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WO2024159093A2 - Diagnostic using crispr rna and cas 13 a enzyme - Google Patents

Diagnostic using crispr rna and cas 13 a enzyme Download PDF

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Publication number
WO2024159093A2
WO2024159093A2 PCT/US2024/013102 US2024013102W WO2024159093A2 WO 2024159093 A2 WO2024159093 A2 WO 2024159093A2 US 2024013102 W US2024013102 W US 2024013102W WO 2024159093 A2 WO2024159093 A2 WO 2024159093A2
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rna
crrna
rsv
seq
crispr guide
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WO2024159093A3 (en
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Gagandeep Renuka KUMAR
Melanie Ott
Daniel A. Fletcher
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J David Gladstone Institutes
University of California Berkeley
University of California San Diego UCSD
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J David Gladstone Institutes
University of California Berkeley
University of California San Diego UCSD
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes

Definitions

  • Detection of respiratory infections is critical for targeting locations and populations that need medical assistance.
  • the estimated U.S. influenza illnesses in the 2019-2020 season was approximately 38 million people.
  • approximately 400,000 people were hospitalized and approximately 22.000 died from the disease.
  • Respiratory Syncytial Virus (RSV) infections are rampant and result in many hospitalizations.
  • RSV Respiratory Syncytial Virus
  • RT-qPCR nucleic-acid based tests include lab-based equipment and personnel or rapid diagnostics (RIDTs) that detect viral antigens. These assays are not quantitative or multiplexed with other relevant respiratory viruses. These assays are also not appropriate for use by inexperienced or untrained personnel, such as for at home use.
  • RSV diagnostic assays include RT-qPCR nucleic-acid based tests or antigen detecting tests that require lab-based equipment and personnel.
  • Descnbed herein are methods, compositions, and devices for detecting and quantifying target viral RNA, such as RSV, that are faster and more readily deployable in the field than currently available methods and devices.
  • the methods, compositions and devices can readily detect and distinguish between strains and variants of the target viral RNA.
  • the methods described herein can include: (a) incubating a sample suspected of containing RSV RNA or virus yvith one or more Casl3 protein(s), at least one CRISPR guide RNA (crRNA), and at least one reporter, e.g., reporter RNA, for a period of time sufficient to form at least one detectable product, such as a detectable RNA cleavage product; and (b) detecting the reporter, e.g., a reporter RNA cleavage product, with a detector.
  • Such methods are useful for detecting whether the sample contains one or more copies of RSV RNA.
  • the methods are also useful for detecting the absence of infection with the virus carry ing the target viral RNA.
  • the methods and compositions described herein can also readily identify whether a variant or mutant strain of virus carrying the target viral RNA is present in a sample, and determining what the variant or mutation is.
  • the methods described herein are useful for diagnosing RSV infections in a variety' of complex biological samples.
  • the samples can include human saliva, sputum, mucus, nasopharyngeal materials, blood, serum, plasma, urine, aspirate, biopsy tissue, or a combination thereof.
  • the assay is a single-step POC, or at-home assay based on CRISPR/Casl3a technology' that enables direct detection of RSV RNA following lysis and provides quantitative information for viral load.
  • the assay does not include reverse transcription and amplification.
  • FIGs. 1A-1B illustrates use of CRISPR-Casl3 and CRISPR guide RNAs (crRNAs) to detect target RNA.
  • FIG. 1A is a schematic diagram illustrating CRISPR-Casl3 detection of target viral RNA using a CRISPR-Casl3 protein that binds CRISPR guide RNAs (crRNA) to form a ribonucleoprotein (RNP) complex.
  • the crRNA targets or guides the CRISPR-Casl3 protein to target viral RNA sequences, where the Cast 3 protein is activated to cleave RNA, including the reporter RNA.
  • FIG. 1A is a schematic diagram illustrating CRISPR-Casl3 detection of target viral RNA using a CRISPR-Casl3 protein that binds CRISPR guide RNAs (crRNA) to form a ribonucleoprotein (RNP) complex.
  • the crRNA targets or guides the CRISPR-Casl
  • IB is a similar schematic diagram further illustrating a Cas 13a: crRNA ribonucleoprotein (RNP) complex binding of target viral RNA, resulting in activation of the Casl3a nuclease (denoted by scissors).
  • RNP crRNA ribonucleoprotein
  • Casl3a indiscriminately cleaves a quenched-fluorophore RNA reporter, allowing for fluorescence detection as a proxy for Casl3a activation and the presence of target RNA.
  • FIG. 2 is a schematic diagram illustrating methods for detection of the SARS-CoV-2 RNA genome and fluorescent detection of reporter RNA.
  • CRISPR guide RNAs crRNA
  • CRISPR guide RNAs crRNA
  • the CRISPR-Casl3 protein binds CRISPR guide RNAs (crRNA) to form a ribonucleoprotein (RNP) complex.
  • the RNP complex is inactive but, when mixed with the sample to be tested, binding of the RNP complex to the SARS-CoV-2 RNA in the sample activates the Cast 3 protein to cut RNA, including reporter RNA molecules added to the assay mixture. Cleavage of the reporter RNA leads to fluorescence, which can be detected by a fluorescence detector.
  • FIG. 3 illustrates a point-of-care (POC) method for detecting viruses.
  • a sample can be collected (e g., a patient’s saliva, sputum, mucus, or nasopharyngeal sample), the cells and/or viruses in the sample can be lysed to release any viral RNA that may be present, and the RNA from the sample can be mixed with reporter RNAs and a CRISPR-Casl3 protein-crRNA ribonucleoprotein (RNP) complex. Background fluorescence from control reactions can be subtracted and the fluorescence of the sample can be detected. Detection can be by a fluorometer or other suitable device. Such point-of-care detection allows mobilization of medical support and medical personnel.
  • RNP CRISPR-Casl3 protein-crRNA ribonucleoprotein
  • FIG. 4 shows the validation and cross-reactivity of exemplary RSV A RNA guides against host RNA and nasal swabs (signal slope over 2 hours).
  • FIG. 5 graphically illustrates that signal slopes from each reaction of target viral RNA for RSV A, or RNP alone with the RNA guides alone or combined.
  • FIG. 6 show the validation and cross-reactivity of exemplary RSV B RNA guides against host RNA and nasal swabs (signal slope over 2 hours).
  • FIG. 7 provides an exemplary RSV A consensus sequence (SEQ ID NO:401) which may be employed to prepare crRNAs specific for a large number of RSV A isolates.
  • FIG. 8 provides an exemplary RSV B consensus sequence (SEQ ID NO:406) which may be employed to prepare crRNAs specific for a large number of RSV B isolates.
  • FIG. 9 provides a summary of exemplary crRNAs (SEQ ID NOS: 1108-1152).
  • FIG. 10 provides results for 96 crRNAs tested for detection of RSV A RNA.
  • FIG. 11 provides results for 99 crRNAs tested for detection of RSV B RNA.
  • FIGS. 12A-12B RSV A virus: 129 crRNAs screened.
  • FIGS. 13A-13B RSV B virus crRNA screen results.
  • FIG. 14 RSV A crRNA off target activity results.
  • FIG. 15. RSV B crRNA off target activity results.
  • FIG. 16 RSV A crRNA off target activity with nasal swabs results.
  • FIG. 17 RSV B crRNA off target activity with nasal swabs results.
  • FIGS. 20 and 21 Tested crRNA off-target activity 7 was tested against non-targeted viral RNA.
  • FIG 20 demonstrates evaluating combinations RSV A
  • FIG. 21 demonstrates evaluating combinations RSV B (10g yields high signal).
  • FIG. 22 RSV B: testing of crRNA off-target activity against non-targeted viral RNA. Most crRNAs have minimal off-target activity against non-targeted viruses.
  • FIG. 23 RSV A and B: crRNA combinations were evaluated, including combinations RSVAB_9g+crl2 and RSVAB 11g.
  • FIGS. 24A-24B RSV A virus LOD: 12 infectious particles (pfu) per ml are detected with both 10g and 11g crRNA combinations.
  • FIGS 25A-25B RSV B virus LOD: 1 to 4.5 infectious particles (pfu) per ml are detected with both 11g and 10g crRNA combinations, respectively.
  • kits and devices are described herein for rapidly detecting and/or quantifying virus infection.
  • the methods can include (a) incubating a sample suspected of containing RNA or virus with one or more Cas 13 protein, at least one CRISPR guide RNA (crRNA) that binds a target site in at least one of RSV A or RSV B nucleic acid, and at least one reporter RNA for a period of time sufficient to form at least one RNA cleavage product(s); and (b) detecting level(s) of reporter RNA cleavage product(s) with a detector.
  • crRNA CRISPR guide RNA
  • Such methods are useful for detecting whether the sample contains one or more copies of RSV RNA.
  • the methods are also useful for detecting the absence of an RSV infection.
  • the disclosure provides methods for identifying the target virus RNA from a sample suspected of containing the target viral RNA.
  • the target virus RNA can be from any RNA vims selected for detection in a sample.
  • the target viral RNA can be from a virus that causes a respiratory 7 infection or establishes its primary 7 infection in the tissues and fluids of the upper respiratory tract.
  • the RNA virus can be RSV, such as RSV A or B. RSV is a negative sense, single stranded RNA virus.
  • the target viral RNA can be common cold coronaviruses, such as strains NL63, OC43, or 229E.
  • the target viral RNA can also be SARS-CoV-2, a hepatitis virus (e.g., HCV), or influenza vims, influenza virus A or influenza virus B.
  • the target viral RNA can be from the human immunodeficiency virus (HIV).
  • the methods can thus be used to detect and identify a combination of viral RNAs, for example, using methods and components described in any of PCT publications WO 2020/051452; WO 2021/188830; and WO 2022/046706, each of which is incorporated by reference herein in its entirety.
  • kits for diagnosing the presence or absence of a RSV infection comprising incubating a mixture comprising a sample suspected of containing RSV RNA, a Cast 3 protein, at least one CRISPR guide RNA (crRNA). and a reporter RNA for a period of time to form any reporter RNA cleavage product(s) that may be present in the mixture; and detecting level(s) of reporter RNA cleavage product(s) that may be present in the mixture with a detector.
  • the RSV RNA in a sample and/or the RNA cleavage products are not reverse transcribed prior to the detecting step.
  • the presence or absence of an RSV infection in patient is detected by qualitatively or quantitatively detecting level of reporter RNA cleavage product(s) that may be present in the mixture.
  • RNA amplification is generally needed, whereas currently available methods (e.g., SHERLOCK) require RNA amplification to be sufficiently sensitive.
  • SHERLOCK RNA amplification-sensitive methods
  • the methods, kits, and devices descnbed herein are rapid, providing results within 30 minutes. Expensive lab equipment and expertise is not needed.
  • the methods described herein are amenable to many different sample types (blood, nasal/oral swab, etc.).
  • the methods, kits, and devices described herein are easily deployable in the field (airport screenings, borders, resource poor areas) so that potentially infected people will not need to go to hospitals and clinics where non-infected patients, vulnerable persons, and highly trained, urgently needed medical people may be. Hence, testing can be isolated from facilities needed for treatment of vulnerable populations and from trained personnel needed for urgent and complex medical procedures.
  • CRISPR-Casl3 is a viable alternative to conventional methods of detecting and quantifying RNA by RT-PCR.
  • the advantages of using CRISPR-Casl3 can be leveraged for RSV diagnostics.
  • the Cast 3 protein targets RNA directly, and it can be programmed with crRNAs to provide a platform for specific RNA sensing. By coupling it to an RNA-based reporter, the collateral or non-specific RNase activity of the Casl3 protein can be harnessed for RS V detection.
  • the present disclosure provides methods and compositions for diagnosing virus infections, such as RSV infections, quantifying RNA concentrations, such as RSV RNA concentrations, and identifying the presence of different virus subtypes and/or mutations, such as RSV subtypes and/or mutations.
  • the methods can be performed in a single tube, for example, the same tube used for collection and RNA extraction. This method provides a single step point of care diagnostic method.
  • the methods can be performed in a two-chamber system.
  • the collection swab containing a biological sample can be directly inserted into chamber one of such a two chamber system. After agitation, removal of the swab, and lysis of biological materials in the sample, the division between the two chambers can be broken or removed, and the contents of the first chamber can be allowed to flow into the second chamber.
  • the second chamber can contain the Casl3 protein, the selected crRNA(s), and the reporter RNA so that the assay for RSV can be performed.
  • Chamber one can contain a buffer that would facilitate lysis of the viral particles and release of genomic material.
  • lysis buffers that can be used include, but are not limited to PBS, commercial lysis buffers such as Qiagen RLT+ buffer or Quick Extract, DNA/RNA Shield, various concentrations of detergents such as Triton X-I00, Tween 20, NP-40, or Oleth-8, or combinations of such reagents.
  • the chamber may be briefly (e.g., 2-5 mins) heated (e.g., 55 °C or 95 °C) to further facilitate lysis. Then, the division between the two chambers would be broken or removed, and the nasal extract buffer would be allowed to flow into and reconstitute the second chamber, which would contain the lyophilized reagents for the Casl3 assay (Casl3 RNPs and reporter RNA molecules).
  • the methods, devices and compositions described herein for diagnosing RSV infection can involve incubating a mixture having a sample suspected of containing RSV RNA, a Cast 3 protein, at least one CRISPR RNA (crRNA), and a reporter RNA for a period of time to form reporter RNA cleavage products that may be present in the mixture and detecting a level of any such reporter RNA cleavage products with a detector.
  • the detector can be a fluorescence detector such as a short quenched-fluorescent RNA detector, or Total Internal Reflection Fluorescence (TIRF) detector.
  • reporter RNA can be used.
  • the reporter RNA can be configured so that upon cleavage by the Casl3 protein, a detectable signal occurs.
  • the reporter RNA can have a fluorophore at one location (e.g.. one end) and a quencher at another location (e.g., the other end).
  • the reporter RNA can have an electrochemical moiety (e.g., ferrocene, or dye), which upon cleavage by a Cast 3 protein can provide electron transfer to a redox probe or transducer.
  • the reporter RNA can have a reporter dye, so that upon cleavage of the reporter RNA the reporter dye is detected by a detector (e.g., spectrophotometer).
  • a detector e.g., spectrophotometer
  • one end of the reporter RNA can be bonded to a solid surface.
  • a reporter RNA can be configured as a cantilever, which upon cleavage releases a signal.
  • a signal may be improved by use of an unattached reporter RNA (e.g.. not covalently bond to a solid surface).
  • a surface of the assay vessel or the assay material can have a detector for sensing release of the signal.
  • the signal can be or can include a light signal (e.g., fluorescence or a detectable dye), an electronic signal, an electrochemical signal, an electrostatic signal, a steric signal, a van der Waals interaction signal, a hydration signal, a Resonant frequency shift signal, or a combination thereof.
  • a light signal e.g., fluorescence or a detectable dye
  • an electronic signal e.g., an electrochemical signal, an electrostatic signal, a steric signal, a van der Waals interaction signal, a hydration signal, a Resonant frequency shift signal, or a combination thereof.
  • the reporter RNA can, for example, be at least one quenched-fluorescent RNA reporter.
  • quenched-fluorescent RNA reporter can optimize fluorescence detection.
  • the quenched- fluorescent RNA reporters include an RNA oligonucleotide with both a fluorophore and a quencher of the fluorophore. The quencher decreases or eliminates the fluorescence of the fluorophore. When the Casl3 protein cleaves the RNA reporter, the fluorophore is separated from the associated quencher, such that a fluorescence signal becomes detectable.
  • RNaseAlert One example of such a fluorophore quencher-labelled RNA reporter is the RNaseAlert (IDT).
  • IDTT RNaseAlert
  • RNaseAlert was developed to detect RNase contaminations in a laboratory, and the substrate sequence is optimized for RNase A species.
  • Another approach is to use lateral flow strips to detect a FAM-biotin reporter that, when cleaved by Cast 3, is detected by anti-FAM antibody- gold nanoparticle conjugates on the strip. Although this allows for instrument-free detection, it requires 90-120 minutes for readout, compared to under 30 minutes for most fluorescence-based assays (Gootenberg et al. Science. 360(6387):439-44 (April 2018)).
  • the sequence of the reporter RNA can be optimized for Cast 3 cleavage.
  • Cast 3 preferentially exerts RNase cleavage activity at exposed uridine or adenosine sites, depending on the Cast 3 homolog.
  • the inventors have tested 5-mer homopolymers for all ribonucleotides. Based on these preferences, various RNA oligonucleotides, labeled at the 5' and 3' ends of the oligonucleotides using an Iowa Black Quencher (IDT) and FAM fluorophore, and systematically test these sequences in the trans- ssRNA cleavage assay as described in the Examples. The best sequence can be moved into the mobile testing.
  • IDTT Iowa Black Quencher
  • FAM fluorophore FAM fluorophore
  • the fluorophores used for the fluorophore quencher-labelled RNA reporters can include Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof.
  • the detector is a fluorescence detector, optionally a short quenched-fluorescent RNA detector, or Total Internal Reflection Fluorescence (TIRF) detector.
  • the fluorescence detector can detect fluorescence from fluorescence dyes such the Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof.
  • Some mechanisms or devices can be used to help eliminate background fluorescence. For example, reducing fluorescence from outside the detection focal plane can improve the signal-to- noise ratio, and consequently, the resolution of signal from the RNA cleavage products of interest.
  • Total internal reflection fluorescence (TIRF) enables very low background fluorescence and single molecule sensitivity with a sufficiently sensitive camera.
  • mobile phones can be used for detection of virus, such as RSV.
  • both Casl3 and reporter RNA can be tethered to a solid surface, upon addition of crRNA and RSV RNA samples, an activated Cast 3 can generate small fluorescent spots on the solid surface when imaged using Total Internal Reflection Fluorescence (TIRF).
  • TIRF Total Internal Reflection Fluorescence
  • the fluorophore side of reporter RNA is tethered to the solid surface as well so that cleavage permits the quencher portion of the reporter RNA to diffuse away.
  • the Cast 3 protein can be tethered to the solid surface with a tether that is long enough to allow it to cleave multiple RNA reporter molecules. Counting the bright spots emerging on the solid surface the viral load can be quantified.
  • Use of TIRF in the portable system facilitates detection and reduces background so that the RNA cleavage product signals can readily be detected.
  • a ribonucleoprotein (RNP) complex of the Cast 3 protein and the crRNA can be tethered to the solid surface.
  • the crRNA would then not need to be added later. Instead, only the sample suspected of containing RSV RNA would need to be contacted with the solid surface.
  • the methods described herein can include direct detection of the target RNA in the sample, without performing further sample preparation steps prior to detection, such as depleting a portion of the sample of protein, enzymes, lipids, nucleic acids, or a combination thereof or inactivating nucleases.
  • the methods described herein can include depleting a portion of the sample prior to other step(s) or inhibiting a nuclease in the sample prior to the other step(s).
  • the sample can be depleted of protein, enzymes, lipids, nucleic acids, or a combination thereof.
  • the depleted portion of the sample is a human nucleic acid portion.
  • RNA extraction of the sample is preferably not performed.
  • the methods can include removing ribonuclease(s) (RNase) from the sample.
  • RNase ribonuclease
  • the RNase is removed from the sample using an RNase inhibitor and/or heat.
  • the Casl3 protein and/or the crRNA can be lyophilized prior to incubation with the sample.
  • the Cast 3 protein, the crRNA, and/or the reporter RNA is lyophilized prior to incubation with the sample.
  • a biological sample is isolated from a patient.
  • suitable biological samples include saliva, sputum, mucus, nasophary ngeal samples, blood, serum, plasma, urine, aspirate, and biopsy samples.
  • sample with respect to a patient can include RNA.
  • Biological samples encompass saliva, sputum, mucus, and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof.
  • the definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, washed, or enrichment for certain cell populations.
  • sample encompasses biological samples such as a clinical sample such as saliva, sputum, mucus, nasopharyngeal samples, blood, plasma, serum, aspirate, cerebral spinal fluid (CSF), and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, and the like.
  • a "biological sample” includes biological fluids derived from cells and/or viruses (e g., from infected cells).
  • a sample containing RNAs can be obtained from such cells (e.g., a cell lysate or other cell extract comprising RNAs).
  • a sample can comprise, or can be obtained from, any of a variety' of bodily fluids (e.g., saliva, mucus, or sputum), cells, tissues, organs, or acellular fluids.
  • the biological sample is isolated from a patient known to have or suspected to have an RSV infection. In other embodiments, the biological sample is isolated from a patient not known have an RSV infection. In other embodiments, the biological sample is isolated from a patient known to have, or suspected to not have, an RSV infection. In other words, the methods and devices described herein can be used to identity subjects that have an RSV infection and to confirm that subjects do not have an RSV infection.
  • biological samples can still be tested using the methods described herein.
  • biological samples can be subjected to lysis, RNA extraction, incubation with Cast 3 and crRNAs, etc. whether or not the sample actually contains RNA, and whether or not a sample contains RSV RNA.
  • Pre-incubation of the crRNA and Cast 3 protein without the sample may be employed, so that the crRNA and the Casl3 protein can form a complex.
  • the reporter RNA can be present while the crRNA and the Cast 3 protein form a complex.
  • the reporter RNA can be added after the crRNA and the Casl3 protein already form a complex.
  • the sample RNA can then be added. The sample RNA acts as an activating RNA.
  • the crRNA/Casl3 complex becomes a non-specific RNase to produce RNA cleavage products that can be detected using a reporter RNA, for example, a short quenched-fluorescent RNA.
  • the Cast 3 and crRNA are incubated for a period of time to form the inactive complex.
  • the Cast 3 and crRNA complexes are formed by incubating together at 37 °C for 30 minutes, 1 hour, or 2 hours (for example, 0.5 to 2 hours) to form an inactive complex.
  • the inactive complex can then be incubated with the reporter RNA.
  • a reporter RNA is provided by the RNase Alert system.
  • the sample RSV RNA can be a ssRNA activator.
  • the Casl3/crRNA with the RSV RNA sample becomes an activated complex that cleaves in cis and trans.
  • the activated complex can cleave RSV RNA.
  • the activated complex can cleave the reporter RNA, thereby releasing a signal such as the fluorophore from the reporter RNA.
  • a CRISPR guide RNA system can be adapted for use in the methods and compositions described herein.
  • the guide RNAs can include: a CRISPR RNA (crRNA or spacer), which is a 17-20 nucleotide sequence complementary to the target DNA, and a trans-activating crRNA (tracrRNA or stem) that is a binding scaffold for the Cas nuclease.
  • the tw o RNAs are fused to make a single guide RNA (sgRNA).
  • the tracrRNA forms a stem loop that is recognized and bound by the Cas nuclease.
  • the term '‘guide RNA" as used herein refers to either a single guide RNA (sgRNA) or a crRNA (spacer).
  • the CRISPR technique is generally described, for example, by Mali et al. Science 339:823-6 (2013); which is incorporated by reference herein in its entirety.
  • the at least one CRISPR guide RNA has a sequence with at least 95% sequence identity to any of SEQ ID NOs: 1-6, 10-199, 201-206, or 210-399, shown below.
  • at least one CRISPR guide RNA has a sequence such as any of SEQ ID NOs: 1-6, 10-199. 201-206. or 210-399, or a nucleotide sequence with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto, or a combination thereof.
  • the sample can be incubated with one or two or more crRNAs.
  • the at least one CRISPR guide RNA include a targeting sequence with at least 80% sequence identity to any of SEQ ID NOs: 11-6, 10-199, 201-206. or 210-399, shown below.
  • at least one CRISPR guide RNA include a targeting sequence such as any of SEQ ID NOs: 10-99 or 100-190, or a combination thereof.
  • the sample can be incubated with one or two or more crRNAs.
  • the sample can be incubated with at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least nine, or at least ten, or more crRNAs.
  • the at least one crRNA has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more sequence identity to any SEQ ID NOs: 1-6, 10-199, 201-206, or 210-399.
  • the targeting sequence in at least one crRNA has at least about 70%. about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more sequence identity to any SEQ ID NO:10-99 or 100-190.
  • 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more crRNAs specific for one or more viruses are employed in a method to detect viruses such as RSV in a physiological sample.
  • the crRNA(s) can include those with SEQ ID NOs: 100-199 or 300-389, or a nucleotide sequence with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto, or a combination thereof.
  • SEQ ID NOs: 191, 192, 196 or 199 can be combined to improve detection of RSV B.
  • the crRNA(s) can include those with SEQ ID NOs: 1-6, 10-99, 201-206 or 210-299, or a nucleotide sequence with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto, or a combination thereof.
  • the crRNA(s) can include those with SEQ ID NOs: 2, 4 or 6.
  • the amount of reporter RNA cleavage product detected is directly correlated with the amount of the target viral RNA.
  • the target viral RNA cleavage product concentration can be quantified or determined by use of a standard curve of the reporter RNA cleavage product(s).
  • At least one crRNA can bind to a region in any of the eight single-stranded RNAs of the RSV RNA genome.
  • the region is a single stranded region of the RSV RNA genome.
  • the region is a secondary structure in regions of the RSV genome with low viral ribonucleoprotein binding.
  • the crRNAs can include additional sequences such as spacer sequences.
  • crRNAs with a sequence of SEQ ID NOs: 191, 192, 196 or 199 exhibit better signals than crRNAs with a sequence of SEQ ID NOs: 193, 194, 195, 197 or 198.
  • the combination of the crRNAs of SEQ ID NOs: 191, 192, 196 and 199 improves detection of RSV B over using crRNAs of SEQ ID NOs: 191, 192, 196 or 199 alone.
  • crRNAs with a sequence of SEQ ID NOs: 2, 4, or 6 exhibit better signals than crRNAs with a sequence of SEQ ID NOs: 1, 3 or 5. Moreover, the combination of the crRNAs of SEQ ID NOs: 2, 4, and 6 improves detection of RSV A over using the crRNAs of SEQ ID NOs: SEQ ID NOs: 2, 4, or 6 alone.
  • the crRNAs may be employed to detect any RSV A or RSV B genome.
  • Exemplary RSV A and RSV B genomes include but not limited to human respiratory' syncytial virus isolate B05 (gi
  • the RSV genome is RNA.
  • the RSV genome can be a copy of the foregoing DNA sequence, where the thymine (T) residues are uracil (U) residues.
  • the RSV viral genome can be a complement of the foregoing DNA sequence.
  • the Cast 3 protein can complex with at least one CRISPR guide RNA (crRNA) to at least one reporter RNA for a period of time sufficient to form at least one RNA cleavage product.
  • crRNA CRISPR guide RNA
  • the Cast 3 protein can, for example, be a Cas 13a protein, Cas 13b protein, or a combination thereof.
  • Casl3 contains two Higher Eukary otes and Prokaryotes Nucleotide-binding (HEPN) domains for RNA cleavage, consistent with known roles for HEPN domains in other proteins.
  • the Cas 13 proteins can have sequence variation and/or be from other organisms.
  • the Casl3 proteins can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity' to any of the Cas 13 sequences disclosed herein or to a Cas 13 in the following bacteria: Leptotrichia wadei, Leptotrichia buccalis, Rhodobacter capsulatus, Herbinix hemicellulosilytica. Leptotrichia buccalis (Lbu), Listeria seeligeri, Paludibacter propionicigen.es. Lachnospiraceae bacterium, [Eubacterium] rectale, Listeria newyorkensis, Clostridium aminophilum, and/or Leptotrichia shahii.
  • ⁇ Leptotrichia wadei Casl3a endonuclease that can be used has the following sequence (SEQ ID NO: 500; NCBI accession no. WP_036059678. 1).
  • a Herbmix hemicellulosifytica Cast 3a endonuclease that can be used has the following sequence (SEQ ID NO: 501; NCBI accession no. WP_103203632.1).
  • a Leptotrichia buccalis Cast 3a endonuclease that can be used has the following sequence (SEQ ID NO: 502; NCBI accession no. WP_015770004.1).
  • a Leptotrichia seeligeri Casl3a endonuclease that can be used has the following sequence (SEQ ID NO:503; NCBI accession no. WP_012985477.1).
  • a Paludibacter propionicigenes Casl3a endonuclease can be used that has the following sequence (SEQ ID NO: 504; NCB1 accession no. WP_013443710.1).
  • aLachnospiraceae bacterium Casl3a endonuclease can be used that has the following sequence (SEQ ID NO: 505; NCBI accession no. WP_022785443.1).
  • aLeptotrichia shahii Cas 13a endonuclease can be used that has the following sequence (SEQ ID NO: 506; NCBI accession no. BBM39911.1). 1 MGNLFGHKRW YEVRDKKDFK IKRKVKVKRN YDGNKYILNI
  • a Leptotrichia buccalis C-1013-b Casl3a endonuclease can have the following sequence (SEQ ID NO: 507; NCBI accession no. C7NBY4; AltName LbuC2c2).
  • Casl3b may work faster in atarget viral RNA detection assay than Casl3a.
  • a Casl3b from Prevotella buccae can be used in the RNA detection methods, compositions and devices.
  • a sequence for a Prevotella buccae Casl3b protein (NCB1 accession no. WP_004343973.1) that can be used is shown below- (SEQ ID NO:508).
  • Such aPrevotella buccae Casl3b protein can have a K m (Michaelis constant) substrate concentration of about 20 micromoles and a K ca t of about 987/second (see, e.g., Slaymaker et al. Cell Rep 26 (13): 3741-3751 (2019)).
  • NCB1 accession no. WP 004343581.1 Another Prevotella buccae Casl3b protein (NCB1 accession no. WP 004343581.1) that can be used in the RSV RNA detection methods, compositions and devices has the sequence shown below (SEQ ID NO: 509).
  • An example of a Bergeyella zoohelcum Casl3b (R1177A) mutant sequence (NCBI accession no. 6AAY_A) that can be used is shown below (SEQ ID NO: 510).
  • EKSENKN or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity 7 thereto.
  • the sample can be incubated with at least one CRISPR RNA (crRNA) and at least one Casl3 protein.
  • the Casl3 protein can, for example, be a Casl3a protein.
  • Cetobacterium somerae Casl3c sequence (NCBI Ref. Sequence WP 266188461 1) that can be used is shown below (SEQ ID NO: 512).
  • NCBI Halarcobacter ebronensis Casl3c sequence
  • NCBI Malaciobacter hal ophilus Casl3c sequence
  • Tissierella sp. P1 Casl3c sequence (NCBI Reference Sequence: WP 255374260.1) having SEQ ID NO: 515 that can be used is shown below
  • Fusibacter paucivorans Casl3c sequence (NCBI Reference Sequence: WP ...213237188.1 ) having SEQ ID NO: 516 that can be used is:
  • WP . 162611874.1 SEQ ID NO:578 is:
  • Genomic editing has been performed by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems (see e.g., Marraffini and Sontheimer. Nature Reviews Genetics 11: 181-190 (2010); Sorek et al. Nature Reviews Microbiology 2008 6: 181-6; Karginov and Hannon. Mol Cell 2010 1 :7-19; Hale et al. Mol Cell 2010:45:292-302; Jinek et al. Science 2012 337:815-820; Bikard and Marraffini Curr Opin Immunol 2012 24: 15-20; Bikard et al. Cell Host & Microbe 2012 12: 177-186; all of which are incorporated by reference herein in their entireties).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas CRISPR-associated
  • a CRISPR guide RNA system can be adapted for use in the methods and compositions described herein.
  • Two RNAs can be used in CRISPR genomic editing systems: a CRISPR RNA (crRNA), which is a 17-20 nucleotide sequence complementary to the target RNA, and a trans-activating crRNA (tracrRNA) that is a binding scaffold for the Cas nuclease.
  • crRNA CRISPR RNA
  • tracrRNA trans-activating crRNA
  • the tracrRNA forms a stem loop that is recognized and bound by the cas nuclease.
  • the crRNA typically has shorter sequence than the tracrRNA.
  • guide RNA refers to either a single guide RNA (sgRNA) or a crRNA.
  • sgRNA single guide RNA
  • crRNA crRNA
  • the CRISPR technique is generally described, for example, by Mali et al. Science 339:823-6 (2013); which is incorporated by reference herein in its entirety 7 .
  • the guide RNA system used herein is encoded within or adjacent to the ncRNA coding region of the expression cassettes. Hence, upon transcription of the guide RNA, it can target a Cas enzyme to the desired location in the genome, where it can cleave the genomic RNA for generation of a genomic modification.
  • a "guide RNA” or “gRNA” as provided herein refers to a ribonucleotide sequence capable of binding a cas nuclease, thereby forming ribonucleoprotein complex.
  • the gRNA includes a nucleotide sequence complementary to a target site (e.g., near or at a genomic site to be edited).
  • the guide RNA includes one or more RNA molecules. TracrRNAs can be used to facilitate assembly of a ribonucleoprotein complex that includes the gRNA together with the tracrRNA and a cas nuclease.
  • a complementary nucleotide sequence of the guide RNA can mediate binding of the ribonucleoprotein complex to the target site thereby providing the sequence specificity of the ribonucleoprotein complex.
  • the guide RNA includes a sequence that is complementary to a target nucleic acid sequence such that the guide RNA binds a target nucleic acid sequence.
  • the complement of the guide RNA includes a sequence having a sequence identity of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to a target nucleic acid (e.g., a target viral RNA sequence).
  • the guide RNA includes a sequence having sequence identity of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to the target nucleic acid sequence.
  • the guide RNA or complement thereof includes a sequence having a sequence identity of at least about 90%, 95%, or 100% to a target viral RNA sequence.
  • segment bound by a guide RNA within the target nucleic acid is about or at least about 10, 15, 20, 25, or more nucleotides in length.
  • the guide RNA is a single-stranded ribonucleic acid, although in some cases it may form some double-stranded regions by folding onto itself.
  • the guide RNA is about 10, 20. 30. 40. 50, 60, 70, 80, 90, 100 or more nucleic acid residues in length.
  • the guide RNA is from about 10 to about 30 nucleic acid residues in length.
  • the guide RNA is about 20 nucleic acid residues in length.
  • the length of the guide RNA can be at least about 5, 6, 7, 8, 9, 10, 11, 12. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. 24, 25, 26, 27, 28, 29, 30. 31. 32.
  • the guide RNA is from 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 5 to 75, 10 to 75, 15 to 75, 20 to 75, 25 to 75, 30 to 75, 35 to 75, 40 to 75, 45 to 75, 50 to 75, 55 to 75, 60 to 75, 65 to 75, 70 to 75, 5 to 100, 10 to 100, 15 to 100, 20 to 100, 25 to 100, 30 to 100, 35 to 100, 40 to 100, 45 to 100, 50 to 100, 55 to 100, 60 to 100, 65 to 100, 70 to 100, 75 to 100, 80 to 100, 85 to 100, 90 to 100, 95 to 100, or more nucleotides or residues in length. In some cases, the guide RNA is from 10 to 15, 10 to 20, 10 to 30. 10 to 40. or 10 to 50 residues in length.
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, bacterial, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.
  • recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • the polynucleotide of interest is cloned and then expressed in transformed organisms, for example, as described herein.
  • the host organism expresses the foreign nucleic acids to produce the RNA, RT- DNA, or protein under expression conditions.
  • a "cell” refers to any type of cell isolated from a prokaryotic, eukaryotic, or archaeon organism, including bacteria, archaea, fungi, protists, plants, and animals, including cells from tissues, organs, and biopsies, as well as recombinant cells, cells from cell lines cultured in vitro, and cellular fragments, cell components, or organelles comprising nucleic acids.
  • the term also encompasses artificial cells, such as nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids.
  • the methods described herein can be performed, for example, on a sample comprising a single cell or a population of cells.
  • the term also includes genetically modified cells.
  • Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.
  • a "coding sequence” or a sequence which "encodes” a selected polypeptide or a selected RNA is a nucleic acid molecule which is transcribed (in the case of DNA templates) into RNA and/or translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”).
  • the boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence can include, but is not limited to, ncRNAs, tracrRNAs, ncRNAs modified to include heterologous sequences, cDNA from viral, prokaryotic or eukaryotic ncRNA, mRNA, viral or prokaryotic DNA. and even synthetic DNA sequences.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • control elements include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.
  • “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper polymerases are present.
  • the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • Encoded by refers to a nucleic acid sequence which codes for a polypeptide or RNA sequence.
  • the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.
  • the RNA sequence or a portion thereof contains a nucleotide sequence of at least 3 to 5 nucleotides, more preferably at least 8 to 10 nucleotides, and even more preferably at least 15 to 20 nucleotides.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein, DNA, or RNA or cause other adverse consequences.
  • nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when obtained from nature or when produced by recombinant DNA techniques, or free from chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • substantially purified generally refers to isolation of a substance (nucleic acid, compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises 50%, or 80%-85%, or 90-95% of the sample.
  • a “vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • target cells e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • vector construct e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • expression vector e transfer vector
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • “Expression” refers to detectable production of a gene product by a cell.
  • the gene product may be a transcription product (i.e., RNA), which may be referred to as “gene expression”, or the gene product may be a translation product of the transcription product (i.e., a protein), depending on the context.
  • “Mammalian cell” refers to any cell derived from a mammalian subject suitable for transfection with vector systems comprising, as described herein.
  • the cell may be xenogeneic, autologous, or allogeneic.
  • the cell can be a primary’ cell obtained directly from a mammalian subject.
  • the cell may also be a cell derived from the culture and expansion of a cell obtained from a mammalian subject. Immortalized cells are also included within this definition.
  • the cell has been genetically engineered to express a recombinant protein and/or nucleic acid.
  • subject includes animals, including both vertebrates and invertebrates, including, without limitation, invertebrates such as arthropods, mollusks, annelids, and cnidarians; and vertebrates such as amphibians, including frogs, salamanders, and caecillians; reptiles, including lizards, snakes, turtles, crocodiles, and alligators; fish; mammals, including human and non-human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows; and birds such as domestic, wild and game birds, including chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
  • the disclosed methods find use of the disclosed methods, find
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • a “therapeutically effective amount” or “efficacious amount” refers to the amount of a compound that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease.
  • the “therapeutically effective amount” will vary depending on the compound or the cell, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • Treatment includes the administration of antiviral, such as protease inhibitors (darunavir, atazanavir, and ritonavir), viral DNA polymerase inhibitors (acyclovir, valacyclovir, valganciclovir, and tenofovir), and/or an integrase inhibitor (raltegravir).
  • Treatment also includes treatment for RSV, such as monoclonal antibodies (e.g., palivizumab) and/or ribavirin. Treatment can also include oxygen supplementation (e.g., oxygen given through a mask), suctioning of mucus, bronchodilator agents, and/or mechanical ventilation.
  • RSV such as monoclonal antibodies (e.g., palivizumab) and/or ribavirin.
  • Treatment can also include oxygen supplementation (e.g., oxygen given through a mask), suctioning of mucus, bronchodilator agents, and/or mechanical ventilation.
  • Gene transfer refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of nonintegrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g.. episomes). or integration of transferred genetic material into the genomic DNA of host cells.
  • Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non-viral vectors, alphaviruses, pox viruses and vaccinia viruses.
  • derived from is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.
  • a polynucleotide or nucleic acid "derived from” a designated sequence refers to a polynucleotide or nucleic acid that includes a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence.
  • the derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.
  • hybridize and “hybridization” refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson-Crick base pairing.
  • homologous region refers to a region of a nucleic acid with homology to another nucleic acid region. Thus, whether a "homologous region" is present in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since a nucleic acid is often double-stranded, the term “homologous, region.” as used herein, refers to the ability of nucleic acid molecules to hybridize to each other. For example, a single-stranded nucleic acid molecule can have two homologous regions which are capable of hybridizing to each other. Thus, the term “homologous region” includes nucleic acid segments with complementary sequences.
  • Homologous regions may vary in length but will typically be between 4 and 500 nucleotides (e.g., from about 4 to about 40, from about 40 to about 80, from about 80 to about 120, from about 120 to about 160, from about 160 to about 200, from about 200 to about 240, from about 240 to about 280, from about 280 to about 320, from about 320 to about 360, from about 360 to about 400. from about 400 to about 440, etc.).
  • nucleotides e.g., from about 4 to about 40, from about 40 to about 80, from about 80 to about 120, from about 120 to about 160, from about 160 to about 200, from about 200 to about 240, from about 240 to about 280, from about 280 to about 320, from about 320 to about 360, from about 360 to about 400. from about 400 to about 440, etc.
  • complementary refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine.
  • uracil when uracil is denoted in the context of the present invention, the ability’ to substitute a thymine is implied, unless otherwise stated.
  • “Complementarity” may exist between two RNA strands, two DNA strands, or between an RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be “complementary” and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary” or " 100% complementary” if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region.
  • Two or more sequences are considered “perfectly complementary” or “100% complementary” even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other.
  • "Less than perfect” complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity’ are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art.
  • donor polynucleotide or ’‘donor DNA” refers to a nucleic acid or polynucleotide that provides a nucleotide sequence of an intended edit to be integrated into the genome at a target locus by HDR or recombineering.
  • a “target site” or “target sequence” is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide (donor DNA).
  • the target site may be allele-specific (e.g., a major or minor allele).
