Type V Cas proteins and applications thereof
Type V Cas proteins, for example Type V Cas proteins referred to as ZWGD, ZJHK, ZIKV, ZZFT, YYAN, ZZGY, ZKBG, ZZKD, ZXPB, ZPPX, ZXHQ, ZQKH, ZRGM, ZTAE, ZSQQ, ZSYN, ZRBH, ZWPU, ZZQE, and ZRXE Type V Cas proteins; gRNAs for Type V Cas proteins; systems comprising Type V Cas proteins and gRNAs; nucleic acids encoding the Type V Cas proteins, gRNAs and systems; particles comprising the foregoing; pharmaceutical compositions of the foregoing; and uses of the foregoing, for example to alter the genomic DNA of a cell.
1 . A fusion protein comprising:
(a) a Type V Cas amino acid sequence comprising an amino acid sequence that is at least 98% identical to the full length of SEQ ID NO:43 or SEQ ID NO:44; and
(b) one or more nuclear localization signals.
2 . The fusion protein of claim 1 , wherein the Type V Cas amino acid sequence comprises an amino acid sequence that is at least 99% identical to the full length of SEQ ID NO:43.
3 . The fusion protein of claim 1 , wherein the Type V Cas amino acid sequence comprises an amino acid sequence that is identical to SEQ ID NO:43.
4 . The fusion protein of claim 1 , wherein the Type V Cas amino acid sequence comprises an amino acid sequence that is identical to SEQ ID NO:44.
5 . The fusion protein of claim 1 , which comprises a C-terminal nuclear localization signal.
6 . The fusion protein of claim 1 , which comprises an N-terminal nuclear localization signal.
7 . The fusion protein of claim 1 , which comprises a nuclear localization signal comprising the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO:122), PKKKRKV (SEQ ID NO:123), PKKKRRV (SEQ ID NO:124), KRPAATKKAGQAKKKK (SEQ ID NO:125), YGRKKRRQRRR (SEQ ID NO:126), RKKRRQRRR (SEQ ID NO:127), PAAKRVKLD (SEQ ID NO:128), RQRRNELKRSP (SEQ ID NO:129), VSRKRPRP (SEQ ID NO:130), PPKKARED (SEQ ID NO:131), PQPKKKPL (SEQ ID NO:132), SALIKKKKKMAP (SEQ ID NO:133), PKQKKRK (SEQ ID NO:134), RKLKKKIKKL (SEQ ID NO:135), REKKKFLKRR (SEQ ID NO:136), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:137), RKCLQAGMNLEARKTKK (SEQ ID NO:138), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:139), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:140), or SSDDEATADSQHAAPPKKKRKV (SEQ ID NO:178).
8 . The fusion protein of claim 1 , which comprises a nuclear localization signal comprising the amino acid sequence GRSSDDEATADSQHAAPPKKKRKV (SEQ ID NO:180).
9 . The fusion protein of claim 1 , wherein the fusion protein comprises a Type V Cas amino acid sequence that is identical to SEQ ID NO:44 and a C-terminal nuclear localization signal comprising the amino acid sequence GRSSDDEATADSQHAAPPKKKRKV (SEQ ID NO:180).
10 . A system comprising the fusion protein of claim 1 and a guide RNA (gRNA) comprising a spacer positioned 3′ to a crRNA scaffold and capable of forming a complex with the fusion protein and directing the fusion protein to a target DNA.
11 . The system of claim 10 , wherein the nucleotide sequence of the spacer is complementary to a target mammalian genomic sequence that is downstream of a NTTV, VTTV, NCTV, or TTTT protospacer adjacent motif (PAM) sequence.
12 . The system of claim 10 , wherein the crRNA scaffold comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:151 or SEQ ID NO:211.
13 . The system of claim 12 , wherein the crRNA scaffold comprises a nucleotide sequence that is identical to SEQ ID NO:151 or SEQ ID NO:211.
14 . The system of claim 10 , which is a ribonucleoprotein (RNP) comprising the fusion protein complexed to the gRNA.
15 . A nucleic acid encoding the fusion protein of claim 1 .
16 . The nucleic acid of claim 15 , wherein the nucleotide sequence encoding the fusion protein is codon optimized for expression in human cells.
17 . An adeno-associated virus (AAV) genome comprising the nucleic acid of claim 15 .
18 . An adeno-associated virus (AAV) particle comprising the AAV genome of claim 17 .
19 . An ex vivo human cell comprising the system of claim 10 .
20 . The ex vivo human cell of claim 19 , which is a hematopoietic stem cell (HSC), pluripotent stem cell or an induced pluripotent stem cell (iPS).
21 . A method for altering a cell comprising contacting the cell with the system of claim 10 , wherein the contacting alters a genomic sequence of the cell.
22 . An ex vivo human cell comprising the fusion protein of claim 1 .
23 . The ex vivo human cell of claim 22 , which is a hematopoietic stem cell (HSC), pluripotent stem cell or an induced pluripotent stem cell (iPS).