St1cas9 compositions and methods for modulating a genome

Abstract

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.

Description

[0001]Attorney Docket No.2017469-0039 ST1CAS9 COMPOSITIONS AND METHODS FOR MODULATING A GENOME CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 565,398, filed March 14, 2024, and U.S. Provisional Patent Application No. 63 / 642,617, filed May 3, 2024, the titles of which are “ST1CAS9 COMPOSITIONS AND METHODS FOR MODULATING A GENOME” and the entireties of which are incorporated herein by reference. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 14, 2025, is named 2017469_0039_SL.xml and is 16,341,326 bytes in size. BACKGROUND Integration of a nucleic acid of interest into a genome occurs at low frequency and with little site specificity, in the absence of a specialized protein to promote the insertion event. Some existing approaches, like CRISPR / Cas9, are more suited for small edits that rely on host repair pathways, and are less effective at integrating longer sequences. Other existing approaches, like Cre / loxP, require a first step of inserting a loxP site into the genome and then a second step of inserting a sequence of interest into the loxP site. There is a need in the art for improved compositions (e.g., proteins and nucleic acids) and methods for inserting, altering, or deleting sequences of interest in a genome. SUMMARY OF THE INVENTION 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. Features of the compositions or methods can include one or more of the following enumerated embodiments. Enumerated Embodiments 1. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain; an Cas9 nickase domain, wherein the RT domain is C-terminal of the Cas9 nickase domain; and Page 1 of 327 12592906v1 Attorney Docket No.2017469-0039 a linker disposed between the RT domain and the Cas9 nickase domain, wherein the linker comprises an amino acid sequence of Table 6, or a sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 2. The gene modifying polypeptide of embodiment 1, wherein the Cas9 nickase domain is a St1Cas9 nickase domain. 3. The gene modifying polypeptide of embodiment 2, wherein the St1Cas9 nickase domain comprises a sequence according to SEQ ID NO: 10208, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. 4. The gene modifying polypeptide of any of embodiments 1-3, wherein the retroviral RT domain has an amino acid sequence of Table 1, or an amino acid sequence with at least 90%, 95%, 97%, 98%, or 99% identity thereto. 5. The gene modifying polypeptide of any of embodiments 1-4, wherein the linker comprises an amino acid sequence of WQAAESYEV (SEQ ID NO: 10154). 6. The gene modifying polypeptide of any of embodiments 1-4, wherein the linker comprises an amino acid sequence of AEIKYDGV (SEQ ID NO: 10162). 7. The gene modifying polypeptide of any of embodiments 1-4, wherein the linker comprises an amino acid sequence of FGMVQGMSTRKGTVVFLDNILEETK (SEQ ID NO: 10166). 8. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain having an amino acid sequence of Table 1, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto; an St1Cas9 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 an amino acid sequence of Table 5, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 9. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain and the linker are from the same row of Table 10. Page 2 of 327 12592906v1 Attorney Docket No.2017469-0039 10. The gene modifying polypeptide of embodiment 8 or 9, wherein the retroviral RT domain and the linker are from the same row of Table 9. 11. The gene modifying polypeptide of any of embodiments 8-10, wherein the retroviral RT domain and the linker are from the same row of Table 8. 12. The gene modifying polypeptide of any of embodiments 8-11, wherein the retroviral RT domain and the linker are from the same row of Table 7. 13. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10316, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10301, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 14. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10317, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10302, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 15. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10318, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10303, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 16. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10319, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and Page 3 of 327 12592906v1 Attorney Docket No.2017469-0039 the linker has an amino acid sequence of SEQ ID NO: 10304, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 17. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10320, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10305, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 18. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10321, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10306, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 19. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10322, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10307, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 20. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10323, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10308, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid Page 4 of 327 12592906v1 Attorney Docket No.2017469-0039 sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 21. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10324, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10309, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 22. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10325, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10310, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 23. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10326, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10311, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 24. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10327, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10312, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 25. The gene modifying polypeptide of embodiment 8, wherein: Page 5 of 327 12592906v1 Attorney Docket No.2017469-0039 the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10328, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO:10313, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 26. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10329, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10314, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 27. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10330, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10315, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 28. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10316, or an amino acid sequence with at least 95% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10301, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 29. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10317, or an amino acid sequence with at least 95% identity thereto, and Page 6 of 327 12592906v1 Attorney Docket No.2017469-0039 the linker has an amino acid sequence of SEQ ID NO: 10302, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 30. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10318, or an amino acid sequence with at least 95% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10303, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 31. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10319, or an amino acid sequence with at least 95% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10304 or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 32. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10320, or an amino acid sequence with at least 95% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10305, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 33. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10321, or an amino acid sequence with at least 95% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10306, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 34. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10322, or an amino acid sequence with at least 95% identity thereto, and Page 7 of 327 12592906v1 Attorney Docket No.2017469-0039 the linker has an amino acid sequence of SEQ ID NO: 10307, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 35. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10323, or an amino acid sequence with at least 95% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10308, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 36. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO:10324, or an amino acid sequence with at least 95% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10309, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 37. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10325, or an amino acid sequence with at least 95% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10310, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 38. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10326, or an amino acid sequence with at least 95% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10311, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 39. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10327, or an amino acid sequence with at least 95% identity thereto, and Page 8 of 327 12592906v1 Attorney Docket No.2017469-0039 the linker has an amino acid sequence of SEQ ID NO: 10312, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 40. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10328, or an amino acid sequence with at least 95% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10313, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 41. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10329, or an amino acid sequence with at least 95% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10314, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 42. The gene modifying polypeptide of embodiment 8, wherein: the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10330, or an amino acid sequence with at least 95% identity thereto, and the linker has an amino acid sequence of SEQ ID NO: 10315, or an amino acid sequence with at least 90% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 43. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10316 and the linker has an amino acid sequence of SEQ ID NO: 10301. 44. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10317 and the linker has an amino acid sequence of SEQ ID NO: 10302. 45. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10318 and the linker has an amino acid sequence of SEQ ID NO: 10303. Page 9 of 327 12592906v1 Attorney Docket No.2017469-0039 46. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10319 and the linker has an amino acid sequence of SEQ ID NO: 10304. 47. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10320 and the linker has an amino acid sequence of SEQ ID NO: 10305. 48. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10321 and the linker has an amino acid sequence of SEQ ID NO: 10306. 49. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 100322 and the linker has an amino acid sequence of SEQ ID NO: 10307. 50. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10323 and the linker has an amino acid sequence of SEQ ID NO: 10308. 51. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10324 and the linker has an amino acid sequence of SEQ ID NO: 10309. 52. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10325 and the linker has an amino acid sequence of SEQ ID NO: 10310. 53. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10326 and the linker has an amino acid sequence of SEQ ID NO: 10311. 54. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10327 and the linker has an amino acid sequence of SEQ ID NO: 10312. 55. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10328 and the linker has an amino acid sequence of SEQ ID NO: 10313. Page 10 of 327 12592906v1 Attorney Docket No.2017469-0039 56. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10329 and the linker has an amino acid sequence of SEQ ID NO: 10314. 57. The gene modifying polypeptide of embodiment 8, wherein the retroviral RT domain has an amino acid sequence of SEQ ID NO: 10330 and the linker has an amino acid sequence of SEQ ID NO: 10315. 58. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a PERV RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10301, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 59. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a BAEVM RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10302, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 60. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a WMSV RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10303, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 61. A gene modifying polypeptide comprising: Page 11 of 327 12592906v1 Attorney Docket No.2017469-0039 a retroviral reverse transcriptase (RT) domain which is a PERV RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10304, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 62. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a PERV RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10305, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 63. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a MLVMS RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10306, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 64. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a PERV RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10307, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. Page 12 of 327 12592906v1 Attorney Docket No.2017469-0039 65. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a BAEVM RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10308, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 66. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a AVIRE RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10309, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 67. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a BAEVM RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10310, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 68. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a XMRV6 RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10311, or a sequence having at least 75%, Page 13 of 327 12592906v1 Attorney Docket No.2017469-0039 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 69. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a BAEVM RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10312, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 70. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a AVIRE RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10313, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 71. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a PERV RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10314, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 72. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a MLVFF RT domain; an St1Cas9 nickase domain, wherein the RT domain is C-terminal of the Cas9 nickase domain; and Page 14 of 327 12592906v1 Attorney Docket No.2017469-0039 a linker disposed between the RT domain and the Cas9 nickase domain, wherein the linker has an amino acid sequence of SEQ ID NO: 10315, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 73. A retroviral reverse transcriptase (RT) domain, wherein the RT domain: a) is a HTLV2_P03363 domain; or b) comprises an amino acid sequence of HLPPPPQVDQFPLNLPERLQALNDLVSKALEAGHIEPYSGPGNNPVFPVKKPNGKWRFIH DLRATNAITTTLTSPSPGPPDLTSLPTALPHLQTIDLTDAFFQIPLPKQYQPYFAFTIPQPCN YGPGTRYAWTVLPQGFKNSPTLFEQQLAAVLNPMRKMFPTSTIVQYMDDILLASPTNEE LQQLSQLTLQALTTHGLPISQEKTQQTPGQIRFLGQVISPNHITYESTPTIPIKSQWTLTEL QVILGEIQWVSKGTPILRKHLQSLYSALHGYRDPRACITLTPQQLHALHAIQQALQHNCR GRLNPALPLLGLISLSTSGTTSVIFQPKQNWPLAWLHTPHPPTSLCPWGHLLACTILTLDK YTLQHYGQLCQSFHHNMSKQALCDFLRNSPHPSVGILIHHMGRFHNLGSQPSGPWKTLL HLPTLLQEPRLLRPIFTLSPVVLDTAPCLFSDGSPQKAAYVLWDQTILQQDITPLPSHETH SAQKGELLALICGLRAAKPWPSLNIFLDSKYLIKYLHSLAIGAFLGTSAHQTLQAALPPLL QGKTIYLHHVRSHTNLPDPISTFNEYTDSLILAPLVPL (SEQ ID NO: 10223), or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. 74. A gene modifying polypeptide comprising: the RT domain of embodiment 100, a Cas9 nickase domain (e.g., as described herein), wherein the RT domain is C-terminal of the Cas9 nickase domain; and a linker (e.g., as described herein) disposed between the RT domain and the Cas9 nickase domain, optionally wherein the linker comprises an amino acid sequence of Table 6, or a sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto. 75. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises the amino acid sequence of a linker as listed in Table 7, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. Page 15 of 327 12592906v1 Attorney Docket No.2017469-0039 76. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises the amino acid sequence of an RT as listed in Table 7, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 77. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises: (i) the amino acid sequence of a linker as listed in a row of Table 7, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) the amino acid sequence of an RT as listed in the same row of Table 7, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 78. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises an amino acid sequence as listed in column 6 of Table 7, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 79. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 12001 – 21744, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 80. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises the amino acid sequence of a linker as listed in Table 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 81. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises the amino acid sequence of an RT as listed in Table 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 82. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 1 of Table 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 2 in the same row of Table 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 83. The gene modifying polypeptide of any of embodiments 1-81, wherein the gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 3 of Table 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, Page 16 of 327 12592906v1 Attorney Docket No.2017469-0039 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 4 in the same row of Table 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 84. The gene modifying polypeptide of any of embodiments 1-81, wherein the gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 5 of Table 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 6 in the same row of Table 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 85. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises the amino acid sequence of a linker as listed in Table 9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 86. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises the amino acid sequence of an RT as listed in Table 9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 87. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 1 of Table 9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 2 in the same row of Table 9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 88. The gene modifying polypeptide of any of embodiments 1-86, wherein the gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 3 of Table 9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 4 in the same row of Table 9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 89. The gene modifying polypeptide of any of embodiments 1-86, wherein the gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 5 of Table 9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as Page 17 of 327 12592906v1 Attorney Docket No.2017469-0039 listed in column 6 in the same row of Table 9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 90. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises the amino acid sequence of a linker as listed in Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 91. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises the amino acid sequence of an RT as listed in Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 92. The gene modifying polypeptide of any of the preceding embodiments, wherein the gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 1 of Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 2 of the same row of Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 93. The gene modifying polypeptide of any of embodiments 1-91, wherein the gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 4 of Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 5 of the same row of Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 94. The gene modifying polypeptide of any of embodiments 1-91, wherein the gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 7 of Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 8 of the same row of Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 95. The gene modifying polypeptide of any of embodiments 1-92, wherein the gene modifying polypeptide comprises an amino acid sequence as listed in column 3 of Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. Page 18 of 327 12592906v1 Attorney Docket No.2017469-0039 96. The gene modifying polypeptide of any of embodiments 1-91, or 93, wherein the gene modifying polypeptide comprises an amino acid sequence as listed in column 6 of Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 97. The gene modifying polypeptide of any of embodiments 1-91, or 94, wherein the gene modifying polypeptide comprises an amino acid sequence as listed in column 9 of Table 10, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 98. The gene modifying polypeptide of any of the preceding embodiments, which comprises a nuclear localization signal (NLS). 99. The gene modifying polypeptide of any of the preceding embodiments, which comprises a first NLS which is N-terminal of the Cas9 nickase domain. 100. The gene modifying polypeptide of any of the preceding embodiments, which comprises an NLS which is C-terminal of the RT domain. 101. The gene modifying polypeptide of any of the preceding embodiments, 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. 102. The gene modifying polypeptide of any of the preceding embodiments, which comprises a first NLS which is N-terminal of the Cas9 nickase domain, wherein the first NLS comprises the amino acid sequence of PAAKRVKLD (SEQ ID NO: 11095). 103. The gene modifying polypeptide of embodiment 102, wherein the first NLS comprises the amino acid sequence of PAAKRVKLDGG (SEQ ID NO: 10207). 104. The gene modifying polypeptide of any of the preceding embodiments, which comprises an NLS which is C-terminal of the RT domain and comprises the amino acid sequence of KRTADGSEFE (SEQ ID NO: 4650). 105. The gene modifying polypeptide of any of the preceding embodiments, which comprises an NLS which is C-terminal of the RT domain and comprises the amino acid sequence of KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 5349). 106. The gene modifying polypeptide of any of the preceding embodiments, which comprises an NLS which is C-terminal of the RT domain and comprises the amino acid sequence of KRTADGSEFESPKKKAKVE (SEQ ID NO: 4651). Page 19 of 327 12592906v1 Attorney Docket No.2017469-0039 107. The gene modifying polypeptide of any of the preceding embodiments, which comprises the amino acid sequence of SEQ ID NO: 10000, which comprises a first NLS and the Cas9 nickase domain. 108. The gene modifying polypeptide of any of the preceding embodiments, which comprises an NLS having the amino acid sequence according to SEQ ID NO: 4649. 109. The gene modifying polypeptide of any of the proceeding embodiments, which comprises a GG amino acid sequence between the Cas9 nickase domain and the linker. 110. The gene modifying polypeptide of embodiment 101, which comprises an AG amino acid sequence between the RT domain and the second NLS. 111. The gene modifying polypeptide of any of the preceding embodiments, which comprises a GG amino acid sequence between the linker and the RT domain. 112. The gene modifying polypeptide of any of the preceding embodiments, which has an amino acid sequence according to any one of SEQ ID NOs: 10001, 10011, 10018-10020, or 17001-21744, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. 113. A nucleic acid molecule encoding a gene modifying polypeptide according to any of the preceding embodiments. 114. The nucleic acid molecule of embodiment 113, which comprises RNA. 115. A cell comprising: i) a gene modifying polypeptide of any of embodiments 1-112, or ii) a nucleic acid encoding the gene modifying polypeptide. 116. A system comprising: i) a gene modifying polypeptide of any of embodiments 1-112, 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 (e.g., a St1Cas9 gRNA scaffold) that binds the Cas9 nickase domain of the gene modifying polypeptide; c) a heterologous object sequence; and d) a primer binding site (PBS) sequence. 117. The system of embodiment 116, wherein the gene modifying polypeptide comprises: Page 20 of 327 12592906v1 Attorney Docket No.2017469-0039 a retroviral reverse transcriptase (RT) domain having an amino acid sequence of Table 1, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto; an St1Cas9 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 an amino acid sequence of Table 5, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. 118. The system of embodiment 116 or 117, wherein the gRNA scaffold is a variant St1Cas9 scaffold having a deletion of part or all of Stem loop 2. 119. The system of any of embodiments 116-118, wherein the template RNA has a nucleic acid sequence of any of Tables 20, 22, or 24, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. 120. The system of any of embodiments 116-119, wherein the template RNA comprises one or more chemical modifications. 121. The system of any of embodiments 116-120, wherein the template RNA comprises the chemical modifications set out in any template RNA sequence of Tables 19, 21, or 23. 122. The system of any of embodiments 116-121, wherein the target nucleic acid sequence is a first portion of the human SERPINA1 gene. 123. The system of any of embodiments 116-122, wherein the gRNA spacer has a nucleotide sequence of AAGGCUGUGCUGACCAUCGA (SEQ ID NO: 21748). 124. The system of any of embodiments 116-123, wherein the heterologous object sequence is to introduce a mutation into (e.g., to correct a mutation in) a second portion of the human SERPINA1 gene. 125. The system of any of embodiments 116-124, wherein the heterologous object sequence has a nucleotide sequence of UUUCUCGUCG (SEQ ID NO: 21749). 126. The system of any of embodiments 116-125, wherein the PBS sequence comprises at least 3, 4, 5, 6, 7, or 8 bases with 100% identity to a third portion of the human SERPINA1 gene. 127. The system of any of embodiments 116-126, wherein the PBS has a nucleotide sequence of AUGGUCAG (SEQ ID NO: 21750). 128. A lipid nanoparticle formulation comprising the system of any of embodiments 116-127. Page 21 of 327 12592906v1 Attorney Docket No.2017469-0039 129. A method for modifying a target nucleic acid molecule in a cell, the method comprising contacting the cell with the system of any of embodiments 116-127, thereby modifying the target nucleic acid molecule. 130. The method of embodiment 129, wherein the cell is a liver cell. 131. A method for modifying a target nucleic acid molecule in a tissue, the method comprising contacting the tissue with the system of any of embodiments 116-127, thereby modifying the target nucleic acid molecule. 132. The method of embodiment 131, wherein the tissue is liver. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram depicting components of a gene modifying system as described herein. FIG. 1A is a diagram showing a gene modifying polypeptide comprising a Cas nickase domain (e.g., spCas9 N863A) and a reverse transcriptase domain (RT domain) which are linked by a linker. FIG. 1B is a diagram showing a template RNA comprising, from 5’ to 3’, a gRNA spacer, a gRNA scaffold, a heterologous object sequence, and a primer binding site sequence (PBS sequence). A heterologous object sequence can comprise a mutation region that comprises one or more sequence differences relative to a target site. A heterologous object sequence can also comprise a pre-edit homology region and a post-edit homology region, which flank a mutation region. Without wishing to be bound by theory, it is thought that a gRNA spacer of a template RNA binds to a second strand of a target site in the genome, and a gRNA scaffold of the template RNA binds to a gene modifying polypeptide, e.g., localizing the gene modifying polypeptide to a target site in the genome. It is thought that a Cas domain of a gene modifying polypeptide nicks a target site (e.g., a first strand of the target site), e.g., allowing a PBS sequence to bind to a sequence adjacent to the target site to be altered on the first strand of the target site. It is thought that an RT domain of a gene modifying polypeptide uses a first strand of a target site that is bound to a complementary sequence comprising a PBS sequence of a template RNA as a primer and a heterologous object sequence of the template RNA as a template to, e.g., polymerize a sequence complementary to the heterologous object sequence. Without wishing to be bound by theory, it is thought that reverse transcription can then proceed through a pre-edit homology region, then through a mutation region, and then through a post-edit homology region, thereby producing a DNA strand comprising a mutation specified by a heterologous object sequence. Page 22 of 327 12592906v1 Attorney Docket No.2017469-0039 FIG. 2A is a diagram illustrating the positions of the reference dSL2 St1Cas9 scaffold sequence. FIG. 2B is a diagram illustrating the positions of the reference wild-type St1Cas9 scaffold sequence. FIG. 3 is a diagram illustrating the hypothesized secondary structure of a wild-type St1Cas9 gRNA scaffold and is overlaid with description of variants described herein. FIG. 4A is a schematic diagram of an exemplary gene modifying polypeptide, a fusion polypeptide comprising, e.g. from N terminus to C terminus, a first nuclear localization signal (NLS) sequence, a Cas nickase domain (e.g., St1 Cas9), a linker selected from Table 5, a reverse transcriptase domain (RT domain) selected from Table 1, and a second NLS sequence. FIG. 4B is a schematic flow diagram of an exemplary polypeptide screen conducted with pooled elements from a library of gene modifying polypeptide candidates. FIG. 4C is a schematic diagram of an exemplary assay for detecting gene editing. A target reporter gene (LP), which comprises a stop codon that prevents the translation of mKate is expressed in a test cell line. If there is perfect mutation (e.g., edit) in the LP stop codon sequence, cells will express green fluorescent protein and red fluorescent protein (GFP-RFP) as compared to only GFP in the event that a mutation in the LP stop codon sequence has not taken place. FIG. 4D is a graph showing mean enrichment scores of exemplary reverse transcriptase (RT) families screened as described herein. Mean enrichment scores are a representative measure of genome editing activity. FIG. 5A is a bar graph showing the rewriting efficiency in the livers of mice administered with exemplary gene modifying systems comprising different template RNAs comprising scaffold chemical modifications. FIG. 5B is a bar graph showing the % indel levels in the livers of mice administered with the exemplary gene modifying systems evaluated in FIG. 5A. FIG. 6A is a bar graph showing the rewriting efficiency in the livers of mice administered with exemplary gene modifying systems comprising different template RNAs comprising scaffold chemical modifications in combination with fluoro modifications at the heterologous object sequence. Page 23 of 327 12592906v1 Attorney Docket No.2017469-0039 FIG. 6B is a bar graph showing the % indel levels in the livers of mice administered with the exemplary gene modifying systems evaluated in FIG. 6A. FIG. 7A is a bar graph showing the rewriting efficiency in the livers of mice administered with exemplary gene modifying systems comprising different gene modifying polypeptides and different template RNAs comprising scaffold chemical modifications in combination with fluoro modifications at the heterologous object sequence. FIG. 7B is a bar graph showing the % indel levels in the livers of mice administered with the exemplary gene modifying systems evaluated in FIG. 7A. FIG. 8A is a bar graph showing the % corrected genomic DNA in the livers of hSERPINA1 E342K mice by administered with exemplary gene modifying systems comprising RNAIVT6241 or RNAIVT6898 polypeptides over evaluated dosages. FIG. 8B is a bar graph showing the % corrected genomic DNA in the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 7 days post-administration. FIG. 8C is a bar graph showing the % corrected genomic DNA in the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 21 days post- administration. FIG. 9is a bar graph showing the % edited mRNA in the livers of hSERPINA1 E342K and NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 21 days post- administration. FIG. 10A is a bar graph showing the % indels introduced into the livers of hSERPINA1 E342K by exemplary gene modifying polypeptides RNAVT6241 and RNAIVT6838 over evaluated dosages. FIG. 10B is a bar graph showing the % indels introduced into the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 7 days post-administration. FIG. 10C is a bar graph showing the % indels introduced into the livers of NSG-PiZ mice by exemplary RNAIVT6898 systems over evaluated dosages at 21 days post-administration. FIG. 11A is a bar graph showing the serum concentration of human A1AT in hSERPINA1 E342K mice administered with exemplary gene modifying systems comprising RNAIVT6241 or RNAIVT6898 polypeptides over evaluated dosages. Page 24 of 327 12592906v1 Attorney Docket No.2017469-0039 FIG. 11B is a bar graph showing the serum concentration of human A1AT in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 7 days post- administration. FIG. 11C is a bar graph showing the serum concentration of human A1AT in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 21 days post- administration. FIG. 12A is a bar graph showing the % liver area occupied by globules in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 7 days post- administration. FIG. 12B is a bar graph showing the % liver area occupied by globules in NSG-PiZ mice administered with exemplary RNAIVT6898 systems over evaluated dosages at 21 days post- administration. FIG. 13A is a bar graph showing the % corrected genomic DNA in the livers of NSG-PiZ mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages. FIG. 13B is a bar graph showing the % edited mRNA in the livers of NSG-PiZ mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages. FIG. 13C is a bar graph showing the % indels introduced into the livers of NSG-PiZ mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages. FIG. 14A is a bar graph showing the serum concentration of human A1AT in NSG-PiZ mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages. FIG. 14B is a line graph showing the serum concentration of human A1AT as a function of % genomic DNA rewriting in NSG-PiZ mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS22230 template RNA over evaluated dosages. FIG. 15 is a bar a graph showing the % hA1AT isoform present in liver samples from NSG- PiZ mice treated with saline or an exemplary RNAIVT9315-containing gene modifying system at 0.5 mg / kg (mpk) or 1.5 mg / kg. Page 25 of 327 12592906v1 Attorney Docket No.2017469-0039 FIGs. 16A is a bar graph showing the % corrected genomic DNA in the livers of cynomolgus monkeys dosed with an exemplary gene modifying system including an mRNA encoding an exemplary RNAIVT9315 gene modifying polypeptide and an RNACS22390 tgRNA at two different doses (2 mg / kg or 3 mg / kg) and formulated in different LNPs (2 mg / kg in exemplary LNP formulation 1, and 3 mg / kg in exemplary LNP formulation 2) as measured by Amp-Seq. FIG. 16B is a bar graph showing the % indels introduced into the livers of cynomolgus monkeys dosed with an exemplary gene modifying system including an mRNA encoding an exemplary RNAIVT9315 gene modifying polypeptide and an RNACS22390 tgRNA at two different doses (2 mg / kg or 3 mg / kg) and formulated in different LNPs (2 mg / kg in exemplary LNP formulation 1, and 3 mg / kg in exemplary LNP formulation 2) as measured by Amp-Seq. FIG. 17A is a bar graph showing the % corrected genomic DNA in the livers of hSERPINA1 E342K mice by exemplary gene modifying systems comprising RNAIVT9315 or RNAIVT9318 gene modifying polypeptides and RNACS22230 template RNA over evaluated dosages. FIG. 17B is a bar graph showing the % indels introduced in the livers of hSERPINA1 E342K mice by exemplary gene modifying systems comprising RNAIVT9315 or RNAIVT9318 gene modifying polypeptides and RNACS22230 template RNA over evaluated dosages. FIGs. 18A to 18D show bar graphs of the rewriting performance in the livers of hSERPINA1 E342K mice administered with exemplary St1Cas9-based gene modifying systems comprising exemplary RNAIVT9315 gene modifying polypeptide and exemplary template RNAs RNACS24756 or RNACS24757 at 0.012 mg / kg (FIG. 18A), 0.025 mg / kg (FIG. 18B), 0.05 mg / kg (FIG. 18C) and 0.1 mg / kg (FIG. 18D). FIGs. 19A to 19D show bar graphs of the % indel levels in the livers of hSERPINA1 E342K mice administered with exemplary St1Cas9-based gene modifying systems comprising exemplary RNAIVT9315 gene modifying polypeptide and exemplary template RNAs RNACS24756 or RNACS24757 at 0.012 mg / kg (FIG. 19A), 0.025 mg / kg (FIG. 19B), 0.05 mg / kg (FIG. 19C) and 0.1 mg / kg (FIG. 19D). FIG. 20A is a bar graph showing the % corrected genomic DNA in the livers of hSERPINA1 E342K mice by exemplary gene modifying systems comprising RNAIVT9315 Page 26 of 327 12592906v1 Attorney Docket No.2017469-0039 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg / kg) and formulated in LNP. FIG. 20B is a bar graph showing the % edited mRNA in the livers of hSERPINA1 E342K mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg / kg) and formulated in LNP. FIG. 20C is a bar graph showing the % indels introduced in the livers of hSERPINA1 E342K mice by exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg / kg) and formulated in LNP. FIG. 21A is a bar graph showing the serum concentration of human A1AT in hSERPINA1 E342K mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg / kg) and formulated in LNP. FIG. 21B is a line graph showing the serum concentration of human A1AT as a function of % genomic DNA rewriting in hSERPINA1 E342K mice administered with exemplary gene modifying systems comprising RNAIVT9315 gene modifying polypeptide and RNACS27457 template RNA over evaluated dosages (mpk = mg / kg) and formulated in LNP. Definitions The term “Cas9 nickase domain,” as used herein, refers to a domain having the amino acid sequence of a Cas9 protein having nickase activity. In some embodiments, a Cas9 sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identity to a wild-type St1Cas9 or SpCas9 sequence. A Cas9 sequence may have a point mutation relative to the wild-type sequence. In some embodiments, a Cas9 point mutant has nickase activity. The term “St1Cas9 nickase domain” refers to a Cas9 nickase domain having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, identity to a wild-type St1Cas9 sequence. An St1Cas9 nickase domain does not have 100% identity to a wild-type SpCas9 nickase domain. The term “chemically modified nucleotide,” as used herein, refers to a nucleotide comprising one or more structural differences relative to the canonical ribonucleotides (i.e., G, Page 27 of 327 12592906v1 Attorney Docket No.2017469-0039 U, C, and A). A chemically modified nucleotide may have (relative to a canonical nucleotide) a chemically modified nucleobase, a chemically modified sugar, a chemically modified phosphodiester linkage, or a combination thereof. In some embodiments, a chemically modified nucleotide is a 2'-O-methyl nucleotide, e.g., 2'-O-methyl-Adenosine, 2'-O-methyl-Cytidine, 2'-O- methyl-Guanosine, or 2'-O-methyl-Uridine. No particular process of making is implied; for instance, a chemically modified nucleotide can be produced directly by chemical synthesis, or by covalently modifying a canonical nucleotide. The term “chemical modification,” as used herein, refers to a structural difference of a chemical modified nucleotide relative to the canonical ribonucleotides (i.e., G, U, C, and A). A chemical modification may comprise a modification resulting in a chemically modified nucleobase, a chemically modified sugar, a chemically modified phosphodiester linkage, or a combination thereof. In some embodiments, a chemical modification is 2'-O-methylation or 2’- fluoro modification. No particular process of making is implied; for instance, a chemical modification can be produced directly by chemical synthesis, or by covalently modifying a canonical nucleotide. The term “expression cassette,” as used herein, refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the instant invention. A “gRNA spacer,” as used herein, refers to a portion of a nucleic acid that has complementarity to a target nucleic acid and can, together with a gRNA scaffold, target a Cas protein to the target nucleic acid. A “gRNA scaffold,” as used herein, refers to a portion of a nucleic acid that can bind a Cas protein and can, together with a gRNA spacer, target the Cas protein to the target nucleic acid. In some embodiments, the gRNA scaffold comprises a crRNA sequence, tetraloop, and tracrRNA sequence. The term “St1Cas9 scaffold,” as used herein, refers to a gRNA scaffold that can bind an St1Cas9 protein and can, together with a gRNA spacer, target the St1Cas9 protein to the target nucleic acid. In some embodiments, an St1Cas9 scaffold comprises a crRNA sequence, tetraloop, and tracerRNA sequence. An exemplary position of St1Cas9 scaffold within an exemplary template RNA is illustrated in FIG. 1. Page 28 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, an St1Cas9 scaffold comprises a full length wild-type sequence. In some embodiments, an St1Cas9 scaffold comprises a sequence with at least 80%, 85%. 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of GUCUUUGUACUCUGGUACCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCA ACACCCUGUCAUUUUAUGGCAGGGUGUUUU (SEQ ID NO: 25999). In some embodiments, an St1Cas9 scaffold comprises a sequence identical to SEQ ID NO: 25999. In some embodiments, an St1Cas9 scaffold comprises a truncation mutant. In some embodiments, an St1Cas9 scaffold comprises a sequence with at least 80%, 85%. 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of GUCUUUGUACUCUGGUACCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCA (SEQ ID NO: 26000). In some embodiments, an St1Cas9 scaffold comprises a sequence identical to SEQ ID NO: 26000. In some embodiments, an St1Cas9 scaffold comprises an insertion, deletion, or substitution to a reference sequence of SEQ ID NO: 25999 or 26000. In some embodiments, an St1Cas9 scaffold comprises a chemically modified nucleotide. A “variant gRNA scaffold,” as used herein, refers to gRNA scaffold having a non- naturally occurring sequence. In some embodiments, a variant gRNA scaffold sequence comprises one or more substitutions relative to the closest naturally occurring sequence. In some embodiments, a variant gRNA scaffold sequence comprises one or more insertions relative to the closest naturally occurring sequence. In some embodiments, a variant gRNA scaffold sequence comprises one or more deletions relative to the closest naturally occurring sequence. As used herein, the term “position” with respect to an St1Cas9 scaffold refers to a nucleotide of the St1Cas9 scaffold that aligns with the corresponding nucleotide of the reference sequence of SEQ ID NO: 25999. The positions of a reference sequence are illustrated in FIG. 2. Alignments of nucleic acid or polypeptide sequences can be performed by using a sequence analysis tool such as Basic Local Alignment Search Tool (BLAST), for instance NIH megablast using default parameters. In some embodiments, a position of an St1Cas9 scaffold can be identified by providing an alignment of the St1Cas9 scaffold (query sequence) to a reference sequence of SEQ ID NO: 25999 (a full length wild-type sequence, see e.g., FIG 2B) or SEQ ID NO: 26000 (a truncation mutant, see e.g., FIG. 2A), and identifying the position in the query sequence that corresponds to the position in the reference sequence. For example, in an St1Cas9 scaffold consisting of the Page 29 of 327 12592906v1 Attorney Docket No.2017469-0039 sequence of SEQ ID NO: 25999 except that the 5’ most G is substituted with a single nucleotide other than G, the substituted position is position 1. As another example, in an St1Cas9 scaffold consisting of the sequence of SEQ ID NO: 25999 except that a single new nucleotide is inserted just 5’ of the 5’ most G, the G is still position 1. As yet another example, in an St1Cas9 scaffold consisting of the sequence of SEQ ID NO: 25999 except that a sequence of n nucleotides is inserted between the G of position 1 and the U of position 2, nucleotides 3’ of the insert maintain their original position number. For example, the U of position 2 is still position 2 rather than position n+2. A nucleotide that is inserted relative to the reference sequence need not be assigned a position number. A range of nucleotides includes all nucleotides in that range regardless of whether they are assigned a number; for example, if a scaffold comprises a chemically modified nucleotide at each of positions 12 through 21, and the scaffold comprises inserted nucleotides anywhere between positions 12 and 21, then the scaffold comprises chemically modified nucleotides at each of the inserted nucleotides situated anywhere between positions 12 and 21 (which inserted nucleotides do not have a position number in this example), as well as chemically modified nucleotides at positions 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21. A “gene modifying polypeptide,” as used herein, refers to a polypeptide comprising a retroviral reverse transcriptase, or a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to a retroviral reverse transcriptase, which is capable of integrating a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell). In some embodiments, a gene modifying polypeptide is capable of integrating a sequence substantially without relying on host machinery. In some embodiments, a gene modifying polypeptide integrates a sequence into a random position in a genome. In some embodiments, a gene modifying polypeptide integrates a sequence into a specific target site. In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding a template nucleic acid, 2) binding a target DNA molecule, and 3) integration of at least a portion of the template nucleic acid into the target DNA. Gene modifying polypeptides include both naturally occurring polypeptides as well as Page 30 of 327 12592906v1 Attorney Docket No.2017469-0039 engineered variants of the foregoing, e.g., having one or more amino acid substitutions to a naturally occurring sequence. Gene modifying polypeptides also include heterologous constructs, e.g., where one or more of the domains are heterologous to each other, whether through a heterologous fusion (or other conjugate) of otherwise wild-type domains, as well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub- domain or other substituted domain. Exemplary gene modifying polypeptides, and systems comprising the same can be used in methods provided herein and described, e.g., in PCT / US2021 / 020948, which is incorporated herein by reference with respect to gene modifying polypeptides that comprise a retroviral reverse transcriptase domain. In some embodiments, a gene modifying polypeptide integrates a sequence into a gene. In some embodiments, a gene modifying polypeptide integrates a sequence into a sequence outside of a gene. A “gene modifying system,” as used herein, refers to a system comprising a gene modifying polypeptide and a template nucleic acid. The term “domain,” as used herein, refers to a structure of a biomolecule that contributes to a specified function of the biomolecule. A domain may comprise a contiguous region (e.g., a contiguous sequence) or distinct, non-contiguous regions (e.g., non-contiguous sequences) of a biomolecule. Examples of protein domains include, but are not limited to, an endonuclease domain, a DNA binding domain, a reverse transcription domain; an example of a domain of a nucleic acid is a regulatory domain, such as a transcription factor binding domain. In some embodiments, a domain (e.g., a Cas domain) can comprise two or more smaller domains (e.g., a DNA binding domain and an endonuclease domain). As used herein, the term “exogenous,” when used with reference to a biomolecule (such as a nucleic acid sequence or polypeptide) means that the biomolecule was introduced into a host genome, cell or organism by the hand of man. For example, a nucleic acid that is as added into an existing genome, cell, tissue or subject using recombinant DNA techniques or other methods is exogenous to the existing nucleic acid sequence, cell, tissue or subject. As used herein, “first strand” and “second strand,” as used to describe the individual DNA strands of target DNA, distinguish the two DNA strands based upon which strand a reverse transcriptase domain initiates polymerization, e.g., based upon where target primed synthesis initiates. A “first strand” refers to the strand of a target DNA upon which a reverse transcriptase domain initiates polymerization, e.g., where target primed synthesis initiates. A “second strand” Page 31 of 327 12592906v1 Attorney Docket No.2017469-0039 refers to the other strand of the target DNA. First and second strand designations do not describe a target site DNA strands in other respects; for example, in some embodiments the first and second strands are nicked by a polypeptide described herein, but the designations ‘first’ and ‘second’ strand have no bearing on the order in which such nicks occur. The term “heterologous,” as used herein to describe a first element in reference to a second element means that a first element and second element do not exist in nature disposed as described. For example, a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, or (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions. For example, a heterologous regulatory sequence (e.g., promoter, enhancer) may be used to regulate expression of a gene or a nucleic acid molecule in a way that is different than the gene or a nucleic acid molecule is normally expressed in nature. In another example, a heterologous domain of a polypeptide or nucleic acid sequence (e.g., a DNA binding domain of a polypeptide or nucleic acid encoding a DNA binding domain of a polypeptide) may be disposed relative to other domains or may be a different sequence or from a different source, relative to other domains or portions of a polypeptide or its encoding nucleic acid. In some embodiments, a heterologous nucleic acid molecule may exist in a native host cell genome but may have an altered expression level or have a different sequence or both. In other embodiments, heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector). As used herein, “insertion” of a sequence into a target site refers to the net addition of DNA sequence at a target site, e.g., where there are new nucleotides in a heterologous object sequence with no cognate positions in the unedited target site. In some embodiments, a nucleotide alignment of a PBS sequence and heterologous object sequence to a target nucleic acid sequence would result in an alignment gap in the target nucleic acid sequence. Page 32 of 327 12592906v1 Attorney Docket No.2017469-0039 As used herein, a “deletion” generated by a heterologous object sequence in a target site refers to the net deletion of DNA sequence at the target site, e.g., where there are nucleotides in the unedited target site with no cognate positions in the heterologous object sequence. In some embodiments, a nucleotide alignment of the PBS sequence and heterologous object sequence to the target nucleic acid sequence would result in an alignment gap in the molecule comprising the PBS sequence and heterologous object sequence. The term “mutation region,” as used herein, refers to a region in a template RNA having one or more sequence difference relative to the corresponding sequence in a target nucleic acid. The one or more sequence difference may comprise, for example, a substitution, insertion, frameshift, or deletion. The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence are inserted, deleted, or changed compared to a reference (e.g., native) nucleic acid sequence. A single alteration may be made at a locus (a point mutation), or multiple nucleotides may be inserted, deleted, or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art. “Nucleic acid molecule” refers to both RNA and DNA molecules including, without limitation, complementary DNA (“cDNA”), genomic DNA (“gDNA”), and messenger RNA (“mRNA”), and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced, such as RNA templates, as described herein. A nucleic acid molecule can be double-stranded or single-stranded, circular, or linear. If single-stranded, a nucleic acid molecule can be a sense strand or an antisense strand. Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:,” or “nucleic acid comprising SEQ ID NO: 1” refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ ID NO: 1, or (ii) a sequence complimentary to SEQ ID NO: 1. The choice between the two is dictated by the context in which SEQ ID NO:1 is used. For instance, if a nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to a desired target. Nucleic acid sequences of the present disclosure may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally Page 33 of 327 12592906v1 Attorney Docket No.2017469-0039 occurring nucleotides with an analog, inter-nucleotide modifications such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendant moieties, (for example, polypeptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.). Also included are chemically modified bases (see, for example, Table 13), backbones (see, for example, Table 14), and modified caps (see, for example, Table 15). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of a molecule, e.g., peptide nucleic acids (PNAs). Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as modifications found in “locked” nucleic acids (LNAs). In some embodiments, nucleic acids are in operative association with additional genetic elements, such as tissue-specific expression- control sequence(s) (e.g., tissue-specific promoters and tissue-specific microRNA recognition sequences), as well as additional elements, such as inverted repeats (e.g., inverted terminal repeats, such as elements from or derived from viruses, e.g., AAV ITRs) and tandem repeats, inverted repeats / direct repeats, homology regions (segments with various degrees of homology to a target DNA), untranslated regions (UTRs) (5´, 3´, or both 5´ and 3´ UTRs), and various combinations of the foregoing. Nucleic acid elements of systems disclosed in the present application may be provided in a variety of topologies, including single-stranded, double- stranded, circular, linear, linear with open ends, linear with closed ends, and particular versions of these, such as doggybone DNA (dbDNA), and closed-ended DNA (ceDNA). As used herein, a “gene expression unit” is a nucleic acid sequence comprising at least one regulatory nucleic acid sequence operably linked to at least one effector sequence. A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous or non-contiguous. Where necessary to join two protein-coding regions, operably linked sequences may be in the same reading frame. Page 34 of 327 12592906v1 Attorney Docket No.2017469-0039 The terms “host genome” or “host cell,” as used herein, refer to a cell and / or its genome into which protein and / or genetic material has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell and / or genome, but to the progeny of such a cell and / or the genome of the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A host genome or host cell may be an isolated cell or cell line grown in culture, or genomic material isolated from such a cell or cell line, or may be a host cell or host genome which composing living tissue or an organism. In some embodiments, a host cell may be an animal cell or a plant cell, e.g., as described herein. In some embodiments, a host cell may be a mammalian cell, a human cell, avian cell, reptilian cell, bovine cell, horse cell, pig cell, goat cell, sheep cell, chicken cell, or turkey cell. In some embodiments, a host cell may be a corn cell, soy cell, wheat cell, or rice cell. As used herein, “operative association” describes a functional relationship between two nucleic acid sequences, such as a 1) promoter and 2) a heterologous object sequence, and means, in such example, the promoter and heterologous object sequence (e.g., a gene of interest) are oriented such that, under suitable conditions, the promoter drives expression of the heterologous object sequence. For instance, a template nucleic acid carrying a promoter and a heterologous object sequence may be single-stranded, e.g., either the (+) or (-) orientation. An “operative association” between the promoter and the heterologous object sequence in this template means that, regardless of whether the template nucleic acid will be transcribed in a particular state, when it is in the suitable state (e.g., is in the (+) orientation, in the presence of required catalytic factors, and NTPs, etc.), it is accurately transcribed. Operative association applies analogously to other pairs of nucleic acids, including other tissue-specific expression control sequences (such as enhancers, repressors and microRNA recognition sequences), IR / DR, ITRs, UTRs, or homology regions and heterologous object sequences or sequences encoding a retroviral RT domain. The term “primer binding site sequence” or “PBS sequence,” as used herein, refers to a portion of a template RNA capable of binding to a region in a target nucleic acid sequence. In some embodiments, a PBS sequence is a nucleic acid sequence comprising at least 3, 4, 5, 6, 7, or 8 bases with 100% identity to a region in a target nucleic acid sequence. In some Page 35 of 327 12592906v1 Attorney Docket No.2017469-0039 embodiments, a primer region comprises at least 5, 6, 7, 8 bases with 100% identity to a region in a target nucleic acid sequence. Without wishing to be bound by theory, in some embodiments, when a template RNA comprises a PBS sequence and a heterologous object sequence, the PBS sequence binds to a region in a target nucleic acid sequence, allowing a reverse transcriptase domain to use that region as a primer for reverse transcription, and to use the heterologous object sequence as a template for reverse transcription. As used herein, a “stem-loop sequence” refers to a nucleic acid sequence (e.g., RNA sequence) with sufficient self-complementarity to form a stem-loop, e.g., having a stem comprising at least two (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) base pairs, and a loop with at least three (e.g., four) base pairs. The stem may comprise mismatches or bulges. As used herein, a “tissue-specific expression-control sequence” means nucleic acid elements that increase or decrease the level of a transcript comprising the heterologous object sequence in a target tissue in a tissue-specific manner, e.g., preferentially in on-target tissue(s), relative to off-target tissue(s). In some embodiments, a tissue-specific expression-control sequence preferentially drives or represses transcription, activity, or the half-life of a transcript comprising the heterologous object sequence in the target tissue in a tissue-specific manner, e.g., preferentially in an on-target tissue(s), relative to an off-target tissue(s). Exemplary tissue- specific expression-control sequences include tissue-specific promoters, repressors, enhancers, or combinations thereof, as well as tissue-specific microRNA recognition sequences. Tissue specificity refers to on-target (tissue(s) where expression or activity of the template nucleic acid is desired or tolerable) and off-target (tissue(s) where expression or activity of the template nucleic acid is not desired or is not tolerable). For example, a tissue-specific promoter drives expression preferentially in on-target tissues, relative to off-target tissues. In contrast, a microRNA that binds the tissue-specific microRNA recognition sequences is preferentially expressed in off-target tissues, relative to on-target tissues, thereby reducing expression of a template nucleic acid in off-target tissues. Accordingly, a promoter and a microRNA recognition sequence that are specific for the same tissue, such as the target tissue, have contrasting functions (promote and repress, respectively, with concordant expression levels, i.e., high levels of the microRNA in off-target tissues and low levels in on-target tissues, while promoters drive high expression in on-target tissues and low expression in off-target tissues) with regard to the transcription, activity, or half-life of an associated sequence in that tissue. Page 36 of 327 12592906v1 Attorney Docket No.2017469-0039 As used herein, “indel” refers to a mutation resulting from an insertion, deletion, or a combination thereof. As will be appreciated by those skilled in the art, an indel in a coding region of a genomic sequence will result in a frameshift mutation, unless the length of the indel is a multiple of three. In some embodiments, a genetic modification is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one of the nucleotides. A system of the present disclosure can be used to induce an indel of any length or a point mutation in a target polynucleotide sequence. As used herein, the term “retroviral reverse transcriptase domain” or “retroviral RT domain” refers to a reverse transcriptase domain having a sequence of a reverse transcriptase domain from a wild-type retrovirus, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. DETAILED DESCRIPTION This disclosure provides compositions for targeting, editing, modifying or manipulating a DNA sequence (e.g., inserting a heterologous object sequence into a target site of a mammalian genome) at one or more locations in a DNA sequence in a cell, tissue or subject, e.g., in vivo or in vitro. Gene modifying systems In some embodiments, a gene modifying system described herein comprises: (A) a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide, wherein the gene modifying polypeptide comprises (i) a reverse transcriptase domain, and either (x) an endonuclease domain that contains DNA binding functionality or (y) an endonuclease domain and separate DNA binding domain; and (B) a template RNA. A gene modifying polypeptide, in some embodiments, acts as a substantially autonomous protein machine capable of integrating a template nucleic acid sequence into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell), substantially without relying on host machinery. For example, a gene modifying polypeptide may comprise a DNA-binding domain, a reverse transcriptase domain, and an endonuclease domain. In some embodiments, a DNA-binding function may involve an RNA component that directs a gene modifying polypeptide to a DNA sequence, e.g., a gRNA spacer. In other embodiments, a gene modifying polypeptide may comprise a reverse transcriptase domain and an endonuclease domain. An RNA template Page 37 of 327 12592906v1 Attorney Docket No.2017469-0039 element of a gene modifying system may be heterologous to a gene modifying polypeptide element and provides an object sequence to be inserted (reverse transcribed) into a host genome. In some embodiments, a gene modifying polypeptide is capable of target primed reverse transcription. In some embodiments, a gene modifying polypeptide is capable of second-strand synthesis. In some embodiments, a gene modifying system is combined with a second polypeptide. In some embodiments, a second polypeptide may comprise an endonuclease domain. In some embodiments, a second polypeptide may comprise a polymerase domain, e.g., a reverse transcriptase domain. In some embodiments, a second polypeptide may comprise a DNA- dependent DNA polymerase domain. In some embodiments, a second polypeptide aids in completion of a genome edit, e.g., by contributing to second-strand synthesis or DNA repair resolution. A functional gene modifying polypeptide can be made up of unrelated DNA binding, reverse transcription, and endonuclease domains. This modular structure allows combining of functional domains, e.g., dCas9 (DNA binding), MMLV reverse transcriptase (reverse transcription), FokI (endonuclease). In some embodiments, multiple functional domains may arise from a single protein, e.g., Cas9 or Cas9 nickase (DNA binding, endonuclease). In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding a template nucleic acid, 2) binding a target DNA molecule, and 3) integration of at least a portion of the template nucleic acid into the target DNA. In some embodiments, a gene modifying polypeptide is an engineered polypeptide that comprises one or more amino acid substitutions to a