Methods and compositions for detecting a target RNA

Patent metadata

Field	Value
Publication number	US-10494664-B2
Application number	US-201815920222-A
Country	US
Kind code	B2
Filing date	Mar 13, 2018
Priority date	Jun 16, 2016
Publication date	Dec 3, 2019
Grant date	Dec 3, 2019

Abstract

The present disclosure provides methods for detecting a single-stranded target RNA. The present disclosure provides methods of cleaving a precursor C2c2 guide RNA array into two or more C2c2 guide RNAs. The present disclosure provides a kit for detecting a target RNA in a sample.

First claim

What is claimed is:

1. A composition for detecting a target RNA in a sample comprising a plurality of different RNAs, the composition comprising:
(a) a first labeled detector RNA comprising at least one uracil;
(b) a second labeled detector RNA lacking uracil; and
(c) a C2c2 protein, or a nucleic acid encoding the C2c2 protein, wherein the C2c2 protein cleaves the first labeled detector RNA and does not cleave the second labeled detector RNA, and wherein the C2c2 protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:6.

2. The composition of claim 1 , comprising a positive control target RNA.

3. The composition of claim 2 , wherein the positive control target RNA is viral, fungal, bacterial, or archaebacterial RNA.

4. The composition of claim 2 , wherein the positive control target RNA is Candida albicans RNA.

5. The composition of claim 2 , wherein the positive control target RNA is Plasmodium parasite, Plasmodium falciparum, Plasmodium vivax , a Toxoplasma parasite, Toxoplasma gondii, Trypanosoma cruzi, Trichomonas spp., or Giardia spp. RNA.

6. The composition of claim 2 , wherein the positive control target RNA is Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Streptococcus pyogenes, Mycobacterium tuberculosis, Streptococcus spp., Streptococcus agalactiae , methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Escherichia coli, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans , a Lyme disease spirochete, Pseudomonas aeruginosa, Mycobacterium leprae , or Brucella abortus RNA.

7. The composition of claim 2 , wherein the positive control target RNA is Hepatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Herpes virus, Herpes simplex virus 1, Herpes simplex virus 2, human papillomavirus, human immunodeficiency virus, or Zika virus RNA.

8. The composition of claim 1 , wherein the first labeled detector RNA comprises a color-based detection label.

9. The composition of claim 1 , wherein the first labeled detector RNA comprises a label for visual based detection, sensor based detection, color detection, gold nanoparticle based detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, or semiconductor-based sensing.

10. The composition of claim 1 , wherein the first labeled detector RNA comprises a fluorescence-emitting dye pair.

11. The composition of claim 10 , wherein the fluorescence-emitting dye pair is a fluorescence resonance energy transfer (FRET) pair.

12. The composition of claim 10 , wherein the fluorescence-emitting dye pair is a quencher/fluor pair.

13. The composition of claim 1 , wherein the first labeled detector RNA comprises a first fluorescence-emitting dye pair that produces a first detectable signal and a second fluorescence-emitting dye pair that produces a second detectable signal.

14. The composition of claim 1 , wherein the first labeled detector RNA comprises a modified nucleobase, a modified sugar moiety, and/or a modified nucleic acid linkage.

15. The composition of claim 1 , comprising at least one of:
(i) a C2c2 guide RNA and/or a nucleic acid encoding said C2c2 guide RNA;
(ii) a precursor C2c2 guide RNA and/or a nucleic acid encoding said precursor C2c2 guide RNA; and
(iii) a precursor C2c2 guide RNA array, and/or a nucleic acid encoding said precursor C2c2 guide RNA array, wherein the precursor C2c2 guide RNA array comprises two or more C2c2 guide RNAs each of which has a different guide sequence and/or an insertion site for a guide sequence of choice.

16. The composition of claim 1 , comprising a DNA comprising a nucleotide sequence that encodes a constant region of a C2c2 guide RNA.

17. The composition of claim 16 , wherein the DNA comprises an insertion sequence for the insertion of a guide sequence.

18. The composition of claim 16 , wherein the DNA further comprises a nucleotide sequence encoding a guide sequence of the C2c2 guide RNA.

19. The composition of claim 1 , further comprising a C2c2 guide RNA.

20. The composition of claim 1 , wherein the C2c2 protein comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:6.

21. A composition comprising:
(a) a first labeled detector RNA that lacks U and comprises at least one A;
(b) a second labeled detector RNA that lacks A and comprises at least one U;
(c) a first C2c2 protein, or a nucleic acid encoding the first C2c2 protein, wherein the first C2c2 protein cleaves the first labeled detector RNA and does not cleave the second labeled detector RNA, and wherein the first C2c2 protein comprises an amino acid sequence having 80% or more amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:301; and
(d) a second C2c2 protein, or a nucleic acid encoding the second C2c2 protein, wherein the second C2c2 protein cleaves the second labeled detector RNA and does not cleave the first labeled detector RNA, and wherein the second C2c2 protein comprises an amino acid sequence having 80% or more amino acid sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs:2, 6, 299, and 300.

22. The composition of claim 21 , wherein the first labeled detector RNA comprises a first color-based detection label.

23. The composition of claim 21 , wherein the second labeled detector RNA comprises a second color-based detection label.

24. The composition of claim 21 , wherein the first labeled detector RNA comprises a first fluorescence-emitting dye pair.

25. The composition of claim 21 , wherein the second labeled detector RNA comprises a second fluorescence-emitting dye pair.

26. The composition of claim 21 , comprising a nucleic acid comprising a nucleotide sequence encoding a first C2c2 guide RNA, wherein the first C2c2 guide RNA comprises a constant region sequence that binds to the first C2c2 protein.

27. The composition of claim 21 , comprising a nucleic acid comprising a nucleotide sequence encoding a second C2c2 guide RNA, wherein the second C2c2 guide RNA comprises a constant region sequence that binds to the second C2c2 protein.

28. The composition of claim 21 , comprising a nucleic acid comprising a nucleotide sequence encoding a constant region sequence that binds to the first C2c2 protein and an insertion site for a guide sequence of choice.

29. The composition of claim 21 , comprising a nucleic acid comprising a nucleotide sequence encoding a constant region sequence that binds to the second C2c2 protein and an insertion site for a guide sequence of choice.

30. The composition of claim 21 , wherein the first C2c2 protein comprises an amino acid sequence having 80% or more amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:301, and wherein the second C2c2 protein comprises an amino acid sequence having 80% or more amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO:2.

31. The composition of claim 21 , wherein at least one of the first and second labeled detector RNA comprises a modified nucleobase, a modified sugar moiety, and/or a modified nucleic acid linkage.

Assignees

• Univ California

Inventors

• Doudna Jennifer A
• O'Connell Mitchell Ray
• East-Seletsky Alexandra
• Knight Spencer Charles
• Doudna Cate James Harrison

Classifications

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C07K2319/40

  containing a tag for immunodetection, or an epitope for immunisation · CPC title

• C07K2319/24

  containing a MBP (maltose binding protein)-tag · CPC title

• C12Q1/6823 Primary

  Release of bound markers · CPC title

• C12Q1/6818

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title