  • a target site can be a genomic site that is intended to be modified such as by insertion of one or more nucleotides, replacement of one or more nucleotides, deletion of one or more nucleotides, or a combination thereof.
  • a CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas") genes, including sequences encoding a Cas gene, and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence.
  • CRISPR-associated genes including sequences encoding a Cas gene, and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence.
  • one or more elements of a CRISPR system are derived from a type I, type II, or type III CRISPR system.
  • Casl and Cas2 are found in all three types of CRISPR-Cas systems, and they are involved in spacer acquisition. In the I-E system of E. coli, Casl and Cas2 form a complex where a Cas2 dimer bridges two Casl dimers.
  • Cas2 performs anon-enzymatic scaffolding role, binding double-stranded fragments of invading DNA, while Casl binds the single-stranded flanks of the DNA and catalyzes their integration into CRISPR array s.
  • one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes .
  • a CRISPR system can be characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
  • the disclosure provides protospacers that are adjacent to short (3 - 5 bp) DNA sequences termed protospacer adjacent motifs (PAM).
  • PAMs are important for type I and type II systems during acquisition.
  • type I and type II systems protospacers are excised at positions adjacent to a PAM sequence, with the other end of the spacer is cut using a ruler mechanism, thus maintaining the regularity of the spacer size in the CRISPR array.
  • the conservation of the PAM sequence differs between CRISPR-Cas systems and may be evolutionarily linked to Casl and the leader sequence.
  • a regulatory element is operably linked to one or more elements of a CRISPR system so as to drive expression of the one or more elements of the CRISPR system.
  • CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
  • SPIDRs Sacer Interspersed Direct Repeats
  • the CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. coli (Ishino et al, J. BacterioL. 169:5429-5433 (1987); and Nakata et al., J.
  • the CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al, OMICS J. Integ. Biol., 6:23-33 (2002); and Mojica et al, Mol. Microbiol., 36:244-246 (2000)).
  • SRSRs short regularly spaced repeats
  • the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al., (2000), supra).
  • CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al, Mol.
  • an enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog. or non-human primate.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g.
  • Codon bias differences in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • Codon usage tables are readily available, for example, at the "Codon Usage Database", and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).
  • Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available.
  • one or more codons e.g., 1, 2, 3. 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons e.g., 1, 2, 3. 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons e.g., 1, 2, 3. 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • administering comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.
  • crRNAs targeting different genomic regions with: specific on-target binding to viral genomes no off-target binding (human or microbial (viruses, bacteria)) appropriate crRNA properties (structure. GC content, etc.)
  • NCBI Public database
  • crRNAs for RSV A and 9 crRNA for RSV B were selected (see SEQ ID NOs: 1-6 and 191-199).
  • crRNAs CRISPR RNA guides
  • Example 4 Exemplary crRNA sequences GL2022-016-W02; BK-2023-052-2 // 3730.221WO1
  • Example 5 crRNAs specific for RSV A or RSV B may be employed with a point of care (POC) device to provide an independent diagnostic for RSV A and RSV B viruses or one that is multiplexed diagnostic with, for example, SARS-CoV-2 and/or influenza viruses (e.g., SARS-CoV-2 +/- Influenza +/- RSV A and B).
  • the POC diagnostic may include a microfluidics cartridge and a device that may include an insert for a swab, assay reagents, and/or a high intensity light source to excite and measure fluorescence.
  • the cartridge includes 3 reaction chambers, each measuring a specific target (e.g., viral RNA, host RNA, no RNA) from the same sample. Additional reaction chambers may measure multiple targets within a single sample. This enables an at-home diagnostic and may be used in a droplet-based assay or other embodiment of direct detection.
  • a bead comprises Casl3, one or more crRNAs and one or more reagents for a CRSPR/Cas assay.
  • Casl3, one or more crRNAs and/or one or more reagents maybe on the surface of the bead, embedded within the bead, or both.
  • the bead may include lyophilized Casl3, one or more crRNAs and/or one or more reagents.
  • the assay is a droplet assay.
  • the assay is a digital PCR assay.
  • the reporter is a luminescent reporter.
  • the reporter comprises a fluorophore.
  • the reporter comprises a quencher.
  • the reporter comprises a fluorophore and a quencher.
  • the reporter comprises a caged compound.
  • the reporter comprises a metal, e.g., a gold particle.
  • the assay detects fluorescence (a fluorescent signal).
  • the assay detects luminescence (a luminescent signal).
  • luminescence may be detected by employing via a split luciferase system that comes together following RNA reporter cleavage by Cast 3a.
  • the assay detects an electrochemical signal.
  • the assay is PCR-based assay, which employs a reporter that may include hairpin structure such as a molecular beacon where cleavage opens the hairpin to reveal a longer template.
  • a reporter that may include hairpin structure such as a molecular beacon where cleavage opens the hairpin to reveal a longer template.
  • On-target activity screen for was performed for crRNAs targeting RSV A and B viruses.
  • the screen results are summarized in the graphs in Figures 12 and 13.
  • a set of top hits were identified for validation of their on-target activity and evaluation of their off-target activity.
  • the hits ( ⁇ 30 crRNAs with high on-target activity for RSV A and ⁇ 40 crRNAs with high on-target activity for RSV B) are highlighted for each of the viruses in Figures 12A-12B and 13A-13B.
  • crRNA combinations were evaluated for RSV A and B viruses.
  • an in-silico crRNA screen was performed to determine which crRNAs target which subtype or strain of RSV A and B viruses and to analyze off-targets of the crRNAs.
  • microbial interference tests were performed where the genomes of all viruses, other respiratory pathogens and normal nasal flora organisms were analyzed to demonstrate less than 80% homology with guide target sequences.
  • the number of hits w ere reported with different lowest-common ancestor (LCA) levels. If a guide is homologous to genomes from multiple species, the LCA of those genomes was reported. However, if a guide is homologous exclusively to only one species, then the LCA of that guide is that species.
  • LCA lowest-common ancestor
  • HCSt RefSsq compete baswA g «nom$ tmbtjw: 05W2C20 O ⁇ wfea ⁇ m, 17404 baeiehal gensmss..
  • C2c2 is a singlecomponent programmable RNA-guided RNA-targeting CRISPR effector. Science. 353(6299): 353, aaf5573.
  • Cpfl is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 163(3), 759-771. Published online 2015/10/01 DOI: 10. 1016/j.cell.2015.09.038.
  • a method comprising:
  • sample suspected of containing the target viral RNA is saliva, sputum, mucus, nasophary ngeal materials, blood, serum, plasma, urine, aspirate, biopsy tissue, or a combination thereof.
  • sample suspected of containing RNA is a lysed biological sample.
  • the detector comprises a light detector, a fluorescence detector, a color filter, an electronic detector, an electrochemical signal detector, an electrostatic signal detector, a steric signal detector, a van der Waals interaction signal detector, a hydration signal detector, a Resonant frequency shift signal detector, or a combination.
  • control assay contains no target viral RNA.
  • control assay contains viral RNA that is not the target viral RNA.
  • RNA from a common cold coronavirus SARS-CoV2, hepatitis virus, or human immunodeficiency virus (HIV).
  • hepatitis virus is hepatitis C virus (HCV).
  • a kit comprising a package containing at least one Casl3 protein, at least one CR1SPR guide RNA (crRNA) that binds a target site in at least one of an RSV A or RSV B nucleic acid, at least one reporter RNA, and instructions for detecting and/or quantifying the target viral RNA in a sample.
  • crRNA CR1SPR guide RNA
  • kits of statement 32 wherein the at least one CRISPR guide RNA (crRNA) has a sequence with at least 95% sequence identity to any of SEQ ID NO: 1-6, 10-199, 201-206, or 210-399.
  • crRNA CRISPR guide RNA
  • crRNAs CRISPR guide RNAs
  • crRNA CRISPR guide RNA
  • crRNA CRISPR guide RNA
  • kits of statement 32, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 2. 4 and 6.
  • kits of statement 32 wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 191, 192, 196, or 199, or a combination thereof.
  • crRNA CRISPR guide RNA
  • kits of statement 32 wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 191, 192, 196 or 199. or a combination thereof.
  • crRNA CRISPR guide RNA
  • kits of statement 28 wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 191, 192, 196 and 199.
  • crRNA CRISPR guide RNA
  • crRNA CRISPR guide RNA
  • the reporter RNA reporter comprises at least one fluorophore and at least one fluorescence quencher.
  • kit of statement 45 wherein the at least one fluorophore is Alexa 430, STAR 520.
  • kits of statement 32 further comprising a detector.
  • An isolated nucleic acid comprising, consisting of or consisting essentially of any of

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Abstract

The present disclosure relates to methods using CRISPR-Casl3 enzyme, complexed with RSV A or RSV B crRNA guide RNAs to detect and quantify the presence of RS V A or RSV B RNA in a sample with enhanced specificity and sensitivity. These methods can be used to diagnose RSV A or RSV B infection, quantify the concentration of RSV A or RSV B RNA present in a sample, and identify the presence of different RSV A subtypes or mutations.

Description

DIAGNOSTIC USING CRISPR RNA AND CAS 13 A ENZYME
PRIORITY
This application claims the benefit of priority to U.S. Provisional Application Ser. No. 63/441,213, filed January 26, 2023, which is incorporated by reference herein as if fully set forth herein.
BACKGROUND
Detection of respiratory infections, including SARS-CoV-2 and influenza A and B, is critical for targeting locations and populations that need medical assistance. For example, the estimated U.S. influenza illnesses in the 2019-2020 season was approximately 38 million people. In that same influenza season, approximately 400,000 people were hospitalized and approximately 22.000 died from the disease. Additionally. Respiratory Syncytial Virus (RSV) infections are rampant and result in many hospitalizations. For example, in 2015, the estimated global acute lower respiratory infections caused by RSV included 3.2 million young children and 336,000 older adults resulting in over 73,000 deaths.
Current respiratory' virus diagnostic assays include RT-qPCR nucleic-acid based tests (NATs) that require lab-based equipment and personnel or rapid diagnostics (RIDTs) that detect viral antigens. These assays are not quantitative or multiplexed with other relevant respiratory viruses. These assays are also not appropriate for use by inexperienced or untrained personnel, such as for at home use.
SUMMARY
A rapid, easy-to-use detection assay for viral RNA from respiratory' body fluid samples is needed for identifying respiratory infections. For instance, current RSV diagnostic assays include RT-qPCR nucleic-acid based tests or antigen detecting tests that require lab-based equipment and personnel.
Descnbed herein are methods, compositions, and devices for detecting and quantifying target viral RNA, such as RSV, that are faster and more readily deployable in the field than currently available methods and devices. In addition, the methods, compositions and devices can readily detect and distinguish between strains and variants of the target viral RNA.
The methods described herein can include: (a) incubating a sample suspected of containing RSV RNA or virus yvith one or more Casl3 protein(s), at least one CRISPR guide RNA (crRNA), and at least one reporter, e.g., reporter RNA, for a period of time sufficient to form at least one detectable product, such as a detectable RNA cleavage product; and (b) detecting the reporter, e.g., a reporter RNA cleavage product, with a detector. Such methods are useful for detecting whether the sample contains one or more copies of RSV RNA. The methods are also useful for detecting the absence of infection with the virus carry ing the target viral RNA. Moreover, the methods and compositions described herein can also readily identify whether a variant or mutant strain of virus carrying the target viral RNA is present in a sample, and determining what the variant or mutation is.
The methods described herein are useful for diagnosing RSV infections in a variety' of complex biological samples. For example, the samples can include human saliva, sputum, mucus, nasopharyngeal materials, blood, serum, plasma, urine, aspirate, biopsy tissue, or a combination thereof.
In one embodiment, the assay is a single-step POC, or at-home assay based on CRISPR/Casl3a technology' that enables direct detection of RSV RNA following lysis and provides quantitative information for viral load. In one embodiment, the assay does not include reverse transcription and amplification.
DESCRIPTION OF THE FIGURES
Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale. Instead, emphasis is placed on clearly illustrating the principles of the present disclosure. Furthermore, components can be shown as transparent in certain views for clarify of illustration only and not to indicate that the illustrated component is necessarily transparent.
FIGs. 1A-1B illustrates use of CRISPR-Casl3 and CRISPR guide RNAs (crRNAs) to detect target RNA. FIG. 1A is a schematic diagram illustrating CRISPR-Casl3 detection of target viral RNA using a CRISPR-Casl3 protein that binds CRISPR guide RNAs (crRNA) to form a ribonucleoprotein (RNP) complex. The crRNA targets or guides the CRISPR-Casl3 protein to target viral RNA sequences, where the Cast 3 protein is activated to cleave RNA, including the reporter RNA. FIG. IB is a similar schematic diagram further illustrating a Cas 13a: crRNA ribonucleoprotein (RNP) complex binding of target viral RNA, resulting in activation of the Casl3a nuclease (denoted by scissors). Upon target recognition and RNP activation, Casl3a indiscriminately cleaves a quenched-fluorophore RNA reporter, allowing for fluorescence detection as a proxy for Casl3a activation and the presence of target RNA.
FIG. 2 is a schematic diagram illustrating methods for detection of the SARS-CoV-2 RNA genome and fluorescent detection of reporter RNA. CRISPR guide RNAs (crRNA) that can target or bind to SARS-CoV-2 RNA are used. As illustrated, in a first step the CRISPR-Casl3 protein binds CRISPR guide RNAs (crRNA) to form a ribonucleoprotein (RNP) complex. The RNP complex is inactive but, when mixed with the sample to be tested, binding of the RNP complex to the SARS-CoV-2 RNA in the sample activates the Cast 3 protein to cut RNA, including reporter RNA molecules added to the assay mixture. Cleavage of the reporter RNA leads to fluorescence, which can be detected by a fluorescence detector.
FIG. 3 illustrates a point-of-care (POC) method for detecting viruses. As illustrated, a sample can be collected (e g., a patient’s saliva, sputum, mucus, or nasopharyngeal sample), the cells and/or viruses in the sample can be lysed to release any viral RNA that may be present, and the RNA from the sample can be mixed with reporter RNAs and a CRISPR-Casl3 protein-crRNA ribonucleoprotein (RNP) complex. Background fluorescence from control reactions can be subtracted and the fluorescence of the sample can be detected. Detection can be by a fluorometer or other suitable device. Such point-of-care detection allows mobilization of medical support and medical personnel.
FIG. 4 shows the validation and cross-reactivity of exemplary RSV A RNA guides against host RNA and nasal swabs (signal slope over 2 hours).
FIG. 5 graphically illustrates that signal slopes from each reaction of target viral RNA for RSV A, or RNP alone with the RNA guides alone or combined.
FIG. 6 show the validation and cross-reactivity of exemplary RSV B RNA guides against host RNA and nasal swabs (signal slope over 2 hours).
FIG. 7 provides an exemplary RSV A consensus sequence (SEQ ID NO:401) which may be employed to prepare crRNAs specific for a large number of RSV A isolates.
FIG. 8 provides an exemplary RSV B consensus sequence (SEQ ID NO:406) which may be employed to prepare crRNAs specific for a large number of RSV B isolates.
FIG. 9 provides a summary of exemplary crRNAs (SEQ ID NOS: 1108-1152).
FIG. 10 provides results for 96 crRNAs tested for detection of RSV A RNA.
FIG. 11 provides results for 99 crRNAs tested for detection of RSV B RNA.
FIGS. 12A-12B. RSV A virus: 129 crRNAs screened.
FIGS. 13A-13B. RSV B virus crRNA screen results.
FIG. 14. RSV A crRNA off target activity results.
FIG. 15. RSV B crRNA off target activity results.
FIG. 16. RSV A crRNA off target activity with nasal swabs results.
FIG. 17. RSV B crRNA off target activity with nasal swabs results.
FIG. 18. crRNA off-target activity against non-targeted viral RNA RSV A results. FIG. 19. crRNA off-target activity’ against non-targeted viral RNA RSV B results.
FIGS. 20 and 21. Tested crRNA off-target activity7 was tested against non-targeted viral RNA. FIG 20 demonstrates evaluating combinations RSV A, while FIG. 21 demonstrates evaluating combinations RSV B (10g yields high signal).
FIG. 22. RSV B: testing of crRNA off-target activity against non-targeted viral RNA. Most crRNAs have minimal off-target activity against non-targeted viruses.
FIG. 23. RSV A and B: crRNA combinations were evaluated, including combinations RSVAB_9g+crl2 and RSVAB 11g.
FIGS. 24A-24B. RSV A virus LOD: 12 infectious particles (pfu) per ml are detected with both 10g and 11g crRNA combinations.
FIGS 25A-25B. RSV B virus LOD: 1 to 4.5 infectious particles (pfu) per ml are detected with both 11g and 10g crRNA combinations, respectively.
DETAILED DESCRIPTION
Methods, kits and devices are described herein for rapidly detecting and/or quantifying virus infection. The methods can include (a) incubating a sample suspected of containing RNA or virus with one or more Cas 13 protein, at least one CRISPR guide RNA (crRNA) that binds a target site in at least one of RSV A or RSV B nucleic acid, and at least one reporter RNA for a period of time sufficient to form at least one RNA cleavage product(s); and (b) detecting level(s) of reporter RNA cleavage product(s) with a detector. Such methods are useful for detecting whether the sample contains one or more copies of RSV RNA. The methods are also useful for detecting the absence of an RSV infection.
In some aspects, the disclosure provides methods for identifying the target virus RNA from a sample suspected of containing the target viral RNA. The target virus RNA can be from any RNA vims selected for detection in a sample. In some aspects, the target viral RNA can be from a virus that causes a respiratory7 infection or establishes its primary7 infection in the tissues and fluids of the upper respiratory tract. For example, the RNA virus can be RSV, such as RSV A or B. RSV is a negative sense, single stranded RNA virus.
In addition to RSV, other targets may’ be identified. In one embodiment, the target viral RNA can be common cold coronaviruses, such as strains NL63, OC43, or 229E. The target viral RNA can also be SARS-CoV-2, a hepatitis virus (e.g., HCV), or influenza vims, influenza virus A or influenza virus B. In some cases, the target viral RNA can be from the human immunodeficiency virus (HIV). The methods can thus be used to detect and identify a combination of viral RNAs, for example, using methods and components described in any of PCT publications WO 2020/051452; WO 2021/188830; and WO 2022/046706, each of which is incorporated by reference herein in its entirety.
In some aspects, provided herein are methods for diagnosing the presence or absence of a RSV infection comprising incubating a mixture comprising a sample suspected of containing RSV RNA, a Cast 3 protein, at least one CRISPR guide RNA (crRNA). and a reporter RNA for a period of time to form any reporter RNA cleavage product(s) that may be present in the mixture; and detecting level(s) of reporter RNA cleavage product(s) that may be present in the mixture with a detector. In some cases, the RSV RNA in a sample and/or the RNA cleavage products are not reverse transcribed prior to the detecting step. The presence or absence of an RSV infection in patient is detected by qualitatively or quantitatively detecting level of reporter RNA cleavage product(s) that may be present in the mixture.
The methods described herein have various advantages. For example, the methods described herein can directly detect RNA without additional manipulations. No RNA amplification is generally needed, whereas currently available methods (e.g., SHERLOCK) require RNA amplification to be sufficiently sensitive. The methods, kits, and devices descnbed herein are rapid, providing results within 30 minutes. Expensive lab equipment and expertise is not needed. The methods described herein are amenable to many different sample types (blood, nasal/oral swab, etc.). The methods, kits, and devices described herein are easily deployable in the field (airport screenings, borders, resource poor areas) so that potentially infected people will not need to go to hospitals and clinics where non-infected patients, vulnerable persons, and highly trained, urgently needed medical people may be. Hence, testing can be isolated from facilities needed for treatment of vulnerable populations and from trained personnel needed for urgent and complex medical procedures.
CRISPR-Casl3 is a viable alternative to conventional methods of detecting and quantifying RNA by RT-PCR. The advantages of using CRISPR-Casl3 can be leveraged for RSV diagnostics. The Cast 3 protein targets RNA directly, and it can be programmed with crRNAs to provide a platform for specific RNA sensing. By coupling it to an RNA-based reporter, the collateral or non-specific RNase activity of the Casl3 protein can be harnessed for RS V detection.
In 2017 and 2018, the laboratory of Dr. Feng Zhang reported a Casl3-based detection system that reached attomolar and zeptomolar sensitivity in detecting Zika virus, but it included an additional reverse transcription step for isothermal amplification of Zika virus cDNA, which was ultimately back-transcribed into RNA for RNA-based detection, a method referred to as SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) (Gootenberg et al. Science 356(6336):438-42 (2017); Gootenberg et al. Science 360(6387): 439-44 (2018)). Although this method improved the sensitivity of Casl3, it introduced two unwanted steps involving reverse transcription and in vitro transcription, which minimizes its potential as a field- deployable and point-of-care device.
The present disclosure provides methods and compositions for diagnosing virus infections, such as RSV infections, quantifying RNA concentrations, such as RSV RNA concentrations, and identifying the presence of different virus subtypes and/or mutations, such as RSV subtypes and/or mutations.
In some cases, the methods can be performed in a single tube, for example, the same tube used for collection and RNA extraction. This method provides a single step point of care diagnostic method. In other cases, the methods can be performed in a two-chamber system. For example, the collection swab containing a biological sample can be directly inserted into chamber one of such a two chamber system. After agitation, removal of the swab, and lysis of biological materials in the sample, the division between the two chambers can be broken or removed, and the contents of the first chamber can be allowed to flow into the second chamber. The second chamber can contain the Casl3 protein, the selected crRNA(s), and the reporter RNA so that the assay for RSV can be performed.
Chamber one can contain a buffer that would facilitate lysis of the viral particles and release of genomic material. Examples of lysis buffers that can be used include, but are not limited to PBS, commercial lysis buffers such as Qiagen RLT+ buffer or Quick Extract, DNA/RNA Shield, various concentrations of detergents such as Triton X-I00, Tween 20, NP-40, or Oleth-8, or combinations of such reagents.
Following agitation and subsequent removal of the swab, the chamber may be briefly (e.g., 2-5 mins) heated (e.g., 55 °C or 95 °C) to further facilitate lysis. Then, the division between the two chambers would be broken or removed, and the nasal extract buffer would be allowed to flow into and reconstitute the second chamber, which would contain the lyophilized reagents for the Casl3 assay (Casl3 RNPs and reporter RNA molecules).
Use of such assay tubes can provide single step point of care diagnostic methods and devices.
The methods, devices and compositions described herein for diagnosing RSV infection can involve incubating a mixture having a sample suspected of containing RSV RNA, a Cast 3 protein, at least one CRISPR RNA (crRNA), and a reporter RNA for a period of time to form reporter RNA cleavage products that may be present in the mixture and detecting a level of any such reporter RNA cleavage products with a detector. The detector can be a fluorescence detector such as a short quenched-fluorescent RNA detector, or Total Internal Reflection Fluorescence (TIRF) detector.
Exemplary Reporters
A single type of reporter RNA can be used. The reporter RNA can be configured so that upon cleavage by the Casl3 protein, a detectable signal occurs. For example, the reporter RNA can have a fluorophore at one location (e.g.. one end) and a quencher at another location (e.g., the other end). In another example, the reporter RNA can have an electrochemical moiety (e.g., ferrocene, or dye), which upon cleavage by a Cast 3 protein can provide electron transfer to a redox probe or transducer. In another example, the reporter RNA can have a reporter dye, so that upon cleavage of the reporter RNA the reporter dye is detected by a detector (e.g., spectrophotometer). In some cases, one end of the reporter RNA can be bonded to a solid surface. For example, a reporter RNA can be configured as a cantilever, which upon cleavage releases a signal. However, in other cases, a signal may be improved by use of an unattached reporter RNA (e.g.. not covalently bond to a solid surface). A surface of the assay vessel or the assay material can have a detector for sensing release of the signal. The signal can be or can include a light signal (e.g., fluorescence or a detectable dye), an electronic signal, an electrochemical signal, an electrostatic signal, a steric signal, a van der Waals interaction signal, a hydration signal, a Resonant frequency shift signal, or a combination thereof.
The reporter RNA can, for example, be at least one quenched-fluorescent RNA reporter. Such quenched-fluorescent RNA reporter can optimize fluorescence detection. The quenched- fluorescent RNA reporters include an RNA oligonucleotide with both a fluorophore and a quencher of the fluorophore. The quencher decreases or eliminates the fluorescence of the fluorophore. When the Casl3 protein cleaves the RNA reporter, the fluorophore is separated from the associated quencher, such that a fluorescence signal becomes detectable.
One example of such a fluorophore quencher-labelled RNA reporter is the RNaseAlert (IDT). RNaseAlert was developed to detect RNase contaminations in a laboratory, and the substrate sequence is optimized for RNase A species. Another approach is to use lateral flow strips to detect a FAM-biotin reporter that, when cleaved by Cast 3, is detected by anti-FAM antibody- gold nanoparticle conjugates on the strip. Although this allows for instrument-free detection, it requires 90-120 minutes for readout, compared to under 30 minutes for most fluorescence-based assays (Gootenberg et al. Science. 360(6387):439-44 (April 2018)).
The sequence of the reporter RNA can be optimized for Cast 3 cleavage. Cast 3 preferentially exerts RNase cleavage activity at exposed uridine or adenosine sites, depending on the Cast 3 homolog. There are also secondary preferences for highly active homologs. The inventors have tested 5-mer homopolymers for all ribonucleotides. Based on these preferences, various RNA oligonucleotides, labeled at the 5' and 3' ends of the oligonucleotides using an Iowa Black Quencher (IDT) and FAM fluorophore, and systematically test these sequences in the trans- ssRNA cleavage assay as described in the Examples. The best sequence can be moved into the mobile testing.
The fluorophores used for the fluorophore quencher-labelled RNA reporters can include Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof.
Exemplary Detection
Various mechanisms and devices can be employed to detect fluorescence. In some cases, the detector is a fluorescence detector, optionally a short quenched-fluorescent RNA detector, or Total Internal Reflection Fluorescence (TIRF) detector. For example, the fluorescence detector can detect fluorescence from fluorescence dyes such the Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof.
Some mechanisms or devices can be used to help eliminate background fluorescence. For example, reducing fluorescence from outside the detection focal plane can improve the signal-to- noise ratio, and consequently, the resolution of signal from the RNA cleavage products of interest. Total internal reflection fluorescence (TIRF) enables very low background fluorescence and single molecule sensitivity with a sufficiently sensitive camera. In some cases, mobile phones can be used for detection of virus, such as RSV.
In some cases, both Casl3 and reporter RNA can be tethered to a solid surface, upon addition of crRNA and RSV RNA samples, an activated Cast 3 can generate small fluorescent spots on the solid surface when imaged using Total Internal Reflection Fluorescence (TIRF). To optimize this embodiment, the fluorophore side of reporter RNA is tethered to the solid surface as well so that cleavage permits the quencher portion of the reporter RNA to diffuse away. The Cast 3 protein can be tethered to the solid surface with a tether that is long enough to allow it to cleave multiple RNA reporter molecules. Counting the bright spots emerging on the solid surface the viral load can be quantified. Use of TIRF in the portable system facilitates detection and reduces background so that the RNA cleavage product signals can readily be detected.
In some cases, a ribonucleoprotein (RNP) complex of the Cast 3 protein and the crRNA can be tethered to the solid surface. The crRNA would then not need to be added later. Instead, only the sample suspected of containing RSV RNA would need to be contacted with the solid surface. In some cases, the methods described herein can include direct detection of the target RNA in the sample, without performing further sample preparation steps prior to detection, such as depleting a portion of the sample of protein, enzymes, lipids, nucleic acids, or a combination thereof or inactivating nucleases. However, the methods described herein can include depleting a portion of the sample prior to other step(s) or inhibiting a nuclease in the sample prior to the other step(s). For example, the sample can be depleted of protein, enzymes, lipids, nucleic acids, or a combination thereof. In some cases, the depleted portion of the sample is a human nucleic acid portion. However, RNA extraction of the sample is preferably not performed.
In some cases, the methods can include removing ribonuclease(s) (RNase) from the sample. In some cases, the RNase is removed from the sample using an RNase inhibitor and/or heat.
In some cases, the Casl3 protein and/or the crRNA can be lyophilized prior to incubation with the sample. In some cases, the Cast 3 protein, the crRNA, and/or the reporter RNA is lyophilized prior to incubation with the sample.
Exemplary Sample
In some embodiments, a biological sample is isolated from a patient. Non-limiting examples of suitable biological samples include saliva, sputum, mucus, nasophary ngeal samples, blood, serum, plasma, urine, aspirate, and biopsy samples. Thus, the term "sample" with respect to a patient can include RNA. Biological samples encompass saliva, sputum, mucus, and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, washed, or enrichment for certain cell populations. The definition also includes sample that have been enriched for particular types of molecules, e.g., RNAs. The term "sample" encompasses biological samples such as a clinical sample such as saliva, sputum, mucus, nasopharyngeal samples, blood, plasma, serum, aspirate, cerebral spinal fluid (CSF), and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, and the like. A "biological sample" includes biological fluids derived from cells and/or viruses (e g., from infected cells). A sample containing RNAs can be obtained from such cells (e.g., a cell lysate or other cell extract comprising RNAs). A sample can comprise, or can be obtained from, any of a variety' of bodily fluids (e.g., saliva, mucus, or sputum), cells, tissues, organs, or acellular fluids.
In some embodiments, the biological sample is isolated from a patient known to have or suspected to have an RSV infection. In other embodiments, the biological sample is isolated from a patient not known have an RSV infection. In other embodiments, the biological sample is isolated from a patient known to have, or suspected to not have, an RSV infection. In other words, the methods and devices described herein can be used to identity subjects that have an RSV infection and to confirm that subjects do not have an RSV infection.
In some cases, it may not be known whether the biological sample contains RNA. However, such biological samples can still be tested using the methods described herein. For example, biological samples can be subjected to lysis, RNA extraction, incubation with Cast 3 and crRNAs, etc. whether or not the sample actually contains RNA, and whether or not a sample contains RSV RNA.
Pre-incubation of the crRNA and Cast 3 protein without the sample may be employed, so that the crRNA and the Casl3 protein can form a complex. In some cases, the reporter RNA can be present while the crRNA and the Cast 3 protein form a complex. However, in other cases, the reporter RNA can be added after the crRNA and the Casl3 protein already form a complex. Also, after formation of the crRNA/Cas!3 complex, the sample RNA can then be added. The sample RNA acts as an activating RNA. Once activated by the activating RNA, the crRNA/Casl3 complex becomes a non-specific RNase to produce RNA cleavage products that can be detected using a reporter RNA, for example, a short quenched-fluorescent RNA.
For example, the Cast 3 and crRNA are incubated for a period of time to form the inactive complex. In some cases, the Cast 3 and crRNA complexes are formed by incubating together at 37 °C for 30 minutes, 1 hour, or 2 hours (for example, 0.5 to 2 hours) to form an inactive complex. The inactive complex can then be incubated with the reporter RNA. One example of a reporter RNA is provided by the RNase Alert system. The sample RSV RNA can be a ssRNA activator. The Casl3/crRNA with the RSV RNA sample becomes an activated complex that cleaves in cis and trans. When cleaving in cis, for example, the activated complex can cleave RSV RNA. When cleaving in trans, the activated complex can cleave the reporter RNA, thereby releasing a signal such as the fluorophore from the reporter RNA.
CRISPR guide RNA (crRNA)
A CRISPR guide RNA system can be adapted for use in the methods and compositions described herein. The guide RNAs can include: a CRISPR RNA (crRNA or spacer), which is a 17-20 nucleotide sequence complementary to the target DNA, and a trans-activating crRNA (tracrRNA or stem) that is a binding scaffold for the Cas nuclease. In some cases, the tw o RNAs are fused to make a single guide RNA (sgRNA). The tracrRNA forms a stem loop that is recognized and bound by the Cas nuclease. The term '‘guide RNA" as used herein refers to either a single guide RNA (sgRNA) or a crRNA (spacer). The CRISPR technique is generally described, for example, by Mali et al. Science 339:823-6 (2013); which is incorporated by reference herein in its entirety.
In some cases, the at least one CRISPR guide RNA (crRNA) has a sequence with at least 95% sequence identity to any of SEQ ID NOs: 1-6, 10-199, 201-206, or 210-399, shown below. In some cases, at least one CRISPR guide RNA (crRNA) has a sequence such as any of SEQ ID NOs: 1-6, 10-199. 201-206. or 210-399, or a nucleotide sequence with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto, or a combination thereof. In some cases, the sample can be incubated with one or two or more crRNAs. In some cases, the at least one CRISPR guide RNA (crRNA) include a targeting sequence with at least 80% sequence identity to any of SEQ ID NOs: 11-6, 10-199, 201-206. or 210-399, shown below. In some cases, at least one CRISPR guide RNA (crRNA) include a targeting sequence such as any of SEQ ID NOs: 10-99 or 100-190, or a combination thereof. In some cases, the sample can be incubated with one or two or more crRNAs. For example, the sample can be incubated with at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least nine, or at least ten, or more crRNAs. In some cases, the at least one crRNA has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more sequence identity to any SEQ ID NOs: 1-6, 10-199, 201-206, or 210-399. In some cases, the targeting sequence in at least one crRNA has at least about 70%. about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more sequence identity to any SEQ ID NO:10-99 or 100-190.
In one example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more crRNAs specific for one or more viruses are employed in a method to detect viruses such as RSV in a physiological sample.
In various examples of crRNA(s) that can be used for detection of RSV B, the crRNA(s) can include those with SEQ ID NOs: 100-199 or 300-389, or a nucleotide sequence with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto, or a combination thereof. In some cases. SEQ ID NOs: 191, 192, 196 or 199 can be combined to improve detection of RSV B.
In various examples of crRNA(s) that can be used for detection of RSV A, the crRNA(s) can include those with SEQ ID NOs: 1-6, 10-99, 201-206 or 210-299, or a nucleotide sequence with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto, or a combination thereof. In some cases, the crRNA(s) can include those with SEQ ID NOs: 2, 4 or 6. The amount of reporter RNA cleavage product detected is directly correlated with the amount of the target viral RNA. In some cases, the target viral RNA cleavage product concentration can be quantified or determined by use of a standard curve of the reporter RNA cleavage product(s).
At least one crRNA can bind to a region in any of the eight single-stranded RNAs of the RSV RNA genome. In some cases, the region is a single stranded region of the RSV RNA genome. In other cases, the region is a secondary structure in regions of the RSV genome with low viral ribonucleoprotein binding.
In some cases, the crRNAs can include additional sequences such as spacer sequences.
As illustrated herein, for detection of RSV B. crRNAs with a sequence of SEQ ID NOs: 191, 192, 196 or 199 exhibit better signals than crRNAs with a sequence of SEQ ID NOs: 193, 194, 195, 197 or 198. Moreover, the combination of the crRNAs of SEQ ID NOs: 191, 192, 196 and 199 improves detection of RSV B over using crRNAs of SEQ ID NOs: 191, 192, 196 or 199 alone.
To detect RSV A, crRNAs with a sequence of SEQ ID NOs: 2, 4, or 6 exhibit better signals than crRNAs with a sequence of SEQ ID NOs: 1, 3 or 5. Moreover, the combination of the crRNAs of SEQ ID NOs: 2, 4, and 6 improves detection of RSV A over using the crRNAs of SEQ ID NOs: SEQ ID NOs: 2, 4, or 6 alone.
