DNA modifying enzymes and active fragments and variants thereof and methods of use
Compositions and methods comprising novel deaminase polypeptides for targeted editing of nucleic acids are provided. Compositions comprise deaminase polypeptides. Also provided are fusion proteins comprising a DNA-binding polypeptide and a deaminase of the invention. The fusion proteins include RNA-guided nucleases fused to deaminases, optionally in complex with guide RNAs. Compositions also include nucleic acid molecules encoding the deaminases or the fusion proteins. Vectors and host cells comprising the nucleic acid molecules encoding the deaminases or the fusion proteins are also provided.
1. A fusion protein comprising a Type II CRISPR-Cas protein nickase and a deaminase, wherein said deaminase comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 405 and has adenine deaminase activity, and wherein said nickase
(a) is a Cas9 nickase; or
(b) comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NO: 42, 52, 53, 55-59, 61, 397, or 398.
2. The fusion protein of claim 1 , wherein said deaminase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 405 and has adenine deaminase activity.
3. The fusion protein of claim 1 , wherein said deaminase comprises the amino acid sequence of SEQ ID NO: 405.
4. The fusion protein of claim 1 , wherein the nickase has the amino acid sequence of any one of SEQ ID NO: 42, 52, 53, 55-59, 61, 397, or 398.
5. The fusion protein of claim 1 , wherein said fusion protein comprises the amino acid sequence of SEQ ID NO: 494.
6. The fusion protein of claim 1 , wherein the fusion protein further comprises at least one nuclear localization signal.
7. The fusion protein of claim 1 , wherein the fusion protein further comprises a protein tag or a cell penetrating domain.
8. A cell comprising the fusion protein of claim 1 , wherein the cell further comprises a guide RNA.
9. A ribonucleoprotein (RNP) complex comprising the fusion protein of claim 1 and a guide RNA.
10. A system for modifying a target DNA molecule, said system comprising:
a) the fusion protein of claim 1 ; and
b) one or more guide RNAs (gRNAs) capable of hybridizing to said target DNA molecule, or one or more nucleic acids encoding the one or more gRNAs; and
wherein the one or more gRNAs are capable of forming a complex with the fusion protein in order to direct said fusion protein to bind to and modify said target DNA molecule.
11. The system of claim 10 , wherein the nickase has the amino acid sequence of any one of SEQ ID NO: 42, 52, 53, 55-59, 61, 397, or 398.
12. The system of claim 10 , wherein at least one of the one or more nucleic acids encoding the one or more gRNAs is operably linked to a promoter heterologous to said nucleic acid.
13. The system of claim 10 , wherein the target DNA molecule is a eukaryotic target DNA.
14. A polypeptide having adenine deaminase activity and comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 405.
15. The polypeptide of claim 14 , wherein said polypeptide comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 405.
16. The polypeptide of claim 14 , wherein said polypeptide has the amino acid sequence of SEQ ID NO: 405.
17. The polypeptide of claim 14 , wherein said polypeptide further comprises at least one nuclear localization signal.
18. An adenine base editor comprising the polypeptide of claim 14 and a Type II CRISPR-Cas nickase, wherein said nickase
(a) is a Cas9 nickase; or
(b) comprises an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 42, 52, 53, 55-59, 61, 397, or 398.
19. The adenine base editor of claim 18 , wherein the nickase has the amino acid sequence of any one of SEQ ID NO: 42, 52, 53, 55-59, 61, 397, or 398.
20. The adenine base editor of claim 18 , wherein the adenine base editor introduces an A>G mutation in a DNA molecule.