIG. 4C, were included in the test against host RNA and nasal swabs. FIG. 7B graphically illustrates the comparative slope ratio between the RNA guides for Influenza A and the RNP control as obtained by dividing the signal slopes of Influenza A by the RNP control. Influenza A RNA guides identified in the boxes had the best detection of the target viral RNA in the nasal swabs and were selected for a combination experiment shown in FIGS. 8A-B.

FIGS. 8A-B shows the effect of combining the best Influenza A RNA guides of FIG. 7A on Influenza A target viral RNA detection. FIG. 8A shows the signal slopes for combination of seven Influenza A RNA guides (the “7g”: cr04m, cr08, crl3, crl6, crl7, cr21, cr22) and four Influenza RNA guides (the “4g”: cr08, crl6, cr21, cr22) that were tested against target viral RNA for Influenza A (strains H1N1 and H3N2).

The signals from each reaction as measured over two hours. FIG. 8B shows the signal slopes of the RNA guides for Influenza A. The signals were divided by the signal slopes of the RNP control determine a comparative slope ratio between the target viral RNA and the RNP control. The slope ratios for the Influenza A RNA guides in the 7g group were 4.9 and 26.5 for the target viral RNA for H1N1 and H3N2, respectively. The slope ratios for the Influenza A RNA guides in the 4g group were 4.5 and 26.4 for the target viral RNA for H1N1 and H3N2, respectively. The slope ratios for each of the 7g and 4g RNA guide groups were significantly higher than for any of the Influenza A RNA guides alone.

Detailed Description

Methods, kits and devices are described herein for rapidly detecting and/or quantifying Influenza virus infection. The methods can include (a) incubating a sample suspected of containing RNA or virus with one or more Cast 3 protein, at least one CRISPR guide RNA (crRNA) that binds a target site in at least one of an Influenza A or Influenza B nucleic acid, and at least one reporter RNA for a period of time sufficient to form at least one RNA cleavage product(s); and (b) detecting level(s) of reporter RNA cleavage product(s) with a detector. Such methods are useful for detecting whether the sample contains one or more copies of an Influenza RNA. The methods are also useful for detecting the absence of an Influenza infection.

In some aspects, the disclosure provides methods for identifying the target virus RNA from a sample suspected of containing the target viral RNA. The target virus RNA can be from any RNA virus selected for detection in a sample. In some aspects, the target viral RNA can be from a virus that causes a respiratory infection or establishes its primary infection in the tissues and fluids of the upper respiratory tract. For example, the RNA virus can be an Influenza virus, such as Influenza A or B. Influenza is an enveloped, single stranded RNA virus that recognizes and binds to N-acetylneuraminic (sialic) acid on a host cell surface, including human tracheal epithelial and respiratory epithelium cells. Influenza A is the primary cause of flu epidemics. The target virus RNA can be the RNA from any of Influenza’s 18 distinct subtypes of hemagglutinin and 11 distinct subtypes of neuraminidase.

In addition to influenza viruses, the target viral RNA can be common cold coronaviruses, such as strains NL63, OC43, or 229E. The target viral RNA can also be SARS-CoV-2, a hepatitis virus (e.g., HCV), or respiratory syncytial virus (RSV). In some cases, the target viral RNA can be from the human immunodeficiency virus (HIV). The methods can thus be used to detect and identify a combination of viral RNAs, for example, using methods and components described in any of PCT publications WO 2020/051452; WO 2021/188830; and WO 2022/046706, each of which is incorporated by reference herein in its entirety.

In some aspects provided herein are methods for diagnosing the presence or absence of an Influenza infection comprising incubating a mixture comprising a sample suspected of containing Influenza RNA, a Cast 3 protein, at least one CRISPR guide RNA (crRNA), and a reporter RNA for a period of time to form any reporter RNA cleavage product(s) that may be present in the mixture; and detecting level(s) of reporter RNA cleavage product(s) that may be present in the mixture with a detector. In some cases, the Influenza RNA in a sample and/or the RNA cleavage products are not reverse transcribed prior to the detecting step. The presence or absence of an Influenza infection in patient is detected by qualitatively or quantitatively detecting level of reporter RNA cleavage product(s) that may be present in the mixture.

The methods described herein have various advantages. For example, the methods described herein can directly detect RNA without additional manipulations. No RNA amplification is generally needed, whereas currently available methods (e.g., SHERLOCK) require RNA amplification to be sufficiently sensitive. The methods, kits, and devices described herein are rapid, providing results within 30 minutes. Expensive lab equipment and expertise is not needed. The methods described herein are amenable to many different sample types (blood, nasal/oral swab, etc.). The methods, kits, and devices described herein are easily deployable in the field (airport screenings, borders, resource poor areas) so that potentially infected people will not need to go to hospitals and clinics where non-infected patients, vulnerable persons, and highly trained, urgently needed medical people may be. Hence, testing can be isolated from facilities needed for treatment of vulnerable populations and from trained personnel needed for urgent and complex medical procedures.

CRISPR-Cas 13 is a viable alternative to conventional methods of detecting and quantifying RNA by RT-PCR. The advantages of using CRISPR-Cas 13 can be leveraged for Influenza diagnostics. The Cast 3 protein targets RNA directly, and it can be programmed with crRNAs to provide a platform for specific RNA sensing. By coupling Cast 3 protein to an RNA- based reporter, the collateral or non-specific RNase activity of the Cast 3 protein can be harnessed for Influenza detection.

In 2017 and 2018, the laboratory of Dr. Feng Zhang reported a Cas 13 -based detection system that reached attomolar and zeptomolar sensitivity in detecting Zika virus, but it included an additional reverse transcription step for isothermal amplification of Zika virus cDNA, which was ultimately back-transcribed into RNA for RNA-based detection, a method referred to as SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) (Gootenberg et al. Science 356(6336): 438-42 (2017); Gootenberg et al. Science 360(6387): 439-44 (2018)). Although this method improved the sensitivity of Cas 13, it introduced two unwanted steps involving reverse transcription and in vitro transcription, which minimizes its potential as a field- deployable and point-of-care device.

The present disclosure provides methods and compositions for diagnosing Influenza infections, quantifying Influenza RNA concentrations, and identifying the presence of different Influenza A subtypes and/or mutations.

In some cases, the methods can be performed in a single tube, for example, the same tube used for collection and RNA extraction. This method provides a single step point of care diagnostic method. In other cases, the methods can be performed in a two-chamber system. For example, the collection swab containing a biological sample can be directly inserted into chamber one of such a two chamber system. After agitation, removal of the swab, and lysis of biological materials in the sample, the division between the two chambers can be broken or removed, and the contents of the first chamber can be allowed to flow into the second chamber. The second chamber can contain the Cas 13 protein, the selected crRNA(s), and the reporter RNA so that the assay for Influenza can be performed.

Chamber one can contain a buffer that would facilitate lysis of the viral particles and release of genomic material. Examples of lysis buffers that can be used include, but are not limited to PBS, commercial lysis buffers such as Qiagen RLT+ buffer or Quick Extract, DNA/RNA Shield, various concentrations of detergents such as Triton X-100, Tween 20, NP-40, or Oleth-8, or combinations of such reagents.

Following agitation and subsequent removal of the swab, the chamber may be briefly (e.g., 2-5 mins) heated (e.g., 55 °C or 95 °C) to further facilitate lysis. Then, the division between the two chambers would be broken or removed, and the nasal extract buffer would be allowed to flow into and reconstitute the second chamber, which would contain the lyophilized reagents for the Cast 3 assay (Cast 3 RNPs and reporter RNA molecules).

Use of such assay tubes can provide single step point of care diagnostic methods and devices.

The methods, devices and compositions described herein for diagnosing Influenza infection can involve incubating a mixture having a sample suspected of containing Influenza RNA, a Cast 3 protein, at least one CRISPR RNA (crRNA), and a reporter RNA for a period of time to form reporter RNA cleavage products that may be present in the mixture and detecting a level of any such reporter RNA cleavage products with a detector. The detector can be a fluorescence detector such as a short quenched-fluorescent RNA detector, or Total Internal Reflection Fluorescence (TIRF) detector.

Reporter.

A single type of reporter RNA can be used. The reporter RNA can be configured so that upon cleavage by the Cast 3 protein, a detectable signal occurs. For example, the reporter RNA can have a fluorophore at one location (e.g., one end) and a quencher at another location (e.g., the other end). In another example, the reporter RNA can have an electrochemical moiety (e.g., ferrocene, or dye), which upon cleavage by a Cast 3 protein can provide electron transfer to a redox probe or transducer. In another example, the reporter RNA can have a reporter dye, so that upon cleavage of the reporter RNA the reporter dye is detected by a detector (e.g., spectrophotometer). In some cases, one end of the reporter RNA can be bonded to a solid surface. For example, a reporter RNA can be configured as a cantilever, which upon cleavage releases a signal. However, in other cases, a signal may be improved by use of an unattached reporter RNA (e.g., not covalently bond to a solid surface). A surface of the assay vessel or the assay material can have a detector for sensing release of the signal. The signal can be or can include a light signal (e.g., fluorescence or a detectable dye), an electronic signal, an electrochemical signal, an electrostatic signal, a steric signal, a van der Waals interaction signal, a hydration signal, a Resonant frequency shift signal, or a combination thereof.

The reporter RNA can, for example, be at least one quenched-fluorescent RNA reporter. Such quenched-fluorescent RNA reporter can optimize fluorescence detection. The quenched- fluorescent RNA reporters include an RNA oligonucleotide with both a fluorophore and a quencher of the fluorophore. The quencher decreases or eliminates the fluorescence of the fluorophore. When the Cast 3 protein cleaves the RNA reporter, the fluorophore is separated from the associated quencher, such that a fluorescence signal becomes detectable.

One example of such a fluorophore quencher-labelled RNA reporter is the RNaseAlert (IDT). RNaseAlert was developed to detect RNase contaminations in a laboratory, and the substrate sequence is optimized for RNase A species. Another approach is to use lateral flow strips to detect a FAM-biotin reporter that, when cleaved by Casl3, is detected by anti-FAM antibody- gold nanoparticle conjugates on the strip. Although this allows for instrument-free detection, it requires 90-120 minutes for readout, compared to under 30 minutes for most fluorescence-based assays (Gootenberg etal. Science. 360(6387): 439-44 (April 2018)).

The sequence of the reporter RNA can be optimized for Cast 3 cleavage. Cast 3 preferentially exerts RNase cleavage activity at exposed uridine or adenosine sites, depending on the Cast 3 homolog. There are also secondary preferences for highly active homologs. The inventors have tested 5-mer homopolymers for all ribonucleotides. Based on these preferences, various RNA oligonucleotides, labeled at the 5' and 3' ends of the oligonucleotides using an Iowa Black Quencher (IDT) and FAM fluorophore, and systematically test these sequences in the trans- ssRNA cleavage assay as described in the Examples. The best sequence can be moved into the mobile testing.

The fluorophores used for the fluorophore quencher-labelled RNA reporters can include Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof.

Detection'.

Various mechanisms and devices can be employed to detect fluorescence. In some cases, the detector is a fluorescence detector, optionally a short quenched-fluorescent RNA detector, or Total Internal Reflection Fluorescence (TIRF) detector. For example, the fluorescence detector can detect fluorescence from fluorescence dyes such the Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof.

Some mechanisms or devices can be used to help eliminate background fluorescence. For example, reducing fluorescence from outside the detection focal plane can improve the signal-to- noise ratio, and consequently, the resolution of signal from the RNA cleavage products of interest. Total internal reflection fluorescence (TIRF) enables very low background fluorescence and single molecule sensitivity with a sufficiently sensitive camera. In some cases, mobile phones can be used for detection of Influenza.

In some cases, both Casl3 and reporter RNA can be tethered to a solid surface, upon addition of crRNA and Influenza RNA samples, an activated Cast 3 can generate small fluorescent spots on the solid surface when imaged using Total Internal Reflection Fluorescence (TIRF). To optimize this embodiment, the fluorophore side of reporter RNA is tethered to the solid surface as well so that cleavage permits the quencher portion of the reporter RNA to diffuse away. The Casl 3 protein can be tethered to the solid surface with a tether that is long enough to allow it to cleave multiple RNA reporter molecules. Counting the bright spots emerging on the solid surface the viral load can be quantified. Use of TIRF in the portable system facilitates detection and reduces background so that the RNA cleavage product signals can readily be detected.

In some cases, a ribonucleoprotein (RNP) complex of the Casl 3 protein and the crRNA can be tethered to the solid surface. The crRNA would then not need to be added later. Instead, only the sample suspected of containing Influenza RNA would need to be contacted with the solid surface.

In some cases, the methods described herein can include direct detection of the target RNA in the sample, without performing further sample preparation steps prior to detection, such as depleting a portion of the sample of protein, enzymes, lipids, nucleic acids, or a combination thereof or inactivating nucleases. However, the methods described herein can include depleting a portion of the sample prior to other step(s) or inhibiting a nuclease in the sample prior to the other step(s). For example, the sample can be depleted of protein, enzymes, lipids, nucleic acids, or a combination thereof. In some cases, the depleted portion of the sample is a human nucleic acid portion. However, RNA extraction of the sample is preferably not performed. In some cases, the methods can include removing ribonuclease(s) (RNase) from the sample. In some cases, the RNase is removed from the sample using an RNase inhibitor and/or heat.

In some cases, the Cast 3 protein and/or the crRNA can be lyophilized prior to incubation with the sample. In some cases, the Cast 3 protein, the crRNA, and/or the reporter RNA is lyophilized prior to incubation with the sample.

Sample:

In some embodiments, a biological sample is isolated from a patient. Non-limiting examples of suitable biological samples include saliva, sputum, mucus, nasopharyngeal samples, blood, serum, plasma, urine, aspirate, and biopsy samples. Thus, the term "sample" with respect to a patient can include RNA. Biological samples encompass saliva, sputum, mucus, and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, washed, or enrichment for certain cell populations. The definition also includes sample that have been enriched for particular types of molecules, e.g., RNAs. The term "sample" encompasses biological samples such as a clinical sample such as saliva, sputum, mucus, nasopharyngeal samples, blood, plasma, serum, aspirate, cerebral spinal fluid (CSF), and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, and the like. A "biological sample" includes biological fluids derived from cells and/or viruses (e.g., from infected cells). A sample containing RNAs can be obtained from such cells (e.g., a cell lysate or other cell extract comprising RNAs). A sample can comprise, or can be obtained from, any of a variety of bodily fluids (e.g., saliva, mucus, or sputum), cells, tissues, organs, or acellular fluids.

In some embodiments, the biological sample is isolated from a patient known to have or suspected to have an Influenza infection. In other embodiments, the biological sample is isolated from a patient not known have an Influenza infection. In other embodiments, the biological sample is isolated from a patient known to have, or suspected to not have, an Influenza infection. In other words, the methods and devices described herein can be used to identity subjects that have an Influenza infection and to confirm that subjects do not have an Influenza infection. In some cases, it may not be known whether the biological sample contains RNA. However, such biological samples can still be tested using the methods described herein. For example, biological samples can be subjected to lysis, RNA extraction, incubation with Cast 3 and crRNAs, etc. whether or not the sample actually contains RNA, and whether or not a sample contains Influenza RNA.

Pre-incubation of the crRNA and Cast 3 protein without the sample is preferred, so that the crRNA and the Cast 3 protein can form a complex. In some cases, the reporter RNA can be present while the crRNA and the Cast 3 protein form a complex. However, in other cases, the reporter RNA can be added after the crRNA and the Cast 3 protein already form a complex. Also, after formation of the crRNA/Casl3 complex, the sample RNA (e.g., Influenza RNA) can then be added. The sample RNA (e.g., Influenza RNA) acts as an activating RNA. Once activated by the activating RNA, the crRNA/Casl3 complex becomes a non-specific RNase to produce RNA cleavage products that can be detected using a reporter RNA, for example, a short quenched- fluorescent RNA.

For example, the Cast 3 and crRNA are incubated for a period of time to form the inactive complex. In some cases, the Cast 3 and crRNA complexes are formed by incubating together at 37 °C for 30 minutes, 1 hour, or 2 hours (for example, 0.5 to 2 hours) to form an inactive complex. The inactive complex can then be incubated with the reporter RNA. One example of a reporter RNA is provided by the RNase Alert system. The sample Influenza RNA can be a ssRNA activator. The Casl3/crRNA with the Influenza RNA sample becomes an activated complex that cleaves in cis and trans. When cleaving in cis, for example, the activated complex can cleave Influenza RNA. When cleaving in trans, the activated complex can cleave the reporter RNA, thereby releasing a signal such as the fluorophore from the reporter RNA.

CRISPR guide RNA (crRNA) -.

A CRISPR guide RNA system can be adapted for use in the methods and compositions described herein. The guide RNAs can include: a CRISPR RNA (crRNA or spacer), which can be a 17-20 nucleotide sequence complementary to the target DNA, and a trans-activating crRNA (tracrRNA or stem) that is a binding scaffold for the Cas nuclease. In some cases, the two RNAs are fused to make a single guide RNA (sgRNA). The tracrRNA forms a stem loop that is recognized and bound by the Cas nuclease. The term “guide RNA” as used herein refers to either a single guide RNA (sgRNA) or a crRNA (spacer). The CRISPR technique is generally described, for example, by Mali et al. Science 339:823-6 (2013); which is incorporated by reference herein in its entirety.

In some cases, the at least one CRISPR guide RNA (crRNA) has a sequence with at least 95% sequence identity to any of SEQ ID NOs: 1-37, shown below. In some cases, at least one CRISPR guide RNA (crRNA) has a sequence such as any of SEQ ID NOs: 1-37 or in some cases the crRNA(s) can include those with SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, 32, or 34-36, or a combination thereof. In some cases, the sample can be incubated with one or two or more crRNAs. For example, the sample can be incubated with at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least nine, or at least ten, or more crRNAs. In some cases, the at least one crRNA has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%, sequence identity to any SEQ ID NO: 1-37.

In various examples of crRNA(s) that can be used for detection of Influenza B, the crRNA(s) can include those with SEQ ID NOs: 32, 34, 35, 36, or a combination thereof. In some cases, SEQ ID NOs: 34 and 36 can be combined to improve detection of Influenza B.

In various examples of crRNA(s) that can be used for detection of Influenza A, the crRNA(s) can include those with SEQ ID NOs: 4, 8, 13, 16, 17, 21 , or 22, or a combination thereof. In some cases, the crRNA(s) can include those with SEQ ID NOs: 8, 16, 21, and 22.

The amount of reporter RNA cleavage product detected is directly correlated with the amount of the target viral RNA. In some cases, the target viral RNA cleavage product concentration can be quantified or determined by use of a standard curve of the reporter RNA cleavage product(s).

At least one crRNA can bind to a region in any of the eight single stranded RNAs of the Influenza RNA genome. In some cases, the region is a single stranded region of the Influenza RNA genome. In other cases, the region is a secondary structure in regions of the Influenza genome with low viral ribonucleoprotein binding.

In some cases, the crRNAs can include additional sequences such as spacer sequences. Table 1 provides examples of Influenza crRNA sequences.