Exemplary RSV Sequences
The crRNAs may be employed to detect any RSV A or RSV B genome. Exemplary RSV A and RSV B genomes include but not limited to human respiratory' syncytial virus isolate B05 (gi|562744952|gb|KF 640637.1) having
ACG CG AAAAAATG CGTACTACAAACTTGCACACTCG AAAAAAAATG GG G CAAATAAG AATTTG ATAAGTG CTATTTAAGTCTAACCTTTTTAATCAGAAATGGGGTGCAATTCACTGAGCATGATAAAGGTTAGATTACA AAATTTATTTGACAATGACGAAGTAGCATTGTTAAAAATAACATGTTATACTGACAAATTAATTCTTCTG ACTAATGCATTAGCCAAAGCAACAATACATACAATTAAATTAAACGGCATAGTTTTTATACATGTTATAA CAAGCAGTGAAGTGTGCCCTGATAACAATATTGTAGTGAAATCTAACTTTACAACAATGCCAATATTACA AAATGGAGGATACATATGGGAATTGATTGAATTGACACACTGCTCTCAATTAAATGGTCTAATAGATGAT AATTGTGAAATCAAATTTTCTAAAAGACTAAGTGACTCAGTAATGACTGATTATATGAATCAAATATCTG ATTTACTTGGGCTTGATCTCCATTCATGAATTATGTTTAGTCTAATTCAATAGACATGTGTTTATTACCA TTTTAGTTAATATAAAACCTCATCAAAGGGAAATGGGGCAAATAAACTCACCCAATCAATCAAACCATGA GCACTACAAACGACAACACCACCATGCAAAGATTGATGATCACAGACATGAGACCCCTGTCGATGGATTC AATAATAACATCTCTCACCAAAGAAATCATCACACACAAATTCATATACTTGATAAACAATGAATGTATT GTAAGAAAACTCGATGAAAGACAAGCTACATTTACATTCCTAGTCAATTATGAGATGAAGCTATTGCACA AAGTAGGGAGTACCAAATACAAGAAATACACTGAATATAATACAAAATATGGCACATTCCCTATGCCTAT ATTTATCAATCATG GCG G GTTTCTAG AATGTATTG G CATTAAGCCTACAAAACATACTCCTATAATATAC AAATATGACCTCAACCCGTAACTTCCAACAAAAAAACCAACTCATCCAAACCAAGCTATTCTCTAAACAA CAGTGCTCAACAGTTAAGAAGGAGCTAATCCATTTTAGTAATTAAAAATAAGGGTGAAGCCAGTAACATA AATTGGGGCAAATACAAAGATGGCTCTTAGCAAAGTCAAGTTGAATGATACATTAAATAAGGATCAGCTG CTGTCATTCAGCAAATACACTATTCAACGTAGTACAGGAGATAATATTGACACTCCCAATTATGATGTGC AAAAACACCTAAACAAACTATGTGGTATGCTATTAATCACTGAAGATGCAAATCATAAATTCACAGGATT AATAGGTATGCTATATGCTATGTCCAGGTTAGGAAGGGAAGACACTATAAAGATACTTAAAGATGCTGGA TATCATGTTAAAGCTAATGGAGTAGATATAACAACATATCGTCAAGATATAAATGGAAAGGAAATGAAAT TCGAAGTATTAACATTATCAAGCTTGACATCAGAAATACAAGTCAATATTGAGATAGAATCTAGAAAGTC CTACAAAAAAATG CTAAAAG AG ATGG G AG AAGTG G CTCCAG AATATAGG CATGATTCTCCAG ACTGTG G G ATGATAATACTGTGTATAGCTGCCCTTGTAATAACCAAATTAGCAGCAGGAGATAGATCAGGTCTTACAG CAGTAATTAG GAG G GCAAACAATGTCTTAAAAAACG AAATAAAACG CTACAAG G GCCTAATACCAAAG GA CATAGCCAACAGTTTTTATGAAGTGTTTGAAAAACACCCTCATCTTATAGATGTTTTTGTGCACTTTGGC ATTGCACAATCATCCACAAGAGGGGGTAGTAGAGTTGAAGGAATCTTTGCAGGATTGTTTATGAATGCCT ATGGTTCAGGACAAGTAATGCTAAGATGGGGAGTTTTAGCCAAATCTGTAAAAAATATCATGCTAGGACA TGCTAGTGTCCAAGCAGAAATGGAGCAAGTTGTGGAAGTCTATGAGTATGCACAGAAGTTGGGAGGAGAA GCTGGTTTCTACCATATATTGAACAATCCAAAAGCATCATTGCTGTCATTAACTCAATTTCCTAACTTCT CAAGTGTG GTCCTAG GCAATG CAG CAGGTCTAG G CATAATG GG AG AGTATAG AG GTACACCAAG AAACCA AGATCTCTATGATGCAGCCAAAGCATATGCAGAGCAACTCAAAGAAAATGGAGTAATAAACTACAGTGTA TTAGACTTAACAACAGAAGAATTGGAAGCCATAAAGCATCAACTCAACCCCAAAGAAGATGACGTAGAGC TTTAAGTTAACAAAAAATACGGGGCAAATAAGTCAACATGGAGAAGTTTGCACCTGAATTTCATGGAGAA GATGCAAATAACAAAGCTACCAAATTCCTAGAATCAATAAAAGGCAAGTTTGCATCATCCAAAGATCCTA AGAAGAAAGATAGCATAATATCTGTCAACTCAATAGACATAGAAGTCACTAAAGAGAGCCCGATAACATC TG G CACCAACATTATCAATCCAACAAGTG AAG CCG ACAGTACCCCAG AAACCAAAG CCAACTACCCAAG A AAACCCCTAGTAAGCTTCAAAGAAGATCTCACCCCAAGTGATAACCCTTTCTCTAAGTTGTACAAAGAAA CCATAGAAACATTTGATAACAATGAAGAAGAATCTAGCTACTCATATGAGGAGATCAATGATCAAACAAA TGACAACATTACAGCAAGACTAGATAGAATTGATGAAAAATTAAGTGAAATATTAGGAATGCTCCATACA TTAGTAGTTGCAAGTGCAGGACCTACTTCGGCTCGTGATGGAATAAGAGATGCTATGGTTGGTCTAAGAG AAGAAATGATAGAAAAAATAAGAGCAGAAGCATTAATGACCAATGATAGGTTAGAGGCTATGGCAAGACT TAGGAATGAGGAAAGCGAAAAAATGGCAAAAGACACCTCAGATGAAGTGTCTCTCAATCCAACCTCCAAA AAATTGAGTGACTTGTTGGAAGACAACGATAGCGACAATGATCTATCACTTGATGATTTTTGATCAGTGA TCAACTCACTCAGCAATCAACAACATCAATAAAACAGACACCAATCCATTGAATCAATTGCCAGACTGAA AAAACAAACATCCATCAGCAGAACCACCAACCAATCAATCAACCAATTGATCAATCAGCACCCTGACAAA ATTAACAATATAGTAACAAAAAAAGAACAAGATGGGGCAAATATGGAAACATACGTGAACAAGCTTCACG AAGGCTCCACATACACAGCAGCTGTTCAGTACAATGTTCTAGAAAAAGATGATGATCCCGCATCACTAAC AATATGGGTGCCTATGTTCCAGTCATCTGTGCCAGCAGACTTGCTCATAAAAGAACTTGCAAGCATCAAC ATACTAGTAAAGCAGATCTCTACGCCCAAAGGACCTTCACTACGAGTCACGATCAACTCAAGAAGTGCTG TGCTGGCTCAAATGCCTAGTAATTTCACCATAAGTGCAAATGTATCATTAGATGAAAGAAGCAAATTAGC ATATGATGTAACTACACCTTGTGAAATCAAAGCATGCAGTCTAACATGCTTAAAAGTAAAAAGTATGTTA ACTACAGTCAAAGATCTAACCATGAAGACATTCAACCCCACTCATGAGATCATTGCTCTATGTGAATTTG AAAATATTATGACATCAAAAAGAGTAATAATACCAACCTATCTAAGATCAATTAGTGTCAAAAACAAGGA CCTGAACTCACTAGAAAATATAGCAACCACCGAATTCAAAAATGCTATCACCAATGCTAAAATTATTCCC TATGCAGGATTAGTATTAGTTATCACGGTTACTGATAATAAAGGAGCATTCAAATATATCAAGCCACAGA GTCAATTTATAGTGGATCTTGGTGCCTACCTAGAAAAAGAGAGCATATATTATGTGACTACTAATTGGAA GCATACAGCTACACGTTTTTCAATCAAACCACTAGAGGATTAAACCCAATTATCAACATTGAATGACAGG TTCACATATATCCTCAACTGCACACTATATCTAACCATCATAAACATCTACACTACACACTTCATCACAC AAACCAATCCCACTCAAAATCCAAAATCACTTCAAGCCATTGTCTGCCAGACCTAGAGTGCGAATAGGTA AATAAAACCAGAATATGGGGTAAATAGATATCAGTTAGAGTTCAATCAATCTCAACAACCATCTATACCG CCAATTCAATACATATACTGCAAATCTCAAAATGGGAAACACATCCATCACAATAGAATTCACAAGCAAA TTTTGGCCCTATTTTACACTAATACATATGATCTTAACTCTAATCTCTTTACTAATTATAATCACTATTA TGATTGCAATACTAAATAAGCTAAGTGAACATAAAACATTCTGTAACAAAACTCTTGAACAAGGACAGAT GTATCAAATCAACACATAGTGTTCTCCCATTATGCTGTGTCAAATTATAATCTTGTATATATAAATAAAC AAATCCAATCTTCTCACAG AGTCATGGTATCACAAAACCATG CCAACCATCATG GTAG CATAG AGTAGTT ATTTAAAAATTAACATAATGATGAATTATTAGTATGGGATCAAAAACAACATTGGGGCAAATGCAACCAT GTCCAAAAACAAGAATCAACGCACTGCCAGGACTCTAGAAAAGACCTGGGATACTCTTAATCATCTAATT GTAATATCCTCTTGTTTATACAAATTAAATTTAAAATCTATAG CACAAATAG CACTATCAGTTTTG GCAA TGATAATCTCAACCTCTCTCATAATTGCAGCCATAATATTCATCATCTCTGCCAATCACAAAGTTACACT AACAACTGTCACAGTTCAAACAATAAAAAACCACACTGAGAAAAACATAACCACTTACCTTACTCAAGTC TCACCAG AAAG G GTTAG CCCATCCAAACAACCCACAACCACACCACCAATCCACACAAACTCAG CCACAA TATCACCTAATACAAAATCAGAAACACACCATACAACAGCACAAACCAAAGGCAGAACCTCTACTCCAAC ACAGAACAACAAGCCAAGCACAAAACCACGTCCAAAAAATCCACCAAAAAAAGATGATTACCATTTTGAA GTGTTCAACTTCGTTCCCTGTAGTATATGTG GCAACAATCAACTCTG CA AATCCATTTG CAAAACAATAC CAAGCAATAAACCAAAGAAAAAACCAACCATAAAACCCACAAACAAACCACCCACCAAAACCACAAACAA AAGAGACCCTAAAACACTAGCCAAAACACCGAAAAAAGAAACCACCATTAACCCAACAAAAAAACCAACC CCCAAGACCACAGAAAGAGACACCAGCACCCCACAATCCACTGTGCTCGACACAACCACATCAAAACACA CAGAAAGAGACACCAGCACCTCACAATCCATTGCGCTTGACACAACCACATCAAAACACACAATCCAACA GCAATCTCTCTACTCAACCACCCCCGAAAACACACCCAACTCCACACAAACACCCACAGCATCCGAGCCC TCCACATCAAATTCCACCTAAAAACTCCAGTCATATGCTTAGTTATTTAAAAACTACATCTTAGCAGAGA ACCGTGATCCCTCAAGCAAGAACGAAATTAAATCTGGGGCAAATAACCATGGAGTTGCTGATCCATAGAT CAAGTG CAATCTTCCTAACTCTTG CTATTAATG CATTGTACCTCACCTCAAGTCAG AACATAACTG AG GA GTTTTACCAATCGACATGTAGTGCAGTTAGCAGAGGTTACTTGAGTGCTTTAAGAACAGGTTGGTATACC AGTGTCATAACAATAGAATTAAGTAATATAAAAGAAACCAAATGCAATGGAACTGACACTAAAGTAAAAC TTATAAAACAAGAATTAGATAAGTATAAGAATGCAGTAACAGAATTACAGTTACTTATGCAAAACACACC AGCTGCCAACAACCGGGCCAGAAGAGAAGCACCACAGTATATGAACTACACAATCAATACCACTAAAAAC CTAAATGTATCAATAAGCAAGAAGAGGAAACGAAGATTTCTGGGCTTCTTGTTAGGTGTAGGATCTGCAA TAGCAAGTGGTATAGCTGTATCCAAAGTTCTACACCTTGAAGGAGAAGTGAACAAGATCAAAAATGCTTT GCTGTCTACAAACAAAGCTGTAGTCAGTCTATCAAATGGGGTCAGTGTTTTAACCAGCAAAGTGTTAGAT CTCAAGAATTATATAAACAACCAATTATTACCTATAGTAAATCAACAGAGTTGTCGCATTTCCAACATTG AAACAGTTATAGAATTCCAGCAGAAGAACAGCAGATTGTTGGAAATCACCAGAGAATTTAGTGTCAATGC AGGTGTAACGACACCTTTAAGCACTTACATGTTAACAAACAGTGAGTTACTATCATTAATCAATGATATG CCTATAACAAATGATCAGAAAAAATTAATGTCAAGCAATGTTCAGATAGTAAGGCAACAAAGTTATTCTA TCATGTCTATAATAAAGGAAGAAGTCCTTGCATATGTTGTACAGCTACCTATCTATGGTGTAATTGATAC ACCTTGCTGGAAATTACACACATCACCTCTGTGCACCACCAACATCAAAGAAGGATCAAATATTTGTTTA ACAAGGACTGATAGAGGATGGTACTGTGATAATGCAGGATCAGTATCCTTCTTTCCACAGGCTGACACTT GTAAAGTACAGTCCAATCGAGTATTTTGTGACACTATGAACAGTTTGACATTACCAAGTGAAGTCAGCCT TTGTAACACTG ACATATTCAATTCCA AGTATG ACTG CAAAATTATG ACATCAAAAACAG ACATAAG CAG C TCAGTAATTACTTCTCTAGGAGCTATAGTGTCATGCTATGGTAAAACTAAATGCACTGCATCCAACAAAA ATCGTGGAATTATAAAGACATTTTCTAATGGTTGTGATTATGTGTCAAACAAAGGAGTAGATACTGTATC AGTGGGCAACACTTTATACTATGTCAACAAGCTGGAAGGCAAAAACCTTTATGTAAAAGGGGAACCTATA ATAAATTACTATGACCCTCTAGTGTTTCCTTCTGATGAGTTTGATGCATCAATATCTCAAGTCAATGAAA AAATTAATCAAAGTTTAGCTTTTATTCGTAGATCCGATGAATTATTACATAATGTAAATACTGGAAAATC TACTACAAATATTATGATAACTGCAATTATTATAGTAATCATTGTAGTATTGTTATCATTAATAGCTATT G GTTTACTGTTGTATTG CAAAG CCAAAACCACACCAGTTACACTAAG CAAAG ACCAACTAAGTG G AATCA ATAATATTGCATTCAGCAAATAGACAAAAAACCACCTGATCATGTTTCAACAACAATCTGCTGACCACCA ATCCCAAATCAACTTACAACAAATACTTCAACATCACAG CACAG GCTG AATCATTTCCTCG CATCATG CT ACCCACACAACTAAGCTAGATCCTTAACTCATAGTTACATAAAAACCTCAAATATCGCAATCAACACTAA ATCAACACATCATTCACAAAACTAACAGCTGGGGCAAATATGTCGCGAAGAAATCCCTGCAAATTTGAGA TTAGAGGTCATTGCTTGAATGGTAGAAGATGCCACTACAGTCATAATTACTTTGAATGGCCTCCTCATGC ATTACTAGTGAGGCAAAACTTCATGTTAAACAAGATACTCAAGTCAATGGACAAGAGCATAGACACTTTG TCTGAAATAAGTGGAGCTGCTGAACTTGATAGAACAGAAGAATATGCTCTTGGTATAGTTGGAGTGCTAG AGAGTTACATAGGATCTATAAACAACATAACAAAACAATCAGCATGTGTTGCTATGAGTAAACTTCTTAT TGAGATCAATAGTGATGACATTAAAAAGCTGAGAGACAATGAAGAACCCAATTCACCTAAGATAAGAGTG TACAATACTGTTATATCATACATCG AG AG CAATAG AAAAAACAG CAAG CAAACCATCCATCTG CTCAAAC GATTACCAGCAGACGTGCTGAAGAAGACAATAAAGAACACATTAGATATCCACAAAAGCATAACCATAAG CAACCCAAAAGAGTCAACCGTAAGTGATCAAAATGACCAAACCAAAAATAATGATATTACCGGATAAATA TCCTTGTAGTATATCATCCATATTGATCTCAAGTGAAAGCATGATTGCTACATTCAATCATAAAGACATA TTACAATTTAACCACAACCATTTGGATAACCACCAGTGTTTATTAAATCATATATTTGATGAAATTCATT GGACACCTAAAAACTTATTAGATGCCACTCAACAATTTCTCCAACATCTTAACATCCCTGAAGATATATA TACAGTATATATATTAGTGTCATAATGCTTGATCATAACGATTCTATATCATCCAACCATAAAACTGTCT TAATAAGGTTATGGGACAAAATGGATCCCATTATTAATGGAAGCTCTGCTAATGTATATCTAACTGATAG TTATCTAAAAGGTGTTATCTCTTTTTCAGAATGTAATGCTTTAGGGAGTTACCTTTTTAATGGCCCTTAT CTTAAAAATGATTATACCAACTTAATTAGTAGACAAAGCCCACTACTAGAGCATATGAATCTAAAAAAAC TAACTATAACACAGTCATTAATATCTAGATACCATAAAGGTGAACTGAAACTAGAAGAACCAACTTATTT CCAGTCATTACTTATGACATATAAAAGCATGTCCTCGTCTGAACAAATTGCTACAACTAACTTACTTAAA AAAATAATACGAAGAGCTATAGAAATAAGTGATGTAAAGGTGTACGCCATCTTGAATAAACTAGGACTAA AGGAAAAGGACAGAGTTAAGCCCAACAACAATTCAGGTAATGAAAACTCAGTACTTACAACCATAATTAA AGATGATATACTCTCAGCTGTGGAAAACAATCAATCATATACAAATTCAGACAAAAATTACTCAGTAAAT CAAAATATCAATATCAAAACAACACTCTTAAAAAAATTGATGTGTTCAATGCAACATCCTCCATCATGGT TAATACACTGGTTCAATTTATATACAAAATTAAATAACATATTAACACAATATCGATCAAATGAGGTAAA AAGTCATGGGTTTATATTAATAGATAATCAAACTTTGAGTGGTTTTCAGTTTATTTTAAATCAATATGGT TG CATTGTTTATCATAAAGG G CTCAAAAAAATCACAACTACTACATACAATCAATTTTTG ACATG G AAAG ACATCAGCCTTAGCAGATTAAATGTTTGCTTAATTACTTGGATAAGTAATTGTTTAAATACATTAAATAA
AAGCTTAGGGTTGAGATGCGGATTCAATAATGTTGTGCTATCACAATTATTCCTTTATGGAGATTGTATA CTGAAATTATTTCATAATGAAGGCTTTTACATAATAAAAGAAGTAGAAGGATTTATTATGTCTTTAATTC TAAACATAACAGAAGAAGATCAATTTAGGAAACGATTTTATAATAGCATGCTAAATAACATCACAGATGC AGCTATTAAGGCTCAAAAGGACCTACTATCAAGAGTATGTCACACTTTATTAGACAAGACAGTGTCTGAT AATATCATAAATGGTAAATGGATAATTCTATTAAGTAAATTTCTTAAATTGATTAAGCTTGCAGGTGATA ATAATCTCAATAACTTGAGTGAGCTATATTTTCTCTTCAGAATCTTTGGACATCCAATGGTTGATGAAAG ACAAGCAATGGATGCTGTAAGAATTAACTGCAATGAAACTAAGTTCTATTTATTAAGTAGCCTAAGTACG TTGAGAGGTGCTTTCATTTATAGAATCATAAAAGGGTTTGTAAATACCTACAACAGATGGCCCACTTTAA GGAATGCTATTGTCCTACCTCTAAGATGGTTAAACTATTATAAACTTAATACTTATCCATCTCTACTTGA AATCACAGAAAATGATTTGATTATTTTATCAGGATTGCGGTTCTATCGTGAGTTTCATCTGCCTAAAAAA GTG G ATCTTG AAATG ATAATAAATG ACAAAG CTATTTCTCCTCCAAAAG ATCTAATATG G ACTAGTTTTC CTAGAAATTACATGCCATCACATATACAAAATTATATAGAACATGAAAAGTTGAAGTTCTCTGAAAGCGA CAGATCAAGAAGAGTACTAGAGTATTACTTGAGAGATAATAAATTCAATGAATGTGATCTATACAATTGT GTAGTTAATCAAAGCTATCTCAACAACTCTAATCATGTGGTATCACTAACTGGTAAAGAAAGAGAGCTCA GTGTGGGTAGAATGTTTGCTATGCAACCAGGTATGTTTAGGCAAATCCAAATCTTAGCAGAGAAAATGAT AGCCGAAAATATTTTACAATTCTTCCCTGAGAGTTTGACAAGATATGGTGATCTAGAGCTTCAAAAGATA TTAGAATTAAAAGCAGGAATAAGCAACAAGTCAAATCGTTATAATGATAACTACAACAATTATATCAGTA AATGTTCTATAATAACAGATCTTAGCAAATTTAATCAAGCATTTAGATATGAAACATCATGTATCTGCAG TG ATGTATTAG ATG AACTG CATG G G GTACAATCTCTATTCTCTTGGTTG CATTTAACAATACCTCTTG CC ACAATAATATGTACATATAGACATGCACCTCCTTTTATAAAGGATCATGTTGTCAATCTTAATGAAGTTG ATGAACAAAGTGGGTTATACAGATATCATATGGGTGGTATTGAGGGCTGGTGTCAAAAACTGTGGACCAT TGAAGCCATATCATTATTAGATCTAATATCTCTTAAAGGTAAATTCTCCATCACAGCTCTGATAAATGGT GATAATCAGTCAATTGATATAAGTAAACCAGTTAGACTTATAGAGGGTCAGACCCATGCTCAAGCAGATT ATTTGTTAG CATTAAATAGCCTTAAATTG CTATATAAAG AGTATGCAG G CATAG GCCATAAG CTTAAGG G AACTGAGACCTATATATCCCGAGATATGCAGTTCATGAGCAAAACAATCCAGCACAATGGAGTGTACTAT CCAGCCAGTATCAAAAAAGTCCTGAGAGTAGGTCCATGGATAAATACAATACTTGATGATTTTAAAGTTA GTTTAGAATCTATAGGTAGCTTAACACAGGAGTTAGAATACAGAGGAGAAAGCTTATTATGCAGTTTAAT ATTTAGGAACATTTGGTTATACAATCAAATTGCTCTGCAACTCCGAAATCATGCATTATGTAATAATAAG CTATATTTAGATATATTGAAAGTATTAAAACACTTAAAAACCTTTTTTAATCTTGATAGTATCGATACGG CGTTATCATTGTATATGAACTTGCCTATGCTGTTTGGTGGTGGTGATCCTAATTTGTTATATCGAAGCTT TTATAGGAGAACTCCAGACTTCCTTACAGAAGCTATAGTACATTCAGTGTTCGTGTTGAGCTATTATACT GGTCACGATCTACAAGATAAGCTCCAGGATCTTCCAGATGATAGACTGAACAAATTCTTGACTTGTGTCA TCACATTTGATAAAAATCCAAATGCCGAGTTTGTAACATTGATGAGGGATCCACAGGCTTTAGGGTCTGA AAGACAAGCTAAAATTACTAGTGAGATTAATAGATTAGCAGTAACAGAAGTCTTAAGTATAGCTCCAAAC AAAATATTTTCTAAAAGTGCGCAACACTATACTACCACTGAGATTGATCTAAATGACATTATGCAAAATA TAGAACCAACTTACCCTCATGGATTAAGAGTTGTTTATGAAAGTTTACCTTTTTATAAAGCAGAAAAAAT AGTTAATCTTATATCAGGAACAAAATCCATAACTAATATACTTGAAAAAACATCAGCGATAGATACAACT GATATTAATAGGGCTACTGATATGATGAGGAAAAATATAACCTTACTTATAAGGATACTTCCACTAGATT GTAACAAAGACAAAAGAGAGTTATTAAGTTTAGAAAATCTTAGCATAACTGAATTAAGCAAGTATGTAAG AGAAAGATCTTGGTCATTATCCAATATAGTAGGAGTAACATCGCCAAGTATTATGTTCACAATGGACATT AAATATACAACTAGCACTATAGCCAGTGGTATAATTATAGAAAAATATAATGTTAATGGTTTAACTCGTG GTGAAAGAGGACCTACTAAGCCATGGGTAGGTTCATCTACGCAGGAGAAAAAAACAATGCCAGTGTACAA TAGACAAGTTTTAACCAAAAAGCAAAGAGACCAAATAGATTTATTAGCAAAATTAGACTGGGTATATGCA TCCATAGACAACAAAGATGAATTCATGGAAGAACTGAGTACTGGAACACTTGGACTGTCATATGAAAAAG CCAAAAAGTTGTTTCCACAATATCTAAGTGTCAATTATTTACACCGGTTAACAGTCAGTAGTAGACCATG CGAATTCCCTGCCTCAATACCAGCTTATAGAACAACAAATTATCATTTTGATACTAGTCCTATCAATCAT GTATTAACAGAAAAGTATGGAGATGAAGATATCGACATTGTGTTTCAAAACTGCATAAGTTTTGGTCTTA GCTTGATGTCAGTTGTGGAACAATTCACAAACATATGTCCTAATAGAATTATTCTCATACCGAAGCTGAA TGAGATACATTTGATGAAACCTCCTATATTTACAGGAGATGTTGATATCATCAAGTTGAAGCAAGTGATA CAAAAACAGCATATGTTCCTACCAGATAAAATAAGTTTGACCCAATATGTAGAATTATTCTTAAGTAACA AAGCACTTAAATCTGGATCCCACATCAACTCTAATTTAATATTAGTACATAAAATGTCTGATTATTTTCA TAATGCGTATATTTTAAGTACTAATTTAGCTGGACATTGGATTCTGATTATTCAACTTATGAAAGATTCA AAAGGTATTTTTGAAAAAGATTGGGGAGAGGGGTATATAACTGATCATATGTTCATTAATTTGAATGTTT TCTTTAATGCTTATAAGACTTATTTGCTATGTTTTCATAGAGGTTATGGTAAAGCAAAATTAGAATGTGA TATGAACACTTCAGATCTTCTTTGTGTTTTAGAGTTAATAGACAGTAGCTACTGGAAATCTATGTCTAAA GTTTTCCTAGAACAAAAAGTCATAAAATACATAGTCAATCAAGACACAAGTTTGCATAGAATAAAAGGCT GTCACAGTTTTAAGTTGTG GTTTTTAAAACG CCTTAATAATGCTAAATTTACCGTATG CCCTTG G GTTGT TAACATAGATTATCACCCAACACACATGAAAGCTATATTATCTTACATAGATTTAGTTAGAATGGGGTTA ATAAATGTAGATAAATTAACCATTAAAAATAAAAACAAATTCAATGATGAATTTTACACATCAAATCTCT TTTACATTAGTTATAACTTTTCAGACAACACTCATCTGCTAACAAAACAAATAAGAATTGCTAATTCAGA ATTAGAAGATAATTATAACAAACTATATCACCCAACCCCAGAAACTTTAGAAAATATATCATTAATTCCT GTTAAAAGTAATAATAGAAACAAACCTAAATTTTGTATAAGTGGAAATACTGAATCTATGATGACGTCAA CATTCTCTAATAAAATGCATATTAAATCTTCCACTGTTACCACAAGATTCAATTATAGCAGACAAGACTT GTACAATTTATTTCCAATTGTTGTGATAGACAGGATTATAGATCATTCAGGTAATACAGAAAAATCTAAC CAACTTTACACCACCACTTCACATCAGACATCTTTAGTAAGGAATAGTGCATCACTTTATTGCATGCTTC CTTGGCATCATGTCAATAGATTTAACTTTGTATTTAGTTCCACAGGATGCAAGATCAGTATAGAGTATAT TTTAAAAGATCTTAAGATTAAAGATCCCAGTTGTATAGCATTCATAGGTGAAGGAGCTGGTAACTTATTA TTACGTACGGTAGTAGAACTTCATCCTGACATAAGATATATTTACAGAAGTTTAAAAGATTGCAATGATC ATAGTTTACCTATTGAATTTCTAAGGTTATACAACGGGCATATAAACATAGATTATGGTGAGAATTTAAC CATTCCTGCTACAGATGCAACTAACAACATACATTGGTCTTATTTACATATAAAATTTGCAGAACCTATT AGTATCTTTGTCTGCGATGCTGAATTACCTGTCACAGCCAATTGGAGTAAAATTATAATTGAATGGAGTA AG CATGTAAG AAAGTG CAAATACTGTTCTTCTGTAAATAG ATG CATTTTAATTG CAAAATATCATG CTCA AGATGATATTGATTTCAAATTAGATAACATTACTATATTAAAAACTTATGTGTGCCTAGGTAGCAAGTTA AAAGGATCTGAAGTTTACTTAGTCCTTACAATAGGCCCTGCAAATATACTTCCTGTTTTTGATGTTGTGC AAAATG CTAAATTG ATTCTTTCAAG AACTAAAAATTTCATTATG CCTAAAAAG ATTG ACAAG G AATCTAT CGATGCAAATATTAAAAGCTTAATACCTTTCCTTTGTTACCCTATAACAAAAAATGGAATTAAGACTTCA TTGTCAAAATTGAAGAGTGTAGTTAATGGAGATATATTATCATATTCTATAGCTGGACGTAATGAAGTAT TCAG CAACAAG CTTATAAACCACAAGCATATG AATATCTTAAAATG GCTG G ATCATGTTTTAAACTTTAG ATCAGCTGAACTTAATTACAATCATTTATACATGATAGAGTCCACATATCCTTACTTGAGTGAATTGTTA AATAGTTTAACAACCAATGAGCTCAAGAAGCTGATTAAAATAACAGGTAGTGTACTATACAACCTTCCTA ATGAACAGTAACTTAAAATATCATTAACAAGTTTGGTCAAATTTAGATGCTAACACATCATTATATTATA GTTATTAAAAAATATGCAAACTTTTCAATAATTTAGCATATTGATTCCAAAATTATCATTTTAATCTTAA GGGATTAAATAAAAGTCTAAAACTAACAATCACACATGTGCATTTACAACACAACGAGACATTAGTTTTT GACACTTTTTTTCTCGT ( SEQ ID NO : 4 02 ) , or a nucleotide sequence with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto, human respiratory syncytial virus strain 98-25147-X (>gi|262479002|gb|FJ948820. 11) having
ACG CG AAAAAATG CGTACAACAAACTTG CGTAAACCAAAAAAATG G G GCAAATAAG AATTTG ATAAGTAC CACTTAAATTTTACTCCTTTGGTTAGAGATGGGCAGCAACTCATTGAGTATGATAAAAGTTAGATTGCAA AATTTGTTTGACAATGATGAAGTAGCATTGTTAAAAATAACATGCTATACTGACAAATTAATACAGTTAA CTAATGCTTTG G CTAAG G CAGTTATACATACAATTAAATTG AATGG CATTGTATTTGTG CATGTTATTAC AAGTAGTGATATTTGCCCTAATAATAATATTGTAGTGAAATCCAACTTCACAACAATGCCAGTATTACAA AATGGAGGTTATATATGGGAAATGATGGAATTAACGCATTGCTCTCAACCTAATGGCCTAATAGATGACA ATTGTGAAATTAAATTCTCCAAAAAACTAAGTGATTCAACAATGACCAATTATATGAATCAATTATCTGA ATTACTTGGATTTGACCTTAATCCATAAATTATAATAAATATCAACTAGCAAATCAATGTCACTAACACC ATTAGTTAATATAAAACTTGACAGAAGATAAAAATGGGGCAAATAAATCAACTAAGCTGACCCAACCATG GACACAACACACAATGATACCACACCACAAAGACTGATGATCACTGACATGAGACCATTGTCACTTGAGA CTATAATAACATCACTAACCAGAGACATCATAACACATAGATTTATATACTTGATAAATCATGAATGCAT AGTGAGAAAACTTGATGAAAGACAGGCCACATTTACATTCCTGGTCAACTATGAAATGAAACTATTGCAC AAAGTGGGAAGCACTAAATATAAAAAATATACTGAATACAACACAAAATATGGCACTTTCCCTATGCCAA TATTTATCAATCATGATG GGTTCTTAG AATG CATTGG CATTAAG CCTACTAAG CACACACCCATAATATA CAAGTATGATCTCAATCCATGAATTTCAAAACAAGATTCAAACAATCTGAAATAATAACTTCATGCACAA TCACACTCCATAGTCCAAATGGAGCCTGAAAATTATAGTAATTTAAAATTAAGGAGAGACCTGGTAAGAT GAAAGATGGGGCAAATACAAAAATGGCTCTTAGCAAAGTCAAGTTGAATGATACACTCAACAAAGATCAA CTTCTGTCATCCAGCAAATACACTATCCAACGGAGCACAGGAGATAGCATTGACACTCCTAATTATGATG TGCAGAAACACATCAACAAGCTATGTGGCATGCTATTAATCACAGAAGATGCTAATCATAAATTCACTGG GTTAATAGGTATGTTATATGCTATGTCTAGATTAGGAAGAGAAGACACCATAAAAATACTCAAAGATGCG GGATATCATGTTAAAGCAAATGGAGTGGATGTAACAACACATCGTCAAGACATTAATGGGAAAGAAATGA AATTTGAAGTGTTAACATTAGCAAGCTTAACAACTGAAATTCAAATCAACATTGAGATAGAATCTAGAAA ATCCTACAAAAAAATGCTAAAAGAAATGGGAGAGGTGGCTCCAGAATACAGGCATGACTCTCCTGATTGT GGGATGATAATATTATGTATAGCGGCATTAGTAATAACCAAATTAGCAGCAGGGGATAGATCTGGTCTTA CAGCTGTGATTAGGAGAGCTAATAATGTCTTAAAAAATGAAATGAAACGTTATAAAGGCTTACTACCCAA G G ATATAG CAAACAG CTTCTATG AAGTGTTTG AAAAATATCCTCACTTTATAG ATGTTTTTGTTCATTTT GGTATAGCACAATCTTCTACCAGAGGTGGCAGTAGAGTTGAAGGGATCTTTGCAGGATTGTTTATGAATG CCTATGGTGCAGGGCAAGTGATGTTACGGTGGGGGGTCTTAGCAAAATCAGTTAAAAATATTATGTTAGG ACACGCTAGTGTGCAAGCAGAAATGGAACAGGTTGTGGAGGTGTATGAATATGCCCAAAAATTGGGTGGA GAAGCAGGATTCTACCATATATTGAACAACCCAAAAGCATCATTATTATCTTTGACTCAATTTCCTCACT TCTCTAGTGTAGTATTAGGCAATGCTGCTGGCCTAGGCATAATGGGAGAATACAGAGGTACACCAAGGAA TCAAGATCTATATGATGCTGCAAAAGCATATGCTGAACAACTCAAAGAAAATGGTGTGATTAACTACAGT GTATTAGACTTGACAGCAGAAGAACTAGAGGCTATCAAACATCAACTTAATCCAAAAGATAATGATGTAG AG CTTTG AGTTAATAAAAAAAATCCCG G GG CAAATAAAACATCATG G AAAAGTTTG CTCCTG AATTCCAT G G AG AAGATG CAAACAACAGAG CCACCAAATTCCTAG AATCAATAAAGG G CAAATTCACATCACCCAAAG ATCCCAAGAAAAAAGATAGTATCATATCTGTCAACTCAATAGATATAGAAGTAACCAAAGAAAGCCCTAT AACATCAAATTCAACCATTATAAACCCAATAAATGAGACAGATGATACTGTAGGGAACAAGCCCAATTAT CAAAGAAAACCTCTAGTAAGTTTCAAAGAAGACCCTACGCCAAGTGATAATCCCTTTTCAAAACTATACA AAGAAACCATAGAAACATTTGATAACAATGAAGAAGAATCTAGCTATTCATATGAAGAAATTAATGACCA GACAAACGATAATATAACAGCAAGATTAGATAGGATTGATGAGAAATTAAGTGAAATACTAGGAATGCTT CACACATTAGTAGTAGCG AGTGCAG G ACCTACATCTGCTCG G G ATG GTATAAG AG ATG CCATG GTTGGTT TAAGAGAAGAAATGATAGAAAAAATCAGAACTGAAGCATTAATGACCAATGACAGACTAGAAGCTATGGC GAGACTCAGGAATGAGGAAAGTGAAAAGATGGCAAAAGACACATCAGATGAAGTGTCTCTCAATCCAACA TCAGAAAAATTGAACAACCTGTTGGAAGGGAATGATAGTGACAATGATCTATCACTTGAAGATTTCTGAT TAGCTACCTATCTGCACATCAAAACACAACATCAATAGAAGACTAACAAACAAACCAATTCACCCATCCA ACCAAACATCCATTTG CCG ATT AG CCAACCAGCCAAAAAAACAACCAG CCAATCCAAAACTAG CCACCCG GAAAAAATCGATACTATAGTTACAAAAAAAGATGGGGCAAATATGGAAACATACGTGAACAAACTTCACG AG GG CTCCACATACACAG CTG CTGTTCAATACAATGTCCTAG AAAAAG ACG ATG ATCCTG CATCACTTAC AATATGGGTGCCCATGTTCCAATCATCCATGCCAGCAGATTTACTTATAAAAGAACTAGCCAATGTCAAT ATACTAGTGAAACAAATATCCACACCCAAGGGACCATCATTAAGAGTCATGATAAACTCAAGAAGTGCAG TGCTAGCGCAAATGCCCAGCAAATTTACCATATGTGCCAATGTGTCCTTGGATGAAAGAAGCAAGCTGGC ATATGATGTAACCACACCCTGTGAAATTAAGGCATGCAGTCTAACATGCCTAAAATCAAAAAATATGTTA ACTACAGTTAAAGATCTCACTATGAAAACACTCAACCCAACACATGACATCATTGCTTTATGTGAATTTG AAAATATAGTAACATCAAAAAAAGTCATAATACCAACATACCTGAGATCCATCAGTGTCAGAAATAAAGA TCTGAACACACTTGAAAATATAACAACCACTGAATTCAAAAATGCTATTACAAATGCAAAAATCATCCCT TACTCAG G ATTACTGTTAGTCATCACAGTG ACTG ACAACAAAG GAG CATTCAAATACATAAAG CCACAAA GTCAATTCATAGTAGATCTTGGAGCTTACTTAGAAAAAGAAAGTATATATTATGTTACAACAAATTGGAA GCACACAGCTACACGATTTGCAATCAAACCCATGGAAGATTAACCTTTTTCCTCTACATCAGTGAGTTGA TTCATACAAACTTTCTACCTACATTCTTCACTTCACAATCGTAATCACCAACCCTTTGTGGTTCAACCAA TCAAACAAAACTCATCAGGAGTCTCAGATCATCCCAAGTCATTGTTCATCAGATCCAGTACTCAAATAAG TTAATAAAAAATCCACATGGGGCAAATAATCATTGAGAGGAATCCAACTAATCACAACATCTGTCAACAT AGACTAGTCAACACATTAGACAAAATCAACCAATGGAAAATACATCCATAACAATAGAATTCTCAAGCAA ATTCTG G CCTTACTTTACACTAATACACATG ATAACAACAATAATCTCTTTG CTAATCATAATCTCCATC ATGATTGCAATACTAAACAAACTCTGCGAATATAACATATTCCATAACAAAACCTTTGAGCTACCAAGAG CTCGAGTCAATACATAGCATTCACCAATCTGATAGCTCAAAACAGTAACCTTGCATTTGTAAGTGAACTA CCCTCACCTCTTCACAAAACCACATCAACGTCTCACCATGCAAGCCATCATCTATACCATAAAGTAGTTA ATTAAAAATAGTCATAACAATGAACTAAGAATTAAGACTAACAATACGTACGTTGGGGCAAATGCAAACA TGTCCAAAACCAAGGACCAACGCGCCGCCAAGACACTAGAAAGGACTTGGGACACTCTCAATCATCTATT ATTCATATCATCGTG CTTATACAAGTTAAATCTTAAATCTATAGCACAAATCACATTATCTATTTTG GCA ATGATAATCTCAACTTCACTTATAATTGCAGCCATCATATTCATAGCCTCGGCAAACCACAAAGTCACAC CAACAACTGCAATCATACAAGATGCAACAAACCAGATCAAGAACACAACCCCAACATACCTCACCCAGAA TCCCCAGCTTGGAATCAGCCTCTCCAATCTGTCCGAAACTACATCACAACCCACCACCACACTAGCTTCA ACAACACCAAGTGCTAAGTCAACCCCACAATCCACAACAGTCAAGACCAAAAACACAACAACAACCCAAA TACAACCCAGCAAGCCCACCACAAAACAACGCCAAAACAAACCACAAAACAAACCAAATAATGATTTTCA CTTTGAAGTGTTCAACTTTGTACCCTGCAGCATATGCAGCAACAATCCAACTTGCTGGGCCATCTGCAAA AGAATACCAAACAAAAAACCTGGAAAGAAAACCACCACCAAGCCCACAAGAAAACCAACCATCAAGACAA CCAAAAAAGATCTCAAACCTCAAACCACAAAACCAAAGGAAGCACTTACCACCAGGCCCACAGAAAAGCC AACCATCAACACCACCAAAACAAACATCAGAACTACACTGCTCACCTCCAACACCACAGGAAATCCAGAA CACACAAGTCAAAAGGAAACTCTCCACTCAACCACCTCCGAAGGCAATCCAAGCCCTTCACAAGTCTATA CAACATCCGAGTACCTATCACAATCTCTATCTCCATCCAACACAACAAACCAGTAGTCATTAAAAAGCGC G CTATTATTG CAAAAAG CCATG ACCAAATCAAACAG AATCAAAATCAACTATG G GG CAAATAACAATG GA GTTG CCAATCCTCAAAACAAATGCTATTACCACAATCCTTG CTG CAGTCACACTCTGTTTCG CTTCCAGT CAAAACATCACTGAAGAATTCTATCAATCAACATGCAGTGCAGTTAGCAAGGGCTATCTTAGCGCTCTAA
