Viral diagnosis using combinations of CRISPR RNAs and Cas13a enzymes

Abstract

The present disclosure relates to methods for detecting and quantifying the presence of influenza A or B RNA in a sample with high specificity and sensitivity using a CRISPR-Cas13 enzyme complexed with a crRNA guide RNA for influenza A or B. These methods can be used to diagnose influenza A or B infection, quantify the concentration of influenza A or B RNA present in a sample, and identify the presence of different influenza A subtypes or mutations.

Description

Technical Field

[0001] The present invention relates to virus diagnosis using combinations of CRISPR RNAs and Cas13a enzymes.

Background Art

[0002] The detection of respiratory infections, including SARS-CoV-2 and influenza A and B, is extremely important in targeting locations and populations in need of medical support. For example, the number of patients affected by influenza in the United States during the 2019 - 2020 season was estimated to be approximately 38 million. During that same season of influenza, approximately 400,000 people were hospitalized and approximately 22,000 people died from the disease.

[0003] Current respiratory virus diagnostic assays include laboratory-based equipment and personnel-required RT-qPCR nucleic acid-based tests (NAT), or rapid influenza diagnostics (RIDT) that detect viral antigens. These assays are not quantitative or multiplexed with other relevant respiratory viruses. These assays are also not suitable for use by inexperienced or untrained people, such as for home use.

Summary of the Invention

Problems to be Solved by the Invention

[0004] To identify respiratory infections, a rapid and user-friendly detection assay for viral RNA from respiratory fluid samples is needed.

Means for Solving the Problems

[0005] Described herein are methods, compositions, and devices for detecting and quantifying target viral RNAs, such as influenza A and B viruses, which are faster than currently available methods and devices and are more readily deployable in the field. Further, the methods, compositions, and devices can readily detect and distinguish between strains and variants of the target viral RNA.

[0006] Current rapid influenza diagnostic tests (RIDTs) are immunoassays that can identify the presence of influenza A and B virus nucleoprotein antigens in respiratory specimens and display the results in a qualitative manner (positive versus negative). However, RIDTs are known to have limited sensitivity for detecting influenza in respiratory specimens compared to time-consuming RT-PCR or virus culture methods. Negative RIDTs can result in false-negative results, especially during the peak of influenza activity in the community.

[0007] The methods described herein can include (a) incubating a sample suspected of containing influenza A or B RNA or virus with one or more Cas13 proteins, at least one CRISPR guide RNA (crRNA), and at least one reporter RNA for a period sufficient to form at least one RNA cleavage product; and (b) detecting the reporter RNA cleavage product(s) with a detector. Such methods are useful for detecting whether a sample contains one or more copies of influenza A or B viral RNA. The methods are also useful for detecting the absence of infection by a virus carrying the target viral RNA. Further, the methods and compositions described herein can also readily identify whether a variant strain or mutant of a virus carrying the target viral RNA is present in a sample and what the variant or mutation is.

[0008] The methods described herein are useful for diagnosing influenza infection in a variety of complex biological samples. For example, the sample can include human saliva, sputum, mucus, nasopharyngeal substances, blood, serum, plasma, urine, aspirate, biopsy tissue, or combinations thereof.

Brief Description of the Drawings

[0009]

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Best Mode for Carrying Out the Invention

[0010] Many aspects of the present disclosure can be better understood with reference to the following drawings. The components of the drawings are not necessarily drawn to scale. Rather, emphasis has been placed on clearly explaining the principles of the present disclosure. Further, components are shown as transparent in certain figures only for the purpose of clarifying the figure, which does not indicate that the illustrated components are necessarily transparent.

[0011] Methods, kits, and devices for rapidly detecting and / or quantifying influenza virus infection are described herein. The method can include (a) incubating a sample suspected of containing RNA or virus for a period sufficient to form at least one RNA cleavage product with one or more Cas13 proteins, at least one CRISPR guide RNA (crRNA) that binds to a target site in at least one of influenza A or influenza B nucleic acids, and at least one reporter RNA; and (b) detecting the level of the reporter RNA cleavage product with a detector. Such a method is useful for detecting whether a sample contains one or more copies of influenza RNA. This method is also useful for detecting the absence of influenza infection.

[0012] In some aspects, the present disclosure provides a method for identifying a target viral RNA from a sample suspected of containing the target viral RNA. The target viral RNA can be derived from any RNA virus selected for detection in the sample. In some aspects, the target viral RNA can be derived from a virus that causes a respiratory infection or establishes its primary infection in upper respiratory tissues and body fluids. 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 acid (sialic acid) on the surface of host cells including human tracheal epithelial cells and respiratory epithelial cells. Influenza A is a major cause of influenza pandemics. The target viral RNA can be RNA derived from any of 18 different subtypes of hemagglutinin and 11 different subtypes of neuraminidase of influenza.

[0013] In addition to influenza virus, the target viral RNA can be a common cold coronavirus such as NL63 strain, OC43 strain, or 229E strain. The target viral RNA can also be SARS-CoV-2, a hepatitis virus (e.g., HCV), or a respiratory syncytial virus (RSV). In some cases, the target viral RNA can be derived from human immunodeficiency virus (HIV). Thus, the method can be used to detect and identify combinations of viral RNAs using, for example, the methods and components described in any of International Application Publication Nos. WO2020 / 051452; WO2021 / 188830; and WO2022 / 046706, each of which is incorporated herein by reference in its entirety.

[0014] In some embodiments, incubating a mixture comprising a sample suspected of containing influenza RNA, a Cas13 protein, at least one CRISPR guide RNA (crRNA), and a reporter RNA for a period of time to form any reporter RNA cleavage products that may be present in the mixture; and detecting, with a detector, the level(s) of reporter RNA cleavage products that may be present in the mixture, to diagnose the presence or absence of influenza infection. In some cases, the influenza RNA and / or RNA cleavage products in the sample are not reverse transcribed prior to the detecting step. The presence or absence of influenza infection in a patient is detected by qualitatively or quantitatively detecting the level of reporter RNA cleavage products that may be present in the mixture.

[0015] The methods described herein may have various advantages. For example, the methods described herein can directly detect RNA without additional manipulation. RNA amplification is generally not required, whereas with currently available methods (e.g., SHERLOCK), it is necessary for the sensitivity of RNA amplification to be high enough. The methods, kits, and devices described herein are rapid and provide results within 30 minutes. Expensive laboratory equipment and specialized techniques are not required. The methods described herein are suitable for many different sample types (blood, nasal / oral swabs, etc.). The methods, kits, and devices described herein are easily deployable in the field (screening at airports, borders, resource-poor regions), such that people at risk of infection do not need to go to hospitals and clinics where there are non-infected patients, socially vulnerable people, and highly trained emergency healthcare workers in urgent need. Thus, testing enables isolation from facilities required for treating vulnerable people and isolation from trained personnel required for urgent and complex medical procedures.

[0016] CRISPR-Cas13 is a viable alternative to conventional methods for detecting and quantifying RNA by RT-PCR. The advantage of using CRISPR-Cas13 is that it can be utilized for influenza diagnosis. The Cas13 protein directly targets RNA and can be programmed with crRNA to provide a platform for specific RNA sensing. By binding the Cas13 protein to an RNA-based reporter, the secondary or non-specific RNase activity of the Cas13 protein can be exploited for influenza detection.

[0017] In 2017 and 2018, Dr. Feng Zhang's laboratory reported a Cas13-based detection system that achieved detection sensitivity at attomolar and zeptomolar concentrations of Zika virus, which included an additional reverse transcription step for isothermal amplification of Zika virus cDNA, which was ultimately back-transcribed to RNA for RNA-based detection by a method called SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing) (Gootenberg et al., Science, 356(6336):438-442 (2017), Gootenberg et al., Science, 360(6387):439-444 (2018)). This method improved the sensitivity of Cas13, but it introduced two undesirable steps involving reverse transcription and in vitro transcription that minimized its potential as a field-deployable and point-of-care device.

[0018] The present disclosure provides methods and compositions for diagnosing influenza infection, quantifying influenza RNA concentration, and identifying the presence of different influenza A subtypes and / or mutations.

[0019] In some cases, the method can be performed in a single tube, e.g., the same tube used for collection and RNA extraction. The method provides a single-step point-of-care diagnostic method. In other cases, the method can be performed in a two-chamber system. For example, a collection swab containing a biological sample can be directly inserted into chamber 1 of such a two-chamber system. After agitation, removal of the swab, and lysis of the biological material in the sample, the partition between the two chambers can be broken or removed, and the contents of the first chamber can be poured into the second chamber. The second chamber can contain Cas13 protein, a selected crRNA, and reporter RNA so that an influenza assay can be performed.

[0020] Chamber 1 can contain a buffer that facilitates lysis of viral particles and release of genomic material. Examples of lysis buffers that can be used include, but are not limited to, PBS, commercially available lysis buffers (e.g., Qiagen RLT+ buffer or Quick Extract™), DNA / RNA Shield, surfactants at various concentrations (e.g., Triton™ X-100, Tween® 20, NP-40, or Oleth™-8), or combinations of such reagents.