Table 1: Examples of Influenza A and B crRNA Sequences

As illustrated herein, for detection of Influenza B, crRNAs with a sequence of SEQ ID NOs: 32, 34, 35, 36 exhibit better signals than crRNAs with a sequence of SEQ ID NOs: 23-31, 33, or 37. Moreover, the combination of the crRNAs of SEQ ID NOs: 34 and 36 significantly improves detection of Influenza B over using crRNAs of SEQ ID NOs: 34 or 36 alone.

To detect Influenza A, crRNAs with a sequence of SEQ ID NOs: 4, 8, 13, 16, 17, 21, or 22 exhibit better signals than crRNAs with a sequence of SEQ ID NOs: 1-3, 5-7, 9-12, 14, 15, or 18- 20. Moreover, the combination of the seven crRNAs of SEQ ID NOs: 4, 8, 13, 16, 17, 21, and 22 and independently the combination of the four crRNAs of SEQ ID NOs: 8, 16, 21, or 22 significantly improves detection of Influenza A over using the crRNAs of SEQ ID NOs: 4, 8, 13, 16, 17, 21, or 22 alone.

Influenza Sequences

A DNA sequence for the Influenza A genome, strain H1N1, with coding regions for each of the eight single stranded RNA segments, is available under the following accession numbers: Segment 1: NC_002023.1 from the NCBI website (provided as SEQ ID NO:51 herein).

1 AGCGAAAGCA GGTCAATTAT ATTCAATATG GAAAGAATAA AAGAACTAAG AAATCTAATG

61 TCGCAGTCTC GCACCCGCGA GATACTCACA AAAACCACCG TGGACCATAT GGCCATAATC

121 AAGAAGTACA CATCAGGAAG ACAGGAGAAG AACCCAGCAC TTAGGATGAA ATGGATGATG

181 GCAATGAAAT ATCCAATTAC AGCAGACAAG AGGATAACGG AAATGATTCC TGAGAGAAAT

241 GAGCAAGGAC AAACTTTATG GAGTAAAATG AATGATGCCG GATCAGACCG AGTGATGGTA

301 TCACCTCTGG CTGTGACATG GTGGAATAGG AATGGACCAA TGACAAATAC AGTTCATTAT

361 CCAAAAATCT ACAAAACTTA TTTTGAAAGA GTCGAAAGGC TAAAGCATGG AACCTTTGGC

421 CCTGTCCATT TTAGAAACCA AGTCAAAATA CGTCGGAGAG TTGACATAAA TCCTGGTCAT

481 GCAGATCTCA GTGCCAAGGA GGCACAGGAT GTAATCATGG AAGTTGTTTT CCCTAACGAA

541 GTGGGAGCCA GGATACTAAC ATCGGAATCG CAACTAACGA TAACCAAAGA GAAGAAAGAA

601 GAACTCCAGG ATTGCAAAAT TTCTCCTTTG ATGGTTGCAT ACATGTTGGA GAGAGAACTG

661 GTCCGCAAAA CGAGATTCCT CCCAGTGGCT GGTGGAACAA GCAGTGTGTA CATTGAAGTG

721 TTGCATTTGA CTCAAGGAAC ATGCTGGGAA CAGATGTATA CTCCAGGAGG GGAAGTGAAG

781 AATGATGATG TTGATCAAAG CTTGATTATT GCTGCTAGGA ACATAGTGAG AAGAGCTGCA

841 GTATCAGCAG ACCCACTAGC ATCTTTATTG GAGATGTGCC ACAGCACACA GATTGGTGGA

901 ATTAGGATGG TAGACATCCT TAAGCAGAAC CCAACAGAAG AGCAAGCCGT GGGTATATGC

961 AAGGCTGCAA TGGGACTGAG AATTAGCTCA TCCTTCAGTT TTGGTGGATT CACATTTAAG

1021 AGAACAAGCG GATCATCAGT CAAGAGAGAG GAAGAGGTGC TTACGGGCAA TCTTCAAACA

1081 TTGAAGATAA GAGTGCATGA GGGATATGAA GAGTTCACAA TGGTTGGGAG AAGAGCAACA

1141 GCCATACTCA GAAAAGCAAC CAGGAGATTG ATTCAGCTGA TAGTGAGTGG GAGAGACGAA

1201 CAGTCGATTG CCGAAGCAAT AATTGTGGCC ATGGTATTTT CACAAGAGGA TTGTATGATA

1261 AAAGCAGTTA GAGGTGATCT GAATTTCGTC AATAGGGCGA ATCAGCGACT GAATCCTATG

1321 CATCAACTTT TAAGACATTT TCAGAAGGAT GCGAAAGTGC TTTTTCAAAA TTGGGGAGTT

1381 GAACCTATCG ACAATGTGAT GGGAATGATT GGGATATTGC CCGACATGAC TCCAAGCATC

1441 GAGATGTCAA TGAGAGGAGT GAGAATCAGC AAAATGGGTG TAGATGAGTA CTCCAGCACG

1501 GAGAGGGTAG TGGTGAGCAT TGACCGGTTC TTGAGAGTCC GGGACCAACG AGGAAATGTA 1561 CTACTGTCTC CCGAGGAGGT CAGTGAAACA CAGGGAACAG AGAAACTGAC AATAACTTAC

1621 TCATCGTCAA TGATGTGGGA GATTAATGGT CCTGAATCAG TGTTGGTCAA TACCTATCAA

1681 TGGATCATCA GAAACTGGGA AACTGTTAAA ATTCAGTGGT CCCAGAACCC TACAATGCTA

1741 TACAATAAAA TGGAATTTGA ACCATTTCAG TCTTTAGTAC CTAAGGCCAT TAGAGGCCAA

1801 TACAGTGGGT TTGTGAGAAC TCTGTTCCAA CAAATGAGGG ATGTGCTTGG GACATTTGAT

1861 ACCGCACAGA TAATAAAACT TCTTCCCTTC GCAGCCGCTC CACCAAAGCA AAGTAGAATG

1921 CAGTTCTCCT CATTTACTGT GAATGTGAGG GGATCAGGAA TGAGAATACT TGTAAGGGGC

1981 AATTCTCCTG TATTCAACTA CAACAAGGCC ACGAAGAGAC TCACAGTTCT CGGAAAGGAT

2041 GCTGGCACTT TAACCGAAGA CCCAGATGAA GGCACAGCTG GAGTGGAGTC CGCTGTTCTG

2101 AGGGGATTCC TCATTCTGGG CAAAGAAGAC AGGAGATATG GGCCAGCATT AAGCATCAAT

2161 GAACTGAGCA ACCTTGCGAA AGGAGAGAAG GCTAATGTGC TAATTGGGCA AGGAGACGTG

2221 GTGTTGGTAA TGAAACGAAA ACGGGACTCT AGCATACTTA CTGACAGCCA GACAGCGACC

2281 AAAAGAATTC GGATGGCCAT CAATTAGTGT CGAATAGTTT AAAAACGACC TTGTTTCTAC 2341 T

Segment 2: NC_002021.1 from the NCBI website (provided as SEQ ID NO:52 herein).

1 AGCGAAAGCA GGCAAACCAT TTGAATGGAT GTCAATCCGA CCTTACTTTT CTTAAAAGTG

61 CCAGCACAAA ATGCTATAAG CACAACTTTC CCTTATACCG GAGACCCTCC TTACAGCCAT

121 GGGACAGGAA CAGGATACAC CATGGATACT GTCAACAGGA CACATCAGTA CTCAGAAAAG

181 GCAAGATGGA CAACAAACAC CGAAACTGGA GCACCGCAAC TCAACCCGAT TGATGGGCCA

241 CTGCCAGAAG ACAATGAACC AAGTGGTTAT GCCCAAACAG ATTGTGTATT GGAAGCAATG

301 GCTTTCCTTG AGGAATCCCA TCCTGGTATT TTTGAAAACT CGTGTATTGA AACGATGGAG

361 GTTGTTCAGC AAACACGAGT AGACAAGCTG ACACAAGGCC GACAGACCTA TGACTGGACT

421 TTAAATAGAA ACCAGCCTGC TGCAACAGCA TTGGCCAACA CAATAGAAGT GTTCAGATCA

481 AATGGCCTCA CGGCCAATGA GTCTGGAAGG CTCATAGACT TCCTTAAGGA TGTAATGGAG

541 TCAATGAAAA AAGAAGAAAT GGGGATCACA ACTCATTTTC AGAGAAAGAG ACGGGTGAGA

601 GACAATATGA CTAAGAAAAT GATAACACAG AGAACAATAG GTAAAAGGAA ACAGAGATTG

661 AACAAAAGGA GTTATCTAAT TAGAGCATTG ACCCTGAACA CAATGACCAA AGATGCTGAG

721 AGAGGGAAGC TAAAACGGAG AGCAATTGCA ACCCCAGGGA TGCAAATAAG GGGGTTTGTA

781 TACTTTGTTG AGACACTGGC AAGGAGTATA TGTGAGAAAC TTGAACAATC AGGGTTGCCA

841 GTTGGAGGCA ATGAGAAGAA AGCAAAGTTG GCAAATGTTG TAAGGAAGAT GATGACCAAT

901 TCTCAGGACA CCGAACTTTC TTTGACCATC ACTGGAGATA ACACCAAATG GAACGAAAAT 961 CAGAATCCTC GGATGTTTTT GGCCATGATC ACATATATGA CCAGAAATCA GCCCGAATGG

1021 TTCAGAAATG TTCTAAGTAT TGCTCCAATA ATGTTCTCAA ACAAAATGGC GAGACTGGGA

1081 AAAGGGTATA TGTTTGAGAG CAAGAGTATG AAACTTAGAA CTCAAATACC TGCAGAAATG

1141 CTAGCAAGCA TTGATTTGAA ATATTTCAAT GATTCAACAA GAAAGAAGAT TGAAAAAATC

1201 CGACCGCTCT TAATAGAGGG GACTGCATCA TTGAGCCCTG GAATGATGAT GGGCATGTTC

1261 AATATGTTAA GCACTGTATT AGGCGTCTCC ATCCTGAATC TTGGACAAAA GAGATACACC

1321 AAGACTACTT ACTGGTGGGA TGGTCTTCAA TCCTCTGACG ATTTTGCTCT GATTGTGAAT

1381 GCACCCAATC ATGAAGGGAT TCAAGCCGGA GTCGACAGGT TTTATCGAAC CTGTAAGCTA

1441 CATGGAATCA ATATGAGCAA GAAAAAGTCT TACATAAACA GAACAGGTAC ATTTGAATTC

1501 ACAAGTTTTT TCTATCGTTA TGGGTTTGTT GCCAATTTCA GCATGGAGCT TCCCAGTTTT

1561 GGTGTGTCTG GGAGCAACGA GTCAGCGGAC ATGAGTATTG GAGTTACTGT CATCAAAAAC

1621 AATATGATAA ACAATGATCT TGGTCCAGCA ACAGCTCAAA TGGCCCTTCA GTTGTTCATC

1681 AAAGATTACA GGTACACGTA CCGATGCCAT AGAGGTGACA CACAAATACA AACCCGAAGA

1741 TCATTTGAAA TAAAGAAACT GTGGGAGCAA ACCCGTTCCA AAGCTGGACT GCTGGTCTCC

1801 GACGGAGGCC CAAATTTATA CAACATTAGA AATCTCCACA TTCCTGAAGT CTGCCTAAAA

1861 TGGGAATTGA TGGATGAGGA TTACCAGGGG CGTTTATGCA ACCCACTGAA CCCATTTGTC

1921 AGCCATAAAG AAATTGAATC AATGAACAAT GCAGTGATGA TGCCAGCACA TGGTCCAGCC

1981 AAAAACATGG AGTATGATGC TGTTGCAACA ACACACTCCT GGATCCCCAA AAGAAATCGA

2041 TCCATCTTGA ATACAAGTCA AAGAGGAGTA CTTGAAGATG AACAAATGTA CCAAAGGTGC

2101 TGCAATTTAT TTGAAAAATT CTTCCCCAGC AGTTCATACA GAAGACCAGT CGGGATATCC

2161 AGTATGGTGG AGGCTATGGT TTCCAGAGCC CGAATTGATG CACGGATTGA TTTCGAATCT

2221 GGAAGGATAA AGAAAGAAGA GTTCACTGAG ATCATGAAGA TCTGTTCCAC CATTGAAGAG

2281 CTCAGACGGC AAAAATAGTG AATTTAGCTT GTCCTTCATG AAAAAATGCC TTGTTCCTAC 2341 T

Segment 3: NC_002022.1 from the NCBI website (provided as SEQ ID NO:53 herein).

1 AGCGAAAGCA GGTACTGATC CAAAATGGAA GATTTTGTGC GACAATGCTT CAATCCGATG

61 ATTGTCGAGC TTGCGGAAAA AACAATGAAA GAGTATGGGG AGGACCTGAA AATCGAAACA

121 AACAAATTTG CAGCAATATG CACTCACTTG GAAGTATGCT TCATGTATTC AGATTTCCAC

181 TTCATCAATG AGCAAGGCGA GTCAATAATC GTAGAACTTG GTGATCCTAA TGCACTTTTG

241 AAGCACAGAT TTGAAATAAT CGAGGGAAGA GATCGCACAA TGGCCTGGAC AGTAGTAAAC

301 AGTATTTGCA ACACTACAGG GGCTGAGAAA CCAAAGTTTC TACCAGATTT GTATGATTAC 361 AAGGAAAATA GATTCATCGA AATTGGAGTA ACAAGGAGAG AAGTTCACAT ATACTATCTG

421 GAAAAGGCCA ATAAAATTAA ATCTGAGAAA ACACACATCC ACATTTTCTC GTTCACTGGG

481 GAAGAAATGG CCACAAAGGC CGACTACACT CTCGATGAAG AAAGCAGGGC TAGGATCAAA

541 ACCAGGCTAT TCACCATAAG ACAAGAAATG GCCAGCAGAG GCCTCTGGGA TTCCTTTCGT

601 CAGTCCGAGA GAGGAGAAGA GACAATTGAA GAAAGGTTTG AAATCACAGG AACAATGCGC

661 AAGCTTGCCG ACCAAAGTCT CCCGCCGAAC TTCTCCAGCC TTGAAAATTT TAGAGCCTAT

721 GTGGATGGAT TCGAACCGAA CGGCTACATT GAGGGCAAGC TGTCTCAAAT GTCCAAAGAA

781 GTAAATGCTA GAATTGAACC TTTTTTGAAA ACAACACCAC GACCACTTAG ACTTCCGAAT

841 GGGCCTCCCT GTTCTCAGCG GTCCAAATTC CTGCTGATGG ATGCCTTAAA ATTAAGCATT

901 GAGGACCCAA GTCATGAAGG AGAGGGAATA CCGCTATATG ATGCAATCAA ATGCATGAGA

961 ACATTCTTTG GATGGAAGGA ACCCAATGTT GTTAAACCAC ACGAAAAGGG AATAAATCCA

1021 AATTATCTTC TGTCATGGAA GCAAGTACTG GCAGAACTGC AGGACATTGA GAATGAGGAG

1081 AAAATTCCAA AGACTAAAAA TATGAAAAAA ACAAGTCAGC TAAAGTGGGC ACTTGGTGAG

1141 AACATGGCAC CAGAAAAGGT AGACTTTGAC GACTGTAAAG ATGTAGGTGA TTTGAAGCAA

1201 TATGATAGTG ATGAACCAGA ATTGAGGTCG CTTGCAAGTT GGATTCAGAA TGAGTTCAAC

1261 AAGGCATGCG AACTGACAGA TTCAAGCTGG ATAGAGCTTG ATGAGATTGG AGAAGATGTG

1321 GCTCCAATTG AACACATTGC AAGCATGAGA AGGAATTATT TCACATCAGA GGTGTCTCAC

1381 TGCAGAGCCA CAGAATACAT AATGAAGGGG GTGTACATCA ATACTGCCTT ACTTAATGCA

1441 TCTTGTGCAG CAATGGATGA TTTCCAATTA ATTCCAATGA TAAGCAAGTG TAGAACTAAG

1501 GAGGGAAGGC GAAAGACCAA CTTGTATGGT TTCATCATAA AAGGAAGATC CCACTTAAGG

1561 AATGACACCG ACGTGGTAAA CTTTGTGAGC ATGGAGTTTT CTCTCACTGA CCCAAGACTT

1621 GAACCACACA AATGGGAGAA GTACTGTGTT CTTGAGATAG GAGATATGCT TCTAAGAAGT

1681 GCCATAGGCC AGGTTTCAAG GCCCATGTTC TTGTATGTGA GGACAAATGG AACCTCAAAA

1741 ATTAAAATGA AATGGGGAAT GGAGATGAGG CGTTGTCTCC TCCAGTCACT TCAACAAATT

1801 GAGAGTATGA TTGAAGCTGA GTCCTCTGTC AAAGAGAAAG ACATGACCAA AGAGTTCTTT

1861 GAGAACAAAT CAGAAACATG GCCCATTGGA GAGTCTCCCA AAGGAGTGGA GGAAAGTTCC

1921 ATTGGGAAGG TCTGCAGGAC TTTATTAGCA AAGTCGGTAT TTAACAGCTT GTATGCATCT

1981 CCACAACTAG AAGGATTTTC AGCTGAATCA AGAAAACTGC TTCTTATCGT TCAGGCTCTT

2041 AGGGACAATC TGGAACCTGG GACCTTTGAT CTTGGGGGGC TATATGAAGC AATTGAGGAG

2101 TGCCTAATTA ATGATCCCTG GGTTTTGCTT AATGCTTCTT GGTTCAACTC CTTCCTTACA

2161 CATGCATTGA GTTAGTTGTG GCAGTGCTAC TATTTGCTAT CCATACTGTC CAAAAAAGTA

2221 CCTTGTTTCT ACT Segment 4: NC_002017.1 from the NCBI website (provided as SEQ ID NO:54 herein).

1 AGCAAAAGCA GGGGAAAATA AAAACAACCA AAATGAAGGC AAACCTACTG GTCCTGTTAT

61 GTGCACTTGC AGCTGCAGAT GCAGACACAA TATGTATAGG CTACCATGCG AACAATTCAA

121 CCGACACTGT TGACACAGTG CTCGAGAAGA ATGTGACAGT GACACACTCT GTTAACCTGC

181 TCGAAGACAG CCACAACGGA AAACTATGTA GATTAAAAGG AATAGCCCCA CTACAATTGG

241 GGAAATGTAA CATCGCCGGA TGGCTCTTGG GAAACCCAGA ATGCGACCCA CTGCTTCCAG

301 TGAGATCATG GTCCTACATT GTAGAAACAC CAAACTCTGA GAATGGAATA TGTTATCCAG

361 GAGATTTCAT CGACTATGAG GAGCTGAGGG AGCAATTGAG CTCAGTGTCA TCATTCGAAA

421 GATTCGAAAT ATTTCCCAAA GAAAGCTCAT GGCCCAACCA CAACACAACC AAAGGAGTAA

481 CGGCAGCATG CTCCCATGCG GGGAAAAGCA GTTTTTACAG AAATTTGCTA TGGCTGACGG

541 AGAAGGAGGG CTCATACCCA AAGCTGAAAA ATTCTTATGT GAACAAGAAA GGGAAAGAAG

601 TCCTTGTACT GTGGGGTATT CATCACCCGT CTAACAGTAA GGATCAACAG AATATCTATC

661 AGAATGAAAA TGCTTATGTC TCTGTAGTGA CTTCAAATTA TAACAGGAGA TTTACCCCGG

721 AAATAGCAGA AAGACCCAAA GTAAGAGATC AAGCTGGGAG GATGAACTAT TACTGGACCT

781 TGCTAAAACC CGGAGACACA ATAATATTTG AGGCAAATGG AAATCTAATA GCACCAAGGT

841 ATGCTTTCGC ACTGAGTAGA GGCTTTGGGT CCGGCATCAT CACCTCAAAC GCATCAATGC

901 ATGAGTGTAA CACGAAGTGT CAAACACCCC TGGGAGCTAT AAACAGCAGT CTCCCTTTCC

961 AGAATATACA CCCAGTCACA ATAGGAGAGT GCCCAAAATA CGTCAGGAGT GCCAAATTGA

1021 GGATGGTTAC AGGACTAAGG AACATTCCGT CCATTCAATC CAGAGGTCTA TTTGGAGCCA

1081 TTGCCGGTTT TATTGAAGGG GGATGGACTG GAATGATAGA TGGATGGTAC GGTTATCATC

1141 ATCAGAATGA ACAGGGATCA GGCTATGCAG CGGATCAAAA AAGCACACAA AATGCCATTA

1201 ACGGGATTAC AAACAAGGTG AACTCTGTTA TCGAGAAAAT GAACATTCAA TTCACAGCTG

1261 TGGGTAAAGA ATTCAACAAA TTAGAAAAAA GGATGGAAAA TTTAAATAAA AAAGTTGATG

1321 ATGGATTTCT GGACATTTGG ACATATAATG CAGAATTGTT AGTTCTACTG GAAAATGAAA

1381 GGACTCTGGA TTTCCATGAC TCAAATGTGA AGAATCTGTA TGAGAAAGTA AAAAGCCAAT

1441 TAAAGAATAA TGCCAAAGAA ATCGGAAATG GATGTTTTGA GTTCTACCAC AAGTGTGACA

1501 ATGAATGCAT GGAAAGTGTA AGAAATGGGA CTTATGATTA TCCCAAATAT TCAGAAGAGT

1561 CAAAGTTGAA CAGGGAAAAG GTAGATGGAG TGAAATTGGA ATCAATGGGG ATCTATCAGA

1621 TTCTGGCGAT CTACTCAACT GTCGCCAGTT CACTGGTGCT TTTGGTCTCC CTGGGGGCAA

1681 TCAGTTTCTG GATGTGTTCT AATGGATCTT TGCAGTGCAG AA

Segment 5: NC_002019.1 from the NCBI website (provided as SEQ ID NO:55 herein). 1 AGCAAAAGCA GGGTAGATAA TCACTCACTG AGTGACATCA AAATCATGGC GTCCCAAGGC