GAACTGGTTGGTATACTAGTGTTATAACTATAGAATTAAGTACTATCAAGGAAAATAAGTGTAATGGAAC AGACGCTAAGGTAAAATTGATAAAACAAGAATTAGATAAATATAAAAATGCTGTAACAGAATTACAATTG CTCATGCAAAGCACACCAGCAGCCAACAATCGAGCCAGAAGAGAACTACCAAGATTTATGAATTATACAC TCAACAATGCCAAAAACACCAATGTAACATTAAGCAAGAAAAGGAAAAGAAGATTTCTTGGCTTTTTGTT AGGTGTTGGATCTGCAATCGCCAGTGGCATTGCCGTATCCAAGGTCTTGCACCTAGAAGGGGAAGTGAAC AAAATCAAAAGTGCTCTACTATCCACAAACAAGGCTGTAGTCAGCTTATCAAATGGAGTCAGTGTCTTAA CCAGCAAAGTGTTAGATCTCAAAAACTATATAGATAAACAGTTGTTACCTATTGTGAACAAGCAAAGCTG CAGCATATCAAACATTGAAACTGTGATAGAATTCCAACAAAAGAACAACAGACTACTAGAGATTACCAGG GAATTTAGTGTCAATGCAGGTGTAACTACACCTGTAAGCACTTATATGTTAACTAATAGTGAATTATTAT CATTAATCAATGATATGCCTATAACAAATGATCAGAAAAAGTTAATGTCCAACAATGTTCAAATAGTTAG ACAGCAAAGTTACTCTATCATGTCCATAATAAAGGAGGAAGTCTTAGCATATGTAGTACAATTACCACTA TATGGTGTAATAGATACACCTTGTTGGAAACTGCACACATCCCCACTATGTACAACCAACACAAAGGAAG GGTCCAACATCTGCTTAACAAGAACCGACAGAGGATGGTACTGTGACAATGCAGGATCAGTATCTTTCTT CCCACAAGCTGAAACATGTAAAGTTCAATCAAATCGGGTATTTTGTGATACAATGAACAGTTTAACATTA CCAAGTGAGGTAAATCTCTGCAACATTGACATATTCAACCCCAAATATGATTGCAAAATTATGACTTCAA AAACAGATGTAAGCAGCTCCGTTATCACATCTCTAGGAGCCATTGTGTCATGCTATGGCAAAACTAAATG TACAGCATCCAATAAAAATCGTGGGATCATAAAGACATTTTCTAACGGGTGTGATTATGTATCAAATAAG G G G GTG GATACTGTGTCTGTAGGTAATACATTATATTATGTAAATAAG CAAG AAG GCAAAAGTCTCTATG TAAAAGGTGAACCAATAATAAATTTCTATGACCCATTAGTGTTCCCCTCTGATGAATTTGATGCATCAAT ATCTCAAGTCAATGAGAAGATTAACCAGAGTCTAGCATTTATTCGTAAATCAGATGAATTATTACATAAT GTAAATGCTGGTAAATCCACCACAAATATCATGATAACTACTATAATTATAGTGATTATAGTAATATTGT TATCATTAATTGCAGTTGGACTGCTTCTATATTGCAAGGCCAGAAGCACACCAGTCACACTAAGTAAGGA TCAACTGAGTGGTATAAACAATATTGCATTTAGTAGCTGAATAAAAATAGCACCTAATCATATTCTTACA ATGGTTCACTATCTAACCATAGATAACCCATCTATCATTGGATTTTCTTAAAATTTGAACTTCATCACAA CTTTCATCTATAAACCATCTCACTTACACTATTTAAGTAGATTCCTATTTTATAGTTATATAAAACAATT GAATACCAGATTAACTTACTATTTGTAAAAATAAGAACTGGGGCAAATATGTCACGAAGGAATCCTTGCA AATTTGAAATTCGAGGTCATTGCTTGAATGGCAAGAGGTGCCATTTTAGTCATAATTATTTTGAATGGCC ACCCCATGCACTGCTTGTAAGACAAAATTTTATGTTAAACAGAATACTTAAGTCTATGGATAAAAGCATA GATACTTTATCAGAAATAAGTGGAGCTGCAGAGTTGGACAGAACAGAAGAGTATGCCCTCGGTGTAGTTG GAGTGCTAGAGAGTTATATAGGATCAATAAATAATATAACTAAACAATCAGCATGTGTTGCCATGAGCAA ACTCCTCACTGAACTCAACAGTGATGACATCAAAAAACTAAGGGACAATGAAGAGCCAAATTCACCCAAG ATAAGAGTGTACAATACTGTCATATCATATATTGAAAGCAACAGGAAAAACAATAAACAAACTATTCATC TGTTAAAAAGATTGCCGGCAGATGTATTGAAGAAAACCATCAAAAACACATTGGATATCCACAAGAGCAT AACCATCAATAACCCAAAAGAATCAACTGTTAGTGATACGAACGACCATGCCAAAAATAATGATACTACC TGACAAATATCCTTGTAGTATAAATTCCATACTAATAACAAGTAGTTGTAGAGTTACTATGTATAATCAA AAAAACACACTATATATCAATCAAACCAACCAAAATAACCATATATACTCACCGAATCAACCATTCAATG AAATCCATTGGACCTCTCAAGACTTGATTGATGCAACTCAAAATTTTCTACAACATCTAGGTATTAATGA TGATATATACACAATATATATATTAGTGTCATAACACTCAATCCTAATGCTTAACGCGTCATCAAACTAT TAACTCAAACAATTCAAGCTATGGGACAAAATGGATCCCATTATTAATGGAAATTCTGCTAATGTTTATC TAACTGATAGTTATTTAAAAGGTGTTATTTCTTTCTCAGAATGTAATGCTTTAGGAAGTTACATATTCAA TGGTCCTTATCTCAAAAATGATTATACCAACTTAATTAGTAGACAAAATCCATTAATAGAACACATAAAT CTAAAGAAACTAAATATAACACAGTCCTTAATATCTAAGTATCATAAAGGTGAAATAAAAATAGAAGAAC CTACCTATTTTCAGTCATTACTTATGACATACAAGAGTATGACCTCGTCAGAACAGATTACTACCACTAA TTTACTTAAAAAGATAATAAGAAGAGCTATAGAAATTAGTGATGTCAAAGTCTATGCTATACTGAATAAA CTGGGGCTTAAAGAAAAAGACAAGATTAAATCCAACAATGGACAAGATGAAGACAACTCAGTTATTACAA CCATAATCAAAGATGATATACTTTTAGCTGTTAAGGATAATCAATCTCATCTTAAAGCAGACAAAAATCA CTCTACAAAACAAAAAGATACAATCAAAACAACACTCTTGAAGAAATTAATGTGTTCGATGCAACATCCT CCATCATGGTTAATACATTGGTTTAATTTATACACAAAATTAAACAGCATATTAACACAGTATCGATCTA GTGAGGTAAAAAACCATGGTTTTATATTGATAGATAATCATACTCTTAATGGATTCCAATTTATTTTGAA TCAATATGGTTGTATAGTTTATCATAAGGAACTCAAAAGAATTACTGTGACAACCTATAATCAATTCTTG ACATGGAAAGATATTAGCCTTAGTAGATTAAATGTTTGTTTGATTACATGGATTAGTAACTGTTTGAACA CATTAAATAAAAGCTTAGGCTTAAGATGTGGATTCAATAATGTTATCTTGACACAATTATTCCTTTATGG AGATTGTATACTAAAACTATTTCACAATGAGGGGTTCTACATAATAAAAGAGGTAGAGGGATTTATTATG TCTCTAATTTTAAATATAACAGAAGAAGATCAATTCAGAAAACGGTTTTATAATAGTATGCTCAACAACA TCACAGATGCTGCTAATAAAGCTCAGAAAAATCTGCTATCAAGAGTATGTCATACATTATTAGATAAGAC AGTATCCGATAATATAATAAATGGCAGATGGATAATTCTATTAAGTAAGTTCCTAAAATTAATTAAGCTT GCAGGTGACAATAACCTTAACAATCTGAGTGAATTATATTTTTTGTTCAGAATATTTGGACACCCAATGG TAGATGAAAGACAAGCCATGGATGCTGTTAAAGTTAATTGCAACGAGACCAAATTTTACTTGTTAAGCAG TTTGAGTATGTTAAGAGGTGCCTTTATATATAGAATTATAAAAGGGTTTGTAAATAATTACAACAGATGG CCTACTTTAAGGAATGCCATTGTTTTACCCTTAAGATGGTTAACTTACTATAAACTAAACACTTATCCTT CCTTGTTGGAACTTACAGAAAGAGATTTGATTGTTCTATCAGGACTACGTTTCTATCGGGAGTTTCGATT GCCTAAAAAAGTGGATCTCGAAATGATCATAAATGATAAGGCTATATCACCTCCTAAAAATTTAATATGG ACTAGTTTCCCTAGAAATTATATGCCGTCACACATACAAAATTATATAGAACATGAAAAGTTAAAATTCT CTGAGAGTGATAAATCAAGAAGAGTATTAGAGTACTATTTAAGAGATAACAAATTCAATGAATGTGATTT ATACAACTGTGTAGTTAATCAAAGTTATCTTAACAACCCTAATCATGTGGTATCATTGACAGGTAAAGAA AGAGAACTCAGTGTAGGTAGAATGTTTGCAATGCAACCAGGAATGTTCAGACAAGTTCAAATATTAGCAG AGAAAATGATAGCTGAAAACATTTTACAATTTTTCCCTGAAAGTCTTACAAGATATGGTGATCTAGAACT ACAGAAAATATTAGAATTGAAAGCAGGAATAAGTAACAAATCAAATCGTTACAATGATAATTACAACAAT TACATTAGTAAGTGCTCTATCATCACAGATCTCAGCAAATTCAATCAAGCATTTCGATATGAAACATCAT GTATTTGTAGTG ATGTACTG GATG AACTG CATG GTGTACAATCTCTATTTTCCTG GTTACATTTAACTAT TCCTCATGTCACAATAATATGCACATATAGGCATGCACCCCCCTATATAAGGGATCATATTGTAGATCTT AACAATGTAGATGAGCAAAGTGGATTATATAGATATCATATGGGTGGTATCGAAGGGTGGTGTCAAAAAC TATGGACCATAGAAGCTATATCACTATTAGATCTAATATCTCTCAAAGGGAAATTCTCAATTACTGCTTT AATTAATG GTG ACAATCAATCAATAGATATAAGTAAACCAGTCAG ACTCATG G AAG GTCAAACTCATG CT CAAGCAGATTATTTGCTAGCATTAAACAGTCTCAAATTACTGTATAAAGAGTATGCAGGCATAGGCCACA AATTAAAAGGAACTGAGACTTATATATCAAGAGATATGCAATTTATGAGTAAAACGATCCAACATAACGG TGTATATTACCCAGCTAGTATAAAGAAAGTCCTAAGAGTGGGACCGTGGATAAACACTATACTTGATGAT TTCAAAGTGAGTCTAGAATCTATAGGTAGTTTGACACAAGAATTAGAATATAGAGGTGAAAGTCTATTAT G CAGTTTAATATTTAG AAATGTATG GTTATATAATCAA ATTG CTTTACAACTAAAAAATCATG CATTATG TAACAACAAATTATATTTGGACATATTAAAGGTTCTAAAACACTTAAAAACCTTTTTTAATCTTGATAAT ATTG ATACAG CATTAACATTGTATATG AATTTGCCCATGTTATTTG GTG GTG GTG ATCCCAACTTGTTAT ATCGAAGTTTCTATAGAAGAACTCCTGATTTTCTCACAGAGGCTATAGTTCACTCTGTGTTCATACTTAG TTATTATACAAACCATGATTTAAAAGATAAACTTCAAGATCTGTCAGATGATAGATTGAATAAATTCTTA ACATGCATAATCACGTTTGACAAAAACCCTAATGCTGAATTTGTAACATTGATGAGAGATCCTCAAGCTT TAGGATCTGAGAGGCAAGCTAAAATTACTAGTGAAATCAATAGACTGGCAGTTACTGAGGTTTTGAGCAC AGCTCCAAACAAAATATTCTCCAAAAGTGCACAACACTATACCACTACAGAGATAGATCTTAATGATATT ATGCAAAATATAGAACCTACATATCCTCACGGGCTAAGAGTTGTTTATGAAAGTTTACCCTTTTATAAAG CAGAGAAAATAGTAAATCTTATATCCGGTACAAAATCTATAACTAACATACTGGAAAAGACTTCTGCCAT AGACTTAACAGATATTGATAGAGCCACTGAGATGATGAGGAAAAACATAACTTTGCTTATAAGGATATTA CCATTAGATTGTAACAGAGATAAAAGAGAAATATTGAGTATGGAAAACCTAAGTATTACTGAATTAAGTA AATATGTTAGAGAAAGATCTTGGTCTTTATCCAATATAGTTGGTGTTACATCACCCAGTATCATGTATAC AATGGACATCAAATATACAACAAGCACTATAGCTAGTGGCATAATCATAGAGAAATATAATGTCAACAGT TTAACACGTG GTG AG AG AG G ACCCACTAAACCATG G GTTGGTTCATCTACACAAG AG AAAAAAACAATG C CAGTTTATAATAGACAAGTTTTAACCAAAAAACAGAGAGATCAAATAGATCTATTAGCAAAATTGGATTG GGTGTATGCATCTATAGATAACAAGGATGAATTCATGGAGGAACTTAGCATAGGAACTCTTGGGTTAGCA TATGAGAAGGCCAAAAAATTATTTCCACAATATTTAAGTGTTAACTATTTGCATCGTCTTACAGTCAGTA GTAGACCATGTGAATTCCCTGCATCAATACCAGCCTATAGAACTACAAACTATCACTTTGATACTAGCCC TATTAATCGCATATTAACAGAAAAGTATGGTGATGAAGATATTGATATAGTATTCCAAAACTGTATAAGC TTTGGCCTTAGCTTAATGTCAGTAGTAGAACAATTTACTAATGTATGTCCTAACAGAATTATTCTCATAC CCAAGCTTAATGAGATACATTTGATGAAACCTCCCATATTCACAGGTGATGTTGATATTCACAAGTTAAA ACAAGTGATACAAAAACAGCATATGTTTTTACCAGACAAAATAAGTTTGACTCAATATGTGGAATTATTC TTAAGTAATAAAACACTAAAATCTGGATCTAATGTTAATTCTAATTTAATATTGGCGCATAAGATATCTG ACTATTTTCATAATACTTACATTTTAAGTACTAATTTAGCTGGACATTGGATTCTGATTATACAACTTAT GAAAGATTCTAAAGGTATTTTTGAAAAAGATTGGGGAGAGGGATATATAACTGATCATATGTTCATTAAT TTGAAAGTTTTCTTCAATGCTTATAAGACATATCTCTTGTGTTTTCATAAAGGTTACAGCAGAGCAAAGC TGGAGTGTGATATGAATACTTCAGATCTCCTATGTGTATTGGAATTAATAGACAGTAGTTATTGGAAGTC TATGTCTAAGGTATTTTTAGAACAAAAAGTTATCAAATACATTCTTAGCCAGGATGCAAGTTTACATAGA GTAAAAGGATGTCATAGCTTCAAACTATGGTTTCTTAAACGTCTTAATGTAGCAGAATTCACAGTTTGCC CTTG G GTTGTTAACATAG ATTATCATCCAACACATATG AAAG CAATATTAACTTATATAG ATCTTGTTAG AATGGGATTGATAAATATAGATAGAATATACATTAAAAATAAACACAAATTCAATGATGAATTTTATACT TCTAATCTTTTTTACATTAATTATAACTTCTCAGATAATACTCATCTATTAACTAAACATATAAGGATTG CTAATTCTGAATTAGAAAGTAATTACAACAAATTATATCATCCTACACCAGAAACCCTAGAAAATATACT
AACCAATCCGGTTAAAAGTAATGATAAAAAGACACTGAATGACTATTGTATAGGTAAAAATGTTGACTCA ATAATGTTACCATTGTTATCTAATAAGAAGCTTATTAAATCGTCTACAATGATTAGAACCAATTACAGCA GACAAGATTTGTATAATTTATTTCCTACGGTTGTGATTGATAAAATTATAGATCATTCAGGTAATACAGC CAAATCTAACCAACTTTACACTACTACTTCTCATCAAATATCTTTAGTACACAATAGCACATCACTTTAT TG CATGCTTCCTTG G CATCATATTAATAG ATTCAATTTTGTATTTAGTTCTACAGGTTGTAAAATTAGTA TAGAGTATATTTTAAAAGACCTTAAAATTAAAGATCCTAATTGTATAGCATTCATAGGTGAAGGAGCAGG GAATTTATTATTGCGTACAGTAGTGGAACTTCATCCTGATATAAGATATATTTACAGAAGTCTGAAAGAT TGTAATGATCATAGTTTACCAATTGAGTTTTTAAGGCTGTACAATGGACATATCAACATTGATTATGGTG AAAATTTGACCATTCCTGCTACAGATGCAACCAACAACATTCATTGGTCTTATTTACATATAAAGTTTGC TGAACCTATCAGCCTTTTTGTCTGTGATGCTGAATTGCCTGTAACAGTCAACTGGAATAAAATTATAATA GAGTGGAGCAAGCATGTAAGAAAATGCAAGTACTGTTCTTCAGTTAATAAATGTACGTTAATAGTAAAAT ATCATGCTCAAGATGATATCGATTTCAAATTAGACAACATAACTATATTAAAAACTTATGTATGCTTAGG CAGTAAGTTAAAGGGATCTGAAGTTTACTTAGTCCTTACAATAGGTCCTGCAAATGTGTTCCCAGTATTT AATGTAGTACAAAATGCTAAATTGATACTATCAAGAACCAAAAATTTCATCATGCCTAAGAAGGCTGATA AAGAGTCTATTGATGCAAATATTAAGAGTTTGATACCCTTTCTTTGTTACCCTATAACAAAAAAAGGAAT TAATACTGCATTGTCTAAACTAAAGAGTGTTGTTAGTGGAGATATACTATCTTATTCTATAGCTGGACGT AATGAAGTTTTCAGCAATAAACTTATAAATCATAAGCACATGAACATCTTAAAGTGGTTCAATCATGTTT TAAATTTCAGATCAACAGAATTAAACTATAATCATTTATATATGGTAGAATCTACATATCCTCATCTAAG TGAATTGTTAAACAGCTTGACAACCAATGAACTTAAAAAACTGATTAAAATCACAGGTAGTTTGTTATAC AACTTCCATAATGAATAATGAACAAAAATCTTATAATAAAAATTCCCATGGCTACACACTAACACTGTAT TCAATTATAGTTATTTAAAATTAATAATTATATAATTTTTAATAACTTCTAGTGAACTAATCCTAAAATT ATCATTTTGATCTAGGAGGAATAAATTTAAACCCAAATCTAATTGGTTTATATGTATATTAACTAAACTA CGAGATATTAGTTTTTGACACTTTTTTTCTCGT (SEQ ID N0:403), or a nucleotide sequence with at least 80%. 82%. 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto, human orthopneumovirus strain A2 (>gi|961480530|gb|KT992094.1|) having
ACGGGAAAAAATGCGTACAACAAACTTGCATAAACCAAAAAAATGGGGCAAATAAGAATTTGATAAGTAC CACTTAAATTTAACTCCCTTGGTTAGAGATGGGCAGCAATTCATTGAGTATGATAAAAGTTAGATTACAA AATTTGTTTGACAATGATGAAGTAGCATTGTTAAAAATAACATGCTATACTGATAAATTAATACATTTAA CTAATGCTTTG G CTAAG G CAGTG ATACATACAATCAAATTG AATG GCATTGTGTTTGTGCATGTTATTAC AAGTAGTGATATTTGCCCTAATAATAATATTGTAGTAAAATCCAATTTCACAACAATGCCAGTACTACAA AATGGAGGTTATATATGGGAAATGATGGAATTAACACATTGCTCTCAACCTAATGGTCTACTAGATGACA ATTGTGAAATTAAATTCTCCAAAAAACTAAGTGATTCAACAATGACCAATTATATGAATCAATTATCTGA ATTACTTGGATTTGATCTTAATCCATAAATTATAATTAATATCAACTAGCAAATCAATGTCACTAACACC ATTAGTTAATATAAAACTTAACAGAAGACAAAAATGGGGCAAATAAATCAATTCAGCCAACCCAACCATG GACACAACCCACAATGATAATACACCACAAAGACTGATGATCACAGACATGAGACCGTTGTCACTTGAGA CCATAATAACATCACTAACCAGAGACATCATAACACACAAATTTATATACTTGATAAATCATGAATGCAT AGTGAGAAAACTTGATGAAAGACAGGCCACATTTACATTCCTGGTCAACTATGAAATGAAACTATTACAC AAAGTAGGAAGCACTAAATATAAAAAATATACTGAATACAACACAAAATATGGCACTTTCCCTATGCCAA TATTCATCAATCATG ATG GGTTCTTAG AATG CATTG G CATTAAG CCTACAAAG CATACTCCCATAATATA CAAGTATGATCTCAATCCATAAATTTCAACACAATATTCACACAATCTAAAACAACAACTCTATGCATAA CTATACTCCATAGTCCAGATGGAGCCTGAAAATTATAGTAATTTAAAACTTAAGGAGAGATATAAGATAG AAGATGGGGCAAATACAACCATGGCTCTTAGCAAAGTCAAGTTGAATGATACACTCAACAAAGATCAACT TCTGTCATCCAGCAAATACACCATCCAACGGAGCACAGGAGATAGTATTGATACTCCTAATTATGATGTG CAGAAACACATCAATAAGTTATGTGGCATGTTATTAATCACAGAAGATGCTAATCATAAATTCACTGGGT TAATAGGTATGTTATATGCGATGTCTAGGTTAGGAAGAGAAGACACCATAAAAATACTCAGAGATGCGGG ATATCATGTAAAAGCAAATGGAGTAGATGTAACAACACATCGTCAAGACATTAATGGAAAAGAAATGAAA TTTGAAGTGTTAACATTGGCAAGCTTAACAACTGAAATTCAAATCAACATTGAGATAGAATCTAGAAAAT CCTACAAAAAAATGCTAAAAGAAATGGGAGAGGTAGCTCCAGAATACAGGCATGACTCTCCTGATTGTGG GATGATAATATTATGTATAGCAGCATTAGTAATAACTAAATTAGCAGCAGGGGACAGATCTGGTCTTACA GCCGTGATTAGGAGAGCTAATAATGTCCTAAAAAATGAAATGAAACGTTACAAAGGCTTACTACCCAAGG ACATAGCCAACAGCTTCTATGAAGTGTTTGAAAAACATCCCCACTTTATAGATGTTTTTGTTCATTTTGG TATAGCACAATCTTCTACCAGAGGTGGCAGTAGAGTTGAAGGGATTTTTGCAGGATTGTTTATGAATGCC TATGGTGCAGGGCAAGTGATGTTACGGTGGGGAGTCTTAGCAAAATCAGTTAAAAATATTATGTTAGGAC ATGCTAGTGTGCAAGCAGAAATGGAACAAGTTGTTGAGGTTTATGAATATGCCCAAAAATTGGGTGGTGA AGCAGGATTCTACCATATATTGAACAACCCAAAAGCATCATTATTATCTTTGACTCAATTTCCTCACTTC TCCAGTGTAGTATTAGGCAATGCTGCTGGCCTAGGCATAATGGGAGAGTACAGAGGTACACCGAGGAATC AAG ATCTATATG ATG CAG CAAAG G CATATG CTG AACAACTCAAAG AAAATG GTGTG ATTAACTACAGTGT ACTAGACTTGACAGCAGAAGAACTAGAGGCTATCAAACATCAGCTTAATCCAAAAGATAATGATGTAGAG CTTTGAGTTAATAAAAAATGGGGCAAATAAATCATCATGGAAAAGTTTGCTCCTGAATTCCATGGAGAAG ATGCAAACAACAGGGCTACTAAATTCCTAGAATCAATAAAGGGCAAATTCACATCACCCAAAGATCCCAA GAAAAAAGATAGTATCATATCTGTCAACTCAATAGATATAGAAGTAACCAAAGAAAGCCCTATAACATCA AATTCAACTATTATCAACCCAACAAATGAGACAGATGATACTGCAGGGAACAAGCCCAATTATCAAAGAA AACCTCTAGTAAGTTTCAAAGAAGACCCTACACCAAGTGATAATCCCTTTTCTAAACTATACAAAGAAAC CATAGAAACATTTGATAACAATGAAGAAGAATCCAGCTATTCATACGAAGAAATAAATGATCAGACAAAC GATAATATAACAGCAAGATTAGATAGGATTGATGAAAAATTAAGTGAAATACTAGGAATGCTTCACACAT TAGTAGTGGCAAGTGCAGGACCTACATCTGCTCGGGATGGTATAAGAGATGCCATGGTTGGTTTAAGAGA AGAAATGATAGAAAAAATCAGAACTGAAGCATTAATGACCAATGACAGATTAGAAGCTATGGCAAGACTC AGGAATGAGGAAAGTGAAAAGATGGCAAAAGACACATCAGATGAAGTGTCTCTCAATCCAACATCAGAGA AATTGAACAACCTATTGGAAGGGAATGATAGTGACAATGATCTATCACTTGAAGATTTCTGATTAGTTAC CAATCTTCACATCAACACACAATACCAACAGAAGACCAACAAACTAACCAACCCAATCATCCAACCAAAC ATCCATCCGCCAATCAGCCAAACAGCCAACAAAACAACCAGCCAATCCAAAACTAACCACCCGGAAAAAA TCTATAATATAGTTACAAAAAAAGG AAAG G GTG GG G CAAATATG G AAACATACGTG AACAAG CTTCACG A AGGCTCCACATACACAGCTGCTGTTCAATACAATGTCTTAGAAAAAGACGATGACCCTGCATCACTTACA ATATG GGTGCCCATGTTCCAATCATCTATG CCAG CAG ATTTACTTATAAAAG AACTAG CTAATGTCAACA TACTAGTGAAACAAATATCCACACCCAAGGGACCTTCACTAAGAGTCATGATAAACTCAAGAAGTGCAGT GCTAGCACAAATGCCCAGCAAATTTACCATATGCGCTAATGTGTCCTTGGATGAAAGAAGCAAACTAGCA TATGATGTAACCACACCCTGTGAAATCAAGGCATGTAGTCTAACATGCCTAAAATCAAAAAATATGTTGA CTACAGTTAAAGATCTCACTATGAAGACACTCAACCCTACACATGATATTATTGCTTTATGTGAATTTGA AAACATAGTAACATCAAAAAAAGTCATAATACCAACATACCTAAGATCCATCAGTGTCAGAAATAAAGAT CTGAACACACTTGAAAATATAACAACCACTGAATTCAAAAATGCTATCACAAATGCAAAAATCATCCCTT ACTCAGG ATTACTATTAGTCATCACAGTG ACTG AC AACAAAG GAG CATTCAAATACATAAAG CCACAAAG TCAATTCATAGTAGATCTTGGAGCTTACCTAGAAAAAGAAAGTATATATTATGTTACCACAAATTGGAAG CACACAGCTACACGATTTGCAATCAAACCCATGGAAGATTAACCTTTTTCCTCTACATCAGTGTGTTAAT TCATACAAACTTTCTACCTACATTCTTCACTTCACCATCACAATCACAAACACTCTGTGGTTCAACCAAT CAAACAAAACTTATCTGAAGTCCCAGATCATCCCAAGTCATTGTTTATCAGATCTAGTACTCAAATAAGT TAATAAAAAATATACACATGGGGCAAATAATCATTGGAGGAAATCCAACTAATCACAATATCTGTTAACA TAGACAAGTCCACACACCATACAGAATCAACCAATGGAAAATACATCCATAACAATAGAATTCTCAAGCA AATTCTGGCCTTACTTTACACTAATACACATGATCACAACAATAATCTCTTTGCTAATCATAATCTCCAT CATGATTGCAATACTAAACAAACTTTGTGAATATAACGTATTCCATAACAAAACCTTTGAGTTACCAAGA GCTCGAGTCAACACATAGCATTCATCAATCCAACAGCCCAAAACAGTAACCTTGCATTTAAAAATGAACA ACCCCTACCTCTTTACAACACCTCATTAACATCCCACCATGCAAACCACTATCCATACTATAAAGTAGTT AATTAAAAATAGTCATAACAATGAACTAGGATATCAAGACTAACAATAACATTGGGGCAAATGCAAACAT GTCCAAAAACAAGGACCAACGCACCGCTAAGACATTAGAAAGGACCTGGGACACTCTCAATCATTTATTA TTCATATCATCGTG CTTATATAAGTTAAATCTTAAATCTGTAG CACAAATCACATTATCCATTCTGG CAA TG ATAATCTCAACTTCACTTATAATTGCAG CCATC ATATTCATAG CCTCG G CAAACCACAAAGTCACACC AACAACTGCAATCATACAAGATGCAACAAGCCAGATCAAGAACACAACCCCAACATACCTCACCCAGAAT CCTCAGCTTGGAATCAGTCCCTCTAATCCGTCTGAAATTACATCACAAATCACCACCATACTAGCTTCAA CAACACCAGGAGTCAAGTCAACCCTGCAATCCACAACAGTCAAGACCAAAAACACAACAACAACTCAAAC ACAACCCAGCAAGCCCACCACAAAACAACGCCAAAACAAACCACCAAGCAAACCCAATAATGATTTTCAC TTTGAAGTGTTCAACTTTGTACCCTGCAGCATATGCAGCAACAATCCAACCTGCTGGGCTATCTGCAAAA GAATACCAAACAAAAAACCAGGAAAGAAAACCACTACCAAGCCCACAAAAAAACCAACCCTCAAGACAAC CAAAAAAGATCCCAAACCTCAAACCACTAAATCAAAGGAAGTACCCACCACCAAGCCCACAGAAGAGCCA ACCATCAACACCACCAAAACAAACATCATAACTACACTACTCACCTCCAACACCACAGGAAATCCAGAAC TCACAAGTCAAATGGAAACCTTCCACTCAACTTCCTCCGAAGGCAATCCAAGCCCTTCTCAAGTCTCTAC AACATCCGAGTACCCATCACAACCTTCATCTCCACCCAACACACCACGCCAGTAGTTACTTAAAAACATA TTATCACAAAAG G CCTTG ACCAACTTAAACAG AATCAAAATAAACTCTG G GG CAAATAACAATG G AGTTG CTAATCCTCAAAGCAAATG CAATTACCACAATCCTCACTGCAGTCACATTTTGTTTTG CTTCTG GTCAAA ACATCACTGAAGAATTTTATCAATCAACATGCAGTGCAGTTAGCAAAGGCTATCTTAGTGCTCTGAGAAC TGGTTGGTATACCAGTGTTATAACTATAGAATTAAGTAATATCAAGAAAAATAAGTGTAATGGAACAGAT GCTAAGGTAAAATTGATAAAACAAGAATTAGATAAATATAAAAATGCTGTAACAGAATTGCAGTTGCTCA TGCAAAGCACACAAGCAACAAACAATCGAGCCAGAAGAGAACTACCAAGGTTTATGAATTATACACTCAA CAATGCCAAAAAAACCAATGTAACATTAAGCAAGAAAAGGAAAAGAAGATTTCTTGGTTTTTTGTTAGGT GTTGGATCTGCAATCGCCAGTGGCGTTGCTGTATCTAAGGTCCTGCACCTAGAAGGGGAAGTGAACAAGA TCAAAAGTGCTCTACTATCCACAAACAAGGCTGTAGTCAGCTTATCAAATGGAGTTAGTGTTTTAACCAG CAAAGTGTTAGACCTCAAAAACTATATAGATAAACAATTGTTACCTATTGTGAACAAGCAAAGCTGCAGC ATATCAAATATAGAAACTGTGATAGAGTTCCAACAAAAGAACAACAGACTACTAGAGATTACCAGGGAAT TTAGTGTTAATGCAGGCGTAACTACACCTGTAAGCACTTACATGTTAACTAATAGTGAATTATTGTCATT AATCAATGATATGCCTATAACAAATGATCAGAAAAAGTTAATGTCCAACAATGTTCAAATAGTTAGACAG CAAAGTTACTCTATCATGTCCATAATAAAAGAGGAAGTCTTAGCATATGTAGTACAATTACCACTATATG GTGTTATAGATACACCCTGTTGGAAACTACACACATCCCCTCTATGTACAACCAACACAAAAGAAGGGTC CAACATCTGTTTAACAAGAACTGACAGAGGATGGTACTGTGACAATGCAGGATCAGTATCTTTCTTCCCA CAAGCTGAAACATGTAAAGTTCAATCAAATCGAGTATTTTGTGACACAATGAACAGTTTAACATTACCAA GTGAAGTAAATCTCTGCAATGTTGACATATTCAACCCCAAATATGATTGTAAAATTATGACTTCAAAAAC AGATGTAAGCAGCTCCGTTATCACATCTCTAGGAGCCATTGTGTCATGCTATGGCAAAACTAAATGTACA G CATCCAATAAAAATCGTG G AATCATAAAG ACATTTTCTAACGG GTG CG ATTATGTATCAAATAAAGG G G TGGACACTGTGTCTGTAGGTAACACATTATATTATGTAAATAAGCAAGAAGGTAAAAGTCTCTATGTAAA AGGTGAACCAATAATAAATTTCTATGACCCATTAGTATTCCCCTCTGATGAATTTGATGCATCAATATCT CAAGTCAACGAGAAGATTAACCAGAGCCTAGCATTTATTCGTAAATCCGATGAATTATTACATAATGTAA ATGCTGGTAAATCCACCACAAATATCATGATAACTACTATAATTATAGTGATTATAGTAATATTGTTATC ATTAATTGCTGTTGGACTGCTCTTATACTGTAAGGCCAGAAGCACACCAGTCACACTAAGCAAAGATCAA CTGAGTGGTATAAATAATATTGCATTTAGTAACTAAATAAAAATAGCACCTAATCATGTTCTTACAATGG TTTACTATCTGCTCATAGACAACCCATCTGTCATTGGATTTTCTTAAAATCTGAACTTCATCGAAACTCT CATCTATAAACCATCTCACTTACACTATTTAAGTAGATTCCTAGTTTATAGTTATATAAAACACAATTGC ATGCCAGATTAACTTACCATCTGTAAAAATGAAAACTGGGGCAAATATGTCACGAAGGAATCCTTGCAAA TTTGAAATTCGAGGTCATTGCTTAAATGGTAAGAGGTGTCATTTTAGTCATAATTATTTTGAATGGCCAC CCCATGCACTGCTTGTAAGACAAAACTTTATGTTAAACAGAATACTTAAGTCTATGGATAAAAGTATAGA TACCTTATCAGAAATAAGTGGAGCTGCAGAGTTGGACAGAACAGAAGAGTATGCTCTTGGTGTAGTTGGA GTG CTAG AG AGTTATATAGG ATCAATAAACAATATAACTAAACAATCAG CATGTGTTG CCATG AG CAAAC TCCTCACTGAACTCAATAGTGATGATATCAAAAAGCTGAGGGACAATGAAGAGCTAAATTCACCCAAGAT AAGAGTGTACAATACTGTCATATCATATATTGAAAGCAACAGGAAAAACAATAAACAAACTATCCATCTG TTAAAAAGATTGCCAGCAGACGTATTGAAGAAAACCATCAAAAACACATTGGATATCCATAAGAGCATAA CCATCAACAACCCAAAAGAATCAACTGTTAGTGATACAAATGACCATGCCAAAAATAATGATACTACCTG ACAAATATCCTTGTAGTATAACTTCCATACTAATAACAAGTAGATGTAGAGTTACTATGTATAATCAAAA GAACACACTATATTTCAATCAAAACAACCCAAATAACCATATGTACTCACCGAATCAAACATTCAATGAA ATCCATTGGACCTCTCAAGAATTGATTGACACAATTCAAAATTTTCTACAACATCTAGGTATTATTGAGG ATATATATACAATATATATATTAGTGTCATAACACTCAATTCTAACACTCACCACATCGTTACATTATTA ATTCAAACAATTCAAGTTGTGGGACAAAATGGATCCCATTATTAATGGAAATTCTGCTAATGTTTATCTA ACCGATAGTTATTTAAAAGGTGTTATCTCTTTCTCAGAGTGTAATGCTTTAGGAAGTTACATATTCAATG GTCCTTATCTCAAAAATGATTATACCAACTTAATTAGTAGACAAAATCCATTAATAGAACACATGAATCT AAAGAAACTAAATATAACACAGTCCTTAATATCTAAGTATCATAAAGGTGAAATAAAATTAGAAGAACCT ACTTATTTTCAGTCATTACTTATGACATACAAGAGTATGACCTCGTCAGAACAGATTGCTACCACTAATT TACTTAAAAAGATAATAAGAAGAGCTATAGAAATAAGTGATGTCAAAGTCTATGCTATATTGAATAAACT AGGGCTTAAAGAAAAGGACAAGATTAAATCCAACAATGGACAAGATGAAGACAACTCAGTTATTACGACC ATAATCAAAGATGATATACTTTCAGCTGTTAAAGATAATCAATCTCATCTTAAAGCAGACAAAAATCACT CTACAAAACAAAAAGACACAATCAAAACAACACTCTTGAAGAAATTGATGTGTTCAATGCAACATCCTCC ATCATGGTTAATACATTGGTTTAACTTATACACAAAATTAAACAACATATTAACACAGTATCGATCAAAT GAGGTAAAAAACCATGGGTTTACATTGATAGATAATCAAACTCTTAGTGGATTTCAATTTATTTTGAACC AATATGGTTGTATAGTTTATCATAAGGAACTCAAAAGAATTACTGTGACAACCTATAATCAATTCTTGAC ATGGAAAGATATTAGCCTTAGTAGATTAAATGTTTGTTTAATTACATGGATTAGTAACTGCTTGAACACA TTAAATAAAAGCTTAGGCTTAAGATGCGGATTCAATAATGTTATCTTGACACAACTATTCCTTTATGGAG ATTGTATACTAAAGCTATTTCACAATGAGGGGTTCTACATAATAAAAGAGGTAGAGGGATTTATTATGTC TCTAATTTTAAATATAACAGAAGAAGATCAATTCAGAAAACGATTTTATAATAGTATGCTCAACAACATC ACAGATGCTGCTAATAAAGCTCAGAAAAATCTGCTATCAAGAGTATGTCATACATTATTAGATAAGACAG TGTCCG ATAATATAATAAATG G CAG ATG G ATAATTCTATTAAGTAAGTTCCTTAAATTAATTAAG CTTG C AGGTGACAATAACCTTAACAATCTGAGTGAACTATATTTTTTGTTCAGAATATTTGGACACCCAATGGTA GATGAAAGACAAGCCATGGATGCTGTTAAAATTAATTGCAATGAGACCAAATTTTACTTGTTAAGCAGTC TGAGTATGTTAAGAGGTGCCTTTATATATAGAATTATAAAAGGGTTTGTAAATAATTACAACAGATGGCC TACTTTAAGAAATGCTATTGTTTTACCCTTAAGATGGTTAACTTACTATAAACTAAACACTTATCCTTCT TTGTTGGAACTTACAGAAAGAGATTTGATTGTGTTATCAGGACTACGTTTCTATCGTGAGTTTCGGTTGC CTAAAAAAGTGGATCTTGAAATGATTATAAATGATAAAGCTATATCACCTCCTAAAAATTTGATATGGAC TAGTTTCCCTAGAAATTACATGCCATCACACATACAAAACTATATAGAACATGAAAAATTAAAATTTTCC GAGAGTGATAAATCAAGAAGAGTATTAGAGTATTATTTAAGAGATAACAAATTCAATGAATGTGATTTAT ACAACTGTGTAGTTAATCAAAGTTATCTCAACAACCCTAATCATGTGGTATCATTGACAGGCAAAGAAAG AGAACTCAGTGTAGGTAGAATGTTTGCAATGCAACCGGGAATGTTCAGACAGGTTCAAATATTGGCAGAG AAAATGATAGCTGAAAACATTTTACAATTCTTTCCTGAAAGTCTTACAAGATATGGTGATCTAGAACTAC AAAAAATATTAGAACTGAAAGCAGGAATAAGTAACAAATCAAATCGCTACAATGATAATTACAACAATTA CATTAGTAAGTGCTCTATCATCACAGATCTCAGCAAATTCAATCAAGCATTTCGATATGAAACGTCATGT ATTTGTAGTGATGTGCTGGATGAACTGCATGGTGTACAATCTCTATTTTCCTGGTTACATTTAACTATTC CTCATGTCACAATAATATGCACATATAGGCATGCACCCCCCTATATAGGAGATCATATTGTAGATCTTAA CAATGTAGATGAACAAAGTGGATTATATAGATATCACATGGGTGGCATCGAAGGGTGGTGTCAAAAACTA TGGACCATAGAAGCTATATCACTATTGGATCTAATATCTCTCAAAGGGAAATTCTCAATTACTGCTTTAA TTAATGGTGACAATCAATCAATAGATATAAGCAAACCAATCAGACTCATGGAAGGTCAAACTCATGCTCA AGCAGATTATTTGCTAGCATTAAATAGCCTTAAATTACTGTATAAAGAGTATGCAGGCATAGGCCACAAA TTAAAAGGAACTGAGACTTATATATCACGAGATATGCAATTTATGAGTAAAACAATTCAACATAACGGTG TATATTACCCAGCTAGTATAAAGAAAGTCCTAAGAGTGGGACCGTGGATAAACACTATACTTGATGATTT CAAAGTGAGTCTAGAATCTATAGGTAGTTTGACACAAGAATTAGAATATAGAGGTGAAAGTCTATTATGC AGTTTAATATTTAGAAATGTATGGTTATATAATCAGATTGCTCTACAATTAAAAAATCATGCATTATGTA ACAATAAACTATATTTGGACATATTAAAGGTTCTGAAACACTTAAAAACCTTTTTTAATCTTGATAATAT TGATACAGCATTAACATTGTATATGAATTTACCCATGTTATTTGGTGGTGGTGATCCCAACTTGTTATAT CGAAGTTTCTATAGAAGAACTCCTGACTTCCTCACAGAGGCTATAGTTCACTCTGTGTTCATACTTAGTT ATTATACAAACCATGACTTAAAAGATAAACTTCAAGATCTGTCAGATGATAGATTGAATAAGTTCTTAAC ATGCATAATCACGTTTGACAAAAACCCTAATGCTGAATTCGTAACATTGATGAGAGATCCTCAAGCTTTA