Methods and compositions for modulating a genome
The disclosure provides, e.g., compositions, systems, and methods for targeting, editing, modifying, or manipulating a host cell's genome at one or more locations in a DNA sequence in a cell, tissue, or subject.
1. A gene modifying polypeptide comprising: a reverse transcriptase (RT) domain having the sequence of SEQ ID NO: 8,113; a Cas9 nickase domain, wherein the RT domain is C-terminal of the Cas9 nickase domain; and a linker disposed between the RT domain and the Cas9 nickase domain, wherein the linker comprises the sequence of SEQ ID NO: 15,404.
2. The gene modifying polypeptide of claim 1 , wherein the Cas9 nickase domain is a SpyCas9 nickase domain.
3. The gene modifying polypeptide of claim 1 , wherein the Cas9 nickase domain comprises an N863A mutation.
4. The gene modifying polypeptide of claim 1 , wherein the Cas9 nickase domain comprises the amino acid sequence of SEQ ID NO: 11,096.
5. The gene modifying polypeptide of claim 1 , wherein the Cas9 nickase domain is an NmeCas9 domain.
6. The gene modifying polypeptide of claim 1 , wherein the Cas9 nickase domain is an St1Cas9 domain.
7. The gene modifying polypeptide of claim 1 , wherein the Cas9 nickase domain is a SauCas9 domain.
8. The gene modifying polypeptide of claim 1 , which comprises a nuclear localization signal (NLS).
9. The gene modifying polypeptide of claim 1 , which comprises a first NLS which is N-terminal of the Cas9 nickase domain.
10. The gene modifying polypeptide of claim 1 , which comprises an NLS which is C-terminal of the RT domain.
11. The gene modifying polypeptide of claim 1 , which comprises a first NLS which is N-terminal of the Cas9 nickase domain and a second NLS which is C-terminal of the RT domain.
12. The gene modifying polypeptide of claim 1 , which comprises a first NLS which is N-terminal of the Cas9 nickase domain, wherein the first NLS comprises the amino acid sequence of
(SEQ ID NO: 11,095)
PAAKRVKLD.
13. The gene modifying polypeptide of claim 1 , which comprises an NLS which is C-terminal of the RT domain and comprises the amino acid sequence of KRTADGSEFE (SEQ ID NO: 4650).
14. The gene modifying polypeptide of claim 1 , which comprises an NLS which is C-terminal of the RT domain and comprises the amino acid sequence of
(SEQ ID NO: 4651)
KRTADGSEFESPKKKAKVE.
15. The gene modifying polypeptide of claim 1 , which comprises the amino acid sequence of SEQ ID NO: 4000 which comprises a first NLS and the Cas9 nickase domain.
16. The gene modifying polypeptide of claim 1 , which comprises an NLS having the amino acid sequence according to SEQ ID NO: 4649.
17. The gene modifying polypeptide of claim 1 , which comprises a GG amino acid sequence between the Cas9 nickase domain and the linker.
18. The gene modifying polypeptide of claim 11 , which comprises an AG amino acid sequence between the RT domain and the second NLS.
19. The gene modifying polypeptide of claim 1 , which comprises a GG amino acid sequence between the linker and the RT domain.
20. The gene modifying polypeptide of claim 1 , which comprises the amino acid sequence according to SEQ ID NO: 647.
21. A nucleic acid molecule encoding a gene modifying polypeptide comprising: a reverse transcriptase (RT) domain having the sequence of SEQ ID NO: 8.113; a Cas9 nickase domain, wherein the RT domain is C-terminal of the Cas9 nickase domain; and a linker disposed between the RT domain and the Cas9 nickase domain, wherein the linker comprises a sequence of SEQ ID NO: 15,404.
22. The nucleic acid molecule of claim 21 , which comprises RNA.
23. A cell comprising: i) a gene modifying polypeptide comprising: a reverse transcriptase (RT) domain having the sequence of SEQ ID NO: 8.113; a Cas9 nickase domain, wherein the RT domain is C-terminal of the Cas9 nickase domain; and a linker disposed between the RT domain and the Cas9 nickase domain, wherein the linker comprises a sequence of SEQ ID NO: 15,404 or ii) a nucleic acid encoding the gene modifying polypeptide.
24. A system comprising:
i) a gene modifying polypeptide comprising: a reverse transcriptase (RT) domain having the sequence of SEQ ID NO: 8.113; a Cas9 nickase domain, wherein the RT domain is C-terminal of the Cas9 nickase domain; and a linker disposed between the RT domain and the Cas9 nickase domain, wherein the linker comprises a sequence of SEQ ID NO: 15,404, or a nucleic acid molecule encoding the gene modifying polypeptide, and
ii) a template RNA that comprises:
a) a gRNA spacer that is complementary to a portion of a target nucleic acid sequence;
b) a gRNA scaffold that binds the Cas9 nickase domain of the gene modifying polypeptide;
c) a heterologous object sequence; and
d) a primer binding site sequence.
25. A lipid nanoparticle formulation comprising the system of claim 24 .
26. A method for modifying a target nucleic acid molecule in a cell, the method comprising contacting the cell with the system of claim 24 , thereby modifying the target nucleic acid molecule.