61 ACCAAACGGT CTTACGAACA GATGGAGACT GATGGAGAAC GCCAGAATGC CACTGAAATC

121 AGAGCATCCG TCGGAAAAAT GATTGGTGGA ATTGGACGAT TCTACATCCA AATGTGCACA

181 GAACTTAAAC TCAGTGATTA TGAGGGACGG TTGATCCAAA ACAGCTTAAC AATAGAGAGA

241 ATGGTGCTCT CTGCTTTTGA CGAAAGGAGA AATAAATACC TGGAAGAACA TCCCAGTGCG

301 GGGAAGGATC CTAAGAAAAC TGGAGGACCT ATATACAGAA GAGTAAACGG AAAGTGGATG

361 AGAGAACTCA TCCTTTATGA CAAAGAAGAA ATAAGGCGAA TCTGGCGCCA AGCTAATAAT

421 GGTGACGATG CAACGGCTGG TCTGACTCAC ATGATGATCT GGCATTCCAA TTTGAATGAT

481 GCAACTTATC AGAGGACAAG GGCTCTTGTT CGCACCGGAA TGGATCCCAG GATGTGCTCT

541 CTGATGCAAG GTTCAACTCT CCCTAGGAGG TCTGGAGCCG CAGGTGCTGC AGTCAAAGGA

601 GTTGGAACAA TGGTGATGGA ATTGGTCAGG ATGATCAAAC GTGGGATCAA TGATCGGAAC

661 TTCTGGAGGG GTGAGAATGG ACGAAAAACA AGAATTGCTT ATGAAAGAAT GTGCAACATT

721 CTCAAAGGGA AATTTCAAAC TGCTGCACAA AAAGCAATGA TGGATCAAGT GAGAGAGAGC

781 CGGGACCCAG GGAATGCTGA GTTCGAAGAT CTCACTTTTC TAGCACGGTC TGCACTCATA

841 TTGAGAGGGT CGGTTGCTCA CAAGTCCTGC CTGCCTGCCT GTGTGTATGG ACCTGCCGTA

901 GCCAGTGGGT ACGACTTTGA AAGAGAGGGA TACTCTCTAG TCGGAATAGA CCCTTTCAGA

961 CTGCTTCAAA ACAGCCAAGT GTACAGCCTA ATCAGACCAA ATGAGAATCC AGCACACAAG

1021 AGTCAACTGG TGTGGATGGC ATGCCATTCT GCCGCATTTG AAGATCTAAG AGTATTGAGC

1081 TTCATCAAAG GGACGAAGGT GGTCCCAAGA GGGAAGCTTT CCACTAGAGG AGTTCAAATT

1141 GCTTCCAATG AAAATATGGA GACTATGGAA TCAAGTACAC TTGAACTGAG AAGCAGGTAC

1201 TGGGCCATAA GGACCAGAAG TGGAGGAAAC ACCAATCAAC AGAGGGCATC TGCGGGCCAA

1261 ATCAGCATAC AACCTACGTT CTCAGTACAG AGAAATCTCC CTTTTGACAG AACAACCGTT

1321 ATGGCAGCAT TCACTGGGAA TACAGAGGGG AGAACATCTG ACATGAGGAC CGAAATCATA

1381 AGGATGATGG AAAGTGCAAG ACCAGAAGAT GTGTCTTTCC AGGGGCGGGG AGTCTTCGAG

1441 CTCTCGGACG AAAAGGCAGC GAGCCCGATC GTGCCTTCCT TTGACATGAG TAATGAAGGA

1501 TCTTATTTCT TCGGAGACAA TGCAGAGGAG TACGACAATT AAAGAAAAAT ACCCTTGTTT

1561 CTACT

Segment 6: NC_002018.1 from the NCBI website (provided as SEQ ID NO:56 herein).

1 AGCGAAAGCA GGGGTTTAAA ATGAATCCAA ATCAGAAAAT AATAACCATT GGATCAATCT

61 GTCTGGTAGT CGGACTAATT AGCCTAATAT TGCAAATAGG GAATATAATC TCAATATGGA

121 TTAGCCATTC AATTCAAACT GGAAGTCAAA ACCATACTGG AATATGCAAC CAAAACATCA 181 TTACCTATAA AAATAGCACC TGGGTAAAGG ACACAACTTC AGTGATATTA ACCGGCAATT

241 CATCTCTTTG TCCCATCCGT GGGTGGGCTA TATACAGCAA AGACAATAGC ATAAGAATTG

301 GTTCCAAAGG AGACGTTTTT GTCATAAGAG AGCCCTTTAT TTCATGTTCT CACTTGGAAT

361 GCAGGACCTT TTTTCTGACC CAAGGTGCCT TACTGAATGA CAGGCATTCA AATGGGACTG

421 TTAAGGACAG AAGCCCTTAT AGGGCCTTAA TGAGCTGCCC TGTCGGTGAA GCTCCGTCCC

481 CGTACAATTC AAGATTTGAA TCGGTTGCTT GGTCAGCAAG TGCATGTCAT GATGGCATGG

541 GCTGGCTAAC AATCGGAATT TCAGGTCCAG ATAATGGAGC AGTGGCTGTA TTAAAATACA

601 ACGGCATAAT AACTGAAACC ATAAAAAGTT GGAGGAAGAA AATATTGAGG ACACAAGAGT

661 CTGAATGTGC CTGTGTAAAT GGTTCATGTT TTACTATAAT GACTGATGGC CCGAGTGATG

721 GGCTGGCCTC GTACAAAATT TTCAAGATCG AAAAGGGGAA GGTTACTAAA TCAATAGAGT

781 TGAATGCACC TAATTCTCAC TATGAGGAAT GTTCCTGTTA CCCTGATACC GGCAAAGTGA

841 TGTGTGTGTG CAGAGACAAT TGGCATGGTT CGAACCGGCC ATGGGTGTCT TTCGATCAAA

901 ACCTGGATTA TCAAATAGGA TACATCTGCA GTGGGGTTTT CGGTGACAAC CCGCGTCCCA

961 AAGATGGAAC AGGCAGCTGT GGTCCAGTGT ATGTTGATGG AGCAAACGGA GTAAAGGGAT

1021 TTTCATATAG GTATGGTAAT GGTGTTTGGA TAGGAAGGAC CAAAAGTCAC AGTTCCAGAC

1081 ATGGGTTTGA GATGATTTGG GATCCTAATG GATGGACAGA GACTGATAGT AAGTTCTCTG

1141 TGAGGCAAGA TGTTGTGGCA ATGACTGATT GGTCAGGGTA TAGCGGGAGT TTCGTTCAAC

1201 ATCCTGAGCT AACAGGGCTA GACTGTATAA GGCCGTGCTT CTGGGTTGAA TTAATCAGGG

1261 GACGACCTAA AGAAAAAACA ATCTGGACTA GTGCGAGCAG CATTTCTTTT TGTGGCGTGA

1321 ATAGTGATAC TGTAGATTGG TCTTGGCCAG ACGGTGCTGA GTTGCCATTC ACCATTGACA

1381 AGTAGTCTGT TCAAAAAACT CCTTGTTTCT ACT

Segment 7: NC_002016.1 from the NCBI website (provided as SEQ ID NO:57 herein).

1 AGCGAAAGCA GGTAGATATT GAAAGATGAG TCTTCTAACC GAGGTCGAAA CGTACGTTCT

61 CTCTATCATC CCGTCAGGCC CCCTCAAAGC CGAGATCGCA CAGAGACTTG AAGATGTCTT

121 TGCAGGGAAG AACACCGATC TTGAGGTTCT CATGGAATGG CTAAAGACAA GACCAATCCT

181 GTCACCTCTG ACTAAGGGGA TTTTAGGATT TGTGTTCACG CTCACCGTGC CCAGTGAGCG

241 AGGACTGCAG CGTAGACGCT TTGTCCAAAA TGCCCTTAAT GGGAACGGGG ATCCAAATAA

301 CATGGACAAA GCAGTTAAAC TGTATAGGAA GCTCAAGAGG GAGATAACAT TCCATGGGGC

361 CAAAGAAATC TCACTCAGTT ATTCTGCTGG TGCACTTGCC AGTTGTATGG GCCTCATATA

421 CAACAGGATG GGGGCTGTGA CCACTGAAGT GGCATTTGGC CTGGTATGTG CAACCTGTGA

481 ACAGATTGCT GACTCCCAGC ATCGGTCTCA TAGGCAAATG GTGACAACAA CCAACCCACT 541 AATCAGACAT GAGAACAGAA TGGTTTTAGC CAGCACTACA GCTAAGGCTA TGGAGCAAAT

601 GGCTGGATCG AGTGAGCAAG CAGCAGAGGC CATGGAGGTT GCTAGTCAGG CTAGGCAAAT

661 GGTGCAAGCG ATGAGAACCA TTGGGACTCA TCCTAGCTCC AGTGCTGGTC TGAAAAATGA

721 TCTTCTTGAA AATTTGCAGG CCTATCAGAA ACGAATGGGG GTGCAGATGC AACGGTTCAA

781 GTGATCCTCT CGCTATTGCC GCAAATATCA TTGGGATCTT GCACTTGATA TTGTGGATTC

841 TTGATCGTCT TTTTTTCAAA TGCATTTACC GTCGCTTTAA ATACGGACTG AAAGGAGGGC

901 CTTCTACGGA AGGAGTGCCA AAGTCTATGA GGGAAGAATA TCGAAAGGAA CAGCAGAGTG

961 CTGTGGATGC TGACGATGGT CATTTTGTCA GCATAGAGCT GGAGTAAAAA ACTACCTTGT

1021 TTCTACT

Segment 8: NC_002020.1 from the NCBI website (provided as SEQ ID NO:58 herein).

1 AGCAAAAGCA GGGTGACAAA GACATAATGG ATCCAAACAC TGTGTCAAGC TTTCAGGTAG

61 ATTGCTTTCT TTGGCATGTC CGCAAACGAG TTGCAGACCA AGAACTAGGT GATGCCCCAT

121 TCCTTGATCG GCTTCGCCGA GATCAGAAAT CCCTAAGAGG AAGGGGCAGC ACTCTTGGTC

181 TGGACATCGA GACAGCCACA CGTGCTGGAA AGCAGATAGT GGAGCGGATT CTGAAAGAAG

241 AATCCGATGA GGCACTTAAA ATGACCATGG CCTCTGTACC TGCGTCGCGT TACCTAACCG

301 ACATGACTCT TGAGGAAATG TCAAGGGAAT GGTCCATGCT CATACCCAAG CAGAAAGTGG

361 CAGGCCCTCT TTGTATCAGA ATGGACCAGG CGATCATGGA TAAAAACATC ATACTGAAAG

421 CGAACTTCAG TGTGATTTTT GACCGGCTGG AGACTCTAAT ATTGCTAAGG GCTTTCACCG

481 AAGAGGGAGC AATTGTTGGC GAAATTTCAC CATTGCCTTC TCTTCCAGGA CATACTGCTG

541 AGGATGTCAA AAATGCAGTT GGAGTCCTCA TCGGAGGACT TGAATGGAAT GATAACACAG

601 TTCGAGTCTC TGAAACTCTA CAGAGATTCG CTTGGAGAAG CAGTAATGAG AATGGGAGAC

661 CTCCACTCAC TCCAAAACAG AAACGAGAAA TGGCGGGAAC AATTAGGTCA GAAGTTTGAA

721 GAAATAAGAT GGTTGATTGA AGAAGTGAGA CACAAACTGA AGGTAACAGA GAATAGTTTT

781 GAGCAAATAA CATTTATGCA AGCCTTACAT CTATTGCTTG AAGTGGAGCA AGAGATAAGA

841 ACTTTCTCAT TTCAGCTTAT TTAATAATAA AAAACACCCT TGTTTCTACT

A DNA sequence for the Influenza B genome, strain Bisbane, with coding regions for each of the eight single stranded RNA segments, is available under the following accession numbers: Segment 1: CY018707.1 from the NCBI website (provided as SEQ ID NO: 59 herein).

1 GATGAATATA AATCCTTATT TTCTCTTCAT AGATGTGCCC ATACAGGCAG CAATTTCAAC

61 AACATTCCCA TACACTGGTG TTCCCCCTTA TTCCCATGGA ACGGGAACAG GCTACACAAT

121 AGACACAGTG ATCAGAACAC ATGAGTACTC AAACAAGGGG AAACAGTACA TTTCTGATGT 181 TACAGGATGC ACAATGGTAG ATCCAACAAA TGGACCATTA CCCGAAGATA ATGAGCCGAG

241 TGCCTATGCG CAATTAGATT GCGTTTTGGA GGCTTTGGAT AGAATGGATG AAGAACACCC

301 AGGTCTGTTT CAAGCAGCCT CACAGAATGC TATGGAGGCC CTAATGGTCA CAACTGTAGA

361 CAAATTAACC CAGGGGAGAC AGACTTTTGA TTGGACAGTA TGCAGAAACC AACCTGCTGC

421 AACGGCACTG AACACAACAA TAACCTCTTT TAGGTTGAAT GATTTAAATG GAGCCGACAA

481 AGGTGGATTA GTACCTTTTT GCCAGGATAT CATTGATTCA TTAGACAGAC CTGAAATGAC

541 TTTCTTCTCA GTAAAGAATA TAAAGAAAAA ATTGCCTGCC AAAAACAGAA AGGGTTTCCT

601 CATAAAGAGG ATACCAATGA AGGTAAAAGA CAAAATAACC AAAGTGGAAT ACATCAAAAG

661 AGCATTATCA TTAAACACAA TGACAAAAGA CGCTGAAAGA GGCAAACTAA AAAGAAGAGC

721 GATTGCCACC GCTGGAATAC AAATCAGAGG ATTTGTATTA GTAGTTGAAA ACTTGGCTAA

781 AAATATATGT GAAAATCTAG AACAAAGTGG TTTACCAGTA GGTGGAAACG AGAAGAAAGC

841 CAAACTGTCA AACGCAGTGG CCAAAATGCT CAGTAACTGC CCACCAGGAG GGATCAGCAT

901 GACAGTAACA GGAGACAATA CTAAATGGAA TGAATGTTTA AACCCAAGAA TCTTTTTGGC

961 TATGACTGAA AGAATAACCA GAGACAGCCC AATTTGGTTC AGGGATTTTT GTAGTATAGC

1021 ACCGGTCCTG TTCTCCAATA AGATAGCCAG ATTGGGGAAA GGGTTTATGA TAACAAGCAA

1081 AACAAAAAGA CTGAAGGCTC AAATACCTTG TCCTGATCTG TTTAGTATAC CATTAGAAAG

1141 ATATAATGAA GAAACAAGGG CAAAATTGAA AAAGCTAAAA CCATTCTTCA ATGAAGAAGG

1201 AACTGCATCT TTGTCGCCTG GGATGATGAT GGGAATGTTT AACATGCTAT CTACCGTGTT

1261 GGGAGTAGCC GCACTAGGTA TCAAGAACAT TGGAAACAAA GAATACTTAT GGGATGGACT

1321 GCAATCTTCT GATGATTTTG CTCTATTTGT TAATGCAAAG GATGAAGAAA CATGTATGGA

1381 AGGAATAAAC GACTTTTACC GAACATGTAA ATTATTGGGA ATAAACATGA GCAAAAAGAA

1441 AAGTTACTGT AATGAGACTG GAATGTTTGA ATTTACAAGC ATGTTCTACA GAGATGGATT

1501 TGTATCTAAT TTTGCAATGG AACTCCCTTC ATTTGGGGTT GCTGGAGTAA ATGAATCAGC

1561 AGATATGGCA ATAGGGATGA CAATAATAAA GAACAACATG ATCAACAATG GAATGGGTCC

1621 GGCAACAGCA CAAACAGCCA TACAGTTATT CATAGCTGAT TATAGATACA CCTACAAATG

1681 CCACAGGGGA GATTCCAAAG TAGAAGGAAA GAGAATGAAA ATCATAAAGG AGTTATGGGA

1741 AAACACTAAA GGAAGAGATG GTCTATTAGT AGCAGATGGT GGGCCCAACA TTTACAATTT

1801 GAGAAACTTG CATATTCCAG AAATAGTATT AAAGTATAAC CTAATGGACC CTGAATACAA

1861 AGGGCGATTA CTTCATCCTC AAAATCCCTT TGTGGGACAT TTGTCTATTG AGGGCATCAA

1921 AGAGGCAGAT ATAACCCCAG CACATGGTCC AGTAAAGAAA ATGGACTACG ATGCGGTGTC

1981 TGGAACTCAT AGTTGGAGAA CCAAAAGAAA CAGATCTATA CTAAACACTG ATCAGAGGAA

2041 CATGATTCTT GAGGAACAAT GCTACGCTAA GTGTTGCAAC CTATTTGAGG CCTGTTTTAA 2101 CAGTGCATCA TACAGGAAGC CAGTGGGTCA ACATAGCATG CTTGAGGCTA TGGCCCACAG

2161 ATTAAGAATG GATGCACGAT TAGATTATGA ATCAGGAAGA ATGTCAAAGG ATGATTTTGA

2221 GAAAGCAATG GCTCACCTTG GTGAGATTGG GTACATATAA GCTTCGAAGA TGTCTATGGG

2281 GTTATTGGTC ATCATTGAAT ACATGCGATA CACAAATGAT TAAAATGA

Segment 2: CY018708.1 from the NCBI website (provided as SEQ ID NO:60 herein).

