Methods and compositions for modulating a genome
Methods and compositions for modulating a target genome are disclosed. This disclosure relates to novel compositions, systems and methods for altering a genome at one or more locations in a host cell, tissue or subject, in vivo or in vitro. In particular, the invention features compositions, systems and methods for inserting, altering, or deleting sequences of interest in a host genome.
1. A fusion protein comprising:
a) a reverse transcriptase (RT) domain having the amino acid sequence of SEQ ID NO: 3225; and
b) a Cas9 nickase domain,
wherein the RT domain is C-terminal of the Cas9 nickase domain.
2. The fusion protein of claim 1 , wherein the Cas9 nickase domain is a SpyCas9 nickase domain.
3. The fusion protein of claim 1 , wherein the Cas9 nickase domain is a SpyCas9 (N863A) nickase domain.
4. The fusion protein of claim 1 , wherein the Cas9 nickase domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 3269.
5. The fusion protein of claim 1 , wherein the Cas9 nickase domain is an NmeCas9 domain.
6. The fusion protein of claim 1 , wherein the Cas9 nickase domain is an St1Cas9 domain.
7. The fusion protein of claim 1 , wherein the Cas9 nickase domain is a SauCas9 domain.
8. The fusion protein of claim 1 , which further comprises a peptide linker disposed between the RT domain and the Cas9 nickase domain.
9. The fusion protein of claim 8 , wherein the peptide linker is between 2-40 amino acids in length.
10. The fusion protein of claim 8 , wherein the peptide linker has an amino acid sequence according to SEQ ID NO: 1589.
11. The fusion protein of claim 1 , which further comprises a nuclear localization sequence (NLS).
12. The fusion protein of claim 11 , wherein the NLS is fused to the N-terminus of the Cas9 nickase domain.
13. The fusion protein of claim 11 , wherein the NLS is fused to the C-terminus of the fusion protein.
14. The fusion protein of claim 11 , wherein the NLS is a monopartite NLS or a bipartite NLS.
15. The fusion protein of claim 11 , which further comprises a linker disposed between the NLS and the Cas9 nickase domain.
16. The fusion protein of claim 1 , which comprises an amino acid sequence according to SEQ ID NO: 3561.
17. The fusion protein of claim 1 , wherein the Cas9 nickase domain has an activity at least 50% of that of an otherwise similar Cas9 nickase molecule that is not fused to an RT domain.