Systems, methods, and compositions for targeted nucleic acid editing
The disclosure provides for systems, methods, and compositions for targeting and editing nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a RNA-targeting Cas13 protein, at least one guide molecule, and at least one adenosine deaminase protein or catalytic domain thereof.
1. An engineered, non-naturally occurring system comprising a catalytically inactive Cas13 effector protein (dCas13) or a nucleotide sequence encoding the catalytically inactive Cas13 effector protein, wherein the dCas13 is Prevotella sp. P5-125 Cas13b protein or Porphyromonas gulae Cas13b protein,
wherein the truncated form of the Cas13 effector protein has been truncated at:
[1] C-terminal Δ984-1090, C-terminal Δ1026-1090, C-terminal Δ1053-1090, C-terminal Δ934-1090, C-terminal Δ884-1090, C-terminal Δ834-1090, C-terminal Δ784-1090, or C-terminal Δ734-1090, wherein amino acid positions of the truncations correspond to amino acid positions of Prevotella sp. P5-125 Cas13b protein as set forth in SEQ ID NO: 82; or
[2] C-terminal Δ875-1175, C-terminal Δ895-1175, C-terminal Δ915-1175, C-terminal Δ935-1175, C-terminal Δ955-1175, C-terminal Δ975-1175, C-terminal Δ995-1175, C-terminal Δ1015-1175, C-terminal Δ1035-1175, C-terminal Δ1055-1175, C-terminal Δ1075-1175, C-terminal Δ1095-1175, C-terminal Δ1115-1175, C-terminal Δ1135-1175, or C-terminal Δ1155-1175, wherein amino acid positions correspond to amino acid positions of Porphyromonas gulae Cas13b protein as set forth in SEQ ID NO: 81.
2. The system of claim 1 , wherein the dCas13 protein is further truncated at an N terminus.
3. The system of claim 2 , wherein the dCas13 is truncated by at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, or at least 300 amino acids on the N terminus.
4. The system of claim 2 , wherein the truncated form of the Cas13 effector protein has been truncated at N-terminal Δ1-125, N-terminal Δ1-88, or N-terminal Δ1-72, wherein amino acid positions of the truncations correspond to amino acid positions of Prevotella sp. P5-125 Cas13b protein.
5. The system of claim 1 , wherein the dCas13 further comprises a truncated form of a Cas13 effector protein at an HEPN domain of the Cas13 effector protein.
6. The system of claim 1 , further comprising a functional component or wherein the nucleotide sequence further encodes the functional component.
7. The system of claim 6 , wherein the functional component is a base editing component.
8. The system of claim 7 , wherein the base editing component comprises an adenosine deaminase, a cytidine deaminase, or a catalytic domain thereof.
9. The system of claim 8 , wherein the adenosine deaminase, the cytidine deaminase, or the catalytic domain thereof, is fused to the dCas13.
10. The system of claim 9 , wherein the adenosine deaminase, the cytidine deaminase, or the catalytic domain thereof, is fused to the dCas13 by a linker.
11. The system of claim 8 , wherein the adenosine deaminase, the cytidine deaminase, or the catalytic domain thereof, is inserted into an internal loop of the dCas13.
12. The system of claim 8 , wherein the adenosine deaminase, the cytidine deaminase, or the catalytic domain thereof, is linked to an adaptor protein.
13. The system of claim 12 , wherein the adaptor protein is selected from MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1.
14. The system of claim 8 , wherein the adenosine deaminase, the cytidine deaminase, or the catalytic domain thereof, is a human, cephalopod, or Drosophila protein.
15. The system of claim 1 , wherein the dCas13 is a split Cas13 effector protein.
16. The system of claim 15 , wherein the split Cas13 effector protein is a first split Cas 13 effector protein and is capable of fusing to a second split Cas13 effector protein to form a catalytically active Cas13 effector protein.
17. The system of claim 16 , wherein fusing of the first and the second Cas13 effector proteins is inducible.
18. The system of claim 6 , wherein the functional component is a transcription factor or an active domain thereof.
19. The system of claim 18 , wherein the dCas13 is fused with the transcription factor or the active domain thereof.
20. The system of claim 1 , further comprising a guide sequence.