Engineered proteins
Provided herein are engineered proteins comprising a RuvC DNA cleavage domain comprising one or more amino acid modifications, and one or more improved characteristics, relative to a naturally occurring RuvC domain. Also provided are gene editing systems comprising engineered proteins, and methods for use thereof.
1. An engineered protein comprising a RuvC cleavage domain, wherein the RuvC cleavage domain comprises the sequence of amino acids 648-812 of SEQ ID NO: 2 with one or more amino acid modifications relative to said RuvC cleavage domain sequence, and
wherein the engineered protein exhibits one or more improved characteristics compared to SEQ ID NO: 2 selected from the group consisting of improved editing of target DNA, improved target DNA cleavage rate, increased formation of cleavage-competent ribonucleoprotein (RNP) complexes, improved protospacer adjacent motif (PAM) utilization, and improved solubility.
2. The engineered protein of claim 1 , wherein the one or more amino acid modifications comprise a modification at a position selected from the group consisting of I658, A708, and P793.
3. The engineered protein of claim 1 , wherein the one or more amino acid modifications comprise an amino acid substitution.
4. The engineered protein of claim 3 , wherein the substitution is a substitution at amino acid position A708, or is a substitution at amino acid position 1658.
5. The engineered protein of claim 4 , wherein the substitution is A708K.
6. The engineered protein of claim 4 , wherein the substitution is I658V.
7. The engineered protein of claim 1 , wherein the one or more amino acid modifications comprise an amino acid deletion.
8. The engineered protein of claim of claim 7 , wherein the deletion is at amino acid position P793.
9. The engineered protein of claim 1 , further comprising amino acids 922-978 of a RuvC DNA cleavage domain of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto.
10. The engineered protein of claim 1 , further comprising one or more of:
(a) a non-target strand binding (NTSB) domain;
(b) a target strand loading (TSL) domain;
(c) a helical I domain;
(d) a helical II domain; and
(e) an oligonucleotide binding domain (OBD).
11. The engineered protein of claim 1 , comprising a NTSB domain, wherein the NTSB domain comprises amino acids 101-191 of SEQ ID NO: 1, or a sequence having at least 70% sequence identity thereto.
12. The engineered protein of claim 1 , comprising a TSL domain, wherein the TSL domain comprises amino acids 813-921 of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto.
13. The engineered protein of claim 1 , comprising a helical I domain, wherein the helical I domain is a chimeric helical I domain derived from the helical I domain sequences of SEQ ID NO: 1 and SEQ ID NO: 2.
14. The engineered protein of claim 13 , wherein the chimeric helical I domain comprises amino acids 59-102 of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto, and comprises amino acids 192-332 of SEQ ID NO: 1, or at least 70% sequence identity thereto.
15. The engineered protein of claim 1 , comprising a helical II domain, wherein the helical II domain comprises amino acids 334-501 of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto.
16. The engineered protein of claim 15 , wherein the helical II domain comprises a substitution at amino acid position L379 of SEQ ID NO: 2.
17. The engineered protein of claim 16 , wherein the substitution is L379R.
18. The engineered protein of claim 15 , wherein the helical II domain comprises a substitution at amino acid position F399 of SEQ ID NO: 2.
19. The engineered protein of claim 18 , wherein the substitution is F399L.
20. The engineered protein of claim 1 , comprising an OBD, wherein the OBD comprises amino acids 1-58 of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto, and comprises amino acids 502-647 of SEQ ID NO: 2, or at least 70% sequence identity thereto.
21. The engineered protein of claim 1 , comprising:
(a) a non-target strand binding (NTSB) domain;
(b) a target strand loading (TSL) domain;
(c) a chimeric helical I domain;
(d) a helical II domain;
(e) an oligonucleotide binding domain (OBD); and
(f) a RuvC DNA cleavage domain,
wherein the engineered protein is a chimeric protein,
wherein the NTSB domain comprises the amino acids of positions 101-191 of SEQ ID NO: 1, or a sequence with at least 70% sequence identity thereto;
wherein the TSL domain comprises the amino acids of positions 813-921 of SEQ ID NO: 2, or a sequence with at least 70% sequence identity thereto;
wherein the helical II domain comprises the amino acids of positions 334-501 of SEQ ID NO: 2, or a sequence with at least 70% sequence identity thereto;
wherein the OBD domain comprises the amino acids of positions 1-58 of SEQ ID NO: 2, or a sequence with at least 70% sequence identity thereto;
and wherein the RuvC DNA cleavage domain comprises the amino acids of positions 648-812 and 922-978 of SEQ ID NO: 2, or sequences with at least 70% sequence identity thereto.
22. The engineered protein of claim 21 , comprising:
(a) a RuvC DNA cleavage domain comprising a substitution at amino acid position 1658 of SEQ ID NO: 2;
(b) a RuvC DNA cleavage domain comprising a substitution at amino acid position A708 of SEQ ID NO: 2;
(c) a helical II domain comprising a substitution at amino acid position L379 of SEQ ID NO: 2; and/or
(d) a helical II domain comprising a substitution at amino acid position F399 of SEQ ID NO: 2.
23. The engineered protein of claim 22 , wherein the chimeric helical I domain comprises amino acids 59-102 of SEQ ID NO: 2, or a sequence having at least 70% sequence identity thereto, and comprises amino acids 192-332 of SEQ ID NO: 1, or a sequence having at least 70% sequence identity thereto.
24. The engineered protein of claim 23 , comprising:
(a) a RuvC DNA cleavage domain comprising an amino acid substitution of I658V;
(b) a RuvC DNA cleavage domain comprising an amino acid substitution of A708K;
(c) a RuvC DNA cleavage domain comprising a deletion at amino acid position P793;
(d) a helical II domain comprising an amino acid substitution of L379R; and
(e) a helical II domain comprising an amino acid substitution of F399L.
25. The engineered protein of claim 1 , wherein the protein is selected from the group consisting of SEQ ID NO: 3505, SEQ ID NO: 3506, SEQ ID NO: 3507, and SEQ ID NO: 3548, or a protein comprising a sequence having at least 70% sequence identity thereto.