corresponding naturally occurring sequence. In some embodiments, a gene modifying polypeptide comprises two or more domains that are heterologous relative to each other, e.g., through a heterologous fusion (or other conjugate) of otherwise wild-type domains, or well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain. For instance, in some embodiments, one or more of: an RT domain is heterologous to a DNA-binding domain (DBD); a DBD is heterologous to an endonuclease domain; or an RT domain is heterologous to an endonuclease domain. In some embodiments, a template RNA molecule for use in a system of the present disclosure comprises, from 5′ to 3′ (1) a gRNA spacer; (2) a gRNA scaffold; (3) a heterologous Page 38 of 327 12592906v1 Attorney Docket No.2017469-0039 object sequence; and (4) a primer binding site (PBS) sequence. In some embodiments, a gRNA spacer is about18 to -22 nucleotides in length (e.g., about 20 nucleotides in length). In some embodiments, a gRNA scaffold comprises one or more hairpin loops, e.g., 1, 2, or 3 loops for associating a template RNA with a Cas domain, e.g., a nickase Cas9 domain. In some embodiments, a gRNA scaffold comprises the sequence, from 5′ to 3′, GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGGACCGAGTCGGTCC (SEQ ID NO: 5008). In some embodiments, a heterologous object sequence is, e.g., 7-74, e.g., 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 nucleotides or, 80-90 nucleotides in length. In some embodiments, a first (i.e., 5′-most) base of a heterologous object sequence is not C. In some embodiments, a PBS sequence that binds a target priming sequence after nicking occurs is e.g., 3-20 nucleotides, e.g., 7-15 nucleotides, e.g., 12-14 nucleotides in length. In some embodiments, a PBS sequence has 40-60% GC content. In some embodiments, a second gRNA associated with a system of the present disclosure may help drive complete integration. In some embodiments, a second gRNA may target a location that is 0-200 nucleotides away from a first-strand nick, e.g., 0-50, 50-100, 100-200 nucleotides away from the first-strand nick. In some embodiments, a second gRNA can only bind its target sequence after an edit is made, e.g., the gRNA binds a sequence present in a heterologous object sequence, but not in the initial target sequence. In some embodiments, a gene modifying system described herein is used to make an edit in HEK293, K562, U2OS, or HeLa cells. In some embodiment, a gene modifying system is used to make an edit in primary cells, e.g., primary liver cells or primary lung cells. In some embodiments, a gene modifying polypeptide as described herein comprises a reverse transcriptase or RT domain (e.g., as described herein) that comprises a MoMLV RT sequence or variant thereof. In embodiments, a MoMLV RT sequence comprises one or more mutations selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, and K103L. In some embodiments, a MoMLV RT sequence comprises a combination of mutations, such as D200N, L603W, and T330P, optionally further including T306K and / or W313F. In some embodiments, an endonuclease domain (e.g., as described herein) is Cas9. In some embodiments, an endonuclease domain is nCas9. In some embodiments, an endonuclease Page 39 of 327 12592906v1 Attorney Docket No.2017469-0039 domain comprises an N863A mutation (e.g., in spCas9). In some embodiments, an endonuclease domain comprises a H840A mutation. In some embodiments, a heterologous object sequence (e.g., of a system as described herein) is about 1-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, or more, nucleotides in length. In some embodiments, RT and endonuclease domains are joined by a flexible linker. In some embodiments, a linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 5006). In some embodiments, an endonuclease domain is N-terminal relative to an RT domain. In some embodiments, an endonuclease domain is C-terminal relative to an RT domain. In some embodiments, a system of the present disclosure incorporates a heterologous object sequence into a target site by target primed reverse transcription (TPRT), e.g., as described herein. In some embodiments, a gene modifying polypeptide comprises a DNA binding domain (DBD). In some embodiments, a gene modifying polypeptide comprises an RNA binding domain. In some embodiments, an RNA binding domain comprises an RNA binding domain of B-box protein, MS2 coat protein, dCas, or an element of a sequence of a Table herein. In some embodiments, an RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain. In some embodiments, a gene modifying system is capable of producing an insertion of at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides (and optionally no more than 500, no more than 400, no more than 300, no more than 200, or no more than 100 nucleotides) in a target site. In some embodiments, a gene modifying system is capable of producing an insertion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides (and optionally no more than 500, no more than 400, no more than 300, no more than 200, or no more than 100 nucleotides) in a target site. In some embodiments, a gene modifying system is capable of producing an insertion of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.5, at least 2, Page 40 of 327 12592906v1 Attorney Docket No.2017469-0039 at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5 or at least 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases) into a target site. In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides (and optionally no more than 500, no more than 400, no more than 300, or no more than 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 nucleotides (and optionally no more than 500, no more than 400, no more than 300, no more than 200, or no more than 100 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, at least 5, at least 5.5, at least 6, at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, at least 9, at least 9.5 or at least 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a gene modifying system is capable of producing a substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100, or more nucleotides in a target site. In some embodiments, a gene modifying system is capable of producing a substitution of 1-2, 2-3, 3-4, 4-5, 5-10, 10-15, 15-20, 20-30, 30-40, 40- 50, 50-60, 60-70, 70-80, 80-90, or 90-100 nucleotides in a target site. In some embodiments, a substitution is a transition mutation. In some embodiments, a substitution is a transversion mutation. In some embodiments, a substitution converts an adenine to a thymine, an adenine to a guanine, an adenine to a cytosine, a guanine to a thymine, a Page 41 of 327 12592906v1 Attorney Docket No.2017469-0039 guanine to a cytosine, a guanine to an adenine, a thymine to a cytosine, a thymine to an adenine, a thymine to a guanine, a cytosine to an adenine, a cytosine to a guanine, or a cytosine to a thymine. In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g., transcription or translation) of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g., transcription or translation) of a gene by altering, adding, or deleting sequences in a promoter or enhancer, e.g., sequences that bind transcription factors. In some embodiments, an insertion, deletion, substitution, or combination thereof alters translation of a gene (e.g., alters an amino acid sequence), inserts or deletes a start or stop codon, or alters or fixes the translation frame of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof alters splicing of a gene, e.g., by inserting, deleting, or altering a splice acceptor or donor site. In some embodiments, an insertion, deletion, substitution, or combination thereof alters transcript or protein half-life. In some embodiments, an insertion, deletion, substitution, or combination thereof alters protein localization in the cell (e.g., from the cytoplasm to a mitochondria, from the cytoplasm into the extracellular space (e.g., adds a secretion tag)). In some embodiments, an insertion, deletion, substitution, or combination thereof alters (e.g., improves) protein folding (e.g., to prevent accumulation of misfolded proteins). In some embodiments, an insertion, deletion, substitution, or combination thereof, alters, increases, decreases the activity of a gene, e.g., a protein encoded by the gene. Exemplary gene modifying polypeptides, systems comprising the same, and methods of using the same are described, e.g., in PCT / US2021 / 020948, which is incorporated herein by reference with respect to retroviral RT domains, including the amino acid and nucleic acid sequences therein. Exemplary gene modifying polypeptides and retroviral RT domain sequences are also described, e.g., in International Application No. PCT / US21 / 20948, filed March 4, 2021, e.g., at Table 30, Table 31, and Table 44 therein; the entire application is incorporated by reference herein with respect to retroviral RTs, e.g., in said sequences and Tables. Accordingly, a gene modifying polypeptide described herein may comprise an amino acid sequence according to any of the Tables mentioned in this paragraph, or a domain thereof (e.g., a retroviral RT domain), or Page 42 of 327 12592906v1 Attorney Docket No.2017469-0039 a functional fragment or variant of any of the foregoing, or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto. Polypeptide components of gene modifying systems In some embodiments, a gene modifying polypeptide possesses the functions of DNA target site binding, template nucleic acid (e.g., template RNA) binding, DNA target site cleavage, and template nucleic acid (e.g., template RNA) writing (e.g., reverse transcription). In some embodiments, each function is contained within a distinct domain. In some embodiments, a function may be attributed to two or more domains (e.g., two or more domains, together, exhibit the functionality). In some embodiments, two or more domains may have the same or similar function (e.g., two or more domains each independently have DNA-binding functionality, e.g., for two different DNA sequences). In some embodiments, one or more domains may be capable of enabling one or more functions, e.g., a Cas9 domain enabling both DNA binding and target site cleavage. In some embodiments, domains are all located within a single polypeptide. In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C- terminal order, one or more (e.g., 1, 2, 3, 4, 5, or all 6) of an N-terminal methionine residue, a first nuclear localization signal (NLS), a DNA binding domain, a linker, an RT domain, and / or a second NLS. In some embodiments, a gene modifying polypeptide further comprises an N- terminal methionine residue. In some embodiments, a nucleic acid encoding a gene modifying polypeptide (e.g., as described herein) encodes a T2A sequence, e.g., wherein the T2A sequence is situated between a region encoding the gene modifying polypeptide and a second region, wherein the second region optionally encodes a selectable marker, e.g., puromycin. In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between a first NLS and a DNA binding domain. In some embodiments, a spacer sequence between a first NLS and a DNA binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between a first NLS and the DNA binding domain comprises the amino acid sequence GG. In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between a DNA binding domain and a linker. In some embodiments, a spacer sequence between a DNA binding domain and a linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between a DNA binding domain and a linker comprises the amino acid sequence GG. In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between a linker and an RT Page 43 of 327 12592906v1 Attorney Docket No.2017469-0039 domain. In some embodiments, a spacer sequence between a linker and an RT domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between a linker and an RT domain comprises the amino acid sequence GG. In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between an RT domain and a second NLS. In some embodiments, a spacer sequence between an RT domain and a second NLS comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between an RT domain and a second NLS comprises the amino acid sequence AG. In some embodiments, a gene modifying polypeptide further comprises a spacer sequence between a second NLS and a T2A sequence and / or puromycin sequence. In some embodiments, a spacer sequence between a second NLS and a T2A sequence and / or puromycin sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, a spacer sequence between a second NLS and a T2A sequence and / or puromycin sequence comprises the amino acid sequence GSG. RT Domains In some embodiments of the present invention, a writing domain of a gene modifying system possesses reverse transcriptase activity and is also referred to as a reverse transcriptase domain (an RT domain). In some embodiments, an RT domain comprises an RT catalytic portion and RNA-binding region (e.g., a region that binds a template RNA). In some embodiments, a nucleic acid encoding a reverse transcriptase is altered from its natural sequence to have altered codon usage, e.g., improved for human cells. In some embodiments, a reverse transcriptase domain is a heterologous reverse transcriptase from a retrovirus. In some embodiments, an RT domain has been mutated from its original amino acid sequence, e.g., has at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 substitutions. In some embodiments, an RT domain is derived from an RT of a retrovirus, e.g., HIV-1 RT, Moloney Murine Leukemia Virus (MMLV) RT, avian myeloblastosis virus (AMV) RT, or Rous Sarcoma Virus (RSV) RT. In some embodiments, an RT domain has a length of about 400-500, about 500-600, about 600-700, about 700-800, about 800-900, or about 900-1000 amino acids. Page 44 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, a retroviral reverse transcriptase (RT) domain exhibits enhanced stringency of target-primed reverse transcription (TPRT) initiation, e.g., relative to an endogenous RT domain. In some embodiments, an RT domain initiates TPRT when the 3 nucleotides in a target site immediately upstream of a first strand nick, e.g., genomic DNA priming of an RNA template, have at least 66% or 100% complementarity to 3 nucleotides of homology in the RNA template. In some embodiments, an RT domain initiates TPRT when there are less than 5 nucleotides mismatched (e.g., less than 1, less than 2, less than 3, less than 4, or less than 5 nt mismatched) between an RNA template and a target DNA priming reverse transcription. In some embodiments, an RT domain is modified such that the stringency for mismatches in priming a TPRT reaction is increased, e.g., wherein the RT domain does not tolerate any mismatches or tolerates fewer mismatches in a priming region relative to a wild-type (e.g., unmodified) RT domain. In some embodiments, an RT domain comprises a HIV-1 RT domain. In embodiments, an HIV-1 RT domain initiates lower levels of synthesis even with three nucleotide mismatches relative to an alternative RT domain (e.g., as described by Jamburuthugoda and Eickbush J Mol Biol 407(5):661-672 (2011); incorporated herein by reference in its entirety). In some embodiments, an RT domain forms a dimer (e.g., a heterodimer or homodimer). In some embodiments, an RT domain is monomeric. In some embodiments, an RT domain naturally functions as a monomer or as a dimer (e.g., heterodimer or homodimer). In some embodiments, an RT domain naturally functions as a monomer, e.g., is derived from a virus wherein it functions as a monomer. In embodiments, an RT domain is selected from an RT domain from murine leukemia virus (MLV; sometimes referred to as MoMLV) (e.g., P03355), porcine endogenous retrovirus (PERV) (e.g., UniProt Q4VFZ2), mouse mammary tumor virus (MMTV) (e.g., UniProt P03365), Avian reticuloendotheliosis virus (AVIRE) (e.g., UniProtKB accession: P03360); Feline leukemia virus (FLV or FeLV) (e.g., e.g., UniProtKB accession: P10273); Mason-Pfizer monkey virus (MPMV) (e.g., UniProt P07572), bovine leukemia virus (BLV) (e.g., UniProt P03361), human T-cell leukemia virus-1 (HTLV-1) (e.g., UniProt P03362), human foamy virus (HFV) (e.g., UniProt P14350), simian foamy virus (SFV) (e.g., SFV3L) (e.g., UniProt P23074 or P27401), or bovine foamy / syncytial virus (BFV / BSV) (e.g., UniProt O41894), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity thereto). In some embodiments, an RT domain is dimeric in its natural functioning. In Page 45 of 327 12592906v1 Attorney Docket No.2017469-0039 some embodiments, an RT domain is derived from a virus wherein it functions as a dimer. In embodiments, an RT domain is selected from an RT domain from avian sarcoma / leukemia virus (ASLV) (e.g., UniProt A0A142BKH1), Rous sarcoma virus (RSV) (e.g., UniProt P03354), avian myeloblastosis virus (AMV) (e.g., UniProt Q83133), human immunodeficiency virus type I (HIV-1) (e.g., UniProt P03369), human immunodeficiency virus type II (HIV-2) (e.g., UniProt P15833), simian immunodeficiency virus (SIV) (e.g., UniProt P05896), bovine immunodeficiency virus (BIV) (e.g., UniProt P19560), equine infectious anemia virus (EIAV) (e.g., UniProt P03371), or feline immunodeficiency virus (FIV) (e.g., UniProt P16088) (Herschhorn and Hizi Cell Mol Life Sci 67(16):2717-2747 (2010)), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity thereto). Naturally heterodimeric RT domains may, in some embodiments, also be functional as homodimers. In some embodiments, dimeric RT domains are expressed as fusion proteins, e.g., as homodimeric fusion proteins or heterodimeric fusion proteins. In some embodiments, an RT function of a system of the present disclosure is fulfilled by multiple RT domains (e.g., as described herein). In further embodiments, multiple RT domains are fused or separate, e.g., may be on the same polypeptide or on different polypeptides. In some embodiments, a gene modifying system described herein comprises an integrase domain, e.g., wherein the integrase domain may be part of an RT domain. In some embodiments, an RT domain (e.g., as described herein) comprises an integrase domain. In some embodiments, an RT domain (e.g., as described herein) lacks an integrase domain, or comprises an integrase domain that has been inactivated by mutation or deleted. In some embodiment, a gene modifying system described herein comprises an RNase H domain, e.g., wherein the RNase H domain may be part of an RT domain. In some embodiments, an RNase H domain is not part of an RT domain and is covalently linked via a flexible linker. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain, e.g., an endogenous RNAse H domain or a heterologous RNase H domain. In some embodiments, an RT domain (e.g., as described herein) lacks an RNase H domain. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain that has been added, deleted, mutated, or swapped for a heterologous RNase H domain. In some embodiments, a gene modifying polypeptide comprises an inactivated endogenous RNase H domain. In some embodiments, an endogenous RNase H domain of a polypeptide is genetically removed such that it is not included Page 46 of 327 12592906v1 Attorney Docket No.2017469-0039 in the polypeptide, e.g., the endogenous RNase H domain is partially or completely truncated from the polypeptide. In some embodiments, one or more mutations of an RNase H domain yields a polypeptide exhibiting lower RNase activity, e.g., as determined by the methods described in Kotewicz et al. Nucleic Acids Res 16(1):265-277 (1988) (incorporated herein by reference in its entirety), e.g., lower by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to an otherwise similar domain without the one or more mutations. In some embodiments, RNase H activity of a gene modifying polypeptide is abolished. In some embodiments, an RT domain is mutated to increase fidelity compared to an otherwise similar domain without the mutation. In some embodiments, a YADD or YMDD motif in an RT domain (e.g., in a reverse transcriptase) is replaced with YVDD. In some embodiments, replacement of a YADD, YMDD, or YVDD motif results in higher fidelity in retroviral reverse transcriptase activity (e.g., as described in Jamburuthugoda and Eickbush J Mol Biol 2011; incorporated herein by reference in its entirety). In some embodiments, a gene modifying polypeptide described herein comprises an RT domain having an amino acid sequence according to any RT domain described in Table 1, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a nucleic acid described herein encodes an RT domain having an amino acid sequence according to any RT domain described in Table 1, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Table 1: Exemplary reverse transcriptase domains from retroviruses RT name RT amino acid sequence SEQ ID Page 47 of 327 12592906v1 Attorney Docket No.2017469-0039 RT name RT amino acid sequence SEQ ID NOs Page 48 of 327 12592906v1 Attorney Docket No.2017469-0039 RT name RT amino acid sequence SEQ ID NOs Page 49 of 327 12592906v1 Attorney Docket No.2017469-0039 RT name RT amino acid sequence SEQ ID NOs Page 50 of 327 12592906v1 Attorney Docket No.2017469-0039 RT name RT amino acid sequence SEQ ID NOs Page 51 of 327 12592906v1 Attorney Docket No.2017469-0039 RT name RT amino acid sequence SEQ ID NOs Page 52 of 327 12592906v1 Attorney Docket No.2017469-0039 RT name RT amino acid sequence SEQ ID NOs Page 53 of 327 12592906v1 Attorney Docket No.2017469-0039 RT name RT amino acid sequence SEQ ID NOs Page 54 of 327 12592906v1 Attorney Docket No.2017469-0039 RT name RT amino acid sequence SEQ ID NOs Page 55 of 327 12592906v1 Attorney Docket No.2017469-0039 RT name RT amino acid sequence SEQ ID NOs Page 56 of 327 12592906v1 Attorney Docket No.2017469-0039 RT name RT amino acid sequence SEQ ID NOs e- specific mutation. In some embodiments, an RT domain is engineered to have improved properties, e.g., SuperScript IV (SSIV) RT derived from the MMLV RT. In some embodiments, an RT domain may be engineered to have lower error rates as compared to a reference RT domain, e.g., as described in WO2001068895, incorporated herein by reference. In some embodiments, an RT domain may be engineered to be more thermostable as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to be more processive as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to have improved tolerance to inhibitors as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to be faster as compared to a reference RT Page 57 of 327 12592906v1 Attorney Docket No.2017469-0039 domain. In some embodiments, an RT domain may be engineered to better tolerate modified nucleotides in an RNA template as compared to a reference RT domain. In some embodiments, an RT domain may be engineered to be capable of inserting modified DNA nucleotides. In some embodiments, an RT domain is engineered to bind a template RNA. In some embodiments, one or more mutations are chosen from D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K, H8Y, T306K, or D653N in an RT domain of murine leukemia virus reverse transcriptase or a corresponding mutation at a corresponding position of another RT domain. In some embodiments, a gene modifying polypeptide comprises an RT domain from a retroviral reverse transcriptase, e.g., a wild-type M-MLV RT, e.g., comprising the following sequence: M-MLV (WT): TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKA TSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDY RPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLI LLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLG YLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAP LYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYA KGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPAT LLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKA GAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFA TAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAE ARGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO: 5002) In some embodiments, a gene modifying polypeptide comprises an RT domain from a retroviral reverse transcriptase, e.g., an M-MLV RT, e.g., comprising the following sequence: TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKA TSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDY RPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPT SQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLI LLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLG YLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAP LYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYA KGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPAT LLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKA GAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFA Page 58 of 327 12592906v1 Attorney Docket No.2017469-0039 TAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAE ARGNRMADQAARKAAITETPDTSTLL (SEQ ID NO: 5003) In some embodiments, a gene modifying polypeptide comprises an RT domain from a retroviral reverse transcriptase comprising the sequence of amino acids 659-1329 of NP_057933. In some embodiments, a gene modifying polypeptide further comprises one additional amino acid at the N-terminus of the sequence of amino acids 659-1329 of NP_057933, e.g., as shown below: TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATS TPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPV QDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTS QPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPD LILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVK YLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMA APLYPLTKTGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGY AKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMG QPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATL LPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAA VTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYR RRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARK AA (SEQ ID NO: 5004) Core RT (bold), annotated per above RNAseH (italics), annotated per above In some embodiments, a gene modifying polypeptide further comprises one additional amino acid at the C-terminus of the sequence of amino acids 659-1329 of NP_057933. In embodiments, a gene modifying polypeptide comprises an RNaseH1 domain (e.g., amino acids 1178-1318 of NP_057933). In some embodiments, a retroviral reverse transcriptase domain, e.g., M-MLV RT, may comprise one or more mutations from a wild-type sequence that may improve features of the RT, e.g., thermostability, processivity, and / or template binding. In some embodiments, an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, one or more mutations, e.g., selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, K103L, or a combination thereof. In some embodiments, an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, a combination of mutations including D200N, L603W, and T330P, and optionally further including T306K and W313F. In some embodiments, an M-MLV Page 59 of 327 12592906v1 Attorney Docket No.2017469-0039 RT used herein comprises D200N, L603W, T330P, T306K and W313F mutations. In some embodiments, a mutant M-MLV RT comprises the following amino acid sequence: M-MLV (PE2): TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKAT STPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRP VQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQP LFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQ YVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLK EGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLT KPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLT QKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVIL APHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPE EGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVT TETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHG EIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNR MADQAARKAAITETPDTSTLLI (SEQ ID NO: 5005) RT Families and Mutants In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV, XMRV6, BLVAU, BLVJ, HTL1A, HTL1C, HTL1L, HTL32, HTL3P, HTLV2, JSRV, MLVF5, MLVRD, MMTVB, MPMV, SFVCP, SMRVH, SRV1, SRV2, and WDSV. In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV, and XMRV6. In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from an MLVMS RT domain. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 1 of Table 2, or a point mutation corresponding thereto. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 3 of Table 2 (Gen1 MLVMS), or a point mutation corresponding thereto. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations at an amino acid position of the RT domain as listed in columns 1 and 2 of Table 3, or an amino acid position corresponding thereto. Page 60 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from an AVIRE RT domain. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 2 of Table 2, or a point mutation corresponding thereto. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations as listed in column 4 of Table 2 (Gen2 AVIRE), or a point mutation corresponding thereto. In some embodiments, the amino acid sequence of an RT domain sequence comprises one or more point mutations at an amino acid position of the RT domain as listed in columns 3 and 4 of Table 3, or an amino acid position corresponding thereto. In some embodiments, an RT domain comprises an IENSSP (e.g., at the C-terminus). Table 2. Exemplary point mutations in MLVMS and AVIRE RT domains RT-linker filing Corresponding Gen1 MLVMS Gen2 AVIRE (MLVMS) AVIRE (PLV4921) (PLV10990) Page 61 of 327 12592906v1 Attorney Docket No.2017469-0039 Table 3. Positions that can be mutated in exemplary MLVMS and AVIRE RT domains WT residue & position MLVMS aa MLVMS AVIRE aa AVIRE * In some emb gamma retrovirus derived RT domain. In some embodiments, a gamma retrovirus-derived RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from a family selected from: AVIRE, BAEVM, FFV, FLV, FOAMV, GALV, KORV, MLVAV, MLVBM, MLVCB, MLVFF, MLVMS, PERV, SFV1, SFV3L, WMSV, and XMRV6. In some embodiments, a gamma retrovirus-derived RT domain of a gene modifying polypeptide is not derived from PERV. In some embodiments, an RT domain includes one, two, three, four, five, six or more mutations shown in Table 4 and corresponding to mutations D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K, or D653N in the RT domain of murine leukemia virus Page 62 of 327 12592906v1 Attorney Docket No.2017469-0039 reverse transcriptase. In some embodiments, a gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of an AVIRE RT (e.g., an AVIRE_P03360 sequence, e.g., SEQ ID NO: 8001), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of an AVIRE RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, G330P, L605W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an AVIRE RT further comprising one, two, or three mutations selected from the group consisting of D200N, G330P, and L605W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a BAEVM RT (e.g., an BAEVM_P10272 sequence, e.g., SEQ ID NO: 8004), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a BAEVM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L602W, T304K, and W311F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a BAEVM RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L602W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of an FFV RT (e.g., an FFV_O93209 sequence, e.g., SEQ ID NO: 8012), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, three, or four mutations selected from the group consisting of D21N, T293N, T419P, and L393K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, or three mutations selected from the group consisting of D21N, T293N, and T419P, or a corresponding position in a homologous RT Page 63 of 327 12592906v1 Attorney Docket No.2017469-0039 domain. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising the mutation D21N. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one, two, or three mutations selected from the group consisting of T207N, T333P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an FFV RT further comprising one or two mutations selected from the group consisting of T207N and T333P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of an FLV RT (e.g., an FLV_P10273 sequence, e.g., SEQ ID NO: 8019), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of an FLV RT further comprising one, two, three, or four mutations selected from the group consisting of D199N, L602W, T305K, and W312F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an FLV RT further comprising one or two mutations selected from the group consisting of D199N and L602W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a FOAMV RT (e.g., a FOAMV_P14350 sequence, e.g., SEQ ID NO: 8021), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, S420P, and L396K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and S420P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising the mutation D24N, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV RT further comprising one, two, or three mutations selected from the group consisting of T207N, S331P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a FOAMV Page 64 of 327 12592906v1 Attorney Docket No.2017469-0039 RT further comprising one or two mutations selected from the group consisting of T207N and S331P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a GALV RT (e.g., an GALV_P21414 sequence, e.g., SEQ ID NO: 8027), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W311F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or a corresponding position in a homologous RT domain. In embodiments, an RT domain comprises the amino acid sequence of an RT domain of a KORV RT (e.g., an KORV_Q9TTC1 sequence, e.g., SEQ ID NO: 8047), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, four, five, or six mutations selected from the group consisting of D32N, D322N, E452P, L274W, T428K, and W435F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising one, two, three, or four mutations selected from the group consisting of D32N, D322N, E452P, and L274W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a GALV RT further comprising the mutation D32N. In some embodiments, an RT domain comprises the amino acid sequence of a KORV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D231N, E361P, L633W, T337K, and W344F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a KORV RT further comprising one, two, or three mutations selected from the group consisting of D231N, E361P, and L633W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVAV RT (e.g., an MLVAV_P03356 sequence, e.g., SEQ ID NO: 8053), or an Page 65 of 327 12592906v1 Attorney Docket No.2017469-0039 amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVAV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVAV RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVBM RT (e.g., an MLVBM_Q7SVK7 sequence, e.g., SEQ ID NO: 8056), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVBM RT further comprising one, two, three, four, or five mutations selected from the group consisting of D199N, T329P, L602W, T305K, and W312F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVBM RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVCB RT (e.g., an MLVCB_P08361 sequence, e.g., SEQ ID NO: 8062), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVCB RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVCB RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVFF RT, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some Page 66 of 327 12592906v1 Attorney Docket No.2017469-0039 embodiments, an RT domain comprises the amino acid sequence of a MLVFF RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVFF RT further comprising one, two, and three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain. In embodiments, an RT domain comprises the amino acid sequence of an RT domain of a MLVMS RT (e.g., an MLVMS_reference sequence, e.g., SEQ ID NO: 8137; or an MLVMS_P03355 sequence, e.g., SEQ ID NO: 8070), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, three, four, five, or six mutations selected from the group consisting of D200N, T330P, L603W, T306K, W313F, and H8Y, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a MLVMS RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a PERV RT (e.g., an PERV_Q4VFZ2 sequence, e.g., SEQ ID NO: 8099), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a PERV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D196N, E326P, L599W, T302K, and W309F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a PERV RT further comprising one, two, or three mutations selected from the group consisting of D196N, E326P, and L599W, or a corresponding position in a homologous RT domain. Page 67 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a SFV1 RT (e.g., an SFV1_P23074 sequence, e.g., SEQ ID NO: 8105), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a SFV1 RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N420P, and L396K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV1 RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N420P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV1 RT further comprising the D24N, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a SFV3L RT (e.g., an SFV3L_P27401 sequence, e.g., SEQ ID NO: 8111), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, three, or four mutations selected from the group consisting of D24N, T296N, N422P, and L396K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, or three mutations selected from the group consisting of D24N, T296N, and N422P, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising the mutation D24N, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one, two, or three mutations selected from the group consisting of T307N, N333P, and L307K, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a SFV3L RT further comprising one or two mutations selected from the group consisting of T307N and N333P, or a corresponding position in a homologous RT domain. In embodiments, an RT domain comprises the amino acid sequence of an RT domain of a WMSV RT (e.g., an WMSV_P03359 sequence, e.g., SEQ ID NO: 8131), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, Page 68 of 327 12592906v1 Attorney Docket No.2017469-0039 or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a WMSV RT further comprising one, two, three, four, or five mutations selected from the group consisting of D198N, E328P, L600W, T304K, and W311F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a WMSV RT further comprising one, two, or three mutations selected from the group consisting of D198N, E328P, and L600W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of an RT domain of a XMRV6 RT (e.g., an XMRV6_A1Z651 sequence, e.g., SEQ ID NO: 8134), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an RT domain comprises the amino acid sequence of a XMRV6 RT further comprising one, two, three, four, or five mutations selected from the group consisting of D200N, T330P, L603W, T306K, and W313F, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain comprises the amino acid sequence of a XMRV6 RT further comprising one, two, or three mutations selected from the group consisting of D200N, T330P, and L603W, or a corresponding position in a homologous RT domain. In some embodiments, an RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain of an AVIRE RT, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217. In some embodiments, the RT domain of a gene modifying polypeptide comprises the amino acid sequence of an RT domain of an MLVMS RT, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217. In some embodiments, an RT domain (e.g., as listed in Table 1) comprises one or more mutations as listed in Table 4 below. In some embodiments, an RT domain as listed in Table 1 comprises one, two, three, four, five, or six of the mutations listed in the corresponding row of Table 4 below. Page 69 of 327 12592906v1 Attorney Docket No.2017469-0039 Table 4. Exemplary RT domain mutations (relative to corresponding wild-type sequences) RT Domain Name Mutation(s) AVIRE_P03360 Page 70 of 327 12592906v1 Attorney Docket No.2017469-0039 RT Domain Name Mutation(s) HTL32_Q0R5R2 Page 71 of 327 12592906v1 Attorney Docket No.2017469-0039 RT Domain Name Mutation(s) MLVMS_P03355_PLV919 D200N T330P L603W T306K W313F H8Y Page 72 of 327 12592906v1 Attorney Docket No.2017469-0039 RT Domain Name Mutation(s) SFV3L_P27401-Pro Cas domains In some embodiments, a gene modifying polypeptide described herein comprises a Cas domain. In some embodiments, a Cas domain can direct a gene modifying polypeptide to a target site specified by a gRNA spacer, thereby modifying a target nucleic acid sequence in “cis”. In some embodiments, a Cas domain comprises two or more smaller domains, e.g., a DNA binding domain and an endonuclease domain. In some embodiments, a Cas domain possesses DNA target site cleavage functionality via an endonuclease domain. In some embodiments, a Cas domain has DNA binding activity. It is understood that when a Cas domain is said to bind to a target nucleic acid sequence, in some embodiments, the binding is mediated Page 73 of 327 12592906v1 Attorney Docket No.2017469-0039 by a gRNA spacer. In some embodiments, a Cas domain has RNA binding activity, e.g., a Cas domain may bind a gRNA scaffold region of a template RNA. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; An example of a PAM sequences is 5´-NGG (Streptococcus pyogenes). Some endonucleases, e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e. g., 5´- NGG, and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5´ from) the PAM site. In some embodiments, a gene modifying polypeptide comprises a GG amino acid sequence between the Cas domain and the linker, an AG amino acid sequence between the RT domain and the second NLS, and / or a GG amino acid sequence between the linker and the RT domain. In some embodiments, a gene modifying polypeptide may comprise an St1Cas9 domain. In some embodiments, a gene modifying polypeptide comprises the amino acid sequence of SEQ ID NO: 10208, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. St1Cas9 Domain: SDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLAR RKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMV KHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTYGQLRG DFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGKRK YYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFN LLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADI KGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQ EALEHEFADGSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSE EQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEY GDFDNIVIEMARETNEDDEKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPH SVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHILPLSITFDD SLANKVLVYATAAQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKKKE YLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQ FTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDI ETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIY ATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQT FEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKY YDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQFE KGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLS RTMPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVR TDVLGNQHIIKNEGDKPKLDF (SEQ ID NO: 10208) Page 74 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, a gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 4001 below, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the amino acid sequence of SEQ ID NO: 4001 below, or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, is positioned at the C-terminal end of a gene modifying polypeptide. In embodiments, the amino acid sequence of SEQ ID NO: 4001 below, or the sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the C-terminal end of a gene modifying polypeptide. Exemplary C-terminal sequence comprising an NLS: AGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4001) In some embodiments, a Cas protein is catalytically active and cuts one or both strands of a target DNA site. In some embodiments, cutting a target DNA site is followed by formation of an alteration, e.g., an insertion or deletion, e.g., by cellular repair machinery. In some embodiments, a Cas protein is modified to deactivate or partially deactivate the nuclease, e.g., nuclease-deficient Cas9. Whereas wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA, a number of CRISPR endonucleases having modified functionalities are available, for example: a “nickase” version of Cas9 that has been partially deactivated generates only a single-strand break; a catalytically inactive Cas9 (“dCas9”) does not cut target DNA. In some embodiments, an endonuclease domain has nickase activity and cleaves one strand of a target DNA. In some embodiments, nickase activity reduces the formation of double-stranded breaks at a target site. In some embodiments, an endonuclease domain has nickase activity and does not form double-stranded breaks. In some embodiments, an endonuclease domain forms single-stranded breaks at a higher frequency than double- stranded breaks, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the breaks are single-stranded breaks, or less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the breaks are double-stranded breaks. In some embodiments, an endonuclease forms substantially no double-stranded breaks. In some embodiments, an endonuclease does not form detectable levels of double-stranded breaks. Page 75 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, a catalytically inactive or partially inactive CRISPR / Cas domain comprises a Cas protein comprising one or more mutations. Linkers In some embodiments, a gene modifying polypeptide may comprise a linker, e.g., a peptide linker, e.g., a linker as described in Table 5 or Table 6. In some embodiments, a gene modifying polypeptide comprises a linker (e.g., as described herein) and an RT domain (e.g., as described herein). In some embodiments, a gene modifying polypeptide comprises, in N- terminal to C-terminal order, a linker (e.g., as described herein, e.g., in Table 5 or Table 6, or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95, or at least 99% identity thereto) and an RT domain (e.g., as described herein). In some embodiments, a gene modifying polypeptide comprises, in an N-terminal to C-terminal direction, a Cas domain (e.g., a Cas domain as described herein, e.g., an St1Cas9 domain), a linker of Table 5 or Table 6 (or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), and an RT domain (e.g., an RT domain of Table 1). In some embodiments, a gene modifying polypeptide comprises a flexible linker between the endonuclease and the RT domain, e.g., a linker comprising the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 11002). In some embodiments, an RT domain of a gene modifying polypeptide may be located C-terminal to a Cas domain. In some embodiments, an RT domain of a gene modifying polypeptide may be located N-terminal to a Cas domain. In some embodiments, a linker of a gene modifying polypeptide comprises a motif chosen from: (SGGS)n (SEQ ID NO: 5025), (GGGS)n (SEQ ID NO: 5026), (GGGGS)n (SEQ ID NO: 5027), (G)n, (EAAAK)n (SEQ ID NO: 5028), (GGS)n, or (XP)n. In some embodiments, a gene modifying polypeptide may comprise a linker, e.g., a peptide linker, e.g., a linker as described in Table 5. In some embodiments, a gene modifying polypeptide comprises a linker (e.g., as described in Table 5) and an RT domain (e.g., as described herein, e.g., in Table 1). In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a linker (e.g., as described herein, e.g., in Table 5) and an RT domain (e.g., as described herein, e.g., in Table 1). In some embodiments, a gene modifying polypeptide comprises, in an N-terminal to C-terminal direction, a Cas domain (e.g., a Cas domain as described herein, e.g., an St1Cas9 domain, e.g., comprising the amino acid Page 76 of 327 12592906v1 Attorney Docket No.2017469-0039 sequence of SEQ ID NO: 10208, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto), a linker of Table 5 (or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), and an RT domain (e.g., an RT domain of Table 1). Table 5. Exemplary linker amino acid sequences Linker ID Linker Amino Acid Sequence SEQ ID NO Page 77 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker ID Linker Amino Acid Sequence SEQ ID NO Page 78 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker ID Linker Amino Acid Sequence SEQ ID NO Page 79 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker ID Linker Amino Acid Sequence SEQ ID NO Page 80 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker ID Linker Amino Acid Sequence SEQ ID NO p p , .g., . , g y g polypeptide comprises a linker (e.g., as described in Table 6) and an RT domain (e.g., as described herein, e.g., in Table 1). In some embodiments, a gene modifying polypeptide comprises, in N-terminal to C-terminal order, a linker (e.g., as described herein, e.g., in Table 6) and an RT domain (e.g., as described herein, e.g., in Table 1). In some embodiments, a gene modifying polypeptide comprises, in an N-terminal to C-terminal direction, a Cas domain (e.g., a Cas domain as described herein, e.g., an St1Cas9 domain, e.g., comprising the amino acid sequence of SEQ ID NO: 10208, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto), a linker of Table 6 (or a sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto), and an RT domain (e.g., an RT domain of Table 1). Table 6. Exemplary linker amino acid sequences SEQUENCE SEQ ID NO Page 81 of 327 12592906v1 Attorney Docket No.2017469-0039 SEQUENCE SEQ ID NO VNPERRRC10188 Exemplary Gene Modifying Polypeptides In some embodiments, a gene modifying polypeptide (e.g., a gene modifying polypeptide that is part of a system described herein) comprises an amino acid sequence of any one of SEQ ID NOs: 12,001-21,744, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker comprising a linker sequence as listed in Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Page 82 of 327 12592906v1 Attorney Docket No.2017469-0039 Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in the same row of Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in column 6 of Table 7, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. Table 7. Exemplary RT-linker combinations Linker ID Linker amino acid SEQ ID RT name SEQ ID Full length NOs NOs SEQ ID Page 83 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker ID Linker amino acid SEQ ID RT name SEQ ID Full length NOs NOs SEQ ID e , , , , at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker comprising a linker sequence as listed in Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 1 of Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 2 in the same row of Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 3 of Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 4 in the same row of Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 5 of Table 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 6 in the same row of Table 8, or an amino acid sequence having at least 70%, Page 84 of 327 12592906v1 Attorney Docket No.2017469-0039 at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. Table 8. Exemplary linker-RT combinations Linker RT name Linker RT name Linker RT name id id id ut u 3 m m m u u u m m u m m m Page 85 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linker RT name Linker RT name id id id m u u m u m m m u u m u u m u m u u Page 86 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linker RT name Linker RT name id id id m u u m u m m u m m m A 3 m m Page 87 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linker RT name Linker RT name id id id u m e , , , , at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker comprising a linker sequence as listed in Table 9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table 9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 1 of Table 9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 2 in the same row of Table 9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 3 of Table 9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 4 in the same row of Table 9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 5 of Table 9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT Page 88 of 327 12592906v1 Attorney Docket No.2017469-0039 domain sequence as listed in column 6 in the same row of Table 9, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. Table 9. Gene modifying polypeptides with Z score > 1 Linker RT name Linke RT name Linker RT name id r id id _ 3 3 _ 3 _ _ _ _ _ 3 _ Page 89 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id 3 3 _ 3 _ 7 3 3 2 _ _ 7 Page 90 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id _ _ 3 _ ut _ 7 3 _ _ 3 _ _ 3 3 Page 91 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id 3 2 _ 3 _ _ 3 _ 3 3 _ 3 _ ut 3 _ Page 92 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id 3 _ 3 2 _ _ _ _ 7 3 _ _ _ 7 3 _ 3 Page 93 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id 3 _ _ 7 3 3 3 3 _ _ _ 3 _ 3 _ Page 94 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id 3 _ 3 3 7 _ 3 3 7 3 _ 3 _ ut 3 Page 95 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id _ 7 _ 3 3 _ 3 _ 3 _ 3 _ _ 7 3 _ 3 Page 96 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id ut _ _ ut 3 7 _ _ 2 2 _ 3 ut Page 97 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id _ 7 3 3 3 7 3 3 3 3 2 _ ut _ Page 98 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id 3 3 _ 3 2 _ _ 3 _ 3 _ 3 3 3 _ 3 Page 99 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id _ _ 3 _ 3 _ 3 3 3 _ ut _ 3 Page 100 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id 3 3 ut 3 _ _ 7 3 _ 3 _ 3 3 ut Page 101 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id _ 7 3 _ ut _ 3 _ 3 _ 3 _ 3 _ _ 3 3 3 Page 102 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id 3 _ _ 3 _ 3 3 _ 3 _ _ 7 _ _ _ 3 3 Page 103 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id _ 3 3 7 3 3 3 3 2 7 _ _ ut Page 104 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id _ 7 _ 3 3 3 2 _ 3 _ 3 _ 3 2 ut Page 105 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id 3 3 _ _ _ 7 3 3 2 _ 3 3 3 _ _ 7 Page 106 of 327 12592906v1 Attorney Docket No.2017469-0039 Linker RT name Linke RT name Linker RT name id r id id 3 e , , , %, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a linker comprising a linker sequence as listed in Table 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an RT domain comprising an RT domain sequence as listed in Table 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 1 of Table 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 2 of the same row of Table 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in column 3 of Table 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of column 4 of Table 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 5 of the same row of Table 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in column 6 of Table 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker Page 107 of 327 12592906v1 Attorney Docket No.2017469-0039 comprising a linker sequence as listed in a row of column 7 of Table 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and (ii) an RT domain comprising an RT domain sequence as listed in column 8 of the same row of Table 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in column 9 of Table 10, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. Table 10. Linker-RT combination of screened gene modifying polypeptides Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 Page 108 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 Page 109 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 Page 110 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 Page 111 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 Page 112 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Page 113 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Page 114 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 Page 115 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 Page 116 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Page 117 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Page 118 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 Page 119 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 Page 120 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 Page 121 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 Page 122 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 Page 123 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 Page 124 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Page 125 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 Page 126 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 Page 127 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Page 128 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 Page 129 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 Page 130 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 Page 131 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 Page 132 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 Page 133 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 Page 134 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 Page 135 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Page 136 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 Page 137 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 Page 138 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Page 139 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 Page 140 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 Page 141 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 Page 142 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 Page 143 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 Page 144 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 Page 145 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 Page 146 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Page 147 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 Page 148 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 Page 149 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Page 150 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 Page 151 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 Page 152 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 Page 153 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 Page 154 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 Page 155 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 Page 156 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 Page 157 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Page 158 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 Page 159 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 Page 160 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Page 161 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Page 162 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 Page 163 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 Page 164 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Page 165 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 Page 166 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 Page 167 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 Page 168 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Page 169 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 Page 170 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 Page 171 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Page 172 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 Page 173 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 Page 174 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 Page 175 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 Page 176 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 Page 177 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 Page 178 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 Page 179 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Page 180 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 Page 181 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 Page 182 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Page 183 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO 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Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 Page 190 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 Page 191 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 Page 192 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 Page 193 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 Page 194 of 327 12592906v1 Attorney Docket No.2017469-0039 Linke RT Name SEQ Linke RT Name SEQ Linke RT Name SEQ r ID ID NO r ID ID NO r ID ID NO 2 3 4 transcriptase (RT) domain. In some embodiments, a gene modifying polypeptide of the present disclosure includes: (1) a nickase St1Cas9 domain having at least 80% amino acid sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10208; (2) a linker having at least 80% amino acid sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10074; and (3) a BAEVM RT having at least 80% amino acid sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10214. In some embodiments, a gene modifying polypeptide of the present disclosure has at least 80% amino acid sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17194. In some embodiments, a gene modifying polypeptide of the present disclosure includes: (1) a nickase St1Cas9 domain having at least 80% amino acid sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10208; (2) a linker having at least 80% amino acid sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10154; and (3) an AVIRE RT having at least 80% amino acid sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 10213. In some embodiments, a gene Page 195 of 327 12592906v1 Attorney Docket No.2017469-0039 modifying polypeptide of the present disclosure has at least 80% amino acid sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 17125. Systems In some embodiments, the present disclosure provides a system comprising a nucleic acid molecule encoding a gene modifying polypeptide (e.g., as described herein) and a template nucleic acid (e.g., a template RNA, e.g., as described herein). In some embodiments, a nucleic acid molecule encoding a gene modifying polypeptide comprises one or more silent mutations in the coding region (e.g., in a sequence encoding an RT domain) relative to a nucleic acid molecule as described herein. In some embodiments, a system further comprises a gRNA (e.g., a gRNA that binds to a polypeptide that induces a nick, e.g., in the opposite strand of a target DNA bound by a gene modifying polypeptide). In some embodiments, a gene modifying system of the present disclosure includes a template nucleic acid (e.g., a template RNA) and a gRNA at a ratio of 1:1 (w / w), 1.25:1 (w / w), 1.5:1 (w / w), 1.75:1 (w / w), 2:1 (w / w), 2.25:1 (w / w), 2.5:1 (w / w), 2.75:1 (w / w), 3:1 (w / w), 1:3 (w / w), 1:2.75 (w / w), 1:2.5 (w / w), 1:2.25 (w / w), 1:2 (w / w), 1.75 (w / w), 1:1.5 (w / w), or 1:1.25 (w / w). In some embodiments, a gene modifying system of the present disclosure includes a total nucleic acid (e.g., template nucleic acid and gRNA) to gene modifying polypeptide ratio of about 2:1, about 2.25:1, about 2.5:1, about 2.75:1, about 3:1, about 3.25:1, about 3.5:1, about 3.75:1, about 4:1, about 4.25:1, about 4.5:1, about 4.75:1, about 5:1, about 5.25:1, about 5.5:1, about 5.75:1, or about 6:1. Localization sequences for gene modifying systems In some embodiments, a gene modifying system RNA further comprises an intracellular localization sequence, e.g., a nuclear localization sequence (NLS). In some embodiments, a gene modifying polypeptide comprises an NLS as comprised in SEQ ID NO: 5344 and / or SEQ ID NO: 4001, or an NLS having an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises a sequence having at least 80% amino acid sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least Page 196 of 327 12592906v1 Attorney Docket No.2017469-0039 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 4001. A nuclear localization sequence may be an RNA sequence that promotes the import of an RNA into the nucleus. In some embodiments the nuclear localization signal is located on a template RNA. In some embodiments, a gene modifying polypeptide is encoded on a first RNA, and a template RNA is a second, separate, RNA, and a nuclear localization signal is located on the template RNA and not on an RNA encoding the gene modifying polypeptide. While not wishing to be bound by theory, in some embodiments, an RNA encoding a gene modifying polypeptide is targeted primarily to the cytoplasm to promote its translation, while a template RNA is targeted primarily to the nucleus to promote insertion into the genome. In some embodiments a nuclear localization signal is at the 3′ end, 5′ end, or in an internal region of a template RNA. In some embodiments a nuclear localization sequence is situated inside of an intron. In some embodiments, a plurality of the same or different nuclear localization signals are in an RNA, e.g., in a template RNA. In some embodiments, a nuclear localization signal is less than 5, less than 10, less than 25, less than 50, less than 75, less than 100, less than 150, less than 200, less than 250, less than 300, less than 350, less than 400, less than 450, less than 500, less than 600, less than 700, less than 800, less than 900 or less than 1000 bp in length. Various RNA nuclear localization sequences can be used. For example, Lubelsky and Ulitsky, Nature 555 (107-111), 2018 describe RNA sequences which drive RNA localization into the nucleus. In some embodiments, a nuclear localization signal is a