Granted Patent B2

US 12,203,145 · App. 16/494,279 · Granted Jan 21, 2025

Inventors: Omar Abudayyeh (Cambridge, MA); James Joseph Collins (Cambridge, MA); Jonathan Gootenberg (Cambridge, MA); Feng Zhang (Cambridge, MA); Eric S. Lander (Cambridge, MA); Pardis Sabeti (Cambridge, MA); Catherine Amanda Freije (Cambridge, MA); Cameron Myhrvold (Cambridge, MA)

Assignees: THE BROAD INSTITUTE, INC.; MASSACHUSETTS INSTITUTE OF TECHNOLOGY; PRESIDENT AND FELLOWS OF HARVARD COLLEGE

C12Q1/70C12N15/115C12Q1/6851C12N2310/16C12N2310/20C12Q2521/301

Abstract

Claims (43)

1. A method for detecting viruses in samples, comprising:

distributing a sample or set of samples into one or more individual discrete volumes, the individual discrete volumes comprising a nucleic acid detection system comprising: a Type VI Cas effector protein having collateral cleavage activity and one or more guide RNAs capable of forming a complex with the Type VI Cas effector protein and directing sequence-specific binding of the complex to one or more corresponding virus polynucleotides in the sample or set of samples; and an RNA-based masking construct comprising a non-target RNA sequence;

incubating the sample or set of samples under conditions sufficient to allow binding of the one or more guide RNAs to one or more virus target polynucleotides;

activating the Type VI Cas effector protein via binding of the one or more guide RNAs to the one or more virus target polynucleotides, wherein activating the Type VI Cas effector protein results in collateral cleavage of the non-target RNA sequence of the RNA-based masking construct, such that a detectable signal is generated; and

detecting the detectable signal, wherein detection of the detectable signal indicates a presence of one or more viruses in the sample,

wherein, prior to incubating the sample or set of samples, the method further comprises: conducting a nuclease inactivation step and a viral inactivation step on the sample or the set of samples, wherein the sample(s) is/are a crude sample(s) and/or wherein the virus target polynucleotides are not extracted or isolated from the sample(s) after the inactivation steps, and

wherein, prior to conducting a nuclease inactivation step and a viral inactivation step on the sample or the set of samples, or prior to distributing a sample or set of samples into one or more individual discrete volumes, the method further comprises amplifying the virus target polynucleotides in the sample.

2. The method of claim 1 , wherein the sample comprises two or more viruses, and wherein the method distinguishes between the two or more viruses.

3. The method of claim 1 , wherein the guide RNAs detect single nucleotide variants of the one or more viruses; and wherein the guide RNAs optionally further comprise one or more synthetic mismatches.

4. The method of claim 1 , wherein the one or more guide RNAs comprise a pan-viral guide RNA set that detects each virus and/or viral strain in a set of viruses; and wherein the guide RNAs are optionally derived using a set cover approach.

5. The method of claim 1 , wherein the nucleic acid detection system is on a substrate, and wherein the substrate is exposed to the sample; optionally wherein the substrate is a flexible materials substrate; optionally a paper substrate, a fabric substrate, or a flexible polymer-based substrate.

6. The method of claim 5 , wherein the nucleic acid detection system or a different nucleic acid detection system is applied to multiple discrete locations on the substrate.

7. The method of claim 6 , wherein the different nucleic acid detection system detects a different virus at each location.

8. The method of claim 5 , wherein the substrate is exposed to the sample passively, by temporarily immersing the substrate in a fluid to be sampled, by applying a fluid to be tested to the substrate, or by contacting a surface to be tested with the substrate.

9. The method of claim 8 , wherein the sample is a biological or environmental sample.

10. The method of claim 9 , wherein the environmental sample is obtained from a food sample, a beverage sample, a paper surface, a fabric surface, a metal surface, a wood surface, a plastic surface, a soil sample, a freshwater sample, a wastewater sample, a saline water sample, exposure to atmospheric air or other gas sample, or a combination thereof.

11. The method of claim 9 , wherein the biological sample is obtained from a tissue sample, saliva, blood, plasma, sera, stool, urine, sputum, mucous, lymph, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, seroma, pus, or a swab of a skin or a mucosal membrane surface.

12. The method of claim 9 , wherein the one or more virus target polynucleotides are not amplified from the sample prior to the inactivation steps.

13. The method of claim 1 , wherein the virus is a DNA virus comprising one or more viral DNAs, wherein the nucleic acid detection system further comprises a primer that can anneal to a target nucleotide sequence of the one or more viral DNAs and wherein the primer further comprises a nucleotide sequence of an RNA polymerase promoter, such that the viral target nucleotide sequences are viral target ribonucleotide sequences generated by transcription of the viral DNAs, or such that amplifying the viral target nucleotide sequences in the sample comprises amplifying the viral DNAs via the primer, such that the amplified viral target nucleotide sequences are amplified viral target ribonucleotide sequences generated by transcription of the amplified viral DNAs.

14. The method of claim 13 , wherein the DNA virus is a Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae (including human herpes virus, and Varicella Zozter virus), Malocoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae, Baculoviridae, Cicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Maseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae, Poxviridae, Sphaerolipoviridae, Tectiviridae, Turriviridae, Dinodnavirus, Salterprovirus, Rhizidovirus, or combination thereof.

15. The method of claim 13 , wherein the virus is a DNA virus comprising one or more viral DNAs, wherein amplifying the viral target nucleotide sequences in the sample comprises amplifying the viral DNAs via a primer that binds the viral DNAs and comprises a nucleotide sequence of an RNA polymerase promoter, such that the amplified viral target molecules are amplified viral target RNAs generated by transcription of the amplified viral DNAs.