[0021] After agitation and subsequent removal of the swab, the chamber can be heated briefly (e.g., for 2-5 minutes) (e.g., to 55°C or 95°C) to further facilitate lysis. The partition between the two chambers is then broken or removed, and nasal extract buffer is poured into the second chamber to reconstitute this second chamber containing the lyophilized reagents (Cas13 RNP and reporter RNA molecules) for the Cas13 assay.

[0022] The use of such assay tubes can provide a single-step point-of-care diagnostic method and device. The methods, devices, and compositions described herein for diagnosing influenza infection comprise incubating a mixture having a sample suspected of containing influenza RNA, a Cas13 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 the level of any such reporter RNA cleavage products with a detector. The detector can be a fluorescence detector such as a short-chain quenched fluorescent RNA detector or a total internal reflection illumination fluorescence (TIRF) detector.

[0023] Reporter: A single type of reporter RNA can be used. The reporter RNA can be configured to produce a detectable signal upon cleavage by the Cas13 protein. For example, the reporter RNA can have a fluorophore at one position (e.g., one end) and a quencher at another position (e.g., the other end). In another example, the reporter RNA can have an electrochemical moiety (e.g., ferrocene or a dye) that can provide an electron transfer to a redox probe or a transducer upon cleavage by the Cas13 protein. In another example, the reporter RNA can have a reporter dye such that the reporter dye is detected by a detector (e.g., a spectrophotometer) upon cleavage of the reporter RNA. In some cases, one end of the reporter RNA can be bound to a solid surface. For example, the reporter RNA can be configured as a cantilever that emits a signal upon cleavage. However, in other cases, the signal can be improved by using unbound reporter RNA (e.g., not covalently bound to a solid surface). The surface of the assay container or assay material can have a detector for sensing the emission of the signal. The signal can be an optical 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 resonance frequency shift signal, or a combination thereof, or can include them.

[0024] The reporter RNA can be, for example, at least one quenched fluorescent RNA reporter. Such quenched fluorescent RNA reporters can optimize the detection of fluorescence. Quenched fluorescent RNA reporters include RNA oligonucleotides having both a fluorophore and a quencher of the fluorophore. The quencher reduces or eliminates the fluorescence of the fluorophore. When the Cas13 protein cleaves the RNA reporter, the fluorophore separates from the bound quencher, and as a result, a fluorescence signal becomes detectable.

[0025] An example of such a fluorophore quencher-labeled RNA reporter is RNaseAlert™ (IDT). RNaseAlert™ was developed to detect RNase™ contamination in the laboratory, and the substrate sequence is optimized for RNase A species. Another approach is to detect the FAM-biotin reporter using a lateral flow strip, which is detected by an anti-FAM antibody-gold nanoparticle conjugate on the strip when cleaved by Cas13. This enables detection without using equipment, but while most fluorescence-based assays take less than 30 minutes, the readout takes 90 - 120 minutes (Gootenberg et al., Science. 360(6387):439 - 44 (April 2018)).

[0026] The sequence of the reporter RNA can be optimized for Cas13 cleavage. Cas13 preferentially exerts RNase cleavage activity at exposed uridine or adenosine sites, depending on the Cas13 homolog. There is also a secondary preference for highly active homologs. The inventors of the present application tested 5-mer homopolymers for all ribonucleotides. Based on these selections, various RNA oligonucleotides labeled at the 5' and 3' ends of the oligonucleotide with Iowa Black® Quencher (IDT) and FAM fluorophore are systematically tested for these sequences in the trans-ssRNA cleavage assay as described in the examples. The best sequences can be transferred to mobile testing.

[0027] The fluorophore used in the RNA reporter labeled with a fluorophore quencher can include Alexa 430, STAR 520, Brilliant Violet™ 510, Brilliant Violet™ 605, Brilliant Violet™ 610, or combinations thereof.

[0028] Detection: Various mechanisms and devices can be used to detect fluorescence. In some cases, the detector is a fluorescence detector, optionally a short-chain quenched fluorescent RNA detector, or a total internal reflection illumination fluorescence (TIRF) detector. For example, the fluorescence detector can detect fluorescence from a fluorescent dye such as Alexa 430, STAR 520, Brilliant Violet™ 510, Brilliant Violet™ 605, Brilliant Violet™ 610, or combinations thereof.

[0029] To help remove background fluorescence, several mechanisms or devices can be used. For example, reducing fluorescence from outside the detection focal plane can improve the signal-to-noise ratio and, as a result, improve the resolution of the signal from the RNA cleavage product of interest. Total internal reflection fluorescence (TIRF) enables very low background fluorescence and single-molecule sensitivity using a sufficiently sensitive camera. In some cases, a mobile phone can be used for the detection of influenza.

[0030] In some cases, both Cas13 and reporter RNA can be tethered to a solid surface. When the crRNA and influenza RNA sample are added, the activated Cas13 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 the reporter RNA is similarly tethered to the solid surface so that cleavage allows the quencher portion of the reporter RNA to diffuse. The Cas13 protein can be tethered to the solid surface with a tether long enough to allow it to cleave multiple RNA reporter molecules. The viral load can be quantified by counting the bright spots that appear on the solid surface. The use of TIRF in a portable system facilitates detection and reduces background, such that the RNA cleavage product signal can be easily detected.

[0031] In some cases, the ribonucleoprotein (RNP) complex of Cas13 protein and crRNA can be tethered to a solid surface. Then, the crRNA does not need to be added later. Instead, only the sample suspected of containing influenza RNA needs to be contacted with the solid surface.

[0032] In some cases, the methods described herein may include direct detection of target RNA in a sample without performing additional sample preparation steps prior to detection (e.g., depleting a portion of a sample of protein, enzyme, lipid, nucleic acid, or a combination thereof, or inactivating nucleases). However, the methods described herein may include depleting a portion of the sample prior to other step(s) or inhibiting nucleases in the sample prior to other step(s). For example, a sample can be depleted of protein, enzyme, lipid, nucleic acid, or a combination thereof. In some cases, the portion of the sample that is depleted is the human nucleic acid portion. However, RNA extraction of the sample is preferably not performed.

[0033] In some cases, the method may include removing ribonuclease (RNase) from the sample. In some cases, RNase is removed from the sample using an RNase inhibitor and / or heat.

[0034] In some cases, the Cas13 protein and / or crRNA are lyophilized prior to incubation with the sample. In some cases, the Cas13 protein, crRNA, and / or reporter RNA are lyophilized prior to incubation with the sample.

[0035] Sample: In some embodiments, the biological sample is isolated from a patient. Non-limiting examples of suitable biological samples include saliva, sputum, mucus, nasopharyngeal samples, blood, serum, plasma, urine, aspirates, and biopsy samples. Thus, the term "sample" with respect to a patient can include RNA. Biological samples include saliva, sputum, mucus, and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny. This definition also includes samples that have been manipulated in some way, such as treatment with reagents, washing, or enrichment of a particular cell population, after they have been procured. This definition also includes samples in which a particular type of molecule, such as RNA, has been enriched. The term "sample" includes biological samples such as clinical samples such as saliva, sputum, mucus, nasopharyngeal samples, blood, plasma, serum, aspirates, cerebrospinal fluid (CSF), and also includes tissues obtained by surgical resection, tissues obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, and the like. "Biological samples" include biological fluids and / or viruses (e.g., obtained from infected cells) derived from cells. Samples containing RNA can be obtained from such cells (e.g., cell lysates, or other cell extracts containing RNA). A sample can comprise or be obtained from any of a variety of body fluids (e.g., saliva, mucus, or sputum), cells, tissues, organs, or cell-free fluids.

[0036] In some embodiments, the biological sample is isolated from a patient known or suspected to have an influenza infection. In other embodiments, the biological sample is isolated from a patient not known to have an influenza infection. In other embodiments, the biological sample is isolated from a patient known or suspected to have or not have an influenza infection. In other words, the methods and devices described herein can be used to identify a subject having an influenza infection and to confirm that the subject does not have an influenza infection.

[0037] In some cases, it may not be known whether a biological sample contains RNA. However, such biological samples can still be tested using the methods described herein. For example, a biological sample can be subjected to lysis, RNA extraction, incubation with Cas13 and crRNA, regardless of whether the sample actually contains RNA and whether the sample contains influenza RNA.

[0038] Pre-incubation of crRNA and Cas13 protein without the sample is preferred so that the crRNA and Cas13 protein can form a complex. In some cases, reporter RNA may be present while the crRNA and Cas13 protein form a complex. However, in other cases, reporter RNA can be added after the crRNA and Cas13 protein have already formed a complex. Also, after formation of the crRNA / Cas13 complex, sample RNA (e.g., influenza RNA) can then be added. The sample RNA (e.g., influenza RNA) acts as the activating RNA. When activated by the activating RNA, the crRNA / Cas13 complex becomes a non-specific RNase and produces RNA cleavage products that can be detected using a reporter RNA, e.g., a short-strand quenched fluorescent RNA.