GGGTCTGAGAGACAAGCTAAAATTACTAGCGAAATCAATAGACTGGCAGTTACAGAGGTTTTGAGTACAG CTCCAAACAAAATATTCTCCAAAAGTGCACAACATTATACTACTACAGAGATAGATCTAAATGATATTAT GCAAAATATAGAACCTACATATCCTCATGGGCTAAGAGTTGTTTATGAAAGTTTACCCTTTTATAAAGCA GAGAAAATAGTAAATCTTATATCAGGTACAAAATCTATAACTAACATACTGGAAAAAACTTCTGCCATAG ACTTAACAGATATTGATAGAGCCACTGAGATGATGAGGAAAAACATAACTTTGCTTATAAGGATACTTCC ATTGGATTGTAACAGAGATAAAAGAGAGATATTGAGTATGGAAAACCTAAGTATTACTGAATTAAGCAAA TATGTTAGGGAAAGATCTTGGTCTTTATCCAATATAGTTGGTGTTACATCACCCAGTATCATGTATACAA TGGACATCAAATATACTACAAGCACTATATCTAGTGGCATAATTATAGAGAAATATAATGTTAACAGTTT AACACGTGGTGAGAGAGGACCCACTAAACCATGGGTTGGTTCATCTACACAAGAGAAAAAAACAATGCCA GTTTATAATAGACAAGTCTTAACCAAAAAACAGAGAGATCAAATAGATCTATTAGCAAAATTGGATTGGG TGTATGCATCTATAGATAACAAGGATGAATTCATGGAAGAACTCAGCATAGGAACCCTTGGGTTAACATA TGAAAAGGCCAAGAAATTATTTCCACAATATTTAAGTGTCAATTATTTGCATCGCCTTACAGTCAGTAGT AGACCATGTGAATTCCCTGCATCAATACCAGCTTATAGAACAACAAATTATCACTTTGACACTAGCCCTA TTAATCGCATATTAACAGAAAAGTATGGTGATGAAGATATTGACATAGTATTCCAAAACTGTATAAGCTT TGGCCTTAGTTTAATGTCAGTAGTAGAACAATTTACTAATGTATGTCCTAACAGAATTATTCTCATACCT AAGCTTAATGAGATACATTTGATGAAACCTCCCATATTCACAGGTGATGTTGATATTCACAAGTTAAAAC AAGTGATACAAAAACAGCATATGTTTTTACCAGACAAAATAAGTTTGACTCAATATGTGGAATTATTCTT AAGTAATAAAACACTCAAATCTGGATCTCATGTTAATTCTAATTTAATATTGGCACATAAAATATCTGAC TATTTTCATAATACTTACATTTTAAGTACTAATTTAGCTGGACATTGGATTCTGATTATACAACTTATGA AAGATTCTAAAGGTATTTTTGAAAAAGATTGGGGAGAGGGATATATAACTGATCATATGTTTATTAATTT GAAAGTTTTCTTCAATGCTTATAAGACCTATCTCTTGTGTTTTCATAAAGGTTATGGCAAAGCAAAGCTG GAGTGTGATATGAACACTTCAGATCTTCTATGTGTATTGGAATTAATAGACAGTAGTTATTGGAAGTCTA TGTCTAAGGTATTTTTAGAACAAAAAGTTATCAAATACATTCTTAGCCAAGATGCAAGTTTACATAGAGT AAAAG G ATGTCATAG CTTCAAATTATG GTTTCTTAAACGTCTTAATGTAGCAG AATTCACAGTTTG CCCT TGGGTTGTTAACATAGATTATCATCCAACACATATGAAAGCAATATTAACTTATATAGATCTTGTTAGAA TGGGATTGATAAATATAGATAGAATACACATTAAAAATAAACACAAATTCAATGATGAATTTTATACTTC TAATCTCTTCTACATTAATTATAACTTCTCAGATAATACTCATCTATTAACTAAACATATAAGGATTGCT AATTCTGAATTAGAAAATAATTACAACAAATTATATCATCCTACACCAGAAACCCTAGAGAATATACTAG CCAATCCGATTAAAAGTAATGACAAAAAGACACTGAATGACTATTGTATAGGTAAAAATGTTGACTCAAT AATGTTACCATTGTTATCTAATAAGAAGCTTATTAAATCGTCTGCAATGATTAGAACCAATTACAGCAAA CAAGATTTGTATAATTTATTCCCTATGGTTGTGATTGATAGAATTATAGATCATTCAGGCAATACAGCCA AATCCAACCAACTTTACACTACTACTTCCCACCAAATATCCTTAGTGCACAATAGCACATCACTTTACTG CATGCTTCCTTGGCATCATATTAATAGATTCAATTTTGTATTTAGTTCTACAGGTTGTAAAATTAGTATA GAGTATATTTTAAAAGATCTTAAAATTAAAGATCCCAATTGTATAGCATTCATAGGTGAAGGAGCAGGGA ATTTATTATTGCGTACAGTAGTGGAACTTCATCCTGACATAAGATATATTTACAGAAGTCTGAAAGATTG CAATGATCATAGTTTACCTATTGAGTTTTTAAGGCTGTACAATGGACATATCAACATTGATTATGGTGAA AATTTGACCATTCCTGCTACAGATGCAACCAACAACATTCATTGGTCTTATTTACATATAAAGTTTGCTG AACCTATCAGTCTTTTTGTCTGTGATGCCGAATTGTCTGTAACAGTCAACTGGAGTAAAATTATAATAGA ATG GAG CAAG CATGTAAG AAAGTG CAAGTACTGTTCCTCAGTTAATAAATGTATGTTAATAGTAAAATAT CATG CTCAAG ATG ATATTG ATTTCAAATTAG ACAATATAACTATATTAAAAACTTATGTATG CTTAG G CA GTAAGTTAAAGGGATCGGAGGTTTACTTAGTCCTTACAATAGGTCCTGCGAATATATTCCCAGTATTTAA TGTAGTACAAAATGCTAAATTGATACTATCAAGAACCAAAAATTTCATCATGCCTAAGAAAGCTGATAAA GAGTCTATTGATGCAAATATTAAAAGTTTGATACCCTTTCTTTGTTACCCTATAACAAAAAAAGGAATTA ATACTGCATTGTCAAAACTAAAGAGTGTTGTTAGTGGAGATATACTATCATATTCTATAGCTGGACGTAA TGAAGTTTTCAGCAATAAACTTATAAATCATAAGCATATGAACATCTTAAAATGGTTCAATCATGTTTTA AATTTCAGATCAACAGAACTAAACTATAACCATTTATATATGGTAGAATCTACATATCCTTACCTAAGTG AATTGTTAAACAGCTTGACAACCAATGAACTTAAAAAACTGATTAAAATCACAGGTAGTCTGTTATACAA CTTTCATAATGAATAATGAATAAAGATCTTATAATAAAAATTCCCATAGCTATACACTAACACTGTATTC AATTATAGTTATTAAAAATTAAAAATCATATAATTTTTTAAATAACTTTTAGTGAACTAATCCTAAAGTT ATCATTTTAATCTTGGAGGAATAAATTTAAACCCTAATCTAATTGGTTTATATGTGTATTAACTAAATTA CGAGATATTAGTTTTTGACACTTTTTTTCTCGT ( SEQ ID NO : 404 ) , or a nucleotide sequence with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto, human respiratory syncytial virus isolate VN-848-12/10 (>gi|780955193 |gb|KJ939971.11) having
ACGGGAAAAAATGCGTACAACAAACTTGCATAAACCAAAAAAATGGGGCAAATAAGAATTTGATAAGTAC CACTTAAATTTAACTCCTTTGGTTAGAGATGGGCAGCAACTCATTGAGTATGATAAAAGTTAGATTGCAA AATCTGTTTGACAATGATGAAGTAGCATTGTTAAAAATAACATGCTATACTGACAAATTAATACAGTTAA CTAATGCTTTGGCTAAGGCAGTTATACATACAATCAAATTGAATGGCATTGTATTTGTGCATGTTATTAC AAGTAGTGATATTTGCCCTAATAATAATATTGTAGTGAAATCCAATTTCACAACAATGCCAGTATTACAA AATGGAGGTTATATATGGGAAATGATGGAATTAACACACTGTTCTCAACCTAATGGCCTAATAGATGACA ATTGTGAAATTAAATTCTCCAAAAAACTAAGTGATTCAACAATGACCAATTATATGAATCAATTATCTGA ATTACTTGGATTTGACCTCAATCCATAAATCATAATAAATATCAACTAGCAAATCAATGTCACCAACACC ATTAGTTAATATAAAACTTGACAGAAGATAAAAATGGGGCAAATAAATCAATTCAGCCGACCCAACCATG GACACAACACACAATGATACCACACCACAAAGACTGATGATCACAGACATGAGACCATTATCACTTGAGA CTATAATAACATCTCTAACCAGAGACATCATAACACATAAATTTATATACTTGATAAATCATGAATGCAT AGTGAGGAAACTTGATGAAAGACAGGCCACATTTACATTTCTGGTCAACTATGAAATGAAACTATTGCAC AAAGTGGGAAGCACTAAATATAAAAAATATACTGAATACAACACAAAATATGGCACTTTCCCTATGCCAA TATTTATCAATCATGATG GGTTCTTAG AATG CATTGG CATTAAG CCTACCAAG CACACACCCATAATATA CAAGTATGATCTCAATCCATGAATATCAAAACAAGATTCAAACAATCCGAAATAACAACTTTATGCATAA TCACACTCCATAGTCCAAATGGAGCCTGAAAATTATAGTTATTTAAAATTAAGGAGAGACATAAGATGAA AGATGGGGCAAATACAAAAATGGCTCTTAGCAAAGTCAAGTTGAATGATACACTCAACAAAGATCAACTT CTATCATCCAGCAAATATACCATCCAACGGAGCACAGGAGACAGCATTGACACTCCTAATTATGATGTGC AGAAACACATTAATAAGTTATGTGGCATGTTATTAATCACAGAAGATGCTAATCATAAATTCACTGGGTT AATAGGTATGTTATATGCTATGTCTAGATTAGGAAGAGAAGACACCATAAAAATACTCAAAGATGCGGGA TATCATGTTAAGGCAAATGGAGTGGATGTAACAACACATCGTCAAGACATTAATGGGAAAGAAATGAAAT TTGATGTGTTAACATTAGCAAGCTTAACAACTGAAATTCAAATCAACATTGAGATAGAATCTAGAAAATC CTACAAAAAAATG CTAAAAG AAATG G GAG AAGTG G CTCCAG AATACAG G CATG ACTCTCCTG ATTGTG G G ATGATAATATTATGTATAGCAGCATTAGTAATAACCAAATTAGCAGCAGGAGATAGATCTGGTCTTACAG CTGTGATTAGGAGAGCTAATAATGTCCTAAAAAATGAAATGAAACGTTATAAAGGTTTATTACCCAAGGA TATAGCCAACAGCTTCTATGAAGTGTTTGAAAAATATCCTCACTTTATAGATGTTTTTGTTCATTTTGGT ATAGCACAATCTTCTACCAGAGGTGGCAGTAGAGTTGAAGGGATTTTTGCAGGATTGTTTATGAATGCCT ATGGTG CAGG G CAAGTG ATGTTACG GTGG GGG GTCTTAG CAAAATCAGTTAAAAACATTATGTTAG G ACA CGCTAGTGTACAAGCAGAAATGGAACAAGTTGTGGAGGTGTATGAGTATGCTCAGAAATTGGGTGGAGAA GCAGGATTCTACCATATATTGAACAACCCAAAAGCATCACTATTATCTTTGACTCAATTTCCTCACTTCT CTAGTGTAGTATTGGGCAATGCTGCTGGCCTAGGCATAATGGGAGAATACAGAGGTACACCAAGGAATCA AGATTTATATGATGCTGCAAAAGCATATGCTGAACAACTCAAAGAAAATGGTGTGATTAACTACAGTGTA TTAGACTTGACAGCAGAAGAACTAGAGGCTATCAAACATCAGCTTAATCCAAAAGATAATGATGTAGAGC TTTGAGTTAATAAAAAAGTGGGGCAAATAAATCATCATGGAAAAGTTTGCTCCTGAATTCCATGGAGAAG ACGCAAACAACAGAGCCACCAAATTCCTAGAATCAATAAAGGGCAAATTCACATCACCCAAAGATCCCAA GAAAAAAGATAGTATCATATCTGTCAACTCAATAGATATAGAAGTAACCAAAGAAAGCCCTATAACATCA AATTCAACCATTATAAACCCAATAAATGAGACAGATGATACTGTAGGGAACAAGCCCAATTATCAAAGAA AGCCTCTAGTAAGTTTCAAAGAAGACCCTACGCCAAGTGATAATCCTTTTTCAAAACTATACAAAGAAAC CATAGAAACATTTGATAACAATGAAGAAGAATCTAGCTATTCATATGAAGAAATAAATGATCAGACAAAC GATAATATAACAGCAAGATTAGATAGGATTGATGAGAAACTAAGTGAAATACTAGGAATGCTTCACACAT TAGTAGTAGCGAGTGCAGGACCTACATCTGCTCGGGATGGTATAAGAGATGCCATGGTTGGTTTAAGAGA AGAAATGATAGAAAAAATCAGAACTGAAGCATTAATGACCAATGACAGACTAGAAGCTATGGCAAGACTC AGGAATGAAGAAAGTGAAAAGATGGCAAAAGACACATCAGATGAAGTGTCTCTCAATCCAACATCAGAGA AACTGAACAACCTGTTGGAAGGGAATGATAGTGACAATGATCTATCACTTGAGGATTTCTGATTAGCTAC CAAACTGTACATCAAAACACACCAATAGAAAACCAACAAACAAACCAACTCACCCATCCAACCAAACATC TATCTGCCG ATTAG CCAACCAG CCAAAAAACAACCAG CCAATCTAAAACTAG CCACACG G AAAAAATCGA TACTATAGTTACAAAAAAAGATGGGGCAAATATGGAAACATACGTGAACAAGCTTCACGAAGGCTCCACA TACACAGCTGCTGTTCAATACAACGTCCTAGAAAAAGACGATGATCCTGCATCACTTACAATATGGGTGC CCATGTTCCAATCATCCATGCCAGCAGATCTACTCATAAAAGAACTAGCCAATGTCAATATACTAGTGAA ACAAATATCCACACCCAAGGGACCCTCATTAAGAGTCATGATAAACTCAAGAAGTGCAGTGCTAGCACAA ATGCCCAGCAAATTTACCATATGTGCCAATGTGTCCTTGGATGAAAGAAGCAAGCTGGCATATGATGTAA CCACACCCTGTGAAATTAAAGCATGCAGTCTAACATGCCTAAAATCAAAAAATATGTTAACTACAGTTAA AGATCTCACTATGAAAACACTCAACCCAACACATGACATCATTGCTTTATGTGAATTTGAAAATATAGTA ACATCAAAAAAAGTCATAATACCAACATACCTAAGATCTATCAGCGTCAGAAATAAAGATCTGAACACAC TTG AAAATATAACAACCACTG AATTCAAAAATG CCATTACAAATG CAAAAATCATCCCTTACTCAG GATT ACTGTTAGTCATCACAGTGACTGACAACAAAGGAGCATTCAAATACATAAAGCCACAAAGTCAATTCATA GTAGATCTTGGAGCTTACCTAGAAAAAGAAAGTATATATTATGTTACAACAAATTGGAAGCACACAGCTA CACGATTTGCAATCAAACCCATGGAAGATTAACCTTTTTCCTCTACATCAATGAGTAGATTCATACAAAC TTTCTAACTACATTCTTCACTTCACAATCATAATCACCAACCCTCTGTGGTTCAATCAATCAAACAAAAC TCATCAGGAGTTCCAGATCATCCCAAGTCATTGTTCATCAGATCCAGTACTCAAATAAGTTAATAAAAAT CCACATGGGGCAAATAATCATTGAGGGAAACCCAACTAATCACAACATCTGTCAACATAGACTAGTCAAC ACGCTAGATAAAATCAACCAATGGAAAATACATCCATAACTATAGAATTCTCAAGCAAATTCTGGCCTTA CTTTACACTAATACACATGATAACAACAATAATCTCTTTGATAATCATAATCTCCATCATGATTGCAATA CTAAACAAACTCTGCGAATATAATGTATTCCACAACAAGACCTTTGAGCTACCAAGAGCTCGAGTCAATA CATAGCATTCACCAATATGATAGCTCAAAACTGTAACCTTGCATTTGTAAGTGAACTACCCTCACCTCTT CACAAAATCACATCAACATCTCACCATGCAAGCAATCATCTACACCATAAAGTAGTTAATTAAAAATAGT CATAACAATG AACTAGG ATATTAAG ACCAAAAACAACG CTG G GG CAAATG CAAACATGTCCAAAACCAAG GACCAACGCACCGCCAAGACTCTAGAAAGGACCTGGGACACTCTCAATCATCTATTATTCATATCATCGT GCTTATACAAGTTAAATCTTAAATCTATAGCACAAATCACATTATCTATTTTGGCAATGATAATCTCAAC CTCACTTATAATTGCAGCCATCATATTCATATCTTCGGCAAACCACAAAGTCACACTAACAACTGCAATC ATACAAGATGCAACGAACCAGATAAAGAACACAACCCCAACATACCTCACCCAGAATCCCCAGCTTGGAA TCAG CTTCTCCAATCTGTCCG G AACTACATCACAATCCACCACCATACTAG CTTCAACAACG CCAAGTGC CGAGTCAACCCCACAATCCACAACAGTCAAGATCAAAAACACAACAACAACCCAAATACAACCCAGCAAA CCCACCACAAAACAACGCCAAAATAAACCACAAAACAAACCCAACAATGATTTTCACTTTGAAGTGTTCA ATTTTGTACCCTGCAGCATATGCAGCAACAATCCAACCTGCTGGGCCATCTGCAAGAGAATACCAAACAA AAAACCTGGAAAGAAAACTACCACCAAGCCCACAAAAAAACCAACCATCAAGACAACCAAAAAAGATCCC AAACCTCAAACCACAAAACCAAAGGAAGTATTCACCACCAAGTCCACAGAAAAGCCAACCATCGACACCA CCAAAACAAACATCAGAACTACACTGCTCACCTCCAACACCACAGGAAATCCAGAACACACAAGTCAAGA GGAAACCCTCCACTCAACCACCTCCGAAGGCAATCTAAGCCCATCACAAGTCTATACAACATCCGAGTAC CTATCACAATCTCCATCTTCATCCAACACAACAAAATGATAGTCATTAAAAAGCGTATTGTTGCAAAAAG CCATAACCAAATCAAACAG AATCAAAATCAACTCTG GG G CAAATAACAATG G AGTTG CCAATCCTCAAAA CAAATG CTATTACCACAATCCTTG CTG CAGTCACACTCTGTTTCG CTTCCAGTCAAAACATCACTG AAGA ATTTTATCAATCAACATGCAGTGCAGTTAGCAAAGGCTATCTTAGTGCTCTAAGAACTGGTTGGTATACT AGTGTTATAACTATAGAATTAAGTAATATCAAGGAAAATAAGTGTAATGGTACAGATGCTAAGGTAAAAT TAATAAAACAAGAATTAGATAAATATAAAAATGCTGTAACAGAATTGCAGTTGCTCATGCAAAGCACACC AGCAGCCAACAGTCGAGCCAGAAGAGAACTACCAAGATTTATGAATTATACACTCAACAATACCAAAAAC ACCAATGTAACATTAAGCAAGAAAAGGAAAAGAAGATTTCTTGGGTTTTTGTTAGGTGTTGGATCTGCAA TCGCCAGTGGCGTTGCTGTATCTAAGGTCCTGCACCTAGAAGGGGAAGTGAACAAAATCAAAAGTGCTCT ACTATCCACAAACAAGG CTGTAGTCAG CTTATCTAATGG AGTCAGTGTCTTAACCAG CAAAGTGTTAG AC CTCAAAAACTATATAGATAAACAGTTGTTACCTATTGTTAACAAGCAAAGCTGCAGCATATCAAACATTG AAACTGTGATAGAGTTCCAACAAAAGAACAACAGACTACTAGAGATTACCAGAGAATTTAGTGTTAATGC AGGTGTAACTACACCTGTAAGCACTTATATGTTAACTAATAGTGAGTTATTATCATTAATCAATGATATG CCTATAACAAATGATCAGAAAAAGTTAATGTCTAACAATGTTCAAATAGTTAGACAGCAAAGTTACTCTA TCATGTCAATAATAAAAGAGGAAGTCTTAGCATATGTAGTACAATTACCACTATATGGTGTAATAGATAC TCCTTGTTGGAAACTACACACATCCCCTCTATGTACAACCAACACAAAGGAAGGATCCAACATCTGCTTA ACAAGAACCGACAGAGGATGGTACTGTGACAATGCAGGATCAGTATCCTTTTTCCCACAAGCTGAAACAT GTAAAGTTCAATCGAATCGGGTATTTTGTGACACAATGAACAGTTTAACATTACCAAGTGAGGTAAATCT CTGCAACATTGACATATTCAACCCCAAATATGATTGCAAAATTATGACTTCAAAAACAGATGTAAGCAGC TCCGTTATCACATCTCTAGGAGCCATTGTGTCATGCTATGGCAAAACCAAATGTACAGCATCCAATAAAA ATCGTGGGATCATAAAGACATTCTCTAACGGGTGTGATTATGTATCAAATAAGGGGGTGGATACTGTGTC TGTAGGTAATACATTATATTATGTAAATAAGCAAGAAGGCAAAAGTCTCTATGTAAAAGGTGAACCAATA ATAAATTTCTATGATCCATTAGTGTTCCCCTCTGATGAATTTGATGCATCAATATCTCAAGTCAATGAGA AAATTAATCAGAGTCTAGCATTTATCCGTAAATCAGATGAATTATTACATAATGTAAATGCTGGTAAATC CACCACAAATATCATGATAACTACTATAATTATAGTAATTATAGTAATATTGTTATCATTAATTGCAGTT GGACTGCTTCTATACTGCAAGGCCAGAAGCACACCAATCACATTAAGTAAGGATCAACTGAGTGGTATAA ATAATATTGCATTTAGTAACTGAATAAAAATAGCACCTAATCATATTCTTACAATGGTTCACTATTTGAC CATAGATAACCCATCTATCATTGGATTATCCTAAAATTTGAACATCATCACAACTTTCATCTATAAACCA TCTCATTTACACTATTTAAGTAGATTTCTATTTTATAGTTATATAAAACAATTGAATACCAAATTAACTT ACTATTTGTAAAAATG AG AACTG G G GCAAATATGTCACG AAG G AATCCTTG CAAATTCG AAATTCG AG GT CATTGCTTGAATGGTAAAAGGTGTCATTTTAGTCATAATTATTTTGAATGGCCACCCCATGCACTACTTG TAAGACAAAACTTTATGTTAAACAGAATACTTAAGTCTATGGATAAAAGCATAGATACTTTGTCAGAAAT AAGTGGAGCTGCAGAGTTGGACAGAACAGAAGAGTATGCCCTCGGTGTAGTTGGAGTGCTAGAGAGTTAT ATAG G ATCAATAAATAATATAACTAAACAATCAG CATGTGTTG CCATG AG CAAACTCCTCACTG AACTCA ACAGCGATGACATCAAAAAACTAAGGGACAATGAAGAGCCAAACTCACCCAAAGTAAGAGTGTACAATAC TGTCATATCATATATTGAAAGCAACAGGAAGAACAATAAACAAACTATCCATCTGTTAAAAAGATTGCCA GCAGACGTATTGAAGAAAACCATCAAAAACACATTGGATATCCACAAGAGCATAACCATCAATAACCCAA AAGAATCAACTGTTAGTGATACGAACGACCATGCCAAAAATAATGATACTACCTGACAAATATCCTTGTA GTATAAATTCCATACTAATAACAAGTAATTGTAGAGTCACTATGTATAATCAAAAAAACACACTATATAT CAATCAAAACAACCAAAATAACCATATATACCCACCGGATCAACCATTCAATGAAATCCATTGGACCTCT CAAGACTTGATTGATGCAACTAAAAATTTTCTACAATATCTAGGTATTACTGATGATATATACACAATAT ATATATTAGTGTCATAATACTCAATCCTAATATTTACCACATCATCAAATTATTAACTCAAACAATTCAA GCTATGGGACAAAATGGATCCCATTATTAGTGGAAATTCTGCTAATGTTTATCTAACTGATAGTTATTTA AAAGGTGTTATTTCTTTCTCAGAATGTAACGCCTTAGGAAGTTACATATTCAATGGTCCTTATCTCAAAA ATGATTATACCAACTTAATTAGTAGACAAAATCCATTAATAGAACACATAAATCTAAAGAAACTAAATAT AACACAGTCCTTAATATCTAAGTATCATAAAGGTGAAATAAAAATAGAAGAACCTACTTACTTTCAGTCA TTACTTATGACATACAAGAGTATGACCTCGTCAGAACAGACTACTACTACTAATTTACTTAAAAAGATAA TAAGAAGAGCTATAGAAATCAGTGATGTCAAAGTCTATGCTATATTGAATAAACTGGGGCTCAAAGAAAA AGACAAGATTAAATCCTACAATGGACAAGATGAAGACAACTCAGTTATTACTACCATAATCAAAGATGAT ATACTTTTAGCTGTCAAGGATAATCAATCTCATCTTAAAGCAGACAAAAATCAATCCACAAAACAAAAAG ATACAATCAAAACAACACTTCTGAAGAAATTAATGTGTTCGATGCAACATCCTCCATCATGGTTAATACA TTGGTTTAATTTATACACAAAATTAAACAGCATATTAACACAGTATCGATCTAGTGAGGTAAAAAACCAT GGTTTTATATTGATAGATAATCATACTCTTAGTGGATTCCAATTTATTTTGAATCAATATGGTTGTATAG TTTATCATAAGGAACTCAAAAGAATTACTGTGACAACTTATAATCAATTCTTGACATGGAAAGATATTAG CCTTAGTAGATTAAATGTTTGTTTGATTACATGGATTAGTAACTGTTTGAACACATTAAACAAAAGCTTA GGCTTAAGATGTGGATTCAATAATGTTATCTTGACACAATTATTCCTTTATGGAGATTGTATACTAAAAC TATTCCACAATGAGGGGTTCTACATAATAAAAGAGGTAGAGGGATTTATTATGTCTCTAATTTTAAATAT AACAGAAGAAGATCAATTCAGAAAACGGTTTTATAATAGTATGCTCAACAACATCACAGATGCCGCTAAT AAAGCTCAGAAAAATCTGCTATCAAGAGTATGTCATACATTATTAGATAAGACAATATCAGATAATATAA TAAATGGCAGATGGATAATTCTATTGAGTAAGTTCCTTAAATTAATTAAGCTTGCAGGTGACAATAACCT CAACAATCTGAGTGAATTATATTTTTTGTTCAGAATATTTGGACACCCAATGGTAGATGAAAGACAAGCC ATGGATGCTGTTAAAGTTAATTGCAACGAGACCAAATTTTACTTGTTAAGTAGTTTGAGTATGTTAAGAG GAGCTTTTATATATAGAATTATAAAAGGGTTTGTAAATAATTACAACAGATGGCCTACTTTAAGAAATGC CATTGTTTTACCCTTAAGATGGTTAACTTACTATAAACTAAACACTTATCCTTCCTTGTTAGAACTTACA GAAAGAGATTTGATTGTTCTATCAGGACTACGTTTCTATCGAGAGTTTCGGTTGCCTAAAAAAGTGGATC TTGAAATGATCATAAATGATAAGGCTATATCACCTCCTAAAAATTTAATATGGACTAGTTTCCCTAGAAA TTATATGCCGTCACACATACAAAATTATATAGAACATGAAAAATTAAAATTCTCTGATAGTGATAAATCA AGAAGAGTATTAGAGTATTATTTAAGAGATAACAAATTCAATGAATGTGATTTATACAACTGTGTAGTTA ATCAAAGTTATCTTAACAACCCGAATCATGTGGTATCATTGACAGGCAAAGAAAGAGAACTCAGTGTAGG TAGAATGTTTGCAATGCAACCAGGAATGTTCAGACAAGTTCAAATATTAGCAGAGAAAATGATAGCTGAA AACATATTACAATTTTTCCCTGAAAGTCTTACAAGATATGGTGATCTAGAACTACAGAAAATATTAGAAT TGAAAGCAGGAATAAGTAACAAATCAAATCGTTACAATGATAATTACAACAATTACATTAGTAAGTGCTC TATCATCACAGATCTCAGCAAATTCAATCAAGCATTTCGATATGAAACATCATGTATTTGTAGCGATGTA CTG GATG AACTG CATG GTGTACAATCTCTATTTTCCTG GTTACATTTAACTATTCCTCATGTCACAATAA TATGCACATATAGGCATGCTCCCCCCTATATAAAGGATCATATTGTAGATCTTAACAATGTAGATGAGCA AAGTGGATTATATAGATATCATATGGGTGGTATCGAAGGGTGGTGTCAAAAACTATGGACCATAGAAGCT ATATCACTATTAGATCTAATATCTCTCAAAGGGAAATTCTCAATTACTGCTTTAATTAATGGTGACAATC
AATCAATAGATATAAGTAAACCAGTCAGACTCATGGAAGGTCAAACTCATGCTCAAGCAGATTATTTGCT AGCATTAAATAGTCTTAAATTACTGTATAAAGAGTATGCAGGAATAGGCCACAAATTAAAAGGAACTGAG ACTTATATATCAAGAGATATGCAATTTATGAGTAAAACGATCCAACATAACGGTGTATATTACCCAGCTA GTATAAAGAAAGTCCTAAGAGTGGGACCGTGGATAAACACTATACTTGATGACTTCAAAGTGAGTCTAGA ATCTATAGGTAGTTTGACACAAGAATTAGAATATAGAGGTGAAAGTCTATTATGCAGTTTAATATTTAGA AATGTATG GTTATATAATCAAATTG CATTACAACTTAAAAATCATG CATTATGTAACAACAAATTATATT TGGACATATTAAAAGTTCTAAAACACTTAAAAACCTTTTTTAATCTTGATAACATTGACACAGCATTAAC ATTGTATATGAATTTGCCCATGTTATTTGGTGGTGGTGATCCTAACTTGTTATATCGAAGTTTCTATAGA AGAACTCCTGATTTCCTCACAGAGGCTATAGTTCACTCTGTGTTCATACTTAGTTATTATACAAACCATG ATTTAAAAGATAAACTTCAAGATCTGTCAGATGATAGATTGAATAAGTTCTTAACATGCATAATCACGTT TGACAAAAACCCCAATGCTGAATTCGTTACATTGATGAGAGATCCTCAAGCTTTAGGATCTGAGAGGCAA G CTAAAATTACTAG CG AAATCAATAG ACTGG CAGTTACCG AG GTTTTG AG CACAGCTCCAAACAAAATAT TCTCCAAAAGTGCACAACACTATACCACTACAGAGATAGATCTTAATGATATTATGCAAAATATAGAACC TACATATCCTCATGGGCTAAGAGTTGTTTATGAAAGTTTACCCTTTTATAAAGCAGAGAAAATAGTAAAT CTTATATCCGGTACAAAATCTATAACTAACATACTGGAAAAGACTTCTGCCATAGACTTAACAGATATTG ATAGAGCCACTGAGATGATGAGGAAAAACATAACTTTGCTTATAAGGATATTACCATTAGATTGTAACAG AGATAAAAGAGAAATATTGAGTATGGAAAACCTAAGTATTACCGAATTAAGCAAATACGTTAGAGAAAGA TCCTGGTCTTTATCCAATATAGTTGGTGTTACATCACCCAGTATCATGTATACAATGGACATAAAATATA CAACAAGCACTATAGCTAGTGGCATAATCATAGAGAAATATAATGTCAACAGTTTAACACGTGGTGAGAG AGGACCCACTAAACCATGGGTTGGTTCATCTACACAAGAGAAAAAGACAATGCCAGTTTATAATAGACAA GTTTTAACCAAAAAACAAAGAGATCAAATAGATCTATTAGCAAAATTGGATTGGGTGTATGCATCTATAG ATAACAAGGATGAATTTATGGAGGAACTTAGCATAGGAACTCTTGGGTTAACATATGAGAAGGCCAAAAA ATTATTTCCACAATATTTAAGTGTTAACTATTTGCATCGTCTTACAGTCAGTAGTAGACCATGTGAATTC CCTGCATCTATACCAGCTTATAGAACTACAAATTATCACTTTGATACTAGCCCTATTAATCGCATATTAA CAGAAAAGTATGGTGATGAAGATATTGACATAGTATTCCAAAACTGTATAAGCTTTGGCCTTAGCTTAAT GTCAGTAGTAGAACAATTTACTAATGTATGTCCTAACAGAATTATTCTCATACCCAAGCTTAATGAGATA CATTTGATGAAACCTCCCATATTCACAGGTGATGTTGATATTCACAAGTTAAAACAAGTGATACAAAAAC AACATATGTTTTTACCAGACAAAATAAGTTTGACTCAATATGTGGAATTATTCTTAAGTAATAAAACACT CAAATCTGGATCTAATGTTAATTCTAATTTAATATTGGCGCATAAGATATCTGACTATTTTCATAATACT TACATTTTAAGTACTAATTTAGCTGGACATTGGATTCTTATTATACAACTTATGAAAGATTCTAAGGGTA TTTTTGAAAAAGATTGGGGAGAGGGATATATAACTGATCATATGTTCATTAATTTGAAAGTTTTCTTCAA TGCTTATAAGACATATCTCTTGTGTTTTCATAAAGGTTACGGCAGAGCAAAGCTGGAGTGTGATATGAAT ACTTCAGATCTCCTATGTGTATTGGAATTAATAGACAGTAGTTATTGGAAGTCTATGTCTAAGGTGTTTT TAGAACAAAAAGTTATCAAATACATTCTTAGCCAGGATGCAAGTTTACATAGAGTAAAAGGATGTCATAG CTTCAAACTATG GTTTCTTAAACGTCTTAATGTAG CAG AATTCACAGTTTG CCCTTG G GTTGTTAACATA GATTATCATCCAACACATATGAAAGCAATATTAACTTATATTGATCTTGTTAGAATGGGATTGATAAATA TAGATAGAATATACATTAAAAATAAACACAAGTTCAATGATGAATTTTATACTTCTAATCTTTTTTACAT TAATTATAACTTCTCAGATAATACTCATCTATTAACTAAACATATAAGGATTGCTAATTCTGAATTAGAA AGTAATTACAACAAATTATATCATCCTACACCAGAAACCCTAGAAAATATACTAACCAATCCGGTTAAAA GTAATGATAAAAAGACACTGAGTGACTATTGTATAGGTAAAAATGTTGACTCAATAATGTTACCATCGTT ATCTAATAAGAAGCTTATTAAATCGTCTACAATGATTAGAACCAATTACAGCAGACAAGATTTGTATAAT TTATTTCCTACGGTTGTGATTGATAAAATTATAGATCATTCAGGTAATACAGCCAAATCTAACCAACTTT ACACTACTACTTCTCATCAAATATCCTTAGTGCACAATAGCACATCACTTTATTGCATGCTTCCTTGGCA TCATATTAATAGATTCAATTTTGTATTTAGTTCTACAGGTTGTAAAATTAGTATAGAGTATATTTTAAAA GATCTTAAAATTAAGGATCCTAATTGTATAGCATTCATAGGTGAAGGAGCAGGGAATTTATTATTGCGTA CAGTAGTGGAACTTCATCCTGATATAAGATATATTTACAGAAGTCTGAAAGATTGCAATGATCATAGTTT ACCAATTGAGTTTTTAAGGCTGTACAATGGGCATATCAACATTGATTATGGTGAAAATTTGACCATTCCT GCTACAGATGCAACCAACAACATTCATTGGTCTTATTTACATATAAAGTTTGCTGAACCTATCAGTCTTT TTGTCTGTGATGCTGAATTGCCTGTAACAGTCAACTGGAGTAAGATTATAATAGAGTGGAGCAAGCATGT AAGAAAATGCAAGTACTGTTCTTCAGTTAATAAATGTACATTAATAGTAAAATATCATGCTCAAGATGAT ATCGATTTCAAATTAGACAACATAACTATATTAAAAACTTATGTATGCTTAGGCAGTAAGTTAAAGGGAT CTGAAGTTTACTTAGTCCTTACAATAGGTCCTGCAAATGTGTTCCCAGTATTTAATGTAGTACAAAATGC TAAATTGATACTATCAAGAACCAAAAATTTCATCATGCCTAAAAAAGCTGATAAAGAGTCTATTGATGCA AATATTAAGAGTTTGATACCCTTTCTTTGTTACCCTATAACAAAAAAAGGAATTAATACTGCATTGTCTA AATTAAAGAGTGTTGTTAGTGGAGATATACTATCATATTCTATAGCTGGACGTAATGAAGTATTCAGCAA TAAACTTATAAATCATAAGCATATGAACATCTTAAAGTGGTTCAATCATGTTTTAAATTTCAGATCAACA GAATTAAACTATAATCATTTATATATGGTAGAATCTACTTATCCTCATCTAAGTGAATTGTTAAACAGCT TGACAACCAATGAACTTAAAAAACTGATTAAAATCACAGGTAGTTTGTTATACAACTTTAATAATGAATA ATGAGCAAAAATCTTATAACAAAAATAGCTATAGCTACACACTAACATTGTATTCAATTATAGTTATTTA AAATTAATAATTATATAATTTTTTAATAACTTCTAGTGAACTAATCCTAAAATTATCATTTTGATCTAAG AAGAATAAGTTTAAATCCAAATCTAATTGGTTTATATGTATATTAACTAAACTACGAGATATTAGTTTTT GACACTTTTTTTCTCGT ( SEQ ID NO : 4 05 ) , or a nucleotide sequence with at least 80%. 82%. 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto, human respiratory syncytial virus isolate VN-360-6/10 (>gi|780954857|gb|KJ939943. 11) having
