Methods and compositions for detecting a target RNA
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.
1. An in vitro acellular method for detecting one or more target RNAs, comprising one or more target sequences, in one or more lysed cell samples, the method comprising:
a) contacting the one or more lysed cell samples with (i), (ii), and (iii) to generate an in vitro acellular reaction:
i) a Cas13a polypeptide;
ii) at least one guide polynucleotide comprising a guide sequence capable of binding the one or more target sequences, and designed to form a complex with the Cas13a polypeptide; and
iii) a labeled detector RNA comprising a non-target sequence, wherein the labeled detector RNA produces a different detectable signal when the labeled detector RNA is cleaved compared to when the labeled detector RNA is uncleaved; and
wherein the one or more samples comprise more than 5 RNAs that differ from one another in nucleotide sequence, and
wherein the complex comprising the Cas13a polypeptide exhibits collateral RNase activity and trans cleaves the non-target sequence of the labeled detector RNA in the presence of the one or more target RNAs; and
b) detecting whether the one or more target RNAs was present in the one or more lysed cell samples by measuring the different detectable signal from cleavage of the non-target sequence of the labeled detector RNA in the in vitro acellular reaction.
2. The method of claim 1 , wherein the labeled detector RNA comprises a quencher/fluorophore pair.
3. The method of claim 1 , wherein a sample of the one or more samples is a biological sample.
4. The method of claim 1 , wherein a target RNA of the one or more target RNAs is from a virus.
5. The method of claim 4 , wherein the virus is a Hepatitis virus, Hepatitis A virus, Hepatitis B virus, or Hepatitis C virus.
6. The method of claim 4 , wherein the virus is a Herpes virus, Herpes simplex virus 1, or a Herpes simplex virus 2.
7. The method of claim 4 , wherein the virus is human papillomavirus.
8. The method of claim 4 , wherein the virus is human immunodeficiency virus.
9. The method of claim 4 , wherein the virus is Zika virus.
10. The method of claim 4 , wherein the virus is an influenza virus.
11. The method of claim 4 , wherein the virus is rabies virus, cytomegalovirus, human serum parvo-like virus, respiratory syncytial virus, varicella-zoster virus, measles virus, adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, west Nile virus, or yellow fever virus.
12. The method of claim 1 , wherein a target RNA of the one or more target RNAs is present in a sample of the one or more samples at from 1 copy per 10 non-target nucleic acids to 1 copy per 10 7 non-target nucleic acids.
13. The method of claim 1 , wherein a target RNA of the one or more target RNAs is present in a sample of the one or more samples at a concentration of from about 500 fM to about 1 nM.
14. The method of claim 1 , wherein the labeled detector RNA comprises a fluorescence-emitting dye pair.
15. The method of claim 1 , wherein the labeled detector RNA comprises a Förster resonance energy transfer (FRET) pair.
16. The method of claim 1 , wherein detecting a signal comprises one or more of: gold nanoparticle-based detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, fluorescent signal detection, and semiconductor-based sensing.
17. The method of claim 1 , wherein detecting a signal comprises a color detection method.
18. The method of claim 17 , wherein the color detection method comprises a visual-based or sensor-based detection of the presence, absence, or ratio change of a color.
19. The method of claim 1 , wherein detecting occurs within 60 minutes of said contacting.
20. The method according to claim 1 , wherein said labeled detector RNA has a length of from 2 to 100 ribonucleotides.
21. The method according to claim 1 , wherein said labeled detector RNA produces a greater amount of detectable signal when said labeled detector RNA is cleaved compared to when said labeled detector RNA is uncleaved.
22. The method according to claim 1 , wherein said labeled detector RNA produces a lesser amount of detectable signal when said labeled detector RNA is cleaved compared to when said labeled detector RNA is uncleaved.
23. The method according to claim 1 , wherein said labeled detector RNA comprises two moieties that when separated by cleavage of the labeled detector RNA produce a greater amount of the detectable signal than when said moieties are not separated by cleavage of the labeled detector RNA.
24. The method according to claim 1 , wherein said labeled detector RNA comprises two moieties that when separated by cleavage of the labeled detector RNA produce a different detectable signal than when said moieties are not separated by cleavage of the labeled detector RNA.
25. The method according to claim 1 , wherein the measuring does not comprise running a labeled detector RNA on a gel.
26. The method according to claim 1 , wherein the measuring comprises measuring the amount of detectable signal from the labeled detector RNA in the in vitro acellular reaction; and wherein the measuring is not dependent on comparing the size of the cleaved labeled detector molecule to the size of the uncleaved labeled detector molecule.