[0039] For example, Cas13 and crRNA are incubated for a period of time to form an inactive complex. In some cases, the Cas13 and crRNA complex is formed by incubating together at 37°C for 30 minutes, 1 hour, or 2 hours (e.g., 0.5 - 2 hours) to form an inactive complex. Then, the inactive complex can be incubated with reporter RNA. An example of reporter RNA is provided by the RNase Alert™ system. The sample influenza RNA can be an ssRNA activator. The Cas13 / crRNA with the influenza RNA sample becomes an activated complex that cleaves in cis and in trans. When cleaving in cis, for example, the activated complex can cleave the influenza RNA. When cleaving in trans, the activated complex can cleave the reporter RNA, thereby releasing a signal such as a fluorophore from the reporter RNA.

[0040] CRISPR guide RNA (crRNA): The CRISPR guide RNA system can be adapted for use in the methods and compositions described herein. The guide RNA 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), which is a binding scaffold for the Cas nuclease. In some cases, the two RNAs are fused to create a single guide RNA (sgRNA). The tracrRNA forms a stem-loop that is recognized and bound by the Cas nuclease. As used herein, the term "guide RNA" refers to either a single guide RNA (sgRNA) or a crRNA (spacer). The CRISPR technology is generally described, for example, in Mali et al., Science, Vol. 339: p. 823 - 6 (2013), which is incorporated herein by reference in its entirety.

[0041] In some cases, at least one CRISPR guide RNA (crRNA) has a sequence having at least 95% sequence identity with 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 may include those having SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, 32, or 34-36, or combinations thereof. In some cases, a sample can be incubated with one or more crRNAs. For example, the sample can be incubated with at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 9, or at least 10, or more crRNAs. In some cases, 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 with any of SEQ ID NOs: 1-37.

[0042] In various examples of crRNAs that can be used for the detection of influenza B, the crRNA may include those having SEQ ID NO: 32, 34, 35, 36, or combinations thereof. In some cases, SEQ ID NOs: 34 and 36 can be combined to improve the detection of influenza B.

[0043] In various examples of crRNAs that can be used for the detection of influenza A, the crRNA may include those having SEQ ID NO: 4, 8, 13, 16, 17, 21, or 22, or combinations thereof. In some cases, the crRNA may include those having SEQ ID NO: 8, 16, 21 or 22, or combinations thereof.

[0044] The amount of reporter RNA cleavage product detected is directly correlated with the amount of target viral RNA. In some cases, the concentration of the target viral RNA cleavage product can be quantified or determined by use of a standard curve of the reporter RNA cleavage product.

[0045] 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 within a region of the influenza genome that has low viral ribonucleoprotein binding.

[0046] In some cases, the crRNA can include additional sequences such as spacer sequences. Table 1 provides examples of influenza crRNA sequences.

[0047]

Table 1-1

[0048]

Table 1-2

[0049] As exemplified herein, for the detection of influenza B, crRNAs having the sequences of SEQ ID NOs: 32, 34, 35, 36 show better signals than crRNAs having the sequences of SEQ ID NOs: 23-31, 33, or 37. Further, the combination of the crRNAs of SEQ ID NOs: 34 and 36 significantly improves the detection of influenza B as compared to using the crRNAs of SEQ ID NO: 34 or 36 alone.

[0050] To detect influenza A, crRNAs having the sequences of SEQ ID NO: 4, 8, 13, 16, 17, 21, or 22 show better signals than crRNAs having the sequences of SEQ ID NO: 1-3, 5-7, 9-12, 14, 15, or 18-20. Further, a combination of seven crRNAs of SEQ ID NO: 4, 8, 13, 16, 17, 21, and 22, and, independently, a combination of four crRNAs of SEQ ID NO: 8, 16, 21, or 22 significantly improve the detection of influenza A over using the crRNAs of SEQ ID NO: 4, 8, 13, 16, 17, 21, or 22 alone.

[0051] Influenza sequence The influenza A genome having coding regions for each of eight single-stranded RNA segments, the DNA sequence for the H1N1 strain, is available under the following accession numbers: Segment 1: NC_002023.1 from the NCBI website (provided herein as SEQ ID NO: 51).

[0052]

Chem.

[0053]

Chem.

[0054] Segment 2: NC_002021.1 from the NCBI website (provided herein as SEQ ID NO: 52).

[0055]

Chem.

[0056]

Chem.

[0057] Segment 3: NC_002022.1 from the NCBI website (provided as SEQ ID NO: 53 in this specification).

[0058]

Chemical formula

[0059]

Chemical formula

[0060] Segment 4: NC_002017.1 from the NCBI website (provided as SEQ ID NO: 54 in this specification).

[0061]

Chemical formula

[0062] Segment 5: NC_002019.1 from the NCBI website (provided as SEQ ID NO: 55 in this specification).

[0063]

Chemical formula

[0064] Segment 6: NC_002018.1 from the NCBI website (provided as SEQ ID NO: 56 in this specification).

[0065]

Chemical formula

[0066] Segment 7: NC_002016.1 from the NCBI website (provided as SEQ ID NO: 57 in this specification).

[0067]

Chemical formula

[0068] Segment 8: NC_002020.1 from the NCBI website (provided herein as SEQ ID NO: 58).

[0069]

Chemical formula

[0070] The DNA sequences for the B - type influenza virus genome, Bisbane strain, each having a coding region for each of the eight single - stranded RNA segments, are available under the following accession numbers.

[0071] Segment 1: CY018707.1 from the NCBI website (provided herein as SEQ ID NO: 59).

[0072]

Chemical formula

[0073]

Chemical formula

[0074] Segment 2: CY018708.1 from the NCBI website (provided herein as SEQ ID NO: 60).

[0075]

Chemical formula

[0076]

Chemical formula

[0077] Segment 3: CY018706.1 from the NCBI website (provided herein as SEQ ID NO: 61).

[0078]

Chem.

[0079]

Chem.

[0080] Segment 4: CY018701.1 from the NCBI website (provided herein as SEQ ID NO: 62).

[0081]

Chem.

[0082]

Chem.

[0083] Segment 5: CY018704.1 from the NCBI website (provided herein as SEQ ID NO: 63).

[0084]

Chem.

[0085]

Chem.

[0086] Segment 6: CY018703.1 from the NCBI website (provided herein as SEQ ID NO: 64).

[0087]

Chem.

[0088] Segment 7: CY018702.1 from the NCBI website (provided herein as SEQ ID NO: 65).

[0089]

Chem.

[0090] Segment 8: CY018705.1 from the NCBI website (provided herein as SEQ ID NO: 66).

[0091]

Chem.

[0092] The influenza virus genome is RNA. Thus, in some cases, the influenza virus genome can be a copy of the aforementioned DNA sequence where the thymine (T) residues are uracil (U) residues. In some cases, the influenza virus genome can be the complement of the aforementioned DNA sequence.

[0093] However, the influenza virus genome can also have sequence variations. For example, the influenza virus genome can be for various influenza strains that include the aforementioned sequences for any of the 18 different subtypes of hemagglutinin (HA) and 11 different subtypes of neuraminidase (NA) of influenza A, such as the H1N1 strain or other strains like H3N2. Variations in influenza B virus can be any of the B / Lee / 1940 strain, B / Brisbane / 60 / 2008 strain, B / Victoria / 504 / 2000 strain, or other strains.

[0094] Array determination has confirmed that influenza viruses share a common genetic ancestor; however, they are genetically branched, and as a result, 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 the standard nomenclature for influenza viruses, including the virus type; the species of origin (in the case of non - human); the location where isolated; the isolate number; the year of isolation; and, in the case of influenza A viruses only, the HA and NA subtypes. In influenza A and B viruses, genome segments 1, 3, 4, and 5 encode only one protein per segment: the PB2, PA, HA, and NP proteins. All influenza viruses encode the polymerase subunit PB1 of segment 2; in some strains of influenza A viruses, this segment also encodes, in the +1 alternative reading frame, the accessory protein PB1 - F2, a small 87 - amino - acid protein with pro - apoptotic activity. Analogs to PB1 - F2 have not been identified in influenza B or C viruses. Conversely, segment 6 of influenza A viruses encodes only the NA protein, while segment 6 of influenza B viruses encodes both the NA protein and the NB matrix protein in the - 1 alternative reading frame. This NB matrix protein is an integral membrane protein corresponding to the M2 protein of influenza A viruses. Segment 7 of both influenza A and B viruses encodes the M1 matrix protein. In the influenza A genome, the M2 ion channel is also expressed from segment 7 by RNA splicing, while influenza B viruses encode their BM2 membrane protein in the +2 alternative reading frame.Finally, both influenza A and B viruses have segment 8, a single RNA segment, from which they express the interferon antagonist NS1 protein and, by mRNA splicing, NEP / NS2, which is involved in the export of viral RNPs from the host cell nucleus. The genomic organization of influenza C virus is generally similar to that of influenza A and influenza B viruses; however, the HEF protein of influenza C virus replaces the HA and NA proteins, and thus the influenza C virus genome has one fewer segment than the genomes of influenza A or influenza B viruses.

