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. A method of detecting a target RNA in a sample, the method comprising:
a) contacting the sample to:
(i) a guide RNA comprising a nucleotide sequence that hybridizes to the target RNA;
(ii) a first labeled detector RNA;
(iii) a second labeled detector RNA; and
(iv) a programmable nuclease that comprises a HEPN1 domain and a HEPN2 domain and that, when complexed with the guide RNA and contacted with a plurality of the first labeled detector RNA, a plurality of the second labeled detector RNA, and the target RNA, cleaves a higher proportion of the plurality of the first labeled detector RNA as compared to the plurality of the second labeled detector RNA, wherein the programmable nuclease comprises an amino acid sequence having at least 80% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 6, 301, 304, 307, 310, 312, 313, 314, 316, and 321; and
b) measuring the amount, presence, or absence of a signal produced by the first labeled detector RNA after cleavage of the first labeled detector RNA, wherein said measuring provides for detection of the target RNA in the sample, and
wherein the first labeled detector RNA comprises at least one uracil and the second labeled detector RNA lacks a uracil; or wherein the first labeled detector RNA comprises as least one adenosine and the second labeled detector RNA lacks an adenosine.
2. The method of claim 1 , wherein the programmable nuclease comprises an amino acid sequence having at least 90% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 6, 301, 304, 307, 310, 312, 313, 314, 316, and 321.
3. The method of claim 1 , wherein the target RNA is a single stranded target RNA.
4. The method of claim 1 , wherein the programmable nuclease does not cleave the second labeled detector RNA.
5. The method of claim 1 , further comprising determining an amount of the target RNA present in the sample.
6. The method of claim 1 , wherein the target RNA in the sample is present in a range of from 50 fM to 1 nM.
7. The method of claim 1 , wherein the target RNA in the sample is present in a range of from 500 fM to 1 nM.
8. The method of claim 1 , wherein the target RNA in the sample is present in a range of from 1 pM to 1 nM.
9. The method of claim 1 , wherein the target RNA is from a human cell, an animal cell, a plant cell, a cancerous cell, an infected cell, or a diseased cell.
10. The method of claim 1 , wherein the target RNA is from a virus, a parasite, a helminth, a fungus, a protozoan, or a bacterium.
11. The method of claim 1 , wherein measuring the signal comprises one or more of: visual based detection, sensor based detection, color detection, gold nanoparticle based detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, fluorescent signal detection, and semiconductor-based sensing.
12. The method of claim 1 , wherein measuring the signal comprises using a detection method comprising visual or sensor-based detection of a presence, absence, or ratio change of the detectable signal.
13. The method of claim 1 , wherein the first labeled detector RNA comprises a fluorescence-emitting dye pair.
14. The method of claim 1 , wherein the first labeled detector RNA produces an amount of the signal prior to being cleaved and the amount of the signal is reduced when the first labeled detector RNA is cleaved.
15. The method of claim 1 , wherein the first labeled detector RNA produces a first signal prior to being cleaved and a second signal when the first labeled detector RNA is cleaved, and wherein measuring the presence or absence of the signal after cleavage of the first labeled detector RNA comprises measuring the presence or absence of the second signal.
16. The method of claim 15 , wherein the amount of the second signal is greater than the amount of the first signal.
17. The method of claim 1 , wherein the first labeled detector comprises a first FRET pair and the second labeled detector comprises a second FRET pair.
18. The method of claim 1 , wherein the first labeled detector comprises a first quencher/fluor pair and the second labeled detector comprises a second quencher/fluor pair.
19. The method of claim 1 , wherein the programmable nuclease is a homolog of the protein set forth in SEQ ID NO: 2 and, wherein the residues R472, H477, R1048, and H1053 of SEQ ID NO:2 are conserved in the programmable nuclease.
20. The method of claim 1 , wherein the programmable nuclease 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.
21. The method of claim 1 , wherein the first labeled detector RNA comprises at least one uracil and the second labeled detector RNA lacks a uracil, and the programmable nuclease comprises an amino acid sequence having at least 80% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 6, 307, 310, 312, and 313.
22. The method of claim 1 , wherein the first labeled detector RNA comprises as least one adenosine and the second labeled detector RNA lacks an adenosine, and the programmable nuclease comprises an amino acid sequence having at least 80% amino acid sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:301, 304, 314, 316, and 321.