Methods and compositions for regulating genomes

Abstract

To provide methods and compositions for regulating genomes. [Solution] Methods and compositions for modulating a target genome are disclosed. The disclosure relates to compositions, systems and methods for targeting, editing, modifying or manipulating a DNA sequence at one or more locations within a DNA sequence in a cell, tissue or subject, for example, in vivo or in vitro (e.g., inserting a heterologous target sequence into a target site in a mammalian genome). The heterologous target DNA sequence may include, for example, substitutions, deletions, insertions, such as coding sequences, regulatory sequences or gene expression units.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Patent Application No. 62 / 985,285 filed on 4 May 2020, U.S. Patent Application No. 63 / 035,627 filed on 5 June 2020, and U.S. Patent Application No. 63 / 067,828 filed on 19 August 2020, the entire contents of each of those applications being incorporated herein by reference. [Background technology]

[0002] The insertion of target nucleic acids into the genome occurs infrequently and with little site specificity, especially in the absence of specialized proteins to facilitate insertion events. Some existing methods, such as CRISPR / Cas9, are better suited to small edits dependent on host repair pathways and are less effective for inserting longer sequences. Other existing methods, such as Cre / loxP, require a first step of inserting a loxP site into the genome, followed by a second step of inserting the target sequence into the loxP site. In this art, there is a need for improved compositions (e.g., proteins and nucleic acids) and methods for inserting, modifying, or deleting target sequences within the genome. [Overview of the Initiative] [Means for solving the problem]

[0003] This disclosure relates to novel compositions, systems, and methods for modifying the genome at one or more locations in a host cell, tissue, or subject in vivo or in vitro. In particular, the present invention features compositions, systems, and methods for inserting, modifying, or deleting a sequence of interest into the host genome.

[0004] The composition or method may include one or more of the embodiments listed below.

[0005] List of embodiments 1. A system for modifying DNA, (a) polypeptides or nucleic acids encoding polypeptides, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome) (e.g., a CRISPR spacer), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system that includes this.

[0006] 2. A system for modifying DNA, (a) polypeptides or nucleic acids encoding polypeptides, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. Includes; (i) The polypeptide contains a heterologous targeting domain (e.g., within a DBD or endonuclease domain) that specifically binds to a sequence contained within the target site; and / or (ii) A system in which the template RNA contains heterologous sequences that have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to sequences contained within the target site.

[0007] 3. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising, wherein the RT domain contains the sequence of Table 1 or 3, or the reverse transcriptase domain sequence of Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0008] 4. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. Includes, The RT domain includes the sequence in Table 1 or 3, or the reverse transcriptase domain sequence in Table 2. The RT domain is a system that further includes several substitutions to the natural sequence, e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions.

[0009] 5. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising, capable of causing the insertion of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides into a target site.

[0010] 6. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising, capable of causing the insertion of at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 44 nucleotides into a target site.

[0011] 7. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising heterologous target sequences having a length of at least 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 nts.

[0012] 8. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising a heterologous target sequence having a length of at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, or 73 nucleotides.

[0013] 9. A system of any of the preceding embodiments in which the RT domain is heterogeneous to the DBD; the DBD is heterogeneous to the endonuclease domain; or the RT domain is heterogeneous to the endonuclease domain, one or more of the above.

[0014] 10. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising a mechanism capable of causing deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides at a target site.

[0015] 11. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising a mechanism capable of causing a deletion of at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, or 80 nucleotides at a target site.

[0016] 12. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising a system capable of causing nucleotide substitutions, e.g., transpositions and / or conversions, of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides to a target site.

[0017] 13. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template (or DNA encoding template RNA) containing a 3' target homology domain. A system comprising (a)(ii) and / or (a)(iii) a TAL domain; a zinc finger domain; or a CRISPR / Cas domain selected from Table 4 or a functional variant thereof (e.g., a variant).

[0018] 14. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome) (e.g., a CRISPR spacer), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising an endonuclease domain, such as a nickasase domain, which cleaves both the first and second strands of target site DNA, wherein the cleavage is separated from each other by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 nucleotides.

[0019] 15. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) a sequence that specifically binds to the RT domain, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system that includes this.

[0020] 16. A system according to any of the preceding embodiments, wherein the template RNA further comprises sequences that bind to (a)(ii) and / or (a)(iii).

[0021] 17. A system for modifying DNA, (a) A first polypeptide or nucleic acid encoding a first polypeptide, wherein the first polypeptide comprises (i) a reverse transcriptase (RT) domain and (ii) optionally a DNA-binding domain, (b) a second polypeptide or a nucleic acid encoding a second polypeptide, the second polypeptide comprising (i) a DNA-binding domain (DBD); (ii) an endonuclease domain, such as a niccasse domain; and (c) (e.g., from 5' to 3') (i) optionally a sequence that binds to a second polypeptide (e.g., (b)(i) and / or (b)(ii)), (ii) optionally a sequence that binds to a first polypeptide (e.g., specifically to the RT domain), (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding template RNA) containing a 3' target homology domain. A system that includes this.

[0022] 18. A system for modifying DNA, (a) a polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain and (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; (b) A first template RNA (or RNA-coding DNA) (for example, where the first RNA includes gRNA) comprising (i) a sequence that binds to a polypeptide (e.g., from 5' to 3') (i) a sequence that binds to (a)(ii) and / or (a)(iii) and (ii) a sequence that binds to a target site (e.g., the second strand of the site in the target genome); (c) (e.g., from 5' to 3') (i) optionally, a sequence that binds to the polypeptide (e.g., specifically to the RT domain), (ii) a heterologous target sequence, and (iii) a second template RNA (or RNA-coding DNA) containing a 3' target homology domain. A system that includes this.

[0023] 19. A system according to any of the preceding embodiments, wherein the second template RNA comprises (i).

[0024] 20. A system according to any of the preceding embodiments, wherein the first template RNA comprises a first conjugate domain and the second template RNA comprises a second conjugate domain.

[0025] 21. The first and second conjugate domains can hybridize with each other, for example, under stringent conditions, such as any of the systems of the previous embodiments, in which the stringent conditions for hybridization include hybridization in 4 × sodium chloride / sodium citrate (SSC) at about 65°C followed by washing in 1 × SSC at about 65°C.

[0026] 22. The first and second conjugate domains can be covalently joined, for example, by sprint ligation, as described by Moore, MJ, & Query, CCMethods in Enzymology, 317, 109-123, 2000, in any of the systems of the preceding embodiments.

[0027] 23. A system according to any of the previous embodiments, wherein the association of the first conjugate domain and the second conjugate domain causes colocalization of the first template RNA and the second template RNA.

[0028] 24. A system according to any of the preceding embodiments, wherein the reverse transcriptase (RT) domain is derived from a retrotransposon or is a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0029] 25. A system for modifying DNA, (a) polypeptides or nucleic acids encoding polypeptides, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain derived from a retrotransposon or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome) (e.g., a CRISPR spacer), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system that includes this.

[0030] 26. The template RNA is a system of any of the preceding embodiments, comprising (i).

[0031] 27. The template RNA is a system of any of the preceding embodiments, comprising (ii).

[0032] 28. The template RNA is a system of either of the preceding embodiments, comprising (i) and (ii).

[0033] 29. A system according to any of the preceding embodiments, wherein the reverse transcriptase domain comprises an amino acid sequence that conforms to any of the reverse transcriptase domains in Table 30, Table 31, Table 41, Table 44, or Table 50, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or a functional fragment thereof.

[0034] 30. A template RNA (or DNA encoding template RNA) containing a sequence within a target DNA molecule (e.g., genomic DNA), a sequence that specifically binds to the RT domain of a polypeptide, and a targeting domain that specifically binds to a heterologous target sequence (e.g., a heterologous targeting domain).

[0035] 31. (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to the endonuclease and / or DNA-binding domain of the polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain.

[0036] A template RNA of any of the previous embodiments, including 32.(i).

[0037] A template RNA of any of the preceding embodiments, including 33.(ii).

[0038] 34. A template RNA (or DNA encoding a template RNA) comprising (i) a sequence that binds to a target site (e.g., the second strand of a site in the target genome) (5' to 3'), (ii) a sequence that binds to the endonuclease and / or DNA-binding domain of a polypeptide, (iii) a heterologous target sequence, and (iv) a 3' target homology domain, wherein (i) comprises a nucleic acid sequence that is complementary to any of the gene sequences in Tables 9 to 12, or that differs from the complementary sequence by 1, 2, 3, 4 or 5 or fewer.

[0039] 35. (For example, from 5' to 3') (i) a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) a sequence that specifically binds to the RT domain of the polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain.

[0040] 36.(v) A template RNA of any of the preceding embodiments, further comprising a sequence that binds to the DNA-binding domain of an endonuclease and / or polypeptide (e.g., the same polypeptide including an RT domain).

[0041] 37. The RT domain is a template RNA according to any of the preceding embodiments, comprising a selected sequence from Table 1 or Table 3, or a reverse transcriptase domain sequence from Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0042] 38. The RT domain comprises a selected sequence from Table 1 or Table 3 or a reverse transcriptase domain sequence from Table 2, wherein the RT domain further comprises several substitutions relative to the native sequence, e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions, as in any of the preceding embodiments of template RNA.

[0043] The sequence in 39.(ii) is a template RNA from any of the previous embodiments that specifically binds to the RT domain.

[0044] 40. A template RNA of any of the previous embodiments, wherein the sequence that specifically binds to the RT domain is a sequence from the domains in Table 1 or Table 2, for example, a UTR sequence or a sequence having at least 70, 75, 80, 85, 90, 95, or 99% identity thereto.

[0045] 41.5' to 3': (ii) a sequence that binds to the endonuclease and / or DNA-binding domain of the polypeptide, (i) a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing the 3' target homology domain.

[0046] 42.5' to 3': (iii) heterologous target sequence, (iv) 3' target homology domain, (i) sequence that binds to a target site (e.g., the second strand of the site in the target genome), and (ii) template RNA (or DNA encoding template RNA) comprising a sequence that binds to the endonuclease and / or DNA-binding domain of the polypeptide.

[0047] 43. The template RNA, first template RNA, or second template RNA is a system or template RNA of any of the preceding embodiments, comprising a sequence that specifically binds to the RT domain.

[0048] 44. The sequence that specifically binds to the RT domain is the system or template RNA of any of the previous embodiments, located between (i) and (ii).

[0049] 45. The sequence that specifically binds to the RT domain is the system or template RNA of any of the previous embodiments, located between (ii) and (iii).

[0050] 46. ​​The sequence that specifically binds to the RT domain is the system or template RNA of any of the previous embodiments, located between (iii) and (iv).

[0051] 47. The sequence that specifically binds to the RT domain is the system or template RNA of any of the previous embodiments, located between (iv) and (i).

[0052] 48. The sequence that specifically binds to the RT domain is the system or template RNA of any of the previous embodiments, located between (i) and (iii).

[0053] 49. A system for modifying DNA, (a)(i) a sequence that binds to the endonuclease domain of a polypeptide, e.g., the nickase domain and / or the DNA-binding domain (DBD), and (ii) a sequence that binds to a target site (e.g., the second strand of the site in the target genome) (or DNA encoding the first template RNA) (e.g., where the first RNA includes gRNA); (b) (i) a sequence that specifically binds to the reverse transcriptase (RT) domain of a polypeptide (e.g., the polypeptide of (a)), (ii) a heterologous target sequence, and (iii) a second template RNA (or DNA encoding the second template RNA) comprising a 3' target homology domain. A system that includes this.

[0054] 50. A system according to any of the previous embodiments, wherein the nucleic acid encoding the first template RNA and the nucleic acid encoding the second template RNA are two separate nucleic acids.

[0055] 51. A system according to any of the previous embodiments, wherein the nucleic acid encoding the first template RNA and the nucleic acid encoding the second template RNA are parts of the same nucleic acid molecule and, for example, reside on the same vector.

[0056] 52. A system according to any of the preceding embodiments that can cause the insertion of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides into a target site.

[0057] 53. A system of any of the preceding embodiments in which the heterologous target sequence has a length of at least 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 nts.

[0058] 54. Any system of the preceding embodiment that can cause deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides at a target site.

[0059] 55. A system of any of the preceding embodiments, wherein one or both of the template RNA and the RNA encoding the polypeptide of (a) include a chemically modified mRNA, for example, mRNA containing a chemically modified base, for example, mRNA containing 5-methoxyuridine.

[0060] 56. A system of any of the preceding embodiments, wherein one or both of the template RNA and the RNA encoding the polypeptide of (a) include a chemically modified RNA, for example, RNA containing a chemically modified base, for example, RNA containing a 2'-o-methylphosphorothioate.

[0061] 57. A system according to any of the preceding embodiments, wherein one or both of the template RNA and the RNA encoding the polypeptide of (a) contain a chemically modified RNA, for example, RNA containing a chemically modified base, for example, 2'-o-methylphosphorothioate, in one or both of the 3, 4, or 5 bases at the 5' or 3' end of the RNA.

[0062] 58. A polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain; the DBD and / or endonuclease domain comprises a heterologous targeting domain that specifically binds to a sequence contained within a target DNA molecule (e.g., genomic DNA).

[0063] 59. A polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain, and the RT domain has a sequence of Table 1 or 3 or a sequence of reverse transcriptase domain of Table 2 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0064] 60. A polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a nickasase domain, the RT domain having a sequence of Table 1 or 3 or a reverse transcriptase domain sequence of Table 2, and the RT domain further comprising several substitutions relative to the native sequence, such as at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions.

[0065] 61. A polypeptide according to any of the preceding embodiments, encoded by mRNA, for example, chemically modified mRNA, for example, mRNA containing a chemically modified base, for example, mRNA containing 5-methoxyuridine.

[0066] 62. A polypeptide according to any of the preceding embodiments, encoded by mRNA, for example, chemically modified mRNA, for example, mRNA containing a chemically modified base, for example, mRNA containing N1-methyl-pseudridine.

[0067] 63. A system for modifying DNA, (a) A first polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide is a reverse transcriptase (RT) domain having a sequence of Table 1 or 3 or a sequence of Table 2 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and optionally comprising a DNA-binding domain (DBD) (e.g., a first DBD); and (b) A second polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a DBD (e.g., a second DBD); and (ii) an endonuclease domain, e.g., a niccasse domain. A system that includes this.

[0068] 64. A system for modifying DNA, (a) A first polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises a reverse transcriptase (RT) domain having the sequence of Table 1 or 3 or the reverse transcriptase domain sequence of Table 2, and comprising several substitutions relative to the native sequence, e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions, and optionally further comprising a DNA-binding domain (DBD) (e.g., a first DBD); and (b) A second polypeptide or nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a DBD (e.g., a second DBD); and (ii) an endonuclease domain, e.g., a niccasse domain. A system that includes this.

[0069] 65. A system according to any of the previous embodiments, wherein the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are two separate nucleic acids.

[0070] 66. A system according to any of the previous embodiments, in which the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are parts of the same nucleic acid molecule and, for example, reside on the same vector.

[0071] 67. A reaction mixture comprising cells and any system, polypeptide, template RNA or DNA encoding it, as described in any of the preceding embodiments.

[0072] 68. A reaction mixture comprising DNA containing a target site and any system, polypeptide, template RNA, or DNA encoding it, as described in any of the previous embodiments.

[0073] 69. A system, polypeptide, template RNA, or DNA encoding it according to any of the preceding embodiments; Instructions for using the system, polypeptides, template RNA or DNA encoding them; and One or both of the cells or DNA containing the target site A kit that includes this.

[0074] 70. DBD is a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising a TAL domain.

[0075] 71. DBD is a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising a zinc finger domain.

[0076] 72. DBD is a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising a CRISPR / Cas domain.

[0077] 73. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the endonuclease domain is a nickasase domain.

[0078] 74. The endonuclease domain is a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising a CRISPR / Cas domain.

[0079] 75. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising a CRISPR / Cas domain, a domain or polypeptide from Table 4, or a functional variant thereof (e.g., a mutant).

[0080] 76. A CRISPR / Cas domain comprising a domain or polypeptide from a genus / species in Table 4, as a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0081] 77. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising an endonuclease domain including a type IIs nuclease (e.g., FokI), a Holliday junction resolver, or a double-stranded DNA nuclease with modifications that inhibit its ability to cleave a single strand (e.g., converting a double-stranded DNA nuclease to a nickase).

[0082] 78. The RT domain comprises a reverse transcriptase selected from Table 1 or 3, or a functional fragment or variant thereof, or a sequence of a reverse transcriptase domain from Table 2, as a system, kit, polypeptide, or reaction mixture of any of the previous embodiments.

[0083] 79. The RT domain comprises a naturally occurring RT domain or an RT domain or functional fragment selected from Table 1 or 3, or a sequence of a reverse transcriptase domain from Table 2, or one or more mutations (e.g., insertion, deletion, or substitution) in sequence numbers 1-67 of the sequence listing in International Publication No. 2018089860A1, which is incorporated herein by reference, in any of the systems, kits, polypeptides, or reaction mixtures of the preceding embodiments.

[0084] 80. One or more mutations are selected from 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 mouse leukemia virus reverse transcriptase, or from corresponding mutations at corresponding positions in another RT domain, according to any of the preceding embodiments, a system, kit, polypeptide, or reaction mixture.

[0085] 81. One or more mutations in the RT domain of R2Bm retrotransposase, as incorporated herein by reference in International Publication No. 2018089860A1 (e.g., C952S and / or C956S and / or C952S, C956S (double variant) and / or C969S and / or H970Y and / or R979Q and / or R976Q and / or R1071S and / or R328A and / or R329A, and / or Q336A, and / or R328A, R329A, Q336A (triple mutant), and / or G426A, and / or D428A, and / or G426A, D428A (double mutant) mutations and / or any combination thereof; a system, kit, polypeptide, or reaction mixture of any of the prior embodiments, selected from the corresponding mutations at the position relative to Sequence ID No. 52 in International Publication No. 2018089860A1 or at the corresponding position of another RT domain.

[0086] 82. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising a DBD and / or endonuclease domain (e.g., CRISPR / Cas domain) as a domain or polypeptide or a functional variant thereof (e.g., a mutant) as listed in Table 4.

[0087] 83. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising a DBD and / or endonuclease domain (e.g., a CRISPR / Cas domain) as a domain or polypeptide listed in Table 4.

[0088] 84. The RT domain and the DBD and / or endonuclease domain (e.g., CRISPR / Cas domain) are fused via a peptide linker, e.g., a linker in Table 42, in any of the systems, kits, polypeptides, or reaction mixtures of the preceding embodiments.

[0089] 85. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the linker has a length of approximately 6–18, 8–16, 10–14, or 12 amino acids.

[0090] 86. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the linker comprises glycine and serine, for example, the linker comprises a glycine residue and a serine residue individually, for example, the linker comprises the sequence GSSGSS.

[0091] 87. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the linker comprises a sequence according to Table 42, for example, a sequence linked to 10 disclosed in Table 42, or a sequence having one, two, or three or fewer substitutions thereto.

[0092] 88. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising a CRISPR / Cas domain, Cas9, e.g., wild-type Cas9 or nickase Cas9.

[0093] 89. The RT domain is located at the C-terminus of the DBD in the polypeptide, in any of the systems, kits, polypeptides, or reaction mixtures of the preceding embodiments.

[0094] 90. The RT domain is located at the C-terminus of the nickase domain in the polypeptide, in any of the systems, kits, polypeptides, or reaction mixtures of the preceding embodiments.

[0095] 91. The RT domain is located at the N-terminus of the DBD in the polypeptide, in any of the systems, kits, polypeptides, or reaction mixtures of the preceding embodiments.

[0096] 92. The RT domain is located at the N-terminus of the nickase domain in the polypeptide, in any of the systems, kits, polypeptides, or reaction mixtures of the preceding embodiments.

[0097] 93. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising, for example, a linker located between the RT domain and the DBD or between the RT domain and the nickase domain.