[0095] Cas13 protein: Any suitable CRISPR-associated RNA-targeting endonuclease, such as a Cas13 protein variant, can be used in the methods and compositions described herein. The Cas13 protein can form a complex with at least one CRISPR guide RNA (crRNA) and at least one reporter RNA for a time sufficient to form at least one RNA cleavage product.

[0096] The Cas13 protein can be, for example, a Cas13a protein, a Cas13b protein, or a combination thereof. Cas13 has two higher eukaryotic and prokaryotic nucleotide binding (HEPN) domains for RNA cleavage, which is consistent with the known role of the HEPN domain in other proteins. In some embodiments, the Cas13 protein can have sequence variations and / or can be derived from other organisms. For example, the Cas13 protein 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 aforementioned Cas13 sequences, or to the Cas13 of 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.

[0097] For example, the Cas13a endonuclease of Leptotrichia wadei having the following sequence (SEQ ID NO: 38; NCBI accession number WP_036059678.1) can be used.

[0098]

Chem.

[0099] Other sequences of the Cas13a endonuclease of Leptotrichia wadei, such as the sequences of NCBI accession numbers BBM46759.1, BBM48616.1, BBM48974.1, BBM48975.1, and WP_021746003.1 are also available.

[0100] In another example, the Cas13a endonuclease of Herbinix hemicellulosilytica having the following sequence (SEQ ID NO: 39; NCBI accession number WP_103203632.1) can be used.

[0101]

Chem.

[0102] However, in some cases, the Cas13 protein having the sequence of SEQ ID NO: 39 is not used. In another example, the Cas13a endonuclease of Leptotrichia buccalis having the following sequence (SEQ ID NO: 40; NCBI accession number WP_015770004.1) can be used.

[0103]

Chem.

[0104] However, in some cases, the Cas13 protein having the sequence of SEQ ID NO: 40 is not used. In another example, the Cas13a endonuclease of Leptotrichia seeligeri having the following sequence (SEQ ID NO: 41; NCBI accession number WP_012985477.1) can be used.

[0105]

Chem.

[0106] For example, the Cas13a endonuclease of Pardiubacter propionicigenes having the following array (array number 42; NCBI accession number WP_013443710.1) can be used.

[0107]

Chem.

[0108] For example, the Cas13a endonuclease of bacteria of the family Ruminospirillaceae having the following array (array number 43; NCBI accession number WP_022785443.1) can be used.

[0109]

Chem.

[0110] For example, the Cas13a endonuclease of Leptotrichia shahii having the following amino acid sequence (array number 44; NCBI accession number BBM39911.1) can be used.

[0111]

Chem.

[0112] In another example, the Cas13a endonuclease of Leptotrichia buccalis C-1013-b having the following amino acid sequence (array number 45; NCBI accession number C7NBY4; alias LbuC2c2) can be used.

[0113]

Chem.

[0114] The inventors of the present application evaluated the kinetics of other Cas13a and Cas13b proteins. Such studies have shown that, in some cases, Cas13b functions faster than Cas13a in target viral RNA detection assays.

[0115] For example, Cas13b from Prevotella buccae can be used in influenza RNA detection methods, compositions, and devices. The amino acid sequence of the Cas13b protein of Prevotella buccae (NCBI accession number WP_004343973.1) is shown below as SEQ ID NO: 46.

[0116]

Chemical formula

[0117] Such a Cas13b protein of Prevotella buccae can have a Km (Michaelis constant) substrate concentration of about 20 micromolar and a Kcat of about 987 / second (see, for example, Slaymaker et al., Cell Rep, Vol. 26(13): p. 3741 - 3751 (2019)).

[0118] Another Cas13b protein of Prevotella buccae (NCBI accession number WP_004343581.1) that can be used in SARS-CoV-2 RNA detection methods, compositions, and devices has the amino acid sequence shown below as SEQ ID NO: 47.

[0119]

Chemical formula

[0120] An example of the Cas13b (R1177A) mutant amino acid sequence of Bergeyella zoohelcum (NCBI accession number 6AAY_A) is shown below as SEQ ID NO: 48.

[0121]

Chem.

[0122] Another example of the Cas13b protein sequence (NCBI accession number WP_007412163.1) from Prevotella sp. MSX73 that can be used in a target viral RNA detection method, composition, and device is shown below as SEQ ID NO: 49.

[0123]

Chem.

[0124] Thus, the sample can be incubated with at least one CRISPR RNA (crRNA) and at least one Cas13 protein. The Cas13 protein can be, for example, a Cas13a protein, a Cas13b protein, or a combination thereof.

[0125] (CRISPR) / CRISPR-associated (Cas) system Genome editing has been carried out by using the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system (see, for example, Marraffini and Sontheimer, Nature Reviews Genetics, Vol. 11: p. 181-190 (2010); Sorek et al., Nature Reviews Microbiology, 2008, Vol. 6: p. 181-6; Karginov and Hannon, Mol Cell, 2010, Vol. 1: p. 7-19; Hale et al., Mol Cell, 2010: Vol. 45: p. 292-302; Jinek et al., Science, 2012, Vol. 337: p. 815-820; Bikard and Marraffini, Curr Opin Immunol, 2012, Vol. 24: p. 15-20; Bikard et al., Cell Host & Microbe, 2012, Vol. 12: p. 177-186, all of which are incorporated herein by reference in their entirety).

[0126] However, the CRISPR guide RNA system can be adapted for use in the methods and compositions described herein. Two RNAs can be used in the CRISPR genome editing system: CRISPR RNA (crRNA), which is a 17- to 20-nucleotide sequence complementary to the target RNA, and trans-activating crRNA (tracrRNA), which serves as a scaffold for binding to the Cas nuclease. In some cases, the two RNAs are fused to create a single guide RNA (sgRNA). The tracrRNA forms a stem-loop that is recognized and bound by the Cas nuclease. The crRNA typically has a shorter sequence than the tracrRNA. The term "guide RNA" as used herein refers to a single guide RNA (sgRNA) or a crRNA. The CRISPR technology is generally described, for example, by Mali et al., Science, Vol. 339: pp. 823-6 (2013), which is hereby incorporated by reference in its entirety.

[0127] The guide RNA system used herein is encoded within or adjacent to the ncRNA coding region of the expression cassette. Therefore, when the guide RNA is transcribed, it can direct the Cas enzyme to a desired location in the genome, where the Cas enzyme can cleave the genomic RNA and induce genomic modification.

[0128] There are several types of CRISPR systems, some of which are summarized in the following table.

[0129]

Table 2

[0130] As used herein, "guide RNA" or "gRNA" refers to a ribonucleotide sequence that can bind to a Cas nuclease, thereby forming a ribonucleoprotein complex. The gRNA includes a nucleotide sequence complementary to a target site (e.g., a genomic site to be edited or near thereto). Optionally, the guide RNA includes one or more RNA molecules. The tracrRNA can be used to facilitate the assembly of a ribonucleoprotein complex that includes the gRNA together with the tracrRNA and the Cas nuclease. The complementary nucleotide sequence of the guide RNA mediates the binding of the ribonucleoprotein complex to the target site, thereby providing sequence specificity to the ribonucleoprotein complex. Accordingly, the guide RNA includes a sequence that is complementary to a target nucleic acid sequence such that the guide RNA binds to the target nucleic acid sequence.

[0131] Optionally, the complementary region of the guide RNA includes a sequence having about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a target nucleic acid (e.g., a target viral RNA sequence). Optionally, the guide RNA includes a sequence having about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a target nucleic acid sequence. Optionally, the guide RNA or its complementary region includes a sequence having at least about 90%, 95% or 100% sequence identity to a target viral RNA sequence. Optionally, the segment to which the guide RNA binds within the target nucleic acid is at least about 10, 15, 20, 25 or more nucleotides in length.

[0132] The guide RNA is a single-stranded ribonucleic acid, but in some cases, the guide RNA 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 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 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 10 to 15, 10 to 20, 10 to 30, 10 to 40 or 10 to 50 residues in length.

[0133] Definition As used herein, the term "about", when referring to measurable values such as amounts, lengths and the like, is intended to encompass variations of ±20%, ±10%, more preferably ±5%, still more preferably ±1%, and even more preferably ±0.1% from a given value.

[0134] As used herein, "recombinant" when used 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 a polynucleotide that is associated in nature.

[0135] The term "recombinant" when used with respect to a protein or polypeptide means a polypeptide produced by the expression of a recombinant polynucleotide. Generally, a polynucleotide of interest is cloned and then expressed in a transformed organism, as described herein, for example. The host organism expresses the foreign nucleic acid under expression conditions to produce RNA, RT-DNA or protein.

