CRISPR effector system based diagnostics for virus detection

Patent metadata

Field	Value
Publication number	US-12203145-B2
Application number	US-201816494279-A
Country	US
Kind code	B2
Filing date	Mar 15, 2018
Priority date	Mar 15, 2017
Publication date	Jan 21, 2025
Grant date	Jan 21, 2025

Abstract

Provided herein is a nucleic acid detection system comprising: a CRISPR system comprising an effector protein and one or more guide RNAs designed to bind to corresponding target molecules; an RNA-based masking construct; and optionally, nucleic acid amplification reagents to amplify target RNA molecules in a sample. In another aspect, the embodiments provide a polypeptide detection system comprising: a CRISPR system comprising an effector protein and one or more guide RNAs designed to bind a trigger RNA, an RNA-based masking construct; and one or more detection aptamers comprising a masked RNA polymerase promoter binding site or a masked primer binding site. In some embodiments, the system may be used to detect viruses in samples.

First claim

What is claimed is:

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 de

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College

Inventors

• Abudayyeh Omar
• Collins James Joseph
• Gootenberg Jonathan
• Zhang Feng
• Lander Eric S
• Sabeti Pardis
• Freije Catherine Amanda
• Myhrvold Cameron

Classifications

• C12Q2521/301

  Endonuclease · CPC title

• C12Q1/6851

  Quantitative amplification · CPC title

• C12N2310/16

  Aptamers · CPC title

• C12N15/115

  Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title