[0098] 94. The linker is an amino acid with a length of 2 to 50, for example, 2 to 30, as in any of the systems, kits, polypeptides, or reaction mixtures of the preceding embodiments.

[0099] 95. The linker is a flexible linker comprising, for example, Gly and / or Ser residues, the system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0100] A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the 96.3' target homology domain is complementary to a sequence adjacent to the site modified by the system, or includes one, two, three, four, or five mismatches with a sequence complementary to a sequence adjacent to the site modified by the system.

[0101] 97.3' target homology domains are >4, >5, >6, >7, >8, >9, >10, >11, >12, >13, >14, >15, >16, >17, >18, >19, >20, >21, >22, >23, 24+, 25+, 26+, 27+, 28+, 29+, 30+, 35+, 40+, 45+, 50+, 55+, 60+, 65+, 70+, 75+, 80+, 85+, 90+, 95+, 100+, 110+, A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, having a nucleotide length greater than 120, greater than 130, greater than 140, greater than 150, greater than 160, greater than 170, greater than 180, greater than 190, or greater than 200 (for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotide lengths).

[0102] 98.3' The target homology domain has a nucleotide length of 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 21 or less, 22 or less, 23 or less, 24 or less, 25 or less, 26 or less, 27 or less, 28 or less, 29 or less, 30 or less, 35 or less, 40 or less, 45 or less, 50 or less, 55 or less, 60 or less, 65 or less, 70 or less, 75 or less, 80 or less, 85 or less, 90 or less, 95 or less, 100 or less, 110 or less, 120 or less, 130 or less, 140 or less, 150 or less, 160 or less, 170 or less, 180 or less, 190 or less, or 200 or less, according to any of the above embodiments, a system, kit, template RNA, or reaction mixture.

[0103] 99. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous target sequence is complementary to the sites modified by the system, except at one or more sites that are modified.

[0104] 100. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous target sequence is complementary to the site modified by the system, except at the site encoding the sequence to be inserted into the site.

[0105] 101. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous target sequence is complementary to the site modified by the system, except that the heterologous target sequence does not contain a nucleotide encoding the sequence to be deleted at that site.

[0106] 102. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous target sequence has a nucleotide length greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotide lengths).

[0107] 103. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous target sequence has a nucleotide length of 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 21 or less, 22 or less, 23 or less, 24 or less, 25 or less, 26 or less, 27 or less, 28 or less, 29 or less, or 30 or less.

[0108] 104. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous target sequence is one in which the non-target site nucleotides are replaced with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

[0109] 105. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, in which a heterologous target sequence inserts 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 or 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 into the target site.

[0110] 106. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous target sequence deletes 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, 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides.

[0111] 107. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous target sequence is isolated from a polypeptide-binding sequence (e.g., an endonuclease domain and / or DBD domain) by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nucleotides from the sequence.

[0112] 108. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds to the polypeptide (e.g., to the endonuclease domain and / or DBD domain) is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130 nucleotide lengths (and optionally 150 or less, 140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 85 or less, or 80 or less).

[0113] 109. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the polypeptide-binding sequence binds to the endonuclease domain and / or DBD domain.

[0114] 110. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds to the polypeptide is a sequence that follows one or both of the predicted 5'UTR and / or predicted 3'UTR of Table 3 or Table 41, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or a functional fragment thereof.

[0115] 111. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, comprising a sequence that binds to a polypeptide (e.g., to an endonuclease domain and / or DBD domain), including a gRNA.

[0116] 112. The sequence that binds to the target site (e.g., the second strand of the site in the target genome) is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130 nucleotides long (and optionally 150 or less, 140 or less, 130 or less, 120 or less, 110 or less). A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, having a nucleotide length of 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, or 20 or less, for example, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides.

[0117] 113. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds to the target site is complementary to the second strand of the target site, or includes one, two, three, four, or five mismatches with a sequence that is complementary to the second strand of the target site.

[0118] 114. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds to a target site (e.g., the second strand of the site in the target genome) is isolated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nucleotides from the sequence that binds to the polypeptide (e.g., the endonuclease domain and / or DBD domain).

[0119] 115. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, further comprising a second strand targeting gRNA that directs an endonuclease domain (e.g., nickase) domain to nick into a second strand (e.g., in a target genome).

[0120] 116. The template RNA is a system, kit, template RNA, or reaction mixture of any of the preceding embodiments, further comprising a second strand targeting gRNA.

[0121] 117. The second strand targeting gRNA is located on a nucleic acid separate from the template RNA, in any of the systems, kits, template RNA, or reaction mixtures of the preceding embodiments.

[0122] 118. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, which directs the gRNA to insert a nick into the second strand (e.g., in the target genome) at a site where 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 are 5' or 3' on the target site modification (e.g., nick on the first strand).

[0123] 119. gRNA is a system, kit, template RNA, or reaction mixture of any of the previous embodiments that specifically binds to the edited strand.

[0124] 120. A system, method, kit, template RNA, or reaction mixture according to any of the preceding embodiments, comprising a polypeptide containing a heterologous targeting domain that specifically binds to a sequence contained within a target DNA molecule (e.g., genomic DNA).

[0125] 121. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous targeting domain binds to an unmodified polypeptide and a different nucleic acid sequence.

[0126] 122. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the polypeptide does not contain a functional endogenous targeting domain (for example, the polypeptide does not contain an endogenous targeting domain).

[0127] 123. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, comprising a zinc finger (for example, a zinc finger that specifically binds to a sequence contained within a target DNA molecule).

[0128] 124. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, comprising a Cas domain (e.g., a Cas9 domain or a variant or variant thereof, e.g., a Cas9 domain that specifically binds to a sequence contained within a target DNA molecule).

[0129] 125. The Cas domain associates with guide RNA (gRNA) in any of the systems, methods, kits, template RNA, or reaction mixtures of the preceding embodiments.

[0130] 126. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, comprising a heterologous targeting domain (e.g., a heterologous endonuclease domain).

[0131] 127. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, comprising an endonuclease domain (e.g., Cas9, or a variant or mutant thereof).

[0132] 128. The Cas domain associates with guide RNA (gRNA) in any of the systems, methods, kits, template RNA, or reaction mixtures of the preceding embodiments.

[0133] 129. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the endonuclease domain includes the Fok1 domain.

[0134] 130. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the template nucleic acid molecule comprises at least one (e.g., one or two) heterologous sequences having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence contained within a target DNA molecule (e.g., genomic DNA).

[0135] 131. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein at least one heterologous sequence is located at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides at the 5' end of a template nucleic acid molecule, or within the range of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides.

[0136] 132. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein at least one heterologous sequence is located at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides at the 3' end of a template nucleic acid molecule, or within the range of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides.

[0137] 133. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein heterologous sequences are bound to a range of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nick site in a target DNA molecule (e.g., purified by a nickase, e.g., an endonuclease domain, as described herein).

[0138] 134. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous sequence has a sequence identity of 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or less than 1% between the endogenous homologous sequence of the unmodified form of the template RNA and the complementary nucleic acid sequence.

[0139] 135. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous sequence has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the sequence of a different target DNA molecule from the sequence to which the endogenous homologous sequence is joined (e.g., replaced by the heterologous sequence).

[0140] 136. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, comprising (for example, at its 3' end) a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence located at the 5' end of a target DNA molecule (for example, a site nicked by a nickase, e.g., an endonuclease domain as described herein).

[0141] 137. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, comprising a heterologous sequence suitable for initiating targeted primed reverse transcription (TPRT) (e.g., at its 5' end).

[0142] 138. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous sequence has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence located within a range of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides of the target insertion site (e.g., 3' thereof) within a target DNA molecule for, for example, a heterologous target sequence (e.g., as described herein), wherein the homology is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0143] 139. The template nucleic acid molecule is a system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, for example, one described herein, which includes a guide RNA (gRNA).

[0144] 140. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the template nucleic acid molecule contains a gRNA spacer sequence (for example, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides at its 5' end, or within the range of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides).

[0145] 141. The RNA of the system (e.g., template RNA, RNA encoding the polypeptide of (a), or RNA expressed from a heterologous target sequence incorporated into the target DNA) includes, for example, a microRNA binding site within the 3'UTR, in any of the systems, methods, kits, template RNA, or reaction mixtures of the preceding embodiments.

[0146] 142. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the microRNA binding site is recognized by a miRNA that is present in non-target cell types but not in target cell types (or present at reduced levels compared to non-target cells).

[0147] 143. The miRNA is miR-142 and / or the non-target cells are Kupfer cells or blood cells, e.g., immune cells, according to any of the preceding embodiments of a system, method, kit, template RNA, or reaction mixture.

[0148] 144. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the miRNA is miR-182 or miR-183, and / or the non-target cell is a spinal dorsal root ganglion neuron.

[0149] 145. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, comprising a first miRNA binding site recognized by a first miRNA (e.g., miR-142), and further comprising a second miRNA binding site recognized by a second miRNA (e.g., miR-182 or miR-183), wherein the first and second miRNA binding sites are located on the same RNA or on different RNAs in the system.

[0150] 146. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the template RNA comprises at least two, three, or four miRNA binding sites, for example, the miRNA binding sites are recognized by the same miRNA or different miRNAs.

[0151] The RNA encoding the polypeptide of 147.(a) comprises at least two, three, or four miRNA binding sites, for example, the miRNA binding sites are recognized by the same miRNA or different miRNAs, according to any of the systems, methods, kits, template RNA, or reaction mixtures of the preceding embodiments.

[0152] 148. A system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the RNA expressed from a heterologous target sequence incorporated into the target DNA comprises at least two, three, or four miRNA binding sites, for example, the miRNA binding sites are recognized by the same miRNA or different miRNAs.

[0153] 149. mRNA encoding any polypeptide or system of the above embodiments, and template RNA of any of the above embodiments A system that includes this.

[0154] 150. The mRNA encoding the polypeptide or system of any of the prior embodiments and the template RNA of any of the prior embodiments are arranged on different nucleic acid molecules, respectively, in any of the prior embodiments.

[0155] 151. A template RNA (or RNA encoding a template RNA) from any of the above embodiments, and Sequences encoding any of the systems or polypeptides of the previous embodiments A system containing RNA molecules.

[0156] 152. A system of any of the preceding embodiments, in which the RNA molecule includes, for example, an internal ribosome entry site operably linked to a sequence encoding a system or polypeptide.

[0157] 153. The RNA molecule is, for example, a system of any of the preceding embodiments, comprising a template RNA (or RNA encoding template RNA) and a sequence encoding a system or polypeptide, wherein the RNA molecule includes a cleavage site located between the template RNA and the sequence encoding the system or polypeptide.

[0158] 154. A polypeptide comprising a split intein, wherein two or more (e.g., all) of the following are translated as separate proteins that combine into a single polypeptide by protein splicing: for example, an RT domain, a DBD, an endonuclease (e.g., nickase) domain, or a combination thereof.

[0159] 155. The system is a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising one or more circular RNA molecules (circRNA).

[0160] 156.circRNA is a system, kit, polypeptide, or reaction mixture of any of the previous embodiments that encodes a Gene Writer polypeptide.

[0161] 157. CircRNA is a system of any of the previous embodiments, including template RNA.

[0162] 158. CircRNA is delivered to a host cell by any of the systems, kits, polypeptides, or reaction mixtures of the preceding embodiments.

[0163] 159. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, which can be linearized, for example, within a host cell, for example, within the nucleus of a host cell.

[0164] 160.CircRNA is a system, kit, polypeptide, or reaction mixture of any of the previous embodiments, containing a cleavage site.

[0165] 161. A system, kit, polypeptide, or reaction mixture of any of the previous embodiments, further comprising a second cleavage site of circRNA.

[0166] 162. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the cleavage site can be cleaved by a ribozyme, such as a ribozyme contained within circRNA (e.g., by autocleavage).

[0167] 163.circRNA is a system, kit, polypeptide, or reaction mixture of any of the previous embodiments, comprising a ribozyme sequence.

[0168] 164. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments in which a ribozyme sequence can be self-cleaved, for example, within a host cell, for example, within the nucleus of a host cell.

[0169] 165. The ribozyme is an inducible ribozyme, which is a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0170] 166. The ribozyme is a protein-responsive ribozyme, e.g., a nucleoprotein-responsive ribozyme, e.g., a genome-interacting protein, e.g., an epigenetic modifier, e.g., EZH2, as a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0171] 167. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme is a nucleic acid-responsive ribozyme.

[0172] 168. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the catalytic activity (e.g., autocatalytic activity) of the ribozyme is activated in the presence of a target nucleic acid molecule (e.g., RNA molecule, e.g., mRNA, miRNA, ncRNA, lncRNA, tRNA, snRNA, or mtRNA).

[0173] 169. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, in which the ribozyme is responsive to a target protein (e.g., MS2 coat protein).

[0174] 170. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the target protein is localized in the cytoplasm or the nucleus (e.g., an epigenetic modifier or transcription factor).

[0175] 171. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising a ribozyme sequence of a B2 or ALU retrotransposon, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0176] 172. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising a ribozyme, the sequence of the tobacco ring spot virus hammerhead ribozyme, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0177] 173. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising a ribozyme, the sequence of a hepatitis delta virus (HDV) ribozyme, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0178] 174. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, in which a ribozyme is activated by a portion expressed within a target cell or target tissue.

[0179] 175. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, in which a ribozyme is activated by a portion expressed in a target intracellular compartment (e.g., nucleus, nucleolus, cytoplasm, or mitochondria).

[0180] 176. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme is contained within circular RNA or linear RNA.

[0181] 177. The first circular RNA encoding the polypeptide of the Gene Writing system; A second circular RNA containing the template RNA of the Gene Writing system and A system that includes this.

[0182] The nucleus encoding the polypeptide of 178.(a) is a system of any of the preceding embodiments, containing a coding sequence that has been codon-optimized for expression in human cells.

[0183] 179. A system of any of the preceding embodiments in which the template RNA contains a coding sequence that has been codon-optimized for expression in human cells.

[0184] 180. Lipid nanoparticles (LNPs) comprising any of the above embodiments, template RNA, polypeptide (or RNA encoding it), or DNA encoding the system, template RNA, or polypeptide.

[0185] 181. A first lipid nanoparticle comprising a polypeptide (or DNA or RNA encoding it) of the Gene Writing system (as described herein, for example); A second lipid nanoparticle containing a nucleic acid molecule (e.g., as described herein) of the Gene Writing system and A system that includes this.

[0186] 182. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the system, nucleic acid molecules, polypeptides, and / or DNA encoding them are formulated as lipid nanoparticles (LNPs).

[0187] 183. An LNP containing a cationic lipid, according to any of the previous embodiments.

[0188] 184. Cationic lipids have the following structure: [ka] An LNP having any of the above embodiments.

[0189] 185. An LNP of any of the previous embodiments, further comprising one or more neutral lipids, e.g., DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, steroids, e.g., cholesterol, and / or one or more polymer-conjugated lipids, e.g., pegylated lipids, e.g., PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG-dialkyloxypropyl carbamate.

[0190] 186. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the system, polypeptide, and / or the DNA encoding it are formulated as lipid nanoparticles (LNPs).

[0191] 187. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticles (or formulations comprising a plurality of lipid nanoparticles) are devoid of reactive impurities (e.g., aldehydes) or contain reactive impurities (e.g., aldehydes) below a pre-selected level.

[0192] 188. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticles (or formulations comprising multiple lipid nanoparticles) lack an aldehyde or contain an aldehyde below a pre-selected level.

[0193] 189. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, comprising lipid nanoparticles in a formulation containing a plurality of lipid nanoparticles.

[0194] 190. A lipid nanoparticle formulation is produced using one or more lipid reagents comprising a total reactive impurity (e.g., aldehyde) content of 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than 0.1% of a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0195] 191. A lipid nanoparticle formulation is a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, produced using one or more lipid reagents comprising a total reactive impurity (e.g., aldehyde) content of less than 3%.

[0196] 192. A lipid nanoparticle formulation is produced using one or more lipid reagents containing any single reactive impurity species (e.g., aldehyde) in amounts of 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than 0.1%, and is a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0197] 193. A lipid nanoparticle formulation is produced using one or more lipid reagents containing less than 0.3% of any single reactive impurity species (e.g., aldehyde), and is a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0198] 194. A lipid nanoparticle formulation is produced using one or more lipid reagents containing less than 0.1% of any single reactive impurity species (e.g., aldehyde), and is a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0199] 195. A system, kit, polypeptide, or reaction mixture of any of the preceding embodiments comprising a lipid nanoparticle formulation with a total reactive impurity (e.g., aldehyde) content of 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than 0.1%.

[0200] 196. A lipid nanoparticle formulation comprising a total reactive impurity (e.g., aldehyde) content of less than 3% of any of the systems, kits, polypeptides, or reaction mixtures of the preceding embodiments.

[0201] 197. A lipid nanoparticle formulation comprising any single reactive impurity species (e.g., aldehyde) in amounts of 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than 0.1%, as a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0202] 198. A lipid nanoparticle formulation comprising any single reactive impurity species (e.g., aldehyde) in a concentration of less than 0.3% of any system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0203] 199. A lipid nanoparticle formulation comprising any single reactive impurity species (e.g., aldehyde) in a concentration of less than 0.1% of any system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0204] 200. One or more, or optionally all, of the lipid reagents used in the lipid nanoparticles described herein, or their formulations, constitute a total reactive impurity (e.g., aldehyde) content of 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than 0.1% of any of the systems, kits, polypeptides, or reaction mixtures of the preceding embodiments.

[0205] 201. One or more, or optionally all, of the lipid reagents used in the lipid nanoparticles described herein, or their formulations, constitute a total reactive impurity (e.g., aldehyde) content of less than 3% in any of the systems, kits, polypeptides, or reaction mixtures of the preceding embodiments.

[0206] 202. One or more, or optionally all, of the lipid reagents used in the lipid nanoparticles described herein, or their formulations, contain any single reactive impurity species (e.g., aldehyde) in amounts of 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or less than 0.1%, as a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0207] 203. One or more, or optionally all, of the lipid reagents used in the lipid nanoparticles described herein, or the formulation thereof, contains less than 0.3% of any single reactive impurity species (e.g., aldehyde), and is a system, kit, polypeptide, or reaction mixture of any of the preceding embodiments.

[0208] 204. One or more or optionally all of the lipid reagents used in the lipid nanoparticles described herein or their pharmaceutical forms are any system, kit, polypeptide or reaction mixture of any of the previous embodiments that contain less than 0.1% of any single reactive impurity (e.g., aldehyde) species.

[0209] 205. The total aldehyde content and / or amount of any single reactive impurity (e.g., aldehyde) species is determined by liquid chromatography (LC), for example in connection with tandem mass spectrometry (MS / MS), according to the method described in Example 26, of any system, kit, polypeptide or reaction mixture of any of the previous embodiments.

[0210] 206. The total aldehyde content and / or amount of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of nucleic acid molecules (e.g., those described herein) associated with the presence of reactive impurity (e.g., aldehyde) within the lipid reagent, of any system, kit, polypeptide or reaction mixture of any of the previous embodiments.

[0211] 207. The total aldehyde content and / or amount of aldehyde species is determined by detecting one or more chemical modifications of nucleotides or nucleosides (e.g., ribonucleotides or ribonucleosides contained in or isolated from nucleic acid molecules, such as those described herein) associated with the presence of reactive impurity (e.g., aldehyde) within the lipid reagent, as described in Example 41, of any system, kit, polypeptide or reaction mixture of any of the previous embodiments.

[0212] 208. Chemical modifications of nucleic acid molecules, nucleotides, or nucleosides are detected by determining the presence of one or more modified nucleotides or nucleosides, for example using LC-MS / MS analysis as described in Example 41, of any system, kit, polypeptide or reaction mixture of any of the previous embodiments.

[0213] A lipid nanoparticle (LNP) comprising a system, polypeptide (or RNA encoding the same), nucleic acid molecule, or DNA encoding a system or polypeptide of any of the previous embodiments.

