Modified template guide RNA molecules

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.: 2017469-0019 MODIFIED TEMPLATE GUIDE RNA MOLECULES CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 453,662, filed March 21, 2023. The contents of the aforementioned application are hereby incorporated by reference in their entirety. 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 template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence comprising a region of at least 5 contiguous nucleotides comprising 2’-fluoro modifications on alternating nucleotides, and (iv) a primer binding site (PBS) sequence. 2. The template RNA of embodiment 1, wherein the heterologous object sequence comprises 2’-fluoro modifications on alternating nucleotides starting from position +4 of the heterologous object sequence. 1 of 237 11867955v1 Attorney Docket No.: 2017469-0019 3. The template RNA of embodiment 2, wherein the heterologous object sequence comprises 2’-fluoro modifications on alternating nucleotides from position +4 through position +8, +10, +12, +14, +16, or +18 of the heterologous object sequence. 4. The template RNA of embodiment 1, wherein the heterologous object sequence comprises 2’-fluoro modifications on alternating nucleotides in a region having a length of 2-5, 5-10, 10-15, 15-20, 20-25, 25- 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, or 80-81 nucleotides. 5. The template RNA of embodiment 1, wherein the heterologous object sequence comprises 2’-fluoro modifications on alternating nucleotides in a region having a length of 80-90, 90-100, 100-150, 150-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500, 1500-2000, 2000- 2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, or 4500-5000 nucleotides. 6. The template RNA of embodiment 1, wherein the heterologous object sequence comprises 2’-fluoro modifications on alternating nucleotides starting from position +5 of the heterologous object sequence. 7. The template RNA of embodiment 6, wherein the heterologous object sequence comprises 2’-fluoro modifications on alternating nucleotides from position +5 through position +9, +11, +13, +15, or +17 of the heterologous object sequence. 8. The template RNA of any one of the preceding embodiments, wherein the heterologous object sequence further comprises a region between the region of at least 5 contiguous nucleotides and the PBS sequence that does not comprise 2’-fluoro modifications, e.g. comprises unmodified nucleotides. 9. The template RNA of embodiment 8, wherein the region that does not comprise 2’-fluoro modifications has a length of at least 2, 3, 4, 5, 6, 7, or 8 nucleotides. 10. The template RNA of embodiment 8, wherein the region that does not comprise 2’-fluoro modifications has a length of 1-3, 2-4, 3-5, 3-6, 5-7, 6-8, or 7-9 nucleotides. 11. The template RNA of any one of the preceding embodiments, which comprises a 2’-fluoro modified nucleotide at the 5’ end of the heterologous object sequence (e.g., at position +10 or +11 of the heterologous object sequence). 2 of 237 11867955v1 Attorney Docket No.: 2017469-0019 12. The template RNA of any one of the preceding embodiments, which comprises 2’-fluoro modified nucleotides at positions +4, +6, +8, and / or +10 of the heterologous object sequence. 13. The template RNA of any one of the preceding embodiments, which comprises 2’-fluoro modified nucleotides at positions +5, +7, +9, and / or +11 of the heterologous object sequence. 14. The template RNA of any one of the preceding embodiments, wherein the second nucleotide from the 5’ end of the heterologous object sequence comprises a 2’-fluoro modification (e.g., position +9 or +10 of the heterologous object sequence). 15. The template RNA of any one of the preceding embodiments, which comprises 2’-fluoro modified nucleotides at positions +5, +7, and / or +9 of the heterologous object sequence. 16. The template RNA of any one of the preceding embodiments, which comprises 2’-fluoro modified nucleotides at positions +4, +6, +8, and / or +10 of the heterologous object sequence. 17. The template RNA of any one of the preceding embodiments, wherein the PBS sequence further comprises one or more of: a 2’-fluoro modified nucleotide, a 2’-OMe modified nucleotide, and / or one or more (e.g., 1, 2, or 3) nucleotides comprising a phosphorothioate modification. 18. The template RNA of embodiment 17, which comprises at the 3’ end, in 5’ to 3’ order, a 2’-fluoro modified nucleotide, a 2’-OMe modified nucleotide, and one or more (e.g., 1, 2, or 3) nucleotides comprising a phosphorothioate modification. 19. The template RNA of embodiment 17 or 18, wherein the one or more nucleotides comprising a phosphorothioate modification further comprise a 2’-OMe modification. 20. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence, wherein the template RNA comprises a region of at least 5 contiguous nucleotides comprising 2’- fluoro modifications on alternating nucleotides, optionally wherein the number of 3 of 237 11867955v1 Attorney Docket No.: 2017469-0019 nucleotides between the 3’ most nucleotide comprising a 2’-fluoro modification in the region and the 3’ end of the template RNA is 8, 9, 10, 11, 12, 13, 14, or 15. 21. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence comprising a 2’-fluoro modified nucleotide. 22. The template RNA of embodiment 21, wherein the PBS sequence further comprises one or more (e.g. 1, 2, or 3) 2’-OMe modified nucleotides. 23. The template RNA of embodiment 22, wherein the 2’-fluoro modified nucleotide is adjacent to one of the one or more 2’-OMe modified nucleotides. 24. The template RNA of embodiment 22 or 23, wherein the 2’-fluoro modified nucleotide is positioned 5’ relative to the 2’-OMe modified nucleotide(s). 25. The template RNA of any one of embodiments 21-24, wherein the template RNA does not comprise any unmodified nucleotides 3’ of the 2’-fluoro modified nucleotide. 26. The template RNA of any one of embodiments 21-25, wherein the template RNA further comprises (e.g., at the 3’ end of the template RNA) one or more (e.g., 1, 2, or 3) nucleotides each comprising a phosphorothioate modification and a 2’-OMe modification. 27. The template RNA of any one of embodiments 21-26, wherein the number of nucleotides between the 2’-fluoro modified nucleotide and the 3’ end of the template RNA is 2, 3, 4, 5, 6, 7, 8, 9, or 10. 28. The template RNA of any one of embodiments 21-27, wherein the number of nucleotides between the 2’-fluoro modified nucleotide and the 3’ end of the template RNA is at least 4. 29. The template RNA of any one of embodiments 21-28, wherein the number of nucleotides between the 2’-fluoro modified nucleotide and the 3’ end of the template RNA is 4. 4 of 237 11867955v1 Attorney Docket No.: 2017469-0019 30. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence comprising one or more (e.g., 1, 2, or 3) 2’-OMe modified nucleotides. 31. The template RNA of embodiment 30, wherein the PBS sequence further comprises a 2’-fluoro modified nucleotide. 32. The template RNA of embodiment 31, wherein the 2’-fluoro modified nucleotide is adjacent to one of the one or more 2’-OMe modified nucleotides. 33. The template RNA of embodiment 31 or 32, wherein the 2’-fluoro modified nucleotide is positioned 5’ relative to the 2’-OMe modified nucleotide(s). 34. The template RNA of any of embodiments 30-33, wherein the template RNA does not comprise any unmodified nucleotides 3’ of the 2’-OMe modified nucleotides. 35. The template RNA of any of embodiments 30-34, wherein the template RNA further comprises (e.g., at the 3’ end of the template RNA) one or more (e.g., 1, 2, or 3) nucleotides each comprising a phosphorothioate modification and a 2’-OMe modification. 36. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence comprising one or more (e.g., 1, 2, or 3) 2’-OMe modified nucleotides, and (iv) a primer binding site (PBS) sequence. 37. The template RNA of embodiment 36, wherein the heterologous object sequence comprises a plurality of 2’-OMe modified nucleotides (e.g., 2, 3, 4, or 52’-OMe modified nucleotides) positioned adjacent to each other. 5 of 237 11867955v1 Attorney Docket No.: 2017469-0019 38. The template RNA of embodiment 37, wherein the plurality of 2’-OMe modified nucleotides are at least 1, 2, 3, 4, or 5 nucleotides from the 3’ end of the heterologous object sequence. 39. The template RNA of embodiment 37, wherein the plurality of 2’-OMe modified nucleotides are less than 10, 9, 8, 7, 6, or 6 nucleotides from the 3’ end of the heterologous object sequence. 40. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence comprising one or more (e.g., 1, 2, or 3) 2’-OMe modified nucleotides. 41. The template RNA of embodiment 40, wherein the PBS sequence comprises a plurality of 2’-OMe modified nucleotides (e.g., 2, 3, 4, or 52’-OMe modified nucleotides) positioned adjacent to each other. 42. The template RNA of embodiment 41, wherein the plurality of 2’-OMe modified nucleotides are at least 1, 2, 3, 4, or 5 nucleotides from the 5’ end of the PBS sequence. 43. The template RNA of any one of embodiments 40-42, wherein the 2’-OMe modified nucleotides further comprise a phosphorothioate modification. 44. The template RNA of embodiment 40, wherein nucleotides -4, -5, -6, -7, -8, -9, and -10 of the PBS sequence comprise a 2’-OMe modification and / or a phosphorothioate modification. 45. The template RNA of embodiment 40, wherein nucleotides -5, -6, -7, -8, -9, and -10 of the PBS sequence comprise a 2’-OMe modification and / or a phosphorothioate modification. 46. The template RNA of embodiment 44 or 45, wherein nucleotide -10 of the PBS sequence is at the 3’ end of the template RNA. 47. The template RNA of any one of embodiments 36-46, which does not comprise a 2’-OMe modified nucleotide at position +1 of the heterologous object sequence or -1 of the PBS sequence. 6 of 237 11867955v1 Attorney Docket No.: 2017469-0019 48. The template RNA of embodiment 47, which does not comprise a 2’-OMe modified nucleotide at positions -2 or -1 of the PBS sequence or, +1 or +2 of the heterologous object sequence. 49. The template RNA of any one of embodiments 36-48, wherein the nucleotides at positions -1 of the PBS sequence and +1 of the heterologous object sequence are unmodified nucleotides. 50. The template RNA of embodiment 47, wherein the nucleotides at positions -2 or -1 of the PBS sequence and +1 or and +2 of the heterologous object sequence are unmodified nucleotides. 51. The template RNA of any one of embodiments 36-50, wherein the nucleotides at positions -6 to -1, -5 to -1, -4 to -1, -3 to -1, or -2 to -1of the PBS sequence are unmodified nucleotides. 52. The template RNA of any one of embodiments 36-51, wherein the heterologous object sequence and / or PBS sequence comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified nucleotides. 53. The template RNA of any one of embodiments 36-52, wherein the heterologous object sequence and / or PBS sequence comprise 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 unmodified nucleotides. 54. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence comprising one or more (e.g., 1, 2, or 3) 2’-fluoro modified nucleotides, and (iv) a primer binding site (PBS) sequence. 55. The template RNA of embodiment 54, wherein the heterologous object sequence comprises a plurality of 2’-fluoro modified nucleotides (e.g., 2, 3, 4, or 52’-fluoro modified nucleotides) positioned adjacent to each other. 56. The template RNA of embodiment 55, wherein the plurality of 2’-fluoro modified nucleotides are at least 1, 2, 3, 4, or 5 nucleotides from the 3’ end of the heterologous object sequence. 7 of 237 11867955v1 Attorney Docket No.: 2017469-0019 57. The template RNA of embodiment 54, wherein the heterologous object sequence comprises a plurality of 2’-fluoro modified nucleotides (e.g., 2, 3, 4, or 5 2’-fluoro modified nucleotides) alternating with a plurality of nucleotides lacking a 2’-fluoro modification. 58. The template RNA of any one of embodiments 54-57, wherein the nucleotide at the 5’ end of the heterologous object sequence (e.g., at position +10 or +11) comprises a 2’-fluoro modification. 59. The template RNA of embodiment 58, which comprises one or more (e.g., 1, 2, 3, 4, or 5) 2’-fluoro modifications on alternating nucleotides after the 2’-fluoro modified nucleotide at the 5’ end of the heterologous object sequence. 60. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence comprising one or more (e.g., 1, 2, or 3) 2’-fluoro modified nucleotides. 61. The template RNA of embodiment 60, wherein the PBS sequence comprises a plurality of 2’-fluoro modified nucleotides (e.g., 1, 2, 3, 4, or 52’-fluoro modified nucleotides) positioned adjacent to each other. 62. The template RNA of embodiment 61, wherein the plurality of 2’-fluoro modified nucleotides are at least 1, 2, 3, 4, or 5 nucleotides from the 5’ end of the PBS sequence. 63. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence, wherein the template RNA comprises one or more (e.g., 1, 2, or 3) 2’-fluoro modified nucleotides (e.g., one or more adjacent nucleotides) comprising 2’-fluoro modifications, optionally wherein the number of nucleotides between the one of the nucleotides comprising a 2’- fluoro modification and the 3’ end of the template RNA is 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, or 29. 8 of 237 11867955v1 Attorney Docket No.: 2017469-0019 64. The template RNA of any one of embodiments 54-63, wherein the PBS sequence comprises one or more (e.g., 1, 2, or 3) phosphorothioate-modified nucleotides, e.g., at the 3’ end of the PBS sequence. 65. The template RNA of any one of embodiments 54-64, wherein the PBS sequence comprises one or more (e.g., 1, 2, or 3) 2’-OMe-modified nucleotides, e.g., at the 3’ end of the PBS sequence. 66. The template RNA of any one of embodiments 54-65, wherein the PBS sequence comprises one or more (e.g., 1, 2, or 3) nucleotides each comprising phosphorothioate and 2’-OMe modifications, e.g., at the 3’ end of the PBS sequence. 67. The template RNA of any one of embodiments 54-66, which does not comprise a 2’-fluoro modified nucleotide at positions -1 of the PBS sequence or +1 of the heterologous object sequence. 68. The template RNA of embodiment 67, which does not comprise a 2’-fluoro modified nucleotide at positions -2 or -1 of the PBS sequence or +1 or +2 of the heterologous object sequence. 69. The template RNA of any one of embodiments 54-68, wherein the nucleotides at position -1 of the PBS sequence and +1 of the heterologous object sequence are unmodified nucleotides. 70. The template RNA of embodiment 67, wherein the nucleotides at positions -2 or -1 of the PBS sequence or +1 of +2 of the heterologous object sequence are unmodified nucleotides. 71. The template RNA of any one of embodiments 54-70, wherein the heterologous object sequence and / or PBS sequence comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified nucleotides. 72. The template RNA of any one of embodiments 54-71, wherein the heterologous object sequence and / or PBS sequence comprise 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 unmodified nucleotides. 73. The template RNA of any one of the preceding embodiments, wherein the gRNA scaffold binds a gene modifying polypeptide (e.g., binds a Cas domain, e.g., a Cas9 domain of the gene modifying polypeptide). 9 of 237 11867955v1 Attorney Docket No.: 2017469-0019 74. The template RNA of any one of the preceding embodiments, wherein the gRNA scaffold binds a gene modifying polypeptide (e.g., binds a Cas domain, e.g., a Cas9 domain of the gene modifying polypeptide) comprising the amino acid sequence of SEQ ID NO: 101, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. 75. The template RNA of any one of the preceding embodiments, wherein the gRNA scaffold binds a gene modifying polypeptide (e.g., binds a Cas domain, e.g., a Cas9 domain of the gene modifying polypeptide) comprising the amino acid sequence of SEQ ID NO: 102, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. 76. The template RNA of any one of the preceding embodiments, wherein the gRNA scaffold binds a gene modifying polypeptide (e.g., binds a Cas domain, e.g., a Cas9 domain of the gene modifying polypeptide) comprising the amino acid sequence of SEQ ID NO: 103, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. 77. The template RNA of any one of the preceding embodiments, wherein the heterologous object sequence comprises a mutation region to introduce a mutation into (e.g., to correct a mutation in) a portion (e.g., a second portion) of the human PAH, FAH, HBB, TRAC4, B2M, or A1AT gene (wherein optionally the heterologous object sequence comprises, from 5’ to 3’, a post-edit homology region, a mutation region, and a pre-edit homology region). 78. The template RNA of any one of the preceding embodiments, which comprises at least 5, 6, 7, or 8 bases with 100% identity to a third portion of the human PAH, FAH, HBB, TRAC4, B2M, or A1AT gene. 79. The template RNA of any one of the preceding embodiments, which does not comprise a 2’-fluoro modified nucleotide at position -1 of the PBS sequence or +1 of the heterologous object sequence. 80. The template RNA of any one of the preceding embodiments, which does not comprise a 2’-fluoro modified nucleotide at positions -2 or -1 of the PBS sequence or +1 or +2 of the heterologous object sequence. 81. The template RNA of any one of the preceding embodiments, which does not comprise a 2’-OMe modified nucleotide at position -1 of the PBS sequence or +1 of the heterologous object sequence and the PBS sequence. 10 of 237 11867955v1 Attorney Docket No.: 2017469-0019 82. The template RNA of any one of the preceding embodiments, which does not comprise a 2’-OMe modified nucleotide at position -2 or -1 of the PBS sequence or +1 or +2 of the heterologous object sequence. 83. The template RNA of any one of the preceding embodiments, wherein the nucleotides at position -1 of the PBS sequence and / or +1 of the heterologous object sequence are unmodified nucleotides. 84. The template RNA of any one of the preceding embodiments, wherein the nucleotides at positions -2 and -1 of the PBS sequence and / or +1 and +2 of the heterologous object sequence are unmodified nucleotides. 85. The template RNA of any one of the preceding embodiments, wherein the 3’ end of the PBS sequence comprises one or more of: a 2’-fluoro modified nucleotide, a 2’-OMe modified nucleotide, and one or more (e.g., 1, 2, or 3) nucleotides comprising a phosphorothioate modification. 86. The template RNA of any one of the preceding embodiments, wherein the 3’ end of the PBS sequence comprises, in 5’ to 3’ order, a 2’-fluoro modified nucleotide, one or more (e.g., 1, 2, 3, 4, or 5) 2’-OMe modified nucleotides, and one or more (e.g., 1, 2, 3, 4, or 5) nucleotides comprising a phosphorothioate modification. 87. The template RNA of any one of the preceding embodiments, wherein one or more (e.g., 1, 2, 3, 4, or 5) of the nucleotides comprising a phosphorothioate modification further comprise a 2’-OMe modification. 88. The template RNA of any one of the preceding embodiments, which comprises a polynucleotide as listed in column 3 of any of Tables 12-14 or column 2 of Table 15, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. 89. The template RNA of embodiment 88, which comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) the nucleotide modifications for the polynucleotide as listed in column 3 of any of Tables 12-14 or column 2 of Table 15. 11 of 237 11867955v1 Attorney Docket No.: 2017469-0019 90. The template RNA of embodiment 88 or 89, which comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) the 2’-OMe modifications for the polynucleotide as listed in column 3 of any of Tables 12-14 or column 2 of Table 15. 91. The template RNA of embodiment 88 or 89, which comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) the 2’-fluoro modifications for the polynucleotide as listed in column 3 of any of Tables 12- 14 or column 2 of Table 15. 92. The template RNA of any one of the preceding embodiments, which is capable of introducing an alteration (e.g., a nucleic acid substitution, deletion, or insertion) into at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% target nucleic acid molecules (e.g., genomic DNA) in a population of said target nucleic acid molecules (e.g., a population of cells comprising said genomic DNA). 93. A gene modifying system comprising: (i) a template RNA of any of the preceding embodiments, and (ii) a gene modifying polypeptide (e.g., as described herein), or a nucleic acid (e.g., RNA) encoding the gene modifying polypeptide. 94. A method for modifying a target site in a nucleic acid molecule (e.g., genomic DNA) in a cell, the method comprising contacting the cell with the gene modifying system of embodiment 94, or DNA encoding the same, thereby modifying the target site in the nucleic acid molecule in the cell. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 depicts a gene modifying system as described herein. The left hand diagram shows the gene modifying polypeptide, which comprises a Cas nickase domain (e.g., spCas9 N863A) and a reverse transcriptase domain (RT domain) which are linked by a linker. The right hand diagram shows the template RNA which comprises, from 5’ to 3’, a gRNA spacer, a gRNA scaffold, a heterologous object sequence, and a primer binding site sequence (PBS sequence). The heterologous object sequence can comprise a mutation region that comprises one or more sequence differences relative to the target site. The heterologous object sequence can also comprise a pre-edit homology region and a post-edit homology region, which flank the mutation region. Without wishing to be bound by theory, it is thought that the gRNA spacer of the template RNA binds to the second strand of a target site in the genome, and the gRNA scaffold of the template RNA binds to the gene modifying polypeptide, e.g., localizing the gene modifying 12 of 237 11867955v1 Attorney Docket No.: 2017469-0019 polypeptide to the target site in the genome. It is thought that the Cas domain of the gene modifying polypeptide nicks the target site (e.g., the first strand of the target site), e.g., allowing the PBS sequence to bind to a sequence adjacent to the site to be altered on the first strand of the target site. It is thought that the RT domain of the gene modifying polypeptide uses the first strand of the target site that is bound to the complementary sequence comprising the PBS sequence of the template RNA as a primer and the 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 the pre-edit homology region, then through the mutation region, and then through the post-edit homology region, thereby producing a DNA strand comprising a mutation specified by the heterologous object sequence. FIG. 2 is a series of diagrams depicting the chemical structures of exemplary chemical modifications to nucleotides described herein. Shown in the upper left is a nucleotide comprising a 2’- hydroxyl group (e.g., as in a ribonucleotide). Shown in the lower left is a 2’-fluoro (2’-F) modified nucleotide comprising a fluorine at the 2’ position of the sugar. Shown on the upper right is a nucleotide comprising both a 2’-O-methyl (OMe) modification and a phosphorothioate modification. Shown in the lower right is a nucleotide comprising just the 2’-OMe modification. FIG. 3A is a series of diagrams showing the heterologous object sequences and PBS (priming) sequences of a series of FAH1 template RNA variants comprising 2’-OMe modifications (indicated by m) and / or phosphorothioate modifications (indicated by *) at varying positions in the heterologous object sequence and / or priming sequence, as indicated in the table. These template RNA variants were tested for their capacity to rewrite the target nucleic acid sequence, with the resultant rewriting efficiency for each variant shown in the bar graph. FIG. 3B is a series of diagrams showing the heterologous object sequences and PBS (primer) sequences of a series of variants of a GFP template RNA, comprising 2’-OMe modifications (indicated by m) and / or phosphorothioate modifications (indicated by *), as indicated in the table. These template RNA variants were tested for their capacity to rewrite the target nucleic acid sequence, with the resultant rewriting efficiency for each variant shown in the bar graph. FIG. 3C is a series of diagrams showing the heterologous object sequences and PBS (priming) sequences of variants of template RNAs targeting HEK3, FAH, or HBB. The template RNA variants comprised 2’-OMe modifications (indicated by m) and / or phosphorothioate modifications (indicated by *), as indicated in the first table. These template RNA variants were tested for their capacity to rewrite the target nucleic acid sequence, with the resultant rewriting efficiency for each variant against each target relative to base variant A1 shown in the second table. 