[0136] As used herein, "cell" refers to any type of cell isolated from organisms such as prokaryotes, eukaryotes or archaebacteria, including bacteria, archaebacteria, fungi, protists, plants and animals, and includes cells from tissues, organs and biopsy materials, as well as recombinant cells, cells from cell lines cultured in vitro, and cell fragments, cell components or organelles containing 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 containing a single cell or a population of cells. The term also includes genetically modified cells.

[0137] "Recombinant host cell", "host cell", "cell", "cell line", "cell culture", and other such terms that mean a higher eukaryotic cell line cultured as a microorganism or single cell organism refer to cells that can or have been able to serve as recipients of recombinant vectors or other introduced DNA, including the original progeny of the original transfected cells.

[0138] A "coding sequence" or sequence that "encodes" a selected polypeptide or selected RNA is a nucleic acid molecule that is transcribed in vivo to RNA (in the case of a DNA template) and / or translated to a polypeptide (in the case of mRNA) when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, ncRNA, tracrRNA, ncRNA modified to contain heterologous sequences, viruses, prokaryotic or eukaryotic ncRNA, cDNA derived from mRNA, viral or prokaryotic DNA, and also synthetic DNA sequences. Transcription termination sequences may be located 3' to the coding sequence.

[0139] Typical "control elements" include, but are not limited to, a transcription promoter, a transcription enhancer element, a transcription termination signal, a polyadenylation sequence (located 3' to the translation stop codon), a sequence for optimization of translation initiation (located 5' to the coding sequence), and a translation termination sequence.

[0140] "Operably linked" refers to an arrangement of elements so configured that the components so described perform their normal functions. Thus, a particular promoter operably linked to a coding sequence can effect the expression of the coding sequence when an appropriate polymerase is present. The promoter need not be contiguous with the coding sequence so long as it functions to direct the expression of the coding sequence. Thus, for example, intervening non-translated but 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.

[0141] "Encoded by" refers to a nucleic acid sequence that codes for a polypeptide or an RNA sequence. For example, a 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, even more preferably at least 15 to 20 amino acids, derived from a polypeptide encoded by a nucleic acid sequence. An 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, even more preferably at least 15 to 20 nucleotides.

[0142] The terms "isolated," "purified," or "biologically pure" refer to a material that, to some degree, lacks the components normally associated with it as seen in its natural state. "Isolating" means a certain degree of separation from its original source or environment. "Purifying" means a greater degree of separation than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials so that any impurities do not significantly affect the biological properties of the protein, DNA, or RNA or cause other adverse effects. That is, the nucleic acids or peptides of the present invention are purified when obtained from nature or produced by recombinant DNA techniques if substantially free of cellular material, viral material, or culture medium, or when chemically synthesized if free of chemical precursors or other chemicals. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein yields essentially one band in an electrophoretic gel. For proteins that can be modified, such as by phosphorylation or glycosylation, various modifications may yield various isolated proteins, which can be purified separately.

[0143] "Substantially purified" generally refers to the isolation of a substance (nucleic acid, compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance constitutes the majority of the sample in which it is present. Typically, a substantially purified component in a sample constitutes 50% of the sample, preferably 80% - 85%, more preferably 90 - 95%. 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 by density.

[0144] A "vector" is capable of introducing a nucleic acid sequence into a target cell (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector construct", "expression vector", and "gene delivery vector" mean any nucleic acid construct that can direct the expression of a nucleic acid of interest and can introduce a nucleic acid sequence into a target cell. Thus, the term includes cloning vehicles and expression vehicles as well as viral vectors.

[0145] "Expression" refers to the detectable production of a gene product by a cell. Depending on the context, the gene product may be a transcript (i.e., RNA), which may also be referred to as "gene expression", or the gene product may be a translation product of the transcript (i.e., protein).

[0146] "Mammalian cell" refers to any cell derived from a mammalian subject that is suitable for transfection by a vector system as described herein. The cells may be heterologous, autologous, or homologous. The cells can be primary cells obtained directly from a mammalian subject. The cells may also be cells derived from the culture and expansion of cells obtained from a mammalian subject. Immortalized cells are also included within this definition. In some embodiments, the cells are genetically engineered to express recombinant proteins and / or nucleic acids.

[0147] The term "subject" includes animals, including both vertebrates and invertebrates, without limitation, invertebrates such as arthropods, mollusks, annelids, and cnidarians; and amphibians including frogs, salamanders, and newts; reptiles including lizards, snakes, turtles, crocodiles, and alligators; fish; mammals including human mammals and non-human mammals such as chimpanzees, other apes, and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; pets such as dogs and cats; livestock such as sheep, goats, pigs, horses, and cows; and birds including chickens, turkeys, and other game birds, pet birds, wild birds, and avian species such as ducks, geese, and the like. In some cases, the disclosed methods find use in veterinary applications and in the development of animal models for diseases in rodents including mice, rats, and hamsters; laboratory animals including primates and transgenic animals, among others.

[0148] "Gene transfer" or "gene delivery" refers to a method or system for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated introduced DNA, extrachromosomal replication and expression of an introduced replicon (e.g., episome), or integration of the introduced genetic material into the genomic DNA of the host cell. Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non-viral vectors, alphaviruses, poxviruses, and vaccinia viruses.

[0149] The term "derived from" is used herein to identify the original source of a molecule, but is not intended to limit the ways in which the molecule can be made, for example, by chemical synthesis or recombinant means.

[0150] A polynucleotide or nucleic acid "derived from" a specified array refers to a polynucleotide or nucleic acid containing a contiguous sequence of approximately at least 6 nucleotides, preferably at least 8 nucleotides, more preferably at least 10 - 12 nucleotides, and even more preferably at least 15 - 20 nucleotides that corresponds to, i.e., is identical or complementary to, a region of the specified nucleotide sequence. The derived polynucleotide is not necessarily materially derived from the nucleotide sequence of interest, but may be generated by any method including, but not limited to, chemical synthesis, replication, reverse transcription, or transcription, based on the information provided by the sequence of bases in the region from which the polynucleotide is derived. Thus, the polynucleotide may indicate the sense or antisense orientation of the original polynucleotide.

[0151] The terms "hybridize" and "hybridization" refer to the formation of a complex between nucleotide sequences that are sufficiently complementary to form a complex via Watson - Crick base pairing.

[0152] The term "homologous region" refers to a region of a nucleic acid that has homology to another nucleic acid region. Thus, whether a "homologous region" exists in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since nucleic acids are 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 that can hybridize to each other. Thus, the term "homologous region" includes nucleic acid segments having complementary sequences. The homologous region may vary in length but is typically between 4 and 500 nucleotides (e.g., about 4 - about 40, about 40 - about 80, about 80 - about 120, about 120 - about 160, about 160 - about 200, about 200 - about 240, about 240 - about 280, about 280 - about 320, about 320 - about 360, about 360 - about 400, about 400 - about 440, etc.).

[0153] As used herein, the terms "complementary" or "complementarity" refer to polynucleotides that can form base pairs with each other. Base pairs are typically formed by hydrogen bonds between nucleotide units in an antiparallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair according to the Watson-Crick rules (e.g., A pairs with T, A pairs with U, C pairs with G) or any other rule that allows for duplex formation. As recognized by those skilled in the art, when using RNA relative to DNA, uracil (U), rather than thymine (T), is the base considered to be complementary to adenosine. However, when uracil is recited with respect to the present invention, it is implied that thymine can be substituted unless otherwise specified. "Complementarity" may exist between two RNA strands, between two DNA strands, or between an RNA strand and a DNA strand. Despite having incomplete or less than 100% complementarity, two or more polynucleotides may be "complementary" and are generally understood to be able to form duplexes. Two sequences are "fully complementary" or "100% complementary" when at least the continuous portions of each polynucleotide sequence that contain regions of complementarity base pair perfectly with the other polynucleotide without any mismatches or breaks within such regions. Two or more sequences are considered "fully complementary" or "100% complementary" as long as the continuous regions of complementarity within each polynucleotide can hybridize perfectly with the other polynucleotide, even if one or both of the polynucleotides contain other non-complementary sequences. "Incomplete" complementarity refers to a situation where not all, but some, of the consecutive nucleotides within such regions of complementarity can base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is routine in the art.

[0154] The term "donor polynucleotide" or "donor DNA" refers to a nucleic acid or polynucleotide that provides the intended edit to a nucleotide sequence so as to be integrated into the genome at the target locus by HDR or recombination.

[0155] A "target site" or "target sequence" is a nucleic acid sequence recognized by the homology arms of a guide RNA (gRNA) or donor polynucleotide (donor DNA) (i.e., sufficiently complementary for hybridization). The target site may be allele-specific (e.g., major or minor allele). For example, the target site can be a genomic site intended to be modified by, for example, insertion of one or more nucleotides, replacement of one or more nucleotides, deletion of one or more nucleotides, or combinations thereof.