[0214] A first lipid nanoparticle comprising a polypeptide (or DNA or RNA encoding the same) of a Gene Writing system (such as described herein); and a second lipid nanoparticle comprising a nucleic acid molecule of a Gene Writing system (such as described herein) A system comprising.

[0215] A system, kit, polypeptide or reaction mixture of any of the previous embodiments, wherein the system, nucleic acid molecule, polypeptide, and / or DNA encoding them are formulated as lipid nanoparticles (LNPs).

[0216] A first lipid nanoparticle comprising a polypeptide (or DNA or RNA encoding the same) of a system of any of the previous embodiments or a polypeptide of any of the previous embodiments; and A second lipid nanoparticle comprising a template RNA (or DNA encoding the same) of a system of any of the previous embodiments or a template RNA of any of the previous embodiments A system comprising.

[0217] A virus, virus-like particle, fusosome or virosome comprising a system, template RNA, polypeptide (or RNA encoding the same) or DNA encoding a system, template RNA or polypeptide of any of the previous embodiments.

[0218] A first virus, virus-like particle, fusosome or virosome comprising a polypeptide (or DNA or RNA encoding the same) of a system of any of the previous embodiments or a polypeptide of any of the previous embodiments; and A second virus, virus-like particle, or virosome containing the template RNA (or DNA encoding it) of any of the systems of the previous embodiments or the template RNA of any of the previous embodiments. A system that includes this.

[0219] 215. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA has a nucleotide length greater than 100, greater than 125, greater than 150, greater than 175, or greater than 200, or at least 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 kilobase lengths (and optionally less than 15, less than 10, less than 5, or less than 20 kilobase lengths, or less than 500, less than 400, less than 300, or less than 200 nucleotide lengths).

[0220] 216. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains a poly-A tail (e.g., a poly-A tail having a length of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides).

[0221] 217. At least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA is 5' caps, e.g., 7-methylguanosine caps (e.g., O-Me-m7G caps); hypermethylated cap analogs; NAD+-derived cap analogs (e.g., as described in Kiledjian, Trends in Cell Biology 28, 454-464 (2018)); or modified, e.g., biotinylated cap analogs (e.g., Bednarek et al., Phil Trans R (as described in Soc B 373, 20180167 (2018)), and / or Poly-A tail; a 16-nucleotide stem-loop structure flanked by five unpaired nucleotides (e.g., as described by Mannironi et al., Nucleic Acid Research 17, 9113-9126 (1989)); a triple helix structure (e.g., as described by Brown et al., PNAS 109, 19202-19207 (2012)); tRNA, Y RNA, or vault RNA structure (e.g., Labno et al., Biochemica et Biophysica Acta 3' features selected from one or more of the following: as described by 1863,3125-3147 (2016); incorporation of one or more deoxyribonucleotide triphosphates (dNTPs), 2'O-methylated NTPs, or phosphorothioate-NTPs; single nucleotide chemical modifications (e.g., oxidation of the 3'-terminal ribose to a reactive aldehyde, followed by conjugation of an aldehyde-reactive modified nucleotide); or chemical ligation to another nucleic acid molecule. A system, kit, template RNA, or reaction mixture according to any of the previous embodiments, containing the above.

[0222] 218. Template RNAs include, for example, dihydrouridine, inosine, 7-methylguanosine, 5-methylcytidine (5mC), 5'-phosphoribothymidine, 2'-O-methylribothymidine, 2'-O-ethylribothymidine, 2'-fluororibothymidine, C-5 propynyl-deoxycytidine (pdC), C-5 propynyl-deoxyuridine (pdU), C-5 propynyl-cytidine (pC), C-5 propynyl-uridine (pU), 5-methylcytidine, 5-methyluridine, 5-methyldeoxycytidine, 5-methyldeoxyuridine methoxy, 2,6-diaminopurine, 5'-dimethoxytrityl-N4-ethyl- A system, kit, template RNA, or reaction mixture according to any of the preceding embodiments, comprising one or more modified nucleotides selected from 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 (sub-groove binder), pseudouridine (Ψ), 1-N-methylpsoiduridine (1-Me-Ψ), or 5-methoxyuridine (5-MO-U).

[0223] 219. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains one or more modified nucleotides.

[0224] 220. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments in which at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA remains intact after stability testing (e.g., nucleotide lengths greater than 100, 125, 150, 175, or 200 or at least 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 kilobase lengths).

[0225] 221. A system, kit, or reaction mixture of any of the preceding embodiments, wherein at least 1% of the target site is modified after the system has been assayed for potency.

[0226] 222. A system, kit, template RNA, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the system, polypeptide, template RNA, and / or DNA encoding it are formulated as lipid nanoparticles (LNPs).

[0227] 223. A system, kit, template RNA, polypeptide, or reaction mixture of any of the preceding embodiments, in which the DNA encoding the system, polypeptide, and / or template RNA is packaged in a virus, virus-like particle, virosome, liposome, vesicle, exosome, or LNP.

[0228] 224. A system, kit, template RNA, polypeptide, or reaction mixture of any of the preceding embodiments, in which DNA encoding a system, template RNA, or polypeptide is packaged within an adeno-associated virus (AAV).

[0229] 225. The system, kit, template RNA, polypeptide, or reaction mixture of any of the prior embodiments is free from or substantially free from pyrogen, virus, fungal, bacterial pathogen and / or host cell protein contamination.

[0230] 226. A system of any of the above embodiments, a template RNA or polypeptide, or DNA encoding either thereof, and Adeno-associated virus (AAV) capsid protein Viruses, virus-like particles, or viromosomes containing these particles.

[0231] The system, template RNA and / or polypeptide are active in the target tissue and less active (e.g., not active) in the non-target tissue, such as any of the systems, kits, template RNAs, polypeptides, viruses, virus-like particles or virosomes of the previous embodiments.

[0232] Further comprising one or more first tissue-specific expression control sequences specific to the target tissue, the one or more first tissue-specific expression control sequences specific to the target tissue are operably associated with the template RNA, the polypeptide or nucleic acid encoding it or both, such as any of the systems, kits, template RNAs, polypeptides, viruses, virus-like particles or virosomes of the previous embodiments.

[0233] The endonuclease domain, such as the nickase domain, makes a nick in the first strand of the target site DNA and makes a nick in the second strand at a site away from the first nick, such as any of the systems, kits, template RNAs or reaction mixtures of the previous embodiments.

[0234] The nick is formed outwardly, such as any of the systems, kits, template RNAs or reaction mixtures of the previous embodiments.

[0235] The nick is formed outwardly, such as any of the systems, kits, template RNAs or reaction mixtures of the previous embodiments.

[0236] The sequence binding to the target site designates the position of the nick on the first strand, the system further comprises an additional nucleic acid comprising a sequence binding to a site away from the target site, and the sequence binding to a site away from the target site designates the position of the nick on the second strand, such as any of the systems, kits, template RNAs or reaction mixtures of the previous embodiments.

[0237] 233. The additional nucleic acid further comprises a sequence that binds to a polypeptide (e.g., an endonuclease domain and / or DBD), for example, the additional nucleic acid comprising gRNA, a system, kit, template RNA or reaction mixture of any of the preceding embodiments.

[0238] 234. The sequence that binds to a site distant from the target site (for example, to the first strand of a site within the target genome) is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130 nucleotides long (and optionally 1 A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, having a length of 50, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides or less, for example, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides.

[0239] 235. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds to a site distant from the target site is complementary to the first strand of the target site, or contains one, two, three, four, or five or fewer mismatches with the first strand of the target site.

[0240] 236. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, comprising a DBD and / or endonuclease domain, and containing a CRISPR / Cas domain.

[0241] 237. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the CRISPR / Cas domain and template RNA are bound to a target site, and the first strand of the target site contains a first PAM site.

[0242] 238. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the CRISPR / Cas domain and additional nucleic acids are bound to a site distant from the target site, and the second strand of the site distant from the target site contains a second PAM site.

[0243] 239. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the first PAM site and the second PAM site are located between the nick location on the first strand and the nick location on the second strand.

[0244] 240. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the location of the nick on the first strand and the location of the nick on the second strand are located between the first PAM site and the second PAM site.

[0245] 241. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, further comprising an additional polypeptide containing an additional DNA-binding domain (DBD) and an additional endonuclease domain, such as an additional niccasse domain.

[0246] 242. The additional endonuclease domain, e.g., additional nickase domain, comprises any of the systems, kits, template RNA, or reaction mixtures of the preceding embodiments, including an endonuclease or nickase domain described herein, e.g., a CRISPR / Cas domain, a type IIs nuclease (e.g., FokI), a Holliday junction resolver, a meganuclease, or a double-stranded DNA nuclease with modifications that eliminate the ability to introduce nicks into a single strand (e.g., converting a double-stranded DNA nuclease to a nickase).

[0247] 243. Additional DBDs are systems, kits, template RNA, or reaction mixtures of any of the previous embodiments that bind to a site distant from the target site.

[0248] 244. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the endonuclease domain of (a) or (b) nicks the first strand, and an additional endonuclease domain (e.g., an additional nickerse domain) nicks the second strand.

[0249] 245. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, in which the nick is formed outward.

[0250] 246. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, in which the nick is formed inward.

[0251] 247. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein DBD and optionally template RNA (e.g., a polypeptide-binding sequence) specify the location of the nick relative to the first strand, and additional DBDs specify the location of the nick relative to the second strand.

[0252] 248. A polypeptide (e.g., DBD) is included in any of the systems, kits, template RNA, or reaction mixtures of the preceding embodiments, comprising a TAL effector molecule.

[0253] 249. A polypeptide (e.g., DBD) comprising a zinc finger molecule, a system, kit, template RNA, or reaction mixture of any of the preceding embodiments.

[0254] 250. A polypeptide (e.g., DBD) comprising a CRISPR / Cas domain, the system, kit, template RNA, or reaction mixture of any of the preceding embodiments.

[0255] 251. Additional polypeptides (e.g., additional DBDs) are included in any of the systems, kits, template RNAs, or reaction mixtures of the preceding embodiments, comprising a TAL effector molecule.

[0256] 252. Additional polypeptides (e.g., additional DBDs) comprising zinc finger molecules, the systems, kits, template RNA, or reaction mixtures of any of the preceding embodiments.

[0257] 253. Additional polypeptides (e.g., additional DBDs) comprising a CRISPR / Cas domain, the system, kit, template RNA, or reaction mixture of any of the preceding embodiments.

[0258] 254. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the polypeptide and additional polypeptides bind to a site on target DNA between the nick site on the first strand and the nick site on the second strand.

[0259] 255. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the location of the nick relative to the first strand and the location of the nick relative to the second strand are located between the sites where the polypeptide and the additional polypeptide bind to the target DNA.

[0260] 256. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein, on the target DNA, the location of the nick relative to the second strand is located on the opposite side of the polypeptide and additional polypeptide binding sites, compared to the location of the nick relative to the first strand.

[0261] 257. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein, on the target DNA, the location of the nick relative to the second strand is located on the same side of the polypeptide and additional polypeptide binding sites compared to the location of the nick relative to the first strand.

[0262] 258. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the CRISPR / Cas domain of the polypeptide and the template RNA are bound to a target site, and the first strand of the target site contains a PAM site.

[0263] 259. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the PAM site and the site away from the target site are located between the nick location on the first strand and the nick location on the second strand.

[0264] 260. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the location of the nick on the first strand and the location of the nick on the second strand are located between the PAM site and a site away from the target site.

[0265] 261. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, further comprising an additional nucleic acid (e.g., gRNA) containing a sequence that binds to a site distant from the target site, wherein the sequence that binds to the site distant from the target site designates the location of the nick relative to the second strand.

[0266] 262. The additional nucleic acid further comprises a sequence that binds to an additional polypeptide (e.g., a CRISPR / Cas domain), for example, the additional nucleic acid comprises a gRNA, the system, kit, template RNA, or reaction mixture of any of the preceding embodiments.

[0267] 263. Sequences that bind to a site distant from the target site (e.g., to the first strand of a site within the target genome) are at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130 nucleotides long (and optionally 150) A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, having a length of 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides or less, for example, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides.

[0268] 264. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the sequence that binds to a site distant from the target site is complementary to the first strand of the target site, or contains one, two, three, four, or five or fewer mismatches with the first strand of the target site.

[0269] 265. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, including a PAM site, located away from the target site.

[0270] 266. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the PAM site and target site are located between the nick location relative to the first strand and the nick location relative to the second strand.

[0271] 267. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the location of the nick on the second strand (for example, compared to the nick on the first strand) is such that DNA polymerization by the RT domain proceeds toward the location of the nick on the second strand.

[0272] 268. A system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein the location of the nick on the second strand (for example, compared to the nick on the first strand) is such that DNA polymerization by the RT domain proceeds away from the location of the nick on the second strand.

[0273] 269. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the first nick and the second nick are separated by 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.

[0274] 270. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the first nick and the second nick are separated by 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 or less.

[0275] 271. The first and second nicknames 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, 140-1 90, 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, 14 0-180, 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, 60-1 40, 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, 110-1 30, 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 A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, separated by ~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.

[0276] 272. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein when modifying DNA, the system produces fewer double-strand breaks than otherwise similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), and one or more PAM sites, target sites, or sites away from the target site are not located between the nick locations of the first strand and the nick locations of the second strand.

[0277] 273. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein when the system modifies DNA, it produces fewer double-strand breaks than other similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), and the polypeptide and additional polypeptides bind to sites on target DNA that are not between the nick sites on the first strand and the nick sites on the second strand.

[0278] 274. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system, when modifying DNA, produces fewer double-strand breaks than otherwise similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), and on the target DNA, the nick locations relative to the second strand and the nick locations relative to the first strand are positioned between the binding sites of the polypeptide and the additional polypeptide.

[0279] 275. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein when the system modifies DNA, it produces fewer double-strand breaks than otherwise similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), and the location of the nick on the second strand (e.g., compared to the nick on the first strand) is such that the RT domain initiates reverse transcription away from the nick location on the second strand.

[0280] 276. A system, kit, template RNA, or reaction mixture of any of the prior embodiments, which, when modifying DNA, produces fewer deletions not encoded by a heterogeneous target sequence than other similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), where one or more PAM sites, target sites, or sites away from the target site are not located between the nick sites of the first strand and the nick sites of the second strand, as measured by PacBio long-read sequencing, for example, as described in Example 29.

[0281] 277. The system, kit, template RNA, or reaction mixture of any of the previous embodiments, wherein when the system modifies DNA, it produces fewer deletions (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%) than otherwise similar systems, and the polypeptide and additional polypeptides bind to a site on the target DNA that is not between the nick site on the first strand and the nick site on the second strand, as measured by PacBio long-read sequencing, for example, as described in Example 29.

[0282] 278. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein, when modifying DNA, the system produces fewer deletions not encoded by a heterogeneous target sequence than other similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), and on the target DNA, the nick locations relative to the second strand and the nick locations relative to the first strand are positioned between the binding sites of the polypeptide and the additional polypeptide, as measured by PacBio long-read sequencing, for example, as described in Example 29.

[0283] 279. The system, kit, template RNA, or reaction mixture of any of the prior embodiments, which, when modifying DNA, produces fewer deletions not encoded by heterologous target sequences than otherwise similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), where the location of the nick on the second strand (e.g., compared to the nick on the first strand) is such that the RT domain initiates reverse transcription away from the nick location on the second strand, as measured by PacBio long-read sequencing, for example, as described in Example 29.

[0284] 280. The system, kit, template RNA, or reaction mixture of any of the prior embodiments, which, when modifying DNA, produces fewer insertions not encoded by heterologous target sequences than otherwise similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), and in which one or more PAM sites, target sites, or sites away from the target site are not located between the nick sites of the first strand and the nick sites of the second strand, as measured by PacBio long-read sequencing, for example, as described in Example 29.

[0285] 281. The system, kit, template RNA, or reaction mixture of any of the previous embodiments, which, when modifying DNA, produces fewer insertions not encoded by heterologous target sequences than other similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), and the polypeptide and additional polypeptides bind to a site on target DNA that is not between the nick site on the first strand and the nick site on the second strand, as measured by PacBio long-read sequencing, for example, as described in Example 29.

[0286] 282. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, which, when modifying DNA, produces fewer insertions not encoded by heterologous target sequences than otherwise similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), and on the target DNA, the nick locations relative to the second strand and the nick locations relative to the first strand are positioned between the binding sites of the polypeptide and the additional polypeptide, as measured by PacBio long-read sequencing, for example, as described in Example 29.

[0287] 283. The system, kit, template RNA, or reaction mixture of any of the prior embodiments, which, when modifying DNA, produces fewer insertions not encoded by heterologous target sequences than otherwise similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), where the location of the nick on the second strand (e.g., compared to the nick on the first strand) is such that the RT domain initiates reverse transcription away from the nick location on the second strand, as measured by PacBio long-read sequencing, for example, as described in Example 29.

[0288] 284. The system, kit, template RNA, or reaction mixture of any of the prior embodiments, which, when modifying DNA, produces more desired Gene Writing modifications (e.g., at least 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% more) than otherwise similar systems, wherein one or more of the PAM sites, target sites, or sites away from the target sites are not located between the nick sites of the first strand and the nick sites of the second strand, as measured by PacBio long-read sequencing, for example, as described in Example 29.

[0289] 285. The system, when modifying DNA, produces more desired Gene Writing modifications (e.g., at least 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% more) than other otherwise similar systems, and the polypeptide and additional polypeptides bind to sites on target DNA that are not between the nick sites on the first strand and the nick sites on the second strand, as measured by PacBio long-read sequencing, for example, as described in Example 29, a system, kit, template RNA, or reaction mixture of any of the previous embodiments.

[0290] 286. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein, when modifying DNA, the system produces more desired Gene Writing modifications (e.g., at least 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% more) than otherwise similar systems, and on target DNA, the nick locations relative to the second strand and the nick locations relative to the first strand are positioned between the binding sites of the polypeptide and the additional polypeptide, as measured by PacBio long-read sequencing, for example, as described in Example 29.

[0291] 287. The system, kit, template RNA, or reaction mixture of any of the prior embodiments, which, when modifying DNA, produces more desired Gene Writing modifications (e.g., at least 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% more) than otherwise similar systems, and the location of the nick on the second strand (e.g., compared to the nick on the first strand) is such that the RT domain initiates reverse transcription away from the nick location on the second strand, as measured by PacBio long-read sequencing, for example, as described in Example 29.

[0292] 288. A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the first nick and the second nick are separated by at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350, 400, 450, or 500 nucleotides, for example, at least 100 nucleotides (and optionally 500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, or 120 nucleotides or less).

[0293] 289. The first and second nicknames 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, 13 0-190, 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, 1 A system, kit, template RNA, or reaction mixture of any of the preceding embodiments, separated by 20-170, 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.

[0294] 290. The system, when modifying DNA, produces fewer insertions not encoded by heterologous target sequences than other similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), and the location of the nick relative to the second strand is less than 100 nucleotides away from the location of the nick relative to the first strand (and optionally at least 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides away), as measured by PacBio long-read sequencing, for example, as described in Example 29, of any of the systems, kits, template RNA, or reaction mixtures of the prior embodiments.

[0295] 291. The system, when modifying DNA, produces fewer deletions not encoded by a heterologous target sequence than other similar systems (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%), and the location of the nick relative to the second strand is less than 100 nucleotides away from the location of the nick relative to the first strand (and optionally at least 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides away), as measured by PacBio long-read sequencing, for example, as described in Example 29, of any of the systems, kits, template RNA, or reaction mixtures of the prior embodiments.

[0296] 292. Any system numbered above that does not contain DNA, or does not contain more than 10%, 5%, 4%, 3%, 2%, or 1% DNA by mass or molar amount.

[0297] 293. A method for constructing a system for modifying DNA (for example, as described herein), (a) Prepare a template nucleic acid (e.g., template RNA or DNA) containing heterologous sequences that have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to sequences contained within the target DNA molecule, and / or (b) Prepare a polypeptide system that includes a heterologous targeting domain that specifically binds to a sequence contained within the target DNA molecule (e.g., including a DNA-binding domain (DBD) and / or an endonuclease domain). A method that includes this.