13 of 237 11867955v1 Attorney Docket No.: 2017469-0019 FIG. 4 is a series of diagrams showing the heterologous object sequences and PBS (priming) sequences of a series of GFP-targeting template RNA variants comprising 2’-fluoro modifications (as indicated by f) at varying positions in the heterologous object sequence and / or priming sequence, as indicated in the table. These template RNA variants were tested for their capacity to rewrite the target nucleic acid sequence, with the resultant rewriting efficiency for each variant shown in the bar graph. FIG. 5A is a series of diagrams showing the heterologous object sequences and PBS (priming) sequences of a series of variants of a template RNA, each comprising 2’-fluoro modifications as indicated in the table on the top in gray boxes. Two of the variants included alternating patterns of 2’-fluoro modifications, in which every other nucleotide in a subsequence of the heterologous object sequence comprises a 2’-fluoro modification. These variants further comprised, in 5’ to 3’ order, a 2’-fluoro modified nucleotide, three 2’-OMe modified nucleotides, and three nucleotides each comprising a 2’-OMe and a phosphorothioate modification, at the 3’ end of the priming region. These template RNA variants were tested for their capacity to introduce alterations the target nucleic acid sequence, with the resultant rewriting efficiency and percentage of insertions or deletions (indels) for each variant shown in the graphs on the bottom left and bottom right, respectively. FIG. 5B is a series of diagrams showing the heterologous object sequences and PBS (priming) sequences of a series of variants of a template RNA, each comprising 2’-fluoro modifications as indicated in the table on the top in gray boxes. Two of the variants included alternating patterns of 2’-fluoro modifications, in which every other nucleotide in a subsequence of the heterologous object sequence comprises a 2’-fluoro modification. These variants further comprised, in 5’ to 3’ order, a 2’-fluoro modified nucleotide, three 2’-OMe modified nucleotides, and three nucleotides each comprising a 2’-OMe and / or a phosphorothioate modification, at the 3’ end of the priming region. These template RNA variants were tested for their capacity to introduce alterations the target nucleic acid sequence, with the resultant rewriting efficiency and percentage of indels for each variant shown in the graphs on the bottom left and bottom right, respectively. FIG. 5C is a series of diagrams showing the heterologous object sequence and PBS (priming) sequence of a variant of a template RNA, each comprising 2’-fluoro modifications as indicated in the table on the top in gray boxes. The RNACS6874 variant included an alternating pattern of 2’-fluoro modifications, in which every other nucleotide in a subsequence of the heterologous object sequence comprises a 2’-fluoro modification. This variant further comprised, in 5’ to 3’ order, a 2’-fluoro modified nucleotide, three 2’-OMe modified nucleotides, and three nucleotides each comprising a 2’-OMe and a phosphorothioate modification, at the 3’ end of the priming region. These template RNA variants were tested for their capacity to introduce alterations the target nucleic acid sequence, with the resultant rewriting 14 of 237 11867955v1 Attorney Docket No.: 2017469-0019 efficiency and percentage of indels for each variant shown in the graphs on the bottom left and bottom right, respectively. FIG. 5D is a series of diagrams showing the heterologous object sequences and PBS (priming) sequences of a series of variants of a template RNA, each comprising 2’-fluoro modifications as indicated in the table on the top in gray boxes. Two of the variants included alternating patterns of 2’-fluoro modifications, in which every other nucleotide in a subsequence of the heterologous object sequence comprises a 2’-fluoro modification. These variants further comprised, in 5’ to 3’ order, a 2’-fluoro modified nucleotide, a 2’-OMe modified nucleotide, and three nucleotides each comprising a 2’-OMe and / or a phosphorothioate modification, at the 3’ end of the priming region. These template RNA variants were tested for their capacity to introduce alterations the target nucleic acid sequence, with the resultant rewriting efficiency and percentage of indels for each variant shown in the graphs on the bottom left and bottom right, respectively. FIG. 5E is a series of diagrams showing the heterologous object sequences and PBS (priming) sequences of a series of variants of a template RNA, each comprising 2’-fluoro modifications as indicated in the table on the top in gray boxes. Two of the variants included alternating patterns of 2’-fluoro modifications, in which every other nucleotide in a subsequence of the heterologous object sequence comprises a 2’-fluoro modification. These variants further comprised, in 5’ to 3’ order, a 2’-fluoro modified nucleotide, a 2’-OMe modified nucleotide, and three nucleotides each comprising a 2’-OMe and / or a phosphorothioate modification, at the 3’ end of the priming region. These template RNA variants were tested for their capacity to introduce alterations the target nucleic acid sequence, with the resultant rewriting efficiency and percentage of indels for each variant shown in the graphs on the bottom left and bottom right, respectively. FIG.6 is a diagram showing exemplary modifications for a template RNA as described herein. The heterologous object sequence can, in some instances, comprise an alternating pattern of 2’-fluoro modified nucleotides and unmodified nucleotides (e.g., ribonucleotides comprising a 2’-hydroxyl group). The length of the region showing the alternating pattern can have a length, in some instances, between 0 nucleotides and the full length of the heterologous object sequence minus four nucleotides. The length of the region showing the alternating pattern can have a length, in some instances, between 0 nucleotides and the full length of the heterologous object sequence minus three nucleotides. In some instances, the 5’-most nucleotide of the heterologous object sequence comprises a 2’-fluoro modification and, in certain instances, is the first 2’-fluoro modified nucleotide of the alternating pattern. In some instances, the junction region connecting the heterologous object sequence to the priming sequence only comprises unmodified nucleotides (e.g., comprising the -1, -2, and / or -3, and / or the +1, +2, and / or +3 nucleotides, as numbered relative to the junction of the heterologous object sequence and the priming sequence, e.g., as described 15 of 237 11867955v1 Attorney Docket No.: 2017469-0019 herein). In some instances, the 3’ end of the priming sequence comprises a motif comprising, in 5’ to 3’ order, a 2’-fluoro modified nucleotide, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2’-OMe modified nucleotides, and one or more (e.g., 1, 2, 3, 4, or 5) nucleotides each comprising both a 2’-OMe modification and a phosphorothioate modification. DETAILED DESCRIPTION Definitions The term “alternating nucleotides,” as used herein with respect to chemical modifications, refers to a pattern of nucleotides wherein all of the odd nucleotides of that region have the same chemical modification and all the even nucleotides do not have that chemical modification, or the opposite: all of the even nucleotides of that region have the same chemical modification and all the odd nucleotides do not have that chemical modification. For instance, in a region that is five nucleotides in length and has alternating nucleotides, the first, third, and fifth positions of a region may all comprise 2’F chemical modifications, and the second and fourth positions of a region may comprise unmodified nucleotides or a chemical modification other than 2’F. The second and fourth positions may be the same or different. Furthermore, of the nucleotides that all comprise the same chemical modification, one or more may comprise a second chemical modification. As a non-limiting example, in the region described above having 2’F chemical modifications at the first, third, and fifth positions, if only one of the nucleotides at those positions further comprises a backbone modification, the region still comprises alternating nucleotides with respect to the 2’F chemical modification. The alternating nucleotides may be found in a region of a larger nucleic acid, wherein the larger nucleic acid comprises one or more other, non-alternating, regions. 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. 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%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% 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, the gene modifying polypeptide is capable of integrating the sequence 16 of 237 11867955v1 Attorney Docket No.: 2017469-0019 substantially without relying on host machinery. In some embodiments, the 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 the template nucleic acid, 2) binding the target DNA molecule, and 3) facilitate integration of the at least a portion of the sequence of the template nucleic acid into the target DNA. Gene modifying polypeptides include both naturally occurring polypeptides as well as engineered variants of the foregoing, e.g., having one or more amino acid substitutions to the naturally occurring sequence. Gene modifying polypeptides also include heterologous constructs, e.g., where one or more of the domains recited above 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. 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 the reverse transcriptase domain initiates polymerization, e.g., based upon where target primed synthesis initiates. The first strand refers to the strand of the target DNA upon which the reverse transcriptase domain initiates polymerization, e.g., where target primed synthesis initiates. The second strand refers to the other strand of the target DNA. First and second strand designations do not describe the 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. 17 of 237 11867955v1 Attorney Docket No.: 2017469-0019 The term “heterologous,” as used herein to describe a first element in reference to a second element means that the 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 certain 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 the target site, e.g., where there are new nucleotides in the heterologous object sequence with no cognate positions in the unedited target site. 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 target nucleic acid sequence. 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 sequence difference may comprise, for example, a substitution, insertion, frameshift, or deletion. 18 of 237 11867955v1 Attorney Docket No.: 2017469-0019 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. The nucleic acid molecule can be double-stranded or single-stranded, circular, or linear. If single-stranded, the nucleic acid molecule can be the sense strand or the 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 the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the 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 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 16), backbones (see, for example, Table 17), and modified caps (see, for example, Table 18). 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 various embodiments, the 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 19 of 237 11867955v1 Attorney Docket No.: 2017469-0019 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. The nucleic acid elements of the systems provided by the invention can 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), 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. 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 instances, a host cell may be an animal cell or a plant cell, e.g., as described herein. In certain instances, 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 certain instances, 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 20 of 237 11867955v1 Attorney Docket No.: 2017469-0019 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 comprised in a target nucleic acid sequence. In some instances, a PBS sequence is a nucleic acid sequence comprising at least 3, 4, 5, 6, 7, or 8 bases with 100% identity to the region comprised in the target nucleic acid sequence. In some embodiments the primer region comprises at least 5, 6, 7, 8 bases with 100% identity to the region comprised in the 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 comprised 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. Unless specified otherwise, the following numbering system will be adhered to for describing the position of nucleotides having chemical modifications in the PBS sequence and / or heterologous object sequence of a template RNA. The positions of nucleotides in the heterologous object sequence are numbered +1, +2, +3, and so on, starting from the 3’-most end of the heterologous object sequence. The positions of nucleotides in the PBS sequence are numbered -1, -2, -3, and so on, starting from the 5’-most end of the PBS sequence. Thus, positions +1 and -1 are directly adjacent to each other. Examples of this numbering system are shown, e.g., in Figures 3A-3C, 4, and 5A-5E. Table of Contents 1) Introduction 2) Gene Modifying Systems a) Polypeptide Components of Gene Modifying Systems i) RT Domains ii) RT Families iii) Cas Domains iv) Linkers 21 of 237 11867955v1 Attorney Docket No.: 2017469-0019 v) Exemplary Gene Modifying Polypeptides vi) Systems vii) Localization Sequences for Gene Modifying Systems viii) Inteins ix) Additional Domains b) Template Nucleic Acids i) gRNA Spacer and gRNA Scaffold ii) Heterologous Object Sequence iii) PBS Sequence c) gRNAs with Inducible Activity d) Circular RNAs and Ribozymes in Gene Modifying Systems e) Target Nucleic Acid Site f) Second Strand Nicking g) Chemically Modified Nucleic Acids and Nucleic Acid End Features 3) Production of Compositions and Systems 4) Therapeutic Applications 5) Administration and Delivery a) Tissue Specific Activity / Administration b) UTRs to Modify Protein Expression Levels c) Viral Vectors and Components Thereof d) AAV Administration e) Lipid Nanoparticles 6) Kits, Articles of Manufacture, and Pharmaceutical Compositions 7) Chemistry, Manufacturing, and Controls (CMC) Introduction This disclosure relates to 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 (x) an endonuclease domain that contains 22 of 237 11867955v1 Attorney Docket No.: 2017469-0019 DNA binding functionality; 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. In some embodiments, the DNA-binding function may involve an RNA component that directs the protein to a DNA sequence, e.g., a gRNA spacer. In other embodiments, the gene modifying polypeptide may comprise a reverse transcriptase domain and an endonuclease domain. The RNA template element of a gene modifying system is typically heterologous to the gene modifying polypeptide element and provides an object sequence to be inserted (reverse transcribed) into the host genome. In some embodiments, the gene modifying polypeptide is capable of target primed reverse transcription. In some embodiments, the gene modifying polypeptide is capable of second-strand synthesis. In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding the template nucleic acid, 2) binding the target DNA molecule, and 3) facilitate integration of the at least a portion of the sequence of the template nucleic acid into the target DNA. In some embodiments, the gene modifying polypeptide is an engineered polypeptide that comprises one or more amino acid substitutions to a corresponding naturally occurring sequence. In some embodiments, the 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. In some embodiments, the system incorporates a heterologous object sequence into a target site by TPRT, e.g., as described herein. In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 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 (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a gene modifying system is capable of producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 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 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally 23 of 237 11867955v1 Attorney Docket No.: 2017469-0019 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 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 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 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 (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a gene modifying system is capable of producing a substitution into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more nucleotides. In some embodiments, a gene modifying system is capable of producing a substitution in the target site 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 some embodiments, the substitution is a transition mutation. In some embodiments, the substitution is a transversion mutation. In some embodiments, the substitution converts an adenine to a thymine, an adenine to a guanine, an adenine to a cytosine, a guanine to a thymine, a 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, 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. Polypeptide Components of Gene Modifying Systems In some embodiments, the gene modifying polypeptide possesses the functions of DNA target site binding, template nucleic acid (e.g., RNA) binding, DNA target site cleavage, and template nucleic acid 24 of 237 11867955v1 Attorney Docket No.: 2017469-0019 (e.g., 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 other 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, the domains are all located within a single polypeptide. In some embodiments, the 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, the 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 certain embodiments, the gene modifying polypeptide further comprises a spacer sequence between the first NLS and the DNA binding domain. In certain embodiments, the spacer sequence between the first NLS and the DNA binding domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the first NLS and the DNA binding domain comprises the amino acid sequence GG. In certain embodiments, the gene modifying polypeptide further comprises a spacer sequence between the DNA binding domain and the linker. In certain embodiments, the spacer sequence between the DNA binding domain and the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the DNA binding domain and the linker comprises the amino acid sequence GG. In certain embodiments, the gene modifying polypeptide further comprises a spacer sequence between the linker and the RT domain. In certain embodiments, the spacer sequence between the linker and the RT domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the linker and the RT domain comprises the amino acid sequence GG. In certain embodiments, the gene modifying polypeptide further comprises a spacer sequence between the RT domain and the second NLS. In certain embodiments, the spacer sequence between the RT domain and the second NLS comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence between the RT domain and the second NLS comprises the amino acid sequence AG. In certain embodiments, the gene modifying polypeptide further comprises a spacer sequence between the second NLS and the T2A sequence and / or puromycin sequence. In certain embodiments, the spacer sequence between the second NLS and the T2A sequence and / or puromycin sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In certain embodiments, the spacer sequence 25 of 237 11867955v1 Attorney Docket No.: 2017469-0019 between the second NLS and the T2A sequence and / or puromycin sequence comprises the amino acid sequence GSG. RT Domains In certain aspects of the present invention, the writing domain of the gene modifying system possesses reverse transcriptase activity and is also referred to as a reverse transcriptase domain (a RT domain). In some embodiments, the RT domain comprises an RT catalytic portion and RNA-binding region (e.g., a region that binds the template RNA). In some embodiments, a nucleic acid encoding the reverse transcriptase is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In some embodiments the reverse transcriptase domain is a heterologous reverse transcriptase from a retrovirus. In some embodiments, the RT domain comprising a gene modifying polypeptide has been mutated from its original amino acid sequence, e.g., has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions. In some embodiments, the RT domain is derived from the 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, the RT domain has a length of about 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 amino acids. In some embodiments, the 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, the RT domain initiates TPRT when the 3 nt in the target site immediately upstream of the first strand nick, e.g., the genomic DNA priming the RNA template, have at least 66% or 100% complementarity to the 3 nt of homology in the RNA template. In some embodiments, the RT domain initiates TPRT when there are less than 5 nt mismatched (e.g., less than 1, 2, 3, 4, or 5 nt mismatched) between the template RNA homology and the target DNA priming reverse transcription. In some embodiments, the RT domain is modified such that the stringency for mismatches in priming the TPRT reaction is increased, e.g., wherein the RT domain does not tolerate any mismatches or tolerates fewer mismatches in the priming region relative to a wild-type (e.g., unmodified) RT domain. In some embodiments, the RT domain comprises a HIV-1 RT domain. In embodiments, the 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, the RT domain forms a dimer (e.g., a heterodimer or homodimer). In some embodiments, the 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 26 of 237 11867955v1 Attorney Docket No.: 2017469-0019 functions as a monomer. In embodiments, the 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%, 80%, 90%, 95%, or 99% identity thereto). In some embodiments, an RT domain is dimeric in its natural functioning. In some embodiments, the RT domain is derived from a virus wherein it functions as a dimer. In embodiments, the 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%, 80%, 90%, 95%, or 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, the RT function of the system is fulfilled by multiple RT domains (e.g., as described herein). In further embodiments, the 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 the 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 the RT domain. In some embodiments, the RNase H domain is not part of the 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 27 of 237 11867955v1 Attorney Docket No.: 2017469-0019 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, the polypeptide comprises an inactivated endogenous RNase H domain. In some embodiments, an endogenous RNase H domain from one of the other domains of the polypeptide is genetically removed such that it is not included in the polypeptide, e.g., the endogenous RNase H domain is partially or completely truncated from the comprising domain. In some embodiments, mutation 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%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to an otherwise similar domain without the mutation. In some embodiments, RNase H activity is abolished. In some embodiments, an RT domain is mutated to increase fidelity compared to an otherwise similar domain without the mutation. For instance, in some embodiments, a YADD or YMDD motif in an RT domain (e.g., in a reverse transcriptase) is replaced with YVDD. In embodiments, replacement of the YADD or YMDD or YVDD 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 Table 1, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. In some embodiments, a nucleic acid described herein encodes an RT domain having an amino acid sequence according to Table 1, or a sequence having at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity thereto. 28 of 237 11867955v1 F P L L A P A S F P L L A P A S F P L L A P A S ESQQ A P K A D A L I T K A D R L RPQV L L P R L RPQA L I T K A D V L L P R L RPQA L I T L L Y V L L P F P L LWGI P T A P T E A I P T A P T E A I P T A P T E A K A H P A A N E KGL T N A A N E KGL T N A A N E KGL T N A P PGA H E W AGSW A DVSV E W AGW A DVV E W AGW A DVV I T R K E L D V A E L DSV ASE L DSV ASE D N FPSA V L ALCK R FPSA V L ALCK R FPSA V L ALCE K K R F W PTAQL H W R P A H D A L H W R P A H D A L H W R P A H D A LPSYKV EYD I H V R V E D I H V RSD W R V E D I H V R K A T Y P FGN A ISE Y P FGA IKSYGKSL R P W D E R A LNGE Y P F R P W D E R A LNA I E H R P WRW R F E R AG CF EGD E E P E LGELICL E A E L F E PYLGCL E A E L F E PLCL E ADP FTG I A R T L R EII A R T L R E L L T R D A R LII A R T L RQR A KSL L L T R D A R LG GL L T R D A RLL FEE P AQT A P D K P A T A P D K P A T A PS GM AN Q N QD KNL K A ASWT TGA ASM AWT TGA ASM A T TGVVGL T T R E L YGE L T T R E L YGE L T T R E L YWGE L T T LEQE L 1 0203040,8 0,8 0, 080,8 1_____ v EI0E0E0 2055R63RI 63tRI 63AtVE1P97V3V3u V3u _6A0PA0Pm3A0Pm3A B M27 811 E K P T V L P A L I L L T E A A P NKQL TASV PTGPGP P FQL VSQR A D W T I D T L N L LHGAQD V Y V H A T LAGR A A R N R T T I WGSR D D L PYPSQRSGV D I T A Y E W L T T H H D P A T D I P V L T LEQP D QGK L L D T E L R KTAG S HSL T LS SHSL T LS SHSL P I L V L D T D V V LIIQLQGD T D V V LIIQLQIQA P I L V LIQGD T D V L ALSTQV A L ALSTV K E T E P P A H K E T E P P A H K E T E H T L PLQGH T L P V A Y A I T R K E V A Y A I T R K E V A Y A E LQ LT W E LQT W L L I M D N E K MTS Q SLQV N A F W PQL L I M D N E K M N A F W PTQL L I M M N ALGD P F HSGLQA LLGD PSGLQ9A L DAW AVA L DAF H AQ S Q SW AVQA I A1TARKQRPSK A T TARKQRPSK A T TARKQPHGLASA L R LLGIPL HGALSA L R L0ACR H W R F ACR H W R F ACRS SIQ SAS0A DK-GA ERCEK9YGRCKGPSTEQAPSI TEQGP A D A E EYP A D A PVP L L D PVP L M HKV P F M H V P FGM H V P LQH V V P LQH V6IPSSRDQR A KTSIKPSDQTSKSSSEGSSSR R A K IPSR A A R V R Y A A R V R4R L D L F P R L D L F P R L D D P L Y P N D P L Y P71LCPQELSLEGK ELSLEGK E L I PIQAKQIQLAQVVGLEL TCPQLAQVVGL P AD N L I P A D E L TC QLQVPEL LEP L L02P N A R T LQE L P N A R T LQE L P N A RGKQT LSASLSVGKEQT LES:.o Nt5 6 7 8e0k0c, 00, 00, 00o8 8 8,8Dt 1t vye2nrV0u2u U9m V0m A5U90A550t 59oE1PttA_3_E1P2 _3_V5 5u77A27 L2PVL2Pm268A B M B M A B _ B _ _11 A I P HSA SSHSR V HGV F I A P H R L LSK Y L L A YSP V P D PQL W AGL V L T R L LSY L Y KSD V W I N L P E P P A A P I L V L A P I L V L A P P L V L A I L V I I N A L ALSAQVIQA L ALSAQVIQA L AILQT K VFSSAQV A K EIPYK T K V LGH T L P E LQ LT WLGH T L P E LQ LT WLGH T L P E LQT WLGVGLQHGL N L P I A ATSL D IAQALGDS QS LALDS QS LALDSLQS LA K K D W E K KKN T L P F HG QLGP F HG QLGP F HG QL R K PFG9GK P EGGA I A A L I A A L I A A L E1LGP0ALQHGLSSAR LLGPGLSR LLGPGLSR LLGL K L I L T V I A R L E D LLQ0L D PPSI TLEAQHSAPSI TLEAQHSAPSI TLEAQT W KNSN A T V P D -SP LQL D PSP LQL D PSP LQL D L P P L F Y F A A9E6GV P LVQH V V P LVQH V V P LVQH V V P V N D L L K PLCL Y T A RSSV REGY T A RSSV REGY T A RSSV REGY K4K71 QN D P L Y P N D P L Y P N D P L Y P N LDGI I I P L L K N H T N L I PIQA DKQN L I PIQA DKQN L I PIQA DKQD A E D K N L P T L INGASLSVGE E A L V ELE A L VPEL LE A LD TYT K L P L L P LQK Y LKL A Y02KQT LS S S GKQT LS S S GKQT LS S SM I K Y E ESA F A:.o Nt90011 2ek0c,0, 10, 10,o8 8 8 8D19 vyePnr_P2 J1_J_P 2 1_J_1O5_59ottV6 6t6 Bt97L 33V0L 33 uVL 33 u V F0268A B B0m B0mF 311 I Q A P Y T D P L K T V TGTGI T V A E I T K N H I P R R RGI R D R I KHGL NVQV N Y E Y W VSDQGW EQL ATSGY H K I E T F T VLQN K QQL E R E V PKGE K EKGSQL L LISD N K N P L VCL TTGI DQQA E QSE L N D W L YGK I K T I L V I I N T K VFI L V I I N T K V D L Y KQ QISFSLF Y D L Y KLF K E Q PYGK T K V K EIQPYGK T K V L L D K WGNGSNSL L D K WNVL HPL N LAL H L N L K D E P K H K D E P KSGP I A ASL D IQVGP I A APSL D IAQI N PLSV H P D V I N PLSV H P K K D W E K KKGN T K K D W E K KKN T T I V I Y V H T K T I V I Y V H R K PF9GK P EGGA R K PFGK P EGL TGGA L V I I N T K V E P K T K VFL V I I N T K E L K L I L T E L K L ISE P K T K1V I ALNR L E D LQV I A R L E D LLQLIQHYGL N L LIQHYGL N0T W KSN A T V P D T W KNSN A T V P D P I A ATSL D IAQP I A APSL D0L P P L F Y F A A -9P V N D L L K PLCL P P L F Y F A L P V N D L L K PLCA K D W E K K L K PKGN T K D W E K KKG6K I I I P L L K NFGK P EGGA K PFGK P E4LDGK H T D A E D KDNGI I I P L L K N L K L I L TLL K L I L T71 GL H T D A E D K I T K LNGI A W KNSR L E D LQI A N A T V P D WNR L E D L P T L I T K L L P T LSD TY0QK N A T V K Y L L A Y D TYQK Y L L A Y P P L F Y F A A P P L F Y2E EKA F A M I K Y E EKA F A V N D L L K PLF A M I K YS S CL V N D L L K P:.o Nt3 4 5 6e1k0c, 10, 10, 10o8 8 8,8D19 9 vyeO2 O 2 9 9 O Orm 55n_ _ _ _A_ - _-297ot V9tF0 tF23u V9F0 tF2u V9F02 orV9F0_ 2 ort68A m3mF 3 P F 3 Pu11 N NGY A H P SSD L KQHGP E H T V V V D P R L R K L D A VSK W KSI H K L P K K R N N V FGNSA W V D L E E N Y A K A V Y D L Y KQLF YTLLQQD HS SLQD HS SLQD H NSL L D K WGN NSPGAQK T D HTSPLGAQQK T D HTSPLGAQQK T H K D ELS SSP K H E T I K E V E T T E T I K E V E T T E T I K E V E D V I N P V H P D V P N E K L A Y E I V I Y V H T K E W PTP N E K I L V T E W PTL A Y E P N E K I L V T E WTL A T K TG G GFSI I N T K VFSFPSDAWQAIQT A FAPAI L V L VSPS SSDWAIT A FPDWAI9V E1IP K T K V L R A I L A L R AQIQL A LSR AQIQL LQHYGL N L R W R F A K K R W0IAQP I A APSL D IAQYQCE L ALCK YQCR F A E L ALCK K R KQCW R F ALCN T K D W E K KYGKGYGKGY EYGL A0KN T E P FTV D A E P FTV D A E P FTV D-9G6GA K P K P EGA E K A KGM H E T E K A KGMGL KFGL I L TGGH E T E K A K M H E4LLQI A R L E D LLQL F P R H A D E L F P R H A D E L F P R H I LNGR M D I LNGNG71P D W KNSN A T V P DLQL L TLCNGK ALQR L L TLCNGM D K ALQI L R LLCNG02LCA PGL V N D L L K PLQGQGQL T P L F Y F A C A L L T DQST L P E P PGSL L L A R T DQST L P E P PGSL L L A R T DQST L P E P PGS:.o Nt7 8 9 0e1 1 1 2k0c,o8 0,8 0,8 0,8D1y9eO1 1 1v 5 -mP P3_P35nr_2_ _ _ _ _A 97ot V9tF0o F23rPAtV3L7V32 L7 t2 u V3L7 t2u68A uF 0 F 0mF 0m11NGK AQPSDGKQHGPCH I ISL R K P L F L A E L L A L I E N K N K I E KGEST L WGR R R Y I EGH V H T T P A L F V A T Y L RSSSD H T T IQQPSFS GQQSS GQQSSGL A E A I PFGL A E A I PFGL A EGA L D ELSE Y E E T K L I L T P L E T K L I L T P L E T K L I L T P L I D P I V T A L A N L Y I K A L A N L Y I K A L A N L Y W K AINMV K D P W K AINMI K T I T V Y T A PQ S Q SV K D P W K AIQNMSV K D9L A L P P L F Y F D L A L P P L F Y F D L A L P P L F Y F D L ALQV I VK1K V N D V L K P A L V N D V L K P A L V N DIGQP KYG00K-GKPQV L K P A L L H K D I L K LPQPQA L T P T EPQI K A D I L K L I K A D I L K L I K A P A E H L TPQPQISAQAS9KLCNP T E E HK NL T P T E E HK N QW E T6QPSYQT L V A KGLQPSYQT L V A KC GLQP YT L V A KC G QPFL A A D K L L A K K L L A KS QK L L A K T KGL4M D L L YQQEKSE FQQKSQQKSI K L L LKY E E F L L LKY E E F L L A NIL71K A P LQPKQV A F P E I P LQH F V L V F N I PKV A F P E I P LQH F V L V F N I PKV A F P E I W K AQN L L N IQ QH F V L V F P P L F Y02A R M Y R R E N Y T A V M Y R R E N Y T A V M Y R R E N Y T A V V N D V L:.o Nt1 2 3 4e2k0c, 20, 2020o8 8,8,8D1vye3 3tu3tu3 55nrM41 M41m2M41m2M41 or 97oAPAPttOF_05OF_ _ AP05O__ AP0O_P- 068A V VFV5AFV511 I K H KKCL A E F P E L V T A K A D I Y E H A FQMQI A R T P H D ENGL L Y P T MISW TWQI N W L R IVQP Y L E A P K K R Y E T P A L H P V TIKGQIMGK A T N MSNSV K I T PSP D Y VPSL K I I E A QGSGM D A T L Y D F M AVGK E I Y L AQS K PSN L D E E K PSN L D E E K PSN P PGA H K ESV P PGA H K H H D V I D PLSI H H D V I D PLSI H H D V K T K K E L E D R K T K K E L M K T V T I T V Y M K T V T I T V Y M K T V E N E K E L A TTE N E K E YL K Y L KYSL T V I V I V T V I V I V T V I H W P T L L H W P T L9T P VS Q G S Q GYSASAS1IPYT P VIP T P V L D W A I I L L D W A L L LQHGK L L LQHYGK L L RPSK V