ACG GG AAAAAATG CGTACAACAAACTTG CGTAAACCAAAAAAATG G GGCAAATAAG AATTTG ATAAG CAC CACTTGAGTTTAACTCCCTTGGTTAGAGATGGGCAGCAATTCATTGAGTATGATAAAAGTTAGATTACAA AATTTATTTGACAATGATGAAGTAGCATTGTTAAAAATAACCTGCTATACTGACAAATTGATACATTTAA CTAATGCTTTG G CTAAG G CAGTG ATACATACAATCAAATTG AATG GCATTGTATTTGTGCATGTTATTAC AAGTAGTGATATTTGCCCTAATAATAATATTGTAGTGAAATCCAACTTCACAACAATGCCAGTATTACAA AATGGAGGTTATATATGGGAAATGATGGAATTAACACACTGCTCTCAACCCAATGGCCTAATAGATGACA ATTGTGAAATCAAATTCTCCAAAAAACTAAGTGACTCAACAATGACCAACTATATGAATCAATTATCTGA ATTACTTGGATTTGATCTCAATCCATAAATTATAATAAATATCAACTAGCAAATCAATGTCACTAACACC ATTAGTTAATATAAAACTTGACAGAAGATAAAAATGGGGCAAATAAATAAACTCAGCCGACCCAACCATG GACACAACACACAATGGTACTACACCACAAAGACTGATGATCACAGACATGAGACCATTGTCACTTGAGA CTATAATAACATCACTAACCAGAGACATCATAACACACAGATTTATATACTTGATAAATCATGAATGTAT AGTGAGAAAACTTGATGAAAGACAGGCCACATTTACATTCCTGGTCAACTATGAAATGAAACTATTGCAC AAAGTG G G AAGCACTAAATACAAAAAATATACTG AATACAACACAAAATATGG CACTTTTCCTATGCCAA TATTTATCAATCATG ATG G GTTCTTAG AATG CATTGG CATTAAG CCTACAAAGCACACTCCCATAATATA CAAGTATGATCTCAATCCATGAATTTCAACACAAGATTCACACAATCTGAAATAACAACTTCATGCATAA CTATACTCCATAGTCCAAATGGAGCCTGAAAATTATAGTAATTTAAAATTAAGGAGAGACATAAGATGAA AGATGGGGCAAATACAAAAATGGCTCTTAGCAAAGTCAAGTTGAACGATACACTCAACAAAGATCAACTT CTGTCATCCAGCAAATACACCATCCAACGGAGCACAGGAGATAGTATTGATACTCCTAATTATGATGTGC AGAAACACATCAACAAGTTATGTGGCATGTTATTAATCACAGAAGATGCTAATCATAAATTCACTGGGGT AATAGGTATGTTATATGCTATGTCTAGATTAGGAAGAGAAGACACCATAAAAATACTCAGAGATGCAGGA TATCATGTAAAAGCAAATGGAGTGGATGTAACAACACATCGTCATGACATTAATGGAAAAGAAATGAAAT TTGAAGTATTAACATTGGCAAGCTTAACAACTGAAATTCAAATCAACATTGAGATAGAATCTAGAAAATC CTACAAAAAAATG CTAAAAG AAATG G GAG AG GTG G CTCCAG AATACAGG CATG ATTCTCCTG ATTGTG G A ATG ATAATACTATGTATAG CCG CATTAGTAATAACCAAATTAGCAG CAG G GG ATAG ATCTG GTCTTACAG CTGTGATTAGGAGAGCTAATAATGTTCTAAAAAATGAAATGAAACGTTATAAAGGCTTACTACCAAAGGA TATAGCCAACAGCTTCTATGAAGTGTTTGAAAAATATCCTCACTTTATAGATGTTTTTGTTCATTTTGGT ATAGCACAATCTTCTACCAGAGGTGGCAGTAGAGTTGAAGGGATTTTTGCAGGATTGTTTATGAATGCCT ATGGTGCAGGGCAAGTGATGTTACGGTGGGGAGTCTTAGCAAAATCAGTTAAAAATATTATGCTAGGACA CGCTAGTGTGCAAGCAGAAATGGAACAAGTTGTGGAAGTTTATGAATATGCCCAAAAATTAGGTGGAGAA GCAGGATTCTACCATATATTGAACAACCCAAAAGCATCATTATTATCTTTGACTCAATTTCCCCACTTCT CCAGTGTAGTATTAGGCAATGCTGCTGGCCTAGGCATAATGGGAGAATACAGAGGTACACCAAGGAATCA AGATCTATATGATGCTGCAAAGGCATATGCTGAACAACTCAAAGAAAATGGTGTGATTAACTACAGTGTA TTAGACTTGACAGCAGAAGAACTAGAGGCTATCAAACATCAGCTTAATCCAAAAGATAATGATGTAGAGC TTTGAGTTAACAAAAAGTGGGGCAAATAAATCATCATGGAAAAGTTTGCTCCTGAATTCCATGGAGAAGA CGCAAACAACAGAGCCACTAAATTCCTAGAATCAATAAAGGGCAAGTTCACATCACCTAAAGATCCCAAG AAAAAAGATAGTATCATATCTGTCAACTCAATAGATATAGAAGTAACCAAAGAAAGCCCTATAACTTCAA ATTCAACCATTATAAACCCTACAAATGAGACAGATGATACTGCAGGGAACAAGCCCAATTATCAAAGAAA ACCTCTAGTAAGTTTCAAAGAAGACCCTACGCCAAGTGATAATCCCTTTTCAAAACTATACAAAGAAACC ATAGAAACATTTGATAACAATGAAGAAGAGTCAAGCTATTCATATGAAGAAATAAATGATCAGACAAACG ATAATATAACAGCAAGATTAGATAGGATTGATGAAAAATTAAGTGAAATACTAGGAATGCTTCACACACT AGTAGTAGCAAGTGCAGGACCTACATCTGCTCGGGATGGTATAAGAGATGCCATGGTTGGTTTAAGAGAA GAAATGATAGAAAAAATCAGAACCGAAGCATTAATGACCAATGATAGACTAGAAGCTATGGCAAGACTCA GGAATGAGGAAAGTGAAAAGATGGCAAAAGATACATCAGATGAAGTGTCTCTCAATCCAACATCAGAGAA ATTGAACAACCTGCTGGAAGGAAATGACAGTGACAATGATCTATCACTTGAAGATTTCTGATCAGTCACC AATCTGTACATCAACACACAACACCAACAGAAGACCAACAAACAAAACAACTCACCCATCCAACCAAACA TCTATCTGCTAATCAGCCAACCAGCCAAAAAAACACCCAGCCAATTCAAAATTAGTCACCCGGAAAAAAT CGATACTATAGTTACAAAAAAAGATGGGGCAAATATGGAAACATACGTGAACAAGCTTCACGAAGGCTCC ACATACACAGCTGCTGTTCAATACAATGTCCTAGAAAAAGACGATGACCCAGCATCACTTACAATATGGG TGCCCATGTTCCAATCATCCATGCCAGCAGATTCACTTATAAAAGAACTAGCTAATGTCAACATACTAGT GAAACAAATATCCACACCCAAAGGACCTTCATTAAGAGTCATGATAAACTCAAGAAGTGCAGTGCTAGCA CAAATGCCCAGCAAATTCACCATATGTGCCAATGTGTCCTTGGATGAAAGAAGCAAGCTGGCATATGATG TAACTACACCCTGCGAAATCAAGGCATGTAGTCTAACATGCCTAAAATCAAAAAATATGTTAACTACAGT TAAAGATCTCACTATGAAAACACTCAACCCAACACATGACATCATTGCTTTATGTGAATTTGAAAATATA GTAACATCAAAAAAAGTCATAATACCAACATACTTAAGATCCATCAGTGTCAGAAATAAAGATCTGAACA CACTTGAAAATATAACAACCACCGAATTCAAAAATGCCATCACAAATGCAAAAATTATCCCTTACTCAGG ATTACTGTTAGTCATCACAGTG ACTG ACAACAAAG GAG CATTCAAATACATAAAG CCACAAAGTCAATTC ATAGTAGATCTTGGTGCTTACCTAGAAAAAGAAAGTATATATTATGTTACAACAAATTGGAAGCACACAG CTACACGATTTGCAATCAAACCCATGGAAGATTAACCTTTTTCCTCTACATCAGTTAGTTGATTCATACA CACTTTCTACCTACATTCTTCACTTCACAATCATAATCACCAACCCTCTGTGGTTTAACCAATCAAACAA AACTTATCTGGAATCTCAGATCATCCCAAGTCATTGTTCATCTGATCTAGTACTCAAATAAGTTAATAAA AATACCCACATGGGGCAAATAATCATCGGAGGAAATCCAACCAATCACAACATCTGTCAACATAGACCAG TCAACGCGCCAAACAAAACAATCCAATGGAAAATACATCCATAACAATAGAATTCTCAAGCAAATTTTGG CCTTACTTTACACTAATACATATGATCACAACAATAATCTCTTTGCTAATCATAATTTCCATCATGATTG CAATACTAAACAAACTCTGTGAATACAACGTATTCCATAACAAAACCTTTGAACTACCAAGAGCTCGAGT CAATACATAGCATTCACCAATCTGATGGCTCAAAACAGCAACCTTGCATTTGTAAGTGAACAATCCTCAC CTTTTTACAAAATCACATCAACATCTCACCATGCAAGCCATCATCCATACTATAAAGTAGTTAATTAAAA ATAGTCATAACAATGAACTAAGATATTAAGACTAACAACAACGTTGGGGCAAATGCAAACATGTCCAAAA CCAAGGACCAACGCACCGCCAAGACACTAGAAAAAACCTGGGACACTCTCAATCATCTATTATTCATATC ATCGTGCTTATACAAGTTAAATCTTAAATCTATAGCACAAATCACATTATCCATTCTGGCAATGATAATC TCAACTTCACTTATAATTGTAGCTATCATATTCATAGCCTCAGCAAACAACAAAGTCACACTAACAACTG CAATCATACAAGATGCAACAAGCCAGATCAAGAACACAACCCCAACATACCTGACCCAGAATCCCCAGCT TGGAATCAACTTCTTCAATCTGTCTGGAACTATATCACAAACCACCGCCATACTAGTTTCAACAACACCA AGTGTCGAGTCAATCCCGCAATCTACAACAGCCAAGACCAAAAACACAACAACAACCCAAGTACAACCCA GCAAGCTCACCACAAAACAACGCCAGAACAAACTACCAAACAAACCCAATGATGATTTCCACTTTGAAGT GTTCAACTTTGTACCCTGCAGCATATGTAGCAACAATCCAACTTGCTGGGCCATCTGCAAAAGAATACCA AGCAAAAAACCTGGAAAGAAAACCACCACCAAGCCCACGAAAAAACCAACCACCAAGACAACCAAAAAAG AACTCAAACCTCAAACCACAAAACCCAAGGAAGCACCTACCACCAAGCCCACAGATAAGCCAACCATCAA CACCACCAAACCAAACATCAGAACTACACTGCTCACCAACAGCACCACAGGAAATCTAGAACACACAAGT CAAGAGGAAACCCTCCATTCAACCTCCTCCGAAGGCAATACAAGCCCTTCACAAATCTATACAATATCCG AGTACCTATCACAACCTCCATCTCCATCCAACATAACAGACCAGTAGTCATTAAAAAGCATATTATTGAA AAAAACCATG ACCAAATCAAACAG AATCAAAATAAG ATCTG G G GCAAATAACAATG G AGTTG CCAATCCT CAAAACAAATG CAATTACCACAATCTTTG CTG CAGTCACACTCTGTTTCG CTTCCAGTCAAAACATCACT GAAGAATTTTATCAATCAACATGCAGTGCAGTTAGCAAAGGCTATCTTAGTGCTTTAAGAACTGGTTGGT ATACTAGTGTTATAACTATAGAATTAAGTAATATCAAGGAAAATAAGTGTAATGGAACAGACGCTAAGGT AAAATTGATAAAACAAGAATTAGATAAATATAAAAATGCTGTAACAGAATTGCAGTTGCTCATGCAAAGC ACACCAGCAGCCAACAATCGAGCCAGAAGAGAACTACCAAGGTTTATGAACTATACACTCAACAATACCA AAAATAACAATGTAACATTAAGCAAGAAAAGGAAAAGAAGATTTCTTGGCTTTTTGTTAGGTGTTGGATC TGCAATCGCCAGTGGCGTTGCTGTATCTAAGGTCCTGCACCTAGAAGGGGAAGTGAACAAAATCAAAAGT GCTCTACTATCCACAAACAAAGCTGTAGTCAGCTTATCAAATGGAGTTAGTGTCTTAACTAGCAAAGTGT TAGACCTCAAAAACTATATAGATAAACAGTTGTTACCCATTGTGAACAAGCAAAGCTGCAGCATATCAAA CATTGAAACTGTGATAGAATTCCAACAAAAGAACAACAGACTACTAGAGATTACCAGGGAATTTAGTGTT AATGCAGGTGTAACTACACCTGTAAGCACTTATATGTTAACAAATAGTGAACTATTATCATTAATCAATG ATATGCCTATAACAAATGATCAGAAAAAGTTAATGTCCAACAATGTTCAAATAGTTAGACAGCAAAGTTA CTCGATCATGTCCATAATAAAGGAGGAAGTCTTAGCATATGTAGTACAATTACCACTATATGGTGTAATA GATACACCTTGTTGGAAACTACACACATCCCCTCTATGCACAACCAACACAAAGGAAGGGTCCAACATCT GTTTAACAAGAACCGACAGAGGATGGTACTGTGACAATGCAGGATCAGTGTCTTTCTTCCCACAAGCTGA AACATGCAAAGTTCAATCGAATCGAGTATTTTGTGACACAATGAACAGTTTAACATTACCAAGTGAAGTA AATCTCTGCAACATTGACATATTCAACCCTAAATATGATTGCAAAATTATGACTTCAAAAACAGATGTAA GCAGCTCCGTTATCACATCTCTAGGAGCCATTGTGTCATGCTATGGAAAAACTAAATGTACAGCATCCAA TAAAAATCGTGGAATCATAAAGACATTTTCTAACGGGTGTGATTATGTATCAAATAAGGGGGTGGACACT GTATCTGTAGGTAATACATTATATTATGTAAATAAGCAAGAAGGAAAAAGTCTCTATGTAAAAGGTGAAC CAATAATAAATTTCTATGACCCATTAGTGTTCCCTTCTGATGAATTTGATGCATCAATATCTCAAGTCAA TGAGAAGATTAACCAGAGCTTAGCATTTATTCGTAAATCCGATGAATTATTACATAATGTAAATGTTGGT AAATCCACCACAAATATCATGATAACTACTATAATTATAGTGATTATAGTAATATTGTTATTATTAATTG CAGTTGGGCTGTTCCTATACTGCAAGGCAAGAAGCACACCAGTCACACTAAGTAAGGATCAACTGAGTGG TATAAATAATATTGCATTTAGTAACTGAATAAAAATAGTACCTAATCATGTTCTTACAATGGTTCACTAT CTGACCATAGACAACCCATCTATCATTGGATTTTCTTAAAGTCTGAACTTCATCGCAACTCTCATCTATA AACCATCTCACTTACACTATTTAAGTAGATTCCTATTTTATAGTTATATAAAACTACTGAGTACCAGATT AACTCACTATCTGTAAAAAATTAGAAATGGGGCAAATATGTCACGAAGGAATCCTTGCAAATTTGAAATT CGAGGTCATTGCTTGAATGGTAAGAGGTGTCATTTTAGTCACAATTATTTTGAATGGCCACCCCATGCAC TGCTTGTAAGACAAAACTTTATGTTAAACAGAATACTTAAGTCTATGGATAAAAGCATAGATACTTTATC AGAAATAAGTGGAGCTGCAGAGTTGGACAGAACTGAAGAGTATGCCCTCGGTGTAGTTGGAGTGCTAGAG AGTTATATAG GATCAATAAATAATATAACTAAACAATCAG CATGTGTTG CCATG AG CAAACTCCTCACTG AACTAAACAGTGATGACATCAAAAAACTAAGAGACAATGAAGAGCTAAATTCACCTAAGGTAAGAGTGTA CAATACTGTCATATCATATATTGAAAGCAACAGGAAAAACAATAAACAAACTATCCATCTGTTAAAAAGA TTGCCAGCAGACGTATTGAAGAAAACCATCAAAAACACATTGGATATCCACAAGAGCATAACCATCAACA ACCCAAAAGAATCAACTGTTAATGATACAAACGACCATGCCAAAAATAATGATACTACCTGACAAATATC CTTGTAGTATAAATTCCATACTAATAACAAGTAGTTGTAGAGTTACTATGTATAATCAAAAGAACACACT ATATTTCAATCAAAACAACCAAAATAACCATACATACTCACCAAATCAACCATTCAATGAAATCCATTGG ACCTCTCAAGACTTGATTGATGCAATTCAAAATTTTCTACAACATCTAGGTATTACTGATGATATATATA CAATATATATATTAGTGTCATAACACTCAATACAAGTGCTTACCACATCATCAAACTATTAACTCAAACA ATTCAAACCATGGGACAAAATGGATCCCATTATTAATGGAAATTCTGCTAATGTTTATCTAACCGATAGT TATTTAAAAGGTGTTATTTCTTTCTCAGAATGTAATGCTTTAGGAAGTTACATATTCAATGGTCCTTATC TCAAAAATGATTACACCAACTTAATTAGTAGACAAAATCCATTAATAGAACACATAAATCTAAAGAAATT AAATATAACACAGTCTTTAATATCTAAGTATCATAAAGGTGAGATAAAAATAGAAGAACCTACTTATTTT CAGTCATTACTTATGACATACAAGAGTATGACCTCGTTAGAACAGATTACTACCACTAATTTACTTAAAA AGATAATAAGAAGAGCTATAGAAATTAGTGATGTCAAAGTTTATGCTATATTGAATAAACTGGGGCTTAA AGAAAAAGACAAGATTAAATCTAACAATGGACAAGATGAAAACAACTCAGTTATTACAACCATAATCAAA GATGATATACTTTTAGCTGTTAAGGATAATCAATCTCATCTTAAAGCAGGCAAAAATCACTCTACAAAAC AAAAAGACACTATCAAAACAACACTTTTGAAAAAATTAATGTGTTCGATGCAACATCCTCCATCATGGTT AATACATTGGTTTAATTTATACACAAAATTAAACAACATATTAACACAGTATCGATCTAATGAGGTAAAA AACCATGGTTTTATATTGATAGATAATCATACTCTCAATGGATTCCAATTTATTTTGAATCAATATGGTT GTATAGTTTATCATAAGGAACTCAAAAGAATTACTGTGACAACCTATAATCAATTCTTGACATGGAAAGA TATTAGCCTTAGTAGATTAAATGTTTGTTTAATTACATGGATTAGTAACTGTTTGAACACATTAAACAAA AGCTTAGGCTTAAGATGTGGATTCAATAATGTTATCTTGACACAACTATTTCTTTATGGAGATTGTATAT TAAAACTATTCCACAATGAAGGGTTCTACATAATAAAAGAGGTAGAGGGTTTTATTATGTCTCTAATTTT AAACATAACAGAAGAAGATCAATTCAGAAAACGGTTTTATAATAGTATGCTCAACAACATCACAGATGCT GCTAATAAAGCTCAGAAAAATCTGCTATCAAGAGTATGTCATACATTATTAGATAAGACAGTATCCGATA ATATAATAAATGGCAGATGGATAATTCTACTAAGTAAGTTTCTTAAATTAATTAAGCTTGCAGGTGACAA TAACCTTAACAATCTG AGTG AATTATATTTTTTATTC AG AATATTTG G ACACCCAATG GTAG ATG AAAG A CAAGCCATGGATGCTGTTAAAGTTAATTGCAACGAGACCAAATTTTACTTGTTAAGCAGTTTGAGTATGT TAAGAGGTGCCTTTATATATAGAATTATAAAAGGGTTTGTAAATAATTACAACAGATGGCCTACTTTAAG AAATGCTATTGTTTTACCCTTAAGATGGTTAACTTACTATAAACTAAACACTTATCCTTCCTTATTGGAA CTTACAGAAAGAGATTTGATTGTTTTATCAGGACTACGTTTCTATCGTGAGTTTCGGTTGCCTAAAAAAG TGGATCTTGAAATGATCATAAATGATAAGGCTATATCACCTCCTAAAAATTTGATATGGACTAGTTTCCC TAGAAATTATATGCCGTCACACATACAAAATTATATAGAACATGAAAAATTAAAATTTTCCGAGAGTGAT AAATCAAGAAGAGTATTAGAGTACTATTTAAGAGATAACAAATTCAATGAATGTGATTTATATAACTGTG TAGTTAATCAAAGCTATCTTAACAACCCTAATCATGTGGTATCATTGACTGGCAAAGAAAGAGAACTCAG TGTAGGTAGAATGTTTGCAATGCAACCAGGAATGTTCAGACAAGTTCAAATATTAGCAGAGAAAATGATA GCTGAAAACATTTTACAATTCTTTCCTGAAAGTCTTACAAGATATGGTGATCTAGAATTACAGAAAATAT TAGAATTGAAAGCAGGAATAAGTAACAAATCAAATCGTTACAATGACAATTACAACAATTACATCAGTAA GTG CTCTATCATCACAG ATCTCAG CAAATTCAATCAAG CATTCCGGTATGAAACATCATGTATTTGTAGT GATGTACTGGATGAACTGCATGGTGTACAATCTCTGTTTTCCTGGTTACATTTAACTATTCCACATGTCA CAATAATATGCACATATAGGCATGCACCCCCCTATATAAGAGATCACATTGTAGATCTTAATAATGTAGA TGAACAAAGTGGATTATATAGATATCATATGGGTGGTATCGAAGGGTGGTGTCAAAAACTATGGACCATA GAAGCTATATCACTATTGGATCTAATATCTCTCAAAGGGAAATTCTCAATTACTGCCTTAATTAATGGAG ACAATCAATCAATAGATATAAGCAAACCAGTCAGACTCATGGAAGGTCAAACTCATGCTCAAGCAGATTA TTTGCTGGCACTAAATAGTCTTAAATTGTTGTATAAAGAGTATGCAGGTATAGGCCACAAATTAAAAGGA ACTGAGACTTATATATCAAGGGATATGCAATTTATGAGTAAAACAATTCAACATAACGGTGTATATTACC CAGCTAGTATAAAGAAAGTCCTAAGAGTGGGACCATGGATAAACACTATACTTGATGATTTCAAAGTGAG TCTAGAATCTATAGGTAGTTTGACACAAGAATTAGAATATAGAGGTGAAAGTCTATTATGCAGTTTAATA TTTAGAAATGTGTGGTTATATAATCAAATTGCTTTACAACTAAAAAATCATGCATTATGTAACAATAAAT TATATTTGGACATATTAAAGGTTCTGAAACACTTAAAAACCTTTTTTAATCTTGATAATATTGATACAGC ATTAACATTGTATATGAATTTGCCCATGTTATTTGGTGGTGGTGATCCTAACTTGTTATATCGAAGTTTC TATAGAAGAACTCCTGATTTCCTCACAGAGGCTATAGTTCACTCTGTGTTCATACTTAGTTATTATACAA ACCATGATTTAAAGGATAAACTTCAAGATCTGTCAGACGATAGATTGAATAAGTTCTTAACATGCATAAT CACATTTGACAAAAACCCCAATGCTGAATTCGTAACATTGATGAGAGATCCTCAAGCTTTAGGGTCCGAG
AGGCAAGCTAAAATTACTAGCGAAATCAATAGACTGGCAGTTACTGAGGTTTTGAGCACAGCTCCAAACA AAATATTCTCCAAAAGTGCACAACACTATACCACTACAGAGATAGATTTAAATGATATTATGCAAAATAT AGAACCTACATATCCTCATGGGCTAAGAGTTGTTTATGAAAGTTTACCCTTTTATAAAGCAGAGAAAATA GTAAATCTTATATCCGGTACAAAATCTATAACTAACATACTGGAAAAGACTTCTGCCATAGACTTAACAG ATATTGATAGAGCCACTGAGATGATGAGGAAAAACATAACTTTGCTTATAAGGATATTTCCATTAGATTG TAACAGAGATAAAAGAGAAATATTGAGTATGGAAAACCTAAGTATTACTGAATTAAGCAAATATGTTAGA GAAAGATCTTGGTCTTTATCCAATATAGTTGGTGTTACATCACCCAGTATCATGTATACAATGGACATCA AATATACAACAAGCACTATAGCTAGTGGCATAATCATAGAGAAATATAATGTCAACAGTTTAACACGTGG TGAGAGAGGACCCACTAAACCATGGGTTGGTTCATCTACACAAGAGAAAAAAACAATACCAGTTTATAAT AGACAAGTTTTAACCAAAAAACAGAGAGATCAAATTGATCTATTAGCAAAATTAGATTGGGTGTATGCAT CTATAGATAACAAGGATGAATTCATGGAAGAACTCAGCATAGGAACTCTTGGGTTAACATATGAGAAAGC CAAAAAATTATTCCCACAATATTTAAGTGTTAACTATTTGCATCGCCTTACAGTCAGTAGTAGACCATGT GAATTCCCTGCATCAATACCAGCTTATAGAACTACAAATTATCACTTTGATACTAGCCCTATTAATCGCG TATTAACAGAAAAGTATGGTGATGAAGATATTGATATAGTATTCCAAAACTGTATAAGTTTTGGCCTTAG CTTAATGTCAGTAGTAGAGCAATTTACCAATGTATGTCCTAACAGAATTATTCTCATACCCAAGCTTAAT GAGATACATTTGATGAAACCTCCCATATTCACAGGTGATGTTGATATTCACAAGTTAAAACAAGTGATCC AAAAACAGCATATGTTTTTACCAGACAAAATAAGTTTGACTCAATATGTGGAATTATTCTTAAGTAATAA AACACTCAAATCTGGTTCTCATGTTAATTCTAATTTAATATTGGCACATAAGATATCTGACTATTTTCAT AATACTTACATTTTAAGTACCAATTTAGCTGGACATTGGATTCTGATTATACAACTTATGAAAGATTCTA AAGGTATTTTTGAAAAAGATTGGGGAGAGGGATATATAACTGATCATATGTTCATTAATTTGAAAGTTTT CTTCAATGCTTATAAGACCTATCTCTTGTGTTTTCATAAAGGTTATGGCAGAGCAAAGCTGGAGTGTGAT ATGAATACTTCGGATCTCCTATGTGTATTGGAATTAATAGACAGTAGTTATTGGAAGTCTATGTCTAAGG TATTTTTAGAACAAAAAGTTATCAAATACATTCTCAGCCAGGATGCAAGTTTACATAGAGTAAAAGGATG TCATAGCTTCAAACTATGGTTTCTTAAACGTCTTAATGTAGCAGAATTCACAGTTTGCCCTTGGGTTGTT AACATAGATTATCATCCAACACATATGAAAGCAATATTAACTTATATAGATCTTGTTAGAATGGGATTGA TAAATATAGATAGAATGTACATTAAAAATAACCACAAATTCAATGATGAATTTTATACCTCTAATCTCTT TTACATTAATTATAACTTCTCAGATAATACTCATTTATTAACTAAACATATAAGGATTGCTAATTCTGAA TTAGAAAATAATTACAACAAATTATATCATCCTACACCTGAAACTCTAGAAAATATACTAACCAATCCGG TTAAATGTGATGACAAAAAGACACTGAATGACTATTGTATAGGTAAAAATATTAACTCAATAATGTTACC ATTGTTATCTAATAAGAAGCTTATTAAATCGTCTACAATGATTAGAACCAATTACAGCAAACAAGATTTG TATAATTTATTTCCTACGGTTGTGATTGATAAAATTATTGATCATTCAGGTAATACATCCAAATCTAACC AACTTTACACTACTACTTCTCATCAAATATCTTTAGTACACAATAGCACATCACTTTATTGCATGCTTCC TTGGCATCACATTAATAGATTCAATTTTGTGTTTAGTTCTACAGGTTGTAAAATTAGTATAGAGTATATT TTAAAAG ACCTTAAAATTAAAG ATCCTAATTGTATAG CATTCATAG GTG AAG G AG CAG G G AATTTATTAT TGCGTACAGTAGTGGAACTTCATCCTGATATAAGATATATTTACAGAAGTCTGAAAGATTGCAATGATCA TAGTTTACCTATTGAGTTTTTAAGGCTGTACAATGGACATATCAACATTGATTATGGTGAAAATTTGACC ATTCCCGCTACAGATGCAACCAACAACATTCATTGGTCTTATTTGCATATAAAGTTTGCTGAACCTATCA GTCTTTTTGTTTGTGATGCTGAATTGCCTGTAACAGTCAACTGGAGTAAAATTATAATAGAGTGGAGCAA GCATGTAAGAAAATGCAAGTACTGTACCTCAGTTAATAAATGTACGTTAATAGTAAAATATCATGCTCAA GATGATATCGATTTCAAATTAGACAACATAACTATATTAAAAACTTATGTATGCTTAGGCAGTAAGTTAA AGGGGTCTGAAGTTTACTTAGTCCTTACTATAGGTCCTGCAAATGTGTTCCCAGTATTTAATGTAGTACA AAATGCTAAATTGATACTATCAAGAACCAAAAATTTCATCATGCCTAAGAAGGCTGATAAAGAGTCTATT GATGCAAATATTAAAAGTTTGATACCCTTTCTTTGTTACCCTATAACAAAGAAAGGAATTACTACTGCAT TATCAAAACTAAAGAGTGTTGTTAGCGGAGATATACTATCATATTCTATAGCTGGACGTAATGAAGTATT CAG CAATAAACTTATAAATCATAAG CATATG AACATCTTAAAGTG GTTCAACCATGTTTTAAATTTCAG A TCAACAGAACTTAACTATAATCATTTATATATGGTAGAATCCACATACCCTTATCTAAGTGAATTGTTAA ACAGCTTGACAACTAATGAACTTAAAAAACTAATTAAAATCACAGGTAGTTTGTTATACAACTTTCATAA TGAATAATGAATAAAAATCTTATATTAAAAATTCCCACAGCTACACACTAACACTGTATTCAATTATAGT TATTTAAAATTAAAAATTATATAATTTTTTTAATAACTTTTAGTGGACTAATCCTAAAATTATCATTTTG ATCCAGGAGGAATAAATTTAAATCCAAATCTAATTGGTTTATATGTATATTAACTAAACTACGAGATATT AGTTTTTGACACTTTTTTTCTCGT ( S EQ ID NO : 1153 ) , or a nucleotide sequence with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% nucleic acid sequence identity thereto.
The RSV genome is RNA. Hence, in some cases the RSV genome can be a copy of the foregoing DNA sequence, where the thymine (T) residues are uracil (U) residues. In some cases, the RSV viral genome can be a complement of the foregoing DNA sequence. Exemplary Casl3 proteins
Any suitable CRISPR-associated RNA-targeting endonuclease, such as a Casl3 protein variant, can be used in the methods and compositions described herein. The Cast 3 protein can complex with at least one CRISPR guide RNA (crRNA) to at least one reporter RNA for a period of time sufficient to form at least one RNA cleavage product.
The Cast 3 protein can, for example, be a Cas 13a protein, Cas 13b protein, or a combination thereof. Casl3 contains two Higher Eukary otes and Prokaryotes Nucleotide-binding (HEPN) domains for RNA cleavage, consistent with known roles for HEPN domains in other proteins. In some embodiments, the Cas 13 proteins can have sequence variation and/or be from other organisms. For example, the Casl3 proteins can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity' to any of the Cas 13 sequences disclosed herein or to a Cas 13 in the following bacteria: Leptotrichia wadei, Leptotrichia buccalis, Rhodobacter capsulatus, Herbinix hemicellulosilytica. Leptotrichia buccalis (Lbu), Listeria seeligeri, Paludibacter propionicigen.es. Lachnospiraceae bacterium, [Eubacterium] rectale, Listeria newyorkensis, Clostridium aminophilum, and/or Leptotrichia shahii.
For example. ^Leptotrichia wadei Casl3a endonuclease that can be used has the following sequence (SEQ ID NO: 500; NCBI accession no. WP_036059678. 1).
1 MKITKI DGVS HYKKQDKGIL KKKWKDLDER KQREKIEARY 41 NKQIESKIYK EFFRLKNKKR IEKEEDQNIK SLYFFIKELY 81 LNEKNEEWEL KNINLEILDD KERVIKGYKF KEDVY FFKEG 121 YKEYYLRILF NNLIEKVQNE NREKVRKNKE FLDLKEI FKK 161 YKNRKI DLLL KS INNNKINL EYKKENVNEE IYGINPTNDR 201 EMT FYELLKE I IEKKDEQKS ILEEKLDNFD ITNFLENIEK 241 I FNEETEINI IKGKVLNELR EYIKEKEENN S DNKLKQIYN 281 LELKKYIENN FSYKKQKS KS KNGKNDYLYL NFLKKIMFI E 321 EVDEKKEINK EKFKNKINSN FKNLFVQHIL DYGKLLYYKE 361 NDEYIKNTGQ LETKDLEYIK TKETLIRKMA VLVS FAANS Y 401 YNLFGRVSGD ILGTEWKS S KTNVIKVGSH I FKEKMLNY F
441 FDFEI FDANK IVEILES I SY S IYNVRNGVG HFNKL ILGKY 481 KKKDINTNKR IEEDLNNNEE IKGYFIKKRG EIERKVKEKF 521 LSNNLQYYYS KEKIENYFEV YEFEILKRKI PFAPNFKRI I 561 KKGEDLFNNK NNKKYEYFKN FDKNSAEEKK EFLKTRNFLL 601 KELYYNNFYK EFLSKKEEFE KIVLEVKEEK KSRGNINNKK 641 SGVS FQS I DD YDTKINIS DY IAS IHKKEME RVEKYNEEKQ 681 KDTAKYIRDF VEEI FLTGFI NYLEKDKRLH FLKEE FS ILC 721 NNNNNWDFN ININEEKI KE FLKENDS KTL NLYLFFNMI D 761 SKRI SEFRNE LVKYKQFTKK RLDEEKE FLG IKIELYETL I
801 EFVILTREKL DTKKSEEI DA WLVDKLYVKD SNEYKEYEE I 841 LKLFVDEKIL S SKEAPYYAT DNKT PILLSN FEKTRKYGTQ 881 S FLSEIQSNY KYSKVEKENI EDYNKKEEIE QKKKSNIEKL 921 QDLKVELHKK WEQNKITEKE IEKYNNTTRK INEYNYLKNK 961 EELQNVYLLH EMLSDLLARN VAFFNKWERD FKFIVIAIKQ
1001 FLRENDKEKV NEFLNPPDNS KGKKVYFSVS KYKNTVENI D
1041 GIHKNFMNLI FLNNKFMNRK IDKMNCAIWV YFRNYIAHFL
1081 HLHTKNEKIS LISQMNLLIK LFSYDKKVQN HILKSTKTLL
1121 EKYNIQINFE ISNDKNEVFK YKIKNRLYSK KGKMLGKNNK
1161 LENEFLE NVKAMLEYSE or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
Other sequences for Leptotrichia wadei Casl3a endonucleases are also available, such as those NCBI accession nos. BBM46759.1, BBM48616.1, BBM48974.1, BBM48975.1, and WP 021746003.1.
In another example, a Herbmix hemicellulosifytica Cast 3a endonuclease that can be used has the following sequence (SEQ ID NO: 501; NCBI accession no. WP_103203632.1).
1 MKLTRRRISG NSVDQKITAA FYRDMSQGLL YYDSEDNDCT
41 DKVIESMDFE RSWRGRILKN GEDDKNPFYM FVKGLVGSND
81 KIVCEPIDVD SDPDNLDILI NKNLTGFGRN LKAPDSNDTL
121 ENLIRKIQAG I PEEEVLPEL KKIKEMIQKD IVNRKEQLLK
161 S IKNNRI PFS LEGSKLVPST KKMKWLFKLI DVPNKTFNEK
201 MLEKYWEIYD YDKLKANITN RLDKTDKKAR S ISRAVSEEL
241 REYHKNLRTN YNRFVSGDRP AAGLDNGGSA KYNPDKEEFL
281 LFLKEVEQYF KKYFPVKSKH SNKSKDKSLV DKYKNYCSYK
321 WKKEVNRS I INQLVAGLIQ QGKLLYYFYY NDTWQEDFLN
361 SYGLSYIQVE EAFKKSVMTS LSWGINRLTS FFIDDSNTVK
401 FDDITTKKAK EAIESNYFNK LRTCSRMQDH FKEKLAFFYP
441 VYVKDKKDRP DDDIENLIVL VKNAIESVSY LRNRT FHFKE
481 SSLLELLKEL DDKNSGQNKI DYSVAAEFIK RDIENLYDVF
521 REQIRSLGIA EYYKADMISD CFKTCGLEFA LYSPKNSLMP
561 AFKNVYKRGA NLNKAYIRDK GPKETGDQGQ NSYKALEEYR
601 ELTWYIEVKN NDQSYNAYKN LLQLIYYHAF LPEVRENEAL
641 ITDFINRTKE WNRKETEERL NTKNNKKHKN FDENDDITVN
681 TYRYES I PDY QGESLDDYLK VLQRKQMARA KEVNEKEEGN
721 NNYIQFIRDV WWAFGAYLE NKLKNYKNEL QPPLSKENIG
761 LNDTLKELFP EEKVKS PFNI KCRFSISTFI DNKGKSTDNT
801 SAEAVKTDGK EDEKDKKNIK RKDLLCFYLF LRLLDENEIC
841 KLQHQFIKYR CSLKERRFPG NRTKLEKETE LLAELEELME
881 LVRFTMPS I P EISAKAESGY DTMIKKYFKD FIEKKVFKNP
921 KTSNLYYHSD SKTPVTRKYM ALLMRSAPLH LYKDI FKGYY
961 LITKKECLEY IKLSNI IKDY QNSLNELHEQ LERIKLKSEK
1001 QNGKDSLYLD KKDFYKVKEY VENLEQVARY KHLQHKINFE
1041 SLYRI FRIHV DIAARMVGYT QDWERDMHFL FKALVYNGVL
1081 EERRFEAI FN NNDDNNDGRI VKKIQNNLNN KNRELVSMLC
1121 WNKKLNKNEF GAI IWKRNPI AHLNHFTQTE QNSKS SLESL
1161 INSLRILLAY DRKRQNAVTK TINDLLLNDY HIRIKWEGRV
1201 DEGQIYFNIK EKEDIENEPI IHLKHLHKKD CYIYKNSYMF
1241 DKQKEWICNG IKEEVYDKS I LKCIGNLFKF DYEDKNKSSA
1281 NPKHT or a polypeptide with at least 80%, 82%, 84%, 85%. 87%. 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
In another example, a Leptotrichia buccalis Cast 3a endonuclease that can be used has the following sequence (SEQ ID NO: 502; NCBI accession no. WP_015770004.1).
1 MKVTKVGGIS HKKYTSEGRL VKSESEENRT DERLSALLNM
41 RLDMYIKNPS STETKENQKR IGKLKKFFSN KMVYLKDNTL
81 SLKNGKKENI DREYSETDIL ESDVRDKKNF AVLKKIYLNE 121 NVNSEELEVF RNDIKKKLNK INSLKYSFEK NKANYQKINE 161 NNIEKVEGKS KRNIIYDYYR ESAKRDAYVS NVKEAFDKLY 201 KEEDIAKLVL EIENLTKLEK YKIREFYHEI IGRKNDKENF
241 AKIIYEEIQN VNNMKELIEK VPDMSELKKS QVFYKYYLDK 281 EELNDKNIKY AFCHFVEIEM SQLLKNYVYK RLSNISNDKI 321 KRIFEYQNLK KLIENKLLNK LDTYVRNCGK YNYYLQDGEI 361 ATSDFIARNR QNEAFLRNII GVSSVAYFSL RNILETENEN 401 DITGRMRGKT VKNNKGEEKY VSGEVDKIYN ENKKNEVKEN 441 LKMFYSYDFN MDNKNEIEDF FANIDEAISS IRHGIVHFNL 481 ELEGKDIFAF KNIAPSEISK KMFQNEINEK KLKLKIFRQL 521 NSANVFRYLE KYKILNYLKR TRFEFVNKNI PFVPSFTKLY 561 SRIDDLKNSL GIYWKTPKTN DDNKTKEIID AQIYLLKNIY 601 YGEFLNYFMS NNGNFFEISK EIIELNKNDK RNLKTGFYKL 641 QKFEDIQEKI PKEYLANIQS LYMINAGNQD EEEKDTYIDF 681 IQKIFLKGFM TYLANNGRLS LIYIGSDEET NTSLAEKKQE 721 FDKFLKKYEQ NNNIKIPYEI NEFLREIKLG NILKYTERLN 761 MFYLILKLLN HKELTNLKGS LEKYQSANKE EAFSDQLELI 801 NLLNLDNNRV TEDFELEADE IGKFLDFNGN KVKDNKELKK
841 FDTNKIYFDG ENIIKHRAFY NIKKYGMLNL LEKIADKAGY
881 KISIEELKKY SNKKNEIEKN HKMQENLHRK YARPRKDEKF
921 TDEDYESYKQ AIENIEEYTH LKNKVEFNEL NLLQGLLLRI
961 LHRLVGYTSI WERDLRFRLK GEFPENQYIE EIFNFENKKN
1001 VKYKGGQIVE KYIKFYKELH QNDEVKINKY SSANIKVLKQ
1041 EKKDLYIRNY IAHFNYIPHA EISLLEVLEN LRKLLSYDRK
1081 LKNAVMKSW DILKEYGFVA TFKIGADKKI GIQTLESEKI
1121 VHLKNLKKKK LMTDRNSEEL CKLVKIMFEY KMEEKKSEN or a polypeptide with at least 80%, 82%, 84%, 85%, 87%. 89%, 90%, 92%, 94%, 95%,
97%, 98% or 99% amino acid sequence identity thereto.
In another example, a Leptotrichia seeligeri Casl3a endonuclease that can be used has the following sequence (SEQ ID NO:503; NCBI accession no. WP_012985477.1).
1 MWISIKTLIH HLGVLFFCDY MYNRREKKII EVKTMRITKV
41 EVDRKKVLIS RDKNGGKLVY ENEMQDNTEQ IMHHKKSSFY
81 KSWNKTICR PEQKQMKKLV HGLLQENSQE KIKVSDVTKL
121 NISNFLNHRF KKSLYYFPEN SPDKSEEYRI EINLSQLLED
161 SLKKQQGTFI CWESFSKDME LYINWAENYI SSKTKLIKKS
201 IRNNRIQSTE SRSGQLMDRY MKDILNKNKP FDIQSVSEKY
241 QLEKLTSALK ATFKEAKKND KEINYKLKST LQNHERQIIE
281 ELKENSELNQ FNIEIRKHLE TYFPIKKTNR KVGDIRNLEI 321 GEIQKIVNHR LKNKIVQRIL QEGKLASYEI ESTVNSNSLQ
361 KIKIEEAFAL KFINACLFAS NNLRNMVYPV CKKDILMIGE
401 FKNS FKEIKH KKFIRQWSQF FSQEITVDDI ELASWGLRGA
441 IAPIRNEI IH LKKHSWKKFF NNPTFKVKKS KI INGKTKDV
481 TSEFLYKETL FKDYFYSELD SVPELI INKM ESSKILDYYS
521 SDQLNQVFTI PNFELSLLTS AVPFAPS FKR VYLKGFDYQN
561 QDEAQPDYNL KLNIYNEKAF NSEAFQAQYS LFKMVYYQVF
601 LPQFTTNNDL FKSSVDFILT LNKERKGYAK AFQDIRKMNK
641 DEKPSEYMSY IQSQLMLYQK KQEEKEKINH FEKFINQVFI
681 KGFNS FIEKN RLTYICHPTK NTVPENDNIE I PFHTDMDDS
721 NIAFWLMCKL LDAKQLSELR NEMIKFSCSL QSTEEISTFT
761 KAREVIGLAL LNGEKGCNDW KELFDDKEAW KKNMSLYVSE
801 ELLQSLPYTQ EDGQTPVINR S IDLVKKYGT ETILEKLFS S
841 SDDYKVSAKD IAKLHEYDVT EKIAQQESLH KQWIEKPGLA
881 RDSAWTKKYQ NVINDISNYQ WAKTKVELTQ VRHLHQLTI D
921 LLSRLAGYMS IADRDFQFSS NYILERENSE YRVTSWILLS
961 ENKNKNKYND YELYNLKNAS IKVSSKNDPQ LKVDLKQLRL
1001 TLEYLELFDN RLKEKRNNIS HFNYLNGQLG NS ILELFDDA
1041 RDVLSYDRKL KNAVSKSLKE ILSSHGMEVT FKPLYQTNHH
1081 LKIDKLQPKK IHHLGEKSTV SSNQVSNEYC QLVRTLLTMK or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%,
97%, 98% or 99% amino acid sequence identity thereto.
For example, a Paludibacter propionicigenes Casl3a endonuclease can be used that has the following sequence (SEQ ID NO: 504; NCB1 accession no. WP_013443710.1).