[0298] 294. (a) comprising introducing a heterologous sequence into a template nucleic acid (e.g., template RNA or DNA) having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence contained within a target DNA molecule, and / or (b) comprising introducing a heterologous targeting domain into a polypeptide of a system (e.g., including a DNA-binding domain (DBD) and / or an endonuclease domain) that specifically binds to a sequence contained within a target DNA molecule. Any of the methods of the previous embodiments.

[0299] The introduction of 295.(a) is a method of any of the preceding embodiments, which includes inserting a homologous sequence into a template nucleic acid.

[0300] The introduction of 296.(a) is a method of any of the preceding embodiments, which includes substituting a segment of a template nucleic acid with a homologous sequence.

[0301] The introduction of 297.(a) is a method of any of the preceding embodiments, comprising generating a segment of a template nucleic acid having a homologous sequence by mutating one or more nucleotides of the template nucleic acid (e.g., at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides).

[0302] The introduction of 298.(b) is a method of any of the preceding embodiments, comprising inserting the amino acid sequence of the targeting domain into the amino acid sequence of the polypeptide.

[0303] The introduction of 299.(b) is a method of any of the preceding embodiments, which involves inserting a nucleic acid sequence encoding a targeting domain into the coding sequence of a polypeptide contained within a nucleic acid molecule.

[0304] The introduction of 300.(b) is any method of the prior embodiments, comprising substituting at least a portion of the polypeptide with a targeting domain.

[0305] The introduction of 301.(a) is a method of any of the preceding embodiments, comprising mutating one or more amino acids of a polypeptide (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, or more amino acids).

[0306] 302. A method for modifying a target site in intracellular genomic DNA, cells, (a) polypeptides or nucleic acids encoding polypeptides (polypeptides include (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a nickas domain); and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. (i) The polypeptide contains a heterologous targeting domain (for example, within a DBD or endonuclease domain) that specifically binds to a sequence located within or adjacent to a target site in genomic DNA; and / or (ii) The template RNA contains heterologous sequences that have at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to sequences contained within or adjacent to the target site of the genomic DNA. A method comprising modifying a target site in intracellular genomic DNA by bringing it into contact with a substance.

[0307] 303. A method for producing template RNA, (a) To provide a template RNA from any of the above embodiments, (b)(i) Length of the template RNA, for example, whether the template RNA has a length greater than the reference length or within the standard length, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA has a nucleotide length greater than 100, greater than 125, greater than 150, greater than 175, or greater than 200; (ii) the presence, absence, and / or length of poly(A) tails on the template RNA, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains poly(A) tails (for example, poly(A) tails having a length of at least 5, 10, 20, or 30 nucleotides); (iii) the presence, absence and / or type of 5' cap on the template RNA, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains a 5' cap, for example, whether the cap is a 7-methylguanosine cap, for example, an O-Me-m7G cap; (iv) One or more modified nucleotides in the template RNA (e.g., dihydrouridine, inosine, 7-methylguanosine, 5-methylcytidine (5mC), 5'-phosphoribothymidine, 2'-O-methylribothymidine, 2'-O-ethylribothymidine, 2'-fluororibothymidine, C-5 propynyl-deoxycytidine (pdC), C-5 propynyl-deoxyuridine (pdU), C-5 propynyl-cytidine (pC), C-5 propynyl-uridine (pU), 5-methylcytidine, 5-methyluridine, 5-methyldeoxycytidine, 5-methyldeoxyuridine methoxy, 2,6-diaminopurine, 5'-dimethoxytrityl-N4-ethyl-2'-deoxycytidine The presence, absence, and / or type of nucleotides (selected from din, 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 (sub-groove binder), pseudouridine (Ψ), 1-N-methylpsoiduridine (1-Me-Ψ), or 5-methoxyuridine (5-MO-U), for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains one or more modified nucleotides; (v) Stability of the template RNA (e.g., over time and / or under pre-selected conditions), e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA remains intact (e.g., with nucleotide lengths greater than 100, 125, 150, 175, or 200) after stability testing; (vi) the efficacy of the template RNA in a system for modifying DNA, e.g., whether at least 1% of the target sites are modified after the system containing the template RNA has been assayed for efficacy; or (vii) The presence, absence, and / or level of one or more pyrogens, viruses, fungi, bacterial pathogens, or host cell proteins, e.g., whether the template RNA is free or substantially free of pyrogen, virus, fungus, bacterial pathogen, or host cell protein contamination. Assaying one or more of the following A method that includes this.

[0308] 304. A method for producing a system for modifying DNA, (a) To provide a system for modifying DNA according to any of the previous embodiments, (b)(i) Length of the template RNA, for example, whether the template RNA has a length greater than or within the reference length, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA has a nucleotide length greater than 100, greater than 125, greater than 150, greater than 175, or greater than 200; (ii) the presence, absence, and / or length of poly(A) tails on the template RNA, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains poly(A) tails (for example, poly(A) tails having a length of at least 5, 10, 20, or 30 nucleotides); (iii) the presence, absence and / or type of 5' cap on the template RNA, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains a 5' cap, for example, whether the cap is a 7-methylguanosine cap, for example, an O-Me-m7G cap; (iv) The presence, absence, and / or type of one or more modified nucleotides in the template RNA (e.g., selected from pseudouridine, dihydrouridine, inosine, 7-methylguanosine, 1-N-methylpsoiduridine (1-Me-Ψ), 5-methoxyuridine (5-MO-U), 5-methylcytidine (5mC), or locked nucleotides), e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains one or more modified nucleotides; (v) Stability of the template RNA (e.g., over time and / or under pre-selected conditions), e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA remains intact (e.g., with nucleotide lengths greater than 100, 125, 150, 175, or 200) after stability testing; (vi) The efficacy of the template RNA in a system for modifying DNA, e.g., whether at least 1% of the target sites are modified after the system containing the template RNA has been assayed for efficacy; (vii) Length of the polypeptide, the first polypeptide or the second polypeptide, for example, whether the polypeptide, the first polypeptide or the second polypeptide has a length greater than or within the reference length, for example, at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present polypeptide, the first polypeptide or the second polypeptide has a length greater than 600, greater than 650, greater than 700, greater than 750, greater than 800, greater than 850, greater than 900, greater than 950, Whether the amino acid length is greater than 1000, greater than 1050, greater than 1100, greater than 1150, greater than 1200, greater than 1250, greater than 1300, greater than 1350, greater than 1400, greater than 1450, greater than 1500, greater than 1600, greater than 1700, greater than 1800, greater than 1900, or greater than 2000 (and optionally, whether the amino acid length is 2500 or less, 2000 or less, 1500 or less, 1400 or less, 1300 or less, 1200 or less, 1100 or less, 1000 or less, 900 or less, 800 or less, 700 or less, or 600 or less); (viii) presence, absence and / or type of post-translational modifications on the polypeptide, the first polypeptide or the second polypeptide, e.g., whether at least 80, 85, 90, 95, 96, 97, 98 or 99% of the polypeptide, the first polypeptide or the second polypeptide contains phosphorylation, methylation, acetylation, myristoylation, palmitoylation, isoprenylation, glipyatyonation or lipoylation; (ix) polypeptides, within the first polypeptide or the second polypeptide, (e.g., ornithine, β-alanine, GABA, δ-aminolevulinic acid, PABA, D-amino acids (e.g., D-alanine or D-glutamate), aminoisobutyric acid, dehydroalanine, cystathionine, lanthionine, gencoric acid, diaminopimelic acid, homoalanine, norvaline, norleucine, homonorleucine, homoserine, O-methyl-homoserine and O-ethyl- The presence, absence, and / or type of one or more artificial, synthetic, or nonstandard amino acids (selected from homoserine, ethionine, selenocysteine, selenohomocysteine, selenomethionine, selenoethionine, tellurocysteine, or telluromethionine), for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present polypeptide, first polypeptide, or second polypeptide contains one or more artificial, synthetic, or nonstandard amino acids; (x) Stability of the polypeptide, first polypeptide or second polypeptide (for example, over time and / or under pre-selected conditions), for example, that at least 80, 85, 90, 95, 96, 97, 98 or 99% of the polypeptide, first polypeptide or second polypeptide remain intact after the stability test (e.g., over 600, over 650, over 700, over 750, over 800, over 850, over 900, over 950, over 1000, over 1050, 11 Whether or not the amino acid length remains above 00, above 1150, above 1200, above 1250, above 1300, above 1350, above 1400, above 1450, above 1500, above 1600, above 1700, above 1800, above 1900, or above 2000 (and optionally below 2500, below 2000, below 1500, below 1400, below 1300, below 1200, below 1100, below 1000, below 900, below 800, below 700, or below 600); (xi) The efficacy of the polypeptide, first polypeptide, or second polypeptide in a system for modifying DNA, for example, whether at least 1% of the target site is modified after the system containing the polypeptide, first polypeptide, or second polypeptide has been assayed for efficacy; or (xii) The presence, absence and / or level of one or more pyrogens, viruses, fungi, bacterial pathogens or host cell proteins, e.g., whether the system is free or substantially free of pyrogen, virus, fungi, bacterial pathogens or host cell protein contamination. Assaying one or more of the following A method that includes this.

[0309] 305. A method for modifying a target site in genomic DNA in cells, cells, (a) polypeptides or nucleic acids encoding polypeptides, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. To bring it into contact with and thereby modify the target site in the genomic DNA of the cell. A method that includes this.

[0310] 306. A method for modifying a target site in genomic DNA in cells, The process involves contacting cells with any of the systems, polypeptides, template RNA, or DNA encoding them, thereby modifying target sites in the genomic DNA of the cells. A method that includes this.

[0311] 307. A method according to any of the preceding embodiments, wherein a system, polypeptide, template RNA, or DNA is delivered to a target site by electroporation, e.g., nucleofection.

[0312] 308. Any method of the preceding embodiments, which does not involve contacting cells with DNA, or which involves contacting cells with a DNA-free composition containing, for example, more than 10%, 5%, 4%, 3%, 2%, or 1% by mass or molar amount.

[0313] 309. Any method of the preceding embodiments, which does not involve contacting cells with proteins, or which involves contacting cells with a composition that does not contain more than 10%, 5%, 4%, 3%, 2%, or 1% by mass or molar amount of proteins.

[0314] 310. Any method according to the previous embodiments, comprising contacting a target cell, or a population of target cells, with at least two template RNAs and / or at least two GeneWriter polypeptides, such that at least two target sites (a first target site and a second target site) are modified within the target cell.

[0315] 311. A method according to any of the previous embodiments, wherein the first target site and the second site are each independently edited at a frequency of at least 5%, 10%, or 15% of copies of the site within the cell population.

[0316] 312. A method according to any of the preceding embodiments, wherein the first target site and the second site are each independently edited at a frequency of at least 50%, 60%, 70%, or 80% of the editing obtained in a otherwise similar cell population that is in contact with a otherwise similar system that targets only one of the target sites.

[0317] 313. Any method of the prior embodiments wherein the resulting cell population contains 5%, 10%, or 20% or less of unwanted indels compared to unwanted indels obtained in otherwise similar cell populations that have come into contact with other otherwise similar systems that target only one of the target sites.

[0318] 314. The cells are primary cells, according to any of the methods of the preceding embodiments.

[0319] 315. The cell is a T cell, according to any of the methods of the preceding embodiments.

[0320] 316. A method for modifying a target site in intracellular genomic DNA, wherein the cell is subjected to, for example, nucleofection or lipid particle delivery. (a) polypeptides or nucleic acids encoding polypeptides (polypeptides include (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a nickas domain); and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. By bringing it into contact with the target site within the genomic DNA of the cell, it modifies the target site. A method comprising cells that are euploid, non-immortalized, part of a tissue, part of an organism, primary cells, non-dividing, haploid (e.g., germline cells), non-cancerous polyploid cells, or from an subject with a genetic disorder.

[0321] 317. The template RNA is a method of any of the preceding embodiments, comprising (i).

[0322] 318. The template RNA is a method of any of the preceding embodiments, comprising (ii).

[0323] 319. The template RNA is a method of any of the preceding embodiments, comprising (i) and (ii).

[0324] 320. A method for treating a subject having a disease or condition associated with a genetic defect, For the target, (a) polypeptides or nucleic acids encoding polypeptides (polypeptides include (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a nickas domain); and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to a polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A method comprising treating a subject having a disease or condition associated with a genetic defect by administering a substance.

[0325] 321. The template RNA is a method of any of the preceding embodiments, comprising (i).

[0326] 322. The template RNA is (ii) of any of the preceding embodiments.

[0327] 323. The template RNA is a method of any of the preceding embodiments, comprising (i) and (ii).

[0328] 324. Methods for treating subjects having diseases or symptoms associated with genetic defects, The subject is administered one of the systems, polypeptides, template RNA, or DNA encoding them according to the previous embodiments, thereby treating the subject who has a disease or symptom associated with a genetic defect. A method that includes this.

[0329] 325. Any method of the preceding embodiment, wherein the disease or condition associated with the genetic defect is one of the indications listed in any of Tables 9 to 12, and / or the genetic defect is a defect in one of the genes listed in any of Tables 9 to 12.

[0330] 326. The subject is a human patient, and the method is one of the embodiments described above.

[0331] definition Domain: As used herein, the term "domain" refers to a structure of a biomolecule that contributes to a specific function of that biomolecule. A domain may include a continuous region (e.g., a continuous sequence) or a distinct discontinuous region (e.g., a discontinuous sequence) of a biomolecule. Examples of protein domains include, but are not limited to, endonuclease domains, DNA-binding domains, and reverse transcription domains; examples of nucleic acid domains include regulatory domains, such as transcription factor-binding domains.

[0332] Extrinsic: As used herein, the term “extrinsic,” when used in relation to biomolecules (e.g., nucleic acid sequences or polypeptides), means that the biomolecule has been artificially introduced into a host genome, cell, or organism. For example, nucleic acids added to an existing genome, cell, tissue, or subject using recombinant DNA technology or other methods are extrinsic to the existing nucleic acid sequence, cell, tissue, or subject.

[0333] First / Second Strand: As used herein, the first and second strands, used to describe individual DNA strands of target DNA, identify two DNA strands on which the reverse transcriptase domain initiates polymerization, for example, based on the location where targeted primed synthesis begins. The first strand refers to the strand of target DNA on which the reverse transcriptase domain initiates polymerization, for example, where targeted primed synthesis begins. The second strand refers to the other strand of target DNA. The names first and second strands do not otherwise describe the target site DNA strands; for example, in some embodiments, the first and second strands are nicked by the polypeptides described herein, but the names "first" and "second" strands are independent of the order in which such nicks appear.

[0334] Genome-safe harbor sites (GSH sites): Genome-safe harbor sites are locations within the host genome that can accommodate the integration of new genetic material, for example, in such a way that the inserted genetic element does not cause significant alteration of the host genome that poses a risk to the host cell or organism. GSH sites generally meet criteria 1, 2, 3, 4, 5, 6, 7, 8, or 9 below: (i) located >300kb from oncological genes; (ii) located >300kb from miRNA / other functional small RNAs; (iii) located >50kb from the 5' gene end; (iv) located >50kb from the origin of replication; (v) located >50kb away from superconserved elements; (vi) having low transcriptional activity (i.e., lacking mRNA+ / -25kb); (vii) not being in a copy number variable region; (viii) being in open chromatin; and / or (ix) having one copy in the human genome and being unique. Examples of GSH sites in the human genome that meet some or all of these criteria include: (i) adenovirus site 1 (AAVS1), a naturally occurring integration site of the AAV virus on chromosome 19; (ii) chemokine (CC motif) receptor 5 (CCR5) gene, a chemokine receptor gene known as the HIV-1 coreceptor; (iii) the human ortholog of the mouse Rosa26 locus; and (iv) rDNA loci. Further GSH sites are known and are described, for example, in Pellenz et al. epub August 20, 2018 (https: / / doi.org / 10.1101 / 396390).

[0335] Heterogeneous: When the term “heterogeneous” is used to describe a first element in relation to a second element, it means that the first and second elements do not naturally exist in the configuration described. For example, heterogeneous polypeptides, nucleic acid molecules, constructs, or sequences refer to (a) a polypeptide, nucleic acid molecule, or portion of a polypeptide or nucleic acid molecule sequence that is not native to the 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 modified or mutated from its native state, or (c) a polypeptide or nucleic acid molecule having modified expression compared to its native expression level under similar conditions. For example, heterogeneous regulatory sequences (e.g., promoters, enhancers) can be used to regulate the expression of a gene or nucleic acid molecule in a manner different from that which is normally expressed in nature. In another example, a heterogeneous domain of a polypeptide, or a nucleic acid sequence (e.g., a DNA-binding domain of a polypeptide, or the nucleic acid encoding the DNA-binding domain of a polypeptide), may be configured in relation to other domains, or may be a different sequence or of a different source compared to other domains or portions of the polypeptide, or the coding nucleic acid thereof. In certain embodiments, heterologous nucleic acid molecules may be present in the native host cell genome, but may have modified expression levels, different sequences, or both. In other embodiments, heterologous nucleic acid molecules may not be endogenous in the host cell or host genome, but may instead be introduced into the host cell by transformation (e.g., transfection, electroporation), where the added molecule may be integrated into the host genome or exist as extrachromosomal genetic material, either transiently (e.g., mRNA) or semi-stable for two or more generations (e.g., episomal viral vector, plasmid, or other self-replicating vector).

[0336] Inverted terminal repeat: As used herein, the term “inverted terminal repeat” or “ITR” refers to an AAV viral cis-element named for its symmetry. This element promotes effective proliferation of the AAV genome. The minimum elements for ITR function are assumed to be a Rep binding site (RBS; 5'-GCGCGCTCGCTCGCTC-3' for AAV2 (SEQ ID NO: 1538) and a terminal separation site (TRS; 5'-AGTTGG-3' for AAV2), as well as a variable palindromic sequence that enables hairpin formation. According to the present invention, an ITR comprises at least these three elements (RBS, TRS, and the sequence that enables hairpin formation). In addition, in the present invention, the term "ITR" refers to ITRs of known native AAV serotypes (e.g., ITRs of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 AAV), chimeric ITRs formed by the fusion of ITR elements derived from different serotypes, and their functional variants. A functional variant of an ITR refers to a sequence that exhibits at least 80%, 85%, 90%, preferably at least 95%, sequence identity with a known ITR, enabling an increase in the sequence containing the ITR in the presence of the Rep protein.

[0337] Mutation or mutant: When the term "(sudden) mutant" is applied to a nucleic acid sequence, it means that nucleotides within the nucleic acid sequence may be inserted, deleted, or altered compared to a reference (e.g., natural) nucleic acid sequence. A single alteration may occur at one locus (point mutation), or multiple nucleotides may be inserted, deleted, or altered at a single locus. In addition, one or more alterations may occur at any number of loci within a single nucleic acid sequence. Nucleic acid sequences can be mutated by any method known in the art.

