Virus diagnostics using CRISPR RNA combinations and Cas13a enzymes

Abstract

The present disclosure relates to methods of detecting and quantifying the presence of influenza A or influenza B RNA in a sample with enhanced specificity and sensitivity using the CRISPR-Cas13 enzyme complexed with influenza A or influenza B crRNA guide RNA. These methods are useful for diagnosing influenza A or influenza B infection, quantifying the concentration of influenza A or influenza B RNA present in a sample, and identifying the presence of different influenza A subtypes or mutations.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 367,988, filed on July 8, 2022, the disclosure of which is incorporated herein by reference.

[0003] Incorporation by Reference to the Sequence Listing

[0004] The sequence listing is provided herewith as an XML file "2349574.xml" created on July 6, 2023, and is 101,264 bytes in size. The contents of the XML file are incorporated herein by reference in their entirety. Background Art

[0005] Testing for respiratory infections, including SARS-CoV-2 and influenza A and B, is critical to identifying where and who needs medical attention. For example, it is estimated that there were approximately 38 million cases of influenza in the United States during the 2019-2020 season. During the same flu season, approximately 400,000 people were hospitalized and approximately 22,000 people died from the disease.

[0006] Current respiratory virus diagnostic assays include nucleic acid-based RT-qPCR tests (NATs) that require laboratory-based equipment and personnel, or rapid influenza diagnostics (RIDTs) that detect viral antigens. These assays are not quantitative and are not multiplexed with other related respiratory viruses. These assays are also not suitable for use by inexperienced or untrained personnel, such as at home.

[0007] There is a need for rapid, easy-to-use detection assays for viral RNA from respiratory fluid samples for the identification of respiratory infections. Summary of the invention

[0008] Described herein are methods, compositions, and devices for detecting and quantifying target viral RNA (e.g., influenza A and influenza B) that are faster and easier to deploy in the field than currently available methods and devices. In addition, the methods, compositions, and devices can easily detect and distinguish strains and variants of target viral RNA.

[0009] Current rapid influenza diagnostic tests (RIDTs) are immunoassays that identify the presence of influenza A and B virus nucleoprotein antigens in respiratory specimens and display results in a qualitative manner (positive vs. negative). However, RIDTs are known to have limited sensitivity for detecting influenza in respiratory specimens compared to time-consuming RT-PCR or viral culture methods. Negative RIDTs have the potential for false-negative results, especially during peak community influenza activity.

[0010] The methods described herein may include: (a) incubating a sample suspected of containing influenza A or influenza 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 of time sufficient to form at least one RNA cleavage product; and (b) detecting the reporter RNA cleavage product with a detector. Such a method can be used to detect whether a sample contains one or more copies of influenza A or influenza B virus RNA. The method can also be used to detect the absence of infection of a virus carrying target viral RNA. In addition, the methods and compositions described herein can also easily identify whether there is a variant or mutant strain of a virus carrying target viral RNA in a sample, and what the variant or mutation is.

[0011] The methods described herein can be used to diagnose influenza infection in a variety of complex biological samples. For example, the sample can include human saliva, sputum, mucus, nasopharyngeal material, blood, serum, plasma, urine, aspirate, biopsy tissue, or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Many aspects of the present disclosure may be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale. Instead, emphasis is placed on clearly illustrating the principles of the present disclosure. In addition, components may be shown as transparent in certain views only for the sake of clarity of illustration and not to indicate that the components shown must be transparent.

[0013] Figures 1A to 1B Shown is the use of CRISPR-Cas13 and CRISPR guide RNA (crRNA) to detect target RNA. Figure 1A Schematic diagram showing CRISPR-Cas13 detection of target viral RNA using CRISPR-Cas13 protein bound to CRISPR guide RNA (crRNA) to form a ribonucleoprotein (RNP) complex. The crRNA targets or guides the CRISPR-Cas13 protein to target a viral RNA sequence (e.g., influenza RNA), where the Cas13 protein is activated to cleave RNA, including reporter RNA. Figure 1B The following similar schematic diagram is further illustrated: Cas13a:crRNA ribonucleoprotein (RNP) complex binding to target viral RNA, resulting in activation of Cas13a nuclease (represented by scissors). After target recognition and RNP activation, Cas13a indiscriminately cuts the quenched fluorophore RNA reporter, allowing fluorescence detection as a proxy for Cas13a activation and target RNA presence.

[0014] Figure 2 Schematic diagram showing a method for detecting the SARS-CoV-2 RNA genome and fluorescent detection of reporter RNA. A CRISPR guide RNA (crRNA) that can target SARS-CoV-2 RNA or bind to SARS-CoV-2 RNA is used. As shown, in the first step, the CRISPR-Cas13 protein binds to the CRISPR guide RNA (crRNA) to form a ribonucleoprotein (RNP) complex. The RNP complex is inactive, but when mixed with a sample to be tested, the binding of the RNP complex to the SARS-CoV-2 RNA in the sample activates the Cas13 protein to cut RNA, including the reporter RNA molecule added to the assay mixture. The cutting of the reporter RNA produces fluorescence, which can be detected by a fluorescence detector.

[0015] Figure 3 Instant (point-of-caring, POC) method for detecting influenza is shown. As shown, samples (for example, saliva, sputum, mucus or nasopharyngeal samples of patients) can be collected, cells and / or viruses in the samples can be lysed to release any viral RNA that may be present, and RNA from the sample can be mixed with reporter RNA and CRISPR-Cas13 protein-crRNA ribonucleoprotein (RNP) complex. The background fluorescence from the control reaction can be subtracted and the fluorescence of the sample can be detected. Detection can be carried out by a fluorometer or other suitable devices. Such instant detection allows the mobilization of medical support and medical staff.

[0016] Figure 4A Figures 4 to 5 show the detection of influenza strains using specific RNA guides. Influenza A and influenza B RNA guides were designed to detect H1N1 or H3N2 strains of influenza A or influenza B (FluB). RNA guides were tested against H1N1, H3N2, FluB target viral RNA and a ribonucleoprotein (RNP) background control without target viral RNA. Figure 4A The signal slope results for different influenza A and influenza B RNA guides are listed. Figure 4AAs shown in , the signal from each reaction was measured over two hours and the signal slope was calculated. The slope ratio was calculated by dividing the slope of the guide RNA + target (i.e., RNP + target viral RNA) reaction by the slope of the guide RNA + no target (i.e., RNP only control) reaction. Figure 4B Signal slope results for different influenza A and influenza B RNA guides are also listed. Figure 4B The comparative slope ratios of the target viral RNA and the RNP control shown in are obtained by dividing the signal slope of the H1N1, H3N2 or FluB RNA guide by the signal slope of the RNP control. When the comparative ratio is high (e.g., greater than 1), the guide RNA used in the assay mixture is more effective in detecting the H1N1, H3N2 or FluB target viral RNA strain. However, when the comparative ratio is low (e.g., less than 1), the guide RNA used in the assay mixture detects the target viral RNA in a manner similar to the RNP control. Figure 4C The RNA guide names for H1N1 and H3N2 strains of influenza A with slope ratios greater than three are listed, as well as the RNA guide name for FluB with slope ratios greater than five.

[0017] Figure 5A B to B show validation and cross-reactivity of influenza B (FluB) RNA guides against host RNA and nasal swabs. Figure 5A Shown when testing for host RNA and nasal swabs, Figure 4C The signal slopes of the RNA guides of FluB shown in Figure 3 were greater than 5. The signal from each reaction was measured over two hours and the signal slope was calculated. Figure 5B The comparative slope ratio between the target viral RNA and the RNP control as obtained by dividing the signal slope of the RNA guide of FluB by the signal slope of the RNP control is shown. c r10 and FluB- c r13 significantly cross-reacted with nasal swab material that was positive for FluB. c r14 did not cross-react to the same extent with nasal swab material that was positive for FluB.

[0018] Fig. 6A B to B show the improved specificity and / or signal obtained for the combined guide RNAs FluB cr12 and FluB-cr14 targeting different viral RNAs. Fig. 6A The signal slope from each reaction against the target viral RNA: H3N1, H1N1, FluB or RNP alone with the RNA guide FluB alone as measured over two hours is shown graphically. cr12, separate FluB- c r14 or combined FluB c r12 and FluB- c r14. Figure 6B The comparative slope ratio between the target viral RNA and the RNP control is shown graphically as obtained by dividing the signal slope of the target viral RNA by the signal slope of the RNP control. c r12 and FluB- c r14 combination improved detection of FluB target viral RNA more than either of these RNA guides alone. c The rl4 combination did not improve detection of H3N1, H1N1, or RNP alone.

[0019] Fig. 7A B to B show validation and cross-reactivity of influenza A (H1N1 and H3N2 strains) RNA guides against host RNA and nasal swabs. Fig. 7A The slope of the signal from each reaction measured over a two-hour period is shown graphically. Figure 4C Influenza A RNA guides with slope ratios greater than three were included in the tests against host RNA and nasal swabs. Figure 7B The comparative slope ratio between the RNA guide for influenza A and the RNP control as obtained by dividing the signal slope for influenza A by the RNP control is shown graphically. The influenza A RNA guide identified in the box has the best detection of the target viral RNA in the nasal swab and was selected for Fig. 8A to the combined experiment shown in B.