[0156] Generally, a "CRISPR system" generically refers to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, and includes a sequence encoding a Cas gene and a CRISPR array nucleic acid sequence comprising a leader sequence and at least one repeat sequence. In some embodiments, one or more elements of the CRISPR system are derived from a type I, type II, or type III CRISPR system. Cas1 and Cas2 are found in all three types of CRISPR-Cas systems and are involved in spacer acquisition. In the I-E system of Escherichia coli (E. coli), Cas1 and Cas2 form a complex, and the Cas2 dimer bridges two Cas1 dimers. In this complex, Cas2 serves as a non-enzymatic scaffold material, binds to the double-stranded fragment of the invading DNA, while Cas1 binds to the single-stranded flank of this DNA and catalyzes their integration into the CRISPR array.

[0157] In some embodiments, one or more elements of the CRISPR system are derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes. Generally, 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 with respect to an endogenous CRISPR system).

[0158] In certain embodiments, the disclosure provides a protospacer that is adjacent to a short (3-5 bp) DNA sequence called a protospacer adjacent motif (PAM). The PAM is important for type I and type II systems during acquisition. In type I and type II systems, the protospacer is excised at a position adjacent to the PAM sequence, and the other end of this spacer is cut using a ruler mechanism, thus maintaining no variation in spacer size in the CRISPR array. The management of the PAM sequence varies between CRISPR-Cas systems and may be evolutionarily related to Casl and the leader sequence.

[0159] In some embodiments, the regulatory element is operably linked to one or more elements of the CRISPR system so as to drive the expression of one or more elements of the CRISPR system. Generally, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is also known as SPIDR (Spacer Interspersed Direct Repeats) and typically constitutes a family of DNA loci that are specific to a particular bacterial species. The CRISPR locus contains discrete classes of short sequence repeats (SSRs) that were recognized in Escherichia coli (Ishino et al., J. Bacteriol., Vol. 169: pp. 5429-5433 (1987); and Nakata et al., J. Bacteriol., Vol. 171: pp. 3553-3556 (1989)) as well as related genes. Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (see Groenen et al., Mol. Microbiol., Vol. 10: pp. 1057-1065 (1993); Hoe et al., Emerg. Infect. Dis., Vol. 5: pp. 254-263 (1999); Masepohl et al., Biochim. Biophys. Acta, Vol. 1307: pp. 26-30 (1996); and Mojica et al., Mol. Microbiol., Vol. 17: pp. 85-93 (1995)). This CRISPR locus typically has a repeat structure called short regularly spaced repeats (SRSRs) that differs from other SSRs (Janssen et al., OMICS J. Integ. Biol., Vol. 6: pp. 23-33 (2002); and Mojica et al., Mol. Microbiol., Vol. 36: pp. 244-246 (2000)). Generally, the repeats areShort elements that exist in clusters regularly spaced by a unique intervening array of substantially constant length (Mohr et al., 2000, supra). Repeat arrays are highly conserved among strains, but the number of interspersed repeats and the sequence of spacer regions typically differ between strains (van Embden et al., J. Bacteriol., Vol. 182: p. 2393-2401 (2000)). CRISPR loci have been identified in over 40 prokaryotes (see, for example, Janssen et al., Mol. Microbiol., Vol. 43: p. 1565-1575 (2002); and Mohr et al. (2005)), including Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Haloarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas,including, but not limited to, Desulfovibrio, Geobacter, Myrococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.,

[0160] In some embodiments, the enzyme coding sequence encoding a CRISPR enzyme (e.g., cas9) is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be or may be derived from the cells of a particular organism, such as a mammal including, but not limited to, humans, mice, rats, rabbits, dogs or non-human primates. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence (e.g., about 1 or about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with a codon that is more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species show a particular bias for particular codons of a particular amino acid. Codon bias (the difference in codon usage frequency between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), and the efficiency of translation is thought to depend, inter alia, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The prevalence of selected tRNAs in a cell generally reflects the codons that are most frequently used in peptide synthesis. Thus, genes can be adjusted to suit optimal gene expression in a particular organism based on codon optimization. Codon usage tables are readily available, for example, in the "Codon Usage Database", and these tables can be adapted in many ways. See Nakamura Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000", Nucl. Acids Res., Vol. 28: p. 292 (2000). Computer algorithms are also available for codon-optimizing a particular sequence for expression in a particular host cell, and Gene Forge (Aptagen; Jacobus, Pennsylvania) is also available, among others.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 the sequence encoding the CRISPR enzyme correspond to the codons most frequently used for a particular amino acid.

[0161] "Administering" a nucleic acid such as an expression cassette includes transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or methods by shock, i.e., any means capable of transporting the nucleic acid across the cell membrane.

[0162] The subject matter disclosed herein is not limited to the specific embodiments described and may, therefore, be subject to change. It should also be understood that the technical terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims.

[0163] Where a range of values is provided, unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of that range to one tenth of the unit of the lower limit, and any other stated value or intervening value in the stated range, is included within the scope of the disclosed subject matter. The upper and lower limits of these smaller ranges may be included within the smaller ranges respectively and further within the scope of the disclosed subject matter, provided that any limit values in the stated range are clearly excluded. Where the stated range includes one or both of the limit values, ranges excluding one or both of the included limit values are also included within the disclosed subject matter.

[0164] Unless otherwise defined, 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 described herein. 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.

[0165] As used in this specification and the appended claims, unless the context clearly dictates otherwise, note that the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a cell" includes reference to a plurality of such cells, reference to "a nucleic acid" includes reference to one or more nucleic acids and equivalents thereof known to those of ordinary skill in the art, and so forth. It should further be noted that the claims may be drafted to exclude optional elements. Thus, this description serves as antecedent basis for the use of exclusive terms such as "solely," "only," and other like terms in connection with the recitation of any feature or element described herein, including the use of "negative" limitations.

[0166] It is recognized that certain features of the disclosed subject matter, which are described with respect to separate embodiments for clarity, may be provided in combination in a single embodiment. Conversely, various features of the disclosed subject matter, which are described with respect to a single embodiment for brevity, may also be provided separately or in any suitable sub-combination. All combinations of embodiments of the present disclosure are clearly encompassed by the disclosed subject matter and are disclosed herein as if each combination were individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and their elements are also clearly encompassed by the present disclosure and are disclosed herein as if each such sub-combination were individually and explicitly disclosed herein.

[0167] 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 by virtue of prior invention. Further, the dates of the publications provided may be different from the actual publication dates which may need to be independently confirmed.

[0168] The following examples illustrate some of the materials, methods, and experiments used or performed in the development of the present invention.

Example

[0169] Example 1: Detection of SARS-CoV-2 Transcripts by Cas13a CRISPR RNA guides (crRNAs) were designed and validated for influenza A, strains H1N2 and H3N2, and influenza B. Twenty-two crRNAs were designed for influenza A and 15 crRNAs were designed for influenza A. Each crRNA contains a crRNA stem derived from a bacterial sequence and the spacer sequence is derived from the influenza genome (reverse complementary region). See Table 1 (reproduced below) for the crRNA sequences.

[0170]

Table 3-1

[0171]

Table 3-2

[0172] Figures 4A - C show the detection of influenza strains by specific RNA guides of Table 1. The RNA guides were tested against H1N1, H3N2, FluB target viral RNAs, and a ribonucleoprotein (RNP) background control without target viral RNA. As shown in Figure 4A, the signals from each reaction were measured over 2 hours and the signal gradients were calculated. The ratio of gradients was calculated by dividing the gradient of the guide RNA + target (i.e., RNP + target viral RNA) reaction by the gradient of the guide RNA + no target (i.e., RNP control only) reaction. As shown in Figure 4B, the signal gradients of the H1N1, H3N2, or FluB RNA guides were divided by the signal gradient of the RNP control to determine the ratio of gradients for comparison between the target viral RNA and the RNP control. When the ratio of comparison is high (greater than 1), the guide RNA used in the assay mixture detects the H1N1, H3N2, or FluB target viral RNA strains more efficiently. However, when the ratio of comparison is low (less than 1), the guide RNA used in the assay mixture detects the target viral RNA similarly to the RNP control. Figure 4C shows the RNA guides for influenza A H1N1 and H3N2 strains with a ratio of gradients greater than 3 and the RNA guide for FluB with a ratio of gradients greater than 5.

[0173] Example 2: Detection of Influenza B RNA in Nasal Swabs by Cas13a Figures 5A - B show the validation and cross - reactivity of influenza B (FluB) RNA guides against host RNA and nasal swabs. RNA guides for FluB with a fold ratio greater than 5 as shown in Figure 4C were tested against host RNA and nasal swabs. Signals from each reaction were measured over 2 hours and signal gradients were calculated. To create the graph shown in Figure 5B, the signal gradient of the RNA guide for FluB was divided by the signal gradient of the RNP control to determine the fold ratio for comparison between the target viral RNA and the RNP control. The FluB RNA guides FluB_cr10 and FluB - cr13 were found to significantly cross - react with nasal swab material that was positive for FluB. FluB_cr12 and FluB - cr14 were found not to cross - react to the same extent with nasal swab material that was positive for FluB.