1 GATGACATTG GCCAAAATTG AATTGTTAAA ACAACTGCTA AGGGACAATG AAGCCAAAAC

61 AGTTTTGAAG CAAACAACGG TAGACCAATA TAACATAATA AGAAAATTCA ATACATCAAG

121 GATTGAAAAG AATCCTTCAC TAAGGATGAA GTGGGCCATG TGTTCTAATT TTCCCTTGGC

181 TCTAACCAAG GGCGATATGG CAAATAGAAT CCCCTTGGAA TACAAAGGAA TACAACTTAA

241 AACAAATGCT GAAGACATAG GAACCAAAGG CCAAATGTGC TCAATAGCAG CAGTTACTTG

301 GTGGAATACA TATGGACCAA TAGGAGATAC TGAAGGTTTC GAAAGGGTCT ACGAAAGCTT

361 TTTTCTCAGA AAAATGAGAC TTGACAACGC CACTTGGGGC CGAATAACTT TTGGCCCAGT

421 TGAAAGAGTG AGAAAAAGGG TACTGCTAAA CCCTCTCACC AAGGAAATGC CTCCAGATGA

481 GGCGAGCAAT GTGATAATGG AAATATTGTT CCCTAAAGAA GCAGGAATAC CAAGAGAATC

541 CACTTGGATA CATAGGGAAC TGATAAAAGA AAAAAGAGAA AAATTGAAAG GAACAATGAT

601 AACTCCAATC GTACTGGCAT ACATGCTTGA AAGAGAACTG GTTGCTCGAA GAAGATTCTT

661 GCCAGTGGCA GGAGCAACAT CAGCTGAGTT CATAGAAATG CTACACTGCT TACAAGGTGA

721 AAATTGGAGA CAAATATATC ACCCAGGAGG GAATAAATTA ACTGAGTCTA GGTCTCAATC

781 AATGATAGTA GCTTGTAGAA AAATAATCAG AAGATCAATA GTCGCTTCAA ACCCACTGGA

841 GCTAGCTGTA GAAATTGCAA ACAAGACTGT GATAGATACT GAACCTTTAA AGTCATGTCT

901 GGCAGCCATA GACGGAGGTG ATGTAGCTTG TGACATAATA AGAGCTGCAT TAGGACTAAA

961 GATCAGACAA AGACAAAGAT TTGGACGGCT TGAGCTAAAA AGAATATCAG GAAGAGGATT

1021 CAAAAATGAT GAAGAAATAT TAATAGGGAA CGGAACAATA CAGAAGATTG GAATATGGGA

1081 CGGGGAAGAG GAGTTCCATG TAAGATGTGG TGAATGCAGG GGAATATTAA AAAAGAGTAA

1141 AATGAAACTG GAAAAACTAC TGATAAATTC AGCCAAAAAG GAGGATATGA GAGATTTAAT

1201 AATCTTATGC ATGGTATTTT CTCAAGACAC TAGGATGTTC CAAGGGGTGA GAGGAGAAAT

1261 AAATTTTCTT AATCGAGCAG GCCAACTTTT ATCTCCAATG TACCAACTCC AACGATATTT

1321 TTTGAATAGG AGCAACGACC TTTTTGATCA ATGGGGGTAT GAGGAATCAC CCAAAGCAAG

1381 TGAACTACAT GGGATAAATG AATCAATGAA TGCATCTGAC TATACATTGA AAGGGGTTGT

1441 AGTGACAAGA AATGTAATTG ACGACTTTAG CTCTACTGAA ACAGAAAAAG TATCCATAAC

1501 AAAAAATCTT AGTTTAATAA AAAGGACTGG GGAAGTCATA ATGGGAGCTA ATGACGTGAG 1561 TGAATTAGAA TCACAAGCAC AGCTGATGAT AACATATGAT ACACCTAAGA TGTGGGAAAT

1621 GGGAACAACC AAAGAACTGG TGCAAAACAC TTATCAATGG GTGCTAAAAA ACTTGGTAAC

1681 ACTGAAGGCT CAGTTTCTTC TAGGAAAAGA GGACATGTTC CAATGGGATG CATTTGAAGC

1741 ATTTGAGAGC ATAATTCCTC AGAAAATGGC TGGTCAGTAC AGTGGATTTG CAAGAGCAGT

1801 GCTCAAACAA ATGAGAGACC AGGAGGTTAT GAAAACTGAC CAGTTCATAA AGTTGTTGCC

1861 TTTTTGTTTC TCACCACCAA AATTAAGGAG CAATGGGGAG CCTTATCAAT TCTTAAAACT

1921 TGTATTGAAA GGAGGAGGGG AAAATTTCAT CGAAGTAAGG AAAGGGTCCC CTCTATTTTC

1981 CTATAATCCA CAAACAGAGG TCCTAACTAT ATGCGGCAGA ATGATGTCAT TAAAAGGGAA

2041 AATTGAAGAT GAAGAAAGGA ATAGATCAAT GGGGAATGCA GTATTAGCAG GCTTTCTCGT

2101 TAGTGGCAAG TATGACCCAG ATCTTGGAGA TTTCAAAACT ATTGAAGAAC TTGAAAAGCT

2161 GAAACCGGGG GAAAAGGCAA ACATCTTACT TTATCAAGGA AAGCCAGTTA AAGTAGTTAA

2221 AAGGAAAAGG TATAGTGCTT TGTCCAATGA CATTTCACAA GGAATTAAGA GACAAAGAAT

2281 GACAGTTGAG TCCATGGGGT GGGCCTTGAG CTAATATAAA TTTATCCATT AATTCAATGA

2341 ACGCAATTGA GT

Segment 3: CY018706.1 from the NCBI website (provided as SEQ ID NO:61 herein).

1 TTTGATTTGT CATAATGGAT ACTTTTATTA CAAGAAACTT CCAGACTACA ATAATACAAA

61 AGGCCAAAAA CACAATGGCA GAATTTAGTG AAGATCCTGA ATTACAACCA GCAATGCTAT

121 TCAATATCTG CGTCCATCTA GAGGTTTGCT ATGTAATAAG TGACATGAAT TTTCTTGACG

181 AAGAAGGAAA AGCATATACA GCATTAGAAG GACAAGGGAA AGAACAAAAT TTGAGACCAC

241 AATATGAAGT AATTGAGGGA ATGCCAAGAA CCATAGCATG GATGGTCCAA AGATCCTTAG

301 CTCAAGAGCA TGGAATAGAG ACTCCCAAGT ATCTGGCTGA TTTGTTTGAT TATAAAACCA

361 AGAGATTTAT AGAAGTTGGA ATAACAAAAG GATTGGCTGA TGATTACTTT TGGAAAAAGA

421 AAGAAAAGTT GGGAAATAGC ATGGAACTGA TGATATTCAG CTACAATCAA GACTACTCGT

481 TAAGTAATGA ATCCTCATTG GATGAGGAAG GGAAAGGGAG AGTGCTAAGC AGACTCACAG

541 AACTTCAGGC TGAATTAAGT CTGAAAAACC TATGGCAAGT TCTCATAGGA GAAGAAGATG

601 TTGAAAAGGG AATTGACTTT AAACTTGGAC AAACAATATC TAGACTAAGG GATATATCTG

661 TTCCAGCTGG TTTCTCCAAT TTTGAAGGAA TGAGGAGCTA CATAGACAAT ATAGACCCAA

721 AAGGAGCAAT AGAGAGAAAT CTAGCAAGGA TGTCTCCCTT AGTATCAGTC ACACCTAAAA

781 AGTTAACATG GGAGGACCTA AGACCAATAG GGCCTCACAT TTACAACCAT GAGCTACCAG

841 AAGTTCCATA TAATGCCTTT CTTCTAATGT CTGATGAACT GGGGCTGGCC AATATGACTG

901 AGGGAAAGTC CAAAAAACCG AAGACATTAG CCAAAGAATG TCTAGAAAAG TACTCAACAC 961 TACGGGATCA AACTGACCCA ATATTAATAA TGAAAAGCGA AAAAGCTAAC GAAAATTTCC

1021 TATGGAAGCT TTGGAGAGAC TGTGTAAATA CAATAAGTAA TGAGGAAATG AATAACGAGT

1081 TACAGAAAAC CAATTATGCC AAGTGGGCCA CAGGGGATGG ATTAACATAC CAGAAAATAA

1141 TGAAAGAAGT AGCAATAGAT GACGAAACAA TGTGCCAAGA AGAGCCTAAA ATCCCTAACA

1201 AATGTAGAGT GGCTGCTTGG GTTCAAACAG AGATGAATCT ATTGAGCACT CTGACAAGTA

1261 AAAGAGCTCT GGACCTACCA GAAATAGGGC CAGACGTAGC ACCCGTGGAG CATGTAGGGA

1321 GTGAAAGAAG GAAATACTTT GTTAATGAAA TCAACTACTG TAAGGCCTCT ACAGTTATGA

1381 TGAAGTATGT GCTTTTTCAC ACTTCATTGT TGAATGAAAG CAATGCCAGC ATGGGAAAAT

1441 ACAAAGTAAT ACCAATAACC AATAGAGTAG TAAATGAAAA AGGAGAAAGT TTCGACATGC

1501 TTTATGGTCT GGCGGTTAAA GGACAATCTC ATCTGAGGGG AGATACTGAT GTTGTAACAG

1561 TTGTAACTTT CGAATTTAGT AGTACAGACC CAAGAGTGGA CTCAGGAAAG TGGCCAAAAT

1621 ATACTGTGTT TAGGATTGGC TCCCTATTTG TGAGTGGGAG GGAAAAATCT GTGTACCTGT

1681 ATTGCCGAGT GAATGGCACA AATAAGATCC AAATGAAATG GGGAATGGAA GCTAGAAGAT

1741 GTCTGCTTCA ATCAATGCAA CAAATGGAAG CAATTGTTGA ACAGGAATCA TCGATACAAG

1801 GATATGACAT GACCAAAGCT TGTTTCAAGG GAGACAGAGT AAATAGCCCC AAAACTTTCA

1861 GTATTGGAAC TCAAGAAGGA AAACTAGTAA AAGGATCCTT TGGAAAAGCA CTAAGAGTAA

1921 TATTTACTAA ATGTTTGATG CACTATGTAT TTGGAAATGC CCAATTGGAG GGGTTTAGTG

1981 CCGAGTCTAG GAGACTTCTA CTGTTGATTC AAGCATTAAA GGACAGAAAG GGCCCTTGGG

2041 TGTTCGACTT AGAGGGAATG TATTCTGGAA TAGAAGAATG TATTAGTAAC AACCCTTGGG

2101 TAATACAGAG TGCATACTGG TTCAATGAAT GGTTGGGCTT TGAAAAGGAG GGGAGTAAAG

2161 TGTTAGAATC AGTGGATGAA ATAATGGATG AATAAAAGGA CATGGTACTC AAT

Segment 4: CY018701.1 from the NCBI website (provided as SEQ ID NO:62 herein).

1 ATATCCACAA AATGAAGGCA ATAATTGTAC TACTCATGGT AGTAACATCC AATGCAGATC

61 GAATCTGCAC TGGGATAACA TCGTCAAACT CACCCCATGT GGTCAAAACT GCTACTCAAG

121 GGGAGGTCAA TGTGACTGGT GTGATACCAC TGACAACAAC ACCCACCAAA TCTCATTTTG

181 CAAATCTCAA AGGAACAAAA ACCAGAGGGA AACTATGCCC AAAATGCCTC AACTGCACAG

241 ATCTGGACGT GGCCTTGGGC AGACCAAAAT GCACGGGGAA CATACCCTCG GCAAAAGTTT

301 CAATACTCCA TGAAGTCAGA CCTGTTACAT CTGGGTGCTT TCCTATAATG CACGACAGAA

361 CAAAAATTAG ACAGCTGCCC AATCTTCTCA GAGGATACGA ACATATCAGG TTATCAACTC

421 ATAACGTTAT CAATGCAGAA AAGGCACCAG GAGGACCCTA CAAAATTGGA ACCTCAGGGT

481 CTTGCCCTAA CGTTACCAAT GGAAACGGAT TTTTCGCAAC AATGGCTTGG GCCGTCCCAA 541 AAAACGACAA CAACAAAACA GCAACAAATT CATTAACAAT AGAAGTACCA TACATTTGTA

601 CAGAAGGAGA AGACCAAATT ACCGTTTGGG GGTTCCACTC TGATAACGAA GCCCAAATGG

661 CAAAACTCTA TGGGGACTCA AAGCCCCAGA AGTTCACCTC ATCTGCCAAC GGAGTGACCA

721 CACATTACGT TTCACAGATT GGTGGCTTCC CAAATCAAAC AGAAGACGGA GGACTACCAC

781 AAAGTGGTAG AATTGTTGTT GATTACATGG TGCAAAAATC TGGGAAAACA GGAACAATTA

841 CCTATCAAAG AGGTATTTTA TTGCCTCAAA AAGTGTGGTG CGCAAGTGGC AGGAGCAAGG

901 TAATAAAAGG ATCCTTGCCT TTAATTGGAG AAGCAGATTG CCTCCACGAA AAATACGGTG

961 GATTAAACAA AAGCAAGCCT TACTACACAG GGGAACATGC AAAGGCCATA GGAAATTGCC

1021 CAATATGGGT GAAAACACCC TTGAAGCTGG CCAATGGAAC CAAATATAGA CCTCCTGCAA

1081 AACTATTAAA GGAAAGAGGT TTCTTCGGAG CTATTGCTGG TTTCTTAGAA GGAGGATGGG

1141 AAGGAATGAT TGCAGGTTGG CACGGATACA CATCCCATGG GGCACATGGA GTAGCAGTGG

1201 CAGCAGACCT TAAGAGTACT CAAGAAGCCA TAAACAAGAT AACAAAAAAT CTCAACTCTT

1261 TGAGTGAGCT GGAAGTAAAG AATCTTCAAA GACTAAGCGG TGCCATGGAT GAACTCCACA

1321 ACGAAATACT AGAACTAGAC GAGAAAGTGG ATGATCTCAG AGCTGATACA ATAAGCTCAC

1381 AAATAGAACT CGCAGTCTTG CTTTCCAATG AAGGAATAAT AAACAGTGAA GATGAGCATC

1441 TCTTGGCGCT TGAAAGAAAG CTGAAGAAAA TGCTGGGCCC CTCTGCTGTA GAGATAGGGA

1501 ATGGATGCTT CGAAACCAAA CACAAGTGCA ACCAGACCTG TCTCGACAGA ATAGCTGCTG

1561 GTACCTTTGA TGCAGGAGAA TTTTCTCTCC CCACTTTTGA TTCACTGAAT ATTACTGCTG

1621 CATCTTTAAA TGACGATGGA TTGGATAATC ATACTATACT GCTTTACTAC TCAACTGCTG

1681 CCTCCAGTTT GGCTGTAACA TTGATGATAG CTATCTTTGT TGTTTATATG GTCTCCAGAG

1741 ACAATGTTTC TTGCTCCATC TGTCTATAAG GAAAGTTAAG CCCTGTATTT TCCTTTATTG

1801 TAGTGCTTGT TTGCTTGTTA CCATTACAAA AAAACGTTAT TGA

Segment 5: CY018704.1 from the NCBI website (provided as SEQ ID NO:63 herein).

1 TTTCTTGTGA ACTTCAAGTG CTAACAAAAG AACTGAAAAT CAAAATGTCC AACATGGATA

61 TTGACGGTAT CAACACTGGG ACAATTGACA AAGCACCGGA AGAAATAACT TCTGGAACCA

121 GTGGGACAAC CAGACCAATC ATCAGACCAG CAACCCTTGC CCCACCAAGC AACAAACGAA

181 CCCGGAACCC ATCCCCGGAA AGAGCAACCA CAATCAGTGA AGCTGATGTC GGAAGGAAAA

241 ACCAAAAGAA ACAGACCCCG ACAGAGATAA AGAAGAGCGT CTACAATATG GTAGTGAAAC

301 TGGGTGAATT CTATAACCAG ATGATGGTCA AAGCTGGACT TAACGATGAC ATGGAGAGAA

361 ACCTAATTCA AAATGCGCAT GCTGTGGAAA GAATTCTATT GGCTGCCACT GATGACAAGA

421 AAACTGAATT CCAGAAGAAA AAGAATGCCA GAGATGTCAA AGAAGGGAAA GAAGAAATAG 481 ATCACAACAA AACAGGGGGC ACCTTTTACA AGATGGTAAG AGATGATAAA ACCATCTACT

541 TCAGCCCTAT AAGAGTCACC TTTTTAAAAG AAGAGGTAAA AACAATGTAC AAAACCACCA

601 TGGGGAGTGA TGGCTTCAGC GGACTAAATC ACATAATGAT TGGGCATTCA CAGATGAATG

661 ATGTCTGTTT CCAAAGATCA AAGGCACTAA AAAGAGTTGG ACTTGACCCT TCATTAATCA

721 GTACCTTTGC AGGAAGCACA CTCCCCAGAA GATCAGGTGC AACTGGTGTT GCGATCAAAG

781 GAGGTGGAAC TCTAGTGGCT GAAGCCATTC GATTTATAGG AAGAGCAATG GCAGACAGAG

841 GGCTATTGAG AGACATCAAA GCTAAGACTG CTTATGAAAA GATTCTTCTG AATCTAAAAA

901 ACAAATGCTC TGCGCCCCAA CAAAAGGCTC TAGTTGATCA AGTGATCGGA AGTAGAAATC

961 CAGGGATCGC AGACATTGAA GACCTAACCC TGCTTGCTCG TAGTATGGTC GTTGTTAGGC

1021 CCTCTGTGGC GAGCAAAGTA GTGCTTCCCA TAAGCATTTA CGCCAAAATA CCTCAACTAG

1081 GGTTCAACGT TGAAGAGTAC TCTATGGTTG GGTATGAAGC CATGGCTCTT TACAATATGG

1141 CAACACCTGT TTCCATATTA AGAGTGGGAG ATGATGCAAA AGACAAATCA CAATTATTCT

1201 TCATGTCTTG CTTCGGAGCT GCCTATGAAG ACCTGAGAGT TTTGTCTGCA TTAACAGGCA

1261 CAGAGTTCAA GCCTAGATCA GCATTAAAAT GCAAGGGTTT CCATGTTCCA GCAAAGGAAC

1321 AGGTGGAAGG AATGGGGGCA GCTCTGATGT CCATCAAGCT CCAGTTTTGG GCTCCAATGA

1381 CCAGATCTGG GGGGAACGAA GTAGGTGGAG ACGGGGGGTC TGGCCAAATA AGTTGCAGCC

1441 CAGTGTTTGC AGTAGAAAGA CCTATTGCTC TAAGCAAGCA AGCTGTAAGA AGAATGCTGT

1501 CAATGAATAT TGAGGGACGT GATGCAGATG TCAAAGGAAA TCTACTCAAG ATGATGAATG

1561 ACTCAATGGC TAAGAAAGCC AATGGAAATG CTTTCATTGG GAAGAAAATG TTTCAAATAT

1621 CAGACAAAAA CAAAACCAAT CCCGTTGAAA TTCCAATTAA GCAAACCATC CCCAATTTCT

1681 TCTTTGGGAG GGACACAGCA GAGGATTATG ATGACCTCGA TTATTAAAGC AACAAAATAG

1741 ACACTATGAC TGTGATTGTT TCAATACGTT TGGAATGTGG GTGTTTACTC TTATTAAAAT

1801 AAATATAAA

Segment 6: CY018703.1 from the NCBI website (provided as SEQ ID NO:64 herein).