[0020] Fig. 8A to B shows the Fig. 7A Effect of optimal influenza A RNA guide combination on influenza A target virus RNA detection. Fig. 8A Signal slopes for a combination of seven influenza A RNA guides ("7g": cr04m, cr08, cr13, cr16, cr17, cr21, cr22) and a combination of four influenza RNA guides ("4g": cr08, cr16, cr21, cr22) tested against target viral RNA of influenza A (strains H1N1 and H3N2) are shown. The signal from each reaction was measured over two hours. Figure 8BThe signal slopes of the RNA guides for influenza A are shown. The signal was divided by the signal slope of the RNP control to determine the comparative slope ratio between the target viral RNA and the RNP control. For the target viral RNAs for H1N1 and H3N2, the slope ratios of the influenza A RNA guides in the 7g group were 4.9 and 26.5, respectively. For the target viral RNAs for H1N1 and H3N2, the slope ratios of the influenza A RNA guides in the 4g group were 4.5 and 26.4, respectively. The slope ratios of each of the 7g and 4g RNA guide groups were significantly higher than the slope ratios of any influenza A RNA guide alone. DETAILED DESCRIPTION

[0021] Methods, kits and devices for rapid detection and / or quantitative influenza virus infection are described herein. The method may include: (a) incubating a sample suspected of containing RNA or virus with one or more Cas13 proteins, at least one CRISPR guide RNA (crRNA) and at least one reporter RNA binding to a target site in at least one of influenza A or influenza B nucleic acids for a period of time sufficient to form at least one RNA cleavage product; and (b) detecting the level of the reporter RNA cleavage product with a detector. Such a method can be used to detect whether a sample contains one or more copies of influenza RNA. The method can also be used to detect the absence of influenza infection.

[0022] 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 may be from any RNA virus selected for detection in a sample. In some aspects, the target viral RNA may be from a virus causing a respiratory infection or establishing its primary infection in the tissues and fluids of the upper respiratory tract. For example, the RNA virus may be an influenza virus, such as influenza A or influenza B. Influenza is an enveloped single-stranded RNA virus that recognizes N-acetylneuraminic acid (sialic acid) on the surface of a host cell (including human tracheal epithelial cells and respiratory epithelial cells) and combines with it. Influenza A is the main cause of influenza epidemics. The target viral RNA may be any RNA from 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes of influenza.

[0023] In addition to influenza virus, the target viral RNA can be a common cold coronavirus, such as strains NL63, OC43 or 229E. 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 may be from a human immunodeficiency virus (HIV). Therefore, the method can be used to detect and identify a combination of viral RNAs, for example, using the methods and components described in any one of PCT publications WO 2020 / 051452; WO 2021 / 188830 and WO 2022 / 046706, each of which is incorporated herein by reference in its entirety.

[0024] In some aspects, provided herein is a method for diagnosing the presence or absence of influenza infection, the method comprising incubating a mixture containing a sample suspected of containing influenza RNA, a Cas13 protein, at least one CRISPR guide RNA (crRNA) and a reporter RNA to form any reporter RNA cleavage product that may be present in the mixture for a period of time; and detecting the level of the reporter RNA cleavage product that may be present in the mixture with a detector. In some cases, before the detection step, the influenza RNA and / or RNA cleavage product in the sample is not reverse transcribed. The presence or absence of influenza infection in the patient is detected by qualitatively or quantitatively detecting the level of the reporter RNA cleavage product that may be present in the mixture.

[0025] The methods described herein have multiple advantages. For example, the methods described herein can directly detect RNA without additional operation. RNA amplification is generally not required, while currently available methods (such as SHERLOCK) require RNA amplification to achieve sufficient sensitivity. The methods, test kits and devices described herein are fast and provide results within 30 minutes. Expensive laboratory equipment and expertise are not required. The methods described herein are applicable to many different sample types (blood, nasal / oral swabs, etc.). The methods, test kits and devices described herein are easy to deploy on site (airport screening, borders, resource-poor areas), so that potential infected people will not need to go to hospitals and clinics, where there can be uninfected patients, susceptible people and trained, urgently needed medical personnel. Therefore, testing can isolate the facilities required for treating susceptible people and the trained personnel required for emergency and complex medical procedures.

[0026] CRISPR-Cas13 is a viable alternative to conventional methods for detecting and quantifying RNA by RT-PCR. The advantages of using CRISPR-Cas13 can be used for influenza diagnosis. The Cas13 protein directly targets RNA, and it can be programmed with crRNA to provide a platform for specific RNA sensing. By coupling the Cas13 protein with an RNA-based reporter, the incidental or non-specific RNase activity of the Cas13 protein can be used for influenza detection.

[0027] In 2017 and 2018, Dr. Feng Zhang's laboratory reported a Cas13-based detection system that achieved attomolar and zeptomolar sensitivities for detecting Zika virus, but the system included an additional reverse transcription step for isothermal amplification of Zika virus cDNA, which was ultimately reverse transcribed into RNA for RNA-based detection, a method known as SHERLOCK (Specific High Sensitivity Enzyme Reporter UnLOCKing) (Gootenberg et al. Science 356 (6336): 438–42 (2017); Gootenberg et al. Science 360 ​​(6387): 439–44 (2018)). Although this approach improves the sensitivity of Cas13, it introduces two undesirable steps involving reverse transcription and in vitro transcription, which minimizes its potential as a field-deployable and instant device.

[0028] The present disclosure provides methods and compositions for diagnosing influenza infection, quantifying influenza RNA concentrations, and identifying the presence of different influenza A subtypes and / or mutations.

[0029] In some cases, the method can be performed in a single tube (e.g., the same tube for collection and RNA extraction). The method provides a single-step instant diagnostic method. In other cases, the method can be performed in a dual-chamber system. For example, a collection swab containing a biological sample can be directly inserted into one chamber of such a dual-chamber system. After stirring, removing the swab, and lysing 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 allowed to flow into the second chamber. The second chamber may include Cas13 protein, selected crRNA, and reporting RNA, so that influenza can be determined.

[0030] Chamber one may contain a buffer that helps to lyse viral particles and release genomic material. Some examples of usable lysis buffers include, but are not limited to, PBS, commercial lysis buffers such as Qiagen RLT+ buffer or QuickExtract, DNA / RNA Shield, detergents of various concentrations such as Triton X-100, Tween 20, NP-40 or Oleth-8, or a combination of such reagents.

[0031] After stirring and then removing the swab, the chamber may be briefly (e.g., 2 to 5 minutes) heated (e.g., 55° C. or 95° C.) to further promote lysis. The partition between the two chambers is then broken or removed, and the nasal extraction buffer is allowed to flow into the second chamber and reconstitute the second chamber, which will contain lyophilized reagents (Cas13 RNP and reporter RNA molecules) for Cas13 assays.

[0032] Use of such an assay tube may provide a single-step point-of-care diagnostic method and device.

[0033] Methods, devices and compositions for diagnosing influenza infection described herein may involve incubating a mixture of a sample suspected of containing influenza RNA, Cas13 protein, at least one CRISPR RNA (crRNA) and a reporter RNA to form a period of time of 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 may be a fluorescence detector, such as a short quenching-fluorescence RNA detector or a total internal reflection fluorescence (TIRF) detector.

[0034] Reporter :

[0035] A single type of reporter RNA can be used. The reporter RNA can be configured so that a detectable signal appears when it is cut by the Cas13 protein. For example, the reporter RNA may 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 may have an electrochemical part (e.g., ferrocene or dye), which can provide electron transfer to a redox probe or transducer when it is cut by the Cas13 protein. In another example, the reporter RNA may have a reporter dye so that when the reporter RNA is cut, the reporter dye is detected by a detector (e.g., a spectrophotometer). In some cases, one end of the reporter RNA may be bonded to a solid surface. For example, the reporter RNA may be configured to release a cantilever (cantilever) of a signal when cutting. However, in other cases, the signal can be improved by using an unattached reporter RNA (e.g., not covalently bonded to a solid surface). The surface of the assay container or assay material may have a detector for sensing the release of the signal. The signal may be or may include an optical signal (eg, fluorescence or a detectable dye), an electrical 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.

[0036] The reporter RNA can be, for example, at least one quencher-fluorescent RNA reporter. Such a quencher-fluorescent RNA reporter can optimize fluorescence detection. The quencher-fluorescent RNA reporter includes an RNA oligonucleotide having both a fluorophore and a fluorophore quencher. The quencher reduces or eliminates the fluorescence of the fluorophore. When the Cas13 protein cuts the RNA reporter, the fluorophore is separated from the associated quencher, so that the fluorescent signal becomes detectable.

[0037] An example of such a fluorophore quencher-labeled RNA reporter is RNAseAlert (IDT). RNaseAlert was developed for detecting RNAse contamination in the laboratory, and the substrate sequence is optimized for RNase A species. Another method is to use a lateral flow strip to detect FAM-biotin reporters, which are detected by anti-FAM antibody-gold nanoparticle conjugates on the strip when cut by Cas13. Although this allows instrument-free detection, it takes 90 to 120 minutes to read out, compared to most fluorescence-based assays that require less than 30 minutes (Gootenberg et al. Science. 360 (6387): 439–44 (April 2018)).

[0038] The sequence of the reporter RNA can be optimized for Cas13 cutting. Cas13 preferentially exerts RNA enzyme cutting activity at exposed uridine or adenosine sites, depending on the Cas13 homolog. There is also a secondary preference for highly active homologs. The inventors have tested 5-mer homopolymers of all ribonucleotides. Based on these preferences, a variety of RNA oligonucleotides were labeled with Iowa Black Quencher (IDT) and FAM fluorophores at the 5' and 3' ends of the oligonucleotides, and these sequences were systematically tested in trans-ssRNA cutting assays, as described in the examples. The best sequence can be moved into the mobile test.

[0039] Fluorophores for fluorophore quencher-labeled RNA reporters can include Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or combinations thereof.

[0040] Detection :

[0041] A variety of mechanisms and devices can be used to detect fluorescence. In some cases, the detector is a fluorescence detector, optionally a short quenched fluorescent RNA detector or a total internal reflection 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 a combination thereof.

