Methods and compositions for modulating a genome

1. A system for modifying DNA, comprising:

a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain and (ii) an endonuclease domain, and wherein the polypeptide comprises an amino acid sequence at least 95% identical to the sequence of SEQ ID NO: 1988; and

b) a template RNA comprising:

(i) a 5′ UTR sequence that binds the polypeptide,

(ii) a 3′ UTR sequence that binds the polypeptide, and

(ii) a heterologous object sequence.

2. The system of claim 1 , wherein the 5′ UTR comprises a sequence according to SEQ ID NO: 1986, or a sequence having at least 95% identity thereto.

3. The system of claim 1 , wherein the 3′ UTR comprises a sequence according to SEQ ID NO: 1987, or a sequence having at least 95% identity thereto.

4. The system of claim 1 , wherein the heterologous object sequence encodes a therapeutic polypeptide or a human polypeptide, or a fragment or variant thereof.

5. The system of claim 1 , wherein the heterologous object sequence encodes a chimeric antigen receptor (CAR).

6. The system of claim 1 , wherein the heterologous object sequence comprises a regulatory sequence.

7. The system of claim 6 , wherein the regulatory sequence:

(a) is a promoter;

(b) is an enhancer;

(c) is a binding site for an endogenous regulatory component;

(d) is a miRNA binding site; or

(e) alters the expression of an endogenous gene or non-coding RNA.

8. The system of claim 1 , wherein the polypeptide comprises a nuclear localization signal (NLS).

9. The system of claim 8 , wherein the NLS is fused to the N-terminus of the polypeptide.

10. The system of claim 8 , wherein the NLS is fused to the C-terminus of the polypeptide.

11. The system of claim 8 , wherein the NLS sequence has an amino acid sequence of PKKKRKV (SEQ ID NO: 2409).

12. The system of claim 1 , wherein the polypeptide comprises a linker having an amino acid sequence of GGGS (SEQ ID NO: 1024).

13. The system of claim 12 , wherein the linker is disposed between an NLS and the remainder of the polypeptide.

14. The system of claim 1 , wherein the polypeptide comprises a linker having an amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 1023).

15. The system of claim 14 , wherein the linker is disposed between an NLS and the remainder of the polypeptide.

16. The system of claim 1 , wherein the template RNA comprises:

(a) a polyA site;

(b) a regulatory element of Woodchuck Hepatitis Virus (WPRE); and/or

(c) a Kozak sequence.

17. The system of claim 1 , wherein the nucleic acid encoding the polypeptide and the template RNA are two separate nucleic acids.

18. The system of claim 1 , wherein (a) comprises RNA encoding the polypeptide and wherein (b) comprises template RNA.

19. The system of claim 1 , wherein the nucleic acid encoding the polypeptide comprises a coding sequence that is codon-optimized for expression in human cells.

20. The system of claim 1 , which is capable of inducing an insertion, deletion, or alteration of a protein coding sequence to a genome of a mammalian cell.

21. The system of claim 20 , wherein the insertion is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length.

22. The system of claim 1 , which is capable of inducing an insertion, deletion, or alteration of a non-coding sequence to a genome of a mammalian cell.

23. The system of claim 22 , wherein the insertion is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length.

24. A method of modifying a target DNA strand in a cell, tissue, or subject, the method comprising administering the system of claim 1 to the cell, tissue, or subject, wherein the system reverse transcribes the template RNA sequence into the target DNA strand, thereby modifying the target DNA strand.

25. The method of claim 24 , wherein the cell is:

(a) a human cell;

(b) a primary cell; and/or

(c) a T cell.

26. A lipid nanoparticle (LNP) comprising the system of claim 1 .