SINE-derived nuclear RNA localization (SIRLOIN) signal. In some embodiments, a nuclear localization signal binds a nuclear-enriched protein. In some embodiments, a nuclear localization signal binds the HNRNPK protein. In some embodiments, a nuclear localization signal is rich in pyrimidines, e.g., is a C / T rich, C / U rich, C rich, T rich, or U rich region. In some embodiments, a nuclear localization signal is derived from a long non-coding RNA. In some embodiments the nuclear localization signal is derived from MALAT1 long non-coding RNA or is the 600 nucleotide M region of MALAT1 (described in Miyagawa et al., RNA 18, (738-751), 2012). In some embodiments, a nuclear localization signal is derived from BORG long non-coding RNA or is a AGCCC motif (described in Zhang et al., Molecular and Cellular Biology 34, 2318-2329 (2014). In some embodiments the nuclear localization sequence is described in Shukla et al., The EMBO Journal e98452 (2018). In some embodiments, a nuclear localization signal is derived from a retrovirus. Page 197 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, a gene modifying polypeptide described herein comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example a nuclear localization sequence (NLS). In some embodiments, an NLS is a bipartite NLS. In some embodiments, an NLS facilitates the import of a protein comprising an NLS into the cell nucleus. In some embodiments, an NLS is fused to the N-terminus of a gene modifying polypeptide as described herein. In some embodiments, an NLS is fused to the C-terminus of a gene modifying polypeptide. In some embodiments, an NLS is fused to the N-terminus or the C-terminus of a Cas domain. In some embodiments, a linker sequence is disposed between an NLS and a neighboring domain of a gene modifying polypeptide. In some embodiments, an NLS comprises the amino acid sequence PKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 5010), RKSGKIAAIWKRPRKPKKKRKV (SEQ ID NO: 5011), KRTADGSEFESPKKKRKV (SEQ ID NO: 5012), KKTELQTTNAENKTKKL (SEQ ID NO: 5013), KRGINDRNFWRGENGRKTR (SEQ ID NO: 5014), KRPAATKKAGQAKKKK (SEQ ID NO: 5015) or a functional fragment or variant thereof. In some embodiments, a first NLS comprises the amino acid sequence PAAKRVKLD (SEQ ID NO: 11095) , or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. Exemplary NLS sequences are also described in PCT / EP2000 / 011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, an NLS comprises an amino acid sequence as disclosed in Table 11. An NLS of this table may be utilized with one or more copies in a polypeptide in one or more locations in a polypeptide, e.g., 1, 2, 3 or more copies of an NLS in an N-terminal domain, between peptide domains, in a C- terminal domain, or in a combination of locations, in order to improve subcellular localization to the nucleus. Multiple unique sequences may be used within a single polypeptide. Sequences may be naturally monopartite or bipartite, e.g., having one or two stretches of basic amino acids, or may be used as chimeric bipartite sequences. Sequence references correspond to UniProt accession numbers, except where indicated as SeqNLS for sequences mined using a subcellular localization prediction algorithm (Lin et al BMC Bioinformat 13:157 (2012), incorporated herein by reference in its entirety). Page 198 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, a NLS sequence (e.g., second NLS sequence) comprises a plurality of partial NLS sequences. In some embodiments, a NLS sequence, e.g., a second NLS sequence, comprises a first partial NLS sequence, e.g., comprising the amino acid sequence KRTADGSEFE (SEQ ID NO: 5350), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, a NLS sequence, e.g., a second NLS sequence, comprises a second partial NLS sequence. In some embodiments, the NLS sequence, e.g., the second NLS sequence, comprises an SV40A5 NLS, e.g., a bipartite SV40A5 NLS, e.g., comprising the amino acid sequence KRTADGSEFESPKKKAKVE (SEQ ID NO: 5351), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, an NLS sequence, e.g., a second NLS sequence, comprises the amino acid sequence KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 5349), or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity thereto. Table 11. Exemplary nuclear localization signals for use in gene modifying systems Sequence Sequence References SEQ ID NO. AHFKI EKRP TDP KKAKN 7 I 7 22 Page 199 of 327 12592906v1 Attorney Docket No.2017469-0039 Sequence Sequence References SEQ ID NO. KKGPSVQKRKKT Q6ZN17 5238 Page 200 of 327 12592906v1 Attorney Docket No.2017469-0039 Sequence Sequence References SEQ ID NO. MPQNEYIELHRKRYGYRLDY SeqNLS 5271 Page 201 of 327 12592906v1 Attorney Docket No.2017469-0039 Sequence Sequence References SEQ ID NO. RKIKRKRAK B9X187 5305 Page 202 of 327 12592906v1 Attorney Docket No.2017469-0039 Sequence Sequence References SEQ ID NO. RRRGKNKVAAQNCRK SeqNLS 5328 s two basic amino acid clusters separated by a spacer sequence (which may be, e.g., about 10 amino acids in length). A monopartite NLS typically lacks a spacer. An example of a bipartite NLS is the nucleoplasmin NLS, having the sequence KR[PAATKKAGQA]KKKK (SEQ ID NO: 5015), wherein the spacer is indicated in square brackets. Another exemplary bipartite NLS has the sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 5016). Exemplary Page 203 of 327 12592906v1 Attorney Docket No.2017469-0039 NLSs are described in International Application WO2020051561, which is herein incorporated by reference in its entirety, including for its disclosures regarding nuclear localization sequences. Inteins In some embodiments, an intein-N (intN) domain may be fused to the N-terminal portion of a first domain of a gene modifying polypeptide described herein, and an intein-C (intC) domain may be fused to the C-terminal portion of a second domain of a gene modifying polypeptide described herein for the joining of the N-terminal portion to the C-terminal portion, thereby joining the first and second domains. nteins can occur as self-splicing protein intron (e.g., peptide), e.g., which ligates flanking N-terminal and C-terminal exteins (e.g., fragments to be joined). An intein may, in some embodiments, comprise a fragment of a protein that is able to excise itself and join the remaining fragments (the exteins) with a peptide bond in a process known as protein splicing. Inteins are also referred to as “protein introns.” The process of an intein excising itself and joining the remaining portions of the protein is herein termed “protein splicing” or “intein-mediated protein splicing.” Additional domains In some embodiments, a gene modifying polypeptide of the present disclosure can bind a target DNA sequence and template nucleic acid (e.g., a template RNA), nick a target site, and write (e.g., reverse transcribe) a template into DNA, resulting in a modification of the target site. In some embodiments, additional domains may be added to a gene modifying polypeptide to enhance the efficiency of the process. In some embodiments, a gene modifying polypeptide may contain an additional DNA ligation domain to join reverse transcribed DNA to the DNA of a target site. In some embodiments, a gene modifying polypeptide may comprise a heterologous RNA-binding domain. In some embodiments, a gene modifying polypeptide may comprise a domain having 5´ to 3´ exonuclease activity (e.g., wherein the 5´ to 3´ exonuclease activity increases repair of the alteration of a target site, e.g., in favor of alteration over the original genomic sequence). In some embodiments, a gene modifying polypeptide may comprise a domain having 3´ to 5´ exonuclease activity, e.g., proof-reading activity. In some embodiments, a writing domain, e.g., an RT domain, has 3´ to 5´ exonuclease activity, e.g., proof-reading activity. Page 204 of 327 12592906v1 Attorney Docket No.2017469-0039 Template nucleic acids Gene modifying systems described herein can modify a host target DNA site using a template nucleic acid sequence. In some embodiments, gene modifying systems described herein transcribe an RNA sequence template into host target DNA sites by target-primed reverse transcription (TPRT). By modifying DNA sequence(s) via reverse transcription of an RNA sequence template directly into the host genome, a gene modifying system can insert an object sequence into a target genome without the need for exogenous DNA sequences to be introduced into the host cell (unlike, for example, CRISPR systems), as well as eliminate an exogenous DNA insertion step. A gene modifying system can also delete a sequence from a target genome or introduce a substitution using an object sequence. Therefore, a gene modifying system provides a platform for the use of customized RNA sequence templates containing object sequences, e.g., sequences comprising heterologous gene coding and / or function information. In some embodiments, a template nucleic acid comprises one or more sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sequences) that binds a gene modifying polypeptide. In some embodiments, a template RNA can comprise a gRNA sequence, e.g., to direct a gene modifying polypeptide to a target site of interest. In some embodiments, a template RNA comprises (e.g., from 5′ to 3′) (i) a gRNA spacer that binds a target site (e.g., a second strand of a site in a target genome), (ii) a gRNA scaffold that binds a polypeptide described herein (e.g., a Cas domain of a gene modifying polypeptide), (iii) a heterologous object sequence comprising a mutation region (optionally the heterologous object sequence comprises, from 5’ to 3’, a first homology region, a mutation region, and a second homology region), and (iv) a primer binding site (PBS) sequence. In some embodiments, a template RNA has a poly-A tail at its 3´ end. In some embodiments, a template RNA does not have a poly-A tail at its 3´ end. In some embodiments, a template nucleic acid is a template RNA. In some embodiments, a template RNA comprises one or more modified nucleotides. For example, in some embodiments, a template RNA comprises one or more deoxyribonucleotides. In some embodiments, regions of a template RNA are replaced by DNA nucleotides, e.g., to enhance stability of the molecule. For example, the 3´ end of a template may comprise DNA nucleotides, while the rest of the template comprises RNA nucleotides that can be reverse transcribed. For instance, in some embodiments, a heterologous object sequence is primarily or wholly made up Page 205 of 327 12592906v1 Attorney Docket No.2017469-0039 of RNA nucleotides (e.g., at least 90%, at least 95%, at least 98%, at least 99%, or 100% RNA nucleotides). In some embodiments, a PBS sequence is primarily or wholly made up of DNA nucleotides (e.g., at least 90%, at least 95%, at least 98%, at least 99%, or 100% DNA nucleotides). A template RNA described herein may comprise, from 5’ to 3’: (1) a gRNA spacer; (2) a gRNA scaffold; (3) heterologous object sequence (4) a primer binding site (PBS) sequence. Each of these components is now described in more detail. Given that a PBS sequence hybridizes to a strand of a target nucleic acid and a heterologous object sequence forms a template for reverse transcriptase activity, the junction between the PBS sequence and the heterologous object sequence (i.e., between positions -1 and +1) corresponds to the site of a break induced in a target nucleic acid sequence where reverse transcription begins. For example, SpCas9 N863A typically nicks a bond in a target nucleic acid molecule that corresponds to the bond between the third and fourth nucleotides from the 3’ end of a gRNA spacer sequence. gRNA spacer and gRNA scaffold A template RNA described herein may comprise a gRNA spacer that directs a gene modifying system to a target nucleic acid, and a gRNA scaffold that promotes association of a template RNA with a Cas domain of a gene modifying polypeptide. In some embodiments, a gRNA scaffold has been engineered for improved performance with St1Cas9. Systems described herein can also comprise a gRNA that is not part of a template nucleic acid. For example, a gRNA that comprises a gRNA spacer and gRNA scaffold, but not a heterologous object sequence or a PBS sequence, can be used, e.g., to induce second strand nicking, e.g., as described in the section herein entitled “Second Strand Nicking”. In some embodiments, a gRNA is a short synthetic RNA composed of a scaffold sequence that participates in CRISPR-associated protein binding and a user-defined ∼20 nucleotide targeting sequence for a genomic target. The structure of a complete gRNA was described by Nishimasu et al. Cell 156, P935-949 (2014). A gRNA (also referred to as sgRNA for single-guide RNA) comprises crRNA- and tracrRNA-derived sequences connected by an artificial tetraloop. The crRNA sequence can be divided into guide (20 nt) and repeat (12 nt) regions, whereas the tracrRNA sequence can be divided into anti-repeat (14 nt) and three tracrRNA stem loops (Nishimasu et al. Cell 156, P935-949 (2014)). In practice, guide RNA Page 206 of 327 12592906v1 Attorney Docket No.2017469-0039 sequences are generally designed to have a length of between 17 – 24 nucleotides (e.g., 19, 20, or 21 nucleotides) and be complementary to a targeted nucleic acid sequence. Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs. In some embodiments, a gRNA comprises two RNA components from the native CRISPR system, e.g., crRNA and tracrRNA. A gRNA may also comprise a chimeric, single guide RNA (sgRNA) containing sequence from both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing / binding). Chemically modified sgRNAs have also been demonstrated to be effective for use with CRISPR- associated proteins; see, for example, Hendel et al. (2015) Nature Biotechnol., 985 – 991. In some embodiments, a gRNA spacer comprises a nucleic acid sequence that is complementary to a DNA sequence associated with a target gene. In some embodiments, the region of a template nucleic acid, e.g., template RNA, comprising a gRNA adopts an underwound ribbon-like structure of gRNA bound to target DNA (e.g., as described in Mulepati et al. Science 19 Sep 2014:Vol. 345, Issue 6203, pp. 1479-1484). Without wishing to be bound by any particular theory, this non-canonical structure is thought to be facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid. Thus, in some embodiments, the region of a template nucleic acid, e.g., template RNA, comprising a gRNA may tolerate increased mismatching with a target site at some interval, e.g., every sixth base. In some embodiments, the region of a template nucleic acid, e.g., template RNA, comprising a gRNA comprising homology to a target site may possess wobble positions at a regular interval, e.g., every sixth base, that do not need to base pair with the target site. In some embodiments, a template nucleic acid (e.g., template RNA) has at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 bases of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% homology to a target site, e.g., at the 5’ end, e.g., comprising a gRNA spacer sequence of length appropriate to the Cas9 domain of a gene modifying polypeptide. Herein, when an RNA sequence (e.g., a template RNA sequence) is said to comprise a particular sequence that comprises thymine (T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil (U) in place of T. Additionally, it is understood that terminal Us and Ts may optionally be added or removed from tracrRNA sequences and may be modified or unmodified when provided as RNA. It is contemplated herein that gRNA scaffold Page 207 of 327 12592906v1 Attorney Docket No.2017469-0039 sequences represent a component of gene modifying systems that can be similarly optimized for a given system, Cas-RT fusion polypeptide, indication, target mutation, template RNA, or delivery vehicle. Variant gRNA scaffolds In some embodiments, a template RNA described herein comprises a gRNA scaffold (e.g., an St1Cas9 gRNA scaffold) that is a variant relative to the wild-type St1Cas9 gRNA scaffold. In some embodiments, a variant scaffold may comprise a deletion of stem loop 2, a lengthened RAR upper stem, a substitution in the RAR upper stem, or a mutation in a tetraloop. In some embodiments, a system described herein comprises a template RNA (tgRNA) comprising (e.g., from 5’ to 3’): (1) a gRNA spacer; (2) a variant St1Cas9 scaffold having a deletion of part or all of stem loop 2; (3) a heterologous object sequence; and (4) a primer binding site (PBS) sequence. In some embodiments, a deletion is between 1-32 (e.g., 2-29, 2-20, 2-10, or 10-20) nucleotides in length. In some embodiments, a deletion is of positions 55 through 84. In some embodiments, a variant St1Cas9 scaffold has one or both of a lengthened RAR upper stem or a substitution resulting in a G-C base pair in the RAR upper stem. In some embodiments, a variant St1Cas9 scaffold has a mutation in the tetraloop. In some embodiments, a system described herein comprises a template RNA (tgRNA) comprising (e.g., from 5’ to 3’): (1) a gRNA spacer; (2) a variant St1Cas9 scaffold having one or both of a lengthened RAR upper stem or a substitution resulting in a G-C base pair in an RAR upper stem; (3) a heterologous object sequence; and (4) a primer binding site (PBS) sequence. In some embodiments, an RAR upper stem is lengthened by 1-8 base pairs (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 base pairs) relative to the wild-type sequence of SEQ ID NO: 25999. In some embodiments, a system described herein comprises a template RNA (tgRNA) comprising (e.g., from 5’ to 3’): (1) a gRNA spacer; (2) a variant St1Cas9 scaffold having a mutation in a tetraloop; (3) a heterologous object sequence; and (4) a primer binding site (PBS) sequence. In some embodiments, a tetraloop is lengthened, e.g., to 5 nucleotides. In some embodiments, a system described herein comprises a template RNA comprising a variant gRNA scaffold that comprises or consists of a sequence according to GUCUUUGUACUCUGGUACCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCA (SEQ ID NO: 26000). In some embodiments, a system described herein comprises a template RNA comprising a variant gRNA scaffold that comprises or consists of a sequence according to Page 208 of 327 12592906v1 Attorney Docket No.2017469-0039 GUCUUUGUACUCUGGGACUUCGGUCCCAGAAGCUACAAAGAUAAGGCUUCAUGCC GAAAUCA (SEQ ID NO: 26001). The wild-type St1Cas9 gRNA scaffold has a hypothesized secondary structure, shown in FIG. 3. Generally, from 5’ to 3’, the gRNA scaffold comprises: a region comprising a lower stem, an upper stem, and tetraloop (also collectively referred to as Repeat:anti-repeat duplex or RAR); a first single stranded region; a stem loop 1, a second single stranded region; a stem loop 2; and a third single stranded region. An upper stem comprises three paired bases (nt 12-14 pair with nt 19-21) and the 4-nucleotide tetraloop is nt 15-18. At the base of the three paired bases of the upper stem is a region with bulges (nt 22 and nt 25 bulge from the region), and at the base of the region with bulges is a lower stem (nt 1-9 pair with nt 26-34). Moving in a 3’ direction, the next region is a first single stranded region which contains nt 35 and 36. Following the first single stranded region is stem loop 1, which comprises nucleotides 37-47. Next is a second single stranded region, comprising nucleotides 48-53. Next is stem loop 2 which comprises nucleotides 54-82. 3’ of stem loop 2 is a third single stranded region which comprises nucleotides 83-84. The hypothesized structure represents the likely secondary structure of the St1Cas9 gRNA scaffold under physiologically relevant conditions. However, even if a St1Cas9 gRNA scaffold were to adopt a different structure from the hypothesized structure described herein, the named regions (such as stem loop 1, stem loop 2, RAR upper stem, RAR lower stem, and tetraloop) of variant scaffolds could still be readily identified based at least on sequence alignments to the wild-type reference sequence, and optionally using additional tools such as RNA folding algorithms. Heterologous object sequence A template RNA described herein may comprise a heterologous object sequence that a gene modifying polypeptide can use as a template for reverse transcription, to write a desired sequence into a target nucleic acid. In some embodiments, a heterologous object sequence comprises, from 5’ to 3’, a post-edit homology region, a mutation region, and a pre-edit homology region. Without wishing to be bound by any particular theory, an RT performing reverse transcription on a template RNA first reverse transcribes a pre-edit homology region, then a mutation region, and then a post-edit homology region, thereby creating a DNA strand comprising a desired mutation with a homology region on either side. Page 209 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, a heterologous object sequence is at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 500, or at least 1,000 nucleotides (nts) in length, or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 kilobases in length. In some embodiments, a heterologous object sequence is no more than 33, no more than 34, no more than 35, no more than 36, no more than 37, no more than 38, no more than 39, no more than 40, no more than 41, no more than 42, no more than 43, no more than 44, no more than 45, no more than 46, no more than 47, no more than 48, no more than 49, no more than 50, no more than 51, no more than 52, no more than 53, no more than 54, no more than 55, no more than 56, no more than 57, no more than 58, no more than 59, no more than 60, no more than 61, no more than 62, no more than 63, no more than 64, no more than 65, no more than 66, no more than 67, no more than 68, no more than 69, no more than 70, no more than 71, no more than 72, no more than 73, no more than 74, no more than 75, no more than 76, no more than 77, no more than 78, no more than 79, no more than 80, no more than 81, no more than 82, no more than 83, no more than 84, no more than 85, no more than 86, no more than 87, no more than 88, no more than 89, no more than 90, no more than 91, no more than 92, no more than 93, no more than 94, no more than 95, no more than 96, no more than 97, no more than 98, no more than 99, no more than 100, no more than 120, no more than 140, no more than 160, no more than 180, no more than 200, no more than 500, no more than 1,000, or no more than 2000 nucleotides (nts) in length, or no more than 20, no more than 15, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, or no more than 3 kilobases in length. In some embodiments, a heterologous object sequence is 30-1000, 40-1000, 50-1000, 60- 1000, 70-1000, 74-1000, 75-1000, 76-1000, 77-1000, 78-1000, 79-1000, 80-1000, 85-1000, 90- 1000, 100-1000, 120-1000, 140-1000, 160-1000, 180-1000, 200-1000, 500-1000, 30-500, 40- Page 210 of 327 12592906v1 Attorney Docket No.2017469-0039 500, 50-500, 60-500, 70-500, 74-500, 75-500, 76-500, 77-500, 78-500, 79-500, 80-500, 85-500, 90-500, 100-500, 120-500, 140-500, 160-500, 180-500, 200-500, 30-200, 40-200, 50-200, 60- 200, 70-200, 74-200, 75-200, 76-200, 77-200, 78-200, 79-200, 80-200, 85-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, 30-100, 40-100, 50-100, 60-100, 70-100, 74-100, 75-100, 76-100, 77-100, 78-100, 79-100, 80-100, 85-100, or 90-100 nucleotides (nts) in length, or 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-15, 6-10, 6-9, 6-8, 6-7, 7-20, 7-15, 7-10, 7-9, 7-8, 8-20, 8-15, 8- 10, 8-9, 9-20, 9-15, 9-10, 10-15, 10-20, or 15-20 kilobases in length. In some embodiments, a heterologous object sequence is 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, or 10- 20 nt in length, e.g., 10-80, 10-50, or 10-20 nt in length, e.g., about10-20 nt in length. In some embodiments, a heterologous object sequence is 8-30, 9-25, 10-20, 11-16, or 12-15 nucleotides in length, e.g., is 11-16 nt in length. Without wishing to be bound by any particular theory, in some embodiments, a larger insertion size, larger region of editing (e.g., the distance between a first edit / substitution and a second edit / substitution in the target region), and / or greater number of desired edits (e.g., mismatches of the heterologous object sequence to the target genome), may result in a longer optimal heterologous object sequence. In some embodiments, a template nucleic acid comprises a customized RNA sequence template which can be identified, designed, engineered and constructed to contain sequences altering or specifying host genome function, for example by introducing a heterologous coding region into a genome; affecting or causing exon structure / alternative splicing, e.g., leading to exon skipping of one or more exons; causing disruption of an endogenous gene, e.g., creating a genetic knockout; causing transcriptional activation of an endogenous gene; causing epigenetic regulation of an endogenous DNA; causing up-regulation of one or more operably linked genes, e.g., leading to gene activation or overexpression; causing down-regulation of one or more operably linked genes, e.g., creating a genetic knock-down; etc. In some embodiments, a customized RNA sequence template can be engineered to contain sequences coding for exons and / or transgenes, provide binding sites for transcription factor activators, repressors, enhancers, etc., and combinations thereof. In some embodiments, a customized template can be engineered to encode a nucleic acid or peptide tag to be expressed in an endogenous RNA transcript or Page 211 of 327 12592906v1 Attorney Docket No.2017469-0039 endogenous protein operably linked to the target site. In other embodiments, the coding sequence can be further customized with splice donor sites, splice acceptor sites, or poly-A tails. A template nucleic acid (e.g., a template RNA) of a system provided herein may comprise an object sequence (e.g., a heterologous object sequence) for writing a desired sequence into a target DNA. An object sequence (e.g., a heterologous object sequence) may be coding or non-coding. A template nucleic acid (e.g., template RNA) can be designed to result in insertions, mutations, or deletions at the target DNA locus. In some embodiments, a template nucleic acid (e.g., template RNA) may be designed to cause an insertion in a target DNA. For example, a template nucleic acid (e.g., template RNA) may contain a heterologous sequence, wherein reverse transcription will result in insertion of the heterologous sequence into a target DNA. In some embodiments, an RNA template may be designed to introduce a deletion into a target DNA. For example, a template nucleic acid (e.g., template RNA) may match a target DNA upstream and downstream of a desired deletion, wherein reverse transcription will result in the copying of the upstream and downstream sequences from the template nucleic acid (e.g., template RNA) without the intervening sequence, e.g., causing deletion of the intervening sequence. In some embodiments, a template nucleic acid (e.g., template RNA) may be designed to introduce an edit into a target DNA. For example, a template RNA may match a target DNA sequence with the exception of one or more nucleotides, wherein reverse transcription will result in the copying of these edits into the target DNA, e.g., resulting in mutations, e.g., transition or transversion mutations. In some embodiments, writing (e.g., reverse transcription) of an object sequence (e.g., a heterologous object sequence) into a target site results in the substitution of nucleotides, e.g., where the full length of the object sequence corresponds to a matching length of the target site with one or more mismatched bases. In some embodiments, a heterologous object sequence may be designed such that a combination of sequence alterations may occur, e.g., a simultaneous addition and deletion, addition and substitution, or deletion and substitution. In some embodiments, a heterologous object sequence may contain an open reading frame or a fragment of an open reading frame. In some embodiments, a heterologous object sequence has a Kozak sequence. In some embodiments, a heterologous object sequence has an internal ribosome entry site. In some embodiments, a heterologous object sequence has a self- cleaving peptide such as a T2A or P2A site. In some embodiments, a heterologous object Page 212 of 327 12592906v1 Attorney Docket No.2017469-0039 sequence has a start codon. In some embodiments, a template RNA has a splice acceptor site. In some embodiments, a template RNA has a splice donor site. Exemplary splice acceptor and splice donor sites are described in WO2016044416, incorporated herein by reference in its entirety. Exemplary splice acceptor site sequences are known to those of skill in the art. In some embodiments, a template RNA has a microRNA binding site downstream of a stop codon. In some embodiments, a template RNA has a polyA tail downstream of a stop codon of an open reading frame. In some embodiments, a template RNA comprises one or more exons. In some embodiments, a template RNA comprises one or more introns. In some embodiments, a template RNA comprises a eukaryotic transcriptional terminator. In some embodiments, a template RNA comprises an enhanced translation element or a translation enhancing element. In some embodiments, a template RNA comprises a human T-cell leukemia virus (HTLV-1) R region. In some embodiments, a template RNA comprises a posttranscriptional regulatory element that enhances nuclear export, such as that of Hepatitis B Virus (HPRE) or Woodchuck Hepatitis Virus (WPRE). In some embodiments, a heterologous object sequence may contain a non-coding sequence. For example, a template nucleic acid (e.g., a template RNA) may comprise a regulatory element, e.g., a promoter or enhancer sequence or miRNA binding site. In some embodiments, integration of an object sequence (e.g., a heterologous object sequence) at a target site will result in upregulation of an endogenous gene. In some embodiments, integration of an object sequence (e.g., a heterologous object sequence)_at a target site will result in downregulation of an endogenous gene. In some embodiments, a template nucleic acid (e.g., template RNA) comprises a tissue specific promoter or enhancer, each of which may be unidirectional or bidirectional. In some embodiments, a promoter is an RNA polymerase I promoter, RNA polymerase II promoter, or RNA polymerase III promoter. In some embodiments, a promoter comprises a TATA element. In some embodiments, a promoter comprises a B recognition element. In some embodiments, a promoter has one or more binding sites for transcription factors. In some embodiments, a template nucleic acid (e.g., a template RNA) comprises a site that coordinates epigenetic modification. In some embodiments, a template nucleic acid (e.g., a template RNA) comprises a chromatin insulator. For example, a template nucleic acid (e.g., a template RNA) may comprise a CTCF site or a site targeted for DNA methylation. Page 213 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, a template nucleic acid (e.g., a template RNA) comprises a gene expression unit composed of at least one regulatory region operably linked to an effector sequence. An effector sequence may be a sequence that is transcribed into RNA (e.g., a coding sequence or a non-coding sequence such as a sequence encoding a micro RNA). In some embodiments, a heterologous object sequence of a template nucleic acid (e.g., a template RNA) is inserted into a target genome in an endogenous intron. In some embodiments, a heterologous object sequence of a template nucleic acid (e.g., a template RNA) is inserted into a target genome and thereby acts as a new exon. In some embodiments, insertion of a heterologous object sequence into a target genome results in replacement of a natural exon or the skipping of a natural exon. A template nucleic acid (e.g., a template RNA) can be designed to result in insertions, mutations, or deletions at a target DNA locus. In some embodiments, a template nucleic acid (e.g., a template RNA) may be designed to cause an insertion in a target DNA. For example, a template nucleic acid (e.g., a template RNA) may contain a heterologous object sequence, wherein reverse transcription will result in insertion of the heterologous object sequence into a target DNA. In some embodiments, an RNA template may be designed to write a deletion into a target DNA. For example, a template nucleic acid (e.g., a template RNA) may match a target DNA upstream and downstream of a desired deletion, wherein reverse transcription will result in copying of the upstream and downstream sequences from the template nucleic acid (e.g., the template RNA) without the intervening sequence, e.g., causing deletion of the intervening sequence. In some embodiments, a template nucleic acid (e.g., a template RNA) may be designed to write (e.g. reverse transcribe) an edit into a target DNA. For example, a template RNA may match a target DNA sequence with the exception of one or more nucleotides, wherein