[0174] Example 3: Improvement in the detection of influenza B by combining the RNA guides of SEQ ID NO: 34 and 36 Figures 6A - B show the effect on target viral RNA detection by combining the RNA guides FluB_cr12 and FluB - cr14 (SEQ ID NO: 34 and 36). Signal gradients from each reaction of the RNA guide FluB_cr12 alone, FluB - cr14 alone, or the combination of FluB_cr12 and FluB - cr14 with the target viral RNA or RNP alone for H3N1, H1N1, FluB were measured over 2 hours, signal gradients were calculated, and are shown in Figure 6A. To create the graph shown in Figure 6B, the signal gradients in Figure 6A were divided by the signal gradient of the RNP control to determine the fold ratio for comparison between the target viral RNA and the RNP control. Combining the RNA guides FluB_cr12 and FluB - cr14 improves the detection of FluB target viral RNA compared to using these RNA guides separately. The detection of H3N1, H1N1, or RNP alone did not increase when the RNA guides FluB_cr12 and FluB - cr14 were combined.

[0175] Example 4: Verification and cross-reactivity of influenza A (H1N1 and H3N2 strains) RNA guides against host RNA and nasal swabs Figures 7A - B show the verification and cross-reactivity of influenza A (H1N1 and H3N2 strains) RNA guides against host RNA and nasal swabs. Signals from each reaction were measured over 2 hours and the signal gradients were calculated as shown in Figure 7A. RNA guides for influenza A with a gradient ratio greater than 3 as shown in Figure 4C were included in the tests against host RNA and nasal swabs. To create the graph shown in Figure 7B, the signal gradient of the RNA guide for FluB was divided by the signal gradient of the RNP control to determine the gradient ratio for comparison between the target viral RNA and the RNP control. The influenza A RNA guides identified by the enclosed lines were selected for the combination experiments shown in Figures 8A - B because they gave the best detection of the target viral RNA in nasal swabs.

[0176] Example 5: Improvement of influenza A detection by combining the RNA guides of SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22 and 8, 16, 21 and 22 respectively.

[0177] Figures 8A - B show the effect on influenza A target virus RNA detection by combining the best influenza A RNAs guides of Figure 7A. Combinations of seven influenza A RNA guides ("7g": cr04m, cr08, cr13, cr16, cr17, cr21, cr22 (SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22 respectively)) and four influenza RNA guides ("4g": cr08, cr16, cr21, cr22 (SEQ ID NOs: 8, 16, 21 and 22 respectively)) were tested against target virus RNA for influenza A (strains H1N1 and H3N2). Signals from each reaction were measured over 2 hours and signal gradients were calculated as shown in Figure 8A. To create the graph shown in Figure 8B, the signal gradients of the RNA guides for influenza A were divided by the signal gradient of the RNP control to determine a ratio for comparison between the target virus RNA and the RNP control. The ratio for the influenza A RNA guides in the 7g group was 4.9 and 26.5 for the target virus RNA for H1N1 and H3N2 respectively. The ratio for the influenza A RNA guides in the 4g group was 4.5 and 26.4 for the target virus RNA for H1N1 and H3N2 respectively. The ratios for the 7g and 4g RNA guide groups were significantly higher than any of the ratios for the influenza A RNA guides alone.

[0178] References

[0179]

Table 4 - 1

[0180]

Table 4 - 2

[0181] All publications, patent applications, patents, and other references mentioned in this specification are hereby expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In case of conflict, the specification, including definitions, will control.

[0182] The following statements provide an overview of some aspects of the nucleic acids and methods of the invention described herein. Statement: 1. (a) Incubating a sample suspected of containing influenza A or B RNA or virus with one or more Cas13 proteins, at least one CRISPR guide RNA (crRNA), and at least one reporter RNA for a time sufficient to form at least one RNA cleavage product, and (b) Detecting the reporter RNA cleavage product with a detector, A method comprising.

[0183] 2. The method of statement 1, wherein at least one CRISPR guide RNA (crRNA) binds to at least one target site of influenza A or influenza B nucleic acid.

[0184] 3. The method according to statement 1 or 2, wherein one or more of the Cas13 proteins have a protein sequence having at least 95% sequence identity with any of SEQ ID NOs: 38 - 49.

[0185] 4. The method according to any one of statements 1 - 3, wherein one or more of the Cas13 proteins have any of SEQ ID NOs: 38 - 49. 5. The method according to any one of statements 1 or 2, wherein the influenza A RNA is derived from an influenza A variant.

[0186] 6. A method according to any one of statements 1 to 5, wherein at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity with any one of SEQ ID NOs: 1 to 37.

[0187] 7. A method according to any one of statements 1 to 6, wherein at least one CRISPR guide RNA (crRNA) has a sequence of any one of SEQ ID NOs: 1 to 37. 8. A method according to any one of statements 1 to 7, wherein at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity with any one of SEQ ID NOs: 32, 34, 35, 36, or a combination thereof.

[0188] 9. A method according to any one of statements 1 to 8, wherein at least one CRISPR guide RNA (crRNA) has a sequence of any one of SEQ ID NOs: 32, 34, 35, 36, or a combination thereof.

[0189] 10. The method of statement 9, wherein at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 34 and 36. 11. A method according to any one of statements 1 to 10, wherein at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity with any one of SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, or a combination thereof.

[0190] 12. The method of statement 1, wherein at least one CRISPR guide RNA (crRNA) has a sequence of any one of SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, or a combination thereof.

[0191] 13. The method of statement 12, wherein at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 8, 16, 21, and 22. 14. A method of any one of statements 1, 2, 5-13, wherein one or more Cas13 proteins are Cas13a or Cas13b proteins.

[0192] 15. The method of statement 1, wherein at least one CRISPR guide RNA (crRNA) is two or more CRISPR guide RNAs (crRNAs). 16. The method of statement 1, wherein the Cas13 protein is complexed with at least one CRISPR guide RNA (crRNA) prior to incubation with a sample suspected of containing the target viral RNA.

[0193] 17. The method of statement 16, wherein one or more Cas13 proteins are complexed with at least one CRISPR guide RNA (crRNA) and prepared as lyophilized beads.

[0194] 18. The method of statement 1, wherein the sample suspected of containing the target viral RNA is saliva, sputum, mucus, nasopharyngeal substance, blood, serum, plasma, urine, aspirate, biopsy tissue, or a combination thereof.

[0195] 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 generates an optical signal, an electronic signal, an electrochemical signal, an electrostatic signal, a steric signal, a van der Waals interaction signal, a hydration signal, a resonance frequency shift signal, or a combination thereof.

[0196] 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 at least one fluorophore is Alexa 430, STAR 520, Brilliant Violet™ 510, Brilliant Violet™ 605, Brilliant Violet™ 610, or a combination thereof.

[0197] 23. The method of any one of statements 1, 21, or 22, wherein the detector comprises a photodetector, a fluorescence detector, a color filter, an electronic detector, an electrochemical signal detector, an electrostatic signal detector, a three-dimensional signal detector, a van der Waals interaction signal detector, a hydration signal detector, a resonance frequency shift signal detector, or a combination.

[0198] 24. The method of statement 1, wherein the target viral RNA is detected when the signal from the reporter RNA cleavage product is distinguishable from the control assay signal. 25. The method of statement 24, wherein the control assay does not contain the target viral RNA.

[0199] 26. The method of statement 24, wherein the control assay contains a viral RNA that is not the target viral RNA. 27. The method of statement 1, wherein the sample comprises at least one RNA derived from an influenza coronavirus, SARS-CoV-2, a hepatitis virus, a respiratory syncytial virus (RSV), or a human immunodeficiency virus (HIV).

[0200] 28. The method of statement 27, wherein the influenza coronavirus is at least one of the NL63 strain, the OC43 strain, or the 229E strain. 29. The method of statement 27, wherein the hepatitis virus is hepatitis C virus (HCV).

[0201] 30. A method according to any one of statements 27-29, wherein at least one CRISPR guide RNA is capable of binding to at least one RNA derived from influenza A virus, SARS-CoV-2, hepatitis virus, respiratory syncytial virus (RSV), or human immunodeficiency virus (HIV).

[0202] 31. A method comprising treating a subject having a detectable influenza A or B infection detected by a method according to any one of statements 1-26. 32. A kit comprising a package containing at least one Cas13 protein, at least one CRISPR guide RNA (crRNA) that binds to at least one target site of influenza A or influenza B nucleic acid, at least one reporter RNA, and instructions for detecting and / or quantifying target viral RNA in a sample.