[0338] Nucleic acid molecules: Nucleic acid molecules refer to, but are not limited to, both RNA and DNA molecules, including cDNA, genomic DNA and mRNA, and also include synthetic nucleic acid molecules, such as those chemically synthesized or produced by recombination, as described herein, such as RNA templates. Nucleic acid molecules may be double-stranded or single-stranded, cyclic or linear. If single-stranded, the nucleic acid molecule may be a sense strand or an antisense strand. Unless otherwise stated, and as an example of all sequences described herein in the general format “Sequence ID,” a nucleic acid including “Sequence ID 1” means a nucleic acid in which at least a portion has either (i) the sequence of Sequence ID 1, or (ii) a sequence complementary to Sequence ID 1. The choice between the two depends on the context in which Sequence ID 1 is used. For example, when a nucleic acid is used as a probe, the choice between the two depends on the requirement that the probe is complementary to the desired target. The nucleic acid sequences of this disclosure may be chemically or biochemically modified, or may contain unnatural or derivatized nucleotide bases, as will be readily apparent to those skilled in the art. Examples of such modifications include labeling, methylation, substitution of one or more spontaneously occurring nucleotides by analogs, internucleotide modifications such as uncharged bonds (e.g., methylphosphonic acid, triester phosphate, phosphoramidates, carbamates, etc.), charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), insertants (e.g., acridine, psoralens, etc.), chelating agents, alkylating agents, and modifying bonds (e.g., α-anomeric nucleic acids, etc.). Synthetic molecules that mimic polynucleotides in their ability to bind to a specified sequence through hydrogen bonding and other chemical interactions are also included. Such molecules are known in the art and, for example, use peptide bonds instead of phosphate bonds in the molecular backbone. Other modifications include analogs that include other structures, such as modifications found in bridging moieties or "locked" nucleic acids, where the ribose ring is located.In various embodiments, nucleic acids are associated with the action of additional genetic elements, e.g., tissue-specific expression-regulatory sequences (e.g., tissue-specific promoters and tissue-specific microRNA recognition sequences), as well as additional elements, e.g., inverted repeats (e.g., inverted terminal repeats, e.g., viral elements (e.g., AAV ITR)) and tandem repeats, inverted repeats / direct repeats (e.g., transposon inverted repeats, e.g., transposon inverted repeats also containing direct repeats, e.g., inverted repeats also containing direct repeats), homologous regions (segments with different degrees of homology to target DNA), UTRs (5', 3', or both 5' and 3' UTRs), and various combinations of the aforementioned. The nucleic acid elements of the systems provided by the present invention may be provided in various topologies, including single-stranded, double-stranded, circular, linear, open-ended linear, closed-ended linear, and specific versions thereof, e.g., doggybone DNA (dbDNA), closed-ended DNA (ceDNA).

[0339] Gene expression unit: A gene expression unit is a nucleic acid sequence comprising at least one regulatory nucleic acid sequence operably ligated to at least one effector sequence. The first nucleic acid sequence is operably ligated to the second nucleic acid sequence when the first nucleic acid sequence is positioned functionally in relation to the second nucleic acid sequence. For example, a promoter or enhancer is operably ligated to a coding sequence if the promoter or enhancer affects the transcription or expression of the coding sequence. The operably ligated DNA sequences may be continuous or discontinuous. If it is necessary to ligate two protein coding regions, the operably ligated sequences may reside within the same reading frame.

[0340] Host: The terms host genome or host cell, as used herein, refer to the cell into which the protein and / or genetic material has been introduced and / or its genome. These terms refer not only to a specific target cell and / or genome, but also to the offspring of such cells and / or the genomes of such offspring. It should be understood that such offspring may not be identical to the parent cell in fact, as certain modifications may occur in later generations due to mutation or environmental influences, but are still included in the scope of the term “host cell” as used herein. A host genome or host cell may be an isolated cell or a cell line grown in culture or genomic material isolated from such cells or cell lines, or it may be a host cell or host genome constituting a living tissue or organism. In some cases, the host cell may be an animal cell or a plant cell, as described herein, for example. In certain cases, the host cell may be a bovine cell, a horse cell, a pig cell, a goat cell, a sheep cell, a chicken cell, or a turkey cell. In certain cases, the host cell may be a maize cell, a soybean cell, a wheat cell, or a rice cell.

[0341] Action-Related: As used herein, “action-related” describes a functional relationship between two nucleic acid sequences, for example, 1) a promoter and 2) a heterologous target sequence, where, in such examples, the promoter and the heterologous target sequence (e.g., the gene of interest) are oriented such that, under appropriate conditions, the promoter drives the expression of the heterologous target sequence. For example, the template nucleic acid may be single-stranded, for example, oriented (+) or (-), but the action-related relationship between the promoter and the heterologous target sequence means that, regardless of whether the template nucleic acid is to be transcribed in a particular state, it will be transcribed correctly if it is in an appropriate state (e.g., oriented (+) in the presence of the required catalyst and NTP, etc.). Action-related relationships apply similarly to other pairs of nucleic acids, including other tissue-specific expression regulatory sequences (e.g., enhancers, repressors, and microRNA recognition sequences), IR / DR, ITR, UTR, or homologous regions, and heterologous target sequences or sequences encoding transposases.

[0342] Pseudoknot: As used herein, “pseudoknot sequence” refers to a nucleic acid (e.g., RNA) having, for example, a first segment, a second segment between the first and third segments (the third segment being complementary to the first segment), and a fourth segment (the fourth segment being complementary to the second segment), the self-complementarity of which is suitable for forming a pseudoknot structure. The pseudoknot may optionally have additional secondary structures, such as a stem-loop located within the second segment, a stem-loop located between the second and third segments, a sequence preceding the first segment, or a sequence following the fourth segment. The pseudoknot may have additional sequences between the first and second segments, between the second and third segments, or between the third and fourth segments. In some embodiments, the segments are arranged 5' to 3' as: first, second, third, and fourth. In some embodiments, the first and third segments contain five fully complementary base pairs. In some embodiments, the second and fourth segments optionally contain ten base pairs having one or more (e.g., two) bulges. In some embodiments, the second segment contains one or more unpaired nucleotides, for example, forming a loop. In some embodiments, the third segment contains one or more unpaired nucleotides, for example, forming a loop.

[0343] Stem-loop sequence: As used herein, “stem-loop sequence” refers to a nucleic acid sequence (e.g., RNA sequence) having a stem containing, for example, at least 2 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) base pairs and a loop containing at least 3 (e.g., 4) base pairs, where self-complementarity is sufficient to form a stem-loop. The stem may contain mismatches or bulges.

[0344] Tissue-Specific Expression-Regulatory Sequences: As used herein, “tissue-specific expression-regulatory sequence” means a nucleic acid element that increases or decreases the level of a transcript containing a heterologous target sequence in a tissue-specific manner within a target tissue, for example, preferentially in an on-target tissue compared to an off-target tissue. In some embodiments, tissue-specific expression-regulatory sequences preferentially drive or suppress the transcription, activity, or half-life of a transcript containing a heterologous target sequence in a tissue-specific manner within a target tissue, for example, preferentially in an on-target tissue compared to an off-target tissue. Exemplary tissue-specific expression-regulatory sequences include tissue-specific promoters, repressors, enhancers, or combinations thereof, and tissue-specific microRNA recognition sequences. Tissue specificity refers to on-target (tissues in which the expression or activity of the template nucleic acid is desired or acceptable) and off-target (tissues in which the expression or activity of the template nucleic acid is undesirable or unacceptable). For example, a tissue-specific promoter (e.g., a promoter that controls the expression of a template nucleic acid or a transposase) preferentially drives expression in an on-target tissue compared to an off-target tissue. In contrast, microRNAs that bind to tissue-specific microRNA recognition sequences (on the transposase-encoding nucleic acid, or on the template nucleic acid, or both) are preferentially expressed in off-target tissues compared to on-target tissues, thereby reducing the expression of the template nucleic acid (or transposase) in off-target tissues. Therefore, promoters and microRNA recognition sequences specific to the same tissue, e.g., target tissue, have contrasting functions with respect to the transcription, activity, or half-life of the related sequences within the tissue (matching expression levels, i.e., promoting and suppressing high levels of microRNA in off-target tissues and low levels in on-target tissues, respectively, while promoters drive high expression in on-target tissues and low expression in off-target tissues).