reverse transcription will result in copying of these edits into the target DNA, e.g., resulting in mutations, e.g., transition or transversion mutations. In some embodiments, a pre-edit homology domain comprises a nucleic acid sequence having 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule. In some embodiments, a post-edit homology domain comprises a nucleic acid sequence having 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule. Page 214 of 327 12592906v1 Attorney Docket No.2017469-0039 PBS sequence In some embodiments, a template nucleic acid (e.g., a template RNA) comprises a primer binding site (PBS) sequence. In some embodiments, a PBS sequence is disposed 3′ of a heterologous object sequence and is complementary to a sequence adjacent to a site to be modified by a system described herein, or comprises no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches to a sequence complementary to a sequence adjacent to a site to be modified by a system / gene modifying polypeptide. In some embodiments, a PBS sequence binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nick site in a target nucleic acid molecule. In some embodiments, binding of a PBS sequence to a target nucleic acid molecule permits initiation of target-primed reverse transcription (TPRT), e.g., with the 3′ homology domain acting as a primer for TPRT. In some embodiments, a PBS sequence is 3-5, 5-10, 10-30, 10-25, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10- 12, 10-11, 11-30, 11-25, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-30, 12-25, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-30, 13-25, 13-20, 13-19, 13- 18, 13-17, 13-16, 13-15, 13-14, 14-30, 14-25, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-30, 15-25, 15-20, 15-19, 15-18, 15-17, 15-16, 16-30, 16-25, 16-20, 16-19, 16-18, 16-17, 17-30, 17- 25, 17-20, 17-19, 17-18, 18-30, 18-25, 18-20, 18-19, 19-30, 19-25, 19-20, 20-30, 20-25, or 25-30 nucleotides in length, e.g., 10-17, 12-16, or 12-14 nucleotides in length. In some embodiments, a PBS sequence is 5-20, 8-16, 8-14, 8-13, 9-13, 9-12, or 10-12 nucleotides in length, e.g., 9-12 nucleotides in length. A template nucleic acid (e.g., a template RNA) may have some homology to a target DNA. In some embodiments, a template nucleic acid (e.g., a template RNA) PBS sequence domain may serve as an annealing region to a target DNA, such that the target DNA is positioned to prime the reverse transcription of the template nucleic acid (e.g., the template RNA). In some embodiments, a template nucleic acid (e.g., a template RNA) has at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200 or more bases of exact homology to a target DNA at the 3′ end of the RNA. In some embodiments, a template nucleic acid (e.g., a template RNA) has at least 2, at least 3, at least 4, at least 5, at least 6, at Page 215 of 327 12592906v1 Attorney Docket No.2017469-0039 least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 175, at least 200 or more bases of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% homology to a target DNA, e.g., at the 5′ end of the template nucleic acid (e.g., the template RNA). Template RNA sequences may be customized depending on the cell being targeted. For example, in some embodiments it may be desired to inactivate a PAM sequence upon editing (e.g., using a “PAM-kill” modification) to decrease the potential for further gene editing (e.g., by Cas retargeting) following an initial edit. Consequently, certain template RNAs described herein are designed to write a mutation (e.g., a substitution) into the PAM of a target site, such that upon editing, the PAM site will be mutated to a sequence no longer recognized by a gene modifying polypeptide. Thus, a mutation region within a heterologous object sequence of a template RNA may comprise a PAM-kill sequence. Without wishing to be bound by any particular theory, in some embodiments, a PAM-kill sequence prevents re-engagement of a gene modifying polypeptide upon completion of a gene modification, or decreases re-engagement relative to a template RNA lacking a PAM-kill sequence. In some embodiments, a PAM-kill sequence does not alter the amino acid sequence encoded by a gene, e.g., the PAM-kill sequence results in a silent mutation. In some embodiments, it may be desired to leave a PAM sequence intact (no PAM-kill). Similarly, in some embodiments, to decrease the potential for further gene editing (e.g., by Cas retargeting) following an initial edit, it may be desirable to alter the first three nucleotides of an RT template sequence via a “seed-kill” motif. Consequently, in some embodiments, template RNAs described herein are designed to write (e.g., reverse transcribe) a mutation (e.g., a substitution) into a portion of a target site corresponding to the first three nucleotides of an RT template sequence, such that upon editing, the target site will be mutated to a sequence with lower homology to the RT template sequence. Thus, a mutation region within a heterologous object sequence of a template RNA may comprise a seed-kill sequence. Without wishing to be bound by any particular theory, in some embodiments, a seed-kill sequence prevents re- engagement of a gene modifying polypeptide upon completion of genetic modification, or Page 216 of 327 12592906v1 Attorney Docket No.2017469-0039 decreases re-engagement relative to an otherwise similar template RNA lacking a seed-kill sequence. In some embodiments, a seed-kill sequence does not alter the amino acid sequence encoded by a gene, e.g., the seed-kill sequence results in a silent mutation. In other embodiments, it is desired to leave a seed region intact, and a seed-kill sequence is not used. In some embodiments, to optimize or improve gene editing efficiency, it may be desirable to evade the target cell’s mismatch repair or nucleotide repair pathways or to bias the target cell’s repair pathways toward preservation of the edited strand. In some embodiments, multiple silent mutations (for example, silent substitutions) may be introduced within an RT template sequence to evade the target cell’s mismatch repair or nucleotide repair pathways or to bias the target cell’s repair pathways toward preservation of the edited strand. Exemplary template nucleic acids In some embodiments, a template nucleic acid is a polyribonucleotide (e.g., RNA) having a nucleotide sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any sequence listed in Table 12. Table 12. Exemplary Template RNAs RNACS NAME SEQUENCE SEQ ID NO RNACS13597 St1 dSL2 A1A AAGGCUGUGCUGACCAUCGAGUCUUUGUACUCU 10010 Page 217 of 327 12592906v1 Attorney Docket No.2017469-0039 RNACS NAME SEQUENCE SEQ ID NO RNACS19672 St1_t- AAGGCUGUGCUGACCAUCGAGUCUUUGUACUCU 10010 Page 218 of 327 12592906v1 Attorney Docket No.2017469-0039 gRNAs with inducible activity In some embodiments, a gRNA described herein (e.g., a gRNA that is part of a template RNA or a gRNA used for second strand nicking) has inducible activity. Inducible activity may be achieved by a template nucleic acid, e.g., a template RNA, further comprising (in addition to a gRNA) a blocking domain, wherein the sequence of a portion of or all of the blocking domain is at least partially complementary to a portion or all of the gRNA. In some embodiments, a gRNA that coordinates a second nick has inducible activity. In some embodiments, a gRNA that coordinates a second nick is induced after a template is reverse transcribed. In some embodiments, hybridization of a gRNA to a blocking domain can be disrupted using an opener molecule. Exemplary blocking domains, opener molecules, and uses thereof are described in PCT App. Publication WO2020044039A1, which is incorporated herein by reference in its entirety. Circular RNAs and Ribozymes in Gene Modifying Systems It is contemplated that it may be useful to employ circular and / or linear RNA states during the formulation, delivery, or gene modifying reaction within the target cell. Thus, in some embodiments, a gene modifying system comprises one or more circular RNAs (circRNAs). In some embodiments, a gene modifying system comprises one or more linear RNAs. In some embodiments, a circRNA comprises one or more ribozyme sequences. In some embodiments, a ribozyme sequence is activated for autocleavage, e.g., in a host cell, e.g., thereby resulting in linearization of the circRNA. Target Nucleic Acid Site In some embodiments, after gene modification, a target site surrounding an edited sequence contains a limited number of insertions or deletions, for example, in less than about 50% or 10% of editing events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. (2020) bioRxiv doi.org / 10.1101 / 645903 (incorporated by reference herein in its entirety). In some embodiments, a target site does not show multiple consecutive editing events, e.g., head-to-tail or head-to-head duplications, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org / 10.1101 / 645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, a target site contains an integrated sequence corresponding to a template RNA. In some embodiments, a target site does not contain insertions resulting from endogenous RNA in Page 219 of 327 12592906v1 Attorney Docket No.2017469-0039 more than about 1% or 10% of events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org / 10.1101 / 645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, a target site contains an integrated sequence corresponding to the template RNA. In some embodiments, a host DNA-binding site integrated into by a gene modifying system of the present disclosure can be in a gene, in an intron, in an exon, an ORF, outside of a coding region of any gene, in a regulatory region of a gene, or outside of a regulatory region of a gene. In some embodiments, a gene modifying polypeptide may bind to one or more than one host DNA sequence. In some embodiments, a gene modifying system is used to edit a target locus in multiple alleles. In some embodiments, a gene modifying system is designed to edit a specific allele. For example, a gene modifying polypeptide may be directed to a specific sequence that is only present on one allele, e.g., comprises a template RNA with homology to a target allele, e.g., a gRNA or annealing domain, but not to a second cognate allele. In some embodiments, a gene modifying system can alter a haplotype-specific allele. In some embodiments, a gene modifying system that targets a specific allele preferentially targets that allele, e.g., has at least a 2-fold, at least a 4-fold, at least a 6-fold, at least a 8-fold, or at least a 10-fold preference for a target allele. Second Strand Nicking In some embodiments, a gene modifying system described herein comprises a nickase activity (e.g., in a gene modifying polypeptide) that nicks the first strand, and a nickase activity (e.g., in the gene modifying polypeptide or in a polypeptide separate from the gene modifying polypeptide) that nicks the second strand of target DNA. As discussed herein, without wishing to be bound by any particular theory, nicking of the first strand of a target site DNA is thought to provide a 3´ OH that can be used by an RT domain to reverse transcribe a sequence of a template RNA, e.g., a heterologous object sequence. Without wishing to be bound by any particular theory, after a writing domain (e.g., RT domain) of a polypeptide described herein polymerizes (e.g., reverse transcribes) from a heterologous object sequence of a template nucleic acid (e.g., a template RNA), cellular DNA repair machinery must repair the nick on the first DNA strand. The target site DNA now contains two different sequences for the first DNA strand: one corresponding to the original genomic DNA (e.g., having a free 5′ end) and a second corresponding to that polymerized from the heterologous object sequence (e.g., having a free 3′ Page 220 of 327 12592906v1 Attorney Docket No.2017469-0039 end). It is thought that the two different sequences equilibrate with one another, first one hybridizing the second strand, then the other, and which sequence the cellular DNA repair apparatus incorporates into its repaired target site may be a stochastic process. Without wishing to be bound by any particular theory, it is thought that introducing an additional nick to the second strand may bias cellular DNA repair machinery to adopt a heterologous object sequence- based sequence more frequently than the original genomic sequence. Alternatively or additionally, without wishing to be bound by any particular theory, it is thought that an additional nick to the second strand may promote second-strand synthesis. In some embodiments, where a gene modifying system has inserted or substituted a portion of the first strand, synthesis of a new sequence corresponding to the insertion / substitution in the second strand is necessary. In some embodiments, a Cas domain is capable of nicking a first strand and a second strand. In some embodiments, first and second strand nicks occur at the same position in a target site but on opposite strands. In some embodiments, a second strand nick occurs in a staggered location, e.g., upstream or downstream, from a first nick. In some embodiments, an endonuclease domain generates a target site deletion if a second strand nick is upstream of a first strand nick. In some embodiments, an endonuclease domain generates a target site duplication if a second strand nick is downstream of a first strand nick. In some embodiments, an endonuclease domain generates no duplication and / or deletion if a first and second strand nicks occur in the same position of a target site. In some embodiments, a Cas domain has altered activity depending on protein conformation or RNA-binding status, e.g., which promotes the nicking of a first or second strand (e.g., as described in Christensen et al. PNAS 2006; incorporated by reference herein in its entirety). In some embodiments, the additional nick to the second strand is made by the same endonuclease domain (e.g., nickase domain) as the endonuclease domain that nicks the first strand. In some embodiments, the same gene modifying polypeptide performs both the nick to the first strand and the nick to the second strand. In some embodiments, a gene modifying polypeptide comprises a CRISPR / Cas domain and an additional nick to the second strand is directed by an additional nucleic acid, e.g., comprising a second gRNA directing the CRISPR / Cas domain to nick the second strand. In some embodiments, an additional second strand nick is made by a different endonuclease domain (e.g., nickase domain) than the Page 221 of 327 12592906v1 Attorney Docket No.2017469-0039 endonuclease domain that nicks the first strand. In some embodiments, a second strand nicking endonuclease domain is situated in an additional polypeptide (e.g., a system of the invention further comprises the additional polypeptide), separate from a gene modifying polypeptide. In some embodiments, an additional polypeptide comprises an endonuclease domain (e.g., nickase domain) described herein. In some embodiments, an additional polypeptide comprises a DNA binding domain, e.g., described herein. It is contemplated herein that the position at which a second strand nick occurs relative to a first strand nick may influence the extent to which one or more of: desired gene modifying DNA modifications are obtained, undesired double-strand breaks (DSBs) occur, undesired insertions occur, or undesired deletions occur. Without wishing to be bound by any particular theory, second strand nicking may occur in two general orientations: inward nicks and outward nicks. In some embodiments, in an inward nick orientation, an RT domain polymerizes (e.g., using a template RNA (e.g., a heterologous object sequence)) away from a second strand nick. In some embodiments, in an inward nick orientation, the location of a nick to the first strand and the location of the nick to the second strand are positioned between the first PAM site and second PAM site (e.g., in a scenario wherein both nicks are made by a polypeptide (e.g., a gene modifying polypeptide) comprising a CRISPR / Cas domain). When there are two PAMs on the outside and two nicks on the inside, this inward nick orientation can also be referred to as “PAM- out”. In some embodiments, in an inward nick orientation, the location of a nick to the first strand and the location of a nick to the second strand are between the sites where the polypeptide and the additional polypeptide bind to a target DNA. In some embodiments, in an inward nick orientation, the location of a nick to the second strand is positioned between the binding sites of a gene modifying polypeptide and an additional polypeptide, and a nick to the first strand is also located between the binding sites of the gene modifying polypeptide and the additional polypeptide. In some embodiments, in an inward nick orientation, the location of a nick to the first strand and the location of a nick to the second strand are positioned between a PAM site and a binding site of a second polypeptide which is at a distance from a target site. An example of a gene modifying system that provides an inward nick orientation comprises a gene modifying polypeptide comprising a CRISPR / Cas domain, a template RNA comprising a gRNA that directs nicking of the target site DNA on the first strand, Page 222 of 327 12592906v1 Attorney Docket No.2017469-0039 and an additional nucleic acid comprising an additional gRNA that directs nicking at a site a distance from the location of the first nick, wherein the location of the first nick and the location of the second nick are between the PAM sites of sequences to which two gRNAs direct the gene modifying polypeptide. In some embodiments, in an outward nick orientation, an RT domain polymerizes (e.g., using a template RNA (e.g., a heterologous object sequence)) toward a second strand nick. In some embodiments, in an outward nick orientation, when both first and second nicks are made by a polypeptide comprising a CRISPR / Cas domain (e.g., a gene modifying polypeptide), a first PAM site and second PAM site are positioned between the location of the nick to the first strand and the location of the nick to the second strand. When there are two PAMs on the inside and two nicks on the outside, this outward nick orientation also can be referred to as “PAM-in”. In some embodiments, in an outward nick orientation, a polypeptide (e.g., a gene modifying polypeptide) and an additional polypeptide bind to sites on the target DNA between the location of the nick to the first strand and the location of the nick to the second. In some embodiments, in an outward nick orientation, the location of a nick to the second strand is positioned on the opposite side of the binding sites of a gene modifying polypeptide and an additional polypeptide relative to the location of a nick to the first strand. In some embodiments, in an outward orientation, a PAM site and a binding site of a second polypeptide which is at a distance from a target site are positioned between the location of a nick to the first strand and the location of a nick to the second strand. In some embodiments, a gene modifying system providing an outward nick orientation comprises a gene modifying polypeptide comprising a Cas domain, a template RNA comprising a gRNA that directs nicking of a target site DNA on the first strand, and an additional nucleic acid comprising an additional gRNA that directs nicking at a site a distance from the location of the first nick, wherein the location of the first nick and the location of the second nick are outside of the PAM sites of the sites to which the two gRNAs direct the gene modifying polypeptide (i.e., the PAM sites are between the location of the first nick and the location of the second nick). Without wishing to be bound by any particular theory, it is thought that, for gene modifying systems where a second strand nick is provided, in some embodiments, an outward nick orientation is preferred. As is described herein, an inward nick may produce a higher number of double-strand breaks (DSBs) than an outward nick orientation. DSBs may be Page 223 of 327 12592906v1 Attorney Docket No.2017469-0039 recognized by DSB repair pathways in the nucleus of a cell, which can result in undesired insertions and deletions. An outward nick orientation may provide a decreased risk of DSB formation, and a corresponding lower amount of undesired insertions and deletions. In some embodiments, undesired insertions and deletions are insertions and deletions not encoded by a heterologous object sequence, e.g., an insertion or deletion produced by the double-strand break repair pathway unrelated to a modification encoded by a heterologous object sequence. In some embodiments, a desired gene modification comprises a change to a target DNA (e.g., a substitution, insertion, or deletion) encoded by the heterologous object sequence (e.g., and achieved by gene modifying writing (e.g., reverse transcribing) the heterologous object sequence into the target site). In some embodiments, a first strand nick and a second strand nick are in an outward orientation. In some embodiments, the distance between a first strand nick and a second strand nick may influence the extent to which one or more of: desired gene modifying system DNA modifications are obtained, undesired double-strand breaks (DSBs) occur, undesired insertions occur, or undesired deletions occur. Without wishing to be bound by any particular theory, it is thought that a second strand nick biases DNA repair toward incorporation of the heterologous object sequence into a target DNA, and that this bias increases as the distance between the first strand nick and second strand nick decreases. However, it is thought that the risk of DSB formation also increases as the distance between the first strand nick and second strand nick decreases. Correspondingly, it is thought that the number of undesired insertions and / or deletions may increase as the distance between the first strand nick and second strand nick decreases. In some embodiments, the distance between a first strand nick and a second strand nick is chosen to balance the benefit of biasing DNA repair toward incorporation of a heterologous object sequence into a target DNA and the risk of DSB formation and of undesired deletions and / or insertions. In some embodiments, a system where a first strand nick and a second strand nick are at least a threshold distance apart has an increased level of desired gene modifying system modification outcomes, a decreased level of undesired deletions, and / or a decreased level of undesired insertions relative to an otherwise similar inward nick orientation system where the first nick and the second nick are less than the threshold distance apart. In some embodiments, threshold distance(s) is / are described below. Page 224 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, a first nick and a second nick are at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 nucleotides apart. In some embodiments, a first nick and a second nick are no more than 25, no more than 30, no more than 35, no more than 40, no more than 45, no more than 50, no more than 55, no more than 60, no more than 65, no more than 70, no more than 75, no more than 80, no more than 85, no more than 90, no more than 95, no more than 100, no more than 110, no more than 120, no more than 130, no more than 140, no more than 150, no more than 160, no more than 170, no more than 180, no more than 190, no more than 200, or no more than 250 nucleotides apart. In some embodiments, a first nick and a second nick are 20-200, 30-200, 40- 200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 20-190, 30-190, 40-190, 50-190, 60-190, 70-190, 80-190, 90-190, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160-190, 170-190, 180-190, 20-180, 30-180, 40-180, 50-180, 60-180, 70-180, 80-180, 90-180, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 20-170, 30-170, 40-170, 50-170, 60- 170, 70-170, 80-170, 90-170, 100-170, 110-170, 120-170, 130-170, 140-170, 150-170, 160-170, 20-160, 30-160, 40-160, 50-160, 60-160, 70-160, 80-160, 90-160, 100-160, 110-160, 120-160, 130-160, 140-160, 150-160, 20-150, 30-150, 40-150, 50-150, 60-150, 70-150, 80-150, 90-150, 100-150, 110-150, 120-150, 130-150, 140-150, 20-140, 30-140, 40-140, 50-140, 60-140, 70-140, 80-140, 90-140, 100-140, 110-140, 120-140, 130-140, 20-130, 30-130, 40-130, 50-130, 60-130, 70-130, 80-130, 90-130, 100-130, 110-130, 120-130, 20-120, 30-120, 40-120, 50-120, 60-120, 70-120, 80-120, 90-120, 100-120, 110-120, 20-110, 30-110, 40-110, 50-110, 60-110, 70-110, 80- 110, 90-110, 100-110, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-90, 20-80, 30-80, 40-80, 50-80, 60-80, 70-80, 20-70, 30- 70, 40-70, 50-70, 60-70, 20-60, 30-60, 40-60, 50-60, 20-50, 30-50, 40-50, 20-40, 30-40, or 20-30 nucleotides apart. In some embodiments, a first nick and a second nick are 40-100 nucleotides apart. In some embodiments, a second nick is positioned at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least Page 225 of 327 12592906v1 Attorney Docket No.2017469-0039 145, or at least 150 nucleotides 5´ or 3´ of a target site modification (e.g., an insertion, deletion, or substitution) or to a nick on the first strand. Without wishing to be bound by any particular theory, it is thought that, for gene modifying systems where a second strand nick is provided and an inward nick orientation is selected, increasing the distance between the first strand nick and second strand nick may be preferred. As described herein, an inward nick orientation may produce a higher number of DSBs than an outward nick orientation, and may result in a higher amount of undesired insertions and deletions than an outward nick orientation, but increasing the distance between the nicks may mitigate that increase in DSBs, undesired deletions, and / or undesired insertions. In some embodiments, an inward nick orientation wherein a first nick and a second nick are at least a threshold distance apart has an increased level of desired gene modifying system modification outcomes, a decreased level of undesired deletions, and / or a decreased level of undesired insertions relative to an otherwise similar inward nick orientation system where the first nick and the second nick are less than the threshold distance apart. In some embodiments, threshold distance(s) is / are described below. In some embodiments, a first strand nick and a second strand nick are in an inward orientation. In some embodiments, a first strand nick and a second strand nick are in an inward orientation and the first strand nick and second strand nick are at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 220, at least 240, at least 260, at least 280, at least 300, at least 350, at least 400, at least 450, or at least 500 nucleotides apart, e.g., at least 100 nucleotides apart, (and optionally no more than 500, no more than 400, no more than 300, no more than 200, no more than 190, no more than 180, no more than 170, no more than 160, no more than 150, no more than 140, no more than 130, or no more than 120 nucleotides apart). In some embodiments, a first strand nick and a second strand nick are in an inward orientation and the first strand nick and second strand nick are 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160-190, 170-190, 180-190, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 100-170, 110-170, 120-170, 130-170, 140-170, 150-170, 160-170, 100-160, 110-160, 120-160, 130-160, 140-160, 150-160, 100-150, 110-150, 120-150, 130-150, 140-150, 100-140, 110-140, 120-140, 130-140, 100-130, 110-130, 120-130, 100-120, 110-120, or 100-110 nucleotides apart. Page 226 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, a second gRNA associated with a system may help drive complete integration. In some embodiments, a second gRNA may target a location that is 0-200 nt away from a first-strand nick, e.g., 0-50, 50-100, 100-200 nt away from the first-strand nick. In some embodiments, a second gRNA can only bind its target sequence after an edit is made, e.g., the gRNA binds a sequence present in the heterologous object sequence, but not in the initial target sequence. Chemically modified nucleic acids and nucleic acid end features A nucleic acid described herein (e.g., a template nucleic acid, e.g., a template RNA; or a nucleic acid (e.g., mRNA) encoding a gene modifying polypeptide; or a gRNA) can comprise unmodified or modified nucleobases. Naturally occurring RNAs are synthesized from four basic ribonucleotides: ATP, CTP, UTP and GTP, but may contain post-transcriptionally modified nucleotides. Further, approximately one hundred different nucleoside modifications have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). An RNA can also comprise wholly synthetic nucleotides that do not occur in nature. In some embodiments, a chemical modification is one provided in WO / 2016 / 183482, US Pat. Pub. No. 20090286852, of International Application No. WO / 2012 / 019168, WO / 2012 / 045075, WO / 2012 / 135805, WO / 2012 / 158736, WO / 2013 / 039857, WO / 2013 / 039861, WO / 2013 / 052523, WO / 2013 / 090648, WO / 2013 / 096709, WO / 2013 / 101690, WO / 2013 / 106496, WO / 2013 / 130161, WO / 2013 / 151669, WO / 2013 / 151736, WO / 2013 / 151672, WO / 2013 / 151664, WO / 2013 / 151665, WO / 2013 / 151668, WO / 2013 / 151671, WO / 2013 / 151667, WO / 2013 / 151670, WO / 2013 / 151666, WO / 2013 / 151663, WO / 2014 / 028429, WO / 2014 / 081507, WO / 2014 / 093924, WO / 2014 / 093574, WO / 2014 / 113089, WO / 2014 / 144711, WO / 2014 / 144767, WO / 2014 / 144039, WO / 2014 / 152540, WO / 2014 / 152030, WO / 2014 / 152031, WO / 2014 / 152027, WO / 2014 / 152211, WO / 2014 / 158795, WO / 2014 / 159813, WO / 2014 / 164253, WO / 2015 / 006747, WO / 2015 / 034928, WO / 2015 / 034925, WO / 2015 / 038892, WO / 2015 / 048744, WO / 2015 / 051214, WO / 2015 / 051173, WO / 2015 / 051169, WO / 2015 / 058069, WO / 2015 / 085318, WO / 2015 / 089511, WO / 2015 / 105926, WO / 2015 / 164674, WO / 2015 / 196130, WO / 2015 / 196128, WO / 2015 / 196118, WO / 2016 / 011226, WO / 2016 / 011222, WO / 2016 / 011306, WO / 2016 / 014846, WO / 2016 / 022914, WO / 2016 / 036902, WO / 2016 / 077125, or WO / 2016 / 077123, each of which is herein incorporated by reference in its entirety. It is understood that incorporation of a chemically modified nucleotide into a Page 227 of 327 12592906v1 Attorney Docket No.2017469-0039 polynucleotide can result in the modification being incorporated into a nucleobase, the backbone, or both, depending on the location of the modification in the nucleotide. In some embodiments, a backbone modification is one provided in EP 2813570, which is herein incorporated by reference in its entirety. In some embodiments, a modified cap is one provided in US Pat. Pub. No. 20050287539, which is herein incorporated by reference in its entirety. In some embodiments, a chemically modified nucleic acid (e.g., RNA, e.g., mRNA) comprises one or more of ARCA: anti-reverse cap analog (m27.3´-OGP3G), GP3G (Unmethylated Cap Analog), m7GP3G (Monomethylated Cap Analog), m32.2.7GP3G (Trimethylated Cap Analog), m5CTP (5´-methyl-cytidine triphosphate), m6ATP (N6-methyl- adenosine-5´-triphosphate), s2UTP (2-thio-uridine triphosphate), and Ѱ (pseudouridine triphosphate). In some embodiments, a chemically modified nucleic acid comprises a 5´ cap, e.g.: a 7- methylguanosine cap (e.g., a O-Me-m7G cap); a hypermethylated cap analog; an NAD+-derived cap analog (e.g., as described in Kiledjian, Trends in Cell Biology 28, 454-464 (2018)); or a modified, e.g., biotinylated, cap analog (e.g., as described in Bednarek et al., Phil Trans R Soc B 373, 20180167 (2018)). In some embodiments, a chemically modified nucleic acid comprises a 3´ feature selected from one or more of: a polyA tail; a 16-nucleotide long stem-loop structure flanked by unpaired 5 nucleotides (e.g., as described by Mannironi et al., Nucleic Acid Research 17, 9113-9126 (1989)); a triple-helical structure (e.g., as described by Brown et al., PNAS 109, 19202-19207 (2012)); a tRNA, Y RNA, or vault RNA structure (e.g., as described by Labno et al., Biochemica et Biophysica Acta 1863, 3125-3147 (2016)); incorporation of one or more deoxyribonucleotide triphosphates (dNTPs), 2’O-Methylated NTPs, or phosphorothioate-NTPs; a single nucleotide chemical modification (e.g., oxidation of the 3´ terminal ribose to a reactive aldehyde followed by conjugation of the aldehyde-reactive modified nucleotide); or chemical ligation to another nucleic acid molecule. In some embodiments, a nucleic acid (e.g., a template nucleic acid) comprises one or more modified nucleotides, e.g., selected from dihydrouridine, inosine, 7-methylguanosine, 5- methylcytidine (5mC), 5′ Phosphate ribothymidine, 2′-O-methyl ribothymidine, 2′-O-ethyl ribothymidine, 2′-fluoro ribothymidine, C-5 propynyl-deoxycytidine (pdC), C-5 propynyl- deoxyuridine (pdU), C-5 propynyl-cytidine (pC), C-5 propynyl-uridine (pU), 5-methyl cytidine, Page 228 of 327 12592906v1 Attorney Docket No.2017469-0039 5-methyl uridine, 5-methyl deoxycytidine, 5-methyl deoxyuridine methoxy, 2,6-diaminopurine, 5′-Dimethoxytrityl-N4-ethyl-2′-deoxycytidine, C-5 propynyl-f-cytidine (pfC), C-5 propynyl-f- uridine (pfU), 5-methyl f-cytidine, 5-methyl f-uridine, C-5 propynyl-m-cytidine (pmC), C-5 propynyl-f-uridine (pmU), 5-methyl m-cytidine, 5-methyl m-uridine, LNA (locked nucleic acid), MGB (minor groove binder) pseudouridine (Ψ), 1-N-methylpseudouridine (1-Me-Ψ), or 5- methoxyuridine (5-MO-U). In some embodiments, a nucleic acid comprises a backbone modification, e.g., a modification to a sugar or phosphate group in the backbone. In some embodiments, a nucleic acid comprises a nucleobase modification. In some embodiments, a nucleic acid comprises one or more chemically modified nucleotides of Table 13, one or more chemical backbone modifications of Table 14, one or more chemically modified caps of Table 15. For instance, in some embodiments, a nucleic acid comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of chemical modifications. As an example, the nucleic acid may comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of modified nucleobases, e.g., as described herein, e.g., in Table 13. Alternatively or in combination, a nucleic acid may comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of backbone modifications, e.g., as described herein, e.g., in Table 14. Alternatively or in combination, a nucleic acid may comprise one or more modified cap, e.g., as described herein, e.g., in Table 15. For instance, in some embodiments, a nucleic acid comprises one or more type of modified nucleobase and one or more type of backbone modification; one or more type of modified nucleobase and one or more modified cap; one or more type of modified cap and one or more type of backbone modification; or one or more type of modified nucleobase, one or more type of backbone modification, and one or more type of modified cap. In some embodiments, a nucleic acid comprises one or more (e.g., 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more) modified nucleobases. In some embodiments, all nucleobases of a nucleic acid are modified. In some embodiments, a nucleic acid is modified at one or more (e.g., 1 or more, 2 or more, 3 or Page 229 of 327 12592906v1 Attorney Docket No.2017469-0039 more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more) positions in the backbone. In some embodiments, all backbone positions of the nucleic acid are modified. Table 13. Modified nucleotides 5-aza-uridine N2-methyl-6-thio-guanosine 2-thio-5-aza-midine N2,N2-dimethyl-6-thio-guanosine e Page 230 of 327 12592906v1 Attorney Docket No.2017469-0039 1-methyl-pseudoisocytidine 7-methylguanosine pyrrolo-cytidine N2,2’-O-dimethylguanosine ’ Page 231 of 327 12592906v1 Attorney Docket No.2017469-0039 6-thio-7-deaza-8-aza-guanosine 5-formyl-2’-O-methylcytidine 7-methyl-guanosine 1,2’-O-dimethylguanosine . 