IPS0LSQGQSQGQSQGQV E N A R K V I V V P0KIAAA L V PIA A L V Y W R F A I Y W R F A SSPGTQSQWQP KSSPGTQSWAQP KS PT ERCESYP ACNAQERCEYP A -9E A PF6GL A E AQP P L T KFGL A ES GA E P FGL I L T P L E V A LIV R A HKK E P FGA E V A LIV L T P L T K L I L TS G SA4M71 SY I K L A N L Y I K L A N L Y I K L L F E K V E A L V K D W KIQMSIQMSKSL FKSP K A N V K D W K A N V K D N F D L A P P L F Y F D L A P P L F Y F D L A LVL KLP A E NVL KL0GL TCT L D LGL TC2K P A L V N D V L K P A L V N D V L K P A L V LPQE L T A R A V LPQE L T:.o Nt5 6 7e2k0c, 20, 28020o8 8,8,8D1vye3n434_4_ _ 455rM1 mP2_ M1 m2_VL 14VL 14t97oA ttOF_- 0 o A 5rP tOPF_ - 0o 5r AtA12 A1u 2m 68A V u VPu GPGP 311 L R AQR TNGL T A A V V L P E N ASKGQRAQSHTGGEPCP H L I SSI A A V W R V R T P R L KGH I D A V E I L P L AGA L A I R E R E KGK N E K T I I D FSKGSGASD T T L Y WWGT PRQVGK Y P I LAPGQH D I E H L A D T R A R F T G A Y A P L TLAGY A L I VGT L I VGT L I VGRQQ Q W T Q W T Q W TT T S L V LNQ S Q Q S Q Q S Q QGD TRSL H DWQH I H DWH I H DWH I E V P P A H K E V A D E L L A DQE L L A DQE L L E D R K T K K E L E D RIQL M I KAGLIQL M I KAGLIQL M I KAGL A TTE N E K E L A TLTP TY E SLGA P TLY E SLGA P TLY E SLGA L YSH W P T L L YSP TQ G QN L D P TQ G QN L D P TQ G QN L D9I I L L DASW A I I L R P L L F K L T R P L L F K L T R P L L F K L T1E N A RPSK V I V E N A P L I L V F E L P L I L V F E L P P I L V F E L00SCI NAQY ERCW R F A ESCIAQLSSTAQV R A A LSSTAQV R A A LSSTAV R A A R K E P FYGP A N H V R K E LCQT L THSRQN H L ECQTHSRQN HCQQTHSRQN -96HKGA E V A LISA HKGL L T L E L L T L A4V E A L L F P K V E ALGD P E P F PTGH HASRSLGD P E P F PTGH HASRSLGD P E P FTGHASRS71P AKVSP H E N L K P A E L L T L K I0GLCPGAQLPGAL T L K I LP AL T L K I T L D L L T T L D V A L L H P VQA L L H P VG QA2PL L H P A R A V LQE L T A R A A P R W T I P D A P R W T I P D A P R W T I P D:.o Nt9 0 1 2e2k0c, 30, 30, 30o8 8 8,8D1_2vye_ V41A A1 623 A1 63tA1263Bt55nroL4t1 tutAL3 3u3u972PmT0PLT0Pm2LT0Pm268A G3H _ H _ _ H _ _11SSI PSQSL L I D H I A TLLQSM N E T F A L R T L DPSH N I K F F R WGQTGP L N V A R K W K A V Y P R F VTGP NPGNGY NPCGTQQY P P V E T I F HGA F A Y E LPQA KFQR K V P L L HQP LIQAQF F L A R D E K P L N D L I P FTQQSL H I A DQQQAL L I A DQQQAL L FQLSPGLP FQLSPGLP FQLSP E L M I KGLL E L M I KGLLQL I V WTGTQL I V WTGTQL I V W P T P TYQEGSQLGA P T N L D P TYQEGSQLGASH D N L D I A DWQQQQH ISH DW QQH ISH DW QAL L I A DQ QAL L I A DQ Q9R P L L F K L T R P L L F K L T E L M I KG G1LGL E L M I KLL E L M I K P L I L V F E L P L I L V F E L P T L A P T LGA0L0HSSTAQV R A A LYT N HSSTAQV R A A P TQEGSQN L D P TYQEGSQP T N L D P TYQEGSQT N R P L L F K L T R P L L F E LCQL THSRQCQHSRQK L T R P L L F -9L E L L T L P L I L V F E L P L I L V F E L P P I L V6D P E H D P E H L V V R A A L V V R A A L4LGP F PTGHASRSLGP F PTGHASRSHSSA S A SVAV CQTH QN HS QT N HS QT71LP AL T L K I L L H P VP AL T L K I E LQL TS QL E LCQL THSQQL E LCQL T VG QA LG QA L L H P02A P R W T I P DLD P E P F PTH HAD P E H D P E A P R W T I P DG G SKSLGP F PTGHASKSLGP F PTG:.o Nt3 4 5 6 7e3 3 3 3 3k0c,o8 0,8 0,8 0,8 0,8D1eCnr 18_ _ _ vy78074C104 t1 1uL1 12L1 12t1uL1 1B2t55u9ttL 1 L2LCL0CLC 7oT P T1PmTP T0Pm2 T0Pm268A H _ H _ _ H H _ H _11 K I H P P D P P P L L N P T FSHSR R QSV H L H I L H Y K I D V W K L H I A Y L A L A R QSP L T L SGAQDSF P FALQCPSSA K T NGQI F I ITGPSW A L L T T F R V L P Y T H L Y A L K Y V PKSA D L L P F A I A N T KLCL H F TWS N R P L A N DPP L R P P LPP L R P P L P L R P P L P L R P WSTW WQL N DSPTW WQL N DSPTW WQL N DSW W L LSL L I AGV PSP L I AGV PSP L I AGVSTGKSESP P L I A V P P G I P T LKSLGSKQL P T LKSLGSKQL P T LKSLGSKQL P T LKLG KL LTGP F R L P L F R L P L F R L P L F R LSPS QL Q T H IQSL I V W H DP LPQL I V WP LPQL I V W P L I V W P WKQ GASH DW KQ GASH DPW KQLGAQSH DPW KQLGA A9GL L V P DQ QA L V P DQ QA L V P DQ QA L V PQ QLAGAGAGDAGA L E L M L K L M E L M L K L M E L M L K L M E L M L K L M1LLGA PGQYQEGTQLLGL PGQYQEGTQLLGL PGQYQEGTQLLGL PDQY E TLLL0N L D P N A P N A P N A PQ G QNGA0K L T P P I L F H F D P P I L F H F D P P I L F H F D P P I L F H F D-9F E L PL6SI M L F L T PLI M L F L T PPI M L F L T PLI M L F L T R A A L4LV R A LSV R AS SHQN H TSSQAEHGE H TLSSQAEHGL E H TLSSQV R AEA LL SV REA HGE H TS QAGE71 S QL E L N L TSA02AQN E L N L TSAQN E L N L TSAN E L N L THSAN H SKSLGD P ETGHQLLGD P ETGHLLD P ET HQLLD P ET HQL H P F A A R P F AQA RGP F AG QA RGP F AG QA R:.o Nt83930 1ek0c,o8 0, 480, 480,8D1e_2_ 2 2 _ vy 2nr 3R 2 Rt_ 2BtP X 55ottL5 3TR 0L5TR u3R5u3 090 m2LTR 0m2LTU7468A HQHQ_ HQ_ HQ 611PGA K PSR V PSP A A L L I D RHSTLLQSNQQT F A L R T L DPSH N I K F F R WGQKGP L N V P R K KWSV Y P R F ITGP NPGNGH N P N GQPQY P P L E T I F H A K F A Y E LPQA F RSV A V P L L D P T LIQAQF F L A R D E T P L N D L I P T F R LSPGS QL F R LSPGS QL N I AGA P R L PLSGF F T ISPLF F T QSL I V W P L I V W P L L L L P D D P TQDGD P H DPWQLGQSPQLGTQKSGSRQQGRQQKQA H DW KA P L PGA APN A P F APN A V P DA9GA L V P DQ QKAGA L L MFL I V W H DPCL HQR L R P V F HQR L R E L M L K L M E L M LQ Q1PW0PDQYQEGTQL L P L L D P DQNQI I T L V T P Y T L V P K T N LVQT L V T P V P K T NLGA PDQYQEGTQNLGAS0P P I L F H F D P P I L F H F DVGLSAGAGQL M I K A AP-SA L P A L KPA L P9Y E PL L D M I H L FATSM I H L P I M L F L T P IQ G Q QLLSV R A LPSM L F L T P T N L T V R A P P V L F H YDSH A D I A L M T Y H VDSH A D I64HLSSQEGLSSQEGEGTQISM TTQY T AHE H T AHE PLI I I F ETM ALV DTM A71E L N L TSN E L N L TSN PSNGT VG GLGD P EA0QAQTSVQVSRGKQL W A A T V L2LGP F ATGHQL A RLGD P E P F ATGHQL L T F L E F K N F T L E F K A R H L T L T M A T P N K VWSD L H P N K VWS:.o Nt243444546e 0 0 0 04kc,o8,8,8,8 0,8D1y_eP Xt_ uPt 236ntP_ P 2 v __ 55r 3 0m3X0u m V33u V32 V32Bt97ottLTU 42LQ_6 TU 42LQ_6 T0Pm2R S6R6J1 S 1u68A H H B H _ _3 J 3m11 E N VGVQQV A L SSAGFSP L TQTSA H Y A Y R TQI HHGN F I IFCL P A AHQTGRCNSH L SGV DQQT I F L I T L F T L A N E L P V I E PQPSR T R VTGI I K I P P F V I T F E A I HMSQSL AAFGQV L V L Y KSDSSD P T Y F H L H N T IF SSPRSLQP R H V E PSLQP R H V E PSLQP R H V E W L QGTQLGK N P T A T I L LRGK N P T A T I L LRGK N P T A T I LIQYQT D P F P L L Y N T M A L P L L Y N T M A L P L L Y N T M A L A R D L E P L PVGRAA D L E P L PVRV HD P V FGAA D L E P L PGRAA K VSV9Y T P V N A T E AGL P V N A T E AGL P V N A T E AGL L1N LVQSGA V P R P K NA0QISGA V P R P K NSAGA PYQQI A V P R P K NAI K V P A L K V V I E T E V V I E T E V V I E TQE T L L0F -9AAQTRSPCP TFSK D M R ERSPCP TFSK D M R ERSPCP TFSK D M R E LPSL E P P A M V W K E P P A M V W K E P PSSM H VGQRGGQRGGQA M V WRGK L A V64ISP H L V Y R N P H L V Y R N P H L V Y R N D L V DD71 GIVQI D T L D D KDDGIVQI D T L D D KVDQDSVQGI I D T L D D KRGL W A A V N Q R A A P M I V NDR A A P M I V N R A A P PQDQM I K N P N F T L L A H K F Q I D L L A H K FPI D L L A H K FPI D L L Y N02D L H T F L V L R P F K T F L V L R PQF K T F L V L R PQF K L E P L:.o Nt748 9 0ek0c, 40, 40, 50,o8 8 8 8D1ye_nrVC _tv 5 RTVC T RTu _ VCt _ TmRTu VC TmRT5o97ottO9Q 1O93Q_O93_ OT9rP- 68A K K1KQ 1A KQ 111 E A K N E T D M V W Y R L D A P F PPQK R T V L DISP E L L A N L H A P P A ASI V L L L V A N E KQGI L RGT L F K A D EQA D H K A L E L T L W A T V F A P A V I K A E K R TLCL T P P Y W L PGSA A A V LSP D A L FGK PKSI L W Y L A RCV P FGR R A W VNII YW L A W VNII YW L A W VNISNGK L A Y KSNGK TGPGRSAQ QT D RSAQ QT D RSA T W PPGVPGE A V A RTHDGPGE A V A RKHDGPGE A V E DASL V I T W P W A I N A E DASW L L N W R K VSV L L N W R K VSV L L N W R YPSR A N I E L A YPSR A N9F L V K L A P F L V1T Y TK L A PYF ETL V K L W R F A R E L W R F EQL T P K V PQEQL T P K V PQ QL T P RQCE L ALCR RQCE L0R T L R L T L L R T L R L T L L R T L R L Y P FYGV DKGA Y PYG0-9VQLSVQK H LPSSSMVQLSQK H LPSSMVLSK H E V A LTGF M H ETAE V A LG6GN I L A VVGN I L ASVQVQN I D L FNT R P AQD L FNP R Y D A D L R Y D AG G GP R H V EDSLVQD P R H V EDSL LVQR Y D A E I L K P R H V E L P LLCAGM D E I L K4R71T A T I LRK N P T A T I L NGT K A LGP L T T A T I LGK N PG QTQT L P E L L NQQT L02VM A L L L Y N T M A L L L Y N T M A L L DSL P P E R L DSL P PGR A A L E P L PVGR A A L E P L PVGR A A T L T L Y W PAQN T L T L Y:.o Nt1 2 3e5k0c, 50, 54050o8 8,8,8Dye_t 1v C _ C3n33u55rVmRT3Vm_ RT3A A3_V0 0 m397ottOT9 o Q-1rP tOT9 o Q-1rPAt L P_V6L P_- 668A K u K u M V5M V511 L T P L L A L I T L A E P A N LRQA VASVTPGGA FQP L V A R R D A T W D I L V L E M T AQE T Y T H V T A M RAGA A NSK R L WQGR DEQP L PQFSK VSGL D A T E Y V W A T H H P D A A L D I P V I L PPQQGD T M L T D L P KGR ATGD H K T R E L A Y KSNGK L A Y KSNGK L A Y KSNGK L A Y KSNGK L V L V I T W PPGV L V I T W PPGV L V I T W PPGV L V I T W PPGV A I N A EAPDSW A I N A EAPDSA I N A E DASA I N A E DASA I E L A YSR A N I E L A HSR AWSI E L A HPSR AWSI E L A HPSR AWSI9A R E L W R1LF A R E L W R F A R E L W R F A R E L W R F A ACK0GR RQCEYGL ALCKR RQCELYL ACQKR RCELYL ACQKR RCEYL A V D A Y P F V DGA Y P FGV DGA Y P FGVG G0M H E E V A LT-GM HTGTGD A Y P FTGV9R P AAQE D L F P R P AAQE V A L M H E D L F T R P AAQE V A L M H E D LAQE V A L M M D E INGDGFDGT R P A D L FDGP R64LCAGL K K A LG71 QP L TLCAGM D E I L K M D E I L K M D E I L K K AIQGGQP L TLCAGK ALICAGLCGGP L T K AI GP L T P E L L N T L P E L L L P E L LQL P E L LG QL ASQSTQQ T Q TP P E Q R L D L P P EAR L D P P EAR L DS QP P EAR L DS QP P02W P N T L T L Y W PQN T L T L Y W PQN T L T L Y W PQN T L T L Y W:.o Nt556 7 8ek0c, 50, 50, 50,o8 8 8 8D1t vyenA33u5r0 m3BS 7o V7BS 7 BS 73 _597tt L P_ -VL Q_KVL Q_7KVL Q_7Ktu6M V65A8A M M V M M V M M V m11 T G L R A A I E L EASADRGQKAQSHTGGALCP H L I AGISA L W R I K V P E L T F E L T A T K V L A L AGA L A I K RESQGK N EQK I L E FSRGSGESD T T L Y WWGT R H R D R A Y D I P IEGPQH I D H T A L T D A P F R A TGY HRST D E T A Y KSNGKPL A Y KTTMK D T E T TTTMK D T E T TSN E K L A L V I T W PAGV L V ISNGKPL A Y KSNGKPPL A Y K T W PGV L I N A EPDSWA I N A T W PAGV L V I T W PAGV L V I E DASW A I E L A HSR ASI E L A EPDSWA I N A E DSA I N A HPSR A N I D9R E L W R F AS SPSWS1LCK0GR RQCEYGL ALCR E H R A I E L A H R A I E L A L W R F A KGR LQCW R F ALCR E LQCW R F AQLCLCCR E R EYGL A D A Y P F0H E E V A LTV D A R EYL A R R E L A R Y P F V D-GM H E Y P FGV DKGA Y P FYGV DKGA E V A LTG9P AAQD L FADQTGTGMHQ6GP R P A E V A L M H EAQE V A L M H EAQD L FDT R M D E I L K M D D L F PG4AGK AIP L TLCAGK A E I LDGR P A D L F K M D E I LDGP R P A E I L K K M D ILCLG71 GG L A L A GP L T E L LQEQ TL P E L LI GP L TC GK AI GP L TC GK A NQQ TL P E02AR L DS QP P E L Y W PARG QN L DQ TL P E L LG QL P E L L L DS QP P E PQN T L TQ S QP P E A R L DQSTQP P E A R T L T L Y W P:.o Nt9 0 1 2e5k0c, 60, 60, 60o8 8 8,8D1e1vyS 3 S 3 _ S 3B655nBr7o VQ_7B7Q_7B A7_CQ_7A 3897tt L_ KtuVL_ KtuSVL_KtuSVL0 6M M V m M MP 8A V m W M M V m W M _11 G R A E ASNGKQHGPCH I ISL R K P L F L A K L L A L I E D K N K I E KGEST L LGR R R Y I EGH I H A T L A L F ATSY T RSE P D T E PGAQK A D T E PGAQK A D TGPGAQK A D TGPGAQK A T EKT M K D T E T EKT M K D T E T EKT M K D T E T E T M K D T E Y RSN E KPL A Y RSN E KPL A Y RSN E KKPL A Y RSN E K L A V T T W PAGV L V T T W PAGV L V T T W PAGV L V T T W PPAGV L N A EPDSW A I N A E DSW A I N A E DSW ASL V HPR A N I D L V HPI N A E K A K I D L V HPD W A I L V HSR A N I DS S SK A K I D9K E L W R F A K E L W R F A K E L W R F A K E L1KGR RQCE LLCQCLCQCLCQCW R F ALCA Y P FYGA V DKGR R E A Y P FYGL A V DKGR R E A Y PYGL AKGR R EYGL A00E E V A LT-GMTGFTGV D A Y P FTGV D9AAQD L F P RHQE AAQE V A L M D L F P RHQE AAQE V A L MHQAAQE V A L MHQM D EDGDGD L FDGT R A D L FDGP R6I L K4K A I P L TLCLGM D E I L K K A I P L TLCLGM D E I K A ILSK L TLCLGM D E I K A ILSKLCLG71LGQGQGQGQL T M NQ TL P E L M N L P E L M N L P E L M N L P E AQR L DS QP P EAQR L DQSTQP P EQASTQQSTQQR L D P P EAQR L D P P E02N T L T L Y W P N T L T L Y W P N T L T L Y W P N T L T L Y W P:.o Nt3 4 5 6e606 6 6kc,o8 0,8 0,8 0,8D1yenC1v B6B 1 0 0 5r3t 6uC3 At5151uF8F8t 59o Vtt L 80m VL 80m VL 62VL 6u 2m76 MP 3 P 3 P P 3 8A _ _ M _ _ M _ M _ _11 G R A E A H NGKQHGPCH I ISL R K P L F L A K L L A L I E D K N K I E KGEST L WGR R R Y I EGH I H A T L A L F ATSY T RSE P D TGPGAQK A D TGPGAQK A D TGPGAQK A D T E PGAQK A T EKT M K D T E T EKT M K D T E T EKT M K D T E T E T M K D T E Y RSN E KPL A Y RSN E KPL A Y RSN E KKPL A Y RSN E K L A V T T W PAGV L V T T W PAGV L V T T W PAGV L V T T W PPAGV L N A EPDSW A I N A E DSW A I N A E DSW ASL V HPR A E I D L V HPI N A E R A E I D L V HPD W A I L V HSK A K I DS S SR A N I D9K E L W R F A K E L W R F A K E L W R F A K E L W1KGR RQCE L ALCQCLCQCLCQCR F ALCA Y P FYGV DKGR R E A Y P FYGL A V DKGR R E A Y P FYGL AKGR R EYGL A00A E V A LT-GM ATGTGV D A H P FTGV N9AAQD L F P RHQAAQE V A L M D L F P RHQE AAQE V A L M DHQEAQE V A L MHQM D E IDGDGL FDGP R A D L FNGT R64K A ILSK L TLCLGM D E I K A ILSKLCLGM D E ILSKLCLGM D E I L KLCLG71NG GL T K A IGL T K A IGP L T L MQQ TL P E L M NQQL P E L M NQL P E L M NQL P E AQR L DS QP P E R L DSTQP P E R L DQSTQP P E R L DQSTQP NAQAQAQP E02T L T L Y W P N T L T L Y W P N T L T L Y W P N T L T L Y W P:.o Nt7 8 9 0e60606 7kc,o8,8 0,8 0,8D1yenF0 9v 51rV8 F09 56AtuF8tF0uF8 Atu M33 59tt L 3V6P 3V6P 3V0P 7o 2PmL2 mL2 mL_S5568A M _ _ M _ _ M _ _ M11 G R A E ASHGKQHGPCH I ISL R K P L F L A K L L A L I E D K N K I E KGEST L LGR R R Y I EGH I H A T L A L F ATSY T RSD P D T E PGAQK A D T E PGAQK A D T E PGAQK A D T E PGAQK A T EKT M K D T E T EKT M K D T E T EKT M K D T E T E T M K D T E Y TSN E KPL A Y TSN E KPL A Y TSN E KKPL A Y TSN E K L A V I T W PAGV L V I T W PAGV L V I T W PAGV L V I T W PPGV L N A EPDSW A I N A E DSW A I N A E DSWASD L A HPR A N I D L A HPA I N A E D W A I L A HSR A N IS SR A N I D L A HPSR A N I D9K K L1QW R F ALK K LQW R F ALK K LQW R F ALK K LQW R F A KGR RCEC C C C C CLCA Y P FYGL A V NKGR R E A H P FYGL A V NKGR R E A HYGL AKGR R EYGL A00E E V A LT-GTGP FTGV N A H P FTGV N9AAQM D L F P RHQE AAQE V A L M D L F T RHQE AAQE V A L M E E V A L M D L F P RHQAAQD L F P RHQ6M D E I LNGKNGNGNG4K A IG71 QP L TLCLGM D E I L K M D E I L K M D E I L K K A ILGCLGLCLGLCLGQP L T K A IGQP L T K A IGQP L T L M N L P E L M N L P E L M N L P E L M N L P E AQR L DQSTQP P EAQR L DQSTQP P EQAR L DSTQP P EQAR L DSTQP P E02N T L T L Y W P N T L T L Y W PQN T L T L Y W PQN T L T L Y W P:.o Nt7317273ek1c,o8 0,8 0, 780,8D1yenMer t tv 5rVefM33M33u m3M33u m5397terV0PV0 V0ot L_Sec L_L P_L P_ S5 _ 5 _ 5 68A M n M5MS 5MS 511 G R A E ASHGKQHGPCH I ISL R K P L F L A K L L A L I E D K N K I E KGEST L WGR R R Y I EGH I H A T L A L F ATSY T RSD P D T E PGAQK A D T E PGAQK A D T E PGAQK A D T E PGAQK A T EKST M K D T E T EKST M K D T E T EKT M K D T E T EKT M K D T E Y T N E KPAGL A Y T N E KPGL A Y TSN E KPGL A Y TSN E KPL A V I T W P S V L V I T W PAGSV L V I T W PASV L V I T W PASV L N A E D W A I N A E D W A I N A E D W A I N A E D A HPR A N I D L A HPR A N I D L A HPW A I LS S SR A N I D L A HPSR A N I D9K K L W R F1QALK K L W R F A K K L W R F A K K L W R F A K0GR RCEYGL ACKGR RQCEYGL ALCKGR RQCELYCQCLCGL AKR R EYL A A H P F V N A H P F V N A Y P F V NGA Y P FGV0E -9AAQETGTD L FHGTGTGN V A L M NQEAQE V A L MHQEAQE V A L MHQEAE V A L MH6GP R A D L FNGP R A D L FNGP R AQD L FNQGP R M D E I L K M D E I L K M4K A IGP L TLCLGK A I P L TLCLGD E I L K K A I P L TLCLGM D E I L K K A ILCLG71 QGQQGQGQP L T L M N0STQL P E L M NQSTQL P E L M NQSTL P E L M NQ TL P E2AQR L D P P EAQR L D P P E Y W PAQR L DQP P E N T L T L Y W PAQR L DS QP P E N T L T L Y W P N T L T L N T L T L Y W P:.o Nt475 6 7e 0707 7kc,o8,8 0,8 0,8D1t t vyeM33uSM3uSM3V M3V55nrV0 m3 3WV0 m3 3WV0 LPV30LP97ott L P_ _ S55_L P__ S55_L P_ _9 L PS551 __ 59168A M A M A M9MS 5 911 G R A E ASHGKQHGPCH I ISL R K P L F L A K L L A L I E D K N K I E E KGF E ESSGT D L A WGT R R K R RSGY IGSEGP HSSI N H E A I T L A L F ATSY T RSD P D T E PGAQK T D P E PGAQK T D P L M V F N YQ GL M V F N Y T ETSTMGK D T E T TTSTMGK D T E T T Y I FLRQLQQD D I I Y I FRD D Y T N K L A Y K N K L A Y K I D V Y L P Y Y I DQPPV L V I I W PPV Y L P V I I WG GV L V I H P Y A D T F H P Y A D N A EAPDSW A I N A EAPDSW A I N A LRGT PKQK LRGT PKQL A HSR A N I E L A HSR A N I E L A M W N R IKCKSVSKECKS91Q LR E L W R F A R E P L W K K A FQM W N R I K K L W R F A P L W K K A F KGR RCESYGL ACKGR RQCESYGL ALCKGRRSTPF E E LRGRRTPF E E L0A Y P V D A Y P V D A FSN P E N KSFSN P E0E E V A LT-GM H E E V A LTGM H ETSTS9AAQD L F T R P AAQD L F P R P AAQDSP LNSPST V LKSA H IDSP LNSPSTKS6M DNGNGI T E P V K RSGV L E I L K M D E I L K M D L I T E P KLCAGLCAGV K R4A I L M NGQP L T K A I T L P E LGQP L T K AEQP EVSLGR H DRQEQP EVSLGR H71R L DQ0SL N K P P E R L DQST L P E L L I L K P P E R V V F HTSRGT I L F L V VTSRG2AQAQT L Y W PAQN W FHGA K I K I H W FHF H N T L T L Y W P N T LGA K I K:.o Nt8 9 0 1e707 8 8kc,o8 0,8 0,8 0,8Dyt 1veR21R21u mVT33V3355nro Vtt L1P_V7L1P 30T0 972_ - 7 MPMP2_5_5 68A M D M D M B6M B611 K L E T Y I K T R PSL T A T E I E P F LGT V Y KSD T Y L N F P QSV E E T F L A I T R I T V ALSV D I A E Y A A QQN AGQT A NQL TPGN P E KLGI T I PHGE R R I GH QGL M V F NLYQ GN K IQL M IGN K IQL M I L M V F N Y I I Y I FRDQD I I L M V F N YQHGL M V F N YGQHGY I F DLQD Y Y I D VQY L P Y Y Y I FLRDQD I I Y I FLRDQD I I I D VRQY L P T F HRVGP YKA D T F I D VQY L P Y Y I D VQY L P Y Y HRP YKA D S K L T PQK H P Y A DG QE M W N R IKCKSVSLRGT PKQT F H K LRGP Y T PKQA D T F L T P K M W N R IKCKS91RQGP L W K K A FEQM W N R IKCKSVSM W N R IKCKSVSP L W K R T F E E LRGR P L W K K A FEQP L W K K A FEQK A F T F E E L0N KRSFPSN P E N K T F E E LRGRGRSPS0AT-SRSPSRRSTPSF E E L R F NTSP E H9DPN PTKA H F NTP E N K F NTP E N K P T SGISLS SV LSSGIDSPNSSPSTKSA HDSPN PSTDKA HSLNSPSV LKS6L I T E P V K R L L V L I LS SV LSI I T E P V4DR71 QEQP EVSLGR HSRGSGK R D Q I T E P V K R L I T E P V K R LEQP EVSLGR H T I L F L V VT RTEP EV LR H DR EP EV LR H DRI L02HF HS GF LQI LS GA K I K I H V V H F HT RTQ QI LS GTQV V F HTSRGI H W FG S GF L V V H F HTSRGF L W FHGA K I K:.o Nt2 3 4 5e8k0c, 80, 80, 80,o8 8 8 8Dt 1t vyeVnr T33u3u3tu3tu550m2VT 30m2VT 30m2VT 30m 9tPt_P_S P S P 2 7oM_ M_ M_ _ M_ _68A M B56M B56W _ M B56W _ M B56B11 K L E T Y I K T R PSL T A T E I E P F LGT V Y KSD T Y L N F P QSV E E T F L A I T R I T V ALSV D I A E Y A A QQN AGQT A NQL TPGN P E KLGI T I PHGE R R I GH QGL M V F NLYQ GN K IQL M I N K IQL M I N K IQL M I I I Y I FRDQD I I L M V F N YGQHGL M V F N YGQHGL M V F N Y Y Y I D VQY L P Y Y Y I FLRDQD I I Y I FLRDQD I I Y I FLRDQD T F HRVGP YKA D T F I D VQY L P Y Y I D VQY L P Y Y I D VQY L P S K L T PQKCKSVK HRP YKA D T F HRP YKA D T F HRP YKA D E M W N R ISELGT PQK LGT PQK LGT PQ91RQGP L W K K A FQM W N R IKCKSVSM W N R IKCKSVSM WKCKSR T F E E LRGR P L W K K A FEQP L W K K A FEQN R I P L W K K A0N KRSPSRGRGF F N P E N K T F E E L R T0T-SRSPSRSPSF E E L RRSTPSF E E L A H9DPN PT A H F N P E N K F N P E N K F N P E SGISLS SV LKSSGIDSPTNSSPSTKSA HDSPTN PSTKA HTDPN PSTK6L I T E P V K R L L V L I LS SV LSISLS S S4D P E R HSGSGV L D I T E P V K R L I T E P71TRQEQV K R L I T E P V K R I LVSLGRF L V VT RTQEP EV LR H DR EP EV LR H DR EP E R H02FHF HS GF LQI LS GA K I K I H V V H F HT RTQ QI LS GF L V V H F HTTQ QI LVSLGI H WG S G SRGF L V V H F HTSRG:.o Nt6 7 8 9e8k0c, 80, 80, 80,o8 8 8 8D1t t vyeVnr T33u3u3tu3S550m2VT 30m2 SVT 30m2 SVT 309ottMP_ _ MP__ W_ MP_ _ W_ MPW7__6M56565656 8A B B M B B M B B M B11 H K L E T Y I K T R PSL T A T E I E P F LGT V Y KSD T Y L N F P QSV E A E T F L A I T R I T V ALSV D I A E Y A A QQN AGQT A NQL TPGN P E KLGI T I PHGE R R I GQHG I I Y I FRQDQD I I T R MQRQN P T R MQRQN P T R MQRQN P T R Y Y I D V Y L P Y Y M W I A V E M W I A V E M W I A V E M W T F HRGP YKQA D T F L KGQHLTGGQLGGQLGSK K L K HTSK K L K HTK K L K V9SK L T PKK R P T I T R P T I T R PST I T R1ERQM W N R ICKSVSGESTGGSTGGSTGGSQV L L Y IHV L L Y IHV L L Y IHV0GP L W K K A F K K V L R P PGK K V L R P PGK K V L R P PGK K R0RSTPSF E E LRGR I K K Y E V E R I K K Y E V E R I K K Y E V E R I K N K F N P E N K D K K N V R I D K K N V R I D K K N V R I -9ATSWQWQWQD K HDPNT A H K I L M I K I L M I K I L M I K I64SGISLSPSV LKSGL I T E P V K RSGI M V F N L I F DLQYQHGM V F NGD I I I F DLQYQHGM V F NGD I I I F DLQYQHGM V D I I I F71D P E R H D D VRQY L P Y Y D VRQY L P Y Y D VRQY L P Y Y D V TR0QEQI LVSLGTRQP Y A D T F P Y A2TSRGRGK R KD T F K V KRP Y T PKA D T F K K V KRP F L V V H F H F L T PQK K V KGT PQKG Q GT:.o Nt09192 3 4ek0c,0, 90, 9090o8 8 8,8,8D1vyeV3n3 Sr T0 V33oV33oV33m V3355PWT0PrP T0 rP T02_T0 97ottM__ M_ - MP_ - MP_ - ortMP_68A M B56M B56M B56M B56 Pu M B11 I C K A E E TSP T V L V K LGR HTSK I N P LNASQD I P MSN L T I L E I F E L D P V K ISL ERGST P T H L A K L L L F I P D R D I M W Y N H I V INGYSL L DQKQY F K D D N R L V T T R L L I K A D K T D R V FIQK L QCKQL TYSPSVS N D K DQPSL N D K DQ SL N D K DQP W F T LQI P W MGGPGPGLGLGF T L K Q V RFQT D L K N P T R MGQV RFQT D L K N P T R MGQV RFQT E A D P N PIA PH N TGN PNSE A D PIAH N TGGQI A V ELM W I A V E M W I A V EC QR L L V F ACPQPTGGQL G LR L L V H TSK K L K H T I T RTGPTSK KGL KQH T I T RTGG PSK K T I TQQL V KTQA TFSASV IQQL V KTQA TFS9LGL Y I VSLGL Y I VSLTGL Y I PAGH LAG1V L R P PHGK K V L R P PHGK K V L R P PHGN A M I K FACL K P H L N V T A M I K FAC0K Y E V E R I K K Y E V E R I K K Y E V E R L L A D I A A A L L A D I A0-9WQK N V R I D K K N V R I D K K N V R I I V T K L I I L I V T K L I L M IG HK IWQL M IWG HK IQL M IG HD M A R F P L D D M A R F P6F N YQ GM V F N YQ GM V F N YQ GI T V I I DLQD I I I F DLQD I ILQWAQA I T V I I W4R71 QRQI FRQD D I I A A Y V L MRA A Y V L M Y L P Y Y D V Y L P Y Y D V Y L P Y Y A N K A P E LQA N K A P E YKQA D T FRGP YKQA D T FRGP YKQA D T F T VQCKQLST ENGT VQ K LT02P K K V K T P K K V K T P K K V K L A N V L AC Q SN:.o Nt596 7 8e 090909kc,o8,8,8 0,8D1y_ venrm2V33_ _ mV33V2V2 55_T0P 2_T0 mP2_M75M75Btu97ott- 5 o 6rP tM_- 5 o 6rPBtM_ - 5 o 6rPBt P 70P 7P0 m 68A u M B u M B u M MP 211 Q A I PGP L GSH A R V HGI Y F P ISR N Y I L QQCQL F L K A T ESI H K IQA V T E L L P ISH A L Y ASD T Y I N L T P N Q I K LRQ QD TSL K LQ QD TSL K LQ QDS Q QNGR RT L K LRD T N S V P A H K E D N V PGA H K E D N V PGA H K E D N V PGA H K E P L TTPGK E V E T V L TTK E V E T V L TTK E V E T V L TTK E V E F A N K P L I YG PNGK P L I YG PNGK P L I YG PNGK P L I ASVSW P A I L IQ SW P A I L IQ SW P A IQ SI Y P DASA N AASASL I W PASA I L WLY P DWALN A Y P DWALN A Y P DWAL9L K L1T RSR ASVQW R F AI ALSR ASVQI ALSR ASVQI AL R ASVQVQ S Q Q S Q Q S QSQ0A A Y V E AHCR W R F A K Y V E AHCR W R F A R W R F A K Y V E AHCKHCI L E P FYGEGI DKGA E P FYGEGI DKGA E P FYGEGI DKGY V E AYGEGA0E P F I D -9L D D V A L K A H E V D V A L K A H E V D V A L K A H E V D V A L K A H6 AQA D L F E K T A R D L F E K T A R D L F P K T A R D LDSK V L D LDSK VDSL FDSP K T4R L I L L I LL DL IL KLV L DL IL KLV71LQ Q GP L TCP R AQ GP L TCP R AQ GP L TCP R AQ GP L TCP ENLQ QE L V E LMLQ QE L V E LMLQ QE L V E L L E L V E02VGTQ SL P P D AQTQ SL P P D AQTQ SL P P D AMQTQQQSL P P D:.o Nt9900102ek0 1 101c,o8,8,8,8D1ye_nrVZF_ZF_ZtFu _Ztv F u55V VV VVmVVm 97oR ttEP4R Q2E 4R E43R _E 43_68AP Q 2 P Q 2 P Q 211NGRSI P D K A KQHGPCH I I A L R K P L H L A E L LSL I E E K N K I E RGAST L WGRQK Y I AGH V H A T A F A Y RSL L D N T V P L TIQKQAGRSL P L TI KAGRL I K PFL A E DGA I K PFL A E Y K L D T L K LQ QD TSL E M KGL V ES GIGQV PRGA H K E D N V PRGA H K E D N A L A NISITQL E M K L V T I K A L A NTIQSI N A L T K E V E T V L T K E V E T V E W K T T T V K D E W K T T T91ISAQPSNTGK P L I Y NTGK P L I Y L P P L F Y F N L A L P W P A I L IGQPSW P A I L IGQP L F Y F V N D I L K P A L V N D I L K00K-GK Y P DASA N A Y P DASA N APQIPQA L R AWSVLQL R AWSVLQLDSL K P A I K N P T E K E LDSI L K P A P T9SQISAQSQISAQQQKCNGE K E E V R W R F A6A R Y V EHR W R F AHP T V A P T V A4L D E P FYGEGACI DKGK Y V E A E P FYGEGACLI DKGKQNYQKLQLLA L L Y EKSL A EQNYQKLQL E F T L L YKS71R A D V A L K A H E V D V A L K A H E V P LKQEKSKQEES0GL F P E H P LKL F L D L F P K T A R D LG2MF P K T A R D I P H Y I V L D I P H Y I AQL I LDSK L V L D L I LDSK L V L D M Y R R E N YLSA T M Y R R E N Y:.o Nt3e0405060k1c,o8 1,8 1,8 1,8D1ye_Zt 2 v u _Zt u P P_ 55nrVF S F S_VmVm1 4_71 47t97oR ttEP43W Q_2_ RV EP43W _ _ V F03 V F03 u68A AQ 2AS 2 S 2m11 A T I M E P AQLQQAHGT K H E DGH M L I P I ISI D WQP K H VLQI KLCY E E A P IISFQK W VQKSA V IGH R MGL P ASK K HSK N K N N L V DFGP HNSK K I W A Y SGP L D E T K Y N F VASM E A A F V ESY PFSH D K T T V K I A L I V V P DGA I K PFL A E DGA L D ELSK PSH L D E D PLSK PSH L D ESK ESL E M KGL V ESL V D PSI H H D V VSI H H D V V D PLSI H T I K A L A N ITQT I K T I T V F M K T V T I T V F M K T V T I T V F M V K D E W K TIST T V K DLV I VKT T VY LV I VKT T VYV I V T9N L A L P P L F Y F N L AQ G S Q G SLQKG1IPYT K I L LIP HYT K I P T P A L V N D I L K P A L LQHGN L AQ GN L A L LIQHYGN L0I K NPQ0DI L K P A I K N P A A A L I VGQP A A A L I VGQP A A A L-9QQKCNGLSP T E K E KISW N N K V P T KISW N P K V P T KISW N P K PQQKCNG6L A ELQNYQT V A K P L A E D A K P L A E D A K P L A E KLQL L A EFGGSFGGSFG4E F T L LKY EKSM K L V E F T L A N ITQE L M K L V T I K L A N ITQE L M K L V T I KTQ71 QEKSI IL A NII P E H P L0GL F P E H W K TST T V K D W K TST T V K D W K TST T2LSV L D I P H Y I V L P P L F Y F N L A P P L F Y F N L A P P L F Y F A T M Y R R E N YLSA T V N D I L K P A L V N D I L K P A L V N D I L K:.o Nt7 8 9 0e0k1c, 01, 01, 11,o8 8 8 8D1yeP 2 v _ P- P-nr_1 4_ mP- m 557At 1 4_71 42__1 4 2_97ot VtF0S32u V F0o S32rV7F03 ortV7F03 or At68A mP S 2 PuS 2 Pu11 K N KCNGA E F T E H V L A T A T I M E P AQLQQAHGT K H E DGH M L I P I ISI D WQP K H VLQI KLCY E E A P IISFQK W VQKSA V IGH R MGL P ASK K HSK N K N N L V DFGP HNSK K I W A Y SGP L D E T K Y N F VASM E A A F V ESY PFSH I K AFL A E DGA I K A L A E DGA I K A L A E DGA L D E H D V E M KGL I T D D L E M KFGL I T D D L E M KFGL I T D D L T N PLSK T V A L AN LI N T I K A L A I N T I K A L A I N T I K T I T V T VY9SE W KS SN T V K D E W KNSLSN T V K D E W KNSLSN T V K D L F Y F T L A L P P L F Y F T L A L P P L F Y F T L ALVIV K I L P PQ S1A LG0QPQI N D V L K P A LPQI N D V L K P A LTPQYGQI N D V L K P A L L H I V I L K P V V K N I L K P V V K N I L K P V V K V P T LDSP T E K E LDSPDSN PISA A0QQK NT E K EQ K NL P T E K EQ K NK W N-9DGSALQPSYQTLQV AC GLQPSYQTLQV AQ C GLQPSYTLV AQ C GK AFL6E L K L K L A E K L K L A EQKQL K L A E M KGL4T I K L V Y E T E F T L V Y E T E F T L V71V K D P LKQLESY P E H P LKQLESY P E H P LKQY E LEST E F T L A Y P E HNSLSD I PKH F V L V F D I PKH F V L V F D I PKW K N L A0G G GH F V L V F P P L F2P A L M Y R R E N Y T A T M Y R R E N Y T A T M Y R R E N Y T A T I N D V:.o Nt112 3 4e1 1 1k1c,o8 1,8 1,8 1,8D1_ _ _ _ _ - vyenrL310L310V474tLu31_ 0 4AtLu31055497ottFV7V7V7o S2 F 2 m F 2 m F 2 r 68AP S P 2 S P 2 S P P11 V V E AQQK L T E Y P V L Y T N K L D Y Y M H I PFQR I K R A P F D E E N L Y FYSR M I W YGT I ASW R PSI P T L N A V K K R M E I P L L R P V T E PKQN I R K E T E LMSN V E I ASPSN Y L V MSL I I E IQNGQMLGQL QSD L N A D K T I T WLG T K PSN L D ELT K PSN L D E T K PSN VQQP L A ESS GA VQQP I H H D V T N PSI H H D V T N PLSI H H D V E T KFGL I L T P L E T KFGY I K T V T I T V Y I K T V T I T V Y I K T V A L A NIL Y V K A L A KQMSN G T I VYSLQVISVKGT I VYSLQVISVKGT I VYP W K A N V K D P W K A9T K I T K I T K ISL P P L F Y F D L A L P P L1N L A L LTQHYGP L A L LTQHYGP L A L V N D V L K P A L V N D0A L I VGQP A A A L I VGQP A A A L I VGQPQD I L K L I K APQD0T K IIPSISTQI L G T K W N T K IPGT K W N T K IPGT L T P T E E HKCNGL T P T -9A E D A K A L A E D A K A L A E D A T L V A K T6I T D D L M KFGL I T D D L M KFGL I T D D LLQPSYQKLQPSYQ4I N T I K L AN LI N T I K L AN LI N T I K L LKYQQL EKSL A E K E F T L LKY71N T V K D W KS SN T V K D W KS SN T V K D P LQY F T L A P P L F Y F T L A P P L F Y F T L A N I PKI A F P E H P LQK0QH F V L V N I PQ2L K P A L I N D V L K P A L I N D V L K P A L M Y R R E N Y T AISM Y R R:.o Nt5 6 7 8e1k1c, 11, 11, 11o8 8 8,8D1- vye_1_- 1 _0_0 55nrL3 0m Lo 42 3 0m2PC40PC40tu97t V7_tFo S2PrP tV4F7_ 2 or AtV F78 V F78m268A uS P PuS Q S Q_11 P N I L F Y N M R I A P F K N K LGLSGE L N I Y SQY R LQV KYGL DASK PPSP T T N V V N K L I L V K L T KRQF D T P D L T R LGE E RGL ENSK A T T I N N L FGA E L I F V E K V N T RLQQGK T I N ESD E K T K W LSE I V Y VKYGGP AQSS L A ESS GA VQQP L A ESS GA L D D E K PSN L D D E K PSN L D L E T KFGL I L T P L I D PLSI H H D V I D PLD L I L T PSI H H D V I D P IQL Y V K A L A N L Y V K T I T V Y M K T V T I T V Y M K T V T I T NMSV K D P W K AIQNMSV K DLV I VKT L I V I V T L I V I9F Y F D L A L P P L F Y F D L AQPGT P VYSLQPKGT P VYSLQP1V L K P A L V N D V L K P A L LIQHYGK L P L LIQHYGP L P LIQ0K LPQGQGQL H0ETQI K A D I L K E HTL I K A P A L I V P A L I V P A KCNGL T P T EQE HIKSAQSSISAQSSISCN QW KS PTQW KS PTQW -9L V A K T L V A KG QP L A ES GAQP LS G Q6L L A ELQPSYQK L LFGFGA E A PFG4QQEKSE F T L L YQQEKSA E T K L I L T P L T K L I L T P L T K E F T L A N L Y V K L A71I A F P EKQIKQMSNIQLMSY V K L A N H P L I A F P E H W K A N V K