1 AAACTGAGGC AAATAGGCCA AAAATGAACA ATGCTACCTT CAACTATACA AACGTTAACC

61 CTATTTCTCA CATCAGGGGG AGTATTATTA TCACTATATG TGTCAGCTTC ATTGTCATAC

121 TTACTATATT CGGATATATT GCTAAAATTC TCACCAACAG AAATAACTGC ACCAACAATG

181 CCATTGGATT GTGCAAACGC ATCAAATGTT CAGGCTGTGA ACCGTTCTGC AACAAAAGGG

241 GTGACACTTC TTCTCCCAGA ACCAGAGTGG ACATACCCGC GTTTATCTTG CCCGGGCTCA

301 ACCTTTCAGA AAGCACTCCT AATTAGCCCT CATAGATTCG GAGAAACCAA AGGAAACTCA

361 GCTCCCTTGA TAATAAGGGA ACCTTTTATT GCTTGTGGAC CAAAGGAATG CAAACACTTT 421 GCTCTAACCC ATTATGCAGC CCAACCAGGG GGATACTACA ATGGAACAAG AGGAGACAGA

481 AACAAGCTGA GGCATCTAAT TTCAGTCAAA TTGGGCAAAA TCCCAACAGT AGAAAACTCC

541 ATTTTCCACA TGGCAGCATG GAGCGGGTCC GCATGCCATG ATGGTAAAGA ATGGACATAT

601 ATCGGAGTTG ATGGCCCTGA CAATAATGCA TTGCTCAAAA TAAAATATGG AGAAGCATAT

661 ACTGACACAT ACCATTCCTA TGCAAACAAC ATCCTAAGAA CACAAGAAAG TGCCTGCAAT

721 TGCATCGGGG GAAATTGTTA TCTTATGATA ACTGATGGCT CAGCTTCAGG TATTAGTGAA

781 TGCAGATTTC TTAAAATTCG AGAGGGCCGA ATAATAAAAG AAATATTTCC AACAGGAAGA

841 GTAAAACATA CTGAAGAATG CACATGCGGA TTTGCCAGCA ATAAGACCAT AGAATGTGCC

901 TGTAGAGATA ACAGTTACAC AGCAAAAAGA CCCTTTGTCA AATTAAACGT GGAGACTGAT

961 ACAGCAGAAA TAAGATTGAT GTGCACAGAG ACTTATTTGG ACACCCCCAG ACCAGATGAT

1021 GGAAGCATAA CAGGGCCTTG TGAATCTAAT GGGGACAAAG GGAGTGGAGG CATCAAGGGA

1081 GGATTTGTTC ATCAAAGAAT GGCATCCAAG ATTGGAAGGT GGTACTCTCG AACGATGTCT

1141 AAAACTAAAA GGATGGGGAT GGGACTGTAT GTCAAGTATG ATGGAGACCC ATGGGCTGAC

1201 AGTGATGCCC TTGCTCTTAG TGGAGTAATG GTTTCAATGG AAGAACCTGG TTGGTACTCC

1261 TTTGGCTTCG AAATAAAAGA TAAGAAATGT GATGTCCCCT GTATTGGAAT AGAGATGGTA

1321 CATGATGGTG GAAAAGAGAC TTGGCACTCA GCAGCAACAG CCATTTACTG TTTAATGGGC

1381 TCAGGACAGC TGCTGTGGGA CACTGTCACA GGTGTTGATA TGGCTCTGTA ATGGAGGAAT

1441 GGTTGAGTCT GTTCTAAACC CTTTGTTCCT ATTTTGTTTG AACAATTGTC CTTACTGAAC

1501 TTAATTGTTT CTGAAA

Segment 7: CY018702.1 from the NCBI website (provided as SEQ ID NO:65 herein).

1 AAAATGTCGC TGTTTGGAGA CACAATTGCC TACCTGCTTT CATTGACAGA AGATGGAGAA

61 GGCAAAGCAG AACTAGCAGA AAAATTACAC TGTTGGTTTG GTGGGAAAGA ATTTGACCTA

121 GACTCTGCCT TGGAATGGAT AAAAAACAAA AGATGCTTAA CTGATATACA AAAAGCACTA

181 ATTGGTGCCT CTATCTGCTT TTTAAAACCC AAAGACCAGG AAAGAAAAAG AAGATTCATC

241 ACAGAGCCCT TATCAGGAAT GGGAACAACA GCAACAAAAA AGAAAGGCCT GATTCTGGCT

301 GAGAGAAAAA TGAGAAGATG TGTGAGCTTT CATGAAGCAT TTGAAATAGC AGAAGGCCAT

361 GAAAGCTCAG CGCTACTATA CTGTCTCATG GTCATGTACC TGAATCCTGG AAATTATTCA

421 ATGCAAGTAA AACTAGGAAC GCTCTGTGCT TTGTGCGAGA AACAAGCATC ACATTCACAC

481 AGGGCTCATA GCAGAGCAGC GAGATCTTCA GTGCCTGGAG TGAGACGAGA AATGCAGATG

541 GTCTCAGCTA TGAACACAGC AAAAACAATG AATGGAATGG GAAAAGGAGA AGACGTCCAA

601 AAGCTGGCAG AAGAGCTGCA AAGCAACATT GGAGTGCTGA GATCTCTTGG GGCAAGTCAA 661 AAGAATGGGG AAGGAATTGC AAAGGATGTA ATGGAGGTGC TAAAGCAGAG CTCTATGGGA

721 AATTCAGCTC TTGTGAAGAA ATATCTATAA TGCTCGAACC ATTTCAGATT CTTTCAATTT

781 GTTCTTTTAT CTTATCAGCT CTCCATTTCA TGGCTTGGAC AATAGGGCAT TTGAATCAAA

841 TAAAAAGAGG AGTAAACATG AAGATACGAA TAAAAAGTCC AAACAAAGAG ACAATAAACA

901 GAGAGGTATC AATTTTGAGA CACAGTTACC AAAAAGAAAT CCAGGCCAAA GAAACAATGA

961 AGGAAGTACT CTCTGACAAC ATGGAGGTAT TGAGTGACCA CATGGTGATT GAGGGGCTTT

1021 CTGCCGAAGA GATAATAAAA ATGGGTGAAA CAGTTTTGGA GATAGAAGAA TTGCATTAAA

1081 TTCAATTTTT ACTGTATTTC TTACCATGCA TTTAAGCAAA TTGTAATCAA TGTCAGCAAA 1141 TAAACT

Segment 8: CY018705.1 from the NCBI website (provided as SEQ ID NO:66 herein).

1 TCACTGGCAA ACAGGAAAAA TGGCGAACAA CATGACCACA ACACAAATTG AGGTGGGTCC

61 GGGAGCAACC AATGCCACCA TAAACTTTGA AGCAGGAATT CTGGAGTGCT ATGAAAGGCT

121 TTCATGGCAA AGAGCCCTTG ACTACCCTGG ACAAGACCGC CTAAACAGAC TAAAGAGAAA

181 ATTAGAGTCA AGAATAAAGA CTCACAACAA AAGTGAGCCT GAAAGTAAAA GGATGTCCCT

241 TGAAGAGAGA AAAGCAATTG GAGTAAAAAT GATGAAAGTA CTCCTATTTA TGAATCCGTC

301 TGCTGGAATT GAAGGGTTTG AGCCATACTG TATGAAAAGT TCCTCAAATA GCAACTGTAC

361 GAAATACAAT TGGACCGATT ACCCTTCAAC ACCAGGGAGG TGCCTTGATG ACATAGAAGA

421 AGAACCAGAG GATGTTGATG GCCCAACTGA AATAGTATTA AGGGACATGA ACAACAAAGA

481 TGCAAGGCAA AAGATAAAGG AGGAAGTAAA CACTCAGAAA GAAGGGAAGT TCCGTTTGAC

541 AATAAAAAGG GATATGCGTA ATGTATTGTC CTTGAGAGTG TTGGTAAACG GAACATTCCT

601 CAAACACCCC AATGGATACA AGTCCTTATC AACTCTGCAT AGATTGAATG CATATGACCA

661 GAGTGGAAGG CTTGTTGCTA AACTTGTTGC TACTGATGAT CTTACAGTGG AGGATGAAGA

721 AGATGGCCAT CGGATCCTCA ACTCACTCTT CGAGCGTCTT AATGAAGGAC ATTCAAAGCC

781 AATTCGAGCA GCTGAAACTG CGGTGGGAGT CTTATCCCAA TTTGGTCAAG AGCACCGATT

841 ATCACCAGAA GAGGGAGACA ATTAGACTGG TCACGGAAGA ACTTTATCTT TTAAGTAAAA

901 GAATTGATGA TAACATATTG TTCCACAAAA CAGTAATAGC TAACAGCTCC ATAATAGCTG

961 ACATGGTTGT ATCATTATCA TTATTAGAAA CATTGTATGA AATGAAGGAT GTGGTTGAAG

1021 TGTACAGCAG GCAGTGCTTG TGAATTTAAA ATAAA

The Influenza viral genome is RNA. Hence, in some cases the Influenza viral genome can be a copy of the foregoing DNA sequence, where the thymine (T) residues are uracil (U) residues. In some cases, the Influenza viral genome can be a complement of the foregoing DNA sequence. However, the Influenza viral genome can also have sequence variation. For example, the Influenza viral genome can be for various Influenza strains including the foregoing sequence for strain H1N1, or other strains such as H3N2, or any of the Influenza A 18 distinct subtypes of hemagglutinin (HA) and 11 distinct subtypes of neuraminidase (NA). Variations in the Influenza B virus can be any of strains B/Lee/1940, B/Brisbane/60/2008, B/Victoria/504/2000, or other strains.

Sequencing has confirmed that Influenza viruses share a common genetic ancestry; however, they have genetically diverged, such that reassortment - the exchange of viral RNA segments between viruses - has been reported to occur within each genus, or type, but not across types. This genetic reassortment has led to a standard naming convention for Influenza viruses that includes virus type; species from which it was isolated (if non-human); location at which it was isolated; isolate number; isolate year; and, for influenza A viruses only, HA and NA subtype. In Influenza A and B viruses, genome segments 1, 3, 4, and 5 encode just one protein per segment: the PB2, PA, HA and NP proteins. All Influenza viruses encode the polymerase subunit PB1 on segment 2; in some strains of Influenza A virus, this segment also codes for the accessory protein PB1-F2, a small, 87-amino acid protein with pro-apoptotic activity, in a +1 alternate reading frame. No analogue to PB1-F2 has been identified in influenza B or C viruses. Conversely, segment 6 of the Influenza A virus encodes only the NA protein, while that of Influenza B virus encodes both the NA protein and, in a -1 alternate reading frame, the NB matrix protein, which is an integral membrane protein corresponding to the influenza A virus M2 protein. Segment 7 of both influenza A and B viruses code for the Ml matrix protein. In the influenza A genome, the M2 ion channel is also expressed from segment 7 by RNA splicing, while influenza B virus encodes its BM2 membrane protein in a +2 alternate reading frame. Finally, both influenza A and B viruses possess a single RNA segment, segment 8, from which they express the interferon-antagonist NS1 protein and, by mRNA splicing, the NEP/NS2, which is involved in viral RNP export from the host cell nucleus. The genomic organization of influenza C viruses is generally similar to that of influenza A and B viruses; however, the HEF protein of influenza C replaces the HA and NA proteins, and thus the influenza C virus genome has one fewer segment than that of influenza A or B viruses. Cas 13 protein'.

Any suitable CRISPR-associated RNA-targeting endonuclease, such as a Cas 13 protein variant, can be used in the methods and compositions described herein. The Cas 13 protein can complex with at least one CRISPR guide RNA (crRNA) to at least one reporter RNA for a period of time sufficient to form at least one RNA cleavage product.

The Cast 3 protein can, for example, be a Cast 3a protein, Cast 3b protein, or a combination thereof. Cast 3 contains two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains for RNA cleavage, consistent with known roles for HEPN domains in other proteins. In some embodiments, the Cast 3 proteins can have sequence variation and/or be from other organisms. For example, the Casl3 proteins can have at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any of the foregoing Cas 13 sequences or to a Cas 13 in the following bacteria: Leptotrichia wadei, Leptotrichia buccalis, Rhodobacter capsulatus, Herbinix hemicellulosilytica, Leptotrichia buccalis (Lbu), Listeria seeligeri, Paludibacter propionicigenes, Lachnospiraceae bacterium, [Eubacterium] rectale, Listeria newyorkensis, Clostridium aminophilum, and/or Leptotrichia shahii.

For example, Leptotrichia wadei Cas 13a endonuclease can be used that has the following sequence (SEQ ID NO: 38; NCBI accession no. WP 036059678.1).

1 MKITKIDGVS HYKKQDKGIL KKKWKDLDER KQREKIEARY 41 NKQIESKIYK EFFRLKNKKR IEKEEDQNIK SLYFFIKELY 81 LNEKNEEWEL KNINLEILDD KERVIKGYKF KEDVYFFKEG 121 YKEYYLRILF NNLIEKVQNE NREKVRKNKE FLDLKEIFKK 161 YKNRKIDLLL KSINNNKINL EYKKENVNEE IYGINPTNDR 201 EMTFYELLKE I IEKKDEQKS ILEEKLDNFD ITNFLENIEK 241 IFNEETEINI IKGKVLNELR EYIKEKEENN SDNKLKQIYN 281 LELKKYIENN FSYKKQKSKS KNGKNDYLYL NFLKKIMFIE 321 EVDEKKEINK EKFKNKINSN FKNLFVQHIL DYGKLLYYKE 361 NDEYIKNTGQ LETKDLEYIK TKETLIRKMA VLVSFAANSY 401 YNLFGRVSGD ILGTEWKSS KTNVIKVGSH IFKEKMLNYF 441 FDFEIFDANK IVEILESISY SIYNVRNGVG HFNKLILGKY 481 KKKDINTNKR IEEDLNNNEE IKGYFIKKRG EIERKVKEKF 521 LSNNLQYYYS KEKIENYFEV YEFEILKRKI PFAPNFKRI I

561 KKGEDLFNNK NNKKYEYFKN FDKNSAEEKK EFLKTRNFLL 601 KELYYNNFYK EFLSKKEEFE KIVLEVKEEK KSRGNINNKK 641 SGVSFQSIDD YDTKINISDY IASIHKKEME RVEKYNEEKQ 681 KDTAKYIRDF VEEIFLTGFI NYLEKDKRLH FLKEEFSILC 721 NNNNNWDFN ININEEKIKE FLKENDSKTL NLYLFFNMID 761 SKRISEFRNE LVKYKQFTKK RLDEEKEFLG IKIELYETLI 801 EFVILTREKL DTKKSEEIDA WLVDKLYVKD SNEYKEYEEI 841 LKLFVDEKIL SSKEAPYYAT DNKTPILLSN FEKTRKYGTQ 881 SFLSEIQSNY KYSKVEKENI EDYNKKEEIE QKKKSNIEKL 921 QDLKVELHKK WEQNKITEKE IEKYNNTTRK INEYNYLKNK 961 EELQNVYLLH EMLSDLLARN VAFFNKWERD FKFIVIAIKQ 1001 FLRENDKEKV NEFLNPPDNS KGKKVYFSVS KYKNTVENID 1041 GIHKNFMNLI FLNNKFMNRK IDKMNCAIWV YFRNYIAHFL 1081 HLHTKNEKIS LISQMNLLIK LFSYDKKVQN HILKSTKTLL 1121 EKYNIQINFE ISNDKNEVFK YKIKNRLYSK KGKMLGKNNK 1161 LENEFLE NVKAMLEYSE

Other sequences for Leptotrichia wadei Cast 3a endonucleases are also available, such as those NCBI accession nos. BBM46759.1, BBM48616.1, BBM48974.1, BBM48975.1, and

WP_021746003.1.

In another example, a Herbinix hemicellulosilytica Casl3a endonuclease can be used that has the following sequence (SEQ ID NO: 39; NCBI accession no. WP_103203632.1).

1 MKLTRRRI SG NSVDQKITAA FYRDMSQGLL YYDSEDNDCT

41 DKVIESMDFE RSWRGRILKN GEDDKNPFYM FVKGLVGSND

81 KIVCEPIDVD SDPDNLDILI NKNLTGFGRN LKAPDSNDTL

121 ENLIRKIQAG IPEEEVLPEL KKIKEMIQKD IVNRKEQLLK

161 SIKNNRIPFS LEGSKLVPST KKMKWLFKLI DVPNKTFNEK

201 MLEKYWEIYD YDKLKANITN RLDKTDKKAR SISRAVSEEL

241 REYHKNLRTN YNRFVSGDRP AAGLDNGGSA KYNPDKEEFL

281 LFLKEVEQYF KKYFPVKSKH SNKSKDKSLV DKYKNYCSYK

321 WKKEVNRSI INQLVAGLIQ QGKLLYYFYY NDTWQEDFLN

361 SYGLSYIQVE EAFKKSVMTS LSWGINRLTS FFIDDSNTVK

401 FDDITTKKAK EAIESNYFNK LRTCSRMQDH FKEKLAFFYP

441 VYVKDKKDRP DDDIENLIVL VKNAIESVSY LRNRTFHFKE

481 SSLLELLKEL DDKNSGQNKI DYSVAAEFIK RDIENLYDVF

521 REQIRSLGIA EYYKADMISD CFKTCGLEFA LYSPKNSLMP

561 AFKNVYKRGA NLNKAYIRDK GPKETGDQGQ NSYKALEEYR

601 ELTWYIEVKN NDQSYNAYKN LLQLIYYHAF LPEVRENEAL

641 ITDFINRTKE WNRKETEERL NTKNNKKHKN FDENDDITVN

681 TYRYESIPDY QGESLDDYLK VLQRKQMARA KEVNEKEEGN

721 NNYIQFIRDV WWAFGAYLE NKLKNYKNEL QPPLSKENIG

761 LNDTLKELFP EEKVKSPFNI KCRFS ISTFI DNKGKSTDNT

801 SAEAVKTDGK EDEKDKKNIK RKDLLCFYLF LRLLDENEIC

841 KLQHQFIKYR CSLKERRFPG NRTKLEKETE LLAELEELME

881 LVRFTMPS IP EISAKAESGY DTMIKKYFKD FIEKKVFKNP

921 KTSNLYYHSD SKTPVTRKYM ALLMRSAPLH LYKDIFKGYY

961 LITKKECLEY IKLSNI IKDY QNSLNELHEQ LERIKLKSEK

1001 QNGKDSLYLD KKDFYKVKEY VENLEQVARY KHLQHKINFE

1041 SLYRIFRIHV DIAARMVGYT QDWERDMHFL FKALVYNGVL

1081 EERRFEAI FN NNDDNNDGRI VKKIQNNLNN KNRELVSMLC

1121 WNKKLNKNEF GAI IWKRNPI AHLNHFTQTE QNSKSSLESL

1161 INSLRILLAY DRKRQNAVTK TINDLLLNDY HIRIKWEGRV

1201 DEGQIYFNIK EKEDIENEPI IHLKHLHKKD CYIYKNSYMF

1241 DKQKEWICNG IKEEVYDKSI LKCIGNLFKF DYEDKNKSSA 1281 NPKHT

However, in some cases the Casl3 proteins with the SEQ ID NO: 39 sequence are not used.