2’-O-Methyl backbone Peptide Nucleic Acid (PNA) backbone Table 15. Modified caps m7GpppA Page 232 of 327 12592906v1 Attorney Docket No.2017469-0039 GppppG m7GppppG of. p , p y p , y , , methylguanosine, or other modified bases. In some embodiments, a template nucleic acid may contain locked nucleic acid nucleotides. In some embodiments, modified bases used in a template nucleic acid do not inhibit the reverse transcription of the template. In some embodiments, modified bases used in a template nucleic acid may improve reverse transcription, e.g., specificity or fidelity. In some embodiments, an RNA component of a system of the present disclosure (e.g., a template RNA or a gRNA) comprises one or more nucleotide modifications. In some embodiments, the modification pattern of a gRNA can significantly affect in vivo activity compared to unmodified or end-modified guides (e.g., as shown in Figure 1D from Finn et al. Cell Rep 22(9):2227-2235 (2018); incorporated herein by reference in its entirety). Without wishing to be bound by any particular theory, this process may be due, at least in part, to a stabilization of the RNA conferred by the modifications. Non-limiting examples of such modifications may include 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-MOE), 2'- fluoro (2'-F), phosphorothioate (PS) bond between nucleotides, G-C substitutions, and inverted abasic linkages between nucleotides and equivalents thereof. In some embodiments, a template RNA (e.g., a portion thereof that binds a target site) or a guide RNA comprises a 5´ terminus region. In some embodiments, a template RNA or a guide RNA does not comprise a 5´ terminus region. In some embodiments, a 5´ terminus region comprises a gRNA spacer region, e.g., as described with respect to sgRNA in Briner AE et al, Molecular Cell 56: 333-339 (2014) (incorporated herein by reference in its entirety; applicable herein, e.g., to all guide RNAs). In some embodiments, a 5´ terminus region comprises a 5´ end modification. In some embodiments, a 5´ terminus region with or without a spacer region may Page 233 of 327 12592906v1 Attorney Docket No.2017469-0039 be associated with a crRNA, trRNA, sgRNA and / or dgRNA. A gRNA spacer region can, in some instances, comprise a guide region, guide domain, or targeting domain. In some embodiments, a template RNA (e.g., a portion thereof that binds a target site) or a guide RNA described herein comprises any of the sequences shown in Table 4 of WO2018107028A1, incorporated herein by reference in its entirety. In some embodiments, where a sequence shows a guide and / or spacer region, a template RNA may comprise this region or not. In some embodiments, a guide RNA comprises one or more of the modifications of any of the sequences shown in Table 4 of WO2018107028A1, e.g., as identified therein by a SEQ ID NO. In some embodiments, nucleotides may be the same or different, and / or the modification pattern shown may be the same or similar to a modification pattern of a guide sequence as shown in Table 4 of WO2018107028A1. In some embodiments, a modification pattern includes the relative position and identity of modifications of a gRNA or a region of the gRNA (e.g., 5´ terminus region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, 3´ terminus region). In some embodiments, a modification pattern contains at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the modifications of any one of the sequences shown in the sequence column of Table 4 of WO2018107028A1, and / or over one or more regions of the sequence. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the modification pattern of any one of the sequences shown in the sequence column of Table 4 of WO2018107028A1. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical over one or more regions of the sequence shown in Table 4 of WO2018107028A1, e.g., in a 5 ' terminus region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, and / or 3´ terminus region. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the modification pattern of a sequence over the 5 ' terminus region. In some embodiments, a modification pattern is at least 50%, at least 55%, at Page 234 of 327 12592906v1 Attorney Docket No.2017469-0039 least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over the lower stem. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical over the bulge. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over the upper stem. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over the nexus. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical over the hairpin 1. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over the hairpin 2. In some embodiments, a modification pattern is at least 50%, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical over the 3 ' terminus. In some embodiments, a modification pattern differs from the modification pattern of a sequence of Table 4 of WO2018107028A1, or a region (e.g. 5´ terminus, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3´ terminus) of such a sequence, e.g., at 0, 1, 2, 3, 4, 5, 6, or more nucleotides. In some embodiments, a gRNA comprises modifications that differ from the modifications of a sequence of Table 4 of WO2018107028A1, e.g., at 0, 1, 2, 3, 4, 5, 6, or more nucleotides. In some embodiments, a gRNA comprises modifications that differ from modifications of a region (e.g. 5 ' terminus, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3´ terminus) of a sequence of Table 4 of WO2018107028A1, e.g., at 0, 1, 2, 3, 4, 5, 6, or more nucleotides. In some embodiments, a template RNA (e.g., a portion thereof that binds a target site) or a gRNA comprises a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, a gRNA comprises a 2'-O-(2-methoxy ethyl) (2'-O-moe) modified nucleotide. In some embodiments, a gRNA comprises a 2'-fluoro (2'- F) modified nucleotide. In some embodiments, Page 235 of 327 12592906v1 Attorney Docket No.2017469-0039 a gRNA comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, a gRNA comprises a 5´ end modification, a 3´ end modification, or 5´ and 3´ end modifications. In some embodiments, a 5´ end modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, a 5´ end modification comprises a 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxy ethyl) (2'-O-MOE), and / or 2'-fluoro (2'-F) modified nucleotide. In some embodiments, a 5´ end modification comprises at least one phosphorothioate (PS) bond and one or more of a 2'-O-methyl (2'-O- Me), 2'-O-(2-methoxyethyl) (2'-O-MOE), and / or 2'-fluoro (2'-F) modified nucleotide. An end modification may comprise a phosphorothioate (PS), 2'-O-methyl (2'-O-Me), 2'-O-(2- methoxyethyl) (2'-O-MOE), and / or 2'-fluoro (2'-F) modification. Equivalent end modifications are also encompassed by embodiments described herein. In some embodiments, a template RNA or gRNA comprises an end modification in combination with a modification of one or more regions of the template RNA or the gRNA. Additional exemplary modifications and methods for protecting RNA, e.g., gRNA, and formulae thereof, are described in WO2018126176A1, which is incorporated herein by reference in its entirety. In some embodiments, a template RNA described herein comprises three phosphorothioate linkages at the 5’ end and three phosphorothioate linkages at the 3’ end. In some embodiments, a template RNA described herein comprises three 2’-O-methyl ribonucleotides at the 5’ end and three 2’-O-methyl ribonucleotides at the 3’ end. In some embodiments, the 5’ most three nucleotides of a template RNA are 2’-O-methyl ribonucleotides, the 5’ most three internucleotide linkages of the template RNA are phosphorothioate linkages, the 3’ most three nucleotides of the template RNA are 2’-O-methyl ribonucleotides, and the 3’ most three internucleotide linkages of the template RNA are phosphorothioate linkages. In some embodiments, a template RNA comprises alternating blocks of ribonucleotides and 2’-O-methyl ribonucleotides, for instance, blocks of between 12 and 28 nucleotides in length. In some embodiments, the central portion of a template RNA comprises the alternating blocks and the 5’ and 3’ ends each comprise three 2’-O-methyl ribonucleotides and three phosphorothioate linkages. In some embodiments, structure-guided and systematic approaches are used to introduce modifications (e.g., 2′-OMe-RNA, 2′-F-RNA, and PS modifications) to a template RNA or guide RNA, for example, as described in Mir et al. Nat Commun 9:2641 (2018) (incorporated by reference herein in its entirety). In some embodiments, incorporation of 2′-F-RNAs increases Page 236 of 327 12592906v1 Attorney Docket No.2017469-0039 thermal and nuclease stability of RNA:RNA or RNA:DNA duplexes, e.g., while minimally interfering with C3′-endo sugar puckering. In some embodiments, 2′-F may be better tolerated than 2′-OMe at positions where the 2′-OH is important for RNA:DNA duplex stability. In some embodiments, a crRNA comprises one or more modifications that do not reduce Cas9 activity, e.g., C10, C20, or C21 (fully modified), e.g., as described in Supplementary Table 1 of Mir et al. Nat Commun 9:2641 (2018), incorporated herein by reference in its entirety. In some embodiments, a tracrRNA comprises one or more modifications that do not reduce Cas9 activity, e.g., T2, T6, T7, or T8 (fully modified) of Supplementary Table 1 of Mir et al. Nat Commun 9:2641 (2018). In some embodiments, a crRNA comprises one or more modifications (e.g., as described herein) may be paired with a tracrRNA comprising one or more modifications, e.g., C20 and T2. In some embodiments, a gRNA comprises a chimera, e.g., of a crRNA and a tracrRNA (e.g., Jinek et al. Science 337(6096):816-821 (2012)). In some embodiments, modifications from a crRNA and tracrRNA are mapped onto a single-guide chimera, e.g., to produce a modified gRNA with enhanced stability. In some embodiments, a gRNA molecule may be modified by the addition or subtraction of naturally occurring structural components, e.g., hairpins. In some embodiments, a gRNA may comprise a gRNA with one or more 3´ hairpin elements deleted, e.g., as described in WO2018106727, incorporated herein by reference in its entirety. In some embodiments, a gRNA may contain an added hairpin structure, e.g., an added hairpin structure in the spacer region, which was shown to increase specificity of a CRISPR-Cas system in the teachings of Kocak et al. Nat Biotechnol 37(6):657-666 (2019). Additional modifications, including examples of shortened gRNA and specific modifications improving in vivo activity, can be found in US20190316121, incorporated herein by reference in its entirety. In some embodiments, structure-guided and systematic approaches (e.g., as described in Mir et al. Nat Commun 9:2641 (2018); incorporated herein by reference in its entirety) are employed to find modifications for a template RNA. In some embodiments, modifications are identified with the inclusion or exclusion of a guide region of template RNA. In some embodiments, a structure of a polypeptide bound to a template RNA is used to determine non- protein-contacted nucleotides of the template RNA that may then be selected for modifications, e.g., with lower risk of disrupting the association of the RNA with the polypeptide. Secondary structures in a template RNA can also be predicted in silico by software tools, e.g., the Page 237 of 327 12592906v1 Attorney Docket No.2017469-0039 RNAstructure tool available at rna.urmc.rochester.edu / RNAstructureWeb (Bellaousov et al. Nucleic Acids Res 41:W471-W474 (2013); incorporated by reference herein in its entirety), e.g., to determine secondary structures for selecting modifications, e.g., hairpins, stems, and / or bulges. An mRNA encoding a gene modifying polypeptide may have a cap, 5′ UTR containing a Kozak, 3′ UTR, and polyA tail containing at least 60 As. An mRNA encoding a gene modifying polypeptide may have a reduced Uridine content through codon selection / optimization. An mRNA encoding a gene modifying polypeptide may have uridines that are about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% substituted with 5-methoxy uridine. An mRNA encoding a gene modifying polypeptide may have uridines that are about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100%substituted with N1-methyl-pseudouridine. An mRNA encoding a gene modifying polypeptide may have cytosines in the mRNA are about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% substituted with 5-methylcytosine. An mRNA encoding a gene modifying polypeptide may have a combination of about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% substitution of cytosine with 5-methylcytosine and about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% substitution of uridine with 5-methoxy uridine. An mRNA encoding a gene modifying polypeptide may have a combination of about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100% substitution of cytosine with 5-methylcytosine and about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100%substitution of uridine with N1-methyl-pseudouridine. A guide RNA may be synthesized by T7 RNA polymerase. A guide RNA may be chemically synthesized and contain modifications such as, e.g., 2′-O-methyl, 2′-Fluoro, and / or phosphorothioate. In some embodiments, the 3 most terminal nucleotides of a guide RNA may contain 2′-O-methyl modifications with 3 phosphorothioate linkages between the nucleotides. A guide RNA may contain 2′-O-methyl modified nucleotides where there are cytosines and Page 238 of 327 12592906v1 Attorney Docket No.2017469-0039 uridines, except at nucleotides found in the “seed” of the guide RNA where cytosines and uridines contain 2′-fluoro modifications. In some embodiments, a template nucleic acid (e.g., RNA) of the present disclosure incorporates at least one nucleotide modification including one or more phosphorothioate linkages, one or more 2′-O-methyl groups, and / or one or more 2-fluororiboses. In some embodiments, a template nucleic acid (e.g., RNA) of the present disclosure incorporates one or more phosphorothioate linkages, one or more 2′-O-methyl groups, and one or more 2- fluororiboses. In some embodiments, a template nucleic acid is a polyribonucleotide (e.g., RNA) having a nucleotide sequence at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any sequence listed in Table 16. Table 16. Exemplary Template RNAs Incorporating Modified Nucleotides. RNACS NAME SEQUENCE SEQ ID NO RNACS1359 St1 dSL2 A1 mA*mA*mG*rGrCrUrGrUrGrCrUrGrArCrCrArUrCrGrArGr 10002 Page 239 of 327 12592906v1 Attorney Docket No.2017469-0039 RNACS NAME SEQUENCE SEQ ID NO G / / i2FC / / i2FC / rGrArArA / i2FU / / i2FC / / i2FA / rUrUrUrCrUrCr Page 240 of 327 12592906v1 Attorney Docket No.2017469-0039 RNACS NAME SEQUENCE SEQ ID NO RNACS2068 St1_t-lock_ mA*mA*mG*rGrCrUrGrUrGrCrUrGrArCrCrArUrCrGrArGr 10016 ‘m’ preceding a nucleotide, and 2-Fluororibose is denoted by an ‘i2F’ preceding a nucleotide. Chemically modified gRNA scaffolds In some embodiments, a template RNA described herein comprises a gRNA scaffold (e.g., an St1Cas9 gRNA scaffold) that is chemically modified. In some embodiments, the St1Cas9 scaffold comprises a chemically modified nucleotide at one or more of (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or all of) positions 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, and 42 relative to SEQ ID NO: 25999. In some embodiments, a nucleic acid molecule described herein comprises an St1Cas9 scaffold comprising: a) a Repeat: anti-repeat (RAR) region, wherein optionally the RAR region comprises a RAR lower stem, a RAR upper stem, and an RAR loop (e.g., a tetraloop); b) a stem- Page 241 of 327 12592906v1 Attorney Docket No.2017469-0039 loop 1 (SL1) region that is optionally 3’ of the RAR region, and c) optionally, a stem loop 2 (SL2) region that is optionally 3’ of the SL1 region; wherein the St1Cas9 scaffold comprises a chemically modified nucleotide in one or both of the RAR region or the SL1 region. In some embodiments, at least 15-20%, at least 20-30%, at least 30-40%, at least 40-50%, at least 50-60%, at least 60-70, or at least 70-75% of nucleotides in a St1Cas9 scaffold are chemically modified. In some embodiments, a St1Cas9 scaffold comprises a RAR region, wherein the RAR region comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 chemically modified nucleotides (e.g., wherein the chemically modified nucleotides have the same chemical modification). In some embodiments, a St1Cas9 scaffold comprises a SL1 region, wherein the SL1 region comprises 1, 2, 3, 4, 5, 6, 7, or 8 chemically modified nucleotides (e.g., wherein the chemically modified nucleotides have the same chemical modification). In some embodiments, positions 1, 2, and 3 (if present) of a nuclide acid do not comprise a 2’-O-methyl chemically modified nucleotide. In some embodiments, positions 43 through 54 (if present) of a nuclide acid do not comprise a 2’-O-methyl chemically modified nucleotide. In some embodiments, a St1Cas9 scaffold comprises a chemically modified nucleotide at each of positions 4 through 6 (if present). In some embodiments, a St1Cas9 scaffold comprises a chemically modified nucleotide at each of positions 13 through 15 (if present). In some embodiments, a St1Cas9 scaffold comprises a chemically modified nucleotide at each of positions 16 through 18 (if present). In some embodiments, a St1Cas9 scaffold comprises a chemically modified nucleotide at each of positions 19 through 21 (if present). In some embodiments, a St1Cas9 scaffold comprises a chemically modified nucleotide at each of positions 22 through 24 (if present). In some embodiments, a St1Cas9 scaffold comprises a chemically modified nucleotide at each of positions 34 through 36 (if present). In some embodiments, a St1Cas9 scaffold comprises a chemically modified nucleotide at each of positions 37 through 39 (if present). In some embodiments, a St1Cas9 scaffold comprises a chemically modified nucleotide at each of positions 40 through 42 (if present). In some embodiments, a St1Cas9 scaffold comprises a chemically modified nucleotide at each of positions 13 through 24 (if present). In some embodiments, a St1Cas9 scaffold comprises a chemically modified nucleotide at each of positions 34 through 42 (if present). Page 242 of 327 12592906v1 Attorney Docket No.2017469-0039 In some embodiments, a chemically modified nucleotide is a modification to a sugar group, e.g., a modification to the 2’-O of ribose, e.g., a 2’-O-Methyl chemically modified nucleotide. In some embodiments, a nucleic acid further comprises a second chemically modified nucleotide. In some embodiments, a nucleic acid described herein comprises a sequence according to SEQ ID NO: 26000, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, a St1Cas9 scaffold binds to an St1Cas9 protein having a sequence described herein, e.g., in Table 7 or Table 8. In some embodiments, a St1Cas9 scaffold comprises a 2’-O-methyl chemically modified nucleotide at each of positions 12 through 21, a 2’-O-methyl chemically modified nucleotide at each of positions 34 through 36, and a 2’-Fluoro chemically modified nucleotide at each of positions 37 through 42. In some embodiments, a St1Cas9 scaffold comprises a 2’-O-methyl chemically modified nucleotide at each of positions 12 through 21, a 2’-O-methyl chemically modified nucleotide at each of positions 34 through 36, a 2’-Fluoro chemically modified nucleotide at each of positions 37 through 42, and a 2’-Fluoro chemically modified nucleotide at each of positions 45 through 47. In some embodiments, a St1Cas9 scaffold comprises a 2’-O- methyl chemically modified nucleotide at each of positions 12 through 21, a 2’-O-methyl chemically modified nucleotide at each of positions 34 through 36, a 2’-Fluoro chemically modified nucleotide at each of positions 37 through 42, a 2’-Fluoro chemically modified nucleotide at each of positions 45 through 47 and a 2’-Fluoro chemically modified nucleotide at each of positions 52 through 54. Production of Compositions and Systems As will be appreciated by one of skill, methods of designing and constructing nucleic acid constructs and proteins or polypeptides (such as the systems, constructs and polypeptides described herein) are routine in the art. Generally, recombinant methods may be used. See, in general, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013). Methods of designing, preparing, evaluating, purifying and manipulating nucleic acid compositions are Page 243 of 327 12592906v1 Attorney Docket No.2017469-0039 described in Green and Sambrook (Eds.), Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012). The present disclosure also provides compositions and methods for the production of template nucleic acid molecules (e.g., template RNAs) with specificity for a gene modifying polypeptide and / or a genomic target site. Therapeutic Applications In some embodiments, a gene modifying system as described herein can be used to modify a cell (e.g., an animal cell, plant cell, or fungal cell). In some embodiments, a gene modifying system as described herein can be used to modify a mammalian cell (e.g., a human cell). In some embodiments, a gene modifying system as described herein can be used to modify a cell from a livestock animal (e.g., a cow, horse, sheep, goat, pig, llama, alpaca, camel, yak, chicken, duck, goose, or ostrich). In some embodiments, a gene modifying system as described herein can be used as a laboratory tool or a research tool, or used in a laboratory method or research method, e.g., to modify an animal cell, e.g., a mammalian cell (e.g., a human cell), a plant cell, or a fungal cell. In some embodiments, a gene modifying system of the present disclosure can be used to correct one or more mutations in vivo, e.g., following administration or delivery, e.g., as described herein. For example, in some embodiments, a gene modifying system can be used to correct one or more point mutations in a gene. In some embodiments, a gene modifying system of the present disclosure can be used to correct a mutation in a human SERPINA1 (hSERPINA1) gene. In some embodiments, a gene modifying system of the present disclosure can be used to correct a G to A mutation at position c.1096 in exon 5 (i.e., correct back to a G). In some embodiments, a gene modifying system of the present disclosure can be used to correct one or more DNA mutations in vivo (e.g., following administration or delivery, e.g., as described herein) with an efficiency (e.g., a “rewrite” percentage) of at least about 40% to 95%, at least about 45% to 95%, at least about 50% to 95%, at least about 55% to 95%, at least about 60% to 95%, at least about 65% to 95%, at least about 70% to 95%, at least about 75% to 95%, at least about 80% to 95%, at least about 85% to 95%, at least about 90% to 95%, at least about 40% to 90%, at least about 45% to 90%, at least about 50% to 90%, at least about 55% to 90%, at least about 60% to 90%, at least about 65% to 90%, at least about 70% to 90%, at least about 75% to 90%, at least about 80% to 90%, at least about 85% to 90%, at least about 40% to 85%, Page 244 of 327 12592906v1 Attorney Docket No.2017469-0039 at least about 45% to 85%, at least about 50% to 85%, at least about 55% to 85%, at least about 60% to 85%, at least about 65% to 85%, at least about 70% to 85%, at least about 75% to 85%, at least about 80% to 85%, at least about 40% to 80%, at least about 45% to 80%, at least about 50% to 80%, at least about 55% to 80%, at least about 60% to 80%, at least about 65% to 80%, at least about 70% to 80%, at least about 75% to 80%, at least about 40% to 75%, at least about 45% to 75%, at least about 50% to 75%, at least about 55% to 75%, at least about 60% to 75%, at least about 65% to 75%, at least about 70% to 75%, at least about 40% to 70%, at least about 45% to 70%, at least about 50% to 70%, at least about 55% to 70%, at least about 60% to 70%, at least about 65% to 70%, at least about 40% to 65%, at least about 45% to 65%, at least about 50% to 65%, at least about 55% to 65%, at least about 60% to 65%, at least about 40% to 60%, at least about 45% to 60%, at least about 50% to 60%, at least about 55% to 60%, at least about 40% to 55%, at least about 45% to 55%, at least about 50% to 55%, at least about 40% to 50%, at least about 45% to 50%, or at least about 40% to 45%. In some embodiments, a correction of one or more DNA mutations in vivo achieved by a gene modifying system of the present disclosure (e.g., following administration or delivery, e.g., as described herein) persists for at least about 1 day to 56 days, at least about 7 days to 56 days, at least about 14 days to 56 days, at least about 21 days to 56 days, at least about 28 days to 56 days, at least about 35 days to 56 days, at least about 42 days to 56 days, at least about 49 days to 56 days, at least about 1 day to 49 days, at least about 7 days to 49 days, at least about 14 days to 49 days, at least about 21 days to 49 days, at least about 28 days to 49 days, at least about 35 days to 49 days, at least about 42 days to 49 days, at least about 1 day to 42 days, at least about 7 days to 42 days, at least about 14 days to 42 days, at least about 21 days to 42 days, at least about 28 days to 42 days, at least about 35 days to 42 days, at least about 1 day to 35 days, at least about 7 days to 35 days, at least about 14 days to 35 days, at least about 21 days to 35 days, at least about 28 days to 35 days, at least about 1 day to 28 days, at least about 7 days to 28 days, at least about 14 days to 28 days, at least about 21 days to 28 days, at least about 1 day to 21 days, at least about 7 days to 21 days, at least about 14 days to 21 days, at least about 1 day to 14 days, at least about 7 days to 14 days, or at least about 1 day to 7 days. In some embodiments, a correction of one or more DNA mutations in vivo achieved by a gene modifying system of the present disclosure (e.g., following administration or delivery, e.g., as described herein) results in a substantial majority of all mRNA containing the correction. For Page 245 of 327 12592906v1 Attorney Docket No.2017469-0039 example, in some embodiments, an in vivo correction of one or more DNA mutations achieved by a gene modifying system of the present disclosure results in at least about 50% to 95%, at least about 55% to 95%, at least about 60% to 95%, at least about 65% to 95%, at least about 70% to 95%, at least about 75% to 95%, at least about 80% to 95%, at least about 85% to 95%, at least about 90% to 95%, at least about 50% to 90%, at least about 55% to 90%, at least about 60% to 90%, at least about 65% to 90%, at least about 70% to 90%, at least about 75% to 90%, at least about 80% to 90%, at least about 85% to 90%, at least about 50% to 85%, at least about 55% to 85%, at least about 60% to 85%, at least about 65% to 85%, at least about 70% to 85%, at least about 75% to 85%, at least about 80% to 85%, at least about 50% to 80%, at least about 55% to 80%, at least about 60% to 80%, at least about 65% to 80%, at least about 70% to 80%, at least about 75% to 80%, at least about 50% to 75%, at least about 55% to 75%, at least about 60% to 75%, at least about 65% to 75%, at least about 70% to 75%, at least about 50% to 70%, at least about 55% to 70%, at least about 60% to 70%, at least about 65% to 70%, at least about 50% to 65%, at least about 55% to 65%, at least about 60% to 65%, at least about 50% to 60%, at least about 55% to 60%, or at least about 50% to 55% of all mRNA containing the correction. In some embodiments, a correction of one or more DNA mutations in vivo achieved by a gene modifying system of the present disclosure (e.g., following administration or delivery, e.g., as described herein) results in increased serum levels of a protein product of the gene in which the correction was made. For example, in some embodiments, a gene modifying system of the present disclosure corrects a mutation in a hSERPINA1 gene resulting in increased serum levels of human alpha 1 antitrypsin (hA1AT) as compared to levels of hA1AT in subjects that have not been treated with the gene modifying system. In some embodiments, a correction of one or more DNA mutations in vivo achieved by a gene modifying system of the present disclosure results in at least about a 1-fold to 10-fold, at least about a 1.5-fold to 10-fold, at least about a 2-fold to 10- fold, at least about a 2.5-fold to 10-fold, at least about a 3-fold to 10-fold, at least about a 3.5- fold to 10-fold, at least about a 4-fold to 10-fold, at least about a 4.5-fold to 10-fold, at least about a 5-fold to 10-fold, at least about a 5.5-fold to 10-fold, at least about a 6-fold to 10-fold, at least about a 6.5-fold to 10-fold, at least about a 7-fold to 10-fold, at least about a 7.5-fold to 10- fold, at least about a 8-fold to 10-fold, at least about a 8.5-fold to 10-fold, at least about a 9-fold to 10-fold, at least about a 9.5-fold to 10-fold, at least about a 1-fold to 9-fold, at least about a 1.5-fold to 9-fold, at least about a 2-fold to 9-fold, at least about a 2.5-fold to 9-fold, at least Page 246 of 327 12592906v1 Attorney Docket No.2017469-0039 about a 3-fold to 9-fold, at least about a 3.5-fold to 9-fold, at least about a 4-fold to 9-fold, at least about a 4.5-fold to 9-fold, at least about a 5-fold to 9-fold, at least about a 5.5-fold to 9-fold, at least about a 6-fold to 9-fold, at least about a 6.5-fold to 9-fold, at least about a 7-fold to 9- fold, at least about a 7.5-fold to 9-fold, at least about a 8-fold to 9-fold, at least about a 8.5-fold to 9-fold, at least about a 1-fold to 8-fold, at least about a 1.5-fold to 8-fold, at least about a 2- fold to 8-fold, at least about a 2.5-fold to 8-fold, at least about a 3-fold to 8-fold, at least about a 3.5-fold to 8-fold, at least about a 4-fold to 8-fold, at least about a 4.5-fold to 8-fold, at least about a 5-fold to 8-fold, at least about a 5.5-fold to 8-fold, at least about a 6-fold to 8-fold, at least about a 6.5-fold to 8-fold, at least about a 7-fold to 8-fold, at least abo...