[0203] 33. The kit of statement 32, wherein the at least one CRISPR guide RNA (crRNA) has a sequence having at least 95% sequence identity with any of SEQ ID NOs: 1-37.

[0204] 34. The kit according to 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 according to any one of statements 32-34, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity with any of SEQ ID NOs: 32, 34, 35, 36, or a combination thereof.

[0205] 36. The kit according to 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.

[0206] 37. The kit of statement 32, wherein at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NO: 34 and 36. 38. The kit of statement 32, wherein at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity with any one of SEQ ID NO: 4, 8, 13, 16, 17, 21, 22 or a combination thereof.

[0207] 39. The kit of statement 32, wherein at least one CRISPR guide RNA (crRNA) has a sequence of any one of SEQ ID NO: 4, 8, 13, 16, 17, 21, 22, or a combination thereof.

[0208] 40. The kit of statement 28, wherein at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NO: 8, 16, 21, and 22. 41. The kit of any one of statements 32 - 40, wherein at least one CRISPR guide RNA (crRNA) is two or more CRISPR guide RNAs (crRNAs).

[0209] 42. The kit of any one of statements 32 - 41, wherein the Cas13 protein forms a complex with at least one CRISPR guide RNA (crRNA). 43. The kit of any one of statements 32 - 42, wherein one or more Cas13 proteins are complexed with at least one CRISPR guide RNA (crRNA) and prepared as lyophilized beads.

[0210] 44. The kit of any one of statements 32, 42, or 43, wherein the Cas13 protein is a Cas13a or Cas13b protein. 45. The kit of statement 32, wherein the reporter RNA reporter comprises at least one fluorophore and at least one fluorescence quencher.

[0211] Kit of statement 45, wherein at least one fluorophore is Alexa 430, STAR 520, Brilliant Violet™ 510, Brilliant Violet™ 605, Brilliant Violet™ 610, or a combination thereof.

[0212] Kit of any one of statements 32 or 43, further comprising a sample chamber, an assay mixture reaction chamber, or a combination thereof. Kit of statement 43, wherein lyophilized beads are included within the assay mixture reaction chamber.

[0213] Kit of statement 32, further comprising a detector. Incorporation by reference of the sequence listing. The sequence listing is provided herein with this specification as an XML file "2349574.xml" created on July 6, 2023, and having a size of 101,264 bytes. The contents of the XML file are hereby incorporated by reference in their entirety.

Claims

**Claim 1** (a) incubating a sample suspected of containing influenza A or B RNA or virus with one or more Cas13 proteins, at least one CRISPR guide RNA (crRNA), and at least one reporter RNA for a time sufficient for at least one RNA cleavage product to form; and (b) detecting the reporter RNA cleavage product with a detector, A method comprising: **Claim 2** The method according to claim 1, wherein the sample contains RNA derived from a variant of influenza A. **Claim 3** The method according to claim 1, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity with any one of SEQ ID NOs: 1-37. **Claim 4** The method according to claim 1, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any one of SEQ ID NOs: 1-37. **Claim 5** The method according to claim 1, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity with any one of SEQ ID NOs: 32, 34, 35, 36, or a combination thereof. **Claim 6** The method according to claim 1, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any one of SEQ ID NOs: 32, 34, 35, 36, or a combination thereof. **Claim 7** The method according to claim 6, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 34 and 36. **Claim 8** The method according to claim 1, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity with any one of SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, or a combination thereof. **Claim 9** The method according to claim 1, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any one of SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, or a combination thereof. **Claim 10** The method according to claim 9, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 8, 16, 21, and 22. **Claim 11** The method according to claim 1, wherein one or more of the Cas13 proteins are Cas13a or Cas13b proteins.

12. The method according to claim 1, wherein the at least one CRISPR guide RNA (crRNA) is two or more CRISPR guide RNAs (crRNAs).

13. The method according to claim 1, wherein the Cas13 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.

14. The method according to claim 13, wherein the one or more Cas13 proteins are complexed with the at least one CRISPR guide RNA (crRNA) and prepared as lyophilized beads.

15. The method according to claim 1, wherein the sample suspected of containing the target viral RNA is saliva, sputum, mucus, nasopharyngeal substance, blood, serum, plasma, urine, aspirate, biopsy tissue, or a combination thereof.

16. The method according to claim 1, wherein the sample suspected of containing RNA is a lysed biological sample.

17. The method according to claim 1, wherein cleavage of the reporter RNA generates an optical signal, an electronic signal, an electrochemical signal, an electrostatic signal, a steric signal, a van der Waals interaction signal, a hydration signal, a resonance frequency shift signal, or a combination thereof.

18. The method according to claim 1, wherein the reporter RNA reporter comprises at least one fluorophore and at least one fluorescence quencher.

19. The method according to claim 18, wherein the at least one fluorophore is Alexa 430, STAR 520, Brilliant Violet (trademark) 510, Brilliant Violet (trademark) 605, Brilliant Violet (trademark) 610, or a combination thereof.

20. The method according to claim 1, wherein the detector comprises an optical 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 resonance frequency shift signal detector, or a combination.

21. The method according to claim 1, wherein the target viral RNA is detected when the signal from the reporter RNA cleavage product is distinguishable from the control assay signal.

22. The method according to claim 21, wherein the control assay does not contain the target viral RNA.

23. The method according to claim 21, wherein the control assay contains a viral RNA that is not the target viral RNA.

24. The method according to claim 1, wherein the sample comprises at least one RNA derived from an influenza A or B virus, a SARS-CoV-2 virus, a hepatitis virus, a respiratory syncytial virus (RSV), or a human immunodeficiency virus (HIV).

25. The method according to claim 24, wherein the influenza A virus is at least one of the NL63 strain, the OC43 strain, or the 229E strain.

26. The method according to claim 24, wherein the hepatitis virus is hepatitis C virus (HCV).

27. The method according to claim 24, wherein the at least one CRISPR guide RNA can bind to the at least one RNA derived from an influenza A or B virus, a SARS-CoV-2 virus, a hepatitis virus, a respiratory syncytial virus (RSV), or a human immunodeficiency virus (HIV).

28. A method comprising treating a subject having a detectable influenza A or B infection detected by the method according to any one of claims 1 to 23.

29. A kit comprising at least one Cas13 protein, at least one CRISPR guide RNA (crRNA) that binds to a target site in at least one of influenza A or influenza B nucleic acids, at least one reporter RNA, and instructions for detecting and / or quantifying the target viral RNA in a sample.

30. The kit according to claim 29, wherein the at least one CRISPR guide RNA (crRNA) has a sequence having at least 95% sequence identity to any of SEQ ID NOs: 1 to 37.

31. The kit according to claim 29, wherein at least one of the CRISPR guide RNAs (crRNAs) has a sequence of any of SEQ ID NOs: 1 to 37.

32. The kit according to claim 29, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity with any one of SEQ ID NO: 32, 34, 35, 36, or a combination thereof.

33. The kit according to claim 29, wherein the at least one CRISPR guide RNA (crRNA) has any one of SEQ ID NO: 32, 34, 35, 36, or a combination thereof.

34. The kit according to claim 29, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NO: 34 and 36.

35. The kit according to claim 29, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity with any one of SEQ ID NO: 4, 8, 13, 16, 17, 21, 22, or a combination thereof.

36. The kit according to claim 29, wherein the at least one CRISPR guide RNA (crRNA) has any one of the sequences of SEQ ID NO: 4, 8, 13, 16, 17, 21, 22, or a combination thereof.

37. The kit according to claim 29, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NO: 8, 16, 21, and 22.

38. The kit according to claim 29, wherein the at least one CRISPR guide RNA (crRNA) is two or more CRISPR guide RNAs (crRNAs).

39. The kit according to claim 29, further comprising at least one CRISPR guide RNA (crRNA) that binds to the RNA of influenza coronavirus, SARS-CoV-2 virus, hepatitis virus, respiratory syncytial virus (RSV), or human immunodeficiency virus (HIV).

40. The kit according to claim 29, wherein the Cas13 protein is complexed with the at least one CRISPR guide RNA (crRNA).

41. The kit according to claim 29, wherein the one or more Cas13 proteins are complexed with the at least one CRISPR guide RNA (crRNA) and prepared as lyophilized beads.

42. The kit according to claim 29, wherein the Cas13 protein is a Cas13a or Cas13b protein.

43. The kit according to claim 29, wherein the reporter RNA reporter comprises at least one fluorophore and at least one fluorescence quencher.

44. The kit according to claim 29, wherein the at least one fluorophore is Alexa 430, STAR 520, Brilliant Violet (trademark) 510, Brilliant Violet (trademark) 605, Brilliant Violet (trademark) 610, or a combination thereof.

45. The kit according to claim 29 or claim 41, further comprising a sample chamber, an assay mixture reaction chamber, or a combination thereof.

46. The kit according to claim 45, wherein the lyophilized beads are contained within the assay mixture reaction chamber.

47. The kit according to claim 29, further comprising a detector.

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