D W K A N V K D W K02IQA H F V L V S N I P H F V L V Y R R E N Y T AIP P L F Y F D L A P P L F Y F D L A P P L E N Y T A MSV N D V L K P A L V N D V L K P A L V N D:.o Nt91021 2ek1 12121c,o8,8,8,8D1y_ - - - vePnr C04A_0_ _50tuPC40PC04m2PC04 59t V7V707_07 7otFS8Qm2F 8orV F8ortV F8 68A _S Q P S Q PuS Q11TQL E V A L EKSA F F V N Y P N I L F Y N M R I A P F K N K LGKSGE L N I Y SQY R LQV KYGL DASK PPSP T T N V V N K L I L V K L T KRQF D T P D L T R LGE E RGL ENSK A T T I N N L FGA E L I F V E K V N T RLQQGK T I N ESD E K T LQSY I P PQA AQA I P PQA AQA I P PQA AQA PLGWSF T I K W A K W F T I L A K W F T I L A K W F T I L A D D LSE K PFSN DLGLGLGPNGI H H D V AQPSD P A I D D D P A I D D D P A I D A K N T N T W A A AQSN T W A A AQSPQE Y L HPR L H E Y L HPN T W A A Y R L L A V Y M K T V HQR L HQ QR L H E Y L VRKQL VTQTYS91YGT L IYSP L V T PDSL A P L V T PDSL A P L V T PDSL AAGP T G T P V V P K T E V P K T E P L P LAGPGA L PNTVGPAGA L P VNGV PAGP K T E A L P VNGP E L N A M I K FAC0 A-QSSL I VQI M I H LQE I M I H LTQE I M I HTQK T D P A D LLQLQLLQE L L A D I A09SSPL A D P A D L L A D P A D L L A I V A R L I L A EGA I V A T L LAL I V A T L L L I V A T L L L6SA M A K L WASA M A K L WAD M A R F P L I L T P L A M A K L WSV LPGPGPGT V I T W4I71 QNMSY V K V V V T L V V V T L V V V T L A A Y V L T V K DRSK FLCL Y PRSK FLCL Y PRSK FLCL Y P T N K A E F Y F D L A R NLKV PQLPL R NKV PQLPL R NKV PQL A VPQT02V L K P A L P VQR L DSN P VQR L DSN P VQR L DPSN L AQCKQT N:.o Nt32425 6e 1 1212kc,o8,8,8 1,8D1yenrmH4 4 4P v 2 V6H5_ R33V6R33 tH6u V R33Btu_2 7 5V1 9t5 7otorPAtMS0PM0Pm2M0Pm2R1 68A u _S_ _S_ _S 511QSL PGP L GSH A R I HGL Y F P ISR N Y I L QQCQL F F K A T DSI H K I HSV T E L L P ISH A L Y ASD T Y V N L T A T R L H T P T APK N TGP T LRI P A Y P I IHL YRI P A Y P F T T I IHL YRI P A Y P F A YQR L L A F A YGF T TG G G GF T T I IHGASV VRQL V T V E A L T I P F E A L T I P F E A L T I P F A9GPTQTYSASV D KGQL I L L D D KGQL I L L D D KGQL I L L D L K P E L N1M I K FAL K I K P V T N I LQA P HDH I K P V N I LQA P HDH I K P V N I LQA P H V T AC G S G S GHDSV0A A L L A D I A A A L P V L KAGLAGAG0I L I V A R L I I L T F T Y L L DVGL P V L K L T F T Y L L DVGL P V L K L T F T Y L L DV-G9L D D M A R F P L D N I W V K I P Y N I W V K I P Y N I W V K I P Y64AQT V T V I T W I A A Y V L TAQT K P T V N IFTSR A K P T V NFR A K P T V NFR A1 QTYQEQF P YTQTYQEQFSP YTQTYQEQFSP Y7L H T N K A E A VP0QE L H T ENCA D ERSRSNCA D ERSRSNCA DRSRS2VNGL AQCKQT N VNGCSPHGQCVAQI DRQCSPHGQCVAQI DRQCSPHGQCE VAQI DRQ:.o Nt7e2829203k1c,o8 1,8 1,8 1,8D1eP _ 2 vynr 2__ 7V5_5 _ 5 S18VS18 tuVS18At55u9oV1t5BttR 2 27S15u D9D9mD29m 6m2 2 8A W O W O _ W O _11 D S L N F P RQH L I K H PLCR F IGF A E D R K A L E D KSL MVSP Y YQW T R VHSA P V P PSL ASSGSL L R N L Y E P V N V P V V P T P P VQH I N ADQLGV R MGK N A I R E E R A L W VDQT V P P F R LQY D L N WSTGP P D K I K SSV P P L TL KAGY A P L TL KAGY A P L TLAGY A E PGAQQK T D V V LNQ QGD TRSL V LNQ QD TRL V LNQKQD TRLKT M K D T E T A P P A H K E V P PGA H K ESV P PGA H K ESVSN E K K E L E D R K T K K E L E D R K T K K E L E D R T W PPL A Y K K T KGA TAV L V M E N E K E LTE N E K E L A T E N E K E L A T E DSW A I N A H W PAT L L YSH W PAT L L YTSH W P T L L YTSHPSR A N I E L A9L1PSDSW A I I L LPSDSW A I I L LAPDSW A I I L L A RQW R F A ELK E R R V I V E N A R R V I V E N A RSR V I V E NCYC00EQGL AKGR Y -CW R F A E P ASCI DAQY EQCW R F A E P ASCI DAQY EQCW R F A E P ASCI DAQY P F E V A LTGV D A M9E P FYGIV R K E P FYGV R K E P FYGH EAQ6SKGISKGISV RKGK D L FNT R P A E V A L A H A E V A L A H A E V A L A H A E I LGK4LCA M D L L F E K V E A L L F P K V E A L L F P N LKSK P A EKSKSK V E A IGQP L T E K A71LV0GL TLCN T L D LVGL K L TLCP A E N T L D LVGL K L TLCP A E N TQSTQL P K L M2PLPE L T A R A V LPL D L D P P E E L T A R A T L T L Y W PAR V LQE L T A R A VQ Q QN:.o Nt1e3233343k1c,o8 1,8 1,8 1,8D1yeV9v 5V95 tV95At6 V5655nr S3M3 S30M3uS30 mM3u 0mRZ197ott P P 3 P 3M A168A W _ W _ _ W _ _ X _11 L P RQH L I R H PLCK F IGF A L D R K A L E D QSL V M T P Y YQWQKHISSP V P P L TSGST L A L K N L Y P P I N I P R P K R E I P R P V FQGENK R E LSR N L V FNQSQGE R N P T D D L AGK P T D D LQGK Q P A M V W NAP A M V W N H L V F REKQH L V F R I R I D T L DEK R I D T L DQ QI V D R E E P L E V D R E E P L E A E H K FPA E LQFSRGH K FPQFSRGGV L R P V L R P R A A K KLGK E E T VSR A A K K MGESM K E E T VSGESGGN N T L L D T N N T L L V FGGD T L D A T L V F L D A T L T E L W P E Y7E R T RWT E L W P E YWLV WGE R T RLV WG 3A L WPGDSQGA T R A LGA T R2L H Y R WPSQGL Hfo AQY R Q N E A P D RDQN E A V K K P A A YAQP D R V K K P A A Y2P P F L A L D I P P F L A L D I6F RGL T I PEF RGL T I P DSYQL VG QEGD PYGI LDSYYGL V I L H D P L L H L N L L PPQV L N L L PPQV WTR YAQD H W R YAQD H TGT K KQGT A TTGT K KQGT A SGP WVQI M L T LSP WVI M L T L K TET D A LGK TQT D A L L K KQL P F L K KEQL P F P VLQQY R ATSP VLQYTSGCK Q KR A V P AGTY T V PGCAGY T D LSIQK AGRSTGSE D LIQK ARSE P LIGQQD H L P LI QD H L E P A K T D V E PGAQK T D V KST M K D T E T A T M K D T E T N EKPSA K AGL A Y K N E K W PPGL A Y K T W P V L V M T V L V M EPSDSW A I N A EAN I E L A HPDSW A I N A H R ASR A N I E L A9L1RQCW R F A E L ALCK E L R RQCW R F A E L ALCK E R0Y P FYGV DKGAYGKGE V A LT-GY P FTGV D A0M H E E V A L M9D L FAQH EAQ6E I LNGP R P A D L F K A M D E I LNGP R P A K A4LCLCM D I P L T E K A I NGQL P K LGQP L T E K A71L DQSTQM N P P EQAR L DSTQL P K L M P P EAR02T L T L Y W PQN T L T L Y W PQN:.o Nt5 6e313kc,o8 1,8D1y6eV56 t6 v u V56t u55nrRZ1mRZ m 97ottM A3_ M1 368A X _1X A __1A11 Attorney Docket No.: 2017469-0019 In some embodiments, reverse transcriptase domains are modified, for example by site-specific mutation. In some embodiments, reverse transcriptase domains are engineered to have improved properties, e.g. SuperScript IV (SSIV) reverse transcriptase derived from the MMLV RT. In some embodiments, the reverse transcriptase domain may be engineered to have lower error rates, e.g., as described in WO2001068895, incorporated herein by reference. In some embodiments, the reverse transcriptase domain may be engineered to be more thermostable. In some embodiments, the reverse transcriptase domain may be engineered to be more processive. In some embodiments, the reverse transcriptase domain may be engineered to have tolerance to inhibitors. In some embodiments, the reverse transcriptase domain may be engineered to be faster. In some embodiments, the reverse transcriptase domain may be engineered to better tolerate modified nucleotides in the RNA template. In some embodiments, the reverse transcriptase domain may be engineered to insert modified DNA nucleotides. In some embodiments, the reverse transcriptase 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 the 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 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, the 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 embodiments, the MoMLV RT sequence comprises a combination of mutations, such as D200N, L603W, and T330P, optionally further including T306K and / or W313F. In some embodiments, a gene modifying polypeptide comprises the RT domain from a retroviral reverse transcriptase, e.g., a wild-type M-MLV RT, e.g., comprising the following sequence: M-MLV (WT): TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEA RLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG 63 of 237 11867955v1 Attorney Docket No.: 2017469-0019 LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO: 5002) In some embodiments, a gene modifying polypeptide comprises the RT domain from a retroviral reverse transcriptase, e.g., an M-MLV RT, e.g., comprising the following sequence: TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEA RLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL (SEQ ID NO: 5003). In some embodiments, a gene modifying polypeptide comprises the RT domain from a retroviral reverse transcriptase comprising the sequence of amino acids 659-1329 of NP_057933. In embodiments, the 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: TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEA RLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TEARKETVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAA (SEQ ID NO: 5004) Core RT (bold), annotated per above RNAseH (underlined), annotated per above 64 of 237 11867955v1 Attorney Docket No.: 2017469-0019 In embodiments, the 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, the 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, e.g., a combination of mutations, such as D200N, L603W, and T330P, optionally further including T306K and W313F. In some embodiments, an M-MLV RT used herein comprises the mutations D200N, L603W, T330P, T306K and W313F. In embodiments, the mutant M- MLV RT comprises the following amino acid sequence: M-MLV (PE2): TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQEA RLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLP PSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADF RIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWL TEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQA LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEG LQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQR AELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRL SIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO: 5005) RT Families and Mutants In certain 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 certain 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. 65 of 237 11867955v1 Attorney Docket No.: 2017469-0019 In certain embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from an MLVMS RT domain. In 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 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 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. In certain embodiments, a gene modifying polypeptide comprises the amino acid sequence of an RT domain sequence from an AVIRE RT domain. In 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 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 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 certain embodiments, the 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 AVIRE Gen1 MLVMS Gen2 AVIRE (MLVMS) (PLV4921) (PLV10990) 66 of 237 11867955v1 Attorney Docket No.: 2017469-0019 D524G D526G E562Q E564Q WT residue & position In certain embodiments, a gene modifying polypeptide comprises a gamma retrovirus derived RT domain. In certain embodiments, the 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, 67 of 237 11867955v1 Attorney Docket No.: 2017469-0019 MLVMS, PERV, SFV1, SFV3L, WMSV, and XMRV6. In some embodiments, the gamma retrovirus- derived RT domain of a gene modifying polypeptide is not derived from PERV. In some embodiments, said RT 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 reverse transcriptase. In some embodiments, the gene modifying polypeptide further comprises a linker having at least 99% or 100% identity to SEQ ID NO: 5217. In embodiments, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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 embodiments, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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 embodiments, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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 domain. In some embodiments, the RT domain comprises the amino acid sequence of an FFV RT further comprising the mutation D21N. In some embodiments, the 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 68 of 237 11867955v1 Attorney Docket No.: 2017469-0019 corresponding position in a homologous RT domain. In some embodiments, the 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 embodiments, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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 embodiments, the RT domain comprises the amino acid sequence of an RT domain of a FOAMV RT (e.g., an FOAMV_P14350 sequence, e.g., SEQ ID NO: 8021), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain comprises the amino acid sequence of an 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, the RT domain comprises the amino acid sequence of an 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, the RT domain comprises the amino acid sequence of an FOAMV RT further comprising the mutation D24N, or a corresponding position in a homologous RT domain. In some embodiments, the RT domain comprises the amino acid sequence of an 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, the RT domain comprises the amino acid sequence of an FOAMV 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 embodiments, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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. 69 of 237 11867955v1 Attorney Docket No.: 2017469-0019 In embodiments, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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, the RT domain comprises the amino acid sequence of a GALV RT further comprising the mutation D32N. In some embodiments, the 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, the 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 embodiments, the 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 amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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 embodiments, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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 embodiments, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain 70 of 237 11867955v1 Attorney Docket No.: 2017469-0019 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, the 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 embodiments, the RT domain comprises the amino acid sequence of an RT domain of a MLVFF RT, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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, the 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 embodiments, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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. 71 of 237 11867955v1 Attorney Docket No.: 2017469-0019 In embodiments, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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, the 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 embodiments, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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, the 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, the 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, the 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, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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. 72 of 237 11867955v1 Attorney Docket No.: 2017469-0019 In embodiments, the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the 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, the 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 certain embodiments, the 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%, 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 certain 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 embodiment, 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. Table 4. Exemplary RT domain mutations (relative to corresponding wild-type sequences as listed in the corresponding row of Table 1) RT Domain Name Mutation(s) 73 of 237 11867955v1 Attorney Docket No.: 2017469-0019 FFV_O93209_2mutA D21N T293N T419P L393K FFV_O93209-Pro F 74 of 237 11867955v1 Attorney Docket No.: 2017469-0019 MLVAV_P03356 MLVAV_P03356_3mut D200N T330P L603W 75 of 237 11867955v1 Attorney Docket No.: 2017469-0019 MMTVB_P03365-Pro MMTVB_P03365-Pro_2mut G309P 76 of 237 11867955v1 Attorney Docket No.: 2017469-0019 WMSV_P03359 WMSV_P03359_3mut D198N E328P L600W In some embodiments, a gene modifying polypeptide described herein comprises a Cas domain. In some embodiments, the Cas domain can direct the gene modifying polypeptide to a target site specified by a gRNA spacer, thereby modifying a target nucleic acid sequence in “cis”. In some embodiments, the Cas domain comprises two or more smaller domains, e.g., a DNA binding domain and an endonuclease domain. In some embodiments, the Cas domain possesses the function of DNA target site cleavage via an endonuclease domain. In some embodiments, the 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 by a gRNA spacer. In some embodiments, the Cas domain has RNA binding activity, e.g., the Cas domain may bind the gRNA scaffold region of the template RNA. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements, e.g., as listed for exemplary Cas enzymes in Table 5; 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 may comprise the amino acid sequence of SEQ ID NO: 4000 below, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the amino acid sequence of SEQ ID NO: 4000 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned at the N-terminal end of the gene modifying polypeptide. In embodiments, the amino acid sequence of SEQ ID NO: 4000 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 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 N-terminal end of the gene modifying polypeptide. In some embodiments, the 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. Exemplary N-terminal sequence comprising an NLS (bold) and an SpCas9 domain with N863A: 77 of 237 11867955v1 Attorney Docket No.: 2017469-0019 MPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYH EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDI VLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFY SNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 4000) A SpCas9 domain with N863A is shown below without a heterologous NLS: DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRR YTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKK LVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAI LSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHA ILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSF IERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTV KQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREM IEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQ KGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHI 78 of 237 11867955v1 Attorney Docket No.: 2017469-0019 VPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEL DKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVK KDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQ HKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 11,096) In certain embodiments, the Cas9 domain comprises the amino acid sequence of SEQ ID NO: 11,096 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. 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%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the amino acid sequence of SEQ ID NO: 4001 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned at the C-terminal end of the gene modifying polypeptide. In embodiments, the amino acid sequence of SEQ ID NO: 4001 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 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 the gene modifying polypeptide. Exemplary C-terminal sequence comprising an NLS: AGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4001) Exemplary benchmarking sequence: MPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETA EATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYH EKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKD TYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGS IPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDI VLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFA 79 of 237 11867955v1 Attorney Docket No.: 2017469-0019 NRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELD INRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKD FQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFY SNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKES ILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPI DFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSSGGSSGSETPGTSES ATPESSGGSSGGSSGGTLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKA TSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVED IHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKN SPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQK QVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLF NWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAA GWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGP VVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTET EVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNK DEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRT ADGSEFEAGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 4002) In some embodiments, a gene modifying polypeptide may comprise a Cas domain as listed in Table 5 or 6, or a functional fragment thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. Table 5. CRISPR / Cas Proteins, Species, and Mutations SEQ Mutations to Mutations to H H 80 of 237 11867955v1es)aCciesaciesaciIsnoiteat cueMdena,s iieetcerpS,snietor PI E E A L A T I R E YQN RSL DQV E A T N K E E D9DLQSGA GLSE KNGYQL P REE K D R D E V R H R IERVL N R D G L D NDQK LSY V L R D D L D W K V MGKSE D H LsaD RTY FDI I K L T F K H V E Y Y T K K N P K F E K VCpISF HSL F DTQLGGH F A RLV DLI V KDNIY R T L LLP N E I H K H TSK KQLKISKQFV EIKSY KSY LfKFV V NQE Y L M H R D KQVGL M RQD ISSKYGo KASGL K A D F W K N K M M E R K T F Y L ESK KASTSD KLQK L Y EEGA P T I V FTQE N K E D V PsKSKGP L L T D I D K I L R L F L K I K N L I I V V K K R Le Ec GEK V Y M L V R F N KQH R V F K D R I R D V L R Y K F K K E K Rneuq seuSccdilo scat)coen A n s oe(rtstpeegoniaPorHtSypm1A v.6 t 9nsa55el ai97braCy6aTVpS 811 A01DA048HA368N900,9N KK AK MISEHNSR FD ITENQGLLVYCK IF RK NKSK RP RV TK YY RE RD RT AI TV RA KWGL RVST AN ETGA TI EDG LSGL D NG QDK LSY V L R D D L D W K VG SGSTDQM K E D H L Y T K K N P K F E K VLGL D D R Y F I I K L T F K H V E Y D RISF HS GH F A R V D I V KTSLTQLGLSLQDSNIQY R T R LLSP N EISF H F DIQH K H T K KLQKIGKFQV EISKSGY K L F DY LFSV V N E Y L M H R D K V L M R D IKYA TY LFVKALLK A D F W K N K M M E R K T F Y L ESK KS SKSLKGD KQK L Y EEGA P T I V FTQE N K E D V P P LAGR L F L K I K N L I I V V K KK KL R K D KD I D K I LS GL V R FER L K V Y D I D K MGN KEQH R V F K D R I R D V L R Y K F K K E K V M L V Rsuscuccocscoeo setpnce otneer g p gtoertoSyp Syp 19 vsa9s 55CaYyCR97pGySN -pSpS6-811 P DLQL DA DI DL YN TE DLRKSRSLQKSL KL AR DASE AL LI DA FKANSD KV FGSN PA TNILGPNLSE LE AF ILQLNN GQK LSY V L R D D L D W K V MGKSE D H LGSL D NG QY FDDQTGI I K L T F K H V E Y Y T K K N P K F E K VLGD RDQQLGH F A RLSV DLQI V KDSNIQY R T R LTLSY FDGSP N EISF HTQLGH IQH K H T K KLQKIGKFV EIKSY K L F DIHV N E Y L M H R D K V L M RQD ISYGY L V V NQLQEW K N K M MTE R K T F Y L EKSK KA TKFE K A D FS S SE N K E D V PALLK A D K L Y EGA P T I V FQKG QESKP L L R K D K K L YI G L REL F L K I K N L I I V V K K RGL K VYD I D K I L V R FEL R F N KQH R V F K D R I R D V L R Y K F K K E KQMGN K succocsoetpneer gt o Syp 19 v sa55C- yraH97pSvR 3 R6- N811IQSHNGP RI AIGLNPGD VF YT YRQP IK RR FL TL LD IE KR EN IL KK EV RL NL DE KE LTGF PD YM FK DELGQI RP R K LSY V L R D D L D W K V MGKSE D H LGLSGL D NG QDQK LSYI I K L T F K H V E Y Y T K K N P K F E K V D R A R V D I V K N Y R T L L PTY F I I K LFSDGH TLK KL D I LN E F H H F A RKS QLS Q SISTQLGLSQKIGKFQV EIKSY K L F DIH K H TY L M H R D K V L M R D ISYGY L V V NQE Y L M EGK M MTE R K T F Y L EKSK KA TKFHF W K NS S SE D V PALLK A D FEW KE A P T I V FQE N KKG Q GESKP L L T K D K K L Y E A PQL F L K I K N L I I V V K K RGL K V Y D I D K I FEL R N KEL F L H R V F K D R I R D V L R Y K F K K E K R M L V RG QH R V succocsoetpneer gt o Syp 19 v saC-55yraHT97pSv3R6- N811 L D L FD LH HY ATGY TLSK LA RN EF ER ID ME EV RGSD EI FE LVST LD TFCL VE II DK EK LF IY DD EE NK ELQE NK D V L R D D L D W K V MGKSE D H LGLSL D NG QK LSY V L R EGDQDT F K H V E Y Y T K K N P K F S K V D RTSY FDGI I K L T F K HV D I V K N Y R T L L N E F KL D I L IH H F A R V D IKQR D KLS Q S STQLGLSLQQK VIGK L M RFQV E D IISK YSGY K L F D Y L V V NIQH K H T K K E Y L M H R DLQTQE R K T F Y L EKSK KA TKFKN K M MS SASLLK A D FEW K N K M MT I V F E N K E D V P I V V K K RKG Q GSKP L L T K D K K L Y E A P T I V FK I K N L IGL K V Y D I D K IEGL REL F L K I K N F K D R I R D V L R Y K F K K E K L M L V R F N KQH R V F K D R succocsoetpneer gt o Syp 19 v saC-55yraHC97pSv3R6- N811 K L MREQE DRSV YN MK DQGRGKQNQT LTQY YN LE YR LA KM EEINQVILQN TE NP EK VH PGGH EM KV LKVIQLESG D L D W K V MGKSE D H LGLSGL D NG QDK LSY V L R D D L TQD WV E Y Y T K K N P K F E K V D R DSY FDI I K L T F K H V E Y YV K S NIQY R T L LLSP N EISF HTGQLGH F A RLSV DLI V KDNKIKFV EIKSY K L F DIH K H T K KQLKISKVGL M RQD ISYGT Y L V V NQE Y L M H RQ GTQK T F Y L EKD K V L M E RSK KAST KFSL K A D E N K E D VAGLQFEGW K N K M MTQE R K P I V V K K RK KP L L N K D K K L Y E A P T I V F E N KL IS GL K V D I D K I L R L F L K I K N L I I Y K F K K E KYQM L V R FEGN KEV I R D V L RQH R V F K D R I R D V succocsoetpneer gt o Syp 19 v sa55Cy1pF97SH6-811 FDDGSYV VL FKSESK EL LT KI PV YK KV KE IR LI ALKTGD VN VE AD NY LK YT AN DM HRSA HD HL YIQN NA IV EH R K V MGKSE D H LGLSGL D NG QDK LSY V L R D D L D W KGTQV MT K K N P K F E K V D RISY FDI I K L T F K H V E Y Y T K K NQY R T L LLSP N EISF HTGQLGH F A RLSV DLQI V KDNIY R T F V EIKSY K L F DIH K H T K KLKISKQFV ERQD ISYGY L V V NQE Y L M H R D KQVGLQF Y L EKK KAM R D ITS STSKFASLLK A D F W K N K M M E R K T F Y LE D V P K RK KP L L T KGD KQK L Y EEGA P T I V FTQE N K E D V PV KS GL K V Y D I D K IEGL REQL F L K I K N L I I V V K K R L R Y K F K K E K R M L V R F N K H R V F K D R I R D V L R Y K succocsoetpneer gt o Syp 19 v sa 155CyR9pSQ7Q6-811 E I V IK LA DV KV KL VVSE KYAYGV KT APSE LD FF DGGI PY NK KK EP FDWSSD RK EK MR IA TILIGK LDSL EN KRKV S E D H LGLSGL D NG QDK LSY V L R D D L D W K V MGKSTQE DP K F E K V D R LSY FDI I K L T F K H V E Y Y T K K N P K F EL L S P N EISF HTGQLGH F A RLSV DLQI V KDSNIY R T L LLPISKSY K L F DIH K H T K KLKIKQFV EIKSSKSYGASTSY LFASV V NQE Y L M H R D KQVGL M RQD ISKGL M E R K T F Y L ESYE K K K L K A D F W K N K M K KKSKGP L L R KGD KQK L Y EEGA P T I V FTQE N K E D V PKSKGP L L K VYQD I D K I L R L F L K I K N L I I V V K K R L L V R FEGN KEK F K K E K MQH R V F K D R I R D V L R Y K F K K E succocsoetpneer gt o Syp 19 v sa55CyG9pSp7S6-811 K RDITTDFYKFAA DPGAGGLLNQST LL DT IF RL TH EI YINLGE TAQIESQR HI IP L K TGTSD F I D L LQT FLQ G S QQE YLV HLA I K E N K TD A I E E ALL A T I R E Y N RSL DQV E A T N K E E D AR D DGE KQN YL P R E K D R D ESGV R H R I R L N R DH LLSGL D NG QDK LESY V L R D D L D W K V MEGKVSE TDQD H LK V D R Y F I I K L T F K H V E Y Y T K K N P K F E K VN E F HS GH F A R V D I V K N Y R T L L P N EY KISL F DTQLGIAQH K H TLSK KLQLQKDISGKIQFQV ELISSKSY KSTSY L V V N E Y L M H R D K VGKFASL M R D I G LLK A D FEW K N K M MKV FTE R K T F Y L ESY K KASTSL R K D KQK L Y EGA P T IQE N K E D V PK KP L L RVYD I D K IEL R L F L K I K N L I I V V K K RS GL K V KQM L V R FGN KEQH R V F K D R I R D V L R Y K F K K E KYQsuccocsoetpneer gt o Syp 19 v sa55CyRQ97pSV6-811 A01DA048HA368N130,9N KK AK MISEHNSR FD ITENQGLLVYCK IF RK NKSK RP RV TK YY RE RD RT AI TV RA KWGL RVST AN ETGA TI EDG LSGL D NG QK LSY V L R D D L D W K V MGKSE D H LGSL D D R Y FDQI I K L T F K H V E Y YLGISF HTSDGT K K N P K F E K V D R H F A R V D I V K NTSL FTQLGLSLQDSIQY R T L LLSP N EISF H D LIH K H T K KLKIKFV EIK Y K L F DYFQ Q G Q SSGSV V N E Y L M H R D K V L M R D IKSYA TY LFVKALLK A D FEW K N K M M E R K T F Y L E K KS SKSLKGD KQK L Y EGA P T I V FTQE N K E D V P P L LAGL F L K I K N L I I V V K K RKSKR K D KD I D K I L RGR FEGN KEL K V Y D I D K M L VQH R V F K D R I R D V L R Y K F K K E K E M L V Rsuscuccoccsoeocsetpneor gtpneetoegSyrpto Syp 19 9 vsaR sa55Cy ECsa97pRySV -pCSx6-811 P DLQL DA DI DL YN TE DLRKSRSLQKSL KL TR DASE AL LI DA FKANSD KV FGSN PA TNILGPNLSE LE AF ILQLNN GQK LSY V L R D D L D W K V MGKSE D H LGSL D NG QY FDDQTGI I K L T F K H V E Y Y T K K N P K F E K VLGD RDQQLGH F A RLSV DLQI V KDSNIQY R T L LTLSY FDGSP N EISF HTQLH IQH K H T K KLQKIKFV EIKSY K L F DGIHV N E Y L M H R D K VGL M RQD ISYGY L V VQLQF W K N K M I E R K T F Y LK A T FN E K A DS S S SK L Y EEGA P T I VTQE K K KAGLLQK A D F E N K E D V PK KP L L T K D K K L YIEL LEL F L K I K N L I I V V K K RS GL K V Y D I D K I L L FGN KQH R V F K D R I R D V L R Y K F K K E K R M L V R FEGN K succocsoetpneer gt o Syp 19 v sa-Cs55ya 97pCSx G6- N811