In another example, a Leptotrichia buccalis Cast 3a endonuclease can be used that has the following sequence (SEQ ID NO: 40; NCBI accession no. WP_015770004.1).

1 MKVTKVGGIS HKKYTSEGRL VKSESEENRT DERLSALLNM

41 RLDMYIKNPS STETKENQKR IGKLKKFFSN KMVYLKDNTL

81 SLKNGKKENI DREYSETDIL ESDVRDKKNF AVLKKIYLNE

121 NVNSEELEVF RNDIKKKLNK INSLKYSFEK NKANYQKINE

161 NNIEKVEGKS KRNI IYDYYR ESAKRDAYVS NVKEAFDKLY

201 KEEDIAKLVL EIENLTKLEK YKIREFYHEI IGRKNDKENF

241 AKI IYEEIQN VNNMKELIEK VPDMSELKKS QVFYKYYLDK

281 EELNDKNIKY AFCHFVEIEM SQLLKNYVYK RLSNISNDKI

321 KRIFEYQNLK KLIENKLLNK LDTYVRNCGK YNYYLQDGEI

361 ATSDFIARNR QNEAFLRNI I GVSSVAYFSL RNILETENEN

401 DITGRMRGKT VKNNKGEEKY VSGEVDKIYN ENKKNEVKEN

441 LKMFYSYDFN MDNKNEIEDF FANIDEAISS IRHGIVHFNL

481 ELEGKDIFAF KNIAPSEISK KMFQNEINEK KLKLKI FRQL

521 NSANVFRYLE KYKILNYLKR TRFEFVNKNI PFVPSFTKLY

561 SRIDDLKNSL GIYWKTPKTN DDNKTKEI ID AQIYLLKNIY

601 YGEFLNYFMS NNGNFFEISK EI IELNKNDK RNLKTGFYKL

641 QKFEDIQEKI PKEYLANIQS LYMINAGNQD EEEKDTYIDF

681 IQKIFLKGFM TYLANNGRLS LIYIGSDEET NTSLAEKKQE

721 FDKFLKKYEQ NNNIKIPYEI NEFLREIKLG NILKYTERLN

761 MFYLILKLLN HKELTNLKGS LEKYQSANKE EAFSDQLELI

801 NLLNLDNNRV TEDFELEADE IGKFLDFNGN KVKDNKELKK

841 FDTNKIYFDG ENI IKHRAFY NIKKYGMLNL LEKIADKAGY

881 KI SIEELKKY SNKKNEIEKN HKMQENLHRK YARPRKDEKF

921 TDEDYESYKQ AIENIEEYTH LKNKVEFNEL NLLQGLLLRI

961 LHRLVGYTSI WERDLRFRLK GEFPENQYIE EIFNFENKKN

1001 VKYKGGQIVE KYIKFYKELH QNDEVKINKY SSANIKVLKQ

1041 EKKDLYIRNY IAHFNYIPHA EISLLEVLEN LRKLLSYDRK

1081 LKNAVMKSW DILKEYGFVA TFKIGADKKI GIQTLESEKI

1121 VHLKNLKKKK LMTDRNSEEL CKLVKIMFEY KMEEKKSEN

However, in some cases the Casl3 proteins with the SEQ ID NO: 40 sequence are not used.

In another example, a Leptotrichia seeligeri Cast 3a endonuclease can be used that has the following sequence (SEQ ID NO: 41; NCBI accession no. WP_012985477.1).

1 MWISIKTLIH HLGVLFFCDY MYNRREKKI I EVKTMRITKV

41 EVDRKKVLIS RDKNGGKLVY ENEMQDNTEQ IMHHKKSSFY 81 KSWNKTICR PEQKQMKKLV HGLLQENSQE KIKVSDVTKL

121 NI SNFLNHRF KKSLYYFPEN SPDKSEEYRI EINLSQLLED

161 SLKKQQGTFI CWESFSKDME LYINWAENYI SSKTKLIKKS

201 IRNNRIQSTE SRSGQLMDRY MKDILNKNKP FDIQSVSEKY

241 QLEKLTSALK ATFKEAKKND KEINYKLKST LQNHERQI IE

281 ELKENSELNQ FNIEIRKHLE TYFPIKKTNR KVGDIRNLEI

321 GEIQKIVNHR LKNKIVQRIL QEGKLASYEI ESTVNSNSLQ

361 KIKIEEAFAL KFINACLFAS NNLRNMVYPV CKKDILMIGE

401 FKNSFKEIKH KKFIRQWSQF FSQEITVDDI ELASWGLRGA

441 IAPIRNEI IH LKKHSWKKFF NNPTFKVKKS KI INGKTKDV

481 TSEFLYKETL FKDYFYSELD SVPELI INKM ESSKILDYYS

521 SDQLNQVFTI PNFELSLLTS AVPFAPSFKR VYLKGFDYQN

561 QDEAQPDYNL KLNIYNEKAF NSEAFQAQYS LFKMVYYQVF

601 LPQFTTNNDL FKSSVDFILT LNKERKGYAK AFQDIRKMNK

641 DEKPSEYMSY IQSQLMLYQK KQEEKEKINH FEKFINQVFI

681 KGFNSFIEKN RLTYICHPTK NTVPENDNIE IPFHTDMDDS

721 NIAFWLMCKL LDAKQLSELR NEMIKFSCSL QSTEEI STFT

761 KAREVIGLAL LNGEKGCNDW KELFDDKEAW KKNMSLYVSE

801 ELLQSLPYTQ EDGQTPVINR SIDLVKKYGT ETILEKLFSS

841 SDDYKVSAKD IAKLHEYDVT EKIAQQESLH KQWIEKPGLA

881 RDSAWTKKYQ NVINDISNYQ WAKTKVELTQ VRHLHQLTID

921 LLSRLAGYMS IADRDFQFSS NYILERENSE YRVTSWILLS

961 ENKNKNKYND YELYNLKNAS IKVSSKNDPQ LKVDLKQLRL

1001 TLEYLELFDN RLKEKRNNIS HFNYLNGQLG NSILELFDDA

1041 RDVLSYDRKL KNAVSKSLKE ILSSHGMEVT FKPLYQTNHH

1081 LKIDKLQPKK IHHLGEKSTV SSNQVSNEYC QLVRTLLTMK

For example, a Paludibacter propionicigenes Casl3a endonuclease can be used that has the following sequence (SEQ ID NO: 42; NCBI accession no. WP_013443710.1).

1 MRVSKVKVKD GGKDKMVLVH RKTTGAQLVY SGQPVSNETS

41 NILPEKKRQS FDLSTLNKTI IKFDTAKKQK LNVDQYKIVE

81 KI FKYPKQEL PKQIKAEEIL PFLNHKFQEP VKYWKNGKEE

121 SFNLTLLIVE AVQAQDKRKL QPYYDWKTWY IQTKSDLLKK

161 SIENNRIDLT ENLSKRKKAL LAWETEFTAS GSIDLTHYHK

201 VYMTDVLCKM LQDVKPLTDD KGKINTNAYH RGLKKALQNH

241 QPAIFGTREV PNEANRADNQ LSIYHLEWK YLEHYFPIKT

281 SKRRNTADDI AHYLKAQTLK TTIEKQLVNA IRANI IQQGK

321 TNHHELKADT TSNDLIRIKT NEAFVLNLTG TCAFAANNIR

361 NMVDNEQTND ILGKGDFIKS LLKDNTNSQL YSFFFGEGLS

401 TNKAEKETQL WGIRGAVQQI RNNVNHYKKD ALKTVFNISN

441 FENPTITDPK QQTNYADTIY KARFINELEK IPEAFAQQLK

481 TGGAVSYYTI ENLKSLLTTF QFSLCRSTIP FAPGFKKVFN

521 GGINYQNAKQ DESFYELMLE QYLRKENFAE ESYNARYFML

561 KLIYNNLFLP GFTTDRKAFA DSVGFVQMQN KKQAEKVNPR

601 KKEAYAFEAV RPMTAADSIA DYMAYVQSEL MQEQNKKEEK 641 VAEETRINFE KFVLQVFIKG FDSFLRAKEF DFVQMPQPQL

681 TATASNQQKA DKLNQLEASI TADCKLTPQY AKADDATHIA

721 FYVFCKLLDA AHLSNLRNEL IKFRESVNEF KFHHLLEI IE

761 ICLLSADWP TDYRDLYSSE ADCLARLRPF IEQGADITNW

801 SDLFVQSDKH SPVIHANIEL SVKYGTTKLL EQI INKDTQF

841 KTTEANFTAW NTAQKSIEQL IKQREDHHEQ WVKAKNADDK

881 EKQERKREKS NFAQKFIEKH GDDYLDICDY INTYNWLDNK

921 MHFVHLNRLH GLTIELLGRM AGFVALFDRD FQFFDEQQIA

961 DEFKLHGFVN LHS IDKKLNE VPTKKIKEIY DIRNKI IQIN

1001 GNKINESVRA NLIQFISSKR NYYNNAFLHV SNDEIKEKQM

1041 YDIRNHIAHF NYLTKDAADF SLIDLINELR ELLHYDRKLK

1081 NAVSKAFIDL FDKHGMILKL KLNADHKLKV ESLEPKKIYH

1121 LGSSAKDKPE YQYCTNQVMM AYCNMCRSLL EMKK

For example, a Lachnospiraceae bacterium Casl3a endonuclease can be used that has the following sequence (SEQ ID NO: 43; NCBI accession no. WP_022785443.1).

1 MKISKVREEN RGAKLTVNAK TAWSENRSQ EGILYNDPSR

41 YGKSRKNDED RDRYIESRLK SSGKLYRIFN EDKNKRETDE

81 LQWFLSEIVK KINRRNGLVL SDMLSVDDRA FEKAFEKYAE

121 LSYTNRRNKV SGSPAFETCG VDAATAERLK GI ISETNFIN

161 RIKNNIDNKV SEDI IDRI IA KYLKKSLCRE RVKRGLKKLL

201 MNAFDLPYSD PDIDVQRDFI DYVLEDFYHV RAKSQVSRSI

241 KNMNMPVQPE GDGKFAITVS KGGTESGNKR SAEKEAFKKF

281 LSDYASLDER VRDDMLRRMR RLWLYFYGS DDSKLSDVNE

321 KFDVWEDHAA RRVDNREFIK LPLENKLANG KTDKDAERIR

361 KNTVKELYRN QNIGCYRQAV KAVEEDNNGR YFDDKMLNMF

401 FIHRIEYGVE KIYANLKQVT EFKARTGYLS EKIWKDLINY

441 IS IKYIAMGK AVYNYAMDEL NASDKKEIEL GKISEEYLSG

481 ISSFDYELIK AEEMLQRETA VYVAFAARHL SSQTVELDSE

521 NSDFLLLKPK GTMDKNDKNK LASNNILNFL KDKETLRDTI

561 LQYFGGHSLW TDFPFDKYLA GGKDDVDFLT DLKDVIYSMR 601 NDSFHYATEN HNNGKWNKEL ISAMFEHETE RMTWMKDKF 641 YSNNLPMFYK NDDLKKLLID LYKDNVERAS QVPSFNKVFV 681 RKNFPALVRD KDNLGIELDL KADADKGENE LKFYNALYYM 721 FKEIYYNAFL NDKNVRERFI TKATKVADNY DRNKERNLKD 761 RIKSAGSDEK KKLREQLQNY IAENDFGQRI KNIVQVNPDY 801 TLAQICQLIM TEYNQQNNGC MQKKSAARKD INKDSYQHYK 841 MLLLVNLRKA FLEFIKENYA FVLKPYKHDL CDKADFVPDF

881 AKYVKPYAGL ISRVAGSSEL QKWYIVSRFL SPAQANHMLG

921 FLHSYKQYVW DIYRRASETG TEINHSIAED KIAGVDITDV

961 DAVIDLSVKL CGTISSEISD YFKDDEVYAE YISSYLDFEY

1001 DGGNYKDSLN RFCNSDAVND QKVALYYDGE HPKLNRNI IL

1041 SKLYGERRFL EKITDRVSRS DIVEYYKLKK ETSQYQTKGI

1081 FDSEDEQKNI KKFQEMKNIV EFRDLMDYSE IADELQGQLI

1121 NWIYLRERDL MNFQLGYHYA CLNNDSNKQA TYVTLDYQGK 1161 KNRKINGAIL YQICAMYING LPLYYVDKDS SEWTVSDGKE

1201 STGAKIGEFY RYAKSFENTS DCYASGLEIF ENISEHDNIT

1241 ELRNYIEHFR YYSSFDRSFL GIYSEVFDRF FTYDLKYRKN

1281 VPTILYNILL QHFVNVRFEF VSGKKMIGID KKDRKIAKEK

1321 ECARITIREK NGVYSEQFTY KLKNGTVYVD ARDKRYLQSI

1361 IRLLFYPEKV NMDEMIEVKE KKKPSDNNTG KGYSKRDRQQ

1401 DRKEYDKYKE KKKKEGNFLS GMGGNINWDE INAQLKN

For example, a Leptotrichia shahii Casl3a endonuclease can be used that has the following amino acid sequence (SEQ ID NO: 44; NCBI accession no. BBM39911.1).

1 MGNLFGHKRW YEVRDKKDFK IKRKVKVKRN YDGNKYILNI

41 NENNNKEKID NNKFIRKYIN YKKNDNILKE FTRKFHAGNI

81 LFKLKGKEGI IRIENNDDFL ETEEWLYIE AYGKSEKLKA

121 LGITKKKI ID EAIRQGITKD DKKIEIKRQE NEEEIEIDIR

161 DEYTNKTLND CSI ILRI IEN DELETKKSIY EIFKNINMSL

201 YKI IEKI IEN ETEKVFENRY YEEHLREKLL KDDKIDVILT

241 NFMEIREKIK SNLEILGFVK FYLNVGGDKK KSKNKKMLVE

281 KILNINVDLT VEDIADFVIK ELEFWNITKR IEKVKKVNNE

321 FLEKRRNRTY IKSYVLLDKH EKFKIERENK KDKIVKFFVE

361 NIKNNSIKEK IEKILAEFKI DELIKKLEKE LKKGNCDTEI

401 FGIFKKHYKV NFDSKKFSKK SDEEKELYKI IYRYLKGRIE

441 KILVNEQKVR LKKMEKIEIE KILNESILSE KILKRVKQYT

481 LEHIMYLGKL RHNDIDMTTV NTDDFSRLHA KEELDLELIT

521 FFASTNMELN KIFSRENINN DENIDFFGGD REKNYVLDKK

561 ILNSKIKI IR DLDFIDNKNN ITNNFIRKFT KIGTNERNRI

601 LHAISKERDL QGTQDDYNKV INI IQNLKIS DEEVSKALNL

641 DWFKDKKNI ITKINDIKIS EENNNDIKYL PSFSKVLPEI

681 LNLYRNNPKN EPFDTIETEK IVLNALIYVN KELYKKLILE

721 DDLEENESKN IFLQELKKTL GNIDEIDENI IENYYKNAQI

761 SASKGNNKAI KKYQKKVIEC YIGYLRKNYE ELFDFSDFKM

801 NIQEIKKQIK DINDNKTYER ITVKTSDKTI VINDDFEYI I

841 SI FALLNSNA VINKIRNRFF ATSVWLNTSE YQNI IDILDE

881 IMQLNTLRNE CITENWNLNL EEFIQKMKEI EKDFDDFKIQ

921 TKKEIFNNYY EDIKNNILTE FKDDINGCDV LEKKLEKIVI

961 FDDETKFEID KKSNILQDEQ RKLSNINKKD LKKKVDQYIK

1001 DKDQEIKSKI LCRI IFNSDF LKKYKKEIDN LIEDMESENE

1041 NKFQEIYYPK ERKNELYIYK KNLFLNIGNP NFDKIYGLIS

1081 NDIKMADAKF LFNIDGKNIR KNKISEIDAI LKNLNDKLNG

1121 YSKEYKEKYI KKLKENDDFF AKNIQNKNYK SFEKDYNRVS

1161 EYKKIRDLVE FNYLNKIESY LIDINWKLAI QMARFERDMH

1201 YIVNGLRELG I IKLSGYNTG ISRAYPKRNG SDGFYTTTAY

1241 YKFFDEESYK KFEKICYGFG IDLSENSEIN KPENES IRNY

1281 ISHFYIVRNP FADYSIAEQI DRVSNLLSYS TRYNNSTYAS

1321 VFEVFKKDVN LDYDELKKKF KLIGNNDILE RLMKPKKVSV 1361 LELESYNSDY IKNLI IELLT KIENTNDTL

In another example, a Leptotrichia buccalis C-1013-b Casl3a endonuclease can have the following amino acid sequence (SEQ ID NO: 45; NCBI accession no. C7NBY4; AltName LbuC2c2).