1 MRVSKVKVKD GGKDKMVLVH RKTTGAQLVY SGQPVSNETS
41 NILPEKKRQS FDLSTLNKTI IKFDTAKKQK LNVDQYKIVE
81 KI FKYPKQEL PKQIKAEEIL PFLNHKFQEP VKYWKNGKEE
121 S FNLTLLIVE AVQAQDKRKL QPYYDWKTWY IQTKS DLLKK
161 S IENNRIDLT ENLSKRKKAL LAWETEFTAS GS IDLTHYHK
201 VYMTDVLCKM LQDVKPLTDD KGKINTNAYH RGLKKALQNH
241 QPAI FGTREV PNEANRADNQ LS IYHLEWK YLEHYFPIKT
281 SKRRNTADDI AHYLKAQTLK TTIEKQLVNA IRANI IQQGK
321 TNHHELKADT TSNDLIRIKT NEAFVLNLTG TCAFAANNIR
361 NMVDNEQTND ILGKGDFIKS LLKDNTNSQL YS FFFGEGLS
401 TNKAEKETQL WGIRGAVQQI RNNVNHYKKD ALKTVFNISN
441 FENPTITDPK QQTNYADTIY KARFINELEK I PEAFAQQLK
481 TGGAVSYYTI ENLKSLLTTF QFSLCRSTI P FAPGFKKVFN
521 GGINYQNAKQ DES FYELMLE QYLRKENFAE ESYNARYFML
561 KLIYNNLFLP GFTTDRKAFA DSVGFVQMQN KKQAEKVNPR
601 KKEAYAFEAV RPMTAADS IA DYMAYVQSEL MQEQNKKEEK
641 VAEETRINFE KFVLQVFIKG FDS FLRAKEF DFVQMPQPQL
681 TATASNQQKA DKLNQLEAS I TADCKLT PQY AKADDATHIA
721 FYVFCKLLDA AHLSNLRNEL IKFRESVNEF KFHHLLEIIE
761 ICLLSADWP TDYRDLYSSE ADCLARLRPF IEQGADITNW
801 SDLFVQSDKH S PVIHANIEL SVKYGTTKLL EQIINKDTQF
841 KT TE AN FT AW NTAQKS IEQL IKQREDHHEQ WVKAKNADDK
881 EKQERKREKS NFAQKFIEKH GDDYLDICDY INTYNWLDNK
921 MHFVHLNRLH GLTIELLGRM AGFVALFDRD FQFFDEQQIA 961 DEFKLHGFVN LHS IDKKLNE VPTKKIKEIY DIRNKI IQIN
1001 GNKINESVRA NLIQFISSKR NYYNNAFLHV SNDEIKEKQM
1041 YDIRNHIAHF NYLTKDAADF SLIDLINELR ELLHYDRKLK
1081 NAVSKAFIDL FDKHGMILKL KLNADHKLKV ESLEPKKIYH
1121 LGSSAKDKPE YQYCTNQVMM AYCNMCRSLL EMKK
For example, aLachnospiraceae bacterium Casl3a endonuclease can be used that has the following sequence (SEQ ID NO: 505; NCBI accession no. WP_022785443.1).
1 MKISKVREEN RGAKLTVNAK TAWSENRSQ EGILYNDPSR
41 YGKSRKNDED RDRYIESRLK SSGKLYRI FN EDKNKRETDE
81 LQWFLSEIVK KINRRNGLVL SDMLSVDDRA FEKAFEKYAE
121 LSYTNRRNKV SGS PAFETCG VDAATAERLK GI ISETNFIN 161 RIKNNIDNKV SEDI IDRI IA KYLKKSLCRE RVKRGLKKLL 201 MNAFDLPYSD PDIDVQRDFI DYVLEDFYHV RAKSQVSRS I 241 KNMNMPVQPE GDGKFAITVS KGGTESGNKR SAEKEAFKKF 281 LSDYASLDER VRDDMLRRMR RLWLYFYGS DDSKLSDVNE
321 KFDVWEDHAA RRVDNREFIK LPLENKLANG KTDKDAERIR 361 KNTVKELYRN QNIGCYRQAV KAVEEDNNGR YFDDKMLNMF 401 FIHRIEYGVE KIYANLKQVT EFKARTGYLS EKIWKDLINY 441 IS IKYIAMGK AVYNYAMDEL NASDKKEIEL GKISEEYLSG 481 ISS FDYELIK AEEMLQRETA VYVAFAARHL SSQTVELDSE
521 NSDFLLLKPK GTMDKNDKNK LASNNILNFL KDKETLRDT I 561 LQYFGGHSLW TDFPFDKYLA GGKDDVDFLT DLKDVIYSMR 601 NDS FHYATEN HNNGKWNKEL ISAMFEHETE RMTWMKDKF 641 YSNNLPMFYK NDDLKKLLID LYKDNVERAS QVPS FNKVFV 681 RKNFPALVRD KDNLGIELDL KADADKGENE LKFYNALYYM
721 FKEIYYNAFL NDKNVRERFI TKATKVADNY DRNKERNLKD
761 RIKSAGSDEK KKLREQLQNY IAENDFGQRI KNIVQVNPDY
801 TLAQICQLIM TEYNQQNNGC MQKKSAARKD INKDSYQHYK
841 MLLLVNLRKA FLEFIKENYA FVLKPYKHDL CDKADFVPDF
881 AKYVKPYAGL ISRVAGSSEL QKWYIVSRFL S PAQANHMLG
921 FLHSYKQYVW DIYRRASETG TEINHS IAED KIAGVDITDV
961 DAVIDLSVKL CGTISSEISD YFKDDEVYAE YISSYLDFEY
1001 DGGNYKDSLN RFCNSDAVND QKVALYYDGE HPKLNRNIIL
1041 SKLYGERRFL EKITDRVSRS DIVEYYKLKK ETSQYQTKGI
1081 FDSEDEQKNI KKFQEMKNIV EFRDLMDYSE IADELQGQLI
1121 NWIYLRERDL MNFQLGYHYA CLNNDSNKQA TYVTLDYQGK
1161 KNRKINGAIL YQICAMYING LPLYYVDKDS SEWTVSDGKE
1201 STGAKIGEFY RYAKS FENTS DCYASGLEI F ENISEHDNIT
1241 ELRNYIEHFR YYSS FDRS FL GIYSEVFDRF FTYDLKYRKN
1281 VPTILYNILL QHFVNVRFEF VSGKKMIGID KKDRKIAKEK
1321 ECARITIREK NGVYSEQFTY KLKNGTVYVD ARDKRYLQS I
1361 IRLLFYPEKV NMDEMIEVKE KKKPSDNNTG KGYSKRDRQQ
1401 DRKEYDKYKE KKKKEGNFLS GMGGNINWDE INAQLKN
For example, aLeptotrichia shahii Cas 13a endonuclease can be used that has the following sequence (SEQ ID NO: 506; NCBI accession no. BBM39911.1). 1 MGNLFGHKRW YEVRDKKDFK IKRKVKVKRN YDGNKYILNI
41 NENNNKEKID NNKFIRKYIN YKKNDNILKE FTRKFHAGNI
81 LFKLKGKEGI IRIENNDDFL ETEEWLYIE AYGKSEKLKA
121 LGITKKKIID EAIRQGITKD DKKIEIKRQE NEEEIEIDIR 161 DEYTNKTLND CSIILRIIEN DELETKKSIY EIFKNINMSL 201 YKIIEKIIEN ETEKVFENRY YEEHLREKLL KDDKIDVILT 241 NFMEIREKIK SNLEILGFVK FYLNVGGDKK KSKNKKMLVE 281 KILNINVDLT VEDIADFVIK ELEFWNITKR IEKVKKVNNE
321 FLEKRRNRTY IKSYVLLDKH EKFKIERENK KDKIVKFFVE 361 NIKNNSIKEK IEKILAEFKI DELIKKLEKE LKKGNCDTEI 401 FGIFKKHYKV NFDSKKFSKK SDEEKELYKI IYRYLKGRIE 441 KILVNEQKVR LKKMEKIEIE KILNESILSE KILKRVKQYT 481 LEHIMYLGKL RHNDIDMTTV NTDDFSRLHA KEELDLELIT 521 FFASTNMELN KIFSRENINN DENIDFFGGD REKNYVLDKK 561 ILNSKIKIIR DLDFIDNKNN ITNNFIRKFT KIGTNERNRI 601 LHAISKERDL QGTQDDYNKV INIIQNLKIS DEEVSKALNL 641 DWFKDKKNI ITKINDIKIS EENNNDIKYL PSFSKVLPEI 681 LNLYRNNPKN EPFDTIETEK IVLNALIYVN KELYKKLILE 721 DDLEENESKN IFLQELKKTL GNIDEIDENI IENYYKNAQI 761 SASKGNNKAI KKYQKKVIEC YIGYLRKNYE ELFDFSDFKM 801 NIQEIKKQIK DINDNKTYER ITVKTSDKTI VINDDFEYII 841 SIFALLNSNA VINKIRNRFF ATSVWLNTSE YQNIIDILDE 881 IMQLNTLRNE CITENWNLNL EEFIQKMKEI EKDFDDFKIQ 921 TKKEIFNNYY EDIKNNILTE FKDDINGCDV LEKKLEKIVI 961 FDDETKFEID KKSNILQDEQ RKLSNINKKD LKKKVDQYIK 1001 DKDQEIKSKI LCRIIFNSDF LKKYKKEIDN LIEDMESENE 1041 NKFQEIYYPK ERKNELYIYK KNLFLNIGNP NFDKIYGLIS 1081 NDIKMADAKF LFNIDGKNIR KNKISEIDAI LKNLNDKLNG 1121 YSKEYKEKYI KKLKENDDFF AKNIQNKNYK SFEKDYNRVS 1161 EYKKIRDLVE FNYLNKIESY LIDINWKLAI QMARFERDMH 1201 YIVNGLRELG IIKLSGYNTG ISRAYPKRNG SDGFYTTTAY 1241 YKFFDEESYK KFEKICYGFG IDLSENSEIN KPENESIRNY 1281 ISHFYIVRNP FADYSIAEQI DRVSNLLSYS TRYNNSTYAS 1321 VFEVFKKDVN LDYDELKKKF KLIGNNDILE RLMKPKKVSV 1361 LELESYNSDY IKNLIIELLT KIENTNDTL or a polypeptide with at least 80%, 82%. 84%. 85%. 87%. 89%, 90%, 92%, 94%, 95%,
97%, 98% or 99% amino acid sequence identity thereto.
In another example, a Leptotrichia buccalis C-1013-b Casl3a endonuclease can have the following sequence (SEQ ID NO: 507; NCBI accession no. C7NBY4; AltName LbuC2c2).
1 MKVTKVGGIS HKKYTSEGRL VKSESEENRT DERLSALLNM
41 RLDMYIKNPS STETKENQKR IGKLKKFFSN KMVYLKDNTL
81 SLKNGKKENI DREYSETDIL ESDVRDKKNF AVLKKIYLNE
121 NVNSEELEVF RNDIKKKLNK INSLKYSFEK NKANYQKINE
161 NNIEKVEGKS KRNIIYDYYR ESAKRDAYVS NVKEAFDKLY
201 KEEDIAKLVL EIENLTKLEK YKIREFYHEI IGRKNDKENF
241 AKIIYEEIQN VNNMKELIEK VPDMSELKKS QVFYKYYLDK
281 EELNDKNIKY AFCHFVEIEM SQLLKNYVYK RLSNISNDKI
321 KRIFEYQNLK KLIENKLLNK LDTYVRNCGK YNYYLQDGEI
361 ATSDFIARNR QNEAFLRNII GVSSVAYFSL RNILETENEN 401 DITGRMRGKT VKNNKGEEKY VSGEVDKIYN ENKKNEVKEN 441 LKMFYSYDFN MDNKNEIEDF FANIDEAISS IRHGIVHFNL 481 ELEGKDI FAF KNIAPSEISK KMFQNEINEK KLKLKI FRQL 521 NSANVFRYLE KYKILNYLKR TRFEFVNKNI PFVPS FTKLY 561 SRIDDLKNSL GIYWKTPKTN DDNKTKEI ID AQIYLLKNIY 601 YGEFLNYFMS NNGNFFEISK EI IELNKNDK RNLKTGFYKL 641 QKFEDIQEKI PKEYLANIQS LYMINAGNQD EEEKDTYIDF 681 IQKI FLKGFM TYLANNGRLS LIYIGSDEET NTSLAEKKQE 721 FDKFLKKYEQ NNNIKI PYEI NEFLREIKLG NILKYTERLN 761 MFYLILKLLN HKELTNLKGS LEKYQSANKE EAFSDQLELI 801 NLLNLDNNRV TEDFELEADE IGKFLDFNGN KVKDNKELKK
841 FDTNKIYFDG ENI IKHRAFY NIKKYGMLNL LEKIADKAGY
881 KIS IEELKKY SNKKNEIEKN HKMQENLHRK YARPRKDEKF
921 TDEDYESYKQ AIENIEEYTH LKNKVEFNEL NLLQGLLLRI
961 LHRLVGYTS I WERDLRFRLK GEFPENQYIE EI FNFENKKN
1001 VKYKGGQIVE KYIKFYKELH QNDEVKINKY SSANIKVLKQ
1041 EKKDLYIRNY IAHFNYIPHA EISLLEVLEN LRKLLSYDRK
1081 LKNAVMKSW DILKEYGFVA TFKIGADKKI GIQTLESEKI
1121 VHLKNLKKKK LMTDRNSEEL CKLVKIMFEY KMEEKKSEN or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%,
97%, 98% or 99% amino acid sequence identity thereto.
In some cases, Casl3b may work faster in atarget viral RNA detection assay than Casl3a.
A Casl3b from Prevotella buccae can be used in the RNA detection methods, compositions and devices. A sequence for a Prevotella buccae Casl3b protein (NCB1 accession no. WP_004343973.1) that can be used is shown below- (SEQ ID NO:508).
1 MQKQDKLFVD RKKNAI FAFP KYITIMENKE KPEPIYYELT
41 DKHFWAAFLN LARHNVYTTI NHINRRLEIA ELKDDGYMMG
81 IKGSWNEQAK KLDKKVRLRD LIMKHFPFLE AAAYEMTNSK
121 S PNNKEQREK EQSEALSLNN LKNVLFI FLE KLQVLRNYYS
161 HYKYSEES PK PI FETSLLKN MYKVFDANVR LVKRDYMHHE
201 NIDMQRDFTH LNRKKQVGRT KNI IDS PNFH YHFADKEGNM
241 TIAGLLFFVS LFLDKKDAIW MQKKLKGFKD GRNLREQMTN
281 EVFCRSRISL PKLKLENVQT KDWMQLDMLN ELVRCPKSLY
321 ERLREKDRES FKVPFDIFSD DYNAEEEPFK NTLVRHQDRF
361 PYFVLRYFDL NEI FEQLRFQ IDLGTYHFS I YNKRIGDEDE
401 VRHLTHHLYG FARIQDFAPQ NQPEEWRKLV KDLDHFETSQ
441 EPYISKTAPH YHLENEKIGI KFCSAHNNLF PSLQTDKTCN
481 GRSKFNLGTQ FTAEAFLSVH ELLPMMFYYL LLTKDYSRKE
521 SADKVEGI IR KEISNIYAIY DAFANNEINS IADLTRRLQN
561 TNILQGHLPK QMIS ILKGRQ KDMGKEAERK IGEMI DDTQR
601 RLDLLCKQTN QKIRIGKRNA GLLKSGKIAD WLVNDMMRFQ
641 PVQKDQNNI P INNSKANSTE YRMLQRALAL FGSENFRLKA
681 YFNQMNLVGN DNPHPFLAET QWEHQTNILS FYRNYLEARK
721 KYLKGLKPQN WKQYQHFLIL KVQKTNRNTL VTGWKNS FNL
761 PRGI FTQPIR EWFEKHNNSK RIYDQILS FD RVGFVAKAI P
801 LYFAEEYKDN VQPFYDYPFN IGNRLKPKKR QFLDKKERVE
841 LWQKNKELFK NYPSEKKKTD LAYLDFLSWK KFERELRLIK
881 NQDIVTWLMF KELFNMATVE GLKIGEIHLR DIDTNTANEE 921 SNNILNRIMP MKLPVKTYET DNKGNILKER PLATFYIEET
961 ETKVLKQGNF KALVKDRRLN GLFS FAETTD LNLEEHPISK
1001 LSVDLELIKY QTTRIS IFEM TLGLEKKLID KYSTLPTDS F
1041 RNMLERWLQC KANRPELKNY VNSLIAVRNA FSHNQYPMYD
1081 ATLFAEVKKF TLFPSVDTKK IELNIAPQLL EIVGKAIKEI
1121 EKSENKN or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto. Such aPrevotella buccae Casl3b protein can have a Km (Michaelis constant) substrate concentration of about 20 micromoles and a Kcat of about 987/second (see, e.g., Slaymaker et al. Cell Rep 26 (13): 3741-3751 (2019)).
Another Prevotella buccae Casl3b protein (NCB1 accession no. WP 004343581.1) that can be used in the RSV RNA detection methods, compositions and devices has the sequence shown below (SEQ ID NO: 509).
1 MQKQDKLFVD RKKNAI FAFP KYITIMENQE KPEPIYYELT
41 DKHFWAAFLN LARHNVYTTI NHINRRLEIA ELKDDGYMMD
81 IKGSWNEQAK KLDKKVRLRD LIMKHFPFLE AAAYEITNSK
121 S PNNKEQREK EQSEALSLNN LKNVLFI FLE KLQVLRNYYS
161 HYKYSEES PK PI FETSLLKN MYKVFDANVR LVKRDYMHHE
201 NIDMQRDFTH LNRKKQVGRT KNI IDS PNFH YHFADKEGNM
241 TIAGLLFFVS LFLDKKDAIW MQKKLKGFKD GRNLREQMTN
281 EVFCRSRISL PKLKLENVQT KDWMQLDMLN ELVRCPKSLY
321 ERLREKDRES FKVPFDIFSD DYDAEEEPFK NTLVRHQDRF
361 PYFVLRYFDL NEI FEQLRFQ IDLGTYHFS I YNKRIGDEDE
401 VRHLTHHLYG FARIQDFAQQ NQPEVWRKLV KDLDYFEASQ
441 EPYI PKTAPH YHLENEKIGI KFCSTHNNLF PSLKTEKTCN
481 GRSKFNLGTQ FTAEAFLSVH ELLPMMFYYL LLTKDYSRKE
521 SADKVEGI IR KEISNIYAIY DAFANGEINS IADLTCRLQK
561 TNILQGHLPK QMIS ILEGRQ KDMEKEAERK IGEMI DDTQR
601 RLDLLCKQTN QKIRIGKRNA GLLKSGKIAD WLVNDMMRFQ
641 PVQKDQNNI P INNSKANSTE YRMLQRALAL FGSENFRLKA
681 YFNQMNLVGN DNPHPFLAET QWEHQTNILS FYRNYLEARK
721 KYLKGLKPQN WKQYQHFLIL KVQKTNRNTL VTGWKNS FNL
761 PRGI FTQPIR EWFEKHNNSK RIYDQILS FD RVGFVAKAI P
801 LYFAEEYKDN VQPFYDYPFN IGNKLKPQKG QFLDKKERVE
841 LWQKNKELFK NYPSEKKKTD LAYLDFLSWK KFERELRLIK
881 NQDIVTWLMF KELFNMATVE GLKIGEIHLR DIDTNTANEE
921 SNNILNRIMP MKLPVKTYET DNKGNILKER PLATFYIEET
961 ETKVLKQGNF KVLAKDRRLN GLLS FAETTD IDLEKNPITK
1001 LSVDHELIKY QTTRIS IFEM TLGLEKKLIN KYPTLPTDS F
1041 RNMLERWLQC KANRPELKNY VNSLIAVRNA FSHNQYPMYD
1081 ATLFAEVKKF TLFPSVDTKK IELNIAPQLL EIVGKAIKEI
1121 EKSENKN or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto. An example of a Bergeyella zoohelcum Casl3b (R1177A) mutant sequence (NCBI accession no. 6AAY_A) that can be used is shown below (SEQ ID NO: 510).
1 XENKTSLGNN IYYNPFKPQD KSYFAGYFNA AXENTDSVFR
41 ELGKRLKGKE YTSENFFDAI FKENISLVEY ERYVKLLSDY
81 FPXARLLDKK EVPIKERKEN FKKNFKGI IK AVRDLRNFYT
121 HKEHGEVEIT DEI FGVLDEX LKSTVLTVKK KKVKTDKTKE
161 ILKKS IEKQL DILCQKKLEY LRDTARKIEE KRRNQRERGE
201 KELVAPFKYS DKRDDLIAAI YNDAFDVYID KKKDSLKES S
241 KAKYNTKSDP QQEEGDLKI P ISKNGWFLL SLFLTKQEIH
281 AFKSKIAGFK ATVIDEATVS EATVSHGKNS ICFXATHEI F
321 SHLAYKKLKR KVRTAEINYG EAENAEQLSV YAKETLXXQX
361 LDELSKVPDV VYQNLSEDVQ KT FIEDWNEY LKENNGDVGT
401 XEEEQVIHPV IRKRYEDKFN YFAIRFLDEF AQFPTLRFQV
441 HLGNYLHDSR PKENLISDRR IKEKITVFGR LSELEHKKAL
481 FIKNTETNED REHYWEIFPN PNYDFPKENI SVNDKDFPIA
521 GS ILDREKQP VAGKIGIKVK LLNQQYVSEV DKAVKAHQLK
561 QRKASKPS IQ NI IEEIVPIN ESNPKEAIVF GGQPTAYLSX
601 NDIHS ILYEF FDKWEKKKEK LEKKGEKELR KEIGKELEKK
641 IVGKIQAQIQ QI IDKDTNAK ILKPYQDGNS TAIDKEKLIK
681 DLKQEQNILQ KLKDEQTVRE KEYNDFIAYQ DKNREINKVR
721 DRNHKQYLKD NLKRKYPEAP ARKEVLYYRE KGKVAVWLAN
761 DIKRFXPTDF KNEWKGEQHS LLQKSLAYYE QCKEELKNLL
801 PEKVFQHLPF KLGGYFQQKY LYQFYTCYLD KRLEYISGLV
841 QQAENFKSEN KVFKKVENEC FKFLKKQNYT HKELDARVQS
881 ILGYPI FLER GFXDEKPTI I KGKTFKGNEA LFADWFRYYK
921 EYQNFQTFYD TENYPLVELE KKQADRKRKT KIYQQKKNDV
961 FTLLXAKHI F KSVFKQDS ID QFSLEDLYQS REERLGNQER
1001 ARQTGERNTN YIWNKTVDLK LCDGKITVEN VKLKNVGDFI
1041 KYEYDQRVQA FLKYEENIEW QAFLIKESKE EENYPYWER
1081 EIEQYEKVRR EELLKEVHLI EEYILEKVKD KEILKKGDNQ
1121 NFKYYILNGL LKQLKNEDVE SYKVFNLNTE PEDVNINQLK
1161 QEATDLEQKA FVLTYIANKF AHNQLPKKEF WDYCQEKYGK
1201 IEKEKTYAEY FAEVFKKEKE ALIKLEHHHH HH or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
Another example of a Casl3b protein sequence from Prevotella sp. MSX73 (NCBI accession no. WP_007412163.1) that can be used in the target viral RNA detection methods, compositions and devices is shown below (SEQ ID NO: 511).
1 MQKQDKLFVD RKKNAI FAFP KYITIMENQE KPEPIYYELT
41 DKHFWAAFLN LARHNVYTTI NHINRRLEIA ELKDDGYMMG
81 IKGSWNEQAK KLDKKVRLRD LIMKHFPFLE AAAYEITNSK
121 S PNNKEQREK EQSEALSLNN LKNVLFI FLE KLQVLRNYYS
161 HYKYSEES PK PI FETSLLKN MYKVFDANVR LVKRDYMHHE
201 NIDMQRDFTH LNRKKQVGRT KNI IDS PNFH YHFADKEGNM
241 TIAGLLFFVS LFLDKKDAIW MQKKLKGFKD GRNLREQMTN
281 EVFCRSRISL PKLKLENVQT KDWMQLDMLN ELVRCPKSLY
321 ERLREKDRES FKVPFDIFSD DYDAEEEPFK NTLVRHQDRF 361 PYFVLRYFDL NEI FEQLRFQ IDLGTYHFS I YNKRIGDEDE
401 VRHLTHHLYG FARIQDFAPQ NQPEEWRKLV KDLDHFETSQ
441 EPYISKTAPH YHLENEKIGI KFCSTHNNLF PSLKREKTCN
481 GRSKFNLGTQ FTAEAFLSVH ELLPMMFYYL LLTKDYSRKE
521 SADKVEGI IR KEISNIYAIY DAFANNEINS IADLTCRLQK
561 TNILQGHLPK QMIS ILEGRQ KDMEKEAERK IGEMI DDTQR
601 RLDLLCKQTN QKIRIGKRNA GLLKSGKIAD WLVSDMMRFQ
641 PVQKDTNNAP INNSKANSTE YRMLQHALAL FGSES SRLKA
681 YFRQMNLVGN ANPHPFLAET QWEHQTNILS FYRNYLEARK
721 KYLKGLKPQN WKQYQHFLIL KVQKTNRNTL VTGWKNS FNL
761 PRGI FTQPIR EWFEKHNNSK RIYDQILS FD RVGFVAKAI P
801 LYFAEEYKDN VQPFYDYPFN IGNKLKPQKG QFLDKKERVE
841 LWQKNKELFK NYPSEKNKTD LAYLDFLSWK KFERELRLIK
881 NQDIVTWLMF KELFKTTTVE GLKIGEIHLR DIDTNTANEE
921 SNNILNRIMP MKLPVKTYET DNKGNILKER PLATFYIEET
961 ETKVLKQGNF KVLAKDRRLN GLLS FAETTD IDLEKNPITK
1001 LSVDYELIKY QTTRIS IFEM TLGLEKKLID KYSTLPTDS F
1041 RNMLERWLQC KANRPELKNY VNSLIAVRNA FSHNQYPMYD
1081 ATLFAEVKKF TLFPSVDTKK IELNIAPQLL EIVGKAIKEI
1121 EKSENKN or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity7 thereto.
Hence, the sample can be incubated with at least one CRISPR RNA (crRNA) and at least one Casl3 protein. The Casl3 protein can, for example, be a Casl3a protein. Casl3b protein, Casl3c protein, or a Casl3d protein, or a combination thereof.
An example of a Cetobacterium somerae Casl3c sequence (NCBI Ref. Sequence WP 266188461 1) that can be used is shown below (SEQ ID NO: 512).
1 MEKNKIYGSK QNRSS IIRI I LSNYDMVGIK ELKVLYQKQG GVDTFNLESY IDLTSRKVII
61 KSFKAKEKER NRYRFSYDTM GNSPGDKNS F IITKFDKILN KEIRRYKVTL SLKEKTTNVI
121 FAEVEDKMEE QKKDVSGERI KLRNRTSQTE RKLLSKEVCR NYSEIARVSV DE IDSLKIYK
181 IKRFLNYRSN LLVYFAL IND FLCAPLKEEG ITEVWKLSKN NTPLLDKRLE KITDYVYSTL
241 SKEVENRVNQ LEKRISKNNK EIEELKVLFS HKNGNKRKIE QLELLNKSLK IKMSELSGYS
301 SKENLKQDLK KIVDIFSEFR HALMHYDYMY FENLFENRGC DNLKNLLDLN FFRYTKLIEE
361 FKIENKTNYL DGDEELS ILG KLKNIKKLYS YYNTLCSNKS GFNKFINTFF TCDGVEDSDF
421 KKL IVENFQN EMDAIKS FSK KNDLSKKNLN RMESHFKLME NTPYFWDIHT SNIYKKLYNK
481 RKNLVEDYNN E INGIKSKTR ITNINSELLE LKKQMEEITK NNSLFRLKYK LQVAYS FLTI
541 EFNGDLKEFK NKFDPTNLEK IQEYLKKKEE YLNFRVPKIK RGEKS IFDLG KLENQLKRMQ
601 DEFNKRDSLY LDVDSKNNLF KFYILNYLLL PVEFKGDFLG FVKTHYYNIK NVDFLDESDE
661 SLSDEKLNEK LKELSDDGFF HKIRLFEKNI KNYE IIKYSI SNHEDMKRYF ELLELNVKHL
721 EYKSTDEVGI FNKNMLL PIF KYYQNVFKLY NDIE IHGLLK LAQNKS INLE ETLTFVKDEG
781 KDGFFNFSNL LKIINKKDYK NKAKIRNS IA HLNLKELIVD LFKNELKLNE NVRSAIEFSL
841 DNKLNKVDLG MDFVNDYYMK KERFIFNQRR LI PSDIIDTK RDLMKKQNDE LMKKYRLNLD
901 YDNLNKVYDR ANKLRS IADG MESIDENKGI FTS IKVDNIP FLKLDEKTRN GILIKDSSDL
961 MGVYKEQWK KLKKKVIEKF IYDEEKIITI NVYKAAAGET ESFIIKVKRD KDSDVYRIDE 1021 KAILENQYYL CSEKGGWTV VPKHSVKNIE FKFDLRSGYV FGKTIVYGTK or a polypeptide with at least 80%, 82%. 84%. 85%. 87%. 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
An example of a Halarcobacter ebronensis Casl3c sequence that can be used (NCBI
Reference Sequence- WP_ 128982150.1 SEQ ID NO- 513) is:
1 MKKLKNPSNR NSLPS IIISK FDSSKIYE IK VKYEKLARLD RLEIGDMSLD ENLNILFKKV
61 NFNGIDLEIL NPLLLDFDSY TISGKLQKNS TNKT ILTLKK DGKIIKYNVL EKDNKYFKNG
121 KEFVI PKDVK EEGKRLVNDK FLLT IEDKKR EENSLPKKRK KETQRDILKD ET IEIYKRIS
181 SNSNIKSEDI YRIKRYMLFR SDMMFFYTFI DNFFYCLYKN KNEQLWNTNF KEKENLGKFI
241 EFTLNDTLKN PRNGILKSYS KDLKWQEDF VKIKDIFEKI RHALAHFDFT FIDNLLSNNI
301 EFDFNIKLLN IVIEDSQDLY YEAKKEFIED EKMDILDEKD IS IKKLYTFY SKIDIKKPAF
361 NKL INSFLIK DGVENSKLKE YIKEKYNCHY FIDIHDNKEY KKIYNEHKKL ISENQNLQLN
421 SKENGQKIKI NNDRLEELKG KMNELTKANS LKRLEFKLRL AFGFIKVEYN IFKDFKNNFS
481 EDIKKDMNID LEKIKSYLDT SYSNNQFFNY KVYNKKTKQK DIDKDIFDDI EKETLKELVE
541 NDSLLKI ILL FYIFT PKELK GEFLGFIKKF YHDTKNIDKD TKDKEEPLEQ IKQEVPLKLK
601 ILEKNLT ILT IFNYS ISLNI EYDKNNNS FY ERGNKFKKIY KDLKISHNQE EFDKSLLAPL
661 LKYYMNLYKL LNDFE IYLLL KYKNKDNLNK ESLNKLINDE QLKHNDHYNF TTLLSEYFNF
721 DPKKNKKYET LTILRNS ISH QKIDNLIYNL DKNKILEQRV KIVEL IKEQR DIKETLKFDP
781 INDFTMKTVQ LLKSLENQSE KRDKIEEILK QQDLSANDFY NIYKLKGVES IKKELFIRLG
841 KTKIEEKIQE DIAKGS I or a polypeptide with at least 80%, 82%. 84%. 85%. 87%. 89%. 90%. 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
An example of a Malaciobacter hal ophilus Casl3c sequence that can be used (NCBI
Reference Sequence: WP 101184979.1 SEQ ID NO: 514) is:
1 MNSIEKIKKP SNRNS I PS II ISDYDENKIK EIKVKYLKLA RLDKIT IQDM E IRDNIVEFK 61 KILLNGIEHT IKDNQKIEFD NYEITAYVRA SKQRRDGKIT QAKYWT ITD KYLRDNEKEK
121 RFKSTERELP NDTLLMRYKQ ISGFDTLTSK DIYKIKRYID FKNEMLFYFQ FIEEFFS PLL
181 PKGTNFYSLN IEQNKDKWK YIVYRLNDDF KNQSLNQFIK KTDTIKYDFL KIQKILSDFR
241 HALAHFDFDF IQKFFDDELD KNRFDIST IS LIKTMLQEKE EKYYQEKNNY IEDSDTLTLF
301 DEKESNFSKI HNFYIKISQK KPAFNKLINS FLSKDGVPNE ELKSYLATKK IDFFEDIHSN
361 KEYKKIYIKH KNLWEKQKE ESQEKPNGQK LKNYNDELQK LKDEMNKITK QNSLNRLEVK
421 LRLAFGFIAN EYNYNFKNFN DKFTLDVKKE QKIKVFKNSS NEKLKEYFES TFIEKRFFHF
481 CVKFFNKKTK KEETKQKNIF NL IENETLEE LVKES PLLQI ITLLYLFIPK ELQGEFVGFI
541 LKIYHHTKNI TNDTKEDEKS IEDTQNSFSL KLKILAKNLR GLQLFNYSLS HNTLYNTKEH
601 FFYEKGNRWQ SVYKSLE ISH NQDEFDIHLV IPVIKYYINL NKL IGDFEIY ALLTYADKNS
661 ITEKLSDITK RDDLKFRGYY NFSTLLFKTF MINTNYEQNQ KSTQYIKQTR NDIAHQNIEN
721 MLKAFENNEI FAQREEIVNY LQKEHKMQEI LHYNPINDFT MKTVQYLKSL NIHSQKESKI
781 ADIHKKESLV PNDYYLIYKL KVIELLKQKV IEAIGETKDE EKIKNAIAKE EQIKKGYNK or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity7 thereto.
An example of a Tissierella sp. P1 Casl3c sequence (NCBI Reference Sequence: WP 255374260.1) having SEQ ID NO: 515 that can be used is shown below
1 MDDKDSQDNA YKDGNKNLIN EIWKYEGKKD DEKNKIIERS YRS IEKS INQ FILDHKYKVE
61 KDKKGEKIDI SQEMIEEDLR KILILFSRLR HSMVHYDYEF YQALYSGKDF VISDKNNLEN
121 RMISQLLDLN IFKELSKVKL IKDKAISNYL DKNTTIHVLG QDIKAIRLLD IYRDICGSKN
181 GFNKFINTMI T ISGEEDREY KEKVIEHFNK KMENLSTYLE KLEKQDNAKR NNKRVYNLLK
241 QKL IEQQKLK EWFGGPYVYD IHSSKRYKEL YIERKKLVDR HSKLFEEGLD EKNKKELTKI
301 NDELSKLNSE MKEMTKLNSK YRLQYKLQLA FGFILEEFDL NIDTFINNFD KDKDLI ISNF
361 MKKRDIYLNR VLDRGDNRLK NI IKEYKFRD TEDIFCNDRD NNLVKLYILM YILLPVEIRG
421 DFLGFVKKNY YDMKHVDFID KKDKEDKDTF FHDLRLFEKN IRKLE ITDYS LS SGFLSKEH
481 KVDIEKKIND FINRNGAMKL PEDITIEEFN KSLILPIMKN YQINFKLLND IE ISALFKIA
541 KDRS ITFKQA IDEIKNEDIK KNSKKNDKNN HKDKNINFTQ LMKRALHEKI PYKAGMYQIR
601 NNISHIDMEQ LYIDPLNSYM NSNKNNIT IS EQIEKIIDVC VTGGVTGKEL NNNIINDYYM
661 KKEKLVFNLK LRKQNDIVS I ESQEKNKREE FVFKKYGLDY KDGEINI IEV IQKVNSLQEE
721 LRNIKETSKE KLKNKETLFR DISL INGT IR KNINFKIKEM VLDIVRMDEI RHINIHIYYK
781 GENYTRSNII KFKYAIDGEN KKYYLKQHEI NDINLELKDK FVTLICNMDK HPNKNKQT IN
841 LESNYIQNVK FIIP or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
An example of a Fusibacter paucivorans Casl3c sequence (NCBI Reference Sequence: WP ...213237188.1 ) having SEQ ID NO: 516 that can be used is:
1 MMQDSAAKQN NGSDKKNGGK QNKNS IVRIV ISNFDHEMIK EIKVLYEKQG GVDTLRIDAM
61 KLLAIDTADS GMIQFQEWG IT PNRIEDRV MRYQPPEITY DFDGDTPYLT VTKVISESND
121 RANRKYKIMG TWRRTNNGE I EVTVTVEDKK KHQRAQSMRN NRKAS SLSSR VLLNEAFQNN
181 IS I ILRRSEY DTFNRTQIDS RMVYKVKRFM TYRSQLSYYL NMINAMIMTM VNDKRLDAIG
241 IENDDAPELR RLYQSGKTRE LDEIWEIVDS EHKANNKANK KANKAKDLTS EDIEILYKGL
301 TKAFNNRIEP FIEHQHQHIE NLKSAADNVY DNEVKKIKKS DRDDAQKEVA IKSCEIERDK
361 KLAAL PVYID KDGFNISEDD VRKI IAVFS S IRHGVMHYEY HLFDDLITGN TIALMQHKTF
421 DINNLELDLF DVLDASYDIK ISHDTTYLTS NDKEMFFGAN QSL IEMNRTY RQVCDHKNGF
481 NAFINGYFVQ DGVENIEVKQ IIKDDFEYQI RKAENYIKYL KNNKKNTKSA EHKLKVLREW
541 LETSDGEVYF QDIHLSKKYK ALYNAHKERV AELQQVMHQP MRKLE IAELN NAINQQKEKM
601 EKMTKANAKW RLYYKLRVAF GYLEEAYNLN YERFKDEFNT DTPSFIGRQR TRASENAYSK
661 EVNAYLDTSL YKRPKNVPYQ SSENNRLQRF INFCDTVPRL KQS IFDES PL LKIYVLFLLF
721 L PKEVRGDFL GFVKHHYYEL KNVDFMSTAY DPENPKDQFF HKMRL IEKHL RHYNLFDFKL
781 EDYWQFSYHN DELEKLQAYV LKEI IDDRAI SNEI INTFNL NGMIIRPFLK VYDNIYQLCN
841 MIELKALLKI ANQDVDGKGP LKTIGDAMKV VGKGHLNFNE LMKTWEMEG EREYITKEYD
901 ENKNNKNKKK NEIYNSAIKM RNKISHFDTV FLFSKFLGFE KTEADS IQKH VKNLMLLYEV
961 LSLKQENLDD FIINDYLMQY DQVLHYLKKS AWEDNTE IK NRFSKKQKNK IKREEDKRDV
1021 ITAFNDFKQL S SYLDDEEAL KKTLMGEIDE HPDI ILDAWI PFKRGKDTAL QLKDMT IAER
1081 KKNINTI IRE LKKDQSDERG ALLSKLNRAI KEKVYEMIAT DRYYIFNIDV YTLVHRAGVK
1141 VDDPDTDLKQ WQLYFDQNG LLQNPWIEQI PHKIKKYDAS LLDEFKGEGI RIMHKPLPYT 1201 LDVTQEEVKA CADKCGWTHF IVLGSVAFPV NLNEKSVFKM R or a polypeptide with at least 80%, 82%, 84%, 85%. 87%. 89%. 90%. 92%. 94%. 95%, 97%, 98% or 99% amino acid sequence identity thereto.
An example of a Ruminococcus biclrculans Cast 3c sequence that can be used is: (NCBI
Reference Sequence: WP J 95551251.1 SEQ ID NO: 517)
1 MAKKNKMKPR ELREAQKKAR QLKAAEINNN AAPAIAAMPA AEAAAPAAEK KKSSVKAAGM 61 KS ILVSENKM YITSFGKGNS AVLEYEVDNN DYNQTQLS SK GSSNIELHGV NEVNITFS SK 121 HGFESGVEIN TSNPTHRSGE SS PVRWDMLG LKSELEKRFF GKTFDDNIHI QL IYNILDIE 181 KILAVYVTNI VYALNNMLGV KGSESHDDFI GYLSTNNTYD VFIDPDNSSL SDDKKANVRK 241 SLSKFNVLLK TKRLGYFGLE EPKTKDTRVS QAYKKRVYHM LAIVGQIRQC VFHDKSGAKR 301 FDLYSFINNI DPEYRETLDY LVDERFDS IN KGFIEGNKIN ISLLIDMMKG YEADDI IRLY 361 YDFIVLKSQK NLGFS IKKLR EKMLDEYGFR FKDKQYDPVR SKMYKLMDFL LFCNHYRNDV 421 AAGEALVRKL RFSMTDDEKE GIYADEAAKL WGKFRNDFEN IADHMNGDVI KELGKADMDF 481 DEKILDSEKK NASDLLYFSK MIYMLTYFLD GKEINDLLTT LISKFDNIKE FLKIMKSSAV 541 DVECELTAGY KLFNDSQRIT NELFIVKNIA SMRKPAASAK LTMFRDALTI LGIDDNITDD 601 RISEILKLKE KGKGIHGLRN FITNNVIES S RFVYLIKYAN AQKIREVAKN EKWMFVLGG 661 I PDTQIERYY KSCVEFPDMN SSLEAKRSEL ARMIKNIS FD DFKNVKQQAK GRENVAKERA 721 KAVIGLYLTV MYLLVKNLVN VNARYVIAIH CLERDFGLYK EII PELASKN LKNDYRILSQ 781 TLCELCDDRD ES PNLFLKKN KRLRKCVEVD INNADSSMTR KYRNC IAHLT WRELKEYIG 841 DIRTVDSYFS IYHYVMQRC I TKRENDTKQE EKIKYEDDLL KNHGYTKDFV KALNS PFGYN 901 I PRFKNLS IE QLFDRNEYLT EK or a polypeptide with at least 80%, 82%. 84%. 85%. 87%. 89%. 90%. 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.