[0345] A patent file or application file must include at least one color-illustrated drawing. A copy of a patent publication or patent application publication containing a color-illustrated drawing will be provided by the Office immediately upon request and payment of the required fees. In embodiments of the present invention, for example, the following items are provided. (Item 1) A system for modifying DNA, (a) a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to the polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising, wherein the RT domain includes the sequence of Table 1 or 3, or the sequence of the reverse transcriptase domain of Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. (Item 2) A system for modifying DNA, (a) a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to the polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. Includes, The RT domain includes the sequence in Table 1 or 3 or the reverse transcriptase domain sequence in Table 2. The RT domain further comprises several substitutions relative to the natural sequence, for example, at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions in the system. (Item 3) A system for modifying DNA, (a) a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to the polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (etRNA) (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising, capable of causing the insertion of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides into the target site. (Item 4) A system for modifying DNA, (a) a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to the polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising, wherein the heterogeneous target sequence has a length of at least 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 nts. (Item 5) The system according to any one of items 1 to 4, wherein the RT domain is heterogeneous with respect to the DBD; the DBD is heterogeneous with respect to the endonuclease domain; or the RT domain is heterogeneous with respect to the endonuclease domain, one or more of the above. (Item 6) A system for modifying DNA, (a) a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to the polypeptide. (iii) a sequence, a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising, capable of causing deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides at the target site. (Item 7) A system for modifying DNA, (a) a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to the polypeptide, (iii) a heterologous target sequence, and (iv) a template (or DNA encoding the template RNA) comprising a 3' target homology domain. A system comprising (a)(ii) and / or (a)(iii) a TAL domain; a zinc finger domain; or a CRISPR / Cas domain or a functional variant thereof (e.g., a variant) selected from Table 3. (Item 8) A system for modifying DNA, (a) a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome) (e.g., a CRISPR spacer), (ii) optionally, a sequence that binds to the polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. A system comprising the endonuclease domain, e.g., nickasase domain, which cleaves both strands of the target site DNA, wherein the cleavage is separated from each other by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 nucleotides. (Item 9) A system for modifying DNA, (a) a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) a sequence that specifically binds to the RT domain, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) comprising a 3' target homology domain. A system that includes this. (Item 10) The system according to item 9, wherein the template RNA further comprises sequences that bind to (a)(ii) and / or (a)(iii). (Item 11) A system for modifying DNA, (a) A first polypeptide or nucleic acid encoding the first polypeptide, wherein the first polypeptide comprises (i) a reverse transcriptase (RT) domain and (ii) optionally a DNA-binding domain, (b) a second polypeptide or nucleic acid encoding the second polypeptide, wherein the second polypeptide comprises (i) a DNA-binding domain (DBD); (ii) an endonuclease domain, such as a niccasse domain; and (c) (e.g., from 5' to 3') (i) optionally a sequence that binds to the second polypeptide (e.g., to (b)(i) and / or to (b)(ii)), (ii) optionally a sequence that binds to the first polypeptide (e.g., to the RT domain), (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) comprising a 3' target homology domain. A system that includes this. (Item 12) A system for modifying DNA, (a) a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain and (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; (b) A first template RNA (or DNA encoding the RNA) comprising (i) a sequence that binds to the polypeptide (e.g., from 5' to 3') (i) a sequence that binds to (a)(ii) and / or (a)(iii) and (ii) a sequence that binds to a target site (e.g., the second strand of the site in the target genome) (e.g., where the first RNA includes gRNA); (c) (e.g., from 5' to 3') (i) optionally, a sequence that binds to the polypeptide (e.g., specifically to the RT domain), (ii) a heterologous target sequence, and (iii) a second template RNA (or DNA encoding the RNA) comprising a 3' target homology domain. A system that includes this. (Item 13) (For example, from 5' to 3') a template RNA (or DNA encoding the template RNA) comprising (i) a sequence that binds to a target site (for example, the second strand of the site in the target genome), (ii) a sequence that specifically binds to the RT domain of the polypeptide, (iii) a heterologous target sequence, and (iv) a 3' target homology domain. (Item 14) (v) The template RNA described in item 13, further comprising a sequence that binds to the endonuclease and / or DNA-binding domain of a polypeptide (e.g., the same polypeptide including the RT domain). (Item 15) The RT domain is a template RNA as described in item 13 or 14, comprising a selected sequence from Table 1 or 3, or a reverse transcriptase domain sequence from Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. (Item 16) The template RNA described in item 13 or 14, wherein the RT domain comprises a selected sequence from Table 1 or 3 or a reverse transcriptase domain sequence from Table 2, and the RT domain further comprises several substitutions relative to the native sequence, e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions. (Item 17) (ii) The sequence is a template RNA according to any one of items 13 to 16 that specifically binds to the RT domain. (Item 18) The sequence that specifically binds to the RT domain is a sequence from the domains in Table 1 or Table 2, for example, a UTR sequence or at least 70, 75, 80, 85, 90, 95 such sequences. Alternatively, a template RNA described in any one of items 13-17, which is a sequence with 99% identity. (Item 19) From 5' to 3': (ii) a sequence that binds to the endonuclease and / or DNA-binding domain of the polypeptide, (i) a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) comprising the 3' target homology domain. (Item 20) From 5' to 3': (iii) a heterologous target sequence, (iv) a 3' target homology domain, (i) a sequence that binds to a target site (e.g., the second strand of the site in the target genome), and (ii) a sequence that binds to the endonuclease and / or DNA-binding domain of the polypeptide (or DNA encoding the template RNA). (Item 21) The template RNA, the first template RNA, or the second template RNA is a system or template RNA according to any one of items 1 to 20, comprising a sequence that specifically binds to the RT domain. (Item 22) A system for modifying DNA, (a)(i) a sequence that binds to the endonuclease domain of a polypeptide, e.g., the nickase domain and / or the DNA-binding domain (DBD), and (ii) a sequence that binds to a target site (e.g., the second strand of the site in the target genome) (or DNA encoding the first template RNA) (e.g., where the first RNA includes gRNA); (b) a second template RNA (or DNA encoding the second template RNA) comprising (i) a sequence that specifically binds to the reverse transcriptase (RT) domain of a polypeptide (e.g., the polypeptide of (a)), (ii) a heterologous target sequence, and (iii) a 3' target homology domain. A system that includes this. (Item 23) The system described in item 14, in which the nucleic acid encoding the first template RNA and the nucleic acid encoding the second template RNA are two separate nucleic acids. (Item 24) The nucleic acid encoding the first template RNA and the nucleic acid encoding the second template RNA are parts of the same nucleic acid molecule, and are, for example, located on the same vector, as described in item 14. (Item 25) A polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a niccasse domain, wherein the RT domain has a sequence from Table 1 or 3, or a sequence of the reverse transcriptase domain from Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. (Item 26) A polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD), and (iii) an endonuclease domain, such as a nickasase domain, the RT domain having a sequence of Table 1 or 3 or a reverse transcriptase domain sequence of Table 2, and the RT domain further comprising several substitutions relative to the native sequence, such as at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions. (Item 27) A system for modifying DNA, (a) A first polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide is a reverse transcriptase (RT) domain having a sequence of Table 1 or 3 or a sequence of Table 2 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and optionally comprising a DNA-binding domain (DBD) (e.g., a first DBD); and (b) A second polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a DBD (e.g., a second DBD); and (ii) an endonuclease domain, e.g., a niccasse domain. A system that includes this. (Item 28) A system for modifying DNA, (a) A first polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide is a reverse transcriptase (RT) domain having a sequence of Table 1 or 3 or a reverse transcriptase domain sequence of Table 2, and further comprising several substitutions relative to the native sequence, e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions; and optionally comprising a DNA-binding domain (DBD) (e.g., a first DBD); and (b) A second polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a DBD (e.g., a second DBD); and (ii) an endonuclease domain, e.g., a niccasse domain. A system that includes this. (Item 29) The system according to item 27 or 28, wherein the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are two separate nucleic acids. (Item 30) The system described in item 27 or 28, wherein the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are parts of the same nucleic acid molecule and, for example, are located on the same vector. (Item 31) A reaction mixture comprising cells and any system, polypeptide, template RNA, or DNA encoding it as described in any one of items 1 to 30. (Item 32) A reaction mixture comprising DNA containing a target site and any system, polypeptide, template RNA, or DNA encoding it as described in any one of items 1 to 30. (Item 33) A system, polypeptide, template RNA, or DNA encoding it, as described in any one of items 1 through 30; Instructions for using the aforementioned system, polypeptide, template RNA or DNA encoding it; and One or both of the cells or DNA containing the target site A kit that includes this. (Item 34) The DBD is a system, kit, polypeptide, or reaction mixture according to any one of items 1 to 33, comprising a TAL domain. (Item 35) The DBD is a system, kit, polypeptide, or reaction mixture according to any one of items 1 to 34, comprising a zinc finger domain. (Item 36) The aforementioned DBD includes a CRISPR / Cas domain and is one of items 1 to 35. The system, kit, polypeptide, or reaction mixture described herein. (Item 37) The system, kit, polypeptide, or reaction mixture according to any one of items 1 to 36, wherein the endonuclease domain is a nickasase domain. (Item 38) The endonuclease domain comprises a CRISPR / Cas domain, and is a system, kit, polypeptide, or reaction mixture as described in any one of items 1 to 37. (Item 39) The CRISPR / Cas domain is a system, kit, polypeptide, or reaction mixture according to any one of items 1 to 38, comprising a domain or polypeptide from Table 4 or a functional variant thereof (e.g., a mutant). (Item 40) The CRISPR / Cas domain is a system, kit, polypeptide, or reaction mixture described in any one of items 1 to 39, comprising a domain or polypeptide from a genus / species in Table 4. (Item 41) The system, kit, polypeptide, or reaction mixture according to any one of items 1 to 40, wherein the endonuclease domain comprises a type IIs nuclease (e.g., FokI), a Holliday junction resolver, or a double-stranded DNA nuclease that has been modified to inhibit its ability to cleave one strand (e.g., converting a double-stranded DNA nuclease to a nickase). (Item 42) The RT domain comprises a reverse transcriptase selected from Table 1 or 3, or a functional fragment or variant thereof, or a sequence of a reverse transcriptase domain from Table 2, as described in any one of items 1 to 41. (Item 43) The RT domain comprises one or more mutations (e.g., insertions, deletions, or substitutions) in the sequence of a naturally occurring RT domain or an RT domain or functional fragment selected from Table 1 or 3 or a reverse transcriptase domain in Table 2, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mutations, as described in any one of items 1 to 42. (Item 44) The system, kit, polypeptide, or reaction mixture described in item 43, wherein the one or more mutations are selected from 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 mouse leukemia virus reverse transcriptase, or the corresponding mutation at the corresponding position in another RT domain. (Item 45) The one or more of the aforementioned mutations in the RT domain of the R2Bm retrotransposase are as described in International Publication No. 2018089860A1, incorporated herein by reference (e.g., C952S and / or C956S and / or C952S, C956S (double mutant), and / or C969S and / or H970Y and / or R979Q and / or R976Q and / or R1071S, and and / or R328A and / or R329A and / or Q336A and / or R328A, R329A, Q336A (triple mutant) and / or G426A and / or D428A and / or G426A, D428A (double mutant) mutations and / or any combination thereof; at the position corresponding to Sequence ID No. 52 in International Publication No. 2018089860A1 or at the corresponding position in another RT domain A system, kit, polypeptide, or reaction mixture as described in item 43, selected from the corresponding mutations. (Item 46) The RT domain is located at the C-terminus of the DBD in the polypeptide, the system, kit, polypeptide, or reaction mixture according to any one of items 1 to 45. (Item 47) The system, kit, polypeptide, or reaction mixture according to any one of items 1 to 46, wherein the RT domain is located at the C-terminus of the nickase domain in the polypeptide. (Item 48) The RT domain is located at the N-terminus of the DBD in the polypeptide, the system, kit, polypeptide, or reaction mixture according to any one of items 1 to 47. (Item 49) The system, kit, polypeptide, or reaction mixture according to any one of items 1 to 48, wherein the RT domain is located at the N-terminus of the nickase domain in the polypeptide. (Item 50) The polypeptide comprises, for example, a linker located between the RT domain and the DBD or between the RT domain and the nickase domain, the system, kit, polypeptide or reaction mixture according to any one of items 1 to 49. (Item 51) The linker is an amino acid having a length of 2 to 50, for example, 2 to 30, as described in any one of items 1 to 50, of the systems, kits, polypeptides, or reaction mixtures. (Item 52) The system, kit, polypeptide, or reaction mixture according to any one of items 1 to 51, wherein the linker is, for example, a flexible linker comprising Gly and / or Ser residues. (Item 53) The system, kit, template RNA, or reaction mixture according to any one of items 1 to 52, wherein the 3' target homology domain is complementary to a sequence adjacent to the site modified by the system, or includes one, two, three, four, or five mismatches with a sequence complementary to the sequence adjacent to the site modified by the system. (Item 54) The 3' target homology domains include >4, >5, >6, >7, >8, >9, >10, >11, >12, >13, >14, >15, >16, >17, >18, >19, >20, >21, >22, >23, >2. >4, >25, >26, >27, >28, >29, >30, >35, >40, >45, >50, >55, >60, >65, >70, >75, >80, >85, >90, >95, >100, >110, 12 A system, kit, template RNA, or reaction mixture described in any one of items 1 to 53, having a nucleotide length greater than 0, greater than 130, greater than 140, greater than 150, greater than 160, greater than 170, greater than 180, greater than 190, or greater than 200 (for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotide lengths). (Item 55) The 3' target homology domain is 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 21 or less, 22 or less, 23 or less, 24 or less, 25 or less, 26 or less, 27 or less, 28 or less, 29 or less, 30 or less, 35 or less, 40 or less, 45 or less, 50 or less, 55 or less, 60 or less, 65 or less, 70 or less, 75 or less, 80 or less, 85 or less, A system, kit, template RNA, or reaction mixture described in any one of items 1 to 54, having a nucleotide length of 90 or less, 95 or less, 100 or less, 110 or less, 120 or less, 130 or less, 140 or less, 150 or less, 160 or less, 170 or less, 180 or less, 190 or less, or 200 or less. (Item 56) The heterologous target sequence is complementary to the sites modified by the system, except at one or more sites to be modified, as described in any one of items 1 to 55. (Item 57) The system, kit, template RNA, or reaction mixture according to any one of items 1 to 56, wherein the heterogeneous target sequence is complementary to the site modified by the system, except at the location where it codes for the sequence to be inserted into the site. (Item 58) The system, kit, template RNA, or reaction mixture according to any one of items 1 to 57, wherein the heterologous target sequence is complementary to the site modified by the system, except that the heterologous target sequence does not contain a nucleotide encoding the sequence to be deleted at the site. (Item 59) The heterologous target sequence is a system, kit, template RNA, or reaction mixture according to any one of items 1 to 58, wherein the heterologous target sequence has a nucleotide length greater than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (for example, a nucleotide length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). (Item 60) The heterologous target sequence has a nucleotide length of 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, 11 or less, 12 or less, 13 or less, 14 or less, 15 or less, 16 or less, 17 or less, 18 or less, 19 or less, 20 or less, 21 or less, 22 or less, 23 or less, 24 or less, 25 or less, 26 or less, 27 or less, 28 or less, 29 or less, or 30 or less, as described in any one of items 1 to 59. (Item 61) The heterologous target sequence is a system, kit, template RNA, or reaction mixture according to any one of items 1 to 60, in which the non-target site nucleotides are replaced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. (Item 62) The heterogeneous target sequence inserts 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 or 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 into the target site, as described in any one of items 1 to 61, a system, kit, template RNA or reaction mixture. (Item 63) The heterologous target sequence is characterized by the 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, 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides, as described in any one of items 1 to 62, a system, kit, template RNA, or reaction mixture. (Item 64) The heterogeneous target sequence is isolated from the sequence that binds to the polypeptide (for example, the endonuclease domain and / or DBD domain) by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nucleotides, according to any one of items 1 to 63, in a system, kit, template RNA, or reaction mixture. (Item 65) The system, kit, template RNA, or reaction mixture described in any one of items 1 to 64, wherein the sequence that binds to the polypeptide (for example, the endonuclease domain and / or DBD domain) is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130 nucleotide lengths (and optionally 150 or less, 140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 85 or less, or 80 or less). (Item 66) The sequence that binds to the polypeptide binds to the endonuclease domain and / or DBD domain, as described in any one of items 1 to 65, in the system, kit, template RNA, or reaction mixture described in any one of items 1 to 65. (Item 67) The sequence that binds to the polypeptide (for example, the endonuclease domain and / or DBD domain) comprises a gRNA, and is part of a system, kit, template RNA, or reaction mixture according to any one of items 1 to 66. (Item 68) The sequence that binds to the target site (for example, the second strand of the site in the target genome) is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120 or 130 nucleotides long (and optionally 150 or less, 140 or less, 130 or less, 120 or less, 110 or less, 1 A system, kit, template RNA, or reaction mixture described in any one of items 1 to 67, having a nucleotide length of 00 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 29 or less, 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, or 20 or less, for example, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. (Item 69) The system, kit, template RNA, or reaction mixture according to any one of items 1 to 68, wherein the sequence that binds to the target site is complementary to the second strand of the target site, or includes one, two, three, four, or five mismatches with a sequence that is complementary to the second strand of the target site. (Item 70) A system, kit, template RNA, or reaction mixture according to any one of items 1 to 69, wherein the sequence that binds to a target site (e.g., the second strand of the site in the target genome) is isolated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nucleotides from the sequence that binds to the polypeptide (e.g., the endonuclease domain and / or DBD domain). (Item 71) A system, kit, template RNA, or reaction mixture according to any one of items 1 to 70, further comprising a second strand targeting gRNA that directs the endonuclease domain (e.g., nickase) domain to nick the second strand (e.g., in the target genome). (Item 72) The system, kit, template RNA, or reaction mixture described in item 71 further comprises the second strand targeting gRNA. (Item 73) The system, kit, template RNA, or reaction mixture described in item 71, wherein the second strand targeting gRNA is located on a nucleic acid separate from the template RNA. (Item 74) A system, kit, template RNA, or reaction mixture according to any one of items 71-73, wherein the gRNA is directed to introduce a nick into the second strand (e.g., in the target genome) at a site where 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 are 5' or 3' to the target site modification (e.g., nick on the first strand). (Item 75) The gRNA specifically binds to the first strand, and is a system, kit, template RNA, or reaction mixture according to any one of items 71 to 74. (Item 76) mRNA encoding a polypeptide or system as described in any one of items 1 to 75, and Template RNA as described in any one of items 1-75 A system that includes this. (Item 77) The mRNA encoding the polypeptide or system described in any one of items 1 to 76 and the template RNA described in any one of items 1 to 76 are arranged on different nucleic acid molecules, as described in item 76. (Item 78) A template RNA (or RNA encoding the template RNA) described in any one of items 1 to 77, and Sequences encoding a system or polypeptide as described in any one of items 1 through 77. A system containing RNA molecules. (Item 79) The system according to item 78, wherein the RNA molecule includes, for example, an internal ribosome entry site operably linked to the sequence encoding the system or polypeptide. (Item 80) The system according to item 78 or 79, wherein the RNA molecule includes, for example, a cleavage site located between the template RNA (or the RNA encoding the template RNA) and the sequence encoding the system or polypeptide. (Item 81) The polypeptide comprises a split intein, and two or more (e.g., all) of the RT domain, DBD, endonuclease (e.g., nickase) domain, or combinations thereof are translated as separate proteins that combine into a single polypeptide by protein splicing, according to any one of items 1 to 80. (Item 82) Lipid nanoparticles (LNPs) comprising a system, template RNA, polypeptide (or RNA encoding the same) as described in any one of items 1 to 81, or DNA encoding the said system, template RNA, or polypeptide. (Item 83) A polypeptide (or DNA or RNA encoding it) of the system described in any one of items 1 to 82, or a first lipid nanoparticle containing the polypeptide described in any one of items 1 to 82; and A template RNA (or DNA encoding it) for the system described in any one of items 1 to 82, or a second lipid nanoparticle containing the template RNA described in any one of items 1 to 82. A system that includes this. (Item 84) A virus, virus-like particle, fusosome, or virosome comprising a system, template RNA, polypeptide (or RNA encoding such system, template RNA, or polypeptide) as described in any one of items 1 to 83. (Item 85) A polypeptide (or DNA or RNA encoding it) of any one of items 1 to 84, or a first virus, virus-like particle, fusosome or virosome containing a polypeptide described in any one of items 1 to 84; and A template RNA (or DNA encoding it) for the system described in any one of items 1-84, or a second virus, virus-like particle, or virosome containing the template RNA described in any one of items 1-84. A system that includes this. (Item 86) A system, kit, template RNA, or reaction mixture according to any one of items 1 to 85, wherein at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA has a nucleotide length greater than 100, greater than 125, greater than 150, greater than 175, or greater than 200, or at least 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 kilobase lengths (and optionally a kilobase length less than 15, less than 10, less than 5, or less than 20, or a nucleotide length less than 500, less than 400, less than 300, or less than 200). (Item 87) The system, kit, template RNA, or reaction mixture according to any one of claims 1 to 86, wherein at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains a poly-A tail (for example, a poly-A tail having a length of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides). (Item 88) At least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA is 5' caps, e.g., 7-methylguanosine caps (e.g., O-Me-m7G caps); hypermethylated cap analogs; NAD+-derived cap analogs (e.g., as described in Kiledjian, Trends in Cell Biology 28, 454-464 (2018)); or modified, e.g., biotinylated cap analogs (e.g., Bednarek et al., Phil Trans R (as described in Soc B 373, 20180167 (2018)), and / or Poly-A tail; a 16-nucleotide stem-loop structure flanked by five unpaired nucleotides (e.g., as described by Mannironi et al., Nucleic Acid Research 17, 9113-9126 (1989)); a triple helix structure (e.g., as described by Brown et al., PNAS 109, 19202-19207 (2012)); tRNA, Y RNA, or vault RNA structure (e.g., Labno et al., Biochemica et Biophysica Acta 3' features selected from one or more of the following: as described by 1863,3125-3147 (2016); incorporation of one or more deoxyribonucleotide triphosphates (dNTPs), 2'O-methylated NTPs, or phosphorothioate-NTPs; single nucleotide chemical modifications (e.g., oxidation of the 3'-terminal ribose to a reactive aldehyde, followed by conjugation of an aldehyde-reactive modified nucleotide); or chemical ligation to another nucleic acid molecule. A system, kit, template RNA, or reaction mixture containing any one of items 1 to 87. (Item 89) The template RNAs include, for example, dihydrouridine, inosine, 7-methylguanosine, 5-methylcytidine (5mC), 5'-phosphate lipothymidine, 2'-O-methyllipothymidine, 2'-O-ethyllipothymidine, 2'-fluorolipothymidine, C-5 propynyl-deoxycytidine (pdC), C-5 propynyl-deoxyuridine (pdU), C-5 propynyl-cytidine (pC), C-5 propynyl-uridine (pU), 5-methylcytidine, 5-methyluridine, 5-methyldeoxycytidine, 5-methyldeoxyuridine methoxy, 2,6-diaminopurine, and 5'-dimethoxytrityl-N4-ethyl-2'- A system, kit, template RNA, or reaction mixture described in any one of items 1 to 88, comprising one or more modified nucleotides selected from oxycytidine, 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 (sub-groove binder), pseudouridine (Ψ), 1-N-methylpsoiduridine (1-Me-Ψ), or 5-methoxyuridine (5-MO-U). (Item 90) A system, kit, template RNA, or reaction mixture according to any one of items 1 to 89, wherein at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains one or more modified nucleotides. (Item 91) A system, kit, template RNA, or reaction mixture according to any one of items 1 to 90, wherein at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA remains intact after stability testing (e.g., nucleotide lengths greater than 100, 125, 150, 175, or 200 or at least 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 kilobase lengths). (Item 92) A system, kit, or reaction mixture according to any one of items 1 to 91, wherein at least 1% of the target site is modified after the system has been assayed for efficacy. (Item 93) The system, polypeptide, template RNA and / or DNA encoding it, is formulated as lipid nanoparticles (LNPs) in any one of the systems, kits, template RNA, polypeptide, or reaction mixtures described in any one of items 1 to 92. (Item 94) The system, the polypeptide and / or the DNA encoding the template RNA, is packaged in a virus, virus-like particle, virosome, liposome, vesicle, exosome or LNP, as described in any one of items 1 to 93. (Item 95) The system, the DNA encoding the template RNA or polypeptide, is packaged in adeno-associated virus (AAV), as described in item 94. (Item 96) The system, template RNA, polypeptide, lipid nanoparticles (LNP), virus, virus-like particles, or virosome described in any one of items 1 to 95 is free from or substantially free from pyrogen, virus, fungal, bacterial pathogen and / or host cell protein contamination. (Item 97) A system, template RNA or polypeptide, or DNA encoding either of the above, as described in any one of items 1 to 96, and Adeno-associated virus (AAV) capsid protein Viruses, virus-like particles, or viromosomes containing these particles. (Item 98) The system, template RNA and / or polypeptide described in any one of items 1 to 97 is active in target tissue and less active (e.g., inactive) in non-target tissue. (Item 99) The system, kit, template RNA, polypeptide, virus, virus-like particle, or virosome according to item 98, further comprising one or more first tissue-specific regulatory sequences specific to the target tissue, wherein the one or more first tissue-specific regulatory sequences specific to the target tissue are operably associated with the template RNA, the polypeptide or nucleic acid encoding it, or both. (Item 100) A method for producing template RNA, (a) To provide a template RNA as described in any one of items 1 to 99, (b)(i) the length of the template RNA, for example, whether the template RNA has a length greater than or within the reference length, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA has a nucleotide length greater than 100, greater than 125, greater than 150, greater than 175, or greater than 200; (ii) the presence, absence, and / or length of the poly(A) tail on the template RNA, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains a poly(A) tail (for example, a poly(A) tail having a length of at least 5, 10, 20, or 30 nucleotides); (iii) the presence, absence and / or type of 5' cap on the template RNA, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains a 5' cap, for example, whether the cap is a 7-methylguanosine cap, for example, an O-Me-m7G cap; (iv) One or more modified nucleotides in the template RNA (e.g., dihydrouridine, inosine, 7-methylguanosine, 5-methylcytidine (5mC), 5'-phosphate lipothymidine, 2'-O-methylribothymidine, 2'-O-ethylribothymidine, 2'-fluororibothymidine, C-5 propynyl-deoxycytidine (pdC), C-5 propynyl-deoxyuridine (pdU), C-5 propynyl-cytidine (pC), C-5 propynyl-uridine (pU), 5-methylcytidine, 5-methyluridine, 5-methyldeoxycytidine, 5-methyldeoxyuridine methoxy, 2,6-diaminopurine, 5'-dimethoxytrityl-N4-ethyl-2'-deoxycytidine The presence, absence, and / or type of din, 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 (sub-groove binder), pseudouridine (Ψ), 1-N-methylpsoiduridine (1-Me-Ψ), or 5-methoxyuridine (5-MO-U), for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains one or more modified nucleotides; (v) Stability of the template RNA (for example, over time and / or under pre-selected conditions), e.g., at least 80, 85, 90, 95, 96, 97, 98 or 99% of the template RNA remains intact after stability testing (e.g., over 100, over 125, over 150, over 175 or Whether or not the nucleotide length remains above 200; (vi) the efficacy of the template RNA in a system for modifying DNA, for example, whether at least 1% of the target sites are modified after the system containing the template RNA has been assayed for efficacy; or (vii) the presence, absence and / or level of one or more pyrogens, viruses, fungi, bacterial pathogens or host cell proteins, for example, whether the template RNA is free or substantially free of pyrogen, virus, fungi, bacterial pathogens or host cell protein contamination. Assaying one or more of the following A method that includes this. (Item 101) A method for manufacturing a system for modifying DNA, (a) To provide a system for modifying DNA as described in any one of items 1 to 99, (b)(i) the length of the template RNA, for example, whether the template RNA has a length greater than or within the reference length, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA has a nucleotide length greater than 100, greater than 125, greater than 150, greater than 175, or greater than 200; (ii) the presence, absence, and / or length of the poly(A) tail on the template RNA, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains a poly(A) tail (for example, a poly(A) tail having a length of at least 5, 10, 20, or 30 nucleotides); (iii) the presence, absence and / or type of 5' cap on the template RNA, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains a 5' cap, for example, whether the cap is a 7-methylguanosine cap, for example, an O-Me-m7G cap; (iv) the presence, absence, and / or type of one or more modified nucleotides in the template RNA (e.g., selected from pseudouridine, dihydrouridine, inosine, 7-methylguanosine, 1-N-methylpsoiduridine (1-Me-Ψ), 5-methoxyuridine (5-MO-U), 5-methylcytidine (5mC), or locked nucleotides), for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present template RNA contains one or more modified nucleotides; (v) The stability of the template RNA (for example, over time and / or under pre-selected conditions), e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA remains intact (e.g., with nucleotide lengths greater than 100, 125, 150, 175, or 200) after stability testing; (vi) the efficacy of the template RNA in a system for modifying DNA, for example, whether at least 1% of the target sites are modified after the system containing the template RNA has been assayed for efficacy; (vii) The length of the polypeptide, the first polypeptide, or the second polypeptide, for example, whether the polypeptide, the first polypeptide, or the second polypeptide has a length greater than or within the reference length, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present polypeptide, the first polypeptide, or the second polypeptide has a length greater than 600, greater than 650, greater than 700, greater than 750, greater than 800, greater than 850, greater than 900, or 95 Whether the amino acid length is greater than 0, greater than 1000, greater than 1050, greater than 1100, greater than 1150, greater than 1200, greater than 1250, greater than 1300, greater than 1350, greater than 1400, greater than 1450, greater than 1500, greater than 1600, greater than 1700, greater than 1800, greater than 1900, or greater than 2000 (and optionally, amino acid lengths of 2500 or less, 2000 or less, 1500 or less, 1400 or less, 1300 or less, 1200 or less, 1100 or less, 1000 or less, 900 or less, 800 or less, 700 or less, or 600 or less); (viii) the presence, absence and / or type of post-translational modifications on the polypeptide, the first polypeptide or the second polypeptide, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, the first polypeptide or the second polypeptide contains the selected post-translational modifications; (ix) the presence, absence and / or type of one or more artificial, synthetic or nonstandard amino acids in the polypeptide, the first polypeptide or the second polypeptide, for example, whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the present polypeptide, the first polypeptide or the second polypeptide contain one or more artificial, synthetic or nonstandard amino acids; (x) The stability of the polypeptide, the first polypeptide or the second polypeptide (for example, over time and / or under pre-selected conditions), for example, that at least 80, 85, 90, 95, 96, 97, 98 or 99% of the polypeptide, the first polypeptide or the second polypeptide remain intact after the stability test (for example, over 600, over 650, over 700, over 750, over 800, over 850, over 900, over 950, over 1000, over 1050, Whether the amino acid length remains above 1100, above 1150, above 1200, above 1250, above 1300, above 1350, above 1400, above 1450, above 1500, above 1600, above 1700, above 1800, above 1900, or above 2000 (and optionally below 2500, below 2000, below 1500, below 1400, below 1300, below 1200, below 1100, below 1000, below 900, below 800, below 700, or below 600); (xi) The efficacy of the polypeptide, the first polypeptide, or the second polypeptide in a system for modifying DNA, for example, whether at least 1% of the target site is modified after the system containing the polypeptide, the first polypeptide, or the second polypeptide has been assayed for efficacy; or (xii) The presence, absence and / or level of one or more pyrogens, viruses, fungi, bacterial pathogens or host cell proteins, for example, whether the system is free or substantially free of pyrogen, virus, fungi, bacterial pathogens or host cell protein contamination. Assaying one or more of the following A method that includes this. (Item 102) A method for modifying target sites in genomic DNA in cells, The aforementioned cells twice (a) a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally a sequence that binds to the target site (e.g., the second strand of the site in the target genome), (ii) optionally a sequence that binds to the polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. To bring into contact with and thereby modify the target site in the genomic DNA of the cell. A method that includes this. (Item 103) A method for modifying target sites in genomic DNA in cells, The cells are brought into contact with the system, polypeptide, template RNA, or DNA encoding it, as described in any one of items 1 to 99, thereby modifying the target site in the genomic DNA of the cells. A method that includes this. (Item 104) A method for modifying target sites in genomic DNA in cells, The aforementioned cells twice (a) a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally a sequence that binds to the target site (e.g., the second strand of the site in the target genome), (ii) optionally a sequence that binds to the polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. To bring into contact with and thereby modify the target site in the genomic DNA of the cell. A method comprising, wherein the cells are euploid, unmortalized, part of a tissue, part of an organism, primary cells, non-dividing, haploid (e.g., germline cells), non-cancerous pluripotent cells, or from an object having a genetic disorder. (Item 105) A method for treating subjects having a disease or condition associated with a genetic defect, The aforementioned target, (a) a polypeptide or nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, such as a niccasse domain; and (b) (e.g., from 5' to 3') (i) optionally, a sequence that binds to a target site (e.g., the second strand of the site in the target genome), (ii) optionally, a sequence that binds to the polypeptide, (iii) a heterologous target sequence, and (iv) a template RNA (or DNA encoding the template RNA) containing a 3' target homology domain. To administer and thereby treat the subject having a disease or condition associated with a genetic defect. A method that includes this. (Item 106) A method for treating subjects having a disease or condition associated with a genetic defect, Administering to the subject a system, polypeptide, template RNA, or DNA encoding the same as described in any one of items 1 to 99, thereby treating the subject who has a disease or condition associated with a genetic defect. A method that includes this. (Item 107) The method according to item 105 or 106, wherein the disease or condition associated with a genetic defect is an indication listed in any of Tables 9 to 12, and / or the genetic defect is a defect in a gene listed in any of Tables 9 to 12. (Item 108) The subject is a human patient, and the method is described in any one of items 105 to 107. [Brief explanation of the drawing]