1 MKVTKVGGIS HKKYTSEGRL VKSESEENRT DERLSALLNM

41 RLDMYIKNPS STETKENQKR IGKLKKFFSN KMVYLKDNTL

81 SLKNGKKENI DREYSETDIL ESDVRDKKNF AVLKKIYLNE

121 NVNSEELEVF RNDIKKKLNK INSLKYSFEK NKANYQKINE

161 NNIEKVEGKS KRNI IYDYYR ESAKRDAYVS NVKEAFDKLY

201 KEEDIAKLVL EIENLTKLEK YKIREFYHEI IGRKNDKENF

241 AKI IYEEIQN VNNMKELIEK VPDMSELKKS QVFYKYYLDK

281 EELNDKNIKY AFCHFVEIEM SQLLKNYVYK RLSNISNDKI

321 KRIFEYQNLK KLIENKLLNK LDTYVRNCGK YNYYLQDGEI

361 ATSDFIARNR QNEAFLRNI I GVSSVAYFSL RNILETENEN

401 DITGRMRGKT VKNNKGEEKY VSGEVDKIYN ENKKNEVKEN

441 LKMFYSYDFN MDNKNEIEDF FANIDEAISS IRHGIVHFNL

481 ELEGKDIFAF KNIAPSEISK KMFQNEINEK KLKLKI FRQL

521 NSANVFRYLE KYKILNYLKR TRFEFVNKNI PFVPSFTKLY

561 SRIDDLKNSL GIYWKTPKTN DDNKTKEI ID AQIYLLKNIY

601 YGEFLNYFMS NNGNFFEISK EI IELNKNDK RNLKTGFYKL

641 QKFEDIQEKI PKEYLANIQS LYMINAGNQD EEEKDTYIDF

681 IQKIFLKGFM TYLANNGRLS LIYIGSDEET NTSLAEKKQE

721 FDKFLKKYEQ NNNIKIPYEI NEFLREIKLG NILKYTERLN

761 MFYLILKLLN HKELTNLKGS LEKYQSANKE EAFSDQLELI

801 NLLNLDNNRV TEDFELEADE IGKFLDFNGN KVKDNKELKK

841 FDTNKIYFDG ENI IKHRAFY NIKKYGMLNL LEKIADKAGY

881 KI SIEELKKY SNKKNEIEKN HKMQENLHRK YARPRKDEKF

921 TDEDYESYKQ AIENIEEYTH LKNKVEFNEL NLLQGLLLRI

961 LHRLVGYTSI WERDLRFRLK GEFPENQYIE EIFNFENKKN

1001 VKYKGGQIVE KYIKFYKELH QNDEVKINKY SSANIKVLKQ

1041 EKKDLYIRNY IAHFNYIPHA EISLLEVLEN LRKLLSYDRK

1081 LKNAVMKSW DILKEYGFVA TFKIGADKKI GIQTLESEKI

1121VHLKNLKKKK LMTDRNSEEL CKLVKIMFEY KMEEKKSEN

The inventors have evaluated the kinetics of other Casl3a and Casl3b proteins. Such work indicates that in some cases Cast 3b works faster in a target viral RNA detection assay than Cast 3 a.

For example, a Casl 3b from Prevotella buccae can be used in the Influenza RNA detection methods, compositions and devices. An amino acid sequence for a Prevotella buccae Casl 3b protein (NCBI accession no. WP_004343973.1) is shown below as SEQ ID NO:46.

1 MQKQDKLFVD RKKNAIFAFP KYITIMENKE KPEPIYYELT

41 DKHFWAAFLN LARHNVYTTI NHINRRLEIA ELKDDGYMMG

81 IKGSWNEQAK KLDKKVRLRD LIMKHFPFLE AAAYEMTNSK 121 SPNNKEQREK EQSEALSLNN LKNVLFIFLE KLQVLRNYYS

161 HYKYSEESPK PIFETSLLKN MYKVFDANVR LVKRDYMHHE

201 NIDMQRDFTH LNRKKQVGRT KNI IDSPNFH YHFADKEGNM

241 TIAGLLFFVS LFLDKKDAIW MQKKLKGFKD GRNLREQMTN

281 EVFCRSRI SL PKLKLENVQT KDWMQLDMLN ELVRCPKSLY

321 ERLREKDRES FKVPFDIFSD DYNAEEEPFK NTLVRHQDRF

361 PYFVLRYFDL NEI FEQLRFQ IDLGTYHFSI YNKRIGDEDE

401 VRHLTHHLYG FARIQDFAPQ NQPEEWRKLV KDLDHFETSQ

441 EPYISKTAPH YHLENEKIGI KFCSAHNNLF PSLQTDKTCN

481 GRSKFNLGTQ FTAEAFLSVH ELLPMMFYYL LLTKDYSRKE

521 SADKVEGI IR KEI SNIYAIY DAFANNEINS IADLTRRLQN

561 TNILQGHLPK QMI SILKGRQ KDMGKEAERK IGEMIDDTQR

601 RLDLLCKQTN QKIRIGKRNA GLLKSGKIAD WLVNDMMRFQ

641 PVQKDQNNIP INNSKANSTE YRMLQRALAL FGSENFRLKA

681 YFNQMNLVGN DNPHPFLAET QWEHQTNILS FYRNYLEARK

721 KYLKGLKPQN WKQYQHFLIL KVQKTNRNTL VTGWKNSFNL

761 PRGIFTQPIR EWFEKHNNSK RIYDQILSFD RVGFVAKAIP

801 LYFAEEYKDN VQPFYDYPFN IGNRLKPKKR QFLDKKERVE

841 LWQKNKELFK NYPSEKKKTD LAYLDFLSWK KFERELRLIK

881 NQDIVTWLMF KELFNMATVE GLKIGEIHLR DIDTNTANEE

921 SNNILNRIMP MKLPVKTYET DNKGNILKER PLATFYIEET

961 ETKVLKQGNF KALVKDRRLN GLFSFAETTD LNLEEHPISK

1001 LSVDLELIKY QTTRISIFEM TLGLEKKLID KYSTLPTDSF

1041 RNMLERWLQC KANRPELKNY VNSLIAVRNA FSHNQYPMYD

1081 ATLFAEVKKF TLFPSVDTKK IELNIAPQLL EIVGKAIKEI

1121 EKSENKN

Such a Prevotella buccae Casl3b protein can have a Km (Michaelis constant) substrate concentration of about 20 micromoles and a Kcat of about 987/second (see, e.g., Slaymaker et al. Cell Rep 26 (13): 3741-3751 (2019)).

Another Prevotella buccae Casl3b protein (NCBI accession no. WP 004343581.1) that can be used in the SARS-CoV-2 RNA detection methods, compositions and devices has the amino acid sequence shown below as SEQ ID NO: 47.

1 MQKQDKLFVD RKKNAIFAFP KYITIMENQE KPEPIYYELT

41 DKHFWAAFLN LARHNVYTTI NHINRRLEIA ELKDDGYMMD

81 IKGSWNEQAK KLDKKVRLRD LIMKHFPFLE AAAYEITNSK

121 SPNNKEQREK EQSEALSLNN LKNVLFIFLE KLQVLRNYYS

161 HYKYSEESPK PIFETSLLKN MYKVFDANVR LVKRDYMHHE

201 NIDMQRDFTH LNRKKQVGRT KNI IDSPNFH YHFADKEGNM

241 TIAGLLFFVS LFLDKKDAIW MQKKLKGFKD GRNLREQMTN

281 EVFCRSRI SL PKLKLENVQT KDWMQLDMLN ELVRCPKSLY

321 ERLREKDRES FKVPFDIFSD DYDAEEEPFK NTLVRHQDRF

361 PYFVLRYFDL NEI FEQLRFQ IDLGTYHFSI YNKRIGDEDE

401 VRHLTHHLYG FARIQDFAQQ NQPEVWRKLV KDLDYFEASQ 441 EPYIPKTAPH YHLENEKIGI KFCSTHNNLF PSLKTEKTCN

481 GRSKFNLGTQ FTAEAFLSVH ELLPMMFYYL LLTKDYSRKE

521 SADKVEGI IR KEI SNIYAIY DAFANGEINS IADLTCRLQK

561 TNILQGHLPK QMI SILEGRQ KDMEKEAERK IGEMIDDTQR

601 RLDLLCKQTN QKIRIGKRNA GLLKSGKIAD WLVNDMMRFQ

641 PVQKDQNNIP INNSKANSTE YRMLQRALAL FGSENFRLKA

681 YFNQMNLVGN DNPHPFLAET QWEHQTNILS FYRNYLEARK

721 KYLKGLKPQN WKQYQHFLIL KVQKTNRNTL VTGWKNSFNL

761 PRGIFTQPIR EWFEKHNNSK RIYDQILSFD RVGFVAKAIP

801 LYFAEEYKDN VQPFYDYPFN IGNKLKPQKG QFLDKKERVE

841 LWQKNKELFK NYPSEKKKTD LAYLDFLSWK KFERELRLIK

881 NQDIVTWLMF KELFNMATVE GLKIGEIHLR DIDTNTANEE

921 SNNILNRIMP MKLPVKTYET DNKGNILKER PLATFYIEET

961 ETKVLKQGNF KVLAKDRRLN GLLSFAETTD IDLEKNPITK

1001 LSVDHELIKY QTTRISIFEM TLGLEKKLIN KYPTLPTDSF

1041 RNMLERWLQC KANRPELKNY VNSLIAVRNA FSHNQYPMYD

1081 ATLFAEVKKF TLFPSVDTKK IELNIAPQLL EIVGKAIKEI

1121 EKSENKN

An example of a Bergeyella zoohelcum Casl3b (R1177A) mutant amino acid sequence (NCBI accession no. 6AAY_A) is shown below as SEQ ID NO: 48.

1 XENKTSLGNN IYYNPFKPQD KSYFAGYFNA AXENTDSVFR

41 ELGKRLKGKE YTSENFFDAI FKENI SLVEY ERYVKLLSDY

81 FPXARLLDKK EVPIKERKEN FKKNFKGI IK AVRDLRNFYT

121 HKEHGEVEIT DEI FGVLDEX LKSTVLTVKK KKVKTDKTKE

161 ILKKSIEKQL DILCQKKLEY LRDTARKIEE KRRNQRERGE

201 KELVAPFKYS DKRDDLIAAI YNDAFDVYID KKKDSLKESS

241 KAKYNTKSDP QQEEGDLKIP ISKNGWFLL SLFLTKQEIH

281 AFKSKIAGFK ATVIDEATVS EATVSHGKNS ICFXATHEIF

321 SHLAYKKLKR KVRTAEINYG EAENAEQLSV YAKETLXXQX

361 LDELSKVPDV VYQNLSEDVQ KTFIEDWNEY LKENNGDVGT

401 XEEEQVIHPV IRKRYEDKFN YFAIRFLDEF AQFPTLRFQV

441 HLGNYLHDSR PKENLISDRR IKEKITVFGR LSELEHKKAL

481 FIKNTETNED REHYWEIFPN PNYDFPKENI SVNDKDFPIA

521 GS ILDREKQP VAGKIGIKVK LLNQQYVSEV DKAVKAHQLK

561 QRKASKPS IQ NI IEEIVPIN ESNPKEAIVF GGQPTAYLSX

601 NDIHSILYEF FDKWEKKKEK LEKKGEKELR KEIGKELEKK

641 IVGKIQAQIQ QI IDKDTNAK ILKPYQDGNS TAIDKEKLIK

681 DLKQEQNILQ KLKDEQTVRE KEYNDFIAYQ DKNREINKVR

721 DRNHKQYLKD NLKRKYPEAP ARKEVLYYRE KGKVAVWLAN

761 DIKRFXPTDF KNEWKGEQHS LLQKSLAYYE QCKEELKNLL

801 PEKVFQHLPF KLGGYFQQKY LYQFYTCYLD KRLEYI SGLV

841 QQAENFKSEN KVFKKVENEC FKFLKKQNYT HKELDARVQS

881 ILGYPIFLER GFXDEKPTI I KGKTFKGNEA LFADWFRYYK

921 EYQNFQTFYD TENYPLVELE KKQADRKRKT KIYQQKKNDV 961 FTLLXAKHIF KSVFKQDSID QFSLEDLYQS REERLGNQER 1001 ARQTGERNTN YIWNKTVDLK LCDGKITVEN VKLKNVGDFI 1041 KYEYDQRVQA FLKYEENIEW QAFLIKESKE EENYPYWER 1081 EIEQYEKVRR EELLKEVHLI EEYILEKVKD KEILKKGDNQ 1121 NFKYYILNGL LKQLKNEDVE SYKVFNLNTE PEDVNINQLK 1161 QEATDLEQKA FVLTYIANKF AHNQLPKKEF WDYCQEKYGK 1201 IEKEKTYAEY FAEVFKKEKE ALIKLEHHHH HH

Another example of a Casl3b protein sequence from Prevotella sp. MSX73 (NCBI accession no. WP_007412163.1) that can be used in the target viral RNA detection methods, compositions and devices is shown below as SEQ ID NO: 49.

1 MQKQDKLFVD RKKNAIFAFP KYITIMENQE KPEPIYYELT

41 DKHFWAAFLN LARHNVYTTI NHINRRLEIA ELKDDGYMMG

81 IKGSWNEQAK KLDKKVRLRD LIMKHFPFLE AAAYEITNSK

121 SPNNKEQREK EQSEALSLNN LKNVLFIFLE KLQVLRNYYS

161 HYKYSEESPK PIFETSLLKN MYKVFDANVR LVKRDYMHHE

201 NIDMQRDFTH LNRKKQVGRT KNI IDSPNFH YHFADKEGNM

241 TIAGLLFFVS LFLDKKDAIW MQKKLKGFKD GRNLREQMTN

281 EVFCRSRI SL PKLKLENVQT KDWMQLDMLN ELVRCPKSLY

321 ERLREKDRES FKVPFDIFSD DYDAEEEPFK NTLVRHQDRF

361 PYFVLRYFDL NEI FEQLRFQ IDLGTYHFSI YNKRIGDEDE

401 VRHLTHHLYG FARIQDFAPQ NQPEEWRKLV KDLDHFETSQ

441 EPYISKTAPH YHLENEKIGI KFCSTHNNLF PSLKREKTCN

481 GRSKFNLGTQ FTAEAFLSVH ELLPMMFYYL LLTKDYSRKE

521 SADKVEGI IR KEI SNIYAIY DAFANNEINS IADLTCRLQK

561 TNILQGHLPK QMI SILEGRQ KDMEKEAERK IGEMIDDTQR

601 RLDLLCKQTN QKIRIGKRNA GLLKSGKIAD WLVSDMMRFQ

641 PVQKDTNNAP INNSKANSTE YRMLQHALAL FGSESSRLKA

681 YFRQMNLVGN ANPHPFLAET QWEHQTNILS FYRNYLEARK

721 KYLKGLKPQN WKQYQHFLIL KVQKTNRNTL VTGWKNSFNL

761 PRGIFTQPIR EWFEKHNNSK RIYDQILSFD RVGFVAKAIP

801 LYFAEEYKDN VQPFYDYPFN IGNKLKPQKG QFLDKKERVE

841 LWQKNKELFK NYPSEKNKTD LAYLDFLSWK KFERELRLIK

881 NQDIVTWLMF KELFKTTTVE GLKIGEIHLR DIDTNTANEE

921 SNNILNRIMP MKLPVKTYET DNKGNILKER PLATFYIEET

961 ETKVLKQGNF KVLAKDRRLN GLLSFAETTD IDLEKNPITK

1001 LSVDYELIKY QTTRISIFEM TLGLEKKLID KYSTLPTDSF

1041 RNMLERWLQC KANRPELKNY VNSLIAVRNA FSHNQYPMYD

1081 ATLFAEVKKF TLFPSVDTKK IELNIAPQLL EIVGKAIKEI

1121 EKSENKN

Hence, the sample can be incubated with at least one CRISPR RNA (crRNA) and at least one Casl3 protein. The Casl3 protein can, for example, be a Casl3a protein, Casl3b protein, or a combination thereof. (CRISPRI/CRISPR-associated (Cas) systems

Genomic editing has been performed by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems (see e.g., Marraffini and Sontheimer. Nature Reviews Genetics 11: 181-190 (2010); Sorek et al. Nature Reviews Microbiology 2008 6: 181-6; Karginov and Hannon. Mol Cell 2010 1 :7-19; Hale et al. Mol Cell 2010:45:292-302; Jinek et al. Science 2012 337:815-820; Bikard and Marraffini Curr Opin Immunol 2012 24:15-20; Bikard et al. Cell Host & Microbe 2012 12: 177-186; all of which are incorporated by reference herein in their entireties).

However, a CRISPR guide RNA system can be adapted for use in the methods and compositions described herein. Two RNAs can be used in CRISPR genomic editing systems: a CRISPR RNA (crRNA), which is a 17-20 nucleotide sequence complementary to the target RNA, and a trans-activating crRNA (tracrRNA) that is a binding scaffold for the Cas nuclease. In some cases, the two RNAs are fused to make a single guide RNA (sgRNA). The tracrRNA forms a stem loop that is recognized and bound by the cas nuclease. The crRNA typically has shorter sequence than the tracrRNA. The term “guide RNA” as used herein refers to either a single guide RNA (sgRNA) or a crRNA. The CRISPR technique is generally described, for example, by Mali et al. Science 339:823-6 (2013); which is incorporated by reference herein in its entirety.

The guide RNA system used herein is encoded within or adjacent to the ncRNA coding region of the expression cassettes. Hence, upon transcription of the guide RNA, it can target a Cas enzyme to the desired location in the genome, where it can cleave the genomic RNA for generation of a genomic modification.

There are several types of CRISPR systems, some of which are summarized in the chart below.

CRISPR System Types Overview

A “guide RNA” or “gRNA” as provided herein refers to a ribonucleotide sequence capable of binding a cas nuclease, thereby forming ribonucleoprotein complex. The gRNA includes a nucleotide sequence complementary to a target site (e.g., near or at a genomic site to be edited). In some cases, the guide RNA includes one or more RNA molecules. TracrRNAs can be used to facilitate assembly of a ribonucleoprotein complex that includes the gRNA together with the tracrRNA and a cas nuclease. A complementary nucleotide sequence of the guide RNA can mediate binding of the ribonucleoprotein complex to the target site thereby providing the sequence specificity of the ribonucleoprotein complex. Thus, the guide RNA includes a sequence that is complementary to a target nucleic acid sequence such that the guide RNA binds a target nucleic acid sequence.

In some cases, the complement of the guide RNA includes a sequence having a sequence identity of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to a target nucleic acid (e.g., a target viral RNA sequence). In some cases, the guide RNA includes a sequence having sequence identity of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the target nucleic acid sequence. In some cases, the guide RNA or complement thereof, includes a sequence having a sequence identity of at least about 90%, 95%, or 100% to a target viral RNA sequence. In some cases, segment bound by a guide RNA within the target nucleic acid is about or at least about 10, 15, 20, 25, or more nucleotides in length.

The guide RNA is a single-stranded ribonucleic acid, although in some cases it may form some double-stranded regions by folding onto itself. In some cases, the guide RNA is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleic acid residues in length. In some cases, the guide RNA is from about 10 to about 30 nucleic acid residues in length. In some cases, the guide RNA is about 20 nucleic acid residues in length. For example, the length of the guide RNA can be at least about 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides or residues in length. In some cases, the guide RNA is from 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 5 to 75, 10 to 75, 15 to 75, 20 to 75, 25 to 75, 30 to 75, 35 to 75, 40 to 75, 45 to 75, 50 to 75, 55 to 75, 60 to 75, 65 to 75, 70 to 75, 5 to 100, 10 to 100, 15 to 100, 20 to 100, 25 to 100, 30 to 100, 35 to 100, 40 to 100, 45 to 100, 50 to 100, 55 to 100, 60 to 100, 65 to 100, 70 to 100, 75 to 100, 80 to 100, 85 to 100, 90 to 100, 95 to 100, or more nucleotides or residues in length. In some cases, the guide RNA is from 10 to 15, 10 to 20, 10 to 30, 10 to 40, or 10 to 50 residues in length.

Definitions

The term "about" as used herein when referring to a measurable value such as an amount, a length, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value.

"Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, bacterial, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.

The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the polynucleotide of interest is cloned and then expressed in transformed organisms, for example, as described herein. The host organism expresses the foreign nucleic acids to produce the RNA, RT- DNA, or protein under expression conditions.

As used herein, a "cell" refers to any type of cell isolated from a prokaryotic, eukaryotic, or archaeon organism, including bacteria, archaea, fungi, protists, plants, and animals, including cells from tissues, organs, and biopsies, as well as recombinant cells, cells from cell lines cultured in vitro, and cellular fragments, cell components, or organelles comprising nucleic acids. The term also encompasses artificial cells, such as nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids. The methods described herein can be performed, for example, on a sample comprising a single cell or a population of cells. The term also includes genetically modified cells. "Recombinant host cells," "host cells", "cells", "cell lines", "cell cultures", and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.