16. The method of claim 1 , wherein the one or more viruses is a double-stranded RNA virus, a positive sense RNA virus, a negative sense RNA virus, a retrovirus, or a combination thereof.

17. The method of claim 16 , wherein the virus is a Coronaviridae virus, a Picornaviridae virus, a Caliciviridae virus, a Flaviviridae virus, a Togaviridae virus, a Bornaviridae, a Filoviridae, a Paramyxoviridae, a Pneumoviridae, a Rhabdoviridae, an Arenaviridae, a Bunyaviridae, an Orthomyxoviridae, or a Deltavirus; optionally Coronavirus, SARS, Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus, Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Zika virus, Rubella virus, Ross River virus, Sindbis virus, Chikungunya virus, Borna disease virus, Ebola virus, Marburg virus, Measles virus, Mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, Human respiratory syncytial virus, Rabies virus, Lassa virus, Hantavirus, Crimean-Congo hemorrhagic fever virus, Influenza, or Hepatitis D virus.

18. The method of claim 1 , wherein the sample is urine, sera, blood, or saliva.

19. The method of claim 1 , wherein the sample volume required for detection is 100 μl to 1 μl.

20. The method of claim 1 , wherein the virus is Zika virus.

21. The method of claim 1 , wherein the RNA-based masking construct comprises an RNA oligonucleotide to which a detectable ligand and a masking component are attached.

22. The method of claim 1 , wherein the nuclease inactivation step is carried out at a temperature ranging from 37° C. to 50° C. and is of a duration selected from 5 minutes, 10 minutes, 15 minutes, and 20 minutes.

23. The method of claim 1 , wherein the viral inactivation step is carried out at a temperature ranging from 64° C. to 95° C. and has a duration of 5 minutes.

24. The method of claim 1 , wherein the nucleic acid detection system further comprises isothermal nucleic acid amplification reagents comprising recombinase polymerase amplification (RPA) reagents, nucleic-acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) reagents, strand displacement amplification (SDA), helicase-dependent amplification (HDA), multiple displacement amplification (MDA), rolling circle amplification (RCA), ligase chain reaction (LCR), or ramification amplification (RAM).

25. The method of claim 1 , wherein the virus is an RNA virus comprising one or more viral RNAs, wherein amplifying the viral target polynucleotides in the sample comprises converting the viral RNAs into viral cDNAs via a reverse transcriptase, such that amplifying the viral target polynucleotides in the sample comprises amplifying the viral cDNAs in the presence of a primer that can anneal to the viral target polynucleotides and comprises a nucleotide sequence of an RNA polymerase promoter, such that the amplified viral target polynucleotides are amplified viral target RNAs generated by transcription of the amplified viral cDNAs.

26. The method of claim 1 , wherein the virus is an RNA virus comprising one or more viral RNAs, wherein amplifying the viral target molecules in the sample comprises converting the viral RNAs into viral cDNAs via reverse transcriptase and amplifying the viral cDNAs via a primer that binds the amplified viral cDNAs and comprises an RNA polymerase promoter, such that the amplified viral target molecules are amplified viral target RNAs generated by transcription of the amplified viral cDNAs.

27. A method for detecting one or more viruses in a sample, comprising:

conducting a nuclease inactivation step and a viral inactivation step on a sample, wherein the sample is a crude sample and/or wherein viral target polynucleotides are not extracted or isolated from the sample after the inactivation steps;

adding a nucleic acid detection system to the sample, the nucleic acid detection system comprising a Type VI Cas effector protein having collateral cleavage activity;

at least one guide RNA comprising a guide sequence capable of forming a complex with the Type VI Cas effector protein and directing sequence-specific binding to one or more viral target polynucleotides in the sample; and

an RNA-based masking construct comprising a non-target RNA sequence, wherein Type VI Cas effector protein collateral activity cleaves the non-target RNA sequence;

applying the sample to a lateral flow immunochromatographic assay; and

detecting a signal generated from cleavage of the non-target RNA sequence of the RNA-based masking construct, thereby detecting one or more viruses that are present in the sample,

wherein said RNA-based masking construct comprises a first and a second molecule separated by the non-target RNA sequence, wherein said lateral flow immunochromatographic assay comprises detecting said first and second molecule at discrete detection sites on a lateral flow strip, wherein uncleaved RNA-based masking construct is bound by said first molecule at a first discrete detection site if the target nucleic acid is not present in said sample, and wherein cleaved RNA-based masking construct is bound by said first molecule at the first discrete detection site and by said second molecule at a second discrete detection site if the target nucleic acid is present in said sample.

28. The method according to claim 27 , wherein said first molecule and said second molecule are detected by binding to an antibody recognizing said first or second molecule and detecting said bound molecule, preferably with sandwich antibodies.

29. The method according to claim 27 , wherein said lateral flow strip comprises an upstream first antibody directed against said first molecule, and a downstream second antibody directed against said second molecule, and wherein uncleaved RNA-based masking construct is bound by said first antibody if the target nucleic acid is not present in said sample, and wherein cleaved RNA-based masking construct is bound both by said first antibody and said second antibody if the target nucleic acid is present in said sample.

30. The method of claim 27 , wherein, prior to conducting a nuclease inactivation step and a viral inactivation step on a sample or prior to adding a nucleic acid detection system to the sample, the method further comprises: amplifying the virus target polynucleotides in the sample.

Assignments (8)

Continuity (7)

References Cited (252)