[0042] Some mechanisms or devices can be used to help eliminate background fluorescence. For example, reducing fluorescence from outside the detection focal plane can improve the signal-to-noise ratio, and thus improve the signal resolution of the target RNA cleavage product. Total internal reflection fluorescence (TIRF) can achieve very low background fluorescence and single molecule sensitivity under sufficiently sensitive video. In some cases, mobile phones can be used for influenza detection.

[0043] In some cases, Cas13 and reporter RNA can be tethered to a solid surface. After adding crRNA and influenza RNA samples, when total internal reflection fluorescence (TIRF) imaging is used, the activated Cas13 can produce small fluorescent spots on the solid surface. In order to optimize this embodiment, the fluorophore side of the reporter RNA is also tethered to the solid surface so that the quencher part of the reporter RNA is dispersed in order to cut. The Cas13 protein can be tethered to the solid surface with a sufficiently long tether to allow it to cut multiple RNA reporter molecules. The bright spots appearing on the solid surface are counted, and the viral load can be quantified. Using TIRF in a portable system helps to detect and reduce the background, so that the RNA cleavage product signal can be easily detected.

[0044] In some cases, the ribonucleoprotein (RNP) complex of Cas13 protein and crRNA can be tethered to a solid surface. In this way, there is no need to add crRNA later. On the contrary, only samples suspected of containing influenza RNA need to be contacted with a solid surface.

[0045] In some cases, the methods described herein may include directly detecting the target RNA in the sample without performing additional sample preparation steps prior to detection, such as depleting a portion of the protein, enzyme, lipid, nucleic acid, or a combination thereof in the sample or inactivating a nuclease. However, the methods described herein may include depleting a portion of the sample prior to other steps or inhibiting the nuclease in the sample prior to other steps. For example, the protein, enzyme, lipid, nucleic acid, or a combination thereof in the sample may be depleted. In some cases, the portion depleted in the sample is a human nucleic acid portion. However, RNA extraction of the sample is preferably not performed.

[0046] In some cases, the method can include removing ribonucleases (RNases) from the sample. In some cases, RNase inhibitors and / or heating are used to remove RNases from the sample.

[0047] In some cases, Cas13 protein and / or crRNA can be lyophilized before incubation with the sample. In some cases, Cas13 protein, crRNA and / or reporter RNA are lyophilized before incubation with the sample.

[0048] sample:

[0049] In some embodiments, biological sample is separated from patient.Some non-limiting examples of suitable biological sample include saliva, sputum, mucus, nasopharyngeal sample, blood, serum, blood plasma, urine, aspirate and biopsy sample.Therefore, the term " sample " related to patient can include RNA.Biological sample covers other liquid samples, solid tissue samples of saliva, sputum, mucus and biological origin, such as biopsy specimens or tissue culture or cells and their offspring derived therefrom.This definition also includes such sample: after it is obtained, it has been operated in any way, such as by treating with reagents, washing or enriching for certain cell groups.This definition also includes samples enriched for molecules (such as RNA) of a specific type.Term " sample " covers biological sample, such as clinical sample, such as saliva, sputum, mucus, nasopharyngeal sample, blood, blood plasma, serum, aspirate, cerebrospinal fluid (cerebral spinal fluid, CSF), and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatant, cell lysate, tissue sample, organ, bone marrow etc. "Biological samples" include biological fluids derived from cells and / or viruses (e.g., from infected cells). Samples containing RNA can be obtained from such cells (e.g., cell lysates or other cell extracts containing RNA). Samples can include or be obtained from any of the following: various body fluids (e.g., saliva, mucus, or sputum), cells, tissues, organs, or cell-free fluids.

[0050] In some embodiments, the biological sample is isolated from a patient known to have or suspected of having an influenza infection. In other embodiments, the biological sample is isolated from a patient known not to have an influenza infection. In other embodiments, the biological sample is isolated from a patient known to have or suspected of not having an influenza infection. In other words, the methods and devices described herein can be used to identify a subject with an influenza infection and to determine that a subject does not have an influenza infection.

[0051] 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, regardless of whether the sample actually contains RNA, and regardless of whether the sample contains influenza RNA, the biological sample can be lysed, RNA extracted, incubated with Cas13 and crRNA, etc.

[0052] Preferably, crRNA and Cas13 protein are pre-incubated in the absence of sample so that crRNA and Cas13 protein can form a complex. In some cases, the reporter RNA may exist when crRNA and Cas13 protein form a complex. However, in other cases, the reporter RNA may be added after crRNA and Cas13 protein have formed a complex. In addition, after the crRNA / Cas13 complex is formed, sample RNA (e.g., influenza RNA) may then be added. Sample RNA (e.g., influenza RNA) serves as activation RNA. Once activated by the activation RNA, the crRNA / Cas13 complex becomes a nonspecific RNA enzyme to produce an RNA cleavage product that can be detected using a reporter RNA (e.g., short quenched fluorescent RNA).

[0053] For example, Cas13 and crRNA are incubated for a period of time to form an inactive complex. In some cases, Cas13 and crRNA complexes are formed by: incubating together at 37 ° C for 30 minutes, 1 hour or 2 hours (e.g., 0.5 to 2 hours) to form an inactive complex. The inactive complex can then be incubated with the reporter RNA. An example of a reporter RNA is provided by the RNase Alert system. Sample influenza RNA can be an ssRNA activator. Cas13 / crRNA and influenza RNA samples become activated complexes cut in cis and trans. For example, when cut in cis, the activated complex can cut influenza RNA. When cut in trans, the activated complex can cut the reporter RNA, thereby releasing a signal, such as a fluorophore from the reporter RNA.

[0054] CRISPR guide RNA (crRNA) :

[0055] The CRISPR guide RNA system may be suitable for the methods and compositions described herein. The guide RNA may include: CRISPR RNA (crRNA or spacer), which may be a 17 to 20 nucleotide sequence complementary to the target DNA, and a trans-activating crRNA (tracrRNA or stem) as a binding scaffold for the Cas nuclease. In some cases, the two RNAs are fused to form a single guide RNA (sgRNA). TracrRNA forms a stem loop that is recognized and bound by the Cas nuclease. The term "guide RNA" used herein refers to a single guide RNA (sgRNA) or crRNA (spacer). CRISPR technology is generally described by, for example, Mali et al. Science 339: 823-6 (2013); it is incorporated herein by reference in its entirety.

[0056] In some cases, at least one CRISPR guide RNA (crRNA) has a sequence with at least 95% sequence identity to any one of SEQ ID NOs: 1 to 37 shown below. In some cases, at least one CRISPR guide RNA (crRNA) has a sequence, for example, of any one of SEQ ID NOs: 1 to 37, or in some cases, the crRNA may include those having SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, 32, or 34 to 36, or a combination thereof. In some cases, the sample may be incubated with one or two or more crRNAs. For example, the sample may be incubated with at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least nine, or at least ten or more crRNAs. In some cases, at least one crRNA has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to any one of SEQ ID NOs: 1-37.

[0057] In multiple examples of crRNAs that can be used to detect influenza B, the crRNAs can include those having SEQ ID NOs: 32, 34, 35, 36, or a combination thereof. In some cases, SEQ ID NOs: 34 and 36 can be combined to improve the detection of influenza B.

[0058] In multiple examples of crRNAs that can be used to detect influenza A, the crRNAs may include those having SEQ ID NOs: 4, 8, 13, 16, 17, 21, or 22, or a combination thereof. In some cases, the crRNAs may include those having SEQ ID NOs: 8, 16, 21, and 22.

[0059] The amount of reporter RNA cleavage product detected is directly related to the amount of target viral RNA. In some cases, the concentration of target viral RNA cleavage product can be quantified or determined by using a standard curve of reporter RNA cleavage product.

[0060] At least one crRNA can bind to a region in any of the eight single-stranded RNAs of the influenza RNA genome. In some cases, the region is a single-stranded region of the influenza RNA genome. In other cases, the region is a secondary structure in a region of the influenza genome where the viral ribonucleoprotein binding rate is low.

[0061] In some cases, the crRNA may include additional sequences, such as spacer sequences. Table 1 provides some examples of influenza crRNA sequences.

[0062] Table 1: Some examples of influenza A and influenza B crRNA sequences

[0063]

[0064]

[0065] As exemplified herein, for the detection of influenza B, crRNAs having sequences of SEQ ID NOs: 32, 34, 35, 36 exhibited better signals than crRNAs having sequences of SEQ ID NOs: 23 to 31, 33, or 37. In addition, the combination of crRNAs of SEQ ID NOs: 34 and 36 significantly improved the detection of influenza B compared to the use of crRNAs of SEQ ID NOs: 34 or 36 alone.

[0066] For the detection of influenza A, crRNAs having the sequences of SEQ ID NOs: 4, 8, 13, 16, 17, 21 or 22 showed better signals than crRNAs having the sequences of SEQ ID NOs: 1 to 3, 5 to 7, 9 to 12, 14, 15 or 18 to 20. In addition, the combination of seven crRNAs of SEQ ID NOs: 4, 8, 13, 16, 17, 21 and 22 and the combination of four crRNAs of SEQ ID NOs: 8, 16, 21 or 22 independently significantly improved the detection of influenza A compared to the use of crRNAs of SEQ ID NOs: 4, 8, 13, 16, 17, 21 or 22 alone.

[0067] Flu sequences

[0068] The DNA sequence of the influenza A genome (H1N1 strain) having the coding regions for each of the eight single-stranded RNA segments is available under the following accession numbers:

[0069] Segment 1: NC_002023.1 from the NCBI website (provided herein as SEQ ID NO:51).

[0070]

[0071]

[0072] Segment 2: NC_002021.1 from the NCBI website (provided herein as SEQ ID NO:52).

[0073]

[0074]

[0075] Segment 3: NC_002022.1 from the NCBI website (provided herein as SEQ ID NO:53).

[0076]

[0077]

[0078] Segment 4: NC_002017.1 from the NCBI website (provided herein as SEQ ID NO:54).