[0002] IQS HNGP R I A IGL NPGD V F Y T Y RQP I K R E F D M R F I A E EKGH ETQY YDSGGV K FQE I N PPSE P L LESPGGL T F P V RAL K DSH N L R R R L M V F AGL7LQE Y T E I A IMSF DIQ 3D ISQRGSE KLSL L R L L A L L I E EQS2fE KAI F D D A K V K KTGNSVGGN P I T L o R ESMGE L R M E KNGN IAGEVSTGIQT D V Y K Y N I L D ENN L N V F R K K29V K EKE T I L K T LGIQD N E A FQD F R K E K V D T NGSVLD D N Y T P F L L FVIQISL T V R D Y L I F Y L K A D F Y H E L N V KCV LQN T D K K Y A F WSSR H I Y L D V V E I K A E N D K K I DRT A T D D R K I E K K ERQASLGSKP E K N D A K V L T M HRK EHN E L E TQKG QTGK M A E T G F F L K L L I H P F IRA K R I L EAI P N E F I D H LSH I DQD Y W N Y DRGERGESQA T M V K D H W ISQE K M K P A L YEQV LIR F E M F P Y DGK R H K D T V E NWGF V L V NLIQQNEI K LRI I EEQI T K E T T KIQI L NSEGGDSLKP L L T FLE Y N RLV H LLA I K E NQK T I R E YQ SSQEGD V E A T N K E E D A P R E K D R D E V R H R I W K V MER KVL N R D K LSY V L R D D L DG SE D H L I I K L T F K H V E Y Y T K K N P K F E K V F A RLV DLI V K N Y R T R L P N E K H TSK KQDLKISKIQLFV EIKSSY K9Y L M H R D KQVGL M RQD IS1F W K N K M I E R K T F Y L EKGSY K KASTS00EEGA P T I V FTQE N K E D V P -9EQL F L K I K N L I I V V K K RKSKGP L L R L K V Y H R V F K D R I R D V L R Y K F K K E K V647102:.o Ntekco D1vye55nr97ott68A11 Attorney Docket No.: 2017469-0019 In some embodiments, a Cas protein comprises one or more mutations altering its PAM. In some embodiments, a Cas protein comprises E1369R, E1449H, and R1556A mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises E782K, N968K, and R1015H mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises D1135V, R1335Q, and T1337R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R and K607R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R, K548V, and N552R mutations or analogous substitutions to the amino acids corresponding to said positions. Exemplary advances in the engineering of Cas enzymes to recognize altered PAM sequences are reviewed in Collias et al Nature Communications 12:555 (2021), incorporated herein by reference in its entirety. In some embodiments, the Cas protein is catalytically active and cuts one or both strands of the target DNA site. In some embodiments, cutting the target DNA site is followed by formation of an alteration, e.g., an insertion or deletion, e.g., by the cellular repair machinery. In some embodiments, the 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, the 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 the target site. In some embodiments, the endonuclease domain has nickase activity and does not form double-stranded breaks. In some embodiments, the endonuclease domain forms single-stranded breaks at a higher frequency than double-stranded breaks, e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% of the breaks are single- stranded breaks, or less than 10%, 5%, 4%, 3%, 2%, or 1% of the breaks are double-stranded breaks. In some embodiments, the endonuclease forms substantially no double-stranded breaks. In some embodiments, the endonuclease does not form detectable levels of double-stranded breaks. In some embodiments, a catalytically inactive or partially inactive CRISPR / Cas domain comprises a Cas protein comprising one or more mutations, e.g., one or more of the mutations listed in Table 5. In some embodiments, a Cas protein described on a given row of Table 5 comprises one, two, three, or all of the mutations listed in the same row of Table 5. In some embodiments, a Cas protein, e.g., not described in Table 5, comprises one, two, three, or all of the mutations listed in a row of Table 5 or a corresponding mutation at a corresponding site in that Cas protein. 93 of 237 11867955v1 Attorney Docket No.: 2017469-0019 In some embodiments, a catalytically inactive, e.g., dCas9, or partially deactivated Cas9 protein comprises a D11 mutation (e.g., D11A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H969 mutation (e.g., H969A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N995 mutation (e.g., N995A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises mutations at one, two, or three of positions D11, H969, and N995 (e.g., D11A, H969A, and N995A mutations) or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D10 mutation (e.g., a D10A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H557 mutation (e.g., a H557A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D10 mutation (e.g., a D10A mutation) and a H557 mutation (e.g., a H557A mutation) or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D839 mutation (e.g., a D839A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H840 mutation (e.g., a H840A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N863 mutation (e.g., a N863A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D10 mutation (e.g., D10A), a D839 mutation (e.g., D839A), a H840 mutation (e.g., H840A), and a N863 mutation (e.g., N863A) or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a E993 mutation (e.g., a E993A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D917 mutation (e.g., a D917A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a E1006 mutation (e.g., a E1006A mutation) or an 94 of 237 11867955v1 Attorney Docket No.: 2017469-0019 analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D1255 mutation (e.g., a D1255A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D917 mutation (e.g., D917A), a E1006 mutation (e.g., E1006A), and a D1255 mutation (e.g., D1255A) or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D16 mutation (e.g., a D16A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D587 mutation (e.g., a D587A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a partially deactivated Cas domain has nickase activity. In some embodiments, a partially deactivated Cas9 domain is a Cas9 nickase domain. In some embodiments, the catalytically inactive Cas domain or dead Cas domain produces no detectable double strand break formation. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H588 mutation (e.g., a H588A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N611 mutation (e.g., a N611A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D16 mutation (e.g., D16A), a D587 mutation (e.g., D587A), a H588 mutation (e.g., H588A), and a N611 mutation (e.g., N611A) or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, an endonuclease domain or DNA binding domain comprises a Streptococcus pyogenes Cas9 (SpCas9) or a functional fragment or variant thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises a modified SpCas9. In embodiments, the modified SpCas9 comprises a modification that alters protospacer-adjacent motif (PAM) specificity. In embodiments, the PAM has specificity for the nucleic acid sequence 5′-NGT-3′. In embodiments, the modified SpCas9 comprises one or more amino acid substitutions, e.g., at one or more of positions L1111, D1135, G1218, E1219, A1322, of R1335, e.g., selected from L1111R, D1135V, G1218R, E1219F, A1322R, R1335V. In embodiments, the modified SpCas9 comprises the amino acid substitution T1337R and one or more additional amino acid substitutions, e.g., selected from L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto. In embodiments, the modified SpCas9 comprises: (i) one or more amino acid substitutions selected from D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, 95 of 237 11867955v1 Attorney Docket No.: 2017469-0019 and T1337; and (ii) one or more amino acid substitutions selected from L1111R, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto. In embodiments, the Cas9 comprises one or more substitutions, e.g., selected from H840A, D10A, P475A, W476A, N477A, D1125A, W1126A, and D1127A. In embodiments, the Cas9 comprises one or more mutations at positions selected from: D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987, e.g., one or more substitutions selected from D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A. In some embodiments, the gene modifying polypeptide comprises spCas9, spCas9-VRQR, spCas9- VRER, spCas9-MQKSER, spCas9-LRKIQK, or spCas9- LRVSQL. 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 7. In some embodiments, a gene modifying polypeptide comprises, in an N-terminal to C-terminal direction, a Cas domain (e.g., a Cas domain of Table 6), a linker of Table 7 (or a sequence having at least 70%, 80%, 85%, 90%, 95%, or 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: 11,002). In some embodiments, an RT domain of a gene modifying polypeptide may be located C-terminal to the Cas domain. In some embodiments, an RT domain of a gene modifying polypeptide may be located N-terminal to the Cas domain. Table 7 Exemplary linker sequences Amino Acid Sequence SEQ ID NO 96 of 237 11867955v1 Attorney Docket No.: 2017469-0019 GGGGSGGGGSGGGGSGGGGS 5110 GGGGSGGGGSGGGGSGGGGSGGGGS 5111 97 of 237 11867955v1 Attorney Docket No.: 2017469-0019 GGGPAP 5149 PAPGGG 5150 98 of 237 11867955v1 Attorney Docket No.: 2017469-0019 GGSPAPEAAAK 5188 EAAAKGGSPAP 5189 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.99 of 237 11867955v1 Attorney Docket No.: 2017469-0019 Exemplary Gene Modifying Polypeptides In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in Table 8, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 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%, 75%, 80%, 85%, 90%, 95%, or 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row 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 the same row of Table 8, 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 comprises a Cas domain according to SEQ ID NO: 11,096, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto, together with an RT and linker of Table 8. In some embodiments, the gene modifying polypeptide comprises a Cas domain according to SEQ ID NO: 11,096 together with an RT and linker of Table 8. Table 8. Selection of exemplary gene modifying polypeptides Linker Sequence SEQ ID NO of linker RT name AEAAAKEAAAKEAAAKEAAAKALE S In some embodiments, a gene modifying polypeptide comprises an amino acid sequence as listed in Table 9, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 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%, 75%, 80%, 85%, 90%, 95%, or 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, a gene modifying polypeptide comprises: (i) a linker comprising a linker sequence as listed in a row of Table 9, or an amino acid sequence 100 of 237 11867955v1 Attorney Docket No.: 2017469-0019 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 the same row of Table 9, 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 comprises a Cas domain according to SEQ ID NO: 11,096, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto, together with an RT and linker of Table 9. In some embodiments, the gene modifying polypeptide comprises a Cas domain according to SEQ ID NO: 11,096 together with an RT and linker of Table 9. Table 9. Selection of exemplary gene modifying polypeptides Linker Sequence SEQ ID NO of linker RT name GGGGSGGGGSGGGGSGGGGS 15,405 MLVCB P08361 3mutA S S 101 of 237 11867955v1 Attorney Docket No.: 2017469-0019 EAAAKGGSPAP 15,432 PERV_Q4VFZ2_3mut EAAAKPAPGGS 15,433 MLVCB_P08361_3mutA S S S S A S S S S S Systems In an aspect, the disclosure relates to a system comprising 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 certain embodiments, the nucleic acid molecule encoding the gene modifying polypeptide comprises one or more silent mutations in the coding region (e.g., in the sequence encoding 102 of 237 11867955v1 Attorney Docket No.: 2017469-0019 the RT domain) relative to a nucleic acid molecule as described herein. In certain embodiments, the 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 the target DNA bound by the gene modifying polypeptide). Localization Sequences for Gene Modifying Systems In certain embodiments, a gene editor 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: 4000 and / or SEQ ID NO: 4001, or an NLS having an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. The nuclear localization sequence may be an RNA sequence that promotes the import of the RNA into the nucleus. In certain embodiments the nuclear localization signal is located on the template RNA. In certain embodiments, the gene modifying polypeptide is encoded on a first RNA, and the template RNA is a second, separate, RNA, and the 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, the RNA encoding the gene modifying polypeptide is targeted primarily to the cytoplasm to promote its translation, while the template RNA is targeted primarily to the nucleus to promote insertion into the genome. In some embodiments the nuclear localization signal is at the 3′ end, 5′ end, or in an internal region of the template RNA. In some embodiments the nuclear localization sequence is situated inside of an intron. In some embodiments a plurality of the same or different nuclear localization signals are in the RNA, e.g., in the template RNA. In some embodiments the nuclear localization signal is less than 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 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, the nuclear localization signal is a SINE-derived nuclear RNA localization (SIRLOIN) signal. In some embodiments the nuclear localization signal binds a nuclear-enriched protein. In some embodiments the nuclear localization signal binds the HNRNPK protein. In some embodiments the 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 the 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 the 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 103 of 237 11867955v1 Attorney Docket No.: 2017469-0019 embodiments the nuclear localization sequence is described in Shukla et al., The EMBO Journal e98452 (2018). In some embodiments the nuclear localization signal is derived from a retrovirus. In some embodiments, a 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, the 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, the NLS is fused to the N-terminus of a gene modifying polypeptide as described herein. In some embodiments, the NLS is fused to the C-terminus of the gene modifying polypeptide. In some embodiments, the NLS is fused to the N-terminus or the C-terminus of a Cas domain. In some embodiments, a linker sequence is disposed between the NLS and the neighboring domain of the 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 certain embodiments, the first NLS comprises the amino acid sequence PAAKRVKLD (SEQ ID NO: 11,095), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 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 10. 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). In certain embodiments, the NLS sequence (e.g., second NLS sequence) comprises a plurality of partial NLS sequences. In embodiments, the NLS sequence, e.g., the 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%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In embodiments, the NLS sequence, e.g., the second NLS sequence, comprises a second partial NLS sequence. In embodiments, the NLS sequence, e.g., the second NLS sequence, comprises an SV40A5 NLS, e.g., a 104 of 237 11867955v1 Attorney Docket No.: 2017469-0019 bipartite SV40A5 NLS, e.g., comprising the amino acid sequence KRTADGSEFESPKKKAKVE (SEQ ID NO: 5351), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. In certain embodiments, the NLS sequence, e.g., the second NLS sequence, comprises the amino acid sequence KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 5349), or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity thereto. Table 10. Exemplary nuclear localization signals for use in gene modifying systems Sequence Sequence References SEQ ID No. AHFKISGEKRPSTDPGKK 105 of 237 11867955v1 Attorney Docket No.: 2017469-0019 KKTGKNRKLKSKRVKTR Q9Z301, O54943, Q8K3T2 5249 KKVSIAGQSGKLWRWKR Q6YUL8 5250 106 of 237 11867955v1 Attorney Docket No.: 2017469-0019 PKKGDKYDKTD Q45FA5 5279 PKKKSRK O35914, Q01954 5280 107 of 237 11867955v1 Attorney Docket No.: 2017469-0019 Q8QPH4, Q809M7, A8C8X1, Q2VNC5, Q38SQ0, O89749, Q6DNQ9, Q809L9, Q0A429, Q20NV3, P16509 P16505 6DN 5 P16506 6XT06 108 of 237 11867955v1 Attorney Docket No.: 2017469-0019 SPKKKRKVE 5342 KRTAD GSEFE5343 , p . p yp y p asic 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 bracketed. Another exemplary bipartite NLS has the sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 5016). Exemplary 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. Inteins 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 instances, 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.” 109 of 237 11867955v1 Attorney Docket No.: 2017469-0019 Additional Domains The gene modifying polypeptide can bind a target DNA sequence and template nucleic acid (e.g., template RNA), nick the target site, and write (e.g., reverse transcribe) the template into DNA, resulting in a modification of the target site. In some embodiments, additional domains may be added to the polypeptide to enhance the efficiency of the process. In some embodiments, the gene modifying polypeptide may contain an additional DNA ligation domain to join reverse transcribed DNA to the DNA of the target site. In some embodiments, the polypeptide may comprise a heterologous RNA-binding domain. In some embodiments, the 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 the target site, e.g., in favor of alteration over the original genomic sequence). In some embodiments, the polypeptide may comprise a domain having 3´ to 5´ exonuclease activity, e.g., proof-reading activity. In some embodiments, the writing domain, e.g., RT domain, has 3´ to 5´ exonuclease activity, e.g., proof-reading activity. Template Nucleic Acids The gene modifying systems described herein can modify a host target DNA site using a template nucleic acid sequence. In some embodiments, the 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 the RNA sequence template directly into the host genome, the 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. The gene modifying system can also delete a sequence from the target genome or introduce a substitution using an object sequence. Therefore, the 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, the template nucleic acid comprises one or more sequence (e.g., 2 sequences) that binds the gene modifying polypeptide. In some embodiments, a template RNA can comprise a gRNA sequence, e.g., to direct the 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. 110 of 237 11867955v1 Attorney Docket No.: 2017469-0019 In some embodiments the template RNA has a poly-A tail at the 3´ end. In some embodiments the template RNA does not have a poly-A tail at the 3´ end. In some embodiments, the template nucleic acid is a template RNA. In some embodiments, the template RNA comprises one or more modified nucleotides. For example, in some embodiments, the template RNA comprises one or more deoxyribonucleotides. In some embodiments, regions of the template RNA are replaced by DNA nucleotides, e.g., to enhance stability of the molecule. For example, the 3´ end of the template may comprise DNA nucleotides, while the rest of the template comprises RNA nucleotides that can be reverse transcribed. For instance, in some embodiments, the heterologous object sequence is primarily or wholly made up of RNA nucleotides (e.g., at least 90%, 95%, 98%, or 99% RNA nucleotides). In some embodiments, the PBS sequence is primarily or wholly made up of DNA nucleotides (e.g., at least 90%, 95%, 98%, or 99% 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 the PBS sequence hybridizes to a strand of the target nucleic acid and the 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 the break induced in the target nucleic acid sequence where reverse transcription begins. For example, SpCas9 N863A typically nicks a bond in the target nucleic acid molecule that corresponds to the bond between the third and fourth nucleotides from the 3’ end of the gRNA spacer sequence. gRNA Spacer and gRNA Scaffold A template RNA described herein may comprise a gRNA spacer that directs the gene modifying system to a target nucleic acid, and a gRNA scaffold that promotes association of the template RNA with the Cas domain of the gene modifying polypeptide. The 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, the 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). The gRNA (also referred to as sgRNA for single-guide RNA) consists of crRNA- and tracrRNA-derived sequences connected by an artificial tetraloop. The crRNA sequence can be divided into 111 of 237 11867955v1 Attorney Docket No.: 2017469-0019 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 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, the gRNA comprises two RNA components from the native CRISPR system, e.g. crRNA and tracrRNA. As is well known in the art, the 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 the template nucleic acid, e.g., template RNA, comprising the 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 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 the template nucleic acid, e.g., template RNA, comprising the gRNA may tolerate increased mismatching with the target site at some interval, e.g., every sixth base. In some embodiments, the region of the template nucleic acid, e.g., template RNA, comprising the gRNA comprising homology to the 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, the template nucleic acid (e.g., template RNA) has at least 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 bases of at least 80%, 85%, 90%, 95%, 99%, or 100% homology to the target site, e.g., at the 5’ end, e.g., comprising a gRNA spacer sequence of length appropriate to the Cas9 domain of the gene modifying polypeptide (Table 6). Table 11 provides parameters to define components for designing gRNA and / or template RNAs to apply Cas variants listed in Table 6 for gene modifying. The cut site indicates the validated or predicted protospacer adjacent motif (PAM) requirements, validated or predicted location of cut site (relative to the most upstream base of the PAM site). The gRNA for a given enzyme can be assembled by concatenating the crRNA, Tetraloop, and tracrRNA sequences, and further adding a 5′ spacer of a length within Spacer (min) and Spacer (max) that matches a protospacer at a target site. Further, the predicted location of the ssDNA nick at the target is important for designing a PBS sequence of a Template RNA that can anneal to the sequence immediately 5′ of the nick in order to initiate target primed reverse transcription. In some embodiments, a gRNA scaffold described herein comprises a nucleic acid sequence comprising, in the 5’ 112 of 237 11867955v1 Attorney Docket No.: 2017469-0019 to 3’ direction, a crRNA of Table 11, a tetraloop from the same row of Table 11, and a tracrRNA from the same row of Table 11, or a sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the gRNA or template RNA comprising the scaffold further comprises a gRNA spacer having a length within the Spacer (min) and Spacer (max) indicated in the same row of Table 11. In some embodiments, the gRNA or template RNA having a sequence according to Table 11 is comprised by a system that further comprises a gene modifying polypeptide, wherein the gene modifying polypeptide comprises a Cas domain described in the same row of Table 11. 113 of 237 11867955v1gniDyI : 8f53 4 5 9i191919 5d QO N, o0, 0 ,1 10 , 0 , 0 menegni6elbaTnidetsilstnairavsaCylppaotsAN RetalpmeTro / dnaANRggningised rofre 1ti1 1 1 1sTnentopuC 3-3-3-3-3-moceDnI : 8if QO090,00,edESN0101otsre)st(= N GeMG= N mGG>r GGGGGGGAa AaPG NGN N N N N(NTG)CP 1.t 99 9 9 9 v11.snaisasasasasa55elbmer CyCy 1vC2vC3vC 97a ts aTysVpSpS_i y_pS_i y_pS_i yG6_pSN -811061, 01A G A ACCA TTGTGA ATCAGCGGA A A T T 0 11, 01A A AG060, 01T TCGTATGGA 0202 12 2 2 2 2 293 3 31- - -3-3-3-3-00-9 06414,2526 7 8 90 0 020202 2710, 0 , 0 , 0 ,00 ,00 , 001 1 1 1 1 1 12:.o N Nt> H HGeN R THCkcR N R R RGGA ANGo N Y N N N N N N D19 9 9 vyesnarCYsa-srHa9 -s9s9sr Ha-r Ha a 19 Rsa5597otyRtpSp CSy apvR 3 RCy apvT3RCy apvC3RCy1pFCyHp Q CQyG ppS68A -S- NS- NS- NS-S-S-11AGCCACCGGGTTGGAGCA ATG T7CTCACA3TGTGTGCGTCGCG2TAfTGT AAGTAGT G TAGT o TTA A AGGAGA TTGA T TTGA T T61102020202020202022 2 2 2931-3-3-3-00-9 0 1 2 36430,30,30,30,710010101012:.o N TtNGAGeAAGkc GCG;G;Go N N N A N D19 9 9 vyesasaR sa9sa- 55nrCyRQCECsaCsa97ottpyRySVpSVpC ySxpCSx G68A - - - - N11 Attorney Docket No.: 2017469-0019 Herein, when an RNA sequence (e.g., a template RNA sequence) is said to comprise a particular sequence (e.g., a sequence of Table 11 or a portion thereof) that comprises thymine (T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil (U) in place of T. For instance, the RNA sequence may comprise U at every position shown as T in the sequence in Table 11. More specifically, the present disclosure provides an RNA sequence according to every gRNA scaffold sequence of Table 11, wherein the RNA sequence has a U in place of each T in the sequence in Table 11. 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. Without wishing to be bound by example, versions of gRNA scaffold sequences alternative to those exemplified in Table 11 may also function with the different Cas9 enzymes or derivatives thereof exemplified in Table 6, e.g., alternate gRNA scaffold sequences with nucleotide additions, substitutions, or deletions, e.g., sequences with stem-loop structures added or removed. It is contemplated herein that the gRNA scaffold 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. Heterologous Object Sequence A template RNA described herein may comprise a heterologous object sequence that the gene modifying polypeptide can use as a template for reverse transcription, to write a desired sequence into the target nucleic acid. In some embodiments, the heterologous object sequence comprises, from 5’ to 3’, a post-edit homology region, the mutation region, and a pre-edit homology region. Without wishing to be bound by theory, an RT performing reverse transcription on the template RNA first reverse transcribes the pre-edit homology region, then the mutation region, and then the post-edit homology region, thereby creating a DNA strand comprising the desired mutation with a homology region on either side. In some embodiments, the heterologous object sequence is at least 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 120, 140, 160, 180, 200, 500, or 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, the heterologous object sequence is no more than 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 120, 140, 160, 180, 200, 500, 1,000, or 2000 nucleotides (nts) in length, or no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 kilobases in length. In some embodiments, the 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, 117 11867955v1 Attorney Docket No.: 2017469-0019 100-1000, 120-1000, 140-1000, 160-1000, 180-1000, 200-1000, 500-1000, 30-500, 40-500, 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, the 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, the 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, the 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 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 certain embodiments, the 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 certain 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 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. 