[0346] [Figure 1] This is a schematic diagram of the Gene Writing (trademark) genome editing system. [Figure 2] This is a schematic diagram of the structure of the Gene Writer (trademark) genome editor polypeptide. [Figure 3] This is a schematic diagram of the structure of an example GeneWriter™ template RNA. [Figure 4A]This is a series of diagrams showing examples of the stereochemistry of GeneWriter using domains derived from various sources. GeneWriter described herein may or may not include all of the domains shown. For example, GeneWrite may optionally lack an RNA-binding domain, or it may have a single domain that satisfies the functions of multiple domains, such as a Cas9 domain for DNA binding and endonuclease activity. Exemplary domains that may be included in a GeneWriter polypeptide include DNA-binding domains (e.g., DNA-binding domains; zinc fingers; TAL domains; Cas9; dCas9; nickase Cas9; transcription factors; or meganucleases, for example, as shown in the table herein), RNA-binding domains (e.g., RNA-binding domains of B-box proteins, MS2 coat proteins, dCas, or elements of sequences in the table herein), reverse transcriptase domains (e.g., reverse transcriptase domains of elements of sequences in the table herein; other retrotransposers) Examples include peptides containing reverse transcriptase domains (e.g., those listed in the table herein); reverse transcriptase domains (e.g., those listed in the table herein); and / or endonuclease domains (e.g., endonuclease domains of the elements in the table herein; Cas9; nickasase Cas9; restriction enzymes (e.g., type II restriction enzymes, e.g., FokI); meganucleases; Holliday junction resolvers; RLE retrotranspases; APE retrotransposases; or GIY-YIG retrotransposases). Exemplary GeneWriter polypeptides containing exemplary combinations of such domains are shown in the bottom panel. [Figure 4B]This is a series of diagrams showing examples of the stereochemistry of GeneWriter using domains derived from various sources. GeneWriter described herein may or may not include all of the domains shown. For example, GeneWrite may optionally lack an RNA-binding domain, or it may have a single domain that satisfies the functions of multiple domains, such as a Cas9 domain for DNA binding and endonuclease activity. Exemplary domains that may be included in a GeneWriter polypeptide include DNA-binding domains (e.g., DNA-binding domains; zinc fingers; TAL domains; Cas9; dCas9; nickase Cas9; transcription factors; or meganucleases, for example, as shown in the table herein), RNA-binding domains (e.g., RNA-binding domains of B-box proteins, MS2 coat proteins, dCas, or elements of sequences in the table herein), reverse transcriptase domains (e.g., reverse transcriptase domains of elements of sequences in the table herein; other retrotransposers) Examples include peptides containing reverse transcriptase domains (e.g., those listed in the table herein); reverse transcriptase domains (e.g., those listed in the table herein); and / or endonuclease domains (e.g., endonuclease domains of the elements in the table herein; Cas9; nickasase Cas9; restriction enzymes (e.g., type II restriction enzymes, e.g., FokI); meganucleases; Holliday junction resolvers; RLE retrotranspases; APE retrotransposases; or GIY-YIG retrotransposases). Exemplary GeneWriter polypeptides containing exemplary combinations of such domains are shown in the bottom panel. [Figure 5] This is a diagram showing the modules of an exemplary GeneWriter RNA template. Individual modules of the exemplary template can be combined, rearranged, and / or omitted to generate, for example, a GeneWriter template. A=5' homologous arm; B=ribozyme; C=5'UTR; D=heterogeneous target sequence; E=3'UTR; F=3' homologous arm. [Figure 6]This is a table showing a list of modules for an exemplary Gene Writer RNA template. Individual modules can be combined, rearranged, and / or omitted to generate, for example, a Gene Writer template. A=5' homologous arm; B=ribozyme; C=5'UTR; D=heterogeneous target sequence; E=3'UTR; F=3' homologous arm. [Figure 7] This is a diagram illustrating an exemplary second strand nicking process. (A) Cas9 nickase fuses with the Gene Writer protein. The Gene Writer protein introduces a nick into the DNA strand via its EN domain (indicated as *), and the fused Cas9 nickase introduces a nick on the upper or lower DNA strand (indicated as X). (B) The Gene Writer targets DNA via its DNA-binding domain and introduces a DNA nick via its EN domain (*). Subsequently, Cas9 nickase is used to generate a second nick (X) on the upper or lower strand, upstream or downstream of the EN-introduced nick. [Figure 8] The C-terminal linker region of the DNA-binding domain of R2Tg can be truncated and modified. Deletion of the native linker from the myb domain at position 1 or 2 in A or B was constructed along with substitution with 3GS (SEQ ID NO: 1024) or XTEN synthetic linker (A). Integration efficiency was measured in HEK293T cells by ddPCR (B). [Figure 9] A landing pad designed to test target site mutations in the R2Tg Gene Writer. [Figure 10A] A ddPCR assay that measures the percentage of lentivirus integration per cell from all landing pads into which the lentivirus has been incorporated. [Figure 10B] Determination of amplicon sequences and NGS analysis of indels present in the landing pad area. [Figure 11] AAVS1 ZFP substitution in the DNA-binding domain of the retrotransposase GeneWriter. This figure discloses the "3GS linker" as sequence number 1024. [Figure 12] Cas9 or Cas9 nickase substitutions (*=mutants) in the DNA-binding domain of retrotransposase GeneWriters, which may or may not have an active EN domain. [Figure 13] AAVS1 ZFP fusion to GeneWriter retrotransposases, with or without a functional DNA-binding domain. [Figure 14] Schematic diagram of the Nicasse Cas9-GeneWriter fusion. (A) Schematic diagram of Nicasse Cas9 fused to the GeneWriter protein. (B) Schematic diagram of the 3' extended gRNA. [Figure 15] Schematic diagram of Nicasse Cas9-GeneWriter fusion. (A) Schematic diagram of Nicasse Cas9 fused to the GeneWriter protein. (B) Schematic diagram of the donor transgene with homology to the UTR and cleavage site located on the side. [Figure 16] Schematic diagrams of the constructs. (A) Schematic diagram of the Gene Writer protein. (B) Schematic diagram of the donor transgene with homology to the UTR and cleavage site located on the side. (C) Schematic diagram of the Cas9 construct used. [Figure 17] Schematic diagram of the mRNA encoding Gene Writer (A). The original untranslated region (UTR) was replaced with 5' and 3' UTRs optimized for protein expression (shown as 5'UTRexp and 3'UTRexp). Gene Writer protein expression was assayed by the HiBit assay, which involves probing HiBit tag expression (B). This figure discloses "3GS" as sequence number 1024. [Figure 18] Genome integration induced by the original UTR and a Gene Writer protein containing an optimized UTR for protein expression. Gene Writing activity induced by an exogenous UTR is stimulated by the presence of an RNA template containing the original retrotransposon UTR. [Figure 19]Delivery of a Gene Writer system using mRNA encoding a polypeptide and plasmid DNA encoding an RNA template for retrotransposition. [Figure 20] A diagram of an example 5'UTR operation strategy. HA = homologous arm; K = Kozak sequence; pA = poly-A signal; AMa = A. maritima; Rx = other type of retrotransposon. [Figure 21] Possible locations of introns (or multiple introns) within the RNA template. Introns are indicated by curves. 5'HA: 5' homologous arm; 3'HA: 3' homologous arm; 5'UTR: retrotransposon-specific 5'UTR; 3'UTR: retrotransposon-specific 3'UTR; GOI: gene of interest. The orange blocks correspond to sequences designed to be expressed from genomic locations that have their own cell-specific promoters, poly(A) signals, and UTRs (5' and 3'UTRexp) for protein expression. Sequences can be oriented sense-wise (as shown above) or antisense-wise with respect to retrotransposon UTRs and homologous arms. Introns can be positioned within GOIs or within UTRexps. [Figure 22] Genomic integration in HEK293T cells reported by 3'ddPCR assay. Gene Writer mRNA was simultaneously transfused at 0.5 μg / well using RNA templates with or without enzymatically added cap 1 and poly(A) tails. The ratio of Gene Writer mRNA to RNA transgene was 1:1. [Figure 23]Genome integration was detected by 3'ddPCR induced by the expression of Gene Writer mRNA generated with unmodified (G0) or modified nucleotides (pseuduridine (Ψ), 1-N-methylpseuduridine (1-Me-Ψ), 5-methoxyuridine (5-MO-U), or 5-methylcytidine (5mC)). 1 μg of Gene Writer mRNA was used per well. An unmodified RNA template was used. Gene Writer RNA and RNA template were simultaneously translocated in a molar ratio of 1:8. [Figure 24] Constructural diagrams of the driver and transgene plasmid. Homologous arms (HAs) and stuffer sequences are variable in experiments using this setup. [Figure 25] (A) Timeline of the experiment. (B) Schematic diagram of the three-dimensional configuration of R2Tg and the transgene construct. (C) Western blot for Rad51 shows loss of Rad51 protein expression on day 3. [Figure 26] U2OS cells were treated with untargeted control siRNA (ctrl) or siRNA against Rad51, along with R2Tg Wt or control RT and EN mutants. Integration efficiency was evaluated on day 3 using ddPCR at the 3'(A) or 5'(B) junction. [Figure 27A] Sequence map of the ribozyme of the R2 element (R2Tg) from the zebra finch (Taeniopygia guttata) in the context of a Gene Writer transgene molecule RNA module. The ribozyme structure is represented as follows: P, base-paired region; P', complementary strand of the base-paired region; L, loop at the end of the P region; J, nucleotide-conjugated base-paired region. The figure discloses sequence number 1734. [Figure 27B] Predicted secondary structure of the ribozyme R2Tg. Shaded boxes indicate predicted catalytic positions that can be used to inactivate the ribozyme. The figure discloses Sequence ID No. 1734. [Figure 28]Sequence map of the ribozyme of the R2 element (R2Tg) from the zebra finch (Taeniopygia guttata) in the context of a Gene Writer transgene molecule RNA module. The ribozyme structure is represented as follows: P, base-paired region; P', complementary strand of the base-paired region; L, loop at the end of the P region; J, nucleotide-conjugated base-paired region. The figure discloses sequence number 1734. [Figure 29] Prediction of the ribozyme secondary structure of the R2 element from the zebra finch (Taeniopygia guttata). The figure discloses sequence number 1734. [Figure 30] A Gene Writing system for treating exemplary repetitive expansion disorder. The figures disclose sequence numbers 1645, 1599, 1645, 1635–1636, 1645, and 1686–1688, respectively, in the order they are viewed. [Figure 31] Diagrams illustrating two orientations of the second chain nicking within an exemplary Gene Writing system. [Figure 32] A diagram illustrating the orientation and position of the second chain nicking in an exemplary Gene Writing system, and their impact on editing. [Figure 33] This report describes the generation and expression of Cas9-RT fusion proteins. To evaluate the expression of novel Gene Writer polypeptides in human cells, U2OS cells were transfused with Cas-RT expression plasmids containing various RT domains listed in Tables 1 and 30, fused to wild-type (WT) or Cas9(N863A) nickase. Cell lysates were collected two days after transfusion and analyzed by Western blotting using a primary antibody against Cas9. A primary antibody against GADPH was included as a loading control. [Figure 34]This study demonstrates the enhancement of Cas-RT fusion expression through linker sequence selection. To evaluate how linkers can modify the expression of a novel Gene Writer polypeptide in human cells, U2OS cells were transfused with Cas-RT expression plasmids containing various linkers listed in Table 42, which fuse Cas9(N863A) nickase to the RT domain of a mutated RNA-binding domain R2Bm retrotransposase. Cell lysates were collected and analyzed by Western blotting using a primary antibody against Cas9. Primary antibodies against vinculin (left) or GADPH (right) were included as loading controls. The Cas9 control on the left represents the titration of the Cas9 expression plasmid. Empty arrows indicate the original linker tested, while filled arrows indicate the linker (linker 10) found to substantially enhance the expression of the fusion polypeptide. Sample numbers correspond to linker sequences in Table 42. [Figure 35] This study demonstrates that Cas / gRNA DNA targeting activity is conserved by Cas-RT fusion. Various RT domains were fused to Cas9(WT) and electroporated into U2OS cells. Genomic DNA was harvested and mutation signatures were analyzed by next-generation sequencing. Where relevant, mutations within the RNA or DNA binding domain (RBD or DBD) of the R2 retrotransposase domain are indicated. Here, indel frequencies are used as a proxy for conserving Cas activity in the context of RT fusion. [Figure 36]We disclose the use of mutations that enhance the reverse transcriptase domain. Conserved reverse transcriptase domains from the retrovirus genera Betaretrovirus, Deltaretrovirus, Gammaretrovirus, Epsilonretrovirus, and Spumavirus were aligned and compared with previously shown mutations that enhance RT activity (Anzalone et al Nat Biotechnol 38(7):824-844(2020); Baranauskas et al Protein Eng Des Sel 25(10):657-668(2012); Arezi and Hogrefe Nucleic Acids Res 37(2):473-481(2009)). Figure 36A shows an identified set of three core mutations used in RTs from the genera shown. Figure 36B discloses that additional mutations were used, with first preference, from the T306K / W313F set, or from L139P / E607K, which was considered untranscribeable from either the first set. The selected mutations are shown in Table 45. [Figure 37] U2OS cells were nucleofected with various Cas-RT fusion vectors, each containing an RT domain selected from a monomeric retroviral reverse transcriptase domain database. Editing of the HEK3 locus using the templates described in Table 43 was evaluated by amplicon sequencing and analysis of accurate edit versus indel signature. Here, the data is expressed as an activity ratio, calculated as the ratio of the frequency of reads with accurately intended edits (CTT insertion at the target nick site) to the frequency of reads with any other mutation (indel). Since the three template RNA configurations assayed yielded similar results, the results for a single template (template P2 from Table 43) are presented. [Figure 38]Targeting multiple loci simultaneously yields effective Gene Writing activity. HEK293 cells were nucleofected with a Gene Writing system containing template plasmids of various compositions to enable the following targeting: 1) HEK3 locus alone, 2) HBB locus alone, or 3) both HBB and HEK3 loci. The percentage of editing is shown for each locus immediately after delivery of one or both locus-specific template RNA expression plasmids. Filled bars represent complete writing events, while empty bars represent indel frequencies. Locus-specific editing was observed when either template was delivered independently, and highly effective and specific editing was observed at both loci when the templates were delivered simultaneously. [Figure 39] This study demonstrates the effect of length on gene writing activity. To test the editing efficiency of a DNA-free approach at the HEK3 locus, HEK293T cells were nucleofected with a total RNA gene writing system containing various template RNAs (Table 48). Template 4, which encodes the same editing as template 1 but has a 20nt addition at the 3' end of the RT template, showed an approximately 3.1-fold decrease in accurate writing activity and an approximately 2.4-fold decrease in the ratio of accurate correction to indels. [Figure 40] This section demonstrates the effects of GeneWriter on total RNA delivery using various mRNA compositions. Nucleofection of various Cas9-RT(MMLV) mRNAs (Table 49) into HEK293T using template 1 (Table 48A) is shown. No significant effects were observed when capping and UTR composition were altered. [Figure 41]HEK293T cells were nucleofected using the Gene Writing system with a set of templates (Template 1, Table 48) for editing the HEK3 locus and two different Cas-RT constructs. Sequence analysis showed that both Cas-RT fusions performed highly accurate and effective editing. In both systems, increased efficiency was observed under conditions including optional secondary nicks. These data demonstrate the success of cloning and accurate writing by the PERV RT domain in the context of these Cas-RT fusions. [Figure 42] This study demonstrates the effect of modified nucleotides on total RNA delivery in Gene Writer. To assess the effect, the composition of the mRNA molecule encoding the Cas-RT(MMLV) polypeptide was altered (Table 49). Using template 1, the HEK3 locus was edited after incorporating modified nucleotides into the mRNA component. Gene Writing activity using the 5moU modified mRNA component was found to be high and accurate. [Figure 43] This section shows the effects of Gene Writer's total RNA delivery using various mRNA compositions delivered into cells via lipid particles. Figure 43A shows total RNA lipofection of various Cas9-RT(MMLV) mRNAs into HEK293T using template 1 (Table 48) and delivered via lipofectamine 3000. Figure 43B shows total RNA lipofection of various Cas9-RT(MMLV) mRNAs into HEK293T using template 1 (Table 48) and delivered via MessengerMax reagent. These data demonstrate higher accuracy editing efficiency with MessengerMax reagent. Figure 43C shows assays using two templates with different full lengths using MessengerMax reagent. No major changes in editing efficiency were found to be associated with template changes in this experiment. When included one-to-one, the addition of a second nick gRNA increased the efficiency of the system. [Figure 44]This report demonstrates total RNA delivery of Cas-RT using a lipid-based system. Cas9-RT(MMLV) and Cas9-RT(PERV) were delivered into HEK293T cells using MessengerMax lipid reagents and template 1 (Table 48). The activity of both enzymes was approximately 5% accurate writing. [Figure 45] This shows the expression of the entire RNA Gene Writer system in primary human CD4+ T cells. Figure 45A shows Gene Writer protein expression from mRNA delivered to primary human CD4+ T cells at varying doses on day 1 post-nucleofection. Gene Writer was detected using an antibody targeting the Cas9 portion of the polypeptide. GAPDH (housekeeping gene) was detected using an antibody against GAPDH. As the dose of nucleofected mRNA encoding the delivered polypeptide increased, for example, from 0, 2.5, 5, and 10 μg Gene Writer mRNA, an increase in expression levels was observed. The data for protein expression detection shown included two replicates. Figure 45B shows cell viability after nucleofection of six template RNAs. This shows the viability of primary CD4+ T cells after RNA delivery of the Gene Rewriter system on day 3 post-nucleofection. Cell viability was evaluated by flow cytometry after viability / death staining of harvested T cells (mean ± sd, n=2 replicates). [Gate: Live cells within the singlet population of cell populations selected by FSC / SSC size plot] [Figure 46]This shows Gene Writing in primary human CD4+ T cells. Figure 46A shows the precise editing of the HEK3 genomic locus by the Gene Writer system in primary human CD4+ T cells without the addition of a second nick gRNA. Figure 46B shows the precise editing of the HEK3 genomic locus by the Gene Writer system in primary human CD4+ T cells. Genomic DNA was extracted from cells 3 days after nucleofection. HEK3 genome editing was investigated by a PCR-based amplicon sequencing assay. DNA amplicons containing expected genomic modifications were identified as precise write events, while amplicons with unintended edits (e.g., insertions, deletions) were counted as indels. Each percentage was calculated based on the total reads per condition (mean ± sd, n=2 repeats). [Figure 47] The use of a second nick gRNA for Gene Writing in primary human CD4+ T cells is shown. Here, data obtained in Figure 46 show a direct comparison of the potential effect of the second nick gRNA on efficiency. Figure 47A shows that in this experiment, the addition of the second nick gRNA did not result in an enhancement of the accurate writing signal. Figure 47B shows, rather, that the use of the second nick gRNA may increase the frequency of indels. Therefore, in some embodiments, the second nick gRNA sequence may be absent from the system described herein. Accurate editing of HEK3 genomic sites by the Gene Writer system in primary human CD4+ T cells, with or without the addition of a second nick gRNA (Figure 47A) or with (Figure 47B). Genomic DNA was extracted from cells 3 days after nucleofection. Genome editing of HEK3 was investigated by a PCR-based amplicon sequencing assay. DNA amplicons containing expected genome modifications by the Gene Writer system were identified as accurate write events, but amplicons with unintended edits (e.g., insertions, deletions) were counted as indels. The percentage for each was calculated based on the total reads per condition (mean ± sd, n=2 repeats). [Figure 48]This paper presents a screening of construct designs for retrotransposon-mediated integration in human cells. A driver plasmid containing a retrotransposase (driver) expression cassette is transfused together with a template plasmid containing a retrotransposon-dependent reporter cassette. Expression from the template plasmid results in non-functional GFP due to the disruption of antisense introns, whereas transcription of the template molecule from the template plasmid results in RNA generation, which can be spliced ​​to remove introns, then reverse-transcribed and integrated by the system. Therefore, reporter cassette expression will occur only from the integrated reporter cassette (integrated gDNA, bottom), and not from the template plasmid. HA = homologous arm (if applicable); CMV = mammalian CMV promoter; HiBit = HiBit tag for protein expression quantification; T7 = T7 RNA polymerase promoter; UTR = untranslated sequence, e.g., natural retrotransposon UTR; pA = poly(A) signal; SD-SA is used to indicate splice donor and splice acceptor sites of antisense introns within the GFP coding sequence. [Figure 49] Screening of candidate retrotransposons identified 25 candidates effective for incorporating transpayloads in human cells. A total of 163 retrotransposon systems were assayed for activity in human cells, as described in Example 39. Integration, measured by ddPCR, is shown as copies / genomes for each retrotransposon driver / template system. The height of each bar represents the mean of two replicates. [Figure 50A] This section describes a luciferase activity assay for primary cells. LNPs formulated according to Example 44 were analyzed for cargo delivery to primary human hepatocytes according to Example 45. The luciferase assay revealed dose-responsive luciferase activity from the cell lysates. This indicates successful RNA delivery from mRNA cargo to cells and successful expression of firefly luciferase. [Figure 50B]A luciferase activity assay for primary cells is shown. LNPs formulated according to Example 44 were analyzed for cargo delivery to mouse hepatocytes according to Example 45. The luciferase assay revealed dose-responsive luciferase activity from the cell lysates. This indicates successful RNA delivery from mRNA cargo to cells and successful expression of firefly luciferase. [Figure 51] This paper discloses LNP-mediated delivery of RNA cargo to mouse liver. Firefly luciferase mRNA-containing LNPs were formulated and delivered to mice via IV. Liver samples were harvested and assayed for luciferase activity at 6, 24, and 48 hours post-administration. Reporter activity across various formulations followed the ranking LIPIDV005 > LIPIDV004 > LIPIDV003. RNA expression was transient, and enzyme levels returned to near vehicle background levels by 48 hours post-administration. [Modes for carrying out the invention]