[0079]

[0080] Segment 5: NC_002019.1 from the NCBI website (provided herein as SEQ ID NO:55).

[0081]

[0082] Segment 6: NC_002018.1 from the NCBI website (provided herein as SEQ ID NO:56).

[0083]

[0084] Segment 7: NC_002016.1 from the NCBI website (provided herein as SEQ ID NO:57).

[0085]

[0086] Segment 8: NC_002020.1 from the NCBI website (provided herein as SEQ ID NO:58).

[0087]

[0088] The DNA sequence of the influenza B genome (strain Bisbane) having the coding regions for each of the eight single-stranded RNA segments is available under the following accession numbers:

[0089] Segment 1: CY018707.1 from the NCBI website (provided herein as SEQ ID NO: 59).

[0090]

[0091]

[0092]

[0093] Segment 2: CY018708.1 from the NCBI website (provided herein as SEQ ID NO: 60).

[0094]

[0095]

[0096] Segment 3: CY018706.1 from the NCBI website (provided herein as SEQ ID NO: 61).

[0097]

[0098]

[0099] Segment 4: CY018701.1 from the NCBI website (provided herein as SEQ ID NO: 62).

[0100]

[0101] Segment 5: CY018704.1 from the NCBI website (provided herein as SEQ ID NO: 63).

[0102]

[0103]

[0104] Segment 6: CY018703.1 from the NCBI website (provided herein as SEQ ID NO: 64).

[0105]

[0106] Segment 7: CY018702.1 from the NCBI website (provided herein as SEQ ID NO: 65).

[0107]

[0108] Segment 8: CY018705.1 from the NCBI website (provided herein as SEQ ID NO: 66).

[0109]

[0110] The influenza virus genome is RNA. Therefore, in some cases, the influenza virus genome can be a copy of the aforementioned DNA sequence, wherein the thymine (T) residue is a uracil (U) residue. In some cases, the influenza virus genome can be a complementary sequence (complement) of the aforementioned DNA sequence.

[0111] However, the influenza virus genome may also have sequence variations. For example, the influenza virus genome may be of a variety of influenza strains, including the aforementioned sequences for: strain H1N1, or other strains such as H3N2, or any of 18 different hemagglutinin (HA) subtypes and 11 different neuraminidase (NA) subtypes of influenza A. The variation of influenza B virus may be any of strains B / Lee / 1940, B / Brisbane / 60 / 2008, B / Victoria / 504 / 2000, or other strains.

[0112] Sequencing has determined that influenza viruses share a common genetic ancestor; however, they differ genetically in ways that are reported to allow reassortment—the exchange of viral RNA segments between viruses—to occur within each genus or type, but not across types. This genetic reassortment leads to a standard naming convention for influenza viruses, including the virus type; the species from which it was isolated (if non-human); its place of isolation; isolation number; year of isolation; and, for influenza A only, the HA and NA subtypes. In influenza A and influenza B viruses, genome segments 1, 3, 4, and 5, each encode only one protein: PB2, PA, HA, and NP proteins. All influenza viruses encode the polymerase subunit PB1 on segment 2; in some strains of influenza A viruses, this segment also encodes the accessory protein PB1-F2, a small 87-amino acid protein with pro-apoptotic activity, located in the +1 alternate reading frame. No analogs of PB1-F2 have been identified in influenza B or influenza C viruses. In contrast, segment 6 of influenza A encodes only the NA protein, while segment 6 of influenza B encodes both the NA protein and the NB matrix protein (in the -1 alternate reading frame), which is an integral membrane protein corresponding to the influenza A M2 protein. Segment 7 of both influenza A and influenza B encodes the M1 matrix protein. In the influenza A genome, the M2 ion channel is also expressed by segment 7 through RNA splicing, while influenza B encodes its BM2 membrane protein in the +2 alternate reading frame. Finally, both influenza A and influenza B have a single RNA segment (segment 8) from which they express the interferon antagonist NS1 protein and, through mRNA splicing, NEP / NS2, which is involved in the export of viral RNPs from the host cell nucleus. The genome organization of influenza C virus is generally similar to that of influenza A and influenza B viruses; however, the HEF protein of influenza C replaces the HA and NA proteins, and thus the influenza C virus genome has one less segment than the genomes of either influenza A or influenza B viruses.

[0113] Cas13 protein :

[0114] 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 be complexed with at least one CRISPR guide RNA (crRNA) to at least one reporter RNA for a period of time sufficient to form at least one RNA cleavage product.

[0115] Cas13 protein can be, for example, Cas13a protein, Cas13b protein or a combination thereof.Cas13 comprises two higher eukaryotic and prokaryotic nucleotide binding (HEPN) domains for RNA cutting, which is consistent with the known role of HEPN domains in other proteins.In some embodiments, Cas13 protein may have sequence changes and / or be 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 with any of the foregoing Cas13 sequences or Cas13 in the following bacteria: Leptotrichia wadei, Leptotrichia buccalis, Rhodobacter capsulatus, Herbinix hemicellulosilytica, Lbu, Listeria seeligeri, Paludibacter propionicigenes, Lachnospiraceae bacterium, [Eubacterium] rectale, Listeria newyorkensis, Clostridium aminophilum aminophilum), and / or Leptotrichia shahii.

[0116] For example, the Leptotrichia wadeii Cas13a endonuclease having the following sequence can be used (SEQ ID NO: 38; NCBI accession no. WP_036059678.1).

[0117]

[0118] Other sequences of the Leptotrichia wadeii Cas13a endonuclease are also available, such as those of NCBI accession nos. BBM46759.1, BBM48616.1, BBM48974.1, BBM48975.1, and WP_021746003.1.

[0119] In another example, the Herbinix hemicellulosilytica Cas13a endonuclease having the following sequence can be used (SEQ ID NO: 39; NCBI accession no. WP_103203632.1).

[0120]

[0121] However, in some cases, the Cas13 protein having the sequence of SEQ ID NO: 39 is not used.

[0122] In another example, the Oral Trichoderma Cas13a endonuclease having the following sequence can be used (SEQ ID NO: 40; NCBI accession no. WP_015770004.1).

[0123]

[0124] However, in some cases, the Cas13 protein having the sequence of SEQ ID NO: 40 is not used.

[0125] In another example, the Leptotrichia seeligeri Cas13a endonuclease having the following sequence can be used (SEQ ID NO: 41; NCBI accession no. WP_012985477.1).

[0126]

[0127]

[0128] For example, the Paludibacter propionicigenes Cas13a endonuclease having the following sequence can be used (SEQ ID NO: 42; NCBI accession no. WP_013443710.1).

[0129]

[0130] For example, the Lachnospiraceae Cas13a endonuclease having the following sequence can be used (SEQ ID NO: 43; NCBI accession no. WP_022785443.1).

[0131]

[0132] For example, the Leptotrichia saboriae Cas13a endonuclease having the following amino acid sequence can be used (SEQ ID NO: 44; NCBI accession no. BBM39911.1).

[0133]

[0134] In another example, the Oral Trichoderma C-1013-b Cas13a endonuclease can have the following amino acid sequence (SEQ ID NO:45; NCBI accession no. C7NBY4; AltName LbuC2c2).

[0135]

[0136] The inventors have evaluated the kinetics of other Cas13a and Cas13b proteins. Such work shows that, in some cases, Cas13b works faster than Cas13a in target viral RNA detection assays.

[0137] For example, Cas13b from Prevotella buccae can be used in influenza RNA detection methods, compositions and devices. The amino acid sequence of Prevotella buccae Cas13b protein (NCBI accession no. WP_004343973.1) is shown in SEQ ID NO:46 below.

[0138]

[0139] Such a Prevotella buccalis Cas13b protein can have a Km (Michelis constant) substrate concentration of about 20 micromolar and a Kcat of about 987 / second (see, e.g., Slaymaker et al. Cell Rep 26(13):3741-3751 (2019)).

[0140] Another Prevotella buccalis Cas13b protein (NCBI accession no. WP_004343581.1) that can be used for SARS-CoV-2 RNA detection methods, compositions and devices has an amino acid sequence as shown in SEQ ID NO:47 below.

[0141]

[0142] An example of a Bergeyella zoohelcum Cas13b (R1177A) mutant amino acid sequence (NCBI accession no. 6AAY_A) is shown below as SEQ ID NO:48.

[0143]

[0144] Another example of a Cas13b protein sequence from Prevotella sp. MSX73 (NCBI accession no. WP_007412163.1) that can be used in target viral RNA detection methods, compositions and devices is shown below as SEQ ID NO:49.

[0145]

[0146] Therefore, 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. (CRISPR) / CRISPR-associated (Cas) systems

[0147] Genome editing has been performed using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems (see, e.g., Marraffini and Sontheimer. Nature Reviews Genetics 11:181-190 (2010); Sorek et al. Nature Reviews Microbiology 2008 6:181-6; Karginov and Hannon. Mol Cell 2010 1:7-19; Hale et al. Mol Cell 2010:45:292-302; Jinek et al. Science 2012 337:815-820; Bikard and Marraffini Curr Opin Immunol 2012 24:15-20; Bikard et al. Cell Host & Microbe 2012 12:177-186; all of which are incorporated herein by reference in their entirety).

[0148] However, the CRISPR guide RNA system may be suitable for 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 is a binding scaffold for the Cas nuclease. In some cases, the two RNAs are fused to form a single guide RNA (sgRNA). TracrRNA forms a stem loop that is recognized and bound by the cas nuclease. CrRNA typically has a shorter sequence than tracrRNA. The term "guide RNA" used herein refers to a single guide RNA (sgRNA) or crRNA. CRISPR technology is generally described by, for example, Mali et al. Science 339: 823-6 (2013); it is incorporated herein by reference in its entirety.