118 of 237 11867955v1 Attorney Docket No.: 2017469-0019 The template nucleic acid (e.g., template RNA) of the system typically comprises an object sequence (e.g., a heterologous object sequence) for writing a desired sequence into a target DNA. The object sequence may be coding or non-coding. The 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, the template nucleic acid (e.g., template RNA) may be designed to cause an insertion in the target DNA. For example, the template nucleic acid (e.g., template RNA) may contain a heterologous sequence, wherein the reverse transcription will result in insertion of the heterologous sequence into the target DNA. In other embodiments, the RNA template may be designed to introduce a deletion into the target DNA. For example, the template nucleic acid (e.g., template RNA) may match the target DNA upstream and downstream of the desired deletion, wherein the 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 other embodiments, the template nucleic acid (e.g., template RNA) may be designed to introduce an edit into the target DNA. For example, the template RNA may match the target DNA sequence with the exception of one or more nucleotides, wherein the 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 of an 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, the heterologous object sequence may contain an open reading frame or a fragment of an open reading frame. In some embodiments the heterologous object sequence has a Kozak sequence. In some embodiments the heterologous object sequence has an internal ribosome entry site. In some embodiments the heterologous object sequence has a self-cleaving peptide such as a T2A or P2A site. In some embodiments the heterologous object sequence has a start codon. In some embodiments the template RNA has a splice acceptor site. In some embodiments the 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 the template RNA has a microRNA binding site downstream of the stop codon. In some embodiments the template RNA has a polyA tail downstream of the stop codon of an open reading frame. In some embodiments the template RNA comprises one or more exons. In some embodiments the template RNA comprises one or more introns. In some embodiments the template RNA comprises a eukaryotic transcriptional terminator. In some embodiments the template RNA comprises an enhanced 119 of 237 11867955v1 Attorney Docket No.: 2017469-0019 translation element or a translation enhancing element. In some embodiments the RNA comprises the human T-cell leukemia virus (HTLV-1) R region. In some embodiments the 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, the heterologous object sequence may contain a non-coding sequence. For example, the template nucleic acid (e.g., template RNA) may comprise a regulatory element, e.g., a promoter or enhancer sequence or miRNA binding site. In some embodiments, integration of the object sequence at a target site will result in upregulation of an endogenous gene. In some embodiments, integration of the object sequence at a target site will result in downregulation of an endogenous gene. In some embodiments the 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 the promoter is an RNA polymerase I promoter, RNA polymerase II promoter, or RNA polymerase III promoter. In some embodiments the promoter comprises a TATA element. In some embodiments the promoter comprises a B recognition element. In some embodiments the promoter has one or more binding sites for transcription factors. In some embodiments, the template nucleic acid (e.g., template RNA) comprises a site that coordinates epigenetic modification. In some embodiments, the template nucleic acid (e.g., template RNA) comprises a chromatin insulator. For example, the template nucleic acid (e.g., template RNA) comprises a CTCF site or a site targeted for DNA methylation. In some embodiments, the template nucleic acid (e.g., template RNA) comprises a gene expression unit composed of at least one regulatory region operably linked to an effector sequence. The 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, the heterologous object sequence of the template nucleic acid (e.g., template RNA) is inserted into a target genome in an endogenous intron. In some embodiments, the heterologous object sequence of the template nucleic acid (e.g., template RNA) is inserted into a target genome and thereby acts as a new exon. In some embodiments, the insertion of the heterologous object sequence into the target genome results in replacement of a natural exon or the skipping of a natural exon. The 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, the template nucleic acid (e.g., template RNA) may be designed to cause an insertion in the target DNA. For example, the template nucleic acid (e.g., template RNA) may contain a heterologous object sequence, wherein the reverse transcription will result in insertion of the heterologous object sequence into the target DNA. In other embodiments, the RNA template may be designed to write a deletion into the target DNA. For example, the template nucleic acid 120 of 237 11867955v1 Attorney Docket No.: 2017469-0019 (e.g., template RNA) may match the target DNA upstream and downstream of the desired deletion, wherein the 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 other embodiments, the template nucleic acid (e.g., template RNA) may be designed to write an edit into the target DNA. For example, the template RNA may match the target DNA sequence with the exception of one or more nucleotides, wherein the 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, the 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, the 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. PBS Sequence In some embodiments, a template nucleic acid (e.g., template RNA) comprises a PBS sequence. In some embodiments, a PBS sequence is disposed 3′ of the 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, 2, 3, 4, or 5 mismatches to a sequence complementary to the sequence adjacent to a site to be modified by the system / gene modifying polypeptide. In some embodiments, the PBS sequence binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nick site in the target nucleic acid molecule. In some embodiments, binding of the PBS sequence to the 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, the 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, the PBS sequence is 5-20, 8-16, 8-14, 8- 13, 8-12, 9-13, 9-12, or 10-12 nucleotides in length, e.g., 9-12 nucleotides in length. The template nucleic acid (e.g., template RNA) may have some homology to the target DNA. In some embodiments, the template nucleic acid (e.g., template RNA) PBS sequence domain may serve as an annealing region to the target DNA, such that the target DNA is positioned to prime the reverse transcription of the template nucleic acid (e.g., template RNA). In some embodiments the template nucleic acid (e.g., 121 of 237 11867955v1 Attorney Docket No.: 2017469-0019 template RNA) has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200 or more bases of exact homology to the target DNA at the 3′ end of the RNA. In some embodiments the template nucleic acid (e.g., template RNA) has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200 or more bases of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology to the target DNA, e.g., at the 5′ end of the template nucleic acid (e.g., template RNA). The template RNA sequences may be customized, e.g., depending on the cell being targeted. For example, in some embodiments it is 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 the initial edit. Consequently, certain template RNAs described herein are designed to write a mutation (e.g., a substitution) into the PAM of the target site, such that upon editing, the PAM site will be mutated to a sequence no longer recognized by the gene modifying polypeptide. Thus, a mutation region within the heterologous object sequence of the template RNA may comprise a PAM-kill sequence. Without wishing to be bound by theory, in some embodiments, a PAM-kill sequence prevents re-engagement of the 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 other embodiments, it is desired to leave the PAM sequence intact (no PAM-kill). Similarly, in some embodiments, to decrease the potential for further gene editing (e.g., by Cas retargeting) following the initial edit, it may be desirable to alter the first three nucleotides of the RT template sequence via a “seed-kill” motif. Consequently, certain template RNAs described herein are designed to write a mutation (e.g., a substitution) into the portion of the target site corresponding to the first three nucleotides of the 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 the heterologous object sequence of the template RNA may comprise a seed-kill sequence. Without wishing to be bound by theory, in some embodiments, a seed-kill sequence prevents re-engagement of the gene modifying polypeptide upon completion of genetic modification, or 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 the seed region intact, and a seed-kill sequence is not used. In further 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 122 of 237 11867955v1 Attorney Docket No.: 2017469-0019 example, silent substitutions) may be introduced within the 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. 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 the template nucleic acid, e.g., template RNA, further comprising (in addition to the 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, the gRNA that coordinates the second nick has inducible activity. In some embodiments, the gRNA that coordinates the second nick is induced after the template is reverse transcribed. In some embodiments, hybridization of the gRNA to the 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 of any of the aspects described herein, a gene modifying system comprises one or more circular RNAs (circRNAs). In some embodiments of any of the aspects described herein, a gene modifying system comprises one or more linear RNAs. In some embodiments, the circRNA comprises one or more ribozyme sequences. In some embodiments, the 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, the target site surrounding the 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, the 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 123 of 237 11867955v1 Attorney Docket No.: 2017469-0019 entirety). In some embodiments, the target site contains an integrated sequence corresponding to the template RNA. In some embodiments, the target site does not contain insertions resulting from endogenous RNA in 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, the target site contains the integrated sequence corresponding to the template RNA. In certain aspects of the present invention, the host DNA-binding site integrated into by the gene modifying system 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 other aspects, the 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, 4, 6, 8, or 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 the 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 theory, nicking of the first strand of the 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 theory, after a writing domain (e.g., RT domain) of a polypeptide described herein polymerizes (e.g., reverse transcribes) from the heterologous object sequence of a template nucleic acid (e.g., template RNA), the 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′ 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 theory, it is thought that introducing an additional nick to the second strand 124 of 237 11867955v1 Attorney Docket No.: 2017469-0019 may bias the cellular DNA repair machinery to adopt the heterologous object sequence-based sequence more frequently than the original genomic sequence. Alternatively or additionally, without wishing to be bound by theory, it is thought that an additional nick to the second strand may promote second-strand synthesis. In some embodiments, where the 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, the Cas domain is capable of nicking a first strand and a second strand. In some embodiments, the first and second strand nicks occur at the same position in the target site but on opposite strands. In some embodiments, the second strand nick occurs in a staggered location, e.g., upstream or downstream, from the first nick. In some embodiments, the endonuclease domain generates a target site deletion if the second strand nick is upstream of the first strand nick. In some embodiments, the endonuclease domain generates a target site duplication if the second strand nick is downstream of the first strand nick. In some embodiments, the endonuclease domain generates no duplication and / or deletion if the first and second strand nicks occur in the same position of the target site. In some embodiments, the Cas domain has altered activity depending on protein conformation or RNA-binding status, e.g., which promotes the nicking of the 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 nick to 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, the gene modifying polypeptide comprises a Cas domain and the additional nick to the second strand is directed by an additional nucleic acid, e.g., comprising a second gRNA directing the Cas domain to nick the second strand. In other embodiments, the additional second strand nick is made by a different endonuclease domain (e.g., nickase domain) than the nick to the first strand. In some embodiments, that different endonuclease domain is situated in an additional polypeptide (e.g., a system of the invention further comprises the additional polypeptide), separate from the gene modifying polypeptide. In some embodiments, the additional polypeptide comprises an endonuclease domain (e.g., nickase domain) described herein. In some embodiments, the additional polypeptide comprises a DNA binding domain, e.g., described herein. It is contemplated herein that the position at which the second strand nick occurs relative to the 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 theory, second strand nicking may occur in two general orientations: inward nicks and outward nicks. 125 of 237 11867955v1 Attorney Docket No.: 2017469-0019 In some embodiments, in the inward nick orientation, the RT domain polymerizes (e.g., using the template RNA (e.g., the heterologous object sequence)) away from the second strand nick. In some embodiments, in the inward nick orientation, the location of the 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 the inward nick orientation, the location of the nick to the first strand and the location of the nick to the second strand are between the sites where the polypeptide and the additional polypeptide bind to the target DNA. In some embodiments, in the inward nick orientation, the location of the nick to the second strand is positioned between the binding sites of the polypeptide and additional polypeptide, and the nick to the first strand is also located between the binding sites of the polypeptide and additional polypeptide. In some embodiments, in the inward nick orientation, the location of the nick to the first strand and the location of the nick to the second strand are positioned between the PAM site and the binding site of the second polypeptide which is at a distance from the 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, 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 the sites to which the two gRNAs direct the gene modifying polypeptide. In some embodiments, in the outward nick orientation, the RT domain polymerizes (e.g., using the template RNA (e.g., the heterologous object sequence)) toward the second strand nick. In some embodiments, in the outward nick orientation when both the first and second nicks are made by a polypeptide comprising a Cas domain (e.g., a gene modifying polypeptide), the 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 the outward nick orientation, the polypeptide (e.g., the gene modifying polypeptide) and the 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 the outward nick orientation, the location of the nick to the second strand is positioned on the opposite side of the binding sites of the polypeptide and additional polypeptide relative to the location of the nick to the first strand. In some embodiments, in the outward orientation, the PAM site and the binding site of the second polypeptide which is at a distance from the target site are positioned between the location of the nick to the first strand and the location of the nick to the second strand. 126 of 237 11867955v1 Attorney Docket No.: 2017469-0019 An example of a gene modifying system that provides an outward nick orientation comprises a gene modifying polypeptide comprising a Cas domain, a template RNA comprising a gRNA that directs nicking of the 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 theory, it is thought that, for gene modifying systems where a second strand nick is provided, an outward nick orientation is preferred in some embodiments. 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 recognized by the 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 the heterologous object sequence, e.g., an insertion or deletion produced by the double-strand break repair pathway unrelated to the modification encoded by the heterologous object sequence. In some embodiments, a desired gene modification comprises a change to the target DNA (e.g., a substitution, insertion, or deletion) encoded by the heterologous object sequence (e.g., and achieved by the gene modifying writing the heterologous object sequence into the target site). In some embodiments, the first strand nick and the second strand nick are in an outward orientation. In addition, the distance between the first strand nick and 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 theory, it is thought the second strand nick benefit, the biasing of DNA repair toward incorporation of the heterologous object sequence into the target DNA, 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 the first strand nick and second strand nick is chosen to balance the benefit of biasing DNA repair toward incorporation of the heterologous object sequence into the target DNA and the risk of DSB formation and of undesired deletions and / or insertions. In some embodiments, a system where the first strand nick and the 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 127 of 237 11867955v1 Attorney Docket No.: 2017469-0019 nick and the second nick are less than the a threshold distance apart. In some embodiments the threshold distance(s) is given below. In some embodiments, the first nick and the second nick are at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides apart. In some embodiments, the first nick and the second nick are no more than 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 250 nucleotides apart. In some embodiments, the first nick and the 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, the first nick and the second nick are 40-100 nucleotides apart. In some embodiments, the second nick is positioned at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides 5´ or 3´ of the target site modification (e.g., the insertion, deletion, or substitution) or to the nick on the first strand. Without wishing to be bound by 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 is 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 the first nick and the 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 128 of 237 11867955v1 Attorney Docket No.: 2017469-0019 to an otherwise similar inward nick orientation system where the first nick and the second nick are less than the a threshold distance apart. In some embodiments the threshold distance is given below. In some embodiments, the first strand nick and the second strand nick are in an inward orientation. In some embodiments, the first strand nick and the second strand nick are in an inward orientation and the first strand nick and second strand nick are at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350, 400, 450, or 500 nucleotides apart, e.g., at least 100 nucleotides apart, (and optionally no more than 500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, or 120 nucleotides apart). In some embodiments, the first strand nick and the 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. In some embodiments, a second gRNA associated with the system may help drive complete integration. In some embodiments, the second gRNA may target a location that is 0-200 nt away from the first-strand nick, e.g., 0-50, 50-100, 100-200 nt away from the first-strand nick. In some embodiments, the second gRNA can only bind its target sequence after the 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. A ribonucleoside having an unmodified sugar comprising a 2’OH as shown below: 129 of 237 11867955v1 Attorney Docket No.: 2017469-0019 O a sugar having a 2’-fluoro (2’F) modification is shown below: a sugar having a 2’-O-Methyl (2’O-Me) modification is shown below: a phosphorothioate modification and a sugar having a 2’-O-Methyl (2’O- Me) modification is shown below: RNA sequences comprising modified nucleotides in the heterologous object sequence and / or the PBS sequence. In some embodiments, the heterologous object sequence comprises one or more 2’-O-methyl (OMe) modified nucleotides. In some embodiments, the heterologous object sequence comprises one or more 2’-fluoro modified nucleotides. In certain embodiments, the heterologous object sequence comprises a region having a pattern in which 2’-fluoro modified nucleotides alternate with unmodified nucleotides (e.g., unmodified ribonucleotides). In certain embodiments, the pattern begins at the 5’ end of the heterologous object sequence (e.g., the 5’-most nucleotide of the 130 of 237 11867955v1 Attorney Docket No.: 2017469-0019 heterologous object sequence comprises a 2’-fluoro modification). In other embodiments, the pattern begins at the second nucleotide from the 5’ end of the heterologous object sequence (e.g., such that the 5’- most nucleotide of the heterologous object sequence is unmodified and the next nucleotide comprises a 2’- fluoro modification). In certain embodiments, the region having the pattern of alternating 2’-fluoro modified and unmodified nucleotides comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 202’-fluoro modified nucleotides. In certain embodiments, the region having the pattern of alternating 2’-fluoro modified and unmodified nucleotides has a length of 0-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, 150-200, 200-300, 300- 400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500, 1500-2000, 2000-2500, 2500- 3000, 3000-3500, 3500-4000, 4000-4500, or 4500-5000 nucleotides. In certain embodiments, the region having the pattern of alternating 2’-fluoro modified and unmodified nucleotides has a length equal to the length of the heterologous object sequence minus 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the PBS sequence of the template RNA comprises one or more 2’-fluoro modified nucleotides. In some embodiments, the PBS sequence of the template RNA comprises one or more 2’-OMe modified nucleotides. In some embodiments, the PBS sequence of the template RNA comprises one or more nucleotides each comprising both a 2’-OMe modification and a phosphorothioate modification. In certain embodiments, the 3’ end of the PBS sequence comprises, in 5’ to 3’ order, a 2’- fluoro modified nucleotide, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) 2’-OMe modified nucleotides, and / or one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) nucleotides each comprising both a 2’-OMe modification and a phosphorothioate modification. In some embodiments, the nucleotides at the junction between the heterologous object sequence and the PBS sequence (e.g., one or more of the nucleotides at positions +3, +2, +1, -1, -2, and / or -3) do not comprise a 2’-fluoro modification. In some embodiments, the nucleotides at the junction between the heterologous object sequence and the PBS sequence (e.g., one or more of the nucleotides at positions +3, +2, +1, -1, -2, and / or -3) do not comprise a 2’-OMe modification. In some embodiments, the nucleotides at the junction between the heterologous object sequence and the PBS sequence (e.g., one or more of the nucleotides at positions +3, +2, +1, -1, -2, and / or -3) are unmodified nucleotides. In some embodiments, the template RNA comprising modified nucleotides (e.g., as described herein) comprises a heterologous object sequence having a mutation region for introducing a mutation into a portion of a human PAH, FAH, HBB, TRAC4, B2M, or A1AT gene. A series of exemplary template RNA sequences comprising 2’-OMe modifications at various positions, e.g., as tested in Example 1, are shown in Table 12 below. A series of exemplary template RNA sequences comprising 2’-fluoro modifications at various positions, e.g., as tested in Example 2, are shown in Table 13 below. 131 of 237 11867955v1 Attorney Docket No.: 2017469-0019 A series of additional exemplary template RNA sequences comprising 2’-fluoro modifications at various positions, e.g., as tested in Example 3, are shown in Table 14 below. A series of additional exemplary template RNA sequences comprising modifications at various positions are shown in Table 15 below. 