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 composition comprising:
(a) a first labeled detector RNA comprising a first label, wherein the first labeled detector RNA comprises at least one uracil and lacks an adenosine;
(b) a second labeled detector RNA comprising a second label, wherein the second labeled detector RNA comprises at least one adenosine and lacks a uracil;
(c) a first programmable endoribonuclease comprising a HEPN1 domain and a HEPN2 domain, or a nucleic acid encoding the first programmable endoribonuclease, wherein the first programmable endoribonuclease, when complexed with a guide RNA and contacted with a plurality of the first labeled detector RNA, a plurality of the second labeled detector RNA, and a target RNA to which the guide RNA hybridizes, 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 HEPN2 domain of the first programmable endonuclease 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:214, 216, 219, 220, 222, and 223; and
(d) a second programmable endoribonuclease comprising a HEPN1 domain and a HEPN2 domain, or a nucleic acid encoding the second programmable endoribonuclease, wherein the second programmable endoribonuclease, when complexed with a guide RNA and contacted with a plurality of the first labeled detector RNA, a plurality of the second labeled detector RNA, and a target RNA to which the guide RNA hybridizes, cleaves a higher proportion of the plurality of the second labeled detector RNA as compared to the plurality of the first labeled detector RNA, wherein the HEPN2 domain of the second programmable endonuclease 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:217, 224, 226, and 231.
2. The composition of claim 1 , further comprising a target RNA, wherein the programmable endoribonuclease is capable of binding to the target RNA when complexed with a guide RNA that comprises a nucleotide sequence that is capable of hybridizing to the target RNA.
3. The composition of claim 2 , where the first programmable endoribonuclease 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.
4. The composition of claim 2 , wherein the target RNA is from a virus, a parasite, a helminth, a fungus, a protozoan, a bacterium, or a pathogenic bacterium.
5. The composition of claim 4 , wherein the target RNA is from an influenza virus.
6. The composition of claim 1 , wherein the first programmable endoribonuclease is a first C2c2 protein and wherein the second programmable endoribonuclease is a second C2c2 protein.
7. The composition of claim 6 , wherein the first C2c2 protein is selected from the group consisting of Lbu C2c2, Lwa C2c2, Ppr C2c2, and Hhe C2c2; and wherein the second C2c2 protein is selected from the group consisting of Lba Cas13a and Ere Cas13a.
8. The composition of claim 1 , wherein the first programmable endoribonuclease is not capable of cleaving the second labeled detector RNA.
9. The composition of claim 1 , wherein the first label provides for visual based detection, sensor based detection, color detection, gold nanoparticle based detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, fluorescent signal detection, or semiconductor-based sensing.
10. The composition of claim 1 , wherein the second label provides for visual based detection, sensor based detection, color detection, gold nanoparticle based detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, fluorescent signal detection, or semiconductor-based sensing.
11. The composition of claim 1 , wherein the first label comprises a first fluorescence-emitting dye pair that is capable of producing a first signal and the second label comprises a second fluorescence-emitting dye pair that is capable of producing a second signal.
12. The composition of claim 1 , further comprising:
i) a first target RNA, wherein the first programmable endoribonuclease is capable of binding to the first target RNA when complexed with a guide RNA that comprises a nucleotide sequence that is capable of hybridizing to the first target RNA and wherein the second programmable endoribonuclease is not capable of binding to the first target RNA; and
ii) a second target RNA, wherein the second programmable endoribonuclease is capable of binding to the second target RNA when complexed with a guide RNA that comprises a nucleotide sequence that is capable of hybridizing to the second target RNA and wherein the second programmable endoribonuclease is not capable of binding to the first target RNA.
13. The composition of claim 1 , wherein the first label comprises a first FRET pair and the second label comprises a second FRET pair.
14. The composition of claim 1 , wherein the first label comprises a first quencher/fluor pair and the second label comprises a second quencher/fluor pair.
15. The composition of claim 1 , wherein the first and second programmable endoribonucleases are homologs of the protein set forth in SEQ ID NO: 2 and wherein residues R472, H477, R1048, and H1053 of SEQ ID NO: 2 are conserved in the first and second programmable endoribonucleases.
16. The composition of claim 1 , wherein the HEPN1 and HEPN2 domains of the first and second programmable endoribonucleases are catalytically active.
17. The composition of claim 1 , wherein the HEPN domain comprises divalent metal-dependent HEPN domains.
18. The composition of claim 1 , wherein the first labeled detector RNA comprises a polyU RNA sequence.
19. The composition of claim 1 , wherein the second labeled detector RNA comprises a polyA RNA sequence.
20. The composition of claim 1 , further comprising from 5 to 107 RNAs that differ from one another in sequence.
21. The composition of claim 1 , further comprising RNA from a cell lysate.
22. The composition of claim 21 , wherein the cell lysate comprises a eukaryotic cell lysate, a mammalian cell lysate, a human cell lysate, a prokaryotic cell lysate, or a plant cell lysate.
23. The composition of claim 1 , wherein the first 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, 305, 307, 308, 310, 312, and 313.
24. The composition of claim 1 , wherein the second 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.