[0149] The guide RNA system used herein is encoded within or near the ncRNA coding region of the expression cassette. Thus, upon transcription of the guide RNA, it can target the Cas enzyme to a desired location in the genome, where it can cleave the genomic RNA to produce a genomic modification.

[0150] There are several types of CRISPR systems, some of which are summarized in the table below.

[0151] Overview of CRISPR System Types

[0152]

[0153] As provided herein, "guide RNA" or "gRNA" refers to a ribonucleoprotein complex that can bind to a cas nuclease to form a ribonucleoprotein complex. The gRNA includes a nucleotide sequence complementary to a target site (e.g., near or at a genomic site to be edited). In some cases, the guide RNA includes one or more RNA molecules. TracrRNA can be used to promote the assembly of a ribonucleoprotein complex including gRNA, tracrRNA, and cas nuclease. The complementary nucleotide sequence of the guide RNA can mediate the binding of the ribonucleoprotein complex to the target site, thereby providing the sequence specificity of the ribonucleoprotein complex. Therefore, the guide RNA includes a sequence complementary to the target nucleic acid sequence, so that the guide RNA binds to the target nucleic acid sequence.

[0154] In some cases, the complementary sequence of the guide RNA includes a sequence with about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the target nucleic acid (e.g., target viral RNA sequence). In some cases, the guide RNA includes a sequence with about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the target nucleic acid sequence. In some cases, the guide RNA or its complementary sequence includes a sequence with at least about 90%, 95% or 100% sequence identity to the target viral RNA sequence. In some cases, the length of the segment bound by the guide RNA in the target nucleic acid is about or at least about 10, 15, 20, 25 or more nucleotides.

[0155] The guide RNA is a single-stranded ribonucleic acid, although in some cases, it can form some double-stranded regions by folding on 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 guide RNA can be at least about

[0156] 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

[0157] 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, 75 to 75, 80 to 80, 85 ... 100, 100, 150, 100, 200, 200, 300, 300, 350, 400, 450, 500, 5500, 600, 6500, 700, 7500, 8000, 8500, 900, 1000, 9500, 1000, or more nucleotides or residues. 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.

[0158] definition

[0159] As used herein, the term "about" when referring to a measurable value such as an amount, length, etc., is meant to cover variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% of a given value.

[0160] "Recombinant" as used herein to describe a nucleic acid molecule refers to a polynucleotide of genomic, cDNA, bacterial, viral, semisynthetic or synthetic origin that, by reason of its origin or manipulation, is not related in whole or in part to polynucleotides with which it is associated in nature.

[0161] The term "recombinant" used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. Generally, for example, a polynucleotide of interest as described herein is cloned and then expressed in a transformed organism. The host organism expresses the exogenous nucleic acid under expression conditions to produce RNA, RT-DNA or protein.

[0162] As used herein, "cell" refers to any type of cell isolated from a prokaryotic, eukaryotic or archaeal organism (including bacteria, archaea, fungi, protists, plants and animals), including cells from tissues, organs and biopsies, as well as recombinant cells, cells from cell lines cultured in vitro, and cell fragments, cell components or organelles containing nucleic acids. The term also encompasses artificial cells, such as nanoparticles, liposomes, polymers, or microcapsules, which encapsulate nucleic acids. The methods described herein can be performed, for example, on a sample comprising a single cell or a cell population. The term also includes genetically modified cells.

[0163] "Recombinant host cell," "host cell," "cell," "cell line," "cell culture" and other such terms referring to microbial or higher eukaryotic cell lines that are cultured as unicellular entities refer to cells that can or have been used as recipients for recombinant vectors or other transfer DNA, and include the original progeny of the original cell that has been transfected.

[0164] A "coding sequence" or a sequence "encoding" a selected polypeptide or selected RNA is a nucleic acid molecule that is transcribed (in the case of a DNA template) into RNA and / or translated (in the case of an mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. The coding sequence may include, but is not limited to, ncRNA, tracrRNA, ncRNA modified to contain heterologous sequences, cDNA from viruses, prokaryotic or eukaryotic ncRNAs, mRNA, viral or prokaryotic DNA, and even synthetic DNA sequences. The transcription termination sequence may be located 3' to the coding sequence.

[0165] Typical "control elements" include, but are not limited to, transcriptional promoters, transcriptional enhancer elements, transcriptional termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimizing translation initiation (located 5' to the coding sequence), and translation termination sequences.

[0166] "Operably linked" refers to an arrangement of elements in which the components so described are configured so as to perform their usual functions. Thus, a given promoter operably linked to a coding sequence is capable of affecting the expression of the coding sequence when an appropriate polymerase is present. The promoter need not be contiguous with the coding sequence, as long as it functions to direct its expression. Thus, for example, there may be an intervening untranslated but transcribed sequence between the promoter sequence and the coding sequence, and the promoter sequence may still be considered to be "operably linked" to the coding sequence.

[0167] "Encoded by" refers to a nucleic acid sequence encoding a polypeptide or 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, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the 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, and even more preferably at least 15 to 20 nucleotides.

[0168] The term "isolated", "purified" or "biologically pure" refers to a substance that is free of components that normally accompany it when it exists in its natural state to varying degrees. "Isolated" means the degree of separation from the original source or environment. "Purity" means a degree of separation higher than separation. A "purified" or "biologically pure" protein is sufficiently free of other substances so that any impurities do not substantially affect the biological properties of the protein, DNA or RNA or cause other adverse consequences. In other words, if the nucleic acid or peptide of the present invention is substantially free of cellular material, viral material or culture medium when obtained from nature or produced by recombinant DNA technology, or does not contain chemical precursors or other chemicals when chemically synthesized, the nucleic acid or peptide of the present invention is purified. Purity and homogeneity are generally determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that the nucleic acid or protein produces essentially one band in an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications can produce different isolated proteins that can be purified separately.

[0169] "Substantially purified" generally refers to the separation of a substance (nucleic acid, compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance accounts for the majority percentage of the sample in which it is present. Generally speaking, in a sample, the substantially purified component accounts for 50%, preferably 80% to 85%, and more preferably 90% to 95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and sedimentation based on density.

[0170] "Vector" is capable of transferring a nucleic acid sequence to a target cell (e.g., viral vectors, non-viral vectors, microparticle vectors, and liposomes). Generally speaking, "vector construct," "expression vector," and "gene transfer vector" mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and capable of transferring a nucleic acid sequence to a target cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0171] "Expression" refers to the production of a gene product detectable by a cell. A gene product can be a transcription product (ie, RNA), which can be referred to as "gene expression," or a gene product can be a translation product of a transcription product (ie, protein), depending on the context.

[0172] As used herein, "mammalian cell" refers to any cell derived from a mammalian subject that is suitable for transfection with the included vector systems. The cell may be xenogeneic, autologous, or allogeneic. The cell may be a primary cell obtained directly from a mammalian subject. The cell may also be a cell derived from the culture and expansion of a cell obtained from a mammalian subject. Immortalized cells are also included in this definition. In some embodiments, the cell has been genetically modified to express a recombinant protein and / or nucleic acid.

[0173] The term "subject" includes animals, including both vertebrates and invertebrates, including but not limited to invertebrates such as arthropods, mollusks, annelids, and cnidarians; and vertebrates such as amphibians, including frogs, salamanders, and caecillians; reptiles, including lizards, snakes, turtles, crocodiles, and alligators; fish; mammals, including humans and non-human mammals, such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals, such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals, such as dogs and cats; farm animals, such as sheep, goats, pigs, horses, and cattle; and birds, such as poultry, wild birds, and game birds, including chickens, turkeys and other quail, ducks, geese, etc. In some cases, the disclosed methods find use in experimental animals, veterinary applications, and for the development of animal models of disease, including but not limited to rodents, including mice, rats, and hamsters; primates, and transgenic animals.

[0174] "Gene transfer" or "gene delivery" refers to a method or system for reliably inserting (inserting into) a host cell a DNA or RNA of interest. Such methods can result in transient expression of non-integrated transferred DNA, expression of extrachromosomal replication and transfer replicons (e.g., episomes), or integration of the transferred genetic material into the genomic DNA of the host cell. Gene delivery expression vectors include, but are not limited to, vectors derived from viral vectors, non-viral vectors, alphaviruses, poxviruses and vaccinia viruses, and bacterial plasmid vectors.

[0175] The term "derived from" is used herein to identify the original source of a molecule, and is not meant to limit the method by which the molecule is made (eg, it may be by chemical synthesis or recombinant means).

[0176] A polynucleotide or nucleic acid "derived from" a specified sequence refers to a polynucleotide or nucleic acid that comprises a continuous sequence of at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10 to 12 nucleotides, and even more preferably at least about 15 to 20 nucleotides corresponding to a region of a specified nucleotide sequence (i.e., having identity or complementarity therewith). The resulting polynucleotide will not necessarily be physically derived from the target nucleotide sequence, but may be generated in any manner (including but not limited to chemical synthesis, replication, reverse transcription or transcription, based on information provided by the base sequence in the region from which the polynucleotide is derived). Therefore, it may represent the sense or antisense orientation of the original polynucleotide.

[0177] 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.

[0178] The term "homologous region" refers to a nucleic acid region homologous to another nucleic acid region. Therefore, it is determined whether a "homologous region" exists in a nucleic acid molecule with reference to another nucleic acid region in the same or different molecule. In addition, since nucleic acid is usually double-stranded, the term "homologous region" used herein refers to the ability of nucleic acid molecules to hybridize with each other. For example, a single-stranded nucleic acid molecule may have two homologous regions that can hybridize with each other. Therefore, the term "homologous region" includes nucleic acid segments with complementary sequences. The length of the homologous region can be different, but generally will be between 4 to 500 nucleotides (for example, about 4 to about 40, about 40 to about 80, about 80 to about 120, about 120 to about 160, about 160 to about 200, about 200 to about 240, about 240 to about 280, about 280 to about 320, about 320 to about 360, about 360 to about 400, about 400 to about 440, etc.).