132 of 237 11867955v1et.aeocdiinht ecaDI :21314151oruq cies e QO lcESN16161616ohs eupn oht ehrpo htU A U G U A U G U A U G U A U ACUGCAU AUGCAU AUGU U ACG:fssestU A sCiUGU ACUGU ACAUGU AUCUlGCU AGUGCU AGUGCU AGUGCU AGllfstrsitifiitl.sitifi-’isirs ecyc Gr U r AGr U r AC Gr UGnhni tetue,at* mr Arr U r A orGU r U r U rrCCU m mrGU r U r U rrGCmrmAGU r U r U rrGCArrmGU r U r U rrCr UqmrUr* * r UrUrCmr UrUrC*r UrUres-ecN OnTU rG GA U U rG G GU rG G GU rG Gr U rrGm m r U rrGmCr UrGrCrG-eA’ uq. DI * r ArrGC**G* r ArrGmGrrGrGr U rrGCm * r ArCr * r ArCN2es sn(A U r r U A U r r A m A U r r A r A U r r mrArU m m mrArU m A m A U r A m A U R,koiece*GGrCCrG**G CC C*GrCrGUrmGrCG*GrCrGUrrGrCG* r rtasirdictanemrCr UrrU r m m m mCr mrr rGrCrGr U r U m mCrU r U r mCU rlpet asccifi uq*GrGA * * rGA m U rGA r U rrGA ArG*C*GrG*GrG*GrGmaideSr r A U A r r A m A r r A r A r r m r A r A m m m m r A r A m A m r A r A r A m r A r Aynelcoet au mr1aybneh et-2-lep dmeethttt aunr eMF-MF 1-2- - - -xostoeEnsilhtitlmaAMEMEMMtMEn3MtEn31.e2dw N v1 s 3e55eelgncbamneu1 297ak l euqma1l2l6TniloCesNMFMFoToT811 5 GAGUACUUGCUGACCUCGCCAUCCU AAGrCr U r UrGrCr UrGr ArCrC Cr mU*rCCmrG* ArU r UGA U r UGA U r UGA U rG GrGrCrGrCr r rUrArU r UrACm r U r m r U r mGr U rCmGr UC G CGr UrCrCrCrrCm r U rrCm *rGUrG*Gr UrGUrG*Gr UrGUrG*Gr UrGUrG*r UrUr *rU rr rU rr rU rrU rGU rG G GCmr U rGrCrr U rGrCrr U rGrrCrrr U rGrrCrrr U rGrrCrG* r ArGCrG* r ArG G G G G G GrGCr * r ArCr * r Arr * r ArrA m A U r r A r A U r r A r A U r r A r A U rCr A r A U rCr A rr A mrGArCU r A mrGArCU r A mrGArCU r A mrGArCU r A mrGArCU r AU rmG*GrrCrrGU rrG*GrrCrrGU rrG*GrCrrGU rrG*GrCrrGU rrG*GrCrrGU rrGU r mCr*GU r U m mCrU r U r mrCrrU r U r mCrrU r U r mCrU r U rr U G rrGA r U*GrGrGA r U*GrGrA r U rGA rGr*A rrU r A r*r A rrA r UA r A r r A m A r r A r A rGr AG G G Gr A r r A m r A r A r A m r A r A r A m r A r A r A m r A r A r A m r A r A r A 3 -4-5-6-7- - - - MMtMn MtMn Mt-M-Mn MtnMtE 3 E 3 E 3 E 3 En3 1v 553l4l5l6 7 97ol lToToToToT68111216U AU AU AGCUAUGGCA AACAGGAU UACA AUACGCAU UGGCGUAAGGGA U0106G rrCU r r A A r r A A rrGA rrGU r r A A rrCU rrCUrrGCrrCA rrGU r rGA r A rCm r U r ArA r A rCmrA r A m m mrA r A mCm rA r A mCm r *Gr U r Ar*Gr U r A**Gr U r AmC Gr U r A *rGU r UGA U rG C GrGr U rrCmrGU r U rrCA mrGU r U r U rmCU mrGU r UmCmrGU r UmCA mrm r U r A r U r mr UrUrC*r UrUrCm*r UrUm* * r UrUmCrmr UrUCU rGr U rrG GGmrCU rGrG Gr r U rGrrCU rGrGr r U rGA U U rGrG G GmG*Gm m m r U rGmmCU r r U rrGrC* r AGmG* r AGrG* r AG GmGmGrGrrr A r A U rr* * r A m * r A rA UC Cr A r A U rrCm U*GA U rrCm A m A U rrCm A rmrGArCU r A mrGArCU m A mrGArCU m m mrGArU m A mrArU r A*GrCrGUrG*GrCrGUrG*GrCrG**C C*GrCCrGUmG CG*Gr rUrrCr rrrCr mrCr UrCr mrC G GCr rm U r U r m U r U r m U m m m m m U m U m m U*GrGmGm mm U rGrrGA r U*rrA m U*rrA * ** *rGrA m U*rGrA r U A r r A rGA rGr A rGA mGm A UC GA mGm A mGA mGm A r m r A r A r A m r A r A m A m r A r A m m m m r A r A m A m r A r A r A 12- 89- - - -Mt- -MFMMtM-MF-n3-En3MtEn3MIMIMI 1 1v 558l9ol 1 21l97ToTMFMFoT68116216U AU AU AGCUAUGGCA AACAGGAU UACA AUACGCAU UGGCGUAAGGGA U5106G mrCU mr AA mr AA mrGArmGU mr AA mrCUrmCUmrGCmrCArrGU rrGA r rGU r UGA U r UGA U r UGA U r UGA U r UGA U r U rmCrrmGr U rmCrrmGr U rmCmrGr U rmCmrGr U rmCmrGrr UrGUmCG*Gr UrGUmC*r UrUrmC*r UrUrmC*r UrrUmC*r UU r rr rU rrGrGU rGrGrGU rGrGrGU rGrGrGU rr UGmCmr U rGmCrr U rGmCrr U rGmCr U rG Cr U* r ArGCrG* r ArGCrG* r ArG G GrGmGrGCr * r ArCr * r A A r A U rrr * rA U r m A m A U r m A r A U r m A r A U r mCm A r A UmrGArCU r A mrGArCU r A mrGArCU r A mrGArCU r A mrArU r A mr* rrrU U U UG CUGG C GrmG*GrCrrGrrG*GrCrrGrrG*GrCrGrG*GrCrGrG*GmrCmU m U r mrCrmU m U m mCU m U r mrCr r U m U r mrCr r U m U r mrC*GrGrGA r U*GrGrGA r U*GrmGrA r U*rmGA rmGmrU m A r*r A mrA r U m A r*r A m m A r A m m A m A mGm A rGAG G G GA m r A r A r A m r A r A r A m r A r A r A m r A r A r A m r A r A r A m r - - - - - -MtMtM M M Mn nt t t t3-3-n3-n3-n3-n3-MI 2MI 3MI 4MI 5MI 6MI 7 1v 552l3l4l5l6 7 97oTol lToToToToT6811 UCUGCAUGACGCUAUGUAUGGCCCCAUCGAGGUCUGGGA AA AA AU AAGA m r A U mrCA mrGA mrGGm r U A m r U A rmCU m r A A r r A A rrCA r r U A r r A U r r UmGA U r UGA U r UGA r U r U A * r U r UC Cr U rU rCrCmrGr U rmCmmrGr U rmCrrm A r U rCrrA A r U rCr r A r UrGUmG*Gr UrGUmCG*Gr UrGUmCG* rUrUrCr rUrUr* *rUrr rU r U rG GrG G C GrG GA AGrGrGmCrr U rrGmmCrr U rrGrmCrrr U rGrrCrrr U rGrr r r U rArGCrG* r ArGCmG* r ArGCrG*Cr ArGCrGA r*GCr A U rrCr*G* *r Ar m A r A U r m A r A U r m A r U r rC G CU rArCU r A mrGArCU r A mrGArCU m A mrGArCUrGU mrGArU r r m mrArrrU rr *U UC* *GC G G GrrCrrGrrG*Gr r r * r r r * r r * * rrCrGmG CrCrGrG CrCrGUGUCrCmGU m U r mCU m U rCA r U rmGm U m U r m U U r U m U U r r r m m U A r U rmGA mrGrCrGrGmrGm A r*GA mrGm A r*GA mrGU m A r*GrGrrGA rrG*rGrGrGA*G*C*G*GrGGr r r A r A r A m r A r A r A m r A r A m A m r A r A r m r A r A r r m m r A - - MtMt-1-51-4n n 4gA4gA4g 13-3-P-P-PA - MI 8MI 9 FGME FGME FGME 1v 558l9ol9ToT 15141 76 A A A811CGG UUGGCGGCGUCGAACGGCCCAAGCUGCGCUCGAAACCAAAGAGUGr CA rrGA rr UArrGGrrGU rrCU rrCUrrGA rr AArrGCrrCU rrCA rr A UrC CCr U r UC Cr U r UC Cr U r U A r U r U A U rrr r A r U rrr r A r U rr * r *r Ur r r A rC C CrGA r U rC CA rUrG* *rGUrGUrG* *rGUrGUr* *rUrUrCmrUrUrrCmrUrGA A r A A rGA AGrG G*GrG G Gr rGr r r U rrGrr r r U rrGrrr r r U rGrrCU r U rrGrrC*Gr UrCr*G*C*Cr AGr AGr AGr m r AGr m r U rrCr*G*C G*CU rrCr*G*C*CU rrCr A*CU rrC*CrCrArU r r m mrArU r r m*r A * U U r r mrArUrGmrArUrU mrrr* U* *GrrCrr* *G C*G C GrG C G GG G C C GU*GU*CrrCrrGU*G G*CrrCrrGr* r r m **C C G G CrrU r r r m m UrU r UCr r r rU r r r m U U r r r m m U m UrU r U m UrA* * * rGrA* *GA *GA rGArCGrG C G G GrGrG C*G*GrGrrGr*G*CU*GrGrrGrrG*GrGrrGrrG*GrGr A r r m m r A r A r r m m r A r A r r m m r A r A r*Gm r A r A rrGm r - 4g 1- 42- 43-21-1PB F -gMPB F -gMPB F -4g4M PB F -gA MPF- GEGEGEGEGMI 1v 55921 716B2B3B1B A811 UCUGCAUGACGCUAUGUAUGGCCCCAUCGAGGUCUGGGA AA AA AU AAGA mr AU mrCA mrGA mrGGmr UA mr UA rmCU mr AA mr AA rrCA rr UA rr AU r r UmA * r U r UC Cr U r UC Cr UC C C Cr r U rmr r A r U rmr U r r A r U rmr U r UU rCA AC C Cr r A r U rmCrCr rGUmGrCrGUrUm* *rUrUm* *rUrUm* *rUrUm* * rGrrGrGA AGrGrGA AGrGrGA AGrGrGA ArmCrr U rGmr r r U rGmr r r U rGmr r r U rGr rArGrG*r ArG*r AGr AGr AmGrCm A rCU rCmG*C G*CU rrCm*G*C*CU rrCm*G*C G*CU rrCm*G*CArCUrGU mrGArCU r r m mrGArCU r r mrGArCU r r m mrArU r rrCrmGrrG*CrrCrmG* U*G* U*CrrCrmG* U*G*CrrCrmG* U*G* U*GCrrCCrmG* U*G GmGU m UrCm UmU m r r m m UmU m r r m UmU m r r m m UmU m r r m r AGAGAGAGA *mGmrG*rGrGmrGm*G*C*G*GrGmrGm*G*C G*GrGmrGm*G*C*G*GrGmrGm*G*CU A r A rGm r A r A r r m m r A r A r r m m r A r A r r m m r A r A r r m - 4 - - - g51PFA4 414142-gPA -gPB -gPB - MF F FGIGMIGMIGMI 1v 55514 9176 A A1B2B8116416U AU AUGA UUGUGCCA AGCCGA ACGU AUCAAAGCGA UAACGGCAGCGA U5306C mrCU mrGA mrCA m r A AmrGCmrGA m r U UmrCCrmGA mrCA rrGGr r A A r r U r UmCA*Gr U r UmCA*CrGU r UrGCmrGU r UrGCmrGU r UrGArUA r U rrA r U r r U r r U r r U rCmGrrGU rrGUmCmrUrUrmCm r UrrUrC*Gr UrrUrC*Gr UrrUrC*r U rGGrC*GrGrGGrC*GU rGrGGrCm U rGrGrm U rGrGrGU rr U rm*GU r U rmGr U rrG*r U rGrC*r U rGrCm r UCr A r m r A rG G G GU rrCm A*CU rrCm * r A m A * A U rrCr * r A r ArCr * r ArCr * * r r A U r r A m A U r r A U A UmrGArCU*rGGmrGArCUrGU mrGArCU r A mrGArCU r A mrGArCU r m mr* r mr* r m m * r rUr * r rUm *U *GCrCrGr m U*C C G G G C G G G C G G GrCrG*GmCr r rrCr rrCr rrCr r ArG U m U m U U m U m U r U r m U r U m mCA rmGArCrrGArGrGU r U m m*GrGmrGmrG*GrGrGrG*GrGr U*GrrGA r U*GrrGA r*C*Gr *Gm 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r A r 1 1 1 11B _ B _ B _ B A 11 1_1_2H H H H H AF 2AFb2AF 7AFb7AF 1v 55b b92712 7 716B1B1B1B A811 CCCGGUGGUCCGUAAUGGU CCCGUA UGGCGU A C rrGUrrGUrrGGrrGU rrCCrrCUrrGGrr UA rrGA rrCA rrCUrrGArrG CrGr U r UrGCmr U r UrG*r U r Urr r r U r Urr rCr U r U rCA r U rGA r U rC CA r U rC* A rCr U UrCrC*GU r **Gm A r U rrGrGrGrGmrGUrGUrGmrGUrGUrGArGUrGUrGA* rUrU r A rr rA rr r *r r r r r rC GrGrGGrCr U rGrGmr U rGrG Cr U rrGrr ** r U rGrr ** m r U rGr * r AGrC* r AGr m * r AG GUG Gr A U rrr A A U rr* r A U * * r Ar AC Cr A A U rrCr r r U A U rrCrrCrCm *r r U A U rU G mrGArCUrCGmrGArCUrCGmrGArCU*G*Cm mrGArCU*G*m mrArr r * r r m * r rm* r r * * r rC*G CGrC C C G C C C G C C G C C C G*CrCGrCrr rrr * rr r rrr r r UrrrrGmrGU rGmGmCrGU rG GmCrGU r **Cm mCrU r **m mCrUA r *r UrA r * A r m * A U *GA UC*GrCrGrCrGrCrG*CrCrG*Cr rG U r r UmGU r r U*GU r r r r U r r r r UCrGA r m r A r A r m r A r A r m r A r A * * m m r A r A * * m m r A r 231 1 A A1__A A B 22_2_2_2H H H H H AFAFAF 4AF 5AF 1v 559 234151 76 A 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/ FrGmCrGAr / rGmCrGAr / FrGmCrGAr / m UF2 r mr m U2mFr mr m U2 r mr m U2*Cr mr m U2 r mr m U A Fi C GA A FiC GA A Fi C GA A F mC GA A Fi C GA A912 / *Gr m r m r mi / F m r m r m r m r m r m r m r m Ai / GA A A A2iU m A A A A2i / GA A A A2iU*GA A A A2i / GA A A A2i00m-G / Cr / rCrGrmG / Cr *rCrGrmG / CrGrCrGrmG / Cr mrCrGrmG / CrrCrGrmG / 9 GU / A U / m U / A U / UCmmU r r r mmCmr r r mGr r r mCr r r mG Cr r r m6UGF m * A A UGFC* A A UmG*CmGmCmGmGm m U r r m m U r r mF2* A A U F * A A UF2m * A A U4271 i*C2imCm m U r r m m2 mU r r m m*U r r m m U U m U A U U m U A U Uim U A U Ui Cm U A U UGm U A U02mCmG / C*CU AmCm / *GG / CU*CU AmCmGU m*Cr r U AmCmG / Cm U*Cr riU m r r m r rm / CmGm*Cr rmCmG:m .mr*CU A U * U A m r r mmr m m r r mmr * m r r mmr m m r r mmr A m r r mo AGm * U A AGA * U A AGA * U A AGA * U A AGmGm / CA r r m / GA r r m / CA r r m / G / Cm * U A A N AmCF*Gm U U AmCFm / m U U AmCFmCm U U AmCFm / A r r m m U U AmCF*CA r r m m U U AmtCekcRkc Rkc RkRkRko2 i 2 i 3ci 1ci 1ciD1n1_oro1n_oro1n_oro1n_or1n_orvyPe_ 4nr 1 sunilPf_14sunilPf_14sunilPf_14o sunilPf_15o su 5n5ilf97ottm2__91m1 v2__71m1 v2__91m1 v2__71vm2__81v68A B _ B _ B _ B1_ B1_11 U A GGU CCGGCGUCA UCCU U U A CGU CUGGGGGCU UGAGm / U F 2i / CrGrGrGr U r A r / GF2i / U r / A / FrmGm AGm / GA m r U m / GGmCrGAr / rGmCrGAr / FrGmCrGr / rCmCrG*GrCmCrGr / CF2m AF2m A / GFAF2 r mr m Ur mr m U2 * r mr m U* r mr m2m r m U2imC GA A FC GA A Fi C C GA A FC G GA ArGmG9r m r mi / r m r m m r m r mi / F r mi / *CA A Fi / 1 / G*GA A A A2iU A A A A2i / GA A A A2iUmCr m A A A A2ir m r m U m A A A A2iA0r m r r*G0- *rCrGUmG / Cr / rCrGUmG / Cr mrCrGr UmG / Cr m rrGrmG / Cr*GrrGrmG / Cr / 9 / G G / G / CU U U / U U A r r r m A r r r m * r rCm * AmGmGr mmGm r r r mmGmCr r r mmGU64F2A U F m * A A U * U r r m m U r r m mF2A * A A U F A U r r m m*GA A U F r r m m m*GA A U r r mF2 C71iC2i*CmC2im 2m0 / m U A U U m U A U Ui / m U A U U / m U A U UiU m U A U Uim r r m r r r / 2UmC*CU AmCmG / G*CU AmCmGUmC*Cr U AmCmG / CU * r r F A U AmCmG / CmC* r r A U AmCmG*G:r m*GU m r r m r m r r m r m m r r UmGmGmmGr2im r r mmGrmGm r r mmGr m.o / C* U A A m A r r m / Gm * U A A * A r r m / CU * U A A m A r r m / G*GU A A r r / G*GU A A / C*CN F A m U U AmCF A m U U AmCF A m U U AmCF / Cm m U U AmCFm / r r m m U U AmCF mtekck k k k koR2ciD1Pn_orR2cio1Pn_orR1cio1n_orR2cio1n_orR0cio1n_or 1o vye_ 5nr 1 sunilf_ 5sulP5sulP4sulP4sul55o _1ni f__1ni f_2ni f_2ni f97ttm2_ 611vm2_ 81m1 v2__01m2 v2__91vm2__72v68A B _ B _ B _ B1_ B1_117636UGUGA A U A CGCGACA U A A CGA AAGGA CGU CCGGAAGUGA U AGU U 7826rGr U r ArGr r U A rCr A rGrCr AmGrCm A r m A U rCm A rGm A r m A A rGmG r mrm2 / rmrm2rrmrm2 / mrmrm2 / *m m / CCACAiACACAiUCACAiA*CACAiACrCArCA2irGUrGA / Cr / rGUrGA / CrGUrGA / CrCA m m m m m r m m / mrGm UrGm A / Cr / mrGm UrGm / Cr / *GrCmCrGUGAr m rm / r r m rm r U* r mm r U*A m rG Gm r U m / FmC C GA / FC C GA / G C CrGA mrCmCrGA rmr mG 2* r mr m U2rr mGF2r m / GF2r m / GF2*CG GA A FC G GA A FGmGA A FmCrGmGA A F*CrGmGA9mim r m r mir m r mim r m r miA F m rim r m r / / / / / 1A A2U*A A A A2A A A A A2U U A A A A2m r m * A Ai i i iU U A A A A2iU U0r0- UrGr UmG / CrGrrGrmCG / Cr / rrGrmG / CrmCrrGrmG / CrmCrrGrmG / Crm9r r r m / mCm U U m U U r r r m U r r / CU U / CU U / CC6 *G*CmG*Gr mmGmGr r r mmGmGr r r mmGmGGA A U F A A U A A U F A A U F A r*GF2*G*G*GA U F4r m m21 im r r m m m r r m m2im / r r m m2imr r m m2 m7m U A U U U m U A U U * r ri0 / *m U A U U m U A U U / m U A U Ui / 2m m / m* r rm mC* r rm m / U * r rm m / U * r r A U AmU A U AC G C CA U AC GU m A U AC G CF A U AC G CFCmG / CF:m r r m .o*GU A AmGrmGm r r m r m r r m r m r r m r m r r m r U A AmG*GU A AmG2iU A AmG2iU AmG2ir r m / G*Gr r m / C*Gr r m / G*G / G*GA / GN m U U AmCFm / m U U AmCFmCm U U AmCF / Gr r m m U U AmCF / Gr r m m U U AmCF / tGekcRkRkRkRkRk o1ciD1nor 2cinor 2cinor 1cinor 0cinor 1vyP_e_nr 24o1suP_ 4o1uP_ 5 o1uP_ 5 o1uP_ 5 o u5nlf_2 snlf_2 sn lf_2 snlf_2 sn lf 59ottmi2__91mi1v2__71vmi2__02vmi2__02vmi2__02 7v68A B _ B1_ B2_ B2_ B2_112736UGUGA A U A CGCGACA U A A CGA AAGGA CGU CCGGAAGUGA U AGU U 2926rGr U r ArGr r U A rCr A rGrCr AmGrCm A r m A U rCm A rGm A r m A A rGmGrCm A rGm U rCmCrGm / G Cr Ar / Fm UmCrGm Ar / r UmCrGm Ar / r UmCrGm Ar / r UmCrGm Ar / r UmCrGm Ar / rGmGr m U2A A F*GrCmGr m U A A FrCmGr m U r m U r m U r m U A A FrCmGrCmGrCmG9miA A F A A F A A F r m r / m m r m m r m m r m m r m1A A A A2iArGA A A2irGA A A2irGA A A2irGA A A2irGm r m A A A2i0r0r- UGr*UmCG / Cr / r m UrGr UmG / Cr UrGr UmG / Cr UrGr UmG / Cr UrGr UmG / Cr UrGr UmG / C9r r r mmU*r r r mmGr r r m r r r m r r r m r r r m6 *GA A UG GA A UGm A A UmGA A UmGA A UmGmGr r m mF2*Gr r m m **Gr r*G*G*GA A U4m m A r r m m r r m m r r m71U Umm m U AiC02m m / m U A U U A m U A U U m m U A U U m U A U U m U A U U * r r m * r rm mm * r rm m ** r r Am mC* r r m U U Am* r r A U AC GU U U U AC GU U AC G CU UC G CmGU UmCmG:m r*.CA r m r m r r m m r r m m m r o*GU A AmGmCmGmGr mmG* m r r mmGU m r r mmGr r m / C*CU A A m r r m **CU A A * r r m U*CU A A U r r m m*CU A A m r r m*CU A A r r mGN m U U AmCFmGm U U AmCU m U U AmCm m U U AmC* m U U AmC*Gm U U AmCmtekck Y5 Y5 5_ _oR2ciY Y D1yPn_orRRp2RR1RR0R5Y R R5R1e_ 5oS1PpS1PpS1PpS9PpS8Pv 55nr 2 sulf_T_ or_T_ or_T_ or_T_ _ _mni_02AoA0oA0oA0or T 0oA oro97ott 2_1DEulf 1DEulf 1DEulf 1DE 1DE68A B81v _ A _ _ A _ _ A _ _ A _ulfA _ulf118736UUGGGA A A ACCGA A GCU A U A AACGCA AGU UCGCGAGUGA U UCU U 8926rGr U rGrGr r A A r r A A rCrCr AmGrGm A rCm U r m U A r m A A rCm A rCmG r mrmrr m m2r m m2r m m2r m m2r m m2A ACACA ArCAriA ACA A ArCA A ArCA A ArCA r mrm rrmrm / rmrmi / irmrm / irmrm / m mi / GUGA UGUGA UGUGA UGUGA UGUGA UrGUrGA U r UmCrGm Ar / r UmCrGm Ar / r UmCrGm Ar / r UmCrGmr / rmCrGmr / rmCrGmr / rGA U A U A CmGr m UrCmGr m Ur mr m Ur mr m Ur mr m Ur mr m U A A F A A F mC GA A FC GA A FC GA A FC GA A F91rGm r m A A A2irGm r m A A A2i* ArGm r m A A A2iA mrGm r m A A A2irGm r m A A A2irGm r m A A A2i0r0- UrGr UmG / Cr UrGr UmG / Cm r UrGr UmG / C*Cr UrGr UmG / CCr m UrGr UmG / Cr UrGr UmG / C9r r r mmr r r m*mCr r r m m r r r m * r r r m U r r r m64*GA A UGA A UGm A A UmG* A A UmGU A A UmGmmGr r m m*Gr r m m **Gr r m m U*Gr r m m m*Gr r m m**GA A U G71m m U A U U*Gr r m m m U A U U m U A U U U m U A U U0Gm U A U U m U A U U m * r r * r r * r r * r r2U U AmCmGU U AmCmGmGU U AmCmG*GU U AmCmG* r r m U U AmCmGm * r r UmCmG*Gm r*GU A:r m .mm r r mm mm r r mm mm r r mm *m r r mmm r r mmmo*CU A AGU A AG GU A AG GU A AG GU A AGm U A AG* r r m*NmCCr r mmCm*Cr r mmCm*Cr r m U AmCm*Cr r m U m U U AmC**Cr r m U m U U AmCU m U U A m U U A U m U U A U m U mtekcY_o R5Y6 Y6 Y6 Y_6Y_6DpR RR7 p2RR1 p1RR1 p0R R 1pR 9pR 81vySe_P Snr T_ _P S_or_P S_or_P S_or_P S_P55A0or T 0 T 0 T 0 T_0 T_0o o o oorooro97ott 1DAEA1D_ulf EulfA1DEulfA1DEulfA1DEulAf1DEulf68A A _ _ A _ _ A _ _ A _ A _114836UUGGGA A A ACCGA A GCU A U A AACGCA AGU UCGCGAGUGA U UCU U 4036rGr U rGrGr r A A r r A A rCrCr AmGrGm A rCm U r m U A r m A A rCm A rCmGr m A A rGmrm / rmrm / m m / m m / m m / / A Ur r r r r r rmrm UGA UGUG G G G G G G G GmGU A U U A U U A U U A U r Crmrrm rm r r m r r m r r m r r m Am r m r m r m rr UGA / UC G C G C G C G C GrCmGr m U r m / Cm U A * r m / CU A A r m / CU A / CU A / CA ArCmGrCmGrCmGrCmGr mrCmGr m F A A F A A A F m A A F9CA A F A A F m r m m r m m m r m m r1rGA A A2irGA A A2irGA A A2i*CrGm A A A2imrGm r m A A A2irGm r m A A A2i0r r r r*Cr0- UrGUmG / CUrGUmG / Cm UrGr UmG / Cm r * UrGr UmG / C* r U UrGr UmG / CU r m UrGr UmG / C9r r r m r r r m * r r r m U r r r m m r r r m r r rG6 *GA A UmGA A UmGU A A UmGm A A UmGmG*GmmGr r m m*Gr r m m*G*G*G*GA A U*GA A U m4r r m m r r m m r r m m m r r71m U A U U m U A U UmGm U A U U*Gm m m U A U U m m U A U U m U*G* r rm mG* r r*m m m* r rm m m* r rm m ** r rmGA U U * r r m02U U AC Gm U U AC G GU U AC G GU U AC G GU U ACmGm U U AmCmG*:m r r m * m r r m m m r r m m m r r m m U A AmGU U A AmGmGmGm r r mmG* m r r mmGU.o*C*CU*CU A A U*CU A A U*CU A A U*CU A A m r r mNmm r r mmm r r mmm r r mmm r r mmm r r m m U U Am* m U U AC* m U U ACA m U U ACA m U U ACA m U U ACACAtekcY_7 7 7_ _o R6Y Y Y Y Y RR R R7 7DpSR 7p2R1R0R S1 pS1 pS1 pSR R 9pR 81vye_PT_ _P_or_P_or_P_or_P S__P_55nrA0or T 0 T 0 T 0 T 0oAoAoAoor T 0ooro97ott 1DAE 1D_ulf Eulf 1DEulf 1DEulfA1DEulAf1DEulf68A A _ _ A _ _ A _ _ A _ A _110936UUGGGA A A ACCGA A GCU A U A AACGCA AGU UCGCGAGUGA U UCU U 0136rGr U rGrGr r A A r r A A rCrCr AmGrGm A rCm U r m U A r m A A rCm A rCmGr m A A rGm UrGm / A UrGm UrGm A / CmGrGm m m m m mGm m m UrGA / CmGrGUrGA / C*GrGUrGA / CmrGUrGA / C* rmCrGm r rmCrGmr / m rmCrGmr / m rmCrGmrm rm rmr* rm rmrU U A U A U U A U U A / U UC GA / U UC GA / m rCmGr m / CrCmr m U mr mr m U mr mr m U mr mr m U mr mr m U * A A FGA A F AC GA A F AC GA A F AC GA A F AC GA A F A91rGm r m A A A2irGm r m A A A2imrGm r m A A A2imrGm r m A A A2imrGm r m m m r m m A A A2irGA2i0r / 0r- UGr UmG / Cr UrGr UmGG / Cr F UrGr / UmGG / Cr F UrGr / UmGG / Cr F UrGr / UmGA A G / Cr F UrGr / UmGG / CF9r r r mmGr r r mmG2ir r r mmG2ir r r mmG2ir r r mmG2ir r r mm 264*GA A U r r m mG*GA A U r r m m* / GA A U r r m m* / GA A U r r m m*A A U*A A UG i / Gr r m m / Gr r m m / 71m U A U U m m U A U UCm U A U UCm U A U UCm U A U UCm U A U UC* r r * * r r r * r r r * r r r * r r r * r r r02U U AmCmGU U U AmCmGU U U AmCmGU U U AmCmGU U U AmCmGU U U AmCmGU:m r r m m m r r m m r r m.mo*GmGrGmGrGm r r mmGrGm r r mmGrm r r mm rC U A A * r r m*CU A A*CU A A*U A A*U A AG*U A AG GNmA r r mm rr r mm rCr r mm rCr r m U U AmCr r m m U U ACm m U U AC Cm U U AC Cm U U AC CmCrCm U U AmtCrCekcY_Y1 Y1 Y1 Y0Y0o R7R R R1 1DpSR R 7p2R1R0R S1 pS1 pS1 pSR R 9pR 81vye_Pnr T_0or_PT_0oro_PT_0oro_PT_0oro_P ST_0or_PT_ or55oululul o0o97ottA1DAEulAf1DE f_A01DE f_A01DE f_A01DEulfA1DEulf68A _ A _ A _ A _ A _ _ A _ _116936UUGGGA A A ACCGA A GCU A U A AACGCA AGU UCGCGAGUGA U UCU U 6136rGr U rGrGr r A A r r A A rCrCr AmGrGm A rCm U r m U A r m A A rCm A rCmGr m A A rGm m * m m / m m m / m m m / m m m / * m m / U UrGA / CUrGUrGA U UrGUrGA U UrGUrGA U UrGUrGA U UrGUrGA U m rmCrGmr / m rmCrGm r m rmCrGm r m rmCrGm r m rmCrGm r m rm rm r * U A * U A / A U A / A U A / A U A / A UC GA A rCmGr m U ArG G G G / GCmr m mr mr m mr mr m mr mr m mr mr m m A A F mGA A F / C GA A F / C GA A F / C GA A F / C GA A F91rm r m2*rm r m2Grm r m2Grm r mGm r mGm r m / GGA A Ai GA A AiFGA A AiFGA A A2iFrGA A A2iFrGA A A2iF0r0r- UGr / UmG / GCr F UrGr UmG / C2ir UrGr UmG / C2ir UrGr UmG / C2ir UrGr UmG / C2ir UrGr UmG / C2i9r r r mmG2ir r r mmG / Cr r r mmG / r r r mm / r r r mm / r r r mm / 64*GA A U r r m m*GA A U r r m m r*GA A UCr r m m r*GA A UG Cr r m m r*GA A UG Cr r m m r*GA A UG Cr r71m U / Cm m r A U U m U A U U U m U A U U U m U A U U U m U A U U U m U A U U U * r rm mr * r r * r r * r r * r r * r r02U U AC GU U U AmCmGrGU U AmCmGrGU U AmCmGrGU U AmCmGrGU U AmCmGrG:m r r m m r r m m r r.mo*GrGmGrCmmGrCm r r mm rm r r mm rm r r mm rC U A A r r m*U A A*U A A*U A AG C*U A AG C*U A AG CNm rCr r mmrCr r m rCr r m rCr r m rCr r m r m U U AC Cm U U ACU m U U AmCU m U U AmCU m U U AmCU m U U AmCUtekcY0Y1 Y1 Y1 Y1Y1o R1R R R1 1DpSR R 7p2R1R0R S1 pS1 pS1 pSR R 9pR 81vye_Pnr T_ or_P_or_P_or_P_or_P S_or_Po 55A0oT 0o ulT 0o ulT 0o ulT 0oT_0 ro97ott 1DAEul _fA1DE f_A11DE f_A11DE f_A11DEulfA1DEulf68A _ A _ A _ A _ A _ _ A _ _112046UUGGGA A A ACCGA A GCU A U A AACGCA AGU UCGCGAGUGA U UCU U 2236rGr U rGrGr r A A r r A A rCrCr AmGrGm A rCm U r m U A r m A A rCm A rCmGr m A A r mrm / mrmrmGm mGm mGm mGm mGGUGA U *GUGA / GFrGUrGA / GFrGUrGA / GFrGUrGA / GFrGUrGA / GF rmm r A r m r m r m r m r m UCrGA m UmCrGAr / 2iUmCrGAr / 2iUmCrGAr / 2iUmCrGAr / 2iUmCrGAr / 2irCmGr m / G* r m A A A FrCmGA A F / CrCmGr m A A A F / CrCmGr m A A A F / CrCmGr m A A A F / CrCmGr m A A A F / C91rGm r m / A A A2GiFrGm r m A A A2ir UrGm r m A A A2ir UrGm r m r m r m r m r m r A A A2iUrGA A A2iUrGA A A2iU0r0r- UGr UmG / C2ir UrGr UmG / CrGr UrGr UmG / CrGr UrGr UmG / CrGr UrGr UmG / CrGr UrGr UmG / CrG9r r r mmG / Cr r r mmGrCr r r mmGrr r r mm rr r r mm rr r r mm r64*GA A U r r m m r*GA A U r r m m r*GA A UCr r m m r*GA A UG Cr r m m r*GA A UG Cr r m m r*GA A UG Cr r m m r71m U A U U U m U A U U U m U A U U U m U A U U U m U A U U U m U A U U U * r rm m r* r r * r r * r r * r r * r r02U U AC G GU U AmCmGrCU U AmCmGrCU U AmCmGrCU U AmCmGrCU U AmCmGrC:m r r m m r r m m r r.mo*GrCmGr / mmGr / m r r mm rm r r mm rm r r mm rC U A A r r m*U A A*U A A*U A AG / *U A AG / *U A AG / NmrCr r mmUCr r m UCr r m UCr r m UCr r m U m U U ACU m U U ACF m U U AmCF m U U AmCF m U U AmCF m U U AmCFtekcY1Y1 Y1 Y1 Y2Y2o R1R R R1 1DpSR R 7p2R1R0R S1 pS1 pS1 pSR R 9pR 81vye_Pnr T_ or_P_or_P_or_P_or_P S_or_Po 55A0oT 0o ulT 0o ulT 0o ulT 0oT_0 ro97ott 1DAEul _fA1DE f_A21DE f_A21DE f_A21DEulfA1DEulf68A _ A _ A _ A _ A _ _ A _ _118046UUGGGA A A ACCGA A GCU A U A AACGCA AGU UCGCGAGUGA U UCU U 8236rGr U rGrGr r A A r r A A rCrCr AmGrGm A rCm U r m U A r m A A rCm A rCmGr m A A r mrm / Grmrm / m m / m m / m m / m m / GUGAGFGUGA A2irGUrGA A2irGUrGA A2irGUrGA A2irGUrGA A2ir m r m r r m r r m r r m r r UmCrGAr / 2iUmCrGA / / CUmCrGA / / CUmCrGA / m r / m rm r / / CUC GA / CUC GACrCmGr m A / C C C C / CCr mr m rr mr m rr mr m rr mr m rr mr m r A A FC GA A F UC GA A F UC GA A F UC GA A F UC GA A F U91rGm r m A A A2ir UrGm r m A A A2irGrGm r m A A A2irGrGm r m A A A2irGrGm r m A A A2irGrGm r m A A A2irG0r0r- UGr UmG / CrGr UrGr UmG / CrCr UrGr UmG / CrCr UrGr UmG / CrCr UrGr UmG / CrCr UrGr UmG / CrC9r r r mmGrCr r r mmGr r r r mmGr r r r mmGr r r r mmr r r r mmr64*GA A U r r m m r*GA A U U r r m m*A A U U*A A U U*A A UGU*A A UGU rGr r m mGr r m mGr r m mGr r m m71m U A U U U m U A U UCm U A U UrCm U A U UrCm U A U UrCm U A U UrC* r r * r r r * r r r * r r r * r r r * r r02mCm rU Am mU U U Am mU U U Am mU U U Am mU U U Am mr U U AG CUC G C G C G C G C GU:m r r m m r r m m r r.mo*Gr / mGr / mmGr / m r r mm rm r r mm rm r r mm rC U A A r r m*U A A*U A A*U A AG / *U A AG / *U A AG / NmUCr r mmUCr r m UCr r m UCr r m UCr r m U m U U ACF m U U ACF m U U AmCF m U U AmCF m U U AmCF m U U AmCFtekcY2Y1 Y1 Y1 Y3Y3o R1R R R1 1DpSR R 7p2R1R0R S1 pS1 pS1 pSR R 9pR 81vye_Pnr T_ or_P_or_P_or_P_or_P S_or_Po 55A0oT 0o ulT 0o ulT 0o ulT 0oT_0 ro97ott 1DAEul _fA1DE f_A31DE f_A31DE f_A31DEulfA1DEulf68A _ A _ A _ A _ A _ _ A _ _114146UUGGGA A A ACCGA A GCU A U A AACGCA AGU UCGCGAGUGA U UCU U 4336rGr U rGrGr r A A r r A A rCrCr AmGrGm A rCm U r m U A r m A A rCm A rCmGr m A A rGm U r m r r m r m r m r UmCrGA / / CUmCrGAr / rCUmCrGAr / rCUmCrGAr / rCUmCrGm Ar / rCr UmCrGmr / rCrCmCA G r m r A A F UrCmGr m U r r m U r r m U r r m U r r m U r A A F UrCmGA A F UrCmGA A F UrCmGA A F UrCmGA A F U91rGm r m A A A2irGrGm r m A A A2irCrGm r m A A A2irCrGm r m A A A2irCrGm r m A A A2irCrGm r m A A A2irC0r0rrm / rrrrm / rrrrm / rrrrm / rrrrm / rr r- UGUG C CUGUG C / UGUG C / UGUG C / UGUG C / UrGUmG / Cr / 9r r r m r r r r m U r r r m U r r r6A A UmGUmGmGmmGU r r r mmGU r r r mmGU4*Gr r m m*GA A U r r m mF2*GA A U r r m mF2*GA A U r r m mF2*GA A U r r m mF2*GA A U r r m mF271m U A U UrCm U A U U m U A U U m U A U U m U A U * r r02U U AmCmGr * r riU U U Am / CmG* r riU U U Am / CmG* r riU U U Am / Ui / m U A U Ui / CmG* r r U U U AmCmG* r r U U U AmCmGU:m r r m .m rm r r mm rm r r mm rm r r mm rm r r mmm r r mo*CU A AG / U A AG / U A AG / U A AG / U AGr / mGr / r r m U*Cr r m UG*Cr r m*C*CA*CU A ANmm U U AmU r r m U r r m U r r m U m U U ACFCF m m U U AmCF m U U AmCF m U U AmCF m U U AmCFtekcY31 o R1Y Y1 Y1 Y4RRDp2RR1RR0R1Y4R1 1ySR 7e_PpS1_ or_PpS1_ or PpS1PpSR 9PpSR 8Pv o_ _ oro_ _ oro_ _ or_ _ or55nr TA0oTA0ulT 0 T 0 T 0fA ulful oT 0o97ott 1DAEul _f 1D_E_4 1DE_ A41DE f_A41DEulfA1DEulf68A A _ A _ A _ A _ _ A _ _110246UUGGGA A A ACCGA A GCU A U A AACGCA AGU UCGCGAGUGA U UCU U 0436rGr U rGrGr r A A r r A A rCrCr AmGrGm A rCm U r m U A r m A A rCm A rCmGr m A A rGm UrGm m m / m m / m m / m m / m m A / CrGrGUrGA UrCrGUrGA UrCrGUrGA UrCrGUrGA UrCrGUrGA rmCrGmr / rCrmCrGmr / r rmCrGmrr rm rmrr rm rmrr rm rm U A U A U U A / U UC GA / U UC GA / U UC GA rCmGr m U rrCmr m Ur r mr m Ur r mr m Ur r mr m Ur r mr m A A F UGA A FC C GA A FC C GA A FC C GA A FC C GA A91rGm r m A A A2irCrGm r m A A A2ir UrGm r m A A A2ir UrGm r m A A A2ir m r m r m r m UrGA A A2iUrGA A A0r0r- UGr UmG / Cr / r UrGr UmG / Cr / r UrGr UmG / Cr / r UrGr UmG / Cr / r UrGr UmG / Cr / r UrGr UmG9r r r m U r r r m U r r r m U6A A UmGr r r m U r r r m U r r r m A AmGmGmGmG4*Gr r m mF2*GU r r m mF2*GA A U r r m mF2*GA A U r r m mF2*GA A U r r m mF2*GA A U r r m71m U A U U * r rim U A U Uim U A U Uim U A U U m U A U U m U A U mm / * r rm m / * r r / * r ri / * r ri / * r r02U U AC GU U U AC GU U U AmCmGU U U AmCmGU U U AmCmGU U U AmC:m r r m m r r m rGm r r m r m r r m o U A AmGr / U A AmGm UmGmGr m r r mmGr m r r m.*Cr r m*C / C*CA A / C*CU A A / C*CU A A / C*U A ANmCU r r mmC* r r mmCA r r mmr r mmCr r m m U U A F m U U A F A m U U A F m m U U ACF m U U ACF m U U AtekcY4Y1 Y1 15 5o R1RRY Y RRRRR1Y R1DpSR 7Pp2S1Pp1S1Pp0S1 pSR 9pSR 81vye_ _ or_ _ or_ _ or_P_or_P_or_P_or55nr TA0oTA0o ulT 0oT 0oT 0 T 0fulul o o97ott 1DAEul _f 1DE_ A51DE f_A51DE f_A51DEulfA1DEulf68A _ A _ A _ A _ A _ _ A _ _11UCU U UC CCCA A G UGGCCUCGAUGU A AUGUGACAAGUCA AACUCCAGGA U UGCGAGU A U G AGU UCGAAGCGU UGAC AU UUA GG GAGCUCGG UA ACCUGU A AGCU UGGCUCU A U UACUGGC64362irGr UrCm U2i / CrGrGrmG / CGU r r r r m r / Cm A A A A / *r r rmC G73FG2A A UGmFm2r rrm2*fi *C CUCiU o / U rm r m / m4U m AG G CU * r *7rm m r A1 / GAGArCA m / m rCmr mG* FGUC C2 / r m rF2 / Gi / F U A UmGF A2ir miA Ar / GmGA2ir / / CrCm r m r C A A U / Cr r / Cr F2UrCmGUmGFU r r m m2GrCriA A AG / UrCrGmiUrGm / A UrCr / r rm rmrr U UC GA / U U r FrCrCmGm U A A FrC9r m r m12iUrGA A A2ir U00 / -Cr / r r UrGUmG / Cr / 9mU r r6Gr mmU4mF2*GA A UGr r m mF271Uim U A U Ui02m / * r rm / GU U U ACmGU:.moGr m r r mmr / U A AGNmC*CCr r m / CF m U U AmCFtekcY5o R1DpR 71vySe_Pnr T_0or55o97ottA1DAEulf68A _ _11 Attorney Docket No.: 2017469-0019 In some embodiments, the 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 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, the backbone modification is one provided in EP 2813570, which is herein incorporated by reference in its entirety. In some embodiments, the modified cap is one provided in US Pat. Pub. No.20050287539, which is herein incorporated by reference in its entirety. In some embodiments, the 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, the 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, the 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)); 175 11867955v1 Attorney Docket No.: 2017469-0019 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, the nucleic acid (e.g., 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, 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, the nucleic acid comprises a backbone modification, e.g., a modification to a sugar or phosphate group in the backbone. In some embodiments, the nucleic acid comprises a nucleobase modification. In some embodiments, the nucleic acid comprises one or more chemically modified nucleotides of Table 16, one or more chemical backbone modifications of Table 17, one or more chemically modified caps of Table 18. For instance, in some embodiments, the nucleic acid comprises two or more (e.g., 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., 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 16. Alternatively or in combination, the nucleic acid may comprise two or more (e.g., 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 17. Alternatively or in combination, the nucleic acid may comprise one or more modified cap, e.g., as described herein, e.g., in Table 18. For instance, in some embodiments, the 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, the nucleic acid comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more) modified nucleobases. In some embodiments, all nucleobases of the nucleic acid are modified. In some embodiments, the nucleic acid is modified at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 176 of 237 11867955v1 Attorney Docket No.: 2017469-0019 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more) positions in the backbone. In some embodiments, all backbone positions of the nucleic acid are modified. Table 16. Modified nucleotides 5-aza-uridine N2-methyl-6-thio-guanosine 2-thio-5-aza-midine N2,N2-dimethyl-6-thio-guanosine 177 of 237 11867955v1 Attorney Docket No.: 2017469-0019 zebularine methylwyosine 5-aza-zebularine queuosine 5 thl bl i i Table 17. Backbone modifications 178 of 237 11867955v1 Attorney Docket No.: 2017469-0019 2’-O-Methyl backbone Peptide Nucleic Acid (PNA) backbone h h thi t b kb a e . o e caps m7GpppA m7GpppC The nucleotides comprising the template of the gene modifying system can be natural or modified bases, or a combination thereof. For example, the template may contain pseudouridine, dihydrouridine, inosine, 7-methylguanosine, or other modified bases. In some embodiments, the template may contain locked nucleic acid nucleotides. In some embodiments, the modified bases used in the template do not 179 of 237 11867955v1 Attorney Docket No.: 2017469-0019 inhibit the reverse transcription of the template. In some embodiments, the modified bases used in the template may improve reverse transcription, e.g., specificity or fidelity. In some embodiments, an RNA component of the system (e.g., a templ...