An example of a Eubacterium sp. Ani l that may be used (NCBI Reference Sequence:
WP . 162611874.1 SEQ ID NO:518) is:
1 MSKKQRPKDI RKRQEEEKRE KYKKQEELRK KQEELRKEQE QRREDQKELE KIKKEVGEEG
61 EKKKSRAKAL GLKSTFILDR DEQKVLMTS F GQGNKAVRDK YIIGDKVSDI NDDRKNKKAA
121 LLVEVCGKSF NISKKENDDC DPVKVNNPW SRNKKDDDLI HCRKKLEELY FGEQFKDNIH
181 IQL IYNILDI EKILAVQVNN IVFALNNLLS WSGEEKFDLI GYLGVNDTYE KFRDAKGKRK
241 GLYEKFSTLI EKKRMRYFGS TFYPLNEKGE EITSNDKKEW EQFEKKCYHL LAVLGMMRQA
301 TAHGDSKRRA E IYKLGKEFD KSEARGCRQE ARKELDDLYR KKIHEMNQSF LKNSKRDILM
361 LFRIYDAESK EAKRKLAQEY YEFIMLKSYK NTGFS IKHLR ETVIDKMDED IKEKIKDDKY
421 NPIRRKLYRI MDFVIYQYYQ ESEQQEEAME LVRKLRNAET KVEKELTYRK EAEKLKEELE
481 KIIRNS ILSV CDRILAEMNE KRHKKVNQES SDTDSEEPLD PEISEGITFI KETAHS FSEM
541 IYLLTVFLDG KEINILLTQL IHCFDNIS S F MDTMKEENLL TKLKEDYEIF EESKEISKEL
601 RIINSFARMT E PVPKTEKTM FIDAAQILGY SNDEKELEGY VDALLDTKNK TKDKERKGFE
661 KYIWNNVIKS TRFRYLVRYA DPKKVRAFAA NKKWAFVLK DIPDEQIKAY YNSCFSQNSD
721 S SSNMS IAFQ DGDSNKKGTS VHDMMRKALT EKITGLNFGD FEEESKKGIR REESDKNI IR
781 LYLTVLYLVQ KNLIYVNSRY FLAFHCAERD EVLYNGET ID NNKEKGSEKD WKKFAKEFII
841 EHPPKKKVKD YLAKNFEYSN KWSLRVFRNS VQHLNVIRDA YKYIKCIDDN KDVQSYFALY
901 HYLVQRYISE MAENLTDKGE LSEGRLQYYL SQVENYRTYC KDFVKALNVP FAYNLPRYKN
961 LS IDELFDRN NYLPNKAKKW ISEKKENGEY VMEDCGNKGA GQVENA or a polypeptide with at least 80%, 82%, 84%, 85%. 87%. 89%. 90%. 92%. 94%. 95%, 97%, 98% or 99% amino acid sequence identity thereto.
An example of aCasl 3d from Hominimer dicola acetithat may be used (NCB1 Reference
Sequence: WP ..267301726.1 SEQ ID NO: 519) is:
1 MLSGIFVNAF S SKHGFESGV EINTSNPTHR SGES SSVRGD MLGLKSELEK RFFGKTFDDN
61 IHIQL IYNIL DIEKILAVYV TNIVYALNNM LGVKGSESYD DFMGYLSAQN TYYIFTHPDK 121 SNLSDKVKGN IKKSLSKFND LLKTKRLGYF GLEE PKTKDK RVSEAYKKRV YHMLAIVGQI 181 RQSVFHDKSN ELDEYLYSFI DI IDSEYRDT LDYLVDERFD S INKGFVQGN KVNISLLIDM 241 MKGYEADDII RLYYDFIVLK SQKNLGFS IK KLREKMLDEY GFRFKDKQYD SVRSKMYKLM 301 DFLLFCNYYR NDWAGEALV RKLRFSMTDD EKEGIYADEA EKLWGKFRND FENIADHMNG 361 DVIKELGKAD MDFDEKILDS EKKNASDLLY FSKMIYMLTY FLDGKEINDL LTTLISKFDN 421 IKEFLKIMKS SAVDVECELT AGYKLFNDSQ RITNELFIVK NIASMRKPAA SAKLTMFRDA 481 LTILGIDDKI TDDRISE ILK LKEKGKGIHG LRNFITNNVI ESSRFVYLIK YANAQKIREV 541 AKNEKWMFV LGGIPDTQIE RYYKSCVEFP DMNS SLEAKC SELARMIKNI SFDDFKNVKQ 601 QAKGRENVAK ERAKAVIGLY LTVMYLLVKN LVNVNARYVI AIHCLERDFG LYKEII PELA 661 SKNLKNDYRI LSQTLCELCD DRDES PNLFL KKNKRLRKCV EVDINNADSS MTRKYRNC IA 721 HLTWRELKE YIGDIRTVDS YFSIYHYVMQ RC ITKREDDT KQEEKIKYED DLLKNHGYTK 781 DFVKALNS PF GYNIPRFKNL S IEQLFDRNE YLTEK or a polypeptide with at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity7 thereto.
(CRISPR)/CRISPR-associated (Cas) systems
Genomic editing has been performed by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems (see e.g., Marraffini and Sontheimer. Nature Reviews Genetics 11: 181-190 (2010); Sorek et al. Nature Reviews Microbiology 2008 6: 181-6; Karginov and Hannon. Mol Cell 2010 1 :7-19; Hale et al. Mol Cell 2010:45:292-302; Jinek et al. Science 2012 337:815-820; Bikard and Marraffini Curr Opin Immunol 2012 24: 15-20; Bikard et al. Cell Host & Microbe 2012 12: 177-186; all of which are incorporated by reference herein in their entireties).
However, a CRISPR guide RNA system can be adapted for use in the methods and compositions described herein. Two RNAs can be used in CRISPR genomic editing systems: a CRISPR RNA (crRNA), which is a 17-20 nucleotide sequence complementary to the target RNA, and a trans-activating crRNA (tracrRNA) that is a binding scaffold for the Cas nuclease. In some cases the two RNAs are fused to make a single guide RNA (sgRNA). The tracrRNA forms a stem loop that is recognized and bound by the cas nuclease. The crRNA typically has shorter sequence than the tracrRNA. The term “guide RNA” as used herein refers to either a single guide RNA (sgRNA) or a crRNA. The CRISPR technique is generally described, for example, by Mali et al. Science 339:823-6 (2013); which is incorporated by reference herein in its entirety7. The guide RNA system used herein is encoded within or adjacent to the ncRNA coding region of the expression cassettes. Hence, upon transcription of the guide RNA, it can target a Cas enzyme to the desired location in the genome, where it can cleave the genomic RNA for generation of a genomic modification.
There are several types of CRISPR systems, some of which are summarized in the chart below.
CRISPR System Types Overview
Figure imgf000047_0001
A "guide RNA” or “gRNA” as provided herein refers to a ribonucleotide sequence capable of binding a cas nuclease, thereby forming ribonucleoprotein complex. The gRNA includes a nucleotide sequence complementary to a target site (e.g., near or at a genomic site to be edited). In some cases, the guide RNA includes one or more RNA molecules. TracrRNAs can be used to facilitate assembly of a ribonucleoprotein complex that includes the gRNA together with the tracrRNA and a cas nuclease. A complementary nucleotide sequence of the guide RNA can mediate binding of the ribonucleoprotein complex to the target site thereby providing the sequence specificity of the ribonucleoprotein complex. Thus, the guide RNA includes a sequence that is complementary to a target nucleic acid sequence such that the guide RNA binds a target nucleic acid sequence.
In some cases, the complement of the guide RNA includes a sequence having a sequence identity of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to a target nucleic acid (e.g., a target viral RNA sequence). In some cases, the guide RNA includes a sequence having sequence identity of about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to the target nucleic acid sequence. In some cases, the guide RNA or complement thereof, includes a sequence having a sequence identity of at least about 90%, 95%, or 100% to a target viral RNA sequence. In some cases, segment bound by a guide RNA within the target nucleic acid is about or at least about 10, 15, 20, 25, or more nucleotides in length.
The guide RNA is a single-stranded ribonucleic acid, although in some cases it may form some double-stranded regions by folding onto itself. In some cases, the guide RNA is about 10, 20. 30. 40. 50, 60, 70, 80, 90, 100 or more nucleic acid residues in length. In some cases, the guide RNA is from about 10 to about 30 nucleic acid residues in length. In some cases, the guide RNA is about 20 nucleic acid residues in length. For example, the length of the guide RNA can be at least about 5, 6, 7, 8, 9, 10, 11, 12. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. 24, 25, 26, 27, 28, 29, 30. 31. 32. 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46. 47. 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides or residues in length. In some cases, the guide RNA is from 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 5 to 75, 10 to 75, 15 to 75, 20 to 75, 25 to 75, 30 to 75, 35 to 75, 40 to 75, 45 to 75, 50 to 75, 55 to 75, 60 to 75, 65 to 75, 70 to 75, 5 to 100, 10 to 100, 15 to 100, 20 to 100, 25 to 100, 30 to 100, 35 to 100, 40 to 100, 45 to 100, 50 to 100, 55 to 100, 60 to 100, 65 to 100, 70 to 100, 75 to 100, 80 to 100, 85 to 100, 90 to 100, 95 to 100, or more nucleotides or residues in length. In some cases, the guide RNA is from 10 to 15, 10 to 20, 10 to 30. 10 to 40. or 10 to 50 residues in length.
Definitions
The term "about" as used herein when referring to a measurable value such as an amount, a length, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value.
"Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, bacterial, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.
The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the polynucleotide of interest is cloned and then expressed in transformed organisms, for example, as described herein. The host organism expresses the foreign nucleic acids to produce the RNA, RT- DNA, or protein under expression conditions.
As used herein, a "cell" refers to any type of cell isolated from a prokaryotic, eukaryotic, or archaeon organism, including bacteria, archaea, fungi, protists, plants, and animals, including cells from tissues, organs, and biopsies, as well as recombinant cells, cells from cell lines cultured in vitro, and cellular fragments, cell components, or organelles comprising nucleic acids. The term also encompasses artificial cells, such as nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids. The methods described herein can be performed, for example, on a sample comprising a single cell or a population of cells. The term also includes genetically modified cells.
"Recombinant host cells," "host cells", "cells", "cell lines", "cell cultures", and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.
A "coding sequence" or a sequence which "encodes" a selected polypeptide or a selected RNA, is a nucleic acid molecule which is transcribed (in the case of DNA templates) into RNA and/or translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, ncRNAs, tracrRNAs, ncRNAs modified to include heterologous sequences, cDNA from viral, prokaryotic or eukaryotic ncRNA, mRNA, viral or prokaryotic DNA. and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.
Typical "control elements," include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.
"Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper polymerases are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.
"Encoded by" refers to a nucleic acid sequence which codes for a polypeptide or RNA sequence. For example, the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. The RNA sequence or a portion thereof contains a nucleotide sequence of at least 3 to 5 nucleotides, more preferably at least 8 to 10 nucleotides, and even more preferably at least 15 to 20 nucleotides.
The terms "isolated." "purified." or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein, DNA, or RNA or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when obtained from nature or when produced by recombinant DNA techniques, or free from chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
"Substantially purified" generally refers to isolation of a substance (nucleic acid, compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically, in a sample, a substantially purified component comprises 50%, or 80%-85%, or 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to densify.
A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector construct." "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.
"Expression" refers to detectable production of a gene product by a cell. The gene product may be a transcription product (i.e., RNA), which may be referred to as "gene expression", or the gene product may be a translation product of the transcription product (i.e., a protein), depending on the context.
"Mammalian cell" refers to any cell derived from a mammalian subject suitable for transfection with vector systems comprising, as described herein. The cell may be xenogeneic, autologous, or allogeneic. The cell can be a primary’ cell obtained directly from a mammalian subject. The cell may also be a cell derived from the culture and expansion of a cell obtained from a mammalian subject. Immortalized cells are also included within this definition. In some embodiments, the cell has been genetically engineered to express a recombinant protein and/or nucleic acid.
The term "subject" includes animals, including both vertebrates and invertebrates, including, without limitation, invertebrates such as arthropods, mollusks, annelids, and cnidarians; and vertebrates such as amphibians, including frogs, salamanders, and caecillians; reptiles, including lizards, snakes, turtles, crocodiles, and alligators; fish; mammals, including human and non-human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows; and birds such as domestic, wild and game birds, including chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. In some cases, the disclosed methods find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; primates, and transgenic animals.
As used herein, the terms "treatment," "treating." and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
A “therapeutically effective amount” or “efficacious amount” refers to the amount of a compound that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the compound or the cell, the disease and its severity and the age, weight, etc., of the subject to be treated. Treatment includes the administration of antiviral, such as protease inhibitors (darunavir, atazanavir, and ritonavir), viral DNA polymerase inhibitors (acyclovir, valacyclovir, valganciclovir, and tenofovir), and/or an integrase inhibitor (raltegravir). Treatment also includes treatment for RSV, such as monoclonal antibodies (e.g., palivizumab) and/or ribavirin. Treatment can also include oxygen supplementation (e.g., oxygen given through a mask), suctioning of mucus, bronchodilator agents, and/or mechanical ventilation.
"Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of nonintegrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g.. episomes). or integration of transferred genetic material into the genomic DNA of host cells. Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non-viral vectors, alphaviruses, pox viruses and vaccinia viruses.
The term "derived from" is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.
A polynucleotide or nucleic acid "derived from" a designated sequence refers to a polynucleotide or nucleic acid that includes a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence. The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.
The terms "hybridize’' and "hybridization" refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson-Crick base pairing.
The term "homologous region" refers to a region of a nucleic acid with homology to another nucleic acid region. Thus, whether a "homologous region" is present in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since a nucleic acid is often double-stranded, the term "homologous, region." as used herein, refers to the ability of nucleic acid molecules to hybridize to each other. For example, a single-stranded nucleic acid molecule can have two homologous regions which are capable of hybridizing to each other. Thus, the term "homologous region" includes nucleic acid segments with complementary sequences. Homologous regions may vary in length but will typically be between 4 and 500 nucleotides (e.g., from about 4 to about 40, from about 40 to about 80, from about 80 to about 120, from about 120 to about 160, from about 160 to about 200, from about 200 to about 240, from about 240 to about 280, from about 280 to about 320, from about 320 to about 360, from about 360 to about 400. from about 400 to about 440, etc.).
As used herein, the terms "complementary" or "complementarity" refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when uracil is denoted in the context of the present invention, the ability’ to substitute a thymine is implied, unless otherwise stated. "Complementarity" may exist between two RNA strands, two DNA strands, or between an RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be "complementary" and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary" or " 100% complementary" if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered "perfectly complementary" or "100% complementary" even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity’ are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art.
The term "donor polynucleotide" or ’‘donor DNA” refers to a nucleic acid or polynucleotide that provides a nucleotide sequence of an intended edit to be integrated into the genome at a target locus by HDR or recombineering.
A "target site" or "target sequence" is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide (donor DNA). The target site may be allele-specific (e.g., a major or minor allele). For example, a target site can be a genomic site that is intended to be modified such as by insertion of one or more nucleotides, replacement of one or more nucleotides, deletion of one or more nucleotides, or a combination thereof.
In general, "a CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, and a CRISPR array nucleic acid sequence including a leader sequence and at least one repeat sequence. In some embodiments, one or more elements of a CRISPR system are derived from a type I, type II, or type III CRISPR system. Casl and Cas2 are found in all three types of CRISPR-Cas systems, and they are involved in spacer acquisition. In the I-E system of E. coli, Casl and Cas2 form a complex where a Cas2 dimer bridges two Casl dimers. In this complex Cas2 performs anon-enzymatic scaffolding role, binding double-stranded fragments of invading DNA, while Casl binds the single-stranded flanks of the DNA and catalyzes their integration into CRISPR array s.
In some embodiments, one or more elements of a CRISPR system are derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes . In general, a CRISPR system can be characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
In certain embodiments, the disclosure provides protospacers that are adjacent to short (3 - 5 bp) DNA sequences termed protospacer adjacent motifs (PAM). The PAMs are important for type I and type II systems during acquisition. In type I and type II systems, protospacers are excised at positions adjacent to a PAM sequence, with the other end of the spacer is cut using a ruler mechanism, thus maintaining the regularity of the spacer size in the CRISPR array. The conservation of the PAM sequence differs between CRISPR-Cas systems and may be evolutionarily linked to Casl and the leader sequence.
In some embodiments, a regulatory element is operably linked to one or more elements of a CRISPR system so as to drive expression of the one or more elements of the CRISPR system. In general. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), also known as SPIDRs (SPacer Interspersed Direct Repeats), constitute a family of DNA loci that are usually specific to a particular bacterial species. The CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. coli (Ishino et al, J. BacterioL. 169:5429-5433 (1987); and Nakata et al., J. BacterioL, 171 :3553-3556 (1989)), and associated genes. Similar interspersed SSRs have been identified in Haloferax mechterranei. Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol., 10: 1057-1065 (1993); Hoe el al., Emerg. Infect. Dis.. 5:254-263 (1999); Masepohl et al, Biochim. Biophys. Acta 1307:26-30 (1996); and Mojica et al, Mol. Microbiol, 17:85-93 (1995)). The CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al, OMICS J. Integ. Biol., 6:23-33 (2002); and Mojica et al, Mol. Microbiol., 36:244-246 (2000)). In general, the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al., (2000), supra). Although the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions ty pically differ from strain to strain (van Embden et al., J. Bacteriol., 182:2393- 2401 (2000)). CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al, Mol. Microbiol., 43: 1565-1575 (2002); and Mojica et al, (2005)) including, but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacteriumn, Methanococcus , Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, 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, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella. Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.
In some embodiments, an enzyme coding sequence encoding a CRISPR enzyme (e.g., cas9) is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog. or non-human primate. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about one or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database", and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available. In some embodiments, one or more codons (e.g., 1, 2, 3. 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.
"Administering" a nucleic acid, such as an expression cassette, comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.
The subject matter disclosed herein is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosed subject matter.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the disclosed subject matter, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the nucleic acid" includes reference to one or more nucleic acids and equivalents thereof know n to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of any features or elements described herein, which includes use of a "negative" limitation.
It is appreciated that certain features of the disclosed subject matter, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed subject matter, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the disclosed subject matter and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the disclosed subject matter is not entitled to antedate such publication. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently- confirmed.
The following Examples illustrate some of the materials, methods, and experiments that were used or performed in the development of the invention.
Examples
Example 1: Design of crRNAs
Methods
Design crRNAs targeting different genomic regions with: specific on-target binding to viral genomes no off-target binding (human or microbial (viruses, bacteria)) appropriate crRNA properties (structure. GC content, etc.)
Synthesize and test crRNAs for on-target and off-target activity:
Screen and validate for activity- against target RNA
Test cross-reactivity with human and nasal background (cellular RNA, nasal swabs)
Test combinations with maximal target detection and minimal background
Evaluate sensitivity in a single-step assay for direct detection of target
Determine limit of detection (LOD) Test performance in single step assay with contrived/clinical samples Virus genome databases
Public database (e.g., NCBI)
Casl3a guide design pipeline
Generate consensus sequence > design guides > filter for sensitivity, secondary structure, human transcriptome
Public code https : // github . com/ amandamok/ cas 13 a_guide_design
In Silico screening of Casl3a guides
Filter guides for cross-reactivity with host and other common microbes
Public code https : //github. com/ czbiohub/sc2-guide-InSilicoSCR#insili co-screening-for-sc2-guides
Example 2: Select crRNAs and test
6 crRNAs for RSV A and 9 crRNA for RSV B were selected (see SEQ ID NOs: 1-6 and 191-199).
Experimental plan
Test detection of RSV A and RSV B RNA
Validate guides
Test cross-reactivity against host background (RNA from cell lines)
Test cross-reactivity against nasal swabs
Test guide combinations to improve detection Resources viral RNA available from BEI resources and ATCC virus from Viratree
Example 3: Cas 13a detection of RSV
CRISPR RNA guides (crRNAs) were designed and validated for RSV A and RSV B. Ninety-six (96) crRNAs were designed for RSV A and ninety -nine (99) crRNAs were designed for RSV B. Each crRNA includes a crRNA stem that is derived from a bacterial sequence while the spacer sequence is derived from the RSV genome (reverse complement). See Tables 1-3 for exemplary crRNA and spacer sequences.
Example 4: Exemplary crRNA sequences GL2022-016-W02; BK-2023-052-2 // 3730.221WO1
Table 1
Figure imgf000059_0001
Figure imgf000060_0001
Table 2
Figure imgf000060_0002
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Table 3
Figure imgf000068_0002
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
GL2022-016-W02; BK-2023-052-2 // 3730.221WO1
Table 4
Figure imgf000076_0001
Table 5
Figure imgf000076_0002
Figure imgf000077_0001
Figure imgf000078_0001
Table 6
Figure imgf000078_0002
Figure imgf000079_0001
Example 5 crRNAs specific for RSV A or RSV B may be employed with a point of care (POC) device to provide an independent diagnostic for RSV A and RSV B viruses or one that is multiplexed diagnostic with, for example, SARS-CoV-2 and/or influenza viruses (e.g., SARS-CoV-2 +/- Influenza +/- RSV A and B). The POC diagnostic may include a microfluidics cartridge and a device that may include an insert for a swab, assay reagents, and/or a high intensity light source to excite and measure fluorescence. In one embodiment, the cartridge includes 3 reaction chambers, each measuring a specific target (e.g., viral RNA, host RNA, no RNA) from the same sample. Additional reaction chambers may measure multiple targets within a single sample. This enables an at-home diagnostic and may be used in a droplet-based assay or other embodiment of direct detection.
In one embodiment, a bead comprises Casl3, one or more crRNAs and one or more reagents for a CRSPR/Cas assay. Casl3, one or more crRNAs and/or one or more reagents maybe on the surface of the bead, embedded within the bead, or both. The bead may include lyophilized Casl3, one or more crRNAs and/or one or more reagents.
In one embodiment, the assay is a droplet assay.
In one embodiment, the assay is a digital PCR assay.
In one embodiment, the reporter is a luminescent reporter.
In one embodiment, the reporter comprises a fluorophore.
In one embodiment, the reporter comprises a quencher.
In one embodiment, the reporter comprises a fluorophore and a quencher.
In one embodiment, the reporter comprises a caged compound.
In one embodiment, the reporter comprises a metal, e.g., a gold particle.
In one embodiment, the assay detects fluorescence (a fluorescent signal).
In one embodiment, the assay detects luminescence (a luminescent signal). For example, luminescence may be detected by employing via a split luciferase system that comes together following RNA reporter cleavage by Cast 3a.
In one embodiment, the assay detects an electrochemical signal.
In one embodiment, the assay is PCR-based assay, which employs a reporter that may include hairpin structure such as a molecular beacon where cleavage opens the hairpin to reveal a longer template.
Example 6
Multiplexing CRISPRCasl3a for an at-home viral diagnostic
A) CRISPR-Casl3a reactions were performed to evaluate the on-target activity of the designed and synthesized crRNAs targeting RSV A and RSV B. Specifically, each crRNA was complexed with a Casl3a, and the complex was then combined with either target RNA (synthetic full length viral RNA) or RNP control (no target RNA) (n=3 or 4 technical replicates per sample). The dynamics of the fluorescence emitted in the reaction w as monitored by measuring it on a Tecan plate reader over 2 hours. The slope of the signal was calculated, as well as the slope ratio (target divided by control) to identify crRNAs hits with high signal. Signal slope and slope ratios are represented in Figures 12A-12B and 13A-13B.
On-target activity screen for was performed for crRNAs targeting RSV A and B viruses. The screen results are summarized in the graphs in Figures 12 and 13. A set of top hits were identified for validation of their on-target activity and evaluation of their off-target activity. The hits (~30 crRNAs with high on-target activity for RSV A and ~40 crRNAs with high on-target activity for RSV B) are highlighted for each of the viruses in Figures 12A-12B and 13A-13B.
B) CRISPR-Casl3a reactions were performed to evaluate the off-target activity of crRNA hits. Specifically, each crRNA was complexed with Casl3a and the complex was combined with either off-target RNA (extracted from cells), nasal swabs (n=3 to 7 individual swab samples) or control (no target RNA or nasal swab) (n=3 or 4 technical replicates per reaction). The dynamics of the fluorescence emitted in the reaction was monitored by measuring it on a Tecan plate reader over 2 hours. The slope of the signal as well as the slope ratio (target divided by control) was calculated to identify crRNAs hits with high off-target signal. Signal slope ratios and the screen results are represented/summarized in the graphs in Figures 14 -17. Off-target activity tests, using cell line RNA and nasal swabs, were completed for crRNAs hits for RSV A, and RSV B viruses. The screen results are summarized in the graphs in Figure 14-17.
C) Off-target activity tests, using non-target viral RNA, for crRNAs hits for influenza B were performed. CRISPR-Casl3a reactions were performed to evaluate the off-target activity’ of crRNA hits. Specifically, each crRNA was complexed with Cast 3a and the complex was combined with either off-target RNA (viral RNA from viruses that are not targeted) or control (no target RNA) (n=3 technical replicates per reaction). The dynamics of the fluorescence emitted in the reaction was monitored by measuring it on a Tecan plate reader over 2 hours. The slope of the signal as well as the slope ratio (target divided by control) was calculated to identify crRNAs hits with high off-target signal. Signal slope ratios are represented in the graphs in Figures 18 and 19.
D) Experiments to evaluate crRNA combinations for and RSV A were conducted. CR1SPR-Casl3a reactions to evaluate crRNA combinations were performed. Specifically, the indicated crRNAs were combined, complexed each combination with Cast 3a, and combined the complex with either on-target RNA (synthetic or extracted viral RNA from the target virus) or control (no target RNA) (n=3 technical replicates per reaction). The dynamics of the fluorescence emitted in the reaction were monitored by measuring it on a Tecan plate reader over 2 hours. The slope of the signal, as well as the slope ratio (target divided by control) was calculated to identify crRNA combinations with the highest detection. Signal slope ratios are represented in the graphs in Figures 20 and 21.
E) crRNA combinations for RSV A and B viruses were evaluated.
CRISPR-Casl3a reactions to evaluate crRNA combinations were performed. Specifically, the indicated crRNAs were combined, complexed each combination with Cast 3a, and combined the complex with either on-target RNA (synthetic or extracted viral RNA from the target virus) or control (no target RNA) (n=3 technical replicates per reaction). The dynamics of the fluorescence emitted in the reaction were monitored by measuring it on a Tecan plate reader over 2 hours. The slope of the signal as well as the slope ratio (target divided by control) were calculated to identify crRNA combinations with the highest detection. Signal slope ratios are represented in the graphs in Figures 22 and 23. Most crRNAs have minimal off-target activity against non-targeted viruses.
F) CRISPR-Cas 13a reactions were performed to evaluate the LOD of virus plaque forming units (pfu, infectious particles) with crRNA combinations Specifically , the indicated crRNAs were combined, each combination complexed with Cast 3a, and combined the complex with either on- target virus (lysed with our lysis buffer) or control (no virus, lysis buffer only) (n=6 technical replicates per reaction). The dynamics of the fluorescence emitted in the reaction was monitored by measuring it on a Tecan plate reader over 2 hours. The slope of the signal and compare it to the control was calculated to identify crRNA combinations with significant detection of a given pfu input. Signal slope is represented in the graphs in Figures 24A-24B and 25A-25B, along with statistical analyses.
G) crRNA combinations were evaluated for RSV A and B viruses. In addition, an in-silico crRNA screen was performed to determine which crRNAs target which subtype or strain of RSV A and B viruses and to analyze off-targets of the crRNAs.
An inclusivity test was performed where published RSV A and B virus sequences were analyzed and compared to guide sequences to demonstrate that the assay design supports detection of all strains and subtypes (NCBI). Sequences for 1,309 complete genomes of RSV A and 807 complete genomes of RSV B deposited in NCBI Virus whose collection dates were between 2000 and 2021 were downloaded and analyzed. The percentage of genomes that contain an exact match to the guides are shown below, results by subtype are only shown for those subtypes that had at least 10 genomes in the database.
Further, microbial interference tests were performed where the genomes of all viruses, other respiratory pathogens and normal nasal flora organisms were analyzed to demonstrate less than 80% homology with guide target sequences. The number of hits w ere reported with different lowest-common ancestor (LCA) levels. If a guide is homologous to genomes from multiple species, the LCA of those genomes was reported. However, if a guide is homologous exclusively to only one species, then the LCA of that guide is that species.
In Silico Guide Screening Report for RSV A
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In Silico Guide Screening Report for RSV B
Figure imgf000087_0001
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All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.
The following statements provide a summary of some aspects of the inventive nucleic acids and methods described herein.
Statements:
1. A method comprising:
(a) incubating a sample suspected of containing RSV A or RSV B RNA or virus with one or more Casl3 protein, at least one CRISPR guide RNA (crRNA), and at least one reporter RNA for a period of time sufficient to form at least one RNA cleavage product; and
(b) detecting reporter RNA cleavage product(s) with a detector.
2. The method of statement 1, wherein the at least one CRISPR guide RNA (crRNA) binds a target site in at least one of an RSV A or RSV B nucleic acid.
3. The method of statement 1 or 2, wherein one or more of the Casl3 proteins has a protein sequence with at least 95% sequence identity to any of SEQ ID NOs: 500-519.
4. The method of any one of statements 1-3, wherein one or more of the Casl3 proteins has any one SEQ ID NOs: 500-519.
5. The method of any one of statements 1 or 2, wherein the RSV A RNA is from a variant of RSV A.
6. The method of any one of statements 1-5, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 1- 6, 10-199, 201-206, or 210-399. 7. The method of any one of statements 1-6, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 2, 4, 6, 191, 192, 196 or 199.
8. The method of any one of statements 1 -7, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 2, 4, or 6, or a combination thereof.
9. The method of any one of statements 1-8, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 2, 4. or 6, or a combination thereof.
10. The method of statement 9, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs:2, 4 and 6.
11. The method of any one of statements 1-10, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 2, 4, 6, 191, 192, 196, 199. or a combination thereof.
12. The method of statement 1, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 191, 192, 196, 199, or a combination thereof.
13. The method of statement 12, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 191, 192, 196 and 199.
14. The method of any one of statements 1, 2, 5-13, wherein one or more of the Casl3 protein is a Casl3a or Casl3b protein.
15. The method of statement 1, wherein the at least one CRISPR guide RNA (crRNA) is two or more CRISPR guide RNAs (crRNAs).
16. The method of statement 1, wherein the Casl3 protein is complexed with the at least one CRISPR guide RNA (crRNA) prior to incubation with the sample suspected of containing the target viral RNA.
17. The method of statement 16, wherein the one or more of the Cast 3 proteins is complexed with the at least one CRISPR guide RNA (crRNA) and prepared as a lyophilized bead.
18. The method of statement 1, wherein the sample suspected of containing the target viral RNA is saliva, sputum, mucus, nasophary ngeal materials, blood, serum, plasma, urine, aspirate, biopsy tissue, or a combination thereof. 19. The method of statement 1, wherein the sample suspected of containing RNA is a lysed biological sample.
20. The method of statement 1 , wherein cleavage of the reporter RNA produces a light signal, an electronic signal, an electrochemical signal, an electrostatic signal, a steric signal, a van der Waals interaction signal, a hydration signal, a Resonant frequency shift signal, or a combination thereof.
21. The method of statement 1, wherein the reporter RNA reporter comprises at least one fluorophore and at least one fluorescence quencher.
22. The method of statement 21, wherein the at least one fluorophore is Alexa 430, STAR 520, Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof.
23. The method of any of statements 1, 21, or 22, wherein the detector comprises a light detector, a fluorescence detector, a color filter, an electronic detector, an electrochemical signal detector, an electrostatic signal detector, a steric signal detector, a van der Waals interaction signal detector, a hydration signal detector, a Resonant frequency shift signal detector, or a combination.
24. The method of statement 1, wherein the target viral RNA is detected when a signal from the reporter RNA cleavage product(s) is distinguishable from a control assay signal.
25. The method of statement 24, wherein the control assay contains no target viral RNA.
26. The method of statement 24, wherein the control assay contains viral RNA that is not the target viral RNA.
27. The method of statement 1, wherein the sample further comprises at least one RNA from a common cold coronavirus, SARS-CoV2, hepatitis virus, or human immunodeficiency virus (HIV).
28. The method of statement 27, wherein the common cold coronavirus is at least one of strain NL63, OC43, or 229E.
29. The method of statement 27, wherein the hepatitis virus is hepatitis C virus (HCV).
30. The method of any one of statements 27-29, wherein at least one CRISPR guide RNAs can bind to at least one RNA from the common cold coronavirus, SARS-CoV-2, hepatitis virus, or human immunodeficiency virus (HIV). 31. A method comprising treating a subject with detectable RSV A or RSV B infection detected by the method of any of statements 1-26.
32. A kit comprising a package containing at least one Casl3 protein, at least one CR1SPR guide RNA (crRNA) that binds a target site in at least one of an RSV A or RSV B nucleic acid, at least one reporter RNA, and instructions for detecting and/or quantifying the target viral RNA in a sample.
33. The kit of statement 32, wherein the at least one CRISPR guide RNA (crRNA) has a sequence with at least 95% sequence identity to any of SEQ ID NO: 1-6, 10-199, 201-206, or 210-399.
34. The kit of any one of statements 32 or 33, wherein at least one of the CRISPR guide RNAs (crRNAs) has a sequence of any of SEQ ID NOs: 1 -6, 10-199, 201-206, or 210-399.
35. The kit of any one of statements 32-34, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identify' to any of SEQ ID NOs: 2. 4, or 6, or a combination thereof.
36. The kit of any one of statements 32-35, wherein the at least one CRISPR guide RNA (crRNA) has any of SEQ ID NOs:2, 4, or 6. or a combination thereof.
37. The kit of statement 32, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 2. 4 and 6.
38. The kit of statement 32, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 191, 192, 196, or 199, or a combination thereof.
39. The kit of statement 32, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 191, 192, 196 or 199. or a combination thereof.
40. The kit of statement 28, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 191, 192, 196 and 199.
41. The kit of any one of statements 32-40, wherein the at least one CRISPR guide RNA (crRNA) is two or more CRISPR guide RNAs (crRNAs).
42. The kit of any one of statements 32-41, wherein the Casl3 protein is complexed with the at least one CRISPR guide RNA (crRNA). 43. The kit of any one of statements 32-42, wherein the one or more of the Cas 13 proteins is complexed with the at least one CRISPR guide RNA (crRNA) and prepared as a lyophilized bead.
44. The kit of any one of statements 32, 42, or 43, wherein the Casl3 protein is a Casl3a or Cas 13b protein.
45. The kit of statement 32, wherein the reporter RNA reporter comprises at least one fluorophore and at least one fluorescence quencher.
46. The kit of statement 45, wherein the at least one fluorophore is Alexa 430, STAR 520.
Brilliant Violet 510, Brilliant Violet 605, Brilliant Violet 610, or a combination thereof. 47. The kit of any one of statements 32 or 43, further comprising a sample chamber, assay mixture reaction chamber, or a combination thereof.
48. The kit of statement 43, wherein the lyophilized bead is included in the assay mixture reaction chamber.
49. The kit of statement 32, further comprising a detector. 50. An isolated nucleic acid comprising, consisting of or consisting essentially of any of
SEQ ID NOs: 1-6 or 10-199, or 210-389.

Claims

WHAT IS CLAIMED IS:
1. A method comprising:
(a) incubating a sample suspected of containing a target viral RNA with one or more Cast 3 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 reporter RNA cleavage product(s) with a detector, wherein the target viral RNA is RSV A or RSV B RNA or viral RNA.
2. The method of claim 1, wherein the sample comprises RNA from a variant of RSV A.
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 of SEQ ID NOs: 1-6, 10-199, 201- 206, or 210-399.
4. The method of claim 1, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 1-6, 10-199, 201-206, or 210-399.
5. The method of any one of claims 1 to 4, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 2, 4 or 6, or a combination thereof.
6. The method of any one of claims 1 to 4, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 2, 4 or 6, 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: 2, 4 and 6.
8. The method of any one of claims 1 to 7, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 191, 192, 196 or 199, or a combination thereof.
9. The method of any one of claims 1 to 7, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 191, 192, 196 or 199, 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: 191, 192, 196 and 199.
11. The method of any one of claims 1 to 10, wherein the one or more of the Cas 13 proteins is a Casl3a or Casl3b protein.
12. The method of any one of claims 1 to 6, 8 to 9 or 11 , wherein the at least one CRISPR guide RNA (crRNA) is two or more CRISPR guide RNAs (crRNAs).
13. The method of any one of claims 1 to 6, 8 to 9 or 11, wherein the one or more Casl3 proteins is complexed with the at least one CRISPR guide RNA (crRNA) prior to incubation with the sample suspected of containing the target viral RNA.
14. The method of claim 13. wherein the one or more of the Cas 13 proteins is complexed with the at least one CRISPR guide RNA (crRNA) and prepared as a lyophilized bead.
15. The method of any one of claims 1 to 14, wherein the sample suspected of containing the target viral RNA is saliva, sputum, mucus, nasopharyngeal materials, blood, serum, plasma, urine, aspirate, biopsy tissue, or a combination thereof.
16. The method of any one of claims 1 to 15, wherein the sample suspected of containing the target viral RNA is a lysed biological sample.
17. The method of any one of claims 1 to 16, wherein cleavage of the at least one reporter RNA produces a light signal, an electronic signal, an electrochemical signal, an electrostatic signal, a steric signal, a van der Waals interaction signal, a hydration signal, a Resonant frequency shift signal, or a combination thereof.
18. The method of any one of claims 1 to 17, wherein the at least one reporter RNA cleavage product 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 any one of claims 1 to 19, wherein the detector comprises a light detector, a fluorescence detector, a color filter, an electronic detector, an electrochemical signal detector, an electrostatic signal detector, a steric signal detector, a van der Waals interaction signal detector, a hydration signal detector, a Resonant frequency shift signal detector, or a combination.
21. The method of any one of claims 1 to 20, wherein the target viral RNA is detected when a signal from the at least one reporter RNA cleavage product(s) is distinguishable from a control assay signal.
22. The method of claim 21. wherein the control assay contains no target viral RNA.
23. The method of claim 21. wherein the control assay contains viral RNA that is not the target viral RNA.
24. The method of any one of claims 1 to 23, wherein the sample further comprises at least one RNA from a common cold coronavirus, SARS-CoV-2, hepatitis virus, or human immunodeficiency virus (HIV).
25. The method of claim 24. wherein the common cold coronavirus is at least one of strain 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 the at least one RNA from the common cold coronavirus, SARS-CoV-2, hepatitis virus, or human immunodeficiency virus (HIV).
28. A method comprising treating a subject with detectable RSV A or RSV B infection detected by the method of any one of claims 1 to 27.
29. A kit comprising a package containing at least one Casl3 protein, at least one CRISPR guide RNA (crRNA) that binds a target site in a target viral RNA in at least one of an RSV A or RSV B nucleic acid, at least one reporter RNA, and instructions for detecting and/or quantifying the target viral RNA in a sample.
30. The kit of claim 29, wherein the at least one CRISPR guide RNA (crRNA) has a sequence with at least 95% sequence identity to any of SEQ ID NO: 1-6, 10-199, 201-206, or 210-399.
31. The kit of claim 29, wherein at least one of the CRISPR guide RNAs (crRNAs) has a sequence of any of SEQ ID NOs: 1-6, 10-199, 201-206, or 210-399.
32. The kit of claim 29, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 2. 4 or 6, or a combination thereof.
33. The kit of claim 29, wherein the at least one CRISPR guide RNA (crRNA) has any of SEQ ID NOs: 2, 4 or 6, or a combination thereof.
34. The kit of claim 33, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 2. 4 and 6.
35. The kit of claim 29, wherein the at least one CRISPR guide RNA (crRNA) has a sequence segment with at least 95% sequence identity to any of SEQ ID NOs: 191, 192, 196 or 199, or a combination thereof.
36. The kit of claim 29, wherein the at least one CRISPR guide RNA (crRNA) has a sequence of any of SEQ ID NOs: 191, 192, 196 or 199, or a combination thereof.
37. The kit of claim 36, wherein the at least one CRISPR guide RNA (crRNA) is a combination of SEQ ID NOs: 191. 192, 196 and 199.
38. The kit of any one of claims 29 to 37. wherein the at least one CRISPR guide RNA (crRNA) is two or more CRISPR guide RNAs (crRNAs).
39. The kit of any one of claims 29 to 38, further comprising at least one CRISPR guide RNA (crRNA) that binds RNA of a common cold coronavirus, SARS-CoV-2, a hepatitis virus, or a human immunodeficiency virus (HIV).
40. The kit of any one of claims 29 to 39, wherein the at least one Cas 13 protein is complexed with the at least one CRISPR guide RNA (crRNA).
41 . The kit of any one of claims 29 to 40, wherein the at least one Cas 13 protein is complexed with the at least one CRISPR guide RNA (crRNA) and prepared as a lyophilized bead.
42. The kit of any one of claims 29 to 41, wherein the at least one Casl3 protein is a Casl3a or Cas 13b protein.
43. The kit of any one of claims 29 to 42, wherein the reporter RNA reporter comprises at least one fluorophore and at least one fluorescence quencher.
44. The kit of claim 43, 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 44, further comprising a sample chamber, assay mixture reaction chamber, or a combination thereof.
46. The kit of claim 45, wherein a lyophilized bead is included in the assay mixture reaction chamber.
47. The kit of any one of claims 29 to 46, further comprising a detector.
48. An isolated nucleic acid comprising any of SEQ ID NOs: 1-6 or 10-199, or 210-389.
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