[0179] The term "complementary" or "complementarity" used herein refers to a polynucleotide that can form base pairs with each other. Base pairs are usually formed by hydrogen bonds between nucleotide units oriented in antiparallel between polynucleotide chains. Complementary polynucleotide chains can be base-paired in a Watson-Crick manner (e.g., A and T, A and U, C and G) or in any other manner that allows the formation of a duplex. As known to those skilled in the art, when RNA is used instead of DNA, it is believed that the base complementary to adenosine is uracil (U) instead of thymine (T). However, unless otherwise stated, when uracil is represented in the context of the present invention, the ability to replace thymine is implied. "Complementarity" can exist between two RNA chains, two DNA chains, or between an RNA chain and a DNA chain. It is generally understood that two or more polynucleotides can be "complementary" and can form a duplex, although with incomplete or less than 100% complementarity. Two sequences are "fully complementary" or "100% complementary" if at least a contiguous portion of each polynucleotide sequence (including the complementary region) is perfectly base paired with the other polynucleotide without any mismatches or interruptions in such region. Two or more sequences are considered to be "fully complementary" or "100% complementary" even if one or both polynucleotides contain additional non-complementary sequences, as long as the contiguous complementary region within each polynucleotide is able to fully hybridize with the other. "Incomplete" complementarity refers to a situation where less than all of the contiguous nucleotides are able to base pair with each other within such a complementary region. Determining the percentage of complementarity between two polynucleotide sequences is a matter for those of ordinary skill in the art.

[0180] The term "donor polynucleotide" or "donor DNA" refers to a nucleic acid or polynucleotide that provides the intended editing nucleotide sequence to be integrated into the genome at a target locus by HDR or recombineering.

[0181] A "target site" or "target sequence" is a nucleic acid sequence that is recognized (i.e., sufficiently complementary for hybridization) by the homology arms of a guide RNA (gRNA) or a donor polynucleotide (donor DNA). A target site can be allele-specific (e.g., a major or minor allele). For example, a target site can be a genomic site that is intended to be modified, such as by inserting one or more nucleotides, replacing one or more nucleotides, deleting one or more nucleotides, or a combination thereof.

[0182] In general, "CRISPR system" refers to transcripts and other elements that participate in the expression of CRISPR-related ("Cas") genes or guide the activity of CRISPR-related ("Cas") genes, including sequences encoding Cas genes and CRISPR array nucleic acid sequences (which include a leader sequence and at least one repeat sequence). In some embodiments, one or more elements of the CRISPR system are derived from type I, type II or type III CRISPR systems. Cas1 and Cas2 are present in all three types of CRISPR-Cas systems, and they are related to spacer acquisition. In the IE system of Escherichia coli, Cas1 and Cas2 form a complex in which a Cas2 dimer bridges two Cas1 dimers. In this complex, Cas2 plays a non-enzymatic scaffolding role, which binds to double-stranded fragments of invading DNA, while Cas1 binds to the single-stranded flanks of DNA and catalyzes its integration into the CRISPR array.

[0183] In some embodiments, one or more elements of a CRISPR system are derived from a specific organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes. Generally speaking, a CRISPR system can be characterized by elements that promote formation of a CRISPR complex at the site of a target sequence (also referred to as protospacers in the context of an endogenous CRISPR system).

[0184] In certain embodiments, the present disclosure provides a protospacer adjacent to a short (3 to 5 bp) DNA sequence called a protospacer adjacent motif (PAM). 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 the spacer is cut using a ruler mechanism to maintain the regularity of the size of the spacer in the CRISPR array. The conservation of the PAM sequence varies between CRISPR-Cas systems and can be associated with Cas1 and the leader sequence in evolution.

[0185] In some embodiments, the regulatory element is operably connected to one or more elements of the CRISPR system to drive the expression of one or more elements of the CRISPR system. Generally speaking, CRISPR (clustered regularly interspaced short palindromic repeats), also known as SPIDR (interspersed direct repeats in the spacer region), constitutes a family of DNA loci that are usually specific to a specific bacterial species. The CRISPR locus contains different types of interspersed short sequence repeats (short sequence repeat, SSR), which are recognized in Escherichia coli (Ishino et al, J. BacterioL, 169: 5429-5433 (1987); and Nakata et al., J. BacterioL, 171: 3553-3556 (1989)), and related genes. Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (see, Groenen et al., Mol. Microbiol., 10:1057-1065 (1993); Hoe et al., Emerg. Infect. Dis., 5:254-263 (1999); Masepohl et al, Biochim. Biophys. Acta 1307:26-30 (1996); and Mojica et al, Mol. Microbiol, 17:85-93 (1995)). CRISPR loci are generally different from other SSRs in the structure of the repeat sequence, which is called short regularly spaced repeat (SRSR) (Janssen et al, OMICS J. Integ. Biol., 6: 23-33 (2002); and Mojica et al, Mol. Microbiol., 36: 244-246 (2000)). Generally speaking, repeats are short elements that appear in clusters that are regularly separated by unique insertion sequences of essentially constant length (Mojica et al., (2000), supra). Although repeat sequences are highly conserved between strains, the number of interspersed repeat and spacer sequences often varies from strain to strain (van Embden et al., J. Bacteriol., 182: 2393-2401 (2000)).CRISPR loci have been identified in more than 40 prokaryotes (see, e.g., Jansen et al, Mol. Microbiol., 43:1565-1575 (2002); and Mojica et al, (2005)), including but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacteriumn, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Acidophilus, Picrophilus), Thernioplasnia, Corynebacterium, Mycobacterium, Streptomyces, Aquifrx, Porphvromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myrococcus, Campylobacter, Wolinella, Acinetobacter The bacteria are classified as follows: Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

[0186] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme (e.g., cas9) is codon-optimized for expression in a specific cell, such as a eukaryotic cell. Eukaryotic cells can be those of a specific organism or derived from the specific organism, such as a mammal, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates. Generally speaking, codon optimization refers to the process of modifying a nucleic acid sequence for enhanced expression in a target host cell, wherein the modification is performed by replacing at least one codon (e.g., about one or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of a native sequence with a codon that is more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. Various species show specific preferences for certain codons of specific amino acids. Codon bias (differences in codon usage between organisms) is generally related to the translation efficiency of messenger RNA (mRNA), which is believed to depend on the properties of the codons translated and the availability of specific transfer RNA (tRNA) molecules, etc. The predominance of selected tRNAs in a cell generally reflects the codons most frequently used in peptide synthesis. Thus, genes can be customized based on codon optimization to achieve optimal gene expression in a given organism. Codon usage tables are readily available, for example, in the "Codon Usage Database", and these tables can be adapted in a variety of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28: 292 (2000). Computer algorithms for codon optimization of specific sequences for expression in specific host cells are also available, such as Gene Forge (Aptagen; Jacobus, Pa.). 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 most frequently used codons for a specific amino acid.

[0187] "Administering" a nucleic acid (eg, an expression cassette) includes transduction, transfection, electroporation, translocation, fusion, engulfment, shooting or ballistic methods, and the like, ie, any means by which a nucleic acid can be transported across a cell membrane.

[0188] The subject matter disclosed herein is not limited to the specific embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0189] When a range of values ​​is provided, it is understood that every intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening values ​​in that stated range, is encompassed within the disclosed subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosed subject matter, subject to any expressly excluded limits in the stated ranges. When the stated range includes one or both limits, ranges excluding either or both of those included limits are also encompassed within the disclosed subject matter.

[0190] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those 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, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications.

[0191] It must be noted that, as used herein and in the appended claims, nouns not modified with quantifiers include one / kind or more / kinds unless the context clearly indicates otherwise. Thus, for example, reference to "cell" includes a plurality of such cells, reference to "nucleic acid" includes reference to one or more nucleic acids and their equivalents known to those skilled in the art, and so on. It should also be noted that the claims may be written to exclude any optional elements. Therefore, this statement is intended to serve as an antecedent basis for the use of such exclusive terms such as "only", "only" and any feature or element described herein (which includes the use of "negative" limitations) in relation to reference.

[0192] It should be understood that, for clarity, certain features of the disclosed subject matter described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed subject matter described in the context of a single embodiment may also be provided individually or in any suitable subcombination. All combinations of embodiments related to the present disclosure are specifically included in the disclosed subject matter and disclosed herein, as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically included in the present disclosure and disclosed herein, as if each and every such subcombination were individually and explicitly disclosed herein.

[0193] The publications discussed herein are provided only for their disclosures prior to the filing date of the present application. Anything herein shall not be construed as admitting that the disclosed subject matter has no right to precede such publications. In addition, the publication date provided may be different from the actual publication date, which may require independent confirmation.

[0194] The following examples illustrate some of the materials, methods, and experiments used or performed in the development of the present invention.

[0195] Example

[0196] Example 1: Cas13a detection of SARS-CoV-2 transcripts

[0197] CRISPR RNA guides (crRNAs) were designed and validated for influenza A (H1N2 and H3N2 strains) and influenza B. Twenty-two (22) crRNAs were designed for influenza A, and fifteen (15) crRNAs were designed for influenza A. Each crRNA contained a crRNA stem derived from a bacterial sequence, and a spacer sequence derived from the influenza genome (reverse complement). The crRNA sequences are shown in Table 1 (copied below).