A "coding sequence" or a sequence which "encodes" a selected polypeptide or a selected RNA, is a nucleic acid molecule which is transcribed (in the case of DNA templates) into RNA and/or translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, ncRNAs, tracrRNAs, ncRNAs modified to include heterologous sequences, cDNA from viral, prokaryotic or eukaryotic ncRNA, mRNA, viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

Typical "control elements," include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.

"Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper polymerases are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

"Encoded by" refers to a nucleic acid sequence which codes for a polypeptide or RNA sequence. For example, the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. The RNA sequence or a portion thereof contains a nucleotide sequence of at least 3 to 5 nucleotides, more preferably at least 8 to 10 nucleotides, and even more preferably at least 15 to 20 nucleotides.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein, DNA, or RNA or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when obtained from nature or when produced by recombinant DNA techniques, or free from chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

"Substantially purified" generally refers to isolation of a substance (nucleic acid, compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically, in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well- known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non- viral vectors, particulate carriers, and liposomes). Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

"Expression" refers to detectable production of a gene product by a cell. The gene product may be a transcription product (i.e., RNA), which may be referred to as "gene expression", or the gene product may be a translation product of the transcription product (i.e., a protein), depending on the context.

"Mammalian cell" refers to any cell derived from a mammalian subject suitable for transfection with vector systems comprising, as described herein. The cell may be xenogeneic, autologous, or allogeneic. The cell can be a primary cell obtained directly from a mammalian subject. The cell may also be a cell derived from the culture and expansion of a cell obtained from a mammalian subject. Immortalized cells are also included within this definition. In some embodiments, the cell has been genetically engineered to express a recombinant protein and/or nucleic acid.

The term "subject" includes animals, including both vertebrates and invertebrates, including, without limitation, invertebrates such as arthropods, mollusks, annelids, and cnidarians; and vertebrates such as amphibians, including frogs, salamanders, and caecillians; reptiles, including lizards, snakes, turtles, crocodiles, and alligators; fish; mammals, including human and non-human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows; and birds such as domestic, wild and game birds, including chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. In some cases, the disclosed methods find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; primates, and transgenic animals.

"Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of nonintegrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non-viral vectors, alphaviruses, pox viruses and vaccinia viruses.

The term "derived from" is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means. A polynucleotide or nucleic acid "derived from" a designated sequence refers to a polynucleotide or nucleic acid that includes a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence. The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.

The terms "hybridize" and "hybridization" refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson-Crick base pairing.

The term "homologous region" refers to a region of a nucleic acid with homology to another nucleic acid region. Thus, whether a "homologous region" is present in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since a nucleic acid is often double-stranded, the term "homologous, region," as used herein, refers to the ability of nucleic acid molecules to hybridize to each other. For example, a single-stranded nucleic acid molecule can have two homologous regions which are capable of hybridizing to each other. Thus, the term "homologous region" includes nucleic acid segments with complementary sequences. Homologous regions may vary in length but will typically be between 4 and 500 nucleotides (e.g., from about 4 to about 40, from about 40 to about 80, from about 80 to about 120, from about 120 to about 160, from about 160 to about 200, from about 200 to about 240, from about 240 to about 280, from about 280 to about 320, from about 320 to about 360, from about 360 to about 400, from about 400 to about 440, etc.).

As used herein, the terms "complementary" or "complementarity" refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated. "Complementarity" may exist between two RNA strands, two DNA strands, or between an RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be "complementary" and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary" or "100% complementary" if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered "perfectly complementary" or "100% complementary" even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art.

The term "donor polynucleotide" or “donor DNA” refers to a nucleic acid or polynucleotide that provides a nucleotide sequence of an intended edit to be integrated into the genome at a target locus by HDR or recombineering.

A "target site" or "target sequence" is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide (donor DNA). The target site may be allele-specific (e.g., a major or minor allele). For example, a target site can be a genomic site that is intended to be modified such as by insertion of one or more nucleotides, replacement of one or more nucleotides, deletion of one or more nucleotides, or a combination thereof.

In general, "a CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence. In some embodiments, one or more elements of a CRISPR system are derived from a type I, type II, or type III CRISPR system. Casl and Cas2 are found in all three types of CRISPR-Cas systems, and they are involved in spacer acquisition. In the I-E system of E. coli, Casl and Cas2 form a complex where a Cas2 dimer bridges two Casl dimers. In this complex Cas2 performs a non-enzymatic scaffolding role, binding double-stranded fragments of invading DNA, while Casl binds the single-stranded flanks of the DNA and catalyzes their integration into CRISPR arrays.

In some embodiments, one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system can be characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).

In certain embodiments, the disclosure provides protospacers that are adjacent to short (3 - 5 bp) DNA sequences termed protospacer adjacent motifs (PAM). The PAMs are important for type I and type II systems during acquisition. In type I and type II systems, protospacers are excised at positions adjacent to a PAM sequence, with the other end of the spacer is cut using a ruler mechanism, thus maintaining the regularity of the spacer size in the CRISPR array. The conservation of the PAM sequence differs between CRISPR-Cas systems and may be evolutionarily linked to Casl and the leader sequence.

In some embodiments, a regulatory element is operably linked to one or more elements of a CRISPR system so as to drive expression of the one or more elements of the CRISPR system. In general, CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), also known as SPIDRs (SPacer Interspersed Direct Repeats), constitute a family of DNA loci that are usually specific to a particular bacterial species. The CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. colt (Ishino et al., J. BacterioL, 169:5429-5433 (1987); and Nakata et al., J. BacterioL, 171:3553-3556 (1989)), and associated genes. Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol., 10: 1057-1065 (1993); Hoe et al., Emerg. Infect. Dis., 5:254-263 (1999); Masepohl et al., Biochim. Biophys. Acta 1307:26-30 (1996); and Mojica et al., Mol. Microbiol, 17:85-93 (1995)). The CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al, OMICS J. Integ. Biol., 6:23-33 (2002); and Mojica etal., Mol. Microbiol., 36:244-246 (2000)). In general, the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al., (2000), supra). Although the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions typically differ from strain to strain (van Embden et al., J. Bacteriol., 182:2393- 2401 (2000)). CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575 (2002); and Mojica et al, (2005)) including, but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacteriumn, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Themioplasnia, Corynebacterium, Mycobacterium, Streptomyces, Aquifrx, Porphvromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myrococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

In some embodiments, an enzyme coding sequence encoding a CRISPR enzyme (e.g., cas9) is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about one or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database", and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.

"Administering" a nucleic acid, such as an expression cassette, comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.

The subject matter disclosed herein is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosed subject matter, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the nucleic acid" includes reference to one or more nucleic acids and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of any features or elements described herein, which includes use of a "negative" limitation.

It is appreciated that certain features of the disclosed subject matter, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed subject matter, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the disclosed subject matter and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the disclosed subject matter is not entitled to antedate such publication. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

The following Examples illustrate some of the materials, methods, and experiments that were used or performed in the development of the invention.

Examples

Example 1: Casl3a detection of SARS-CoV-2 transcripts

CRISPR RNA guides (crRNAs) were designed and validated for Influenza A, strains H1N2 and H3N2, and Influenza B. Twenty-two (22) crRNAs were designed for Influenza A and fifteen (15) crRNAs were designed for Influenza A. Each crRNA includes a crRNA stem that is derived from a bacterial sequence, while the spacer sequence is derived from the Influenza genome (reverse complement). See Table 1 (reproduced below) for crRNA sequences.

Table 1: Examples of Influenza A and B crRNA Sequences

FIGS. 4A-C shows the detection of influenza strains with the specific RNA guides of Table 1. The RNA guides were tested against H1N1, H3N2, FluB target viral RNA, and ribonucleoprotein (RNP) background control with no target viral RNA. As shown in FIG. 4A, the signals from each reaction were measured over two hours and the signal slopes were calculated. Slope ratios were calculated by dividing the slope of a guide RNA + target (i.e. RNP + target viral RNA) reaction by the slope of guide RNA + no target (i.e. RNP control only) reaction. As shown in FIG. 4B, the signal slopes of H1N1, H3N2, or FluB RNA guides was divided by the signal slopes of the RNP control determine comparative slope ratio between the target viral RNA and the RNP control. When the comparative ratio is high (greater than 1), the guide RNAs employed in the assay mixture detect Hl N1 , H3N2, or FluB target viral RNA strains more efficiently. But when the comparative ratio is low (less than 1), the guide RNAs employed in the assay mixture detect the target viral RNA similarly to the RNP control. FIG. 4C shows the RNA guides for H1N1 and H3N2 strains of Influenza A with a slope ratio of more than three and the RNA guides for FluB with a slope ratio of more than five.

Example 2: Casl3a detection of Influenza B RNA in nasal swabs

FIGS. 5A-B show the validation and cross-reactivity of Influenza B (FluB) RNA guides against host RNA and nasal swabs. The RNA guides for FluB having a slope ratio of more than five, as shown in FIG. 4C, were tested against host RNA and nasal swabs. The signals from each reaction were measured over two hours and the signal slopes were calculated. To prepare the graph shown in FIG. 5B, the signal slopes of the RNA guides for FluB was divided by the signal slopes of the RNP control determine a comparative slope ratio between the target viral RNA and the RNP control. FluB RNA guides FluB crlO and FluB-crl3 were found to cross-react significantly with nasal swab material that was positive for FluB. FluB_crl2 and FluB-crl4 were found to not crossreact to the same extent with the nasal swab material that was positive for FluB.

Example 3: Improving detection of Influenza B by combining RNA guides of SEQ. ID. NOs: 34 and 36

FIGS. 6A-B illustrate the effect on target viral RNA detection of combining the RNA guides FluB_crl2 and FluB-crl4 (SEQ. ID. NOs: 34 and 36). The signals slope from each reaction of target viral RNA for H3N1, H1N1, FluB, or RNP alone with the RNA guides FluB_crl2 alone, FluB-crl4 alone, or FluB_crl2 and FluB-crl4 combined were measured over two hours and the signal slopes were calculated and shown in FIG 6A. To prepare the graph shown in FIG. 6B, the signal slopes of FIG. 6A were divided by the signal slopes of the RNP control to determine comparative slope ratio between the target viral RNA and the RNP control. Combining the RNA guides FluB_crl2 and FluB-crl4 improves detection of FluB target viral RNA more than use of these RNA guides separately. Detection of H3N1, H1N1, or RNP alone was not increased by combining the RNA guides FluB_crl2 and FluB-crl4.

Example 4: Validation and cross-reactivity of Influenza A (H1N1 and H3N2 strains) RNA guides against host RNA and nasal swabs

FIGS. 7A-B show the validation and cross-reactivity of Influenza A (H1N1 and H3N2 strains) RNA guides against host RNA and nasal swabs. The signals from each reaction were measured over two hours and the signal slopes were calculated, as shown in FIG. 7A. The RNA guides for Influenza A having a slope ratio of more than three, as shown in FIG. 4C, were included in the test against host RNA and nasal swabs. To prepare the graph shown in FIG. 7B, the signal slopes of the RNA guides for FluB was divided by the signal slopes of the RNP control determine a comparative slope ratio between the target viral RNA and the RNP control. Influenza A RNA guides identified in the boxes had the best detection of the target viral RNA in the nasal swabs and were selected for a combination experiment shown in FIGS. 8A-B.

Example 5: Improving detection of Influenza A by combining RNA guides of SEQ. ID. NOs: 4, 8, 13, 16, 17, 21, 22 and, independently, 8, 16, 21, and 22.

FIGS. 8A-B shows the effect of combining the best Influenza A RNA guides of FIG. 7A on Influenza A target viral RNA detection. A combination of seven Influenza A RNA guides (the “7g”: cr04m, cr08, crl3, crl6, crl7, cr21, cr22 (SEQ. ID. NOs: 4, 8, 13, 16, 17, 21, 22, respectively)) and four Influenza RNA guides (the “4g”: cr08, crl6, cr21, cr22 (SEQ. ID. NOs: 8, 16, 21, and 22, respectively)) were tested against target viral RNA for Influenza A (strains H1N1 and H3N2). The signals from each reaction were measured over two hours and the signal slopes were calculated, as shown in FIG. 8 A. To prepare the graph shown in FIG. 8B, the signal slopes of the RNA guides for Influenza A were divided by the signal slopes of the RNP control determine a comparative slope ratio between the target viral RNA and the RNP control. The slope ratios for the Influenza A RNA guides in the 7g group were 4.9 and 26.5 for the target viral RNA for H1N1 and H3N2, respectively. The slope ratios for the Influenza A RNA guides in the 4g group were 4.5 and 26.4 for the target viral RNA for H1N1 and H3N2, respectively. The slope ratios for each of the 7g and 4g RNA guide groups were significantly higher than for any of the Influenza A RNA guides alone.

References:

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All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

The following statements provide a summary of some aspects of the inventive nucleic acids and methods described herein.

Statements:

1. A method comprising:

(a) incubating a sample suspected of containing Influenza A or B RNA or virus with one or more Cast 3 protein, at least one CRISPR guide RNA (crRNA), and at least one reporter RNA for a period of time sufficient to form at least one RNA cleavage product; and

(b) detecting reporter RNA cleavage product(s) with a detector.

2. The method of statement 1, wherein the at least one CRISPR guide RNA (crRNA) binds a target site in at least one of an Influenza A or Influenza B nucleic acid.

3. The method of statement 1 or 2, wherein one or more of the Cast 3 proteins has a protein sequence with at least 95% sequence identity to any of SEQ ID NOs: 38-49.

4. The method of any one of statements 1-3, wherein one or more of the Cast 3 proteins has any one SEQ ID NOs: 38-49.

5. The method of any one of statements 1 or 2, wherein the Influenza A RNA is from a variant of Influenza A.

6. The method of any one of statements 1-5, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 1- 37.

7. The method of any one of statements 1-6, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 1-37.

8. The method of any one of statements 1-7, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 32, 34, 35, 36, or a combination thereof.

9. The method of any one of statements 1-8, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 32, 34, 35, 36, or a combination thereof.

10. The method of statement 9, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 34 and 36. 11. The method of any one of statements 1-10, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, or a combination thereof.

12. The method of statement 1 , wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, or a combination thereof.

13. The method of statement 12, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 8, 16, 21, and 22.

14. The method of any one of statements 1, 2, 5-13, wherein one or more of the Casl3 protein is a Casl3a or Casl3b protein.

15. The method of statement 1, wherein the at least one CRISPR guide RNA (crRNA) is two or more CRISPR guide RNAs (crRNAs).

16. The method of statement 1, wherein the Casl3 protein is complexed with the at least one CRISPR guide RNA (crRNA) prior to incubation with the sample suspected of containing the target viral RNA.

17. The method of statement 16, wherein the one or more of the Casl3 proteins is complexed with the at least one CRISPR guide RNA (crRNA) and prepared as a lyophilized bead.

18. The method of statement 1 , wherein the sample suspected of containing the target viral RNA is saliva, sputum, mucus, nasopharyngeal materials, blood, serum, plasma, urine, aspirate, biopsy tissue, or a combination thereof.

19. The method of statement 1, wherein the sample suspected of containing RNA is a lysed biological sample.

20. The method of statement 1 , wherein cleavage of the reporter RNA produces a light signal, an electronic signal, an electrochemical signal, an electrostatic signal, a steric signal, a van der Waals interaction signal, a hydration signal, a Resonant frequency shift signal, or a combination thereof.

21. The method of statement 1, wherein the reporter RNA reporter comprises at least one fluorophore and at least one fluorescence quencher. 22. The method of statement 21, wherein the at least one fluorophore is Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof.

23. The method of any of statements 1, 21, or 22, wherein the detector comprises a light detector, a fluorescence detector, a color filter, an electronic detector, an electrochemical signal detector, an electrostatic signal detector, a steric signal detector, a van der Waals interaction signal detector, a hydration signal detector, a Resonant frequency shift signal detector, or a combination.

24. The method of statement 1 , wherein the target viral RNA is detected when a signal from the reporter RNA cleavage product(s) is distinguishable from a control assay signal.

25. The method of statement 24, wherein the control assay contains no target viral RNA.

26. The method of statement 24, wherein the control assay contains viral RNA that is not the target viral RNA.

27. The method of statement 1 , wherein the sample comprises at least one RNA from a common cold coronavirus, SARS-CoV-2, hepatitis virus, respiratory syncytial virus (RSV), or human immunodeficiency virus (HIV).

28. The method of statement 27, wherein the common cold coronavirus is at least one of strain NL63, OC43, or 229E.

29. The method of statement 27, wherein the hepatitis virus is hepatitis C virus (HCV).

30. The method of any one of statements 27-29, wherein at least one CRISPR guide RNAs can bind to at least one RNA from the common cold coronavirus, SARS-CoV-2, hepatitis virus, respiratory syncytial virus (RSV), or human immunodeficiency virus (HIV).

31. A method comprising treating a subject with detectable Influenza A or B infection detected by the method of any of statements 1 -26.

32. A kit comprising a package containing at least one Cast 3 protein, at least one CRISPR guide RNA (crRNA) that binds a target site in at least one of an Influenza A or Influenza B nucleic acid, at least one reporter RNA, and instructions for detecting and/or quantifying the target viral RNA in a sample. 33. The kit of statement 32, wherein the at least one CRISPR guide RNA (crRNA) has a sequence with at least 95% sequence identity to any of SEQ ID NO: 1-37.

34. The kit of any one of statements 32 or 33, wherein at least one of the CRISPR guide RNAs (crRNAs) has a sequence of any of SEQ ID NOs: 1-37.

35. The kit of any one of statements 32-34, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 32, 34, 35, 36, or a combination thereof.

36. The kit of any one of statements 32-35, wherein the at least one CRISPR guide RNA (crRNA) has any of SEQ ID NOs: 32, 34, 35, 36, or a combination thereof.

37. The kit of statement 32, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 34 and 36.

38. The kit of statement 32, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, or a combination thereof.

39. The kit of statement 32, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, or a combination thereof.

40. The kit of statement 28, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 8, 16, 21, and 22.

41. The kit of any one of statements 32-40, wherein the at least one CRISPR guide RNA (crRNA) is two or more CRISPR guide RNAs (crRNAs).

42. The kit of any one of statements 32-41, wherein the Casl3 protein is complexed with the at least one CRISPR guide RNA (crRNA).

43. The kit of any one of statements 32-42, wherein the one or more of the Cast 3 proteins is complexed with the at least one CRISPR guide RNA (crRNA) and prepared as a lyophilized bead.

44. The kit of any one of statements 32, 42, or 43, wherein the Casl3 protein is a Casl3a or Cast 3b protein. 45. The kit of statement 32, wherein the reporter RNA reporter comprises at least one fluorophore and at least one fluorescence quencher.

46. The kit of statement 45, wherein the at least one fluorophore is Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof.

47. The kit of any one of statements 32 or 43, further comprising a sample chamber, assay mixture reaction chamber, or a combination thereof.

48. The kit of statement 43, wherein the lyophilized bead is included in the assay mixture reaction chamber.

49. The kit of statement 32, further comprising a detector.