Claims

Attorney Docket No.2017469-0039 CLAIMS What is claimed is:

1. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain; an St1Cas9 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 an amino acid sequence of Table 6, or a sequence having at least 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto.

2. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain having an amino acid sequence of Table 1, or an amino acid sequence with at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto; an St1Cas9 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 an amino acid sequence of Table 5, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto.

3. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a PERV RT domain; an St1Cas9 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 has an amino acid sequence of any of SEQ ID NOs: 10301, 10304, 10305, 10307, or 10314 or a sequence having at least 75%, 80%, 85%, 90%, 95%, Page 323 of 327 12592906v1Attorney Docket No.2017469-0039 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto.

4. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a BAEVM RT domain; an St1Cas9 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 has an amino acid sequence of any of SEQ ID NOs: 10302, 10308, 10310, or 10312, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto.

5. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a WMSV RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10303, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto.

6. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a MLMS RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10306, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto.

7. A gene modifying polypeptide comprising: Page 324 of 327 12592906v1Attorney Docket No.2017469-0039 a retroviral reverse transcriptase (RT) domain which is a AVIRE RT domain; an St1Cas9 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 has an amino acid sequence of any of SEQ ID NOs: 10309 or 10313, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto.

8. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a XMRV6 RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10311, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto.

9. A gene modifying polypeptide comprising: a retroviral reverse transcriptase (RT) domain which is a MLVFF RT domain; an St1Cas9 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 has an amino acid sequence of SEQ ID NO: 10315, or a sequence having at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto, or an amino acid sequence having up to 1, 2, or 3 amino acid sequence differences (e.g., substitutions) relative thereto.

10. A nucleic acid molecule encoding a gene modifying polypeptide according to any of the preceding claims.

11. A cell comprising: i) a gene modifying polypeptide of any of claims 1-9 or Page 325 of 327 12592906v1Attorney Docket No.2017469-0039 ii) a nucleic acid encoding the gene modifying polypeptide.

12. A system comprising: i) a gene modifying polypeptide of any of claims 1-9, 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 (e.g., a St1Cas9 gRNA scaffold) that binds the Cas9 nickase domain of the gene modifying polypeptide; c) a heterologous object sequence; and d) a primer binding site (PBS) sequence.

13. A lipid nanoparticle formulation comprising the system of claim 12.

14. A method for modifying a target nucleic acid molecule in a cell, the method comprising contacting the cell with the system of claim 12, thereby modifying the target nucleic acid molecule.

15. A method for modifying a target nucleic acid molecule in a tissue, the method comprising contacting the tissue with the system of claim 12, thereby modifying the target nucleic acid molecule. Page 326 of 327 12592906v1