[0198] Table 1: Examples of influenza A and B crRNA sequences

[0199]

[0200] Figure 4A Figures 4 to 5 show the detection of influenza virus strains using specific RNA guides from Table 1. The RNA guides were tested against H1N1, H3N2, FluB target viral RNA and a ribonucleoprotein (RNP) background control without target viral RNA. Figure 4A As shown, the signal of each reaction was measured over two hours and the signal slope was calculated. The slope ratio was calculated by dividing the slope of the guide RNA + target (i.e., RNP + target viral RNA) reaction by the slope of the guide RNA + no target (i.e., RNP only control) reaction. Figure 4B As shown, the signal slope of the H1N1, H3N2 or FluB RNA guide is divided by the signal slope of the RNP control to determine the comparative slope between the target viral RNA and the RNP control. When the comparative ratio is high (greater than 1), the guide RNA used in the assay mixture is more effective in detecting the H1N1, H3N2 or FluB target viral RNA strain. However, when the comparative ratio is low (less than 1), the detection of the target viral RNA by the guide RNA used in the assay mixture is similar to that of the RNP control. Figure 4CRNA guides of influenza A H1N1 and H3N2 strains with slope ratios greater than 3 and an RNA guide of FluB with a slope ratio greater than 5 are shown.

[0201] Example 2: Cas13a detection of influenza B RNA in nasal swabs

[0202] Figure 5A Figures 4 to 5 show validation and cross-reactivity of influenza B (FluB) RNA guides against host RNA and nasal swabs. RNA guides for FluB with slopes greater than 5 were tested against host RNA and nasal swabs (e.g. Figure 4C The signal of each reaction was measured over two hours and the signal slope was calculated. Figure 5B As shown in the graph, the signal slope of the RNA guide of FluB was divided by the signal slope of the RNP control to determine the comparative slope ratio between the target viral RNA and the RNP control. FluB RNA guides FluB_cr10 and FluB-cr13 were found to cross-react significantly with nasal swab material positive for FluB. FluB_cr12 and FluB-cr14 were found to cross-react with nasal swab material positive for FluB to varying degrees.

[0203] Example 3: Improving the detection of influenza B by combining RNA guides of SEQ ID NO: 34 and 36

[0204] Fig. 6A Figures 1 to 3 show the effect of combining RNA guides FluB_cr12 and FluB-cr14 (SEQ ID NOs: 34 and 36) on target viral RNA detection. The signal slopes for each reaction of target viral RNA or RNP alone of H3N1, H1N1, FluB with RNA guides FluB_cr12 alone, FluB-cr14 alone, or FluB_cr12 and FluB-cr14 combined were measured over two hours, and the signal slopes were calculated and shown in Figure 3. Fig. 6A In order to prepare Figure 6B As shown in the figure, Fig. 6A The signal slope of the target viral RNA was divided by the signal slope of the RNP control to determine the comparative slope ratio between the target viral RNA and the RNP control. Combining the RNA guides FluB_cr12 and FluB-cr14 improved detection of the FluB target viral RNA compared to using these RNA guides alone. Detection of H3N1, H1N1, or RNP alone was not improved by combining the RNA guides FluB_cr12 and FluB-cr14.

[0205] Example 4: Validation and cross-reactivity of influenza A (H1N1 and H3N2 strains) RNA guides against host RNA and nasal swabs

[0206] Fig. 7A Validation and cross-reactivity of influenza A (H1N1 and H3N2 strains) RNA guides against host RNA and nasal swabs are shown in Figures B to B. The signal for each reaction was measured over two hours and the signal slope was calculated as Fig. 7A As shown in . The RNA guide of influenza A with a slope ratio greater than 3 (such as Figure 4C In order to prepare Figure 7B As shown in the figure, the signal slope of the RNA guide for FluB was divided by the signal slope of the RNP control to determine the comparative slope ratio between the target viral RNA and the RNP control. The influenza A RNA guide identified in the box had the best detection of the target viral RNA in the nasal swab and was selected for Fig. 8A to the combined experiment shown in B.

[0207] Example 5: Improving detection of influenza A virus by combining RNA guides of SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, and independently combining SEQ ID NOs: 8, 16, 21, and 22.

[0208] Fig. 8A to B shows the Fig. 7A Effect of optimal influenza A RNA guide combinations on influenza A target viral RNA detection. A combination 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 a combination of four influenza RNA guides ("4g": cr08, cr16, cr21, cr22 (SEQ ID NOs: 8, 16, 21, and 22, respectively) were tested against target viral RNA of influenza A (H1N1 and H3N2 strains). The signal for each reaction was measured over two hours, and the signal slope was calculated as Fig. 8A In order to prepare Figure 8B As shown in the figure, the signal slope of the influenza A RNA guide was divided by the signal slope of the RNP control to determine the comparative slope ratio between the target viral RNA and the RNP control. For the target viral RNAs of H1N1 and H3N2, the slope ratios of the influenza A RNA guides in the 7g group were 4.9 and 26.5, respectively. For the target viral RNAs of H1N1 and H3N2, the slope ratios of the influenza A RNA guides in the 4g group were 4.5 and 26.4, respectively. The slope ratios of each of the 7g and 4g RNA guide groups were significantly higher than the slope ratios of any influenza A RNA guide alone.

[0209] References

[0210] Abudayyeh OO,Gootenberg JS,Essletzbichler P,Han S,Joung J,BelantoJJ,Verdine V,Cox DBT,Kellner MJ,Regev A,Lander ES,Voytas DF,Ting AY,ZhangF.RNAtargeting with CRISPR-Cas13.Nature.Nature Publishing Group;2017Oct 12:550(7675):280-4.

[0211] Abudayyeh,OO,Gootenberg,JS,Konermann,S.,Joung,J.,Slaymaker,IM,Cox,DB,Shmakov,S,Makarova,KS,Semenova,E,Minakhin,L,et al.(2016).C2c2is a single-component programmable RNA-guided RNA-targeting CRISPRefiector.Science.353(6299):353,aaf5573.

[0212] Babin, SM, Hsieh, YH, Rothman, RE, and Gaydos, CA(2011).A meta-analysis of point-of-care laboratory tests in the diagnosis of novel2009swine-lineage pandemic influenza A(H1N1).Diagn Microbiol Infect Dis.69(4),410-418.

[0213] Chartrand,C.,Leeflang,M.M.,Minion,J.,Brewer,T.,and Pai,M.(2012).Accuracy of rapid influenza diagnostic tests:a meta-analysis.Ann InternMed.156(7),500-511.Published online 2012 / 03 / 01 DOI:10.7326 / 0003-4819-156-7-201204030-00403.

[0214] Chen,J.S.,Ma,E.,Harrington,L.B.,Da Costa,M.,Tian,X.,Palefsky,J.M.,andDoudna,J.A.(2018).CRISPR-Cas12a target binding unleashes indiscriminatesingle-stranded DNase activity.Science.360(6387),436-439.Published online2018 / 02 / 17DOI:10.1126 / science.aar6245.

[0215] Chu,H.,Lofgren,E.T.,Halloran,M.E.,Kuan,P.F.,Hudgens,M.,and Cole,S.R.(2012).Performance of rapid influenza H1N1 diagnostic tests:a meta-analysis.Influenza Other Respir Viruses.6(2),80-86.Published online 2011 / 09 / 03D0I:10.1111 / j.1750-2659.2011.00284.x.

[0216] East-Seletsky,A.,O′Connell,M.R.,Burstein,D.,Knott,G.J.,and Doudna,J.A.(2017).RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-CasEnzymes.Mol Cell.66(3),373-383e373.

[0217] East-Seletsky,A.,O′Connell,M.R.,Knight,S.C.,Burstein,D.,Cate,J.H.,Tjian,R.,and Doudna,J.A.(2016).Two distinct RNase activities oi CRISPR-C2c2enable guide-RNA processing and RNA detection.Nature.538(7624),270-273.

[0218] Gootenberg,J.S.,Abudayyeh,O.O.,Kellner,M.J.,Joung,J.,Collins,J.J.,andZhang,F.(2018).Multiplexed and portable nucleic acid detection platform withCas13,Cas12a,and Csm6.Science.360(6387),439-444.

[0219] Gootenberg,J.S.,Abudayyeh,O.O.,Lee,J.W.,Essletzbichler,P.,Dy,A.J.,Joung,J.,Verdine,V.,Donghia,N.,Daringer,N.M.,Freije,C.A.,et al.(2017).Nucleicacid detection with CRISPR-Cas13a / C2c2.Science.356(6336),438-442.

[0220] Green,D.A.,and StGeorge,K.(2018).Rapid Antigen Tests for Influenza:Rationale and Significance of the FDA Reclassification.J Clin Microbiol.56(10).Published online 2018 / 06 / 15DOI:10.1128 / JCM.00711-18.

[0221] Gu W,Crawford ED,O’Donovan BD,Wilson MR,Chow ED,Retallack H,DeRisiJL.Depletion of Abundant Sequences by Hybridization(DASH):using Cas9 toremove unwanted high-abundance species in sequencing libraries and molecularcounting applications.Genome Biol.BioMed Central;2016Mar 4;17(1):41.

[0222] Myhrvold,C.,Freije,C.A.,Gootenberg,J.S.,Abudayyeh,O.O.,Metsky,H.C.,Durbin,A.F.,Kellner,M.J.,Tan,A.L.,Paul,L.M.,Parham,L.A.,et al.(2018).Field-deployable viral diagnostics using CRISPR-Cas13.Science.360(6387),444-448.Published online 2018 / 04 / 28DOI:10.1126 / science.aas8836.

[0223] Smith,A.M.,and Perelson,A.S.(2011).Influenza A virus infectionkinetics:quantitative data and models.Wiley Interdiscip Rev Syst Biol Med.3(4),429-445.Published online 2011 / 01 / 05DOI:10.1002 / wsbm.129.

[0224] Zetsche, B., Gootenberg, JS, Abudayyeh, OO, Slaymaker, IM, Makarova, KS, Essletzbichler, P., Volz, SE, Joung, J., van der Oost, J., Regev, A., et al. (2015). Cpfl is a single RNA-guided endonuclease of a class 2CRISPR-Cassystem.Cell.163(3),759-771.Published online 2015 / 10 / 01 DOI: 10.1016 / j.cell.2015.09.038.