Claims

Attorney Docket No.: 2017469-0019 CLAIMS What is claimed is:

1. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence comprising a region of at least 5 contiguous nucleotides comprising 2’-fluoro modifications on alternating nucleotides, and (iv) a primer binding site (PBS) sequence.

2. The template RNA of claim 1, wherein the heterologous object sequence comprises 2’-fluoro modifications on alternating nucleotides starting from position +4 of the heterologous object sequence.

3. The template RNA of claim 2, wherein the heterologous object sequence comprises 2’-fluoro modifications on alternating nucleotides from position +4 through position +8, +10, +12, +14, +16, or +18 of the heterologous object sequence.

4. The template RNA of claim 1, wherein the heterologous object sequence comprises 2’-fluoro modifications on alternating nucleotides starting from position +5 of the heterologous object sequence.

5. The template RNA of claim 2, wherein the heterologous object sequence comprises 2’-fluoro modifications on alternating nucleotides from position +5 through position +9, +11, +13, +15, or +17 of the heterologous object sequence.

6. The template RNA of any of the preceding claims, wherein the heterologous object sequence further comprises a region between the region of at least 5 contiguous nucleotides and the PBS sequence that does not comprise 2’-fluoro modifications, e.g. comprises unmodified nucleotides.

7. The template RNA of claim 6, wherein the region that does not comprise 2’-fluoro modifications has a length of at least 2, 3, 4, 5, 6, 7, or 8 nucleotides.

8. The template RNA of claim 6, wherein the region that does not comprise 2’-fluoro modifications has a length of 1-3, 2-4, 3-5, 3-6, 5-7, 6-8, or 7-9 nucleotides. 226 of 237 11867955v1Attorney Docket No.: 2017469-0019 9. The template RNA of any one of the preceding claims, which comprises a 2’-fluoro modified nucleotide at the 5’ end of the heterologous object sequence (e.g., at position +10 or +11 of the heterologous object sequence).

10. The template RNA of any one of the preceding claims, which comprises 2’-fluoro modified nucleotides at positions +4, +6, +8, and / or +10 of the heterologous object sequence.

11. The template RNA of any one of the preceding claims, which comprises 2’-fluoro modified nucleotides at positions +5, +7, +9, and / or +11 of the heterologous object sequence.

12. The template RNA of any one of the preceding claims, wherein the second nucleotide from the 5’ end of the heterologous object sequence comprises a 2’-fluoro modification (e.g., position +9 or +10 of the heterologous object sequence).

13. The template RNA of any one of the preceding claims, which comprises 2’-fluoro modified nucleotides at positions +5, +7, and / or +9 of the heterologous object sequence.

14. The template RNA of any one of the preceding claims, which comprises 2’-fluoro modified nucleotides at positions +4, +6, +8, and / or +10 of the heterologous object sequence.

15. The template RNA of any one of the preceding claims, wherein the PBS sequence further comprises one or more of: a 2’-fluoro modified nucleotide, a 2’-OMe modified nucleotide, and one or more (e.g., 1, 2, or 3) nucleotides comprising a phosphorothioate modification.

16. The template RNA of claim 15, which comprises at the 3’ end, in 5’ to 3’ order, a 2’-fluoro modified nucleotide, a 2’-OMe modified nucleotide, and one or more (e.g., 1, 2, or 3) nucleotides comprising a phosphorothioate modification.

17. The template RNA of claim 15 or 16, wherein the one or more nucleotides comprising a phosphorothioate modification further comprise a 2’-OMe modification.

18. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, 227 of 237 11867955v1Attorney Docket No.: 2017469-0019 (ii) a gRNA scaffold, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence, wherein the template RNA comprises a region of at least 5 contiguous nucleotides comprising 2’- fluoro modifications on alternating nucleotides, optionally wherein the number of nucleotides between the 3’ most nucleotide comprising a 2’-fluoro modification in the region and the 3’ end of the template RNA is 8, 9, 10, 11, 12, 13, 14, or 15.

19. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence comprising a 2’-fluoro modified nucleotide.

20. The template RNA of claim 19, wherein the PBS sequence further comprises one or more (e.g. 1, 2, or 3) 2’-OMe modified nucleotides.

21. The template RNA of claim 20, wherein the 2’-fluoro modified nucleotide is adjacent to one of the one or more 2’-OMe modified nucleotides.

22. The template RNA of claim 20 or 21, wherein the 2’-fluoro modified nucleotide is positioned 5’ relative to the 2’-OMe modified nucleotide(s).

23. The template RNA of any one of claims 19-22, wherein the template RNA does not comprise any unmodified nucleotides 3’ of the 2’-fluoro modified nucleotide.

24. The template RNA of any one of claims 19-23, wherein the template RNA further comprises (e.g., at the 3’ end of the template RNA) one or more (e.g., 1, 2, or 3) nucleotides each comprising a phosphorothioate modification and a 2’-OMe modification.

25. The template RNA of any one of claims 19-24, wherein the number of nucleotides between the 2’- fluoro modified nucleotide and the 3’ end of the template RNA is 2, 3, 4, 5, 6, 7, 8, 9, or 10. 228 of 237 11867955v1Attorney Docket No.: 2017469-0019 26. The template RNA of any one of claims 19-25, wherein the number of nucleotides between the 2’- fluoro modified nucleotide and the 3’ end of the template RNA is at least 4.

27. The template RNA of any one of claims 19-26, wherein the number of nucleotides between the 2’- fluoro modified nucleotide and the 3’ end of the template RNA is 4.

28. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence comprising one or more (e.g., 1, 2, or 3) 2’-OMe modified nucleotides.

29. The template RNA of claim 28, wherein the PBS sequence further comprises a 2’-fluoro modified nucleotide.

30. The template RNA of claim 29, wherein the 2’-fluoro modified nucleotide is adjacent to one of the one or more 2’-OMe modified nucleotides.

31. The template RNA of claim 29 or 30, wherein the 2’-fluoro modified nucleotide is positioned 5’ relative to the 2’-OMe modified nucleotide(s).

32. The template RNA of any one of claims 28-31, wherein the template RNA does not comprise any unmodified nucleotides 3’ of the 2’-OMe modified nucleotides.

33. The template RNA of any one of claims 28-32, wherein the template RNA further comprises (e.g., at the 3’ end of the template RNA) one or more (e.g., 1, 2, or 3) nucleotides each comprising a phosphorothioate modification and a 2’-OMe modification.

34. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence comprising one or more (e.g., 1, 2, or 3) 2’-OMe modified nucleotides, and 229 of 237 11867955v1Attorney Docket No.: 2017469-0019 (iv) a primer binding site (PBS) sequence.

35. The template RNA of claim 34, wherein the heterologous object sequence comprises a plurality of 2’-OMe modified nucleotides (e.g., 2, 3, 4, or 52’-OMe modified nucleotides) positioned adjacent to each other.

36. The template RNA of claim 35, wherein the plurality of 2’-OMe modified nucleotides are at least 1, 2, 3, 4, or 5 nucleotides from the 3’ end of the heterologous object sequence.

37. The template RNA of claim 35, wherein the plurality of 2’-OMe modified nucleotides are less than 10, 9, 8, 7, 6, or 6 nucleotides from the 3’ end of the heterologous object sequence.

38. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence comprising one or more (e.g., 1, 2, or 3) 2’-OMe modified nucleotides.

39. The template RNA of claim 38, wherein the PBS sequence comprises a plurality of 2’-OMe modified nucleotides (e.g., 2, 3, 4, or 52’-OMe modified nucleotides) positioned adjacent to each other.

40. The template RNA of claim 39, wherein the plurality of 2’-OMe modified nucleotides are at least 1, 2, 3, 4, or 5 nucleotides from the 5’ end of the PBS sequence.

41. The template RNA of any one of claims 38-40, wherein the 2’-OMe modified nucleotides further comprise a phosphorothioate modification.

42. The template RNA of claim 38, wherein nucleotides -4, -5, -6, -7, -8, -9, and -10 of the PBS sequence comprise a 2’-OMe modification and / or a phosphorothioate modification.

43. The template RNA of claim 38, wherein nucleotides -5, -6, -7, -8, -9, and -10 of the PBS sequence comprise a 2’-OMe modification and / or a phosphorothioate modification. 230 of 237 11867955v1Attorney Docket No.: 2017469-0019 44. The template RNA of claim 42 or 43, wherein nucleotide -10 of the PBS sequence is at the 3’ end of the template RNA.

45. The template RNA of any one of claims 34-44, which does not comprise a 2’-OMe modified nucleotide at position +1 of the heterologous object sequence or -1 of the PBS sequence.

46. The template RNA of claim 45, which does not comprise a 2’-OMe modified nucleotide at positions -2 or -1 of the PBS sequence or, +1 or +2 of the heterologous object sequence.

47. The template RNA of any one of claims 34-46, wherein the nucleotides at positions -1 of the PBS sequence and +1 of the heterologous object sequence are unmodified nucleotides.

48. The template RNA of claim 47, wherein the nucleotides at positions -2 or -1 of the PBS sequence and +1 or and +2 of the heterologous object sequence are unmodified nucleotides.

49. The template RNA of any one of claims 34-48, wherein the nucleotides at positions -6 to -1, -5 to -1, -4 to -1, -3 to -1, or -2 to -1of the PBS sequence are unmodified nucleotides.

50. The template RNA of any one of claims 34-49, wherein the heterologous object sequence and / or PBS sequence comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified nucleotides.

51. The template RNA of any one of claims 34-50, wherein the heterologous object sequence and / or PBS sequence comprise 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 unmodified nucleotides.

52. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence comprising one or more (e.g., 1, 2, or 3) 2’-fluoro modified nucleotides, and (iv) a primer binding site (PBS) sequence. 231 of 237 11867955v1Attorney Docket No.: 2017469-0019 53. The template RNA of claim 52, wherein the heterologous object sequence comprises a plurality of 2’-fluoro modified nucleotides (e.g., 2, 3, 4, or 52’-fluoro modified nucleotides) positioned adjacent to each other.

54. The template RNA of claim 53, wherein the plurality of 2’-fluoro modified nucleotides are at least 1, 2, 3, 4, or 5 nucleotides from the 3’ end of the heterologous object sequence.

55. The template RNA of claim 52, wherein the heterologous object sequence comprises a plurality of 2’-fluoro modified nucleotides (e.g., 2, 3, 4, or 52’-fluoro modified nucleotides) alternating with a plurality of nucleotides lacking a 2’-fluoro modification.

56. The template RNA of any one of claims 52-55, wherein the nucleotide at the 5’ end of the heterologous object sequence (e.g., at position +10 or +11) comprises a 2’-fluoro modification.

57. The template RNA of claim 56, which comprises one or more (e.g., 1, 2, 3, 4, or 5) 2’-fluoro modifications on alternating nucleotides after the 2’-fluoro modified nucleotide at the 5’ end of the heterologous object sequence.

58. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence comprising one or more (e.g., 1, 2, or 3) 2’-fluoro modified nucleotides.

59. The template RNA of claim 58, wherein the PBS sequence comprises a plurality of 2’-fluoro modified nucleotides (e.g., 1, 2, 3, 4, or 52’-fluoro modified nucleotides) positioned adjacent to each other.

60. The template RNA of claim 59, wherein the plurality of 2’-fluoro modified nucleotides are at least 1, 2, 3, 4, or 5 nucleotides from the 5’ end of the PBS sequence.

61. A template RNA comprising, from 5’ to 3’: (i) a gRNA spacer, (ii) a gRNA scaffold, 232 of 237 11867955v1Attorney Docket No.: 2017469-0019 (iii) a heterologous object sequence, and (iv) a primer binding site (PBS) sequence, wherein the template RNA comprises one or more (e.g., 1, 2, or 3) 2’-fluoro modified nucleotides (e.g., one or more adjacent nucleotides) comprising 2’-fluoro modifications, optionally wherein the number of nucleotides between the one of the nucleotides comprising a 2’- fluoro modification and the 3’ end of the template RNA is 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, or 29.

62. The template RNA of any one of claims 52-61, wherein the PBS sequence comprises one or more (e.g., 1, 2, or 3) phosphorothioate-modified nucleotides, e.g., at the 3’ end of the PBS sequence.

63. The template RNA of any one of claims 52-62, wherein the PBS sequence comprises one or more (e.g., 1, 2, or 3) 2’-OMe-modified nucleotides, e.g., at the 3’ end of the PBS sequence.

64. The template RNA of any one of claims 52-63, wherein the PBS sequence comprises one or more (e.g., 1, 2, or 3) nucleotides each comprising phosphorothioate and 2’-OMe modifications, e.g., at the 3’ end of the PBS sequence.

65. The template RNA of any one of claims 52-64, which does not comprise a 2’-fluoro modified nucleotide at positions -1 of the PBS sequence or +1 of the heterologous object sequence.

66. The template RNA of claim 65, which does not comprise a 2’-fluoro modified nucleotide at positions -2 or -1 of the PBS sequence or +1 or +2 of the heterologous object sequence.

67. The template RNA of any one of claims 52-66, wherein the nucleotides at position -1 of the PBS sequence and +1 of the heterologous object sequence are unmodified nucleotides.

68. The template RNA of claim 67, wherein the nucleotides at positions -2 or -1 of the PBS sequence or +1 of +2 of the heterologous object sequence are unmodified nucleotides.

69. The template RNA of any one of claims 52-68, wherein the heterologous object sequence and / or PBS sequence comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified nucleotides. 233 of 237 11867955v1Attorney Docket No.: 2017469-0019 70. The template RNA of any one of claims 52-69, wherein the heterologous object sequence and / or PBS sequence comprise 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 unmodified nucleotides.

71. The template RNA of any one of the preceding claims, wherein the gRNA scaffold binds a gene modifying polypeptide (e.g., binds a Cas domain, e.g., a Cas9 domain of the gene modifying polypeptide).

72. The template RNA of any one of the preceding claims, wherein the heterologous object sequence comprises a mutation region to introduce a mutation into (e.g., to correct a mutation in) a portion (e.g., a second portion) of the human PAH, FAH, HBB, TRAC4, B2M, or A1AT gene (wherein optionally the heterologous object sequence comprises, from 5’ to 3’, a post-edit homology region, a mutation region, and a pre-edit homology region) 73. The template RNA of any one of the preceding claims, which comprises at least 5, 6, 7, or 8 bases with 100% identity to a third portion of the human PAH, FAH, HBB, TRAC4, B2M, or A1AT gene.

74. The template RNA of any one of the preceding claims, which does not comprise a 2’-fluoro modified nucleotide at position -1 of the PBS sequence or +1 of the heterologous object sequence.

75. The template RNA of any one of the preceding claims, which does not comprise a 2’-fluoro modified nucleotide at positions -2 or -1 of the PBS sequence or +1 or +2 of the heterologous object sequence.

76. The template RNA of any one of the preceding claims, which does not comprise a 2’-OMe modified nucleotide at position -1 of the PBS sequence or +1 of the heterologous object sequence and the PBS sequence.

77. The template RNA of any one of the preceding claims, which does not comprise a 2’-OMe modified nucleotide at position -2 or -1 of the PBS sequence or +1 or +2 of the heterologous object sequence.

78. The template RNA of any one of the preceding claims, wherein the nucleotides at position -1 of the PBS sequence and +1 of the heterologous object sequence are unmodified nucleotides. 234 of 237 11867955v1Attorney Docket No.: 2017469-0019 79. The template RNA of any one of the preceding claims, wherein the nucleotides at positions -2 and -1 of the PBS sequence and +1 and +2 of the heterologous object sequence are unmodified nucleotides.

80. The template RNA of any one of the preceding claims, wherein the 3’ end of the PBS sequence comprises one or more of: a 2’-fluoro modified nucleotide, a 2’-OMe modified nucleotide, and one or more (e.g., 1, 2, or 3) nucleotides comprising a phosphorothioate modification.

81. The template RNA of any one of the preceding claims, wherein the 3’ end of the PBS sequence comprises, in 5’ to 3’ order, a 2’-fluoro modified nucleotide, one or more (e.g., 1, 2, 3, 4, or 5) 2’-OMe modified nucleotides, and one or more (e.g., 1, 2, 3, 4, or 5) nucleotides comprising a phosphorothioate modification.

82. The template RNA of any one of the preceding claims, wherein one or more (e.g., 1, 2, 3, 4, or 5) of the nucleotides comprising a phosphorothioate modification further comprise a 2’-OMe modification.

83. The template RNA of any one of the preceding claims, which comprises a polynucleotide as listed in column 3 of any of Tables 12-14 or column 2 of Table 15, or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

84. The template RNA of claim 83, which comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) the nucleotide modifications for the polynucleotide as listed in column 3 of any of Tables 12-14 or column 2 of Table 15.

85. The template RNA of claim 83 or 84, which comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) the 2’-OMe modifications for the polynucleotide as listed in column 3 of any of Tables 12-14 or column 2 of Table 15.

86. The template RNA of claim 83 or 84, which comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of) the 2’-fluoro modifications for the polynucleotide as listed in column 3 of any of Tables 12- 14 or column 2 of Table 15.

87. The template RNA of any one of the preceding claims, which is capable of introducing an alteration (e.g., a nucleic acid substitution, deletion, or insertion) into at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 235 of 237 11867955v1Attorney Docket No.: 2017469-0019 60%, 65%, 70%, 75%, 80%, 85%, or 90% target nucleic acid molecules (e.g., genomic DNA) in a population of said target nucleic acid molecules (e.g., a population of cells comprising said genomic DNA).

88. A gene modifying system comprising: (i) a template RNA of any of the preceding claims, and (ii) a gene modifying polypeptide (e.g., as described herein), or a nucleic acid (e.g., RNA) encoding the gene modifying polypeptide.

89. A method for modifying a target site in a nucleic acid molecule (e.g., genomic DNA) in a cell, the method comprising contacting the cell with the gene modifying system of claim 88, or DNA encoding the same, thereby modifying the target site in the nucleic acid molecule in the cell. 236 of 237 11867955v1

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