[0225] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0226] The following statement provides a summary of some aspects of the inventive nucleic acids and methods described herein.

[0227] statement

[0228] 1. A method comprising:

[0229] (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 of time sufficient to form at least one RNA cleavage product; and

[0230] (b) Detection of reporter RNA cleavage products using a detector.

[0231] 2. The method of statement 1, wherein the at least one CRISPR guide RNA (crRNA) binds to a target site in at least one of an influenza A or B nucleic acid.

[0232] 3. The method of statement 1 or 2, wherein one or more of the Cas13 proteins has a protein sequence having at least 95% sequence identity with any one of SEQ ID NOs: 38 to 49.

[0233] 4. The method of any one of statements 1 to 3, wherein one or more of the Cas13 proteins has any one of SEQ ID NOs: 38 to 49.

[0234] 5. The method of any one of statements 1 or 2, wherein the influenza A RNA is from a variant of influenza A.

[0235] 6. The method of any one of statements 1 to 5, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any one of SEQ ID NOs: 1 to 37.

[0236] 7. The method of any one of statements 1 to 6, wherein the at least one CRISPR guide RNA (crRNA) has the sequence of any one of SEQ ID NOs: 1 to 37.

[0237] 8. The method of any one of statements 1 to 7, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity to any one of SEQ ID NO: 32, 34, 35, 36, or a combination thereof.

[0238] 9. The method of any one of statements 1 to 8, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any one of SEQ ID NO: 32, 34, 35, 36, or a combination thereof.

[0239] 10. The method of statement 9, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NO:34 and 36.

[0240] 11. The method of any one of statements 1 to 10, 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.

[0241] 12. The method of statement 1, wherein the 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.

[0242] 13. The method of statement 12, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 8, 16, 21, and 22.

[0243] 14. The method of any one of statements 1, 2, 5 to 13, wherein one or more of the Cas13 proteins is a Cas13a or Cas13b protein.

[0244] 15. The method of statement 1, wherein the at least one CRISPR guide RNA (crRNA) is two or more CRISPR guide RNAs (crRNAs).

[0245] 16. The method of statement 1, wherein the Cas13 protein is complexed with the at least one CRISPR guide RNA (crRNA) prior to incubation with a sample suspected of containing target viral RNA.

[0246] 17. The method of statement 16, wherein one or more of the Cas13 proteins are complexed with the at least one CRISPR guide RNA (crRNA) and prepared into lyophilized beads.

[0247] 18. The method of statement 1, wherein the sample suspected of containing target viral RNA is saliva, sputum, mucus, nasopharyngeal material, blood, serum, plasma, urine, aspirate, biopsy tissue, or a combination thereof.

[0248] 19. The method of statement 1, wherein the sample suspected of containing RNA is a lysed biological sample.

[0249] 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.

[0250] 21. The method of statement 1, wherein the reporter RNA reporter comprises at least one fluorophore and at least one fluorescence quencher.

[0251] 22. The method of statement 21, wherein the at least one fluorophore is Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof.

[0252] 23. The method of any one of statements 1, 21, or 22, wherein the detector comprises a light detector, a fluorescence detector, a color filter, an electronic detector, an electrochemical signal detector, an electrostatic signal detector, a stereo signal detector, a van der Waals interaction signal detector, a hydration signal detector, a resonant frequency shift signal detector, or a combination thereof.

[0253] 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.

[0254] 25. The method of statement 24, wherein the control assay does not contain target viral RNA.

[0255] 26. The method of statement 24, wherein the control assay contains viral RNA that is not the target viral RNA.

[0256] 27. The method of statement 1, wherein the sample comprises at least one RNA from a common cold coronavirus, SARS-CoV-2, a hepatitis virus, respiratory syncytial virus (RSV), or human immunodeficiency virus (HIV).

[0257] 28. The method of statement 27, wherein the common cold coronavirus is at least one of the NL63, OC43, or 229E strains.

[0258] 29. The method of statement 27, wherein the hepatitis virus is hepatitis C virus (HCV).

[0259] 30. The method of any one of statements 27 to 29, wherein at least one CRISPR guide RNA can bind to at least one RNA from a common cold coronavirus, SARS-CoV-2, a hepatitis virus, respiratory syncytial virus (RSV), or human immunodeficiency virus (HIV).

[0260] 31. A method comprising treating a subject having a detectable influenza A or B infection detected by the method of any one of Statements 1 to 26.

[0261] 32. A kit comprising a package containing 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 B nucleic acids, at least one reporter RNA, and instructions for detecting and / or quantifying target viral RNA in a sample.

[0262] 33. The kit of statement 32, wherein the at least one CRISPR guide RNA (crRNA) has a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1 to 37.

[0263] 34. The kit of any one of statements 32 or 33, wherein at least one of the CRISPR guide RNAs (crRNAs) has a sequence of any one of SEQ ID NOs: 1 to 37.

[0264] 35. The kit of any one of statements 32 to 34, 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.

[0265] 36. The kit of any one of statements 32 to 35, wherein the at least one CRISPR guide RNA (crRNA) has any one of SEQ ID NO: 32, 34, 35, 36, or a combination thereof.

[0266] 37. The kit of claim 32, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NO:34 and 36.

[0267] 38. The kit of statement 32, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity to any one of SEQ ID NO: 4, 8, 13, 16, 17, 21, 22, or a combination thereof.

[0268] 39. The kit of statement 32, wherein the 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.

[0269] 40. The kit of statement 28, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 8, 16, 21, and 22.

[0270] 41. The kit of any one of Statements 32 to 40, wherein the at least one CRISPR guide RNA (crRNA) is two or more CRISPR guide RNAs (crRNA).

[0271] 42. The kit of any one of statements 32 to 41, wherein said Cas13 protein is complexed with said at least one CRISPR guide RNA (crRNA).

[0272] 43. The kit of any one of statements 32 to 42, wherein one or more of said Cas13 proteins are complexed with said at least one CRISPR guide RNA (crRNA) and prepared as lyophilized beads.

[0273] 44. The kit of any one of statements 32, 42, or 43, wherein the Cas13 protein is a Cas13a or Cas13b protein.

[0274] 45. The kit of statement 32, wherein said reporter RNA reporter comprises at least one fluorophore and at least one fluorescence quencher.

[0275] 46. ​​The kit of statement 45, wherein the at least one fluorophore is Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof.

[0276] 47. The kit of any of statements 32 or 43, further comprising a sample chamber, an assay mixing reaction chamber, or a combination thereof.

[0277] 48. The kit of statement 43 wherein said lyophilized beads are contained in said assay mixing reaction chamber.

[0278] 49. The kit of statement 32, further comprising a detector.

Claims

1. A method comprising: (a) incubating a sample suspected of containing influenza A or influenza 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 of time sufficient to form at least one RNA cleavage product; and (b) Detection of reporter RNA cleavage products using a detector.

2. The method of claim 1, wherein the sample comprises RNA from an influenza A variant.

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

4. The method of claim 1, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any one of SEQ ID NOs: 1 to 37.

5. The method of claim 1, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity to any one of SEQ ID NO: 32, 34, 35, 36, or a combination thereof.

6. The method of claim 1, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any one of SEQ ID NO: 32, 34, 35, 36, or a combination thereof.

7. The method of claim 6, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 34 and 36.

8. The method of claim 1, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity to any one of SEQ ID NO: 4, 8, 13, 16, 17, 21, 22, or a combination thereof.

9. The method of claim 1, wherein the 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.

10. The method of claim 9, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 8, 16, 21, and 22.

11. The method of claim 1, wherein one or more of the Cas13 proteins are Cas13a or Cas13b proteins.

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

13. The method of claim 1, wherein the Cas13 protein is complexed with the at least one CRISPR guide RNA (crRNA) prior to incubation with a sample suspected of containing target viral RNA.

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

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

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

17. The method of claim 1, wherein cleavage of the reporter RNA generates an optical signal, an electrical 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 of claim 1, wherein the reporter RNA reporter comprises at least one fluorophore and at least one fluorescence quencher.

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

20. The method of claim 1, wherein the detector comprises a light detector, a fluorescence detector, a color filter, an electrical detector, an electrochemical signal detector, an electrostatic signal detector, a stereo signal detector, a van der Waals interaction signal detector, a hydration signal detector, a resonance frequency shift signal detector, or a combination thereof.

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

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

23. The method of claim 21, wherein the control assay comprises viral RNA that is not the target viral RNA.

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

25. The method of claim 24, wherein the common cold coronavirus is at least one of strains NL63, OC43, or 229E.

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

27. The method of claim 24, wherein the at least one CRISPR guide RNA can bind to at least one RNA from the common cold coronavirus, SARS-CoV-2, a hepatitis virus, respiratory syncytial virus (RSV), or human immunodeficiency virus (HIV).

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

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

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

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

32. The kit of claim 29, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity to any one of SEQ ID NOs: 32, 34, 35, 36, or a combination thereof.

33. The kit of 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 of claim 29, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 34 and 36.

35. The kit of claim 29, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment having at least 95% sequence identity to any one of SEQ ID NOs: 4, 8, 13, 16, 17, 21, 22, or a combination thereof.

36. The kit of claim 29, wherein the 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.

37. The kit of claim 29, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 8, 16, 21, and 22.

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

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

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

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

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

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

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

45. The kit of any one of claims 29 or 41, further comprising a sample chamber, an assay mixture reaction chamber, or a combination thereof.

46. ​​The kit of claim 45, wherein lyophilized beads are contained in the assay mixture reaction chamber.

47. The kit of claim 29, further comprising a detector.

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