[0347] This disclosure relates to compositions, systems, and methods for targeting, editing, modifying, or manipulating DNA sequences at one or more locations within a DNA sequence in a cell, tissue, or subject, for example, in vivo or in vitro (e.g., inserting a heterologous target sequence into a target site in a mammalian genome). The heterologous target DNA sequence may include, for example, substitutions, deletions, insertions, such as coding sequences, regulatory sequences, or gene expression units.

[0348] More specifically, this disclosure provides a reverse transcriptase-based system for modifying a genomic DNA sequence of interest, for example, by inserting, deleting, or substituting one or more nucleotides into or from the sequence of interest. This disclosure is partly based on bioinformatics analysis to identify reverse transcriptase sequences in retrotransposons of various biological origins, for example (see Table 1 or Table 3).

[0349] This disclosure provides the Gene Writer® genome editor, which comprises a polypeptide component and a template nucleic acid (e.g., template RNA) component. In some embodiments, the Gene Writer® genome editor can be used to introduce modifications into target sites within the genome. In some embodiments, the polypeptide component comprises a writing domain (e.g., reverse transcriptase domain), a DNA-binding domain, and an endonuclease domain (e.g., nickase domain). In some embodiments, the template nucleic acid (e.g., template RNA) comprises a sequence that binds to a target site within the genome (e.g., binds to the second strand of the target site), a sequence that binds to the polypeptide component, a heterologous target sequence, and a 3' target homology domain. While we do not wish to be constrained by theory, it is assumed that the template nucleic acid (e.g., template RNA) binds to the second strand of the target site within the genome and then binds to the polypeptide component (e.g., localizes the polypeptide component to the target site within the genome). The endonuclease (e.g., nickase) of the polypeptide component is thought to cleave the target site (e.g., the first chain of the target site), allowing, for example, the 3' homologous domain to bind to a sequence adjacent to the site to be modified on the first chain of the target site. The writing domain (e.g., reverse transcriptase domain) of the polypeptide component is thought to polymerize, for example, a sequence complementary to the heterologous sequence, using the 3' target homology domain as a primer and the heterologous target sequence as a template. While we do not wish to be constrained by theory, it is thought that the appropriate selection of the heterologous target sequence may result in the substitution, deletion, or insertion of one or more nucleotides at the target site.

[0350] In embodiments, the Disclosure provides nucleic acid molecules or systems for retargeting, for example, Gene Writer polypeptides or nucleic acid molecules, or systems described herein. Retargeting (for example, Gene Writer polypeptides or nucleic acid molecules, or systems described herein) generally involves: (i) instructing the polypeptide to bind and cleave at a target site; and / or (ii) designing a template RNA to have complementarity to a target sequence. In some embodiments, the template RNA has complementarity to the target sequence at the 5' end of a nick on the first strand, for example, such that the 3' end of the template RNA anneals and the 5' end of the target site acts as a primer for targeted primed reverse transcription (TPRT). In some embodiments, the endonuclease domain of the polypeptide and the 5' end of the RNA template are also modified as described.

[0351] Gene Writer (trademark) genome editor The Gene Writer® genome editor is a system capable of modifying the genome of a host cell and can be used for mutations, deletions, or other modifications of genomic target sequences, including the insertion of heterologous payloads. In some embodiments, the system is inspired by a group of naturally evolved mobile genetic elements known as retrotransposons. The Gene Writer® polypeptide can also contain RT domains derived from sources other than retrotransposons, such as viruses.

[0352] Non-long terminal repeat (LTR) retrotransposons are a type of mobile genetic element found throughout the eukaryotic genome. They comprise two classes: aprine / apyrimidine endonuclease (APE) types and restriction enzyme-like endonuclease (RLE) types. APE-class retrotransposons consist of two functional domains: an endonuclease / DNA-binding domain and a reverse transcriptase domain. RLE-class retrotransposons consist of three functional domains: a DNA-binding domain, a reverse transcription domain, and an endonuclease domain. The reverse transcriptase domain of non-LTR retrotransposons functions by binding to an RNA sequence template and reverse transcribing it to target DNA in the host genome. The RNA sequence template has a 3' untranslated region that specifically binds to the transposase and a variable 5' region that typically contains an open reading frame ("ORF") encoding the transposase protein. Alternatively, the RNA sequence template may also include a 5' untranslated region that specifically binds to the retrotransposase.

[0353] In some embodiments, elements of such non-LTR retrotransposons described herein can be functionally modularized and / or modified, for example by reverse transcription, to target, edit, modify, or manipulate a target DNA sequence, to insert a target (e.g., heterologous) nucleic acid sequence into a target genome, e.g., a mammalian genome. Such modularized and modified nucleic acids, polypeptide compositions, and systems are described herein and referred to as Gene Writer® gene editors. A Gene Writer® gene editor system comprises: (A) a polypeptide or nucleic acid encoding a polypeptide (the polypeptide comprises (i) a reverse transcriptase domain and (x) an endonuclease domain containing DNA-binding function, or (y) an endonuclease domain and a separate DNA-binding domain); and (B) a template RNA comprising (i) a sequence that binds to the polypeptide and (ii) a heterologous insertion sequence. For example, a Gene Writer® genome editor protein may comprise a DNA-binding domain, a reverse transcriptase domain, and an endonuclease domain. In some embodiments, the DNA binding function may include an RNA component that directs the protein to a DNA sequence, such as gRNA. In other embodiments, the Gene Writer® genome editor protein may include a reverse transcriptase domain and an endonuclease domain. In certain embodiments, elements of the Gene Writer® gene editor polypeptide may be derived from non-LTR retrotransposons, such as APE-type or RLE-type retrotransposons, or sequences of parts or domains thereof. In some embodiments, the RLE-type non-LTR retrotransposon is derived from the R2, NeSL, HERO, R4, or CRE clades. In some embodiments, the Gene Writer® genome editor is derived from the R4 element X4_Line, which is found in the human genome. In some embodiments, the APE-type non-LTR retrotransposon is derived from the R1 or Tx1 clade.In some embodiments, the Gene Writer® genome editor is derived from the Tx1 element Mare6, which is found in the human genome. The RNA template element of the Gene Writer® gene editor system is typically heterogeneous to the polypeptide element and provides a target sequence to be inserted (reverse transcribed) into the host genome. In some embodiments, the Gene Writer® genome editor protein is capable of targeted primed reverse transcription. In some embodiments, Gene... The Writer genome editor protein enables the synthesis of a second strand. Table 50 shows representative Gene Writer proteins and relevant sequences identified using data mining from various retrotransposases. Column 1 shows the family to which the retrotransposon belongs. Column 2 shows a list of element names. Column 3 shows the accession number, if present. Column 4 shows a list of organisms in which retrotransposases are found. Column 5 shows a list of predicted 5' untranslated regions, and column 6 shows a list of predicted 3' untranslated regions; both are segments that are predicted to allow the template RNA to bind to the retrotransposase in column 7. (It is understood that columns 5-6 show DNA sequences, and RNA sequences following either column 5-6 typically contain uracil rather than thymidine.) Column 7 shows a list of predicted retrotransposase amino acid sequences. Column 8 lists the predicted RT domains based on sequence analysis, column 9 lists the start codon locations, and column 10 lists the stop codon locations.

[0354] [Table 1]

[0355] [Table 2]

[0356] Table 3

[0357] Table 4

[0358] Table 5

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[0361] Table 8

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[0363] Table 10

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[0476]

Table 123

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[0524] Table 171

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[0543] [Table 190]

[0544] [Table 191]

[0545] In some embodiments, the Gene Writer® genome editor is combined with the second polypeptide. In some embodiments, the second polypeptide is obtained from an APE-type non-LTR retrotransposon. In some embodiments, the second polypeptide has a zinc knuckle-like motif. In some embodiments, the second polypeptide is a homolog of the Gag protein.

[0546] Essentially, the success of retrotransposons has prompted the consideration herein that the intrinsic functions of retrotransposons can be replicated using functional parts derived from completely independent systems. For example, a functional GeneWriter™ may consist of unrelated DNA-binding, reverse transcription, and endonuclease domains. This modular structure allows for combinations of functional domains, e.g., dCas9 (DNA binding), MMLV reverse transcriptase (reverse transcription), and FokI (endonuclease). In some embodiments, multiple functional domains may arise from a single protein, e.g., Cas9 nickase (DNA binding, endonuclease) and R2 retrotransposon (DNA binding, reverse transcription, endonuclease).

[0547] In some embodiments, the Gene Writer® system can generate insertions at target sites of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally 500 or fewer, 400, 300, 200, or 100 nucleotides). In some embodiments, the Gene Writer® system can generate insertions at target sites 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 500, 400, 300, 200, or 100 nucleotides). In some embodiments, the Gene Writer® system can generate insertions at target sites 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 1, 5, 10, or 20 kilobases or less). In some embodiments, the Gene Writer® system can generate deletions of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally 500, 400, 300, or 200 nucleotides or less). In some embodiments, the Gene Writer® system can generate deletions of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally 500, 400, 300, or 200 nucleotides or less).In some embodiments, the Gene Writer® system can generate deletions 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 500, 400, 300, or 200 nucleotides or less). In some embodiments, the Gene Writer® system can generate deletions 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 1, 5, 10, or 20 kilobases or less). In some embodiments, the Gene Writer system can generate substitutions at target sites 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, the substitutions are transposition mutations. In some embodiments, the substitutions are transversion mutations. In some embodiments, the substitution converts adenine to thymine, adenine to guanine, adenine to cytosine, guanine to thymine, guanine to cytosine, guanine to adenine, thymine to cytosine, thymine to adenine, thymine to guanine, cytosine to adenine, cytosine to guanine, or cytosine to thymine.

[0548] Polypeptide components of the Gene Writer® gene editor system Domain and function: In some embodiments, the Gene Writer® polypeptide has the functions of DNA target site binding, template nucleic acid (e.g., RNA) binding, DNA target site cleavage, and template nucleic acid (e.g., RNA) writing, e.g., reverse transcription. In some embodiments, each function is contained within a different domain. In some embodiments, the function may be attributed to two or more domains (e.g., two or more domains exhibit functionality together). In some embodiments, two or more domains may have the same or similar functions (e.g., two or more domains independently have DNA binding functionality, e.g., in two different DNA sequences). In other embodiments, one or more domains may perform one or more functions; for example, the Cas9 domain may be capable of both DNA binding and target site cleavage. In some embodiments, all domains are located within a single polypeptide. In some embodiments, the first domain is present in the first polypeptide, and the second domain is present in the second polypeptide. For example, in some embodiments, the Gene Writer® polypeptide may be split between a first polypeptide and a second polypeptide, for example, the first polypeptide comprising a reverse transcriptase (RT) domain and the second polypeptide comprising a DNA-binding domain and an endonuclease domain, such as a nickasase domain. Further examples include, in some embodiments, each of the first and second polypeptides comprising a DNA-binding domain (e.g., a first DNA-binding domain and a second DNA-binding domain). In some embodiments, the first and second polypeptides may be associated post-translation via a split intein.

[0549] Writing domain: In certain embodiments of the present invention, the writing domain of the Gene Writer® system has reverse transcriptase activity and is also referred to as the reverse transcriptase domain (RT domain). In some embodiments, the RT domain includes an RT catalytic moiety and an RNA-binding region (e.g., a region that binds to template RNA).

[0550] In certain embodiments of the present invention, the writing domain is based on the reverse transcriptase domain of an APE-type or RLE-type non-LTR retrotransposon. The wild-type reverse transcriptase domain of an APE-type or RLE-type non-LTR retrotransposon may be used in the Gene Writer® system or may be modified (e.g., by insertion, deletion, or substitution of one or more residues) to alter the reverse transcriptase activity in a target DNA sequence. In some embodiments, the reverse transcriptase is modified from its native sequence to have improved, modified codon use, for example, in human cells. In some embodiments, the reverse transcriptase domain is a heterologous reverse transcriptase from a different retrovirus, LTR retrotransposon, or non-LTR retrotransposon. In certain embodiments, the Gene Writer® system comprises a polypeptide containing the reverse transcriptase domain of an RLE-type non-LTR retrotransposon from the R2, NeSL, HERO, R4, or CRE clade, or an APE-type non-LTR retrotransposon from the R1 or Tx1 clade. In certain embodiments, the Gene Writer® system includes polypeptides comprising a reverse transcriptase domain of a non-LTR retrotransposon, an LTR retrotransposon, a group II intron, a diversity-generating element, a retron, a telomerase, a retroplasmid, a retrovirus, or a modified polymerase listed in Table 1 or Table 3. In some embodiments, the Gene Writer® system includes polypeptides comprising a reverse transcriptase domain listed in Table 2.In several embodiments, the amino acid sequence of the reverse transcriptase domain of the Gene Writer® system is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identical to the amino acid sequence of the reverse transcriptase domain of a non-LTR retrotransposon, LTR retrotransposon, group II intron, diversity-generating element, retroron, telomerase, retroplasm, retrovirus, or modified polymerase as the DNA sequence is referenced in Table 1 or Table 3, or the amino acid sequence of a peptide containing the RT domain referenced in Table 2. In some embodiments, the RT domain has a sequence selected from Table 1 or 3 or a sequence of a peptide containing the RT domain selected from Table 2, or a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the RT domain containing the Gene Writer polypeptide is mutated from its original amino acid sequence, for example, having substitutions of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100. In some embodiments, the RT domain is derived from retrovirus RTs, such as HIV-1 RT, Moloney's mouse leukemia virus (MMLV) RT, avian myeloblastosis virus (AMV) RT, or Rous sarcoma virus (RSV) RT. In some embodiments, the RT domain is derived from the RT of a group II intron, e.g., group II intron maturase RT (MarathonRT) from Eubacterium rectale (Zhao et al. RNA 24:22018), the RT domain from LtrA, or RT TGIRT (or trt). In some embodiments, the RT domain is derived from the RT of a retron, e.g., reverse transcriptase (RT86) from Ec86.In some embodiments, the RT domain is derived from a diversity-generating retroelement, e.g., the RT of Brt. In some embodiments, the RT domain is derived from the RT of a retroplasmid, e.g., the RT from the Mauriceville plasmid. In some embodiments, the RT domain is derived from a non-LTR retrotransposon, e.g., the RT from R2Bm, the RT from R2Tg, the RT from LINE-1, or the RT from a Penelope or Penelope-like element (PLE). In some embodiments, the RT domain is derived from a reverse transcriptase from an LTR retrotransposon, e.g., Ty1. In some embodiments, the RT domain is derived from telomerase, e.g., TERT. A person with common skills in the art can identify the reverse transcriptase domain using a common tool such as the Basic Local Alignment Search Tool (BLAST) based on homology with other known reverse transcriptase domains. In some embodiments, the reverse transcriptase contains the InterPro domain IPR000477. In some embodiments, the reverse transcriptase contains the pfam domain PF00078. In some embodiments, the RT contains the InterPro domain IPR013103. In some embodiments, RT includes the pfam domain PF07727. In some embodiments, the reverse transcriptase includes conserved protein domains of the cd00304 RT_like family, e.g., cd01644 (RT_pepA17), cd01645 (RT_Rtv), cd01646 (RT_Bac_retron_I), cd01647 (RT_LTR), cd01648 (TERT), cd01650 (RT_nLTR_like), cd01651 (RT_G2_intron), cd01699 (RNA_dep_RNAP), cd01709 (RT_like_1), cd03487 (RT_Bac_retron_II), cd03714 (RT_DIRS1), cd03715 (RT_ZFREV_like).Proteins containing these domains can be found in protein databases such as InterPro (Mitchell et al. Nucleic Acids Res 47, D351-360 (2019)) and UniProt (The UniProt Consortium Nucleic Acids). Further discoveries can be made by searching for domains in Res 47, D506-515 (2019) or in the preserved domain database (Lu et al. Nucleic Acids Res 48, D265-268 (2020)), or by scanning open reading frames for reverse transcriptase domains using a prediction tool, such as InterProScan. The diversity of reverse transcriptases is not limited to their use by prokaryotes (Zimmerly et al. Microbiol Spectr3(2):MDNA3-0058-2014(2015); Lampson BC(2007) Prokaryotic Peverse Transcriptases. In:Polaina J., MacCabe AP(eds) Industrial Enzymes.Springer,Dordrecht), viruses (Herschhorn et al. Cell Mol Life Sci 67(16):2717-2747(2010); Menendez-Arias et al. Virus Res 234:153-176(2017)), and mobile elements (Eickbush et al. Virus Res 134(1-2):221-234(2008); Craig et al. Mobile DNA III 3rd) They are described in Ed.DOI:10.1128 / 9781555819217(2015)) (each of which is incorporated herein by reference).

[0551] In some embodiments, the reverse transcriptase (RT) domain exhibits increased stringency for target-primed reverse transcription (TPRT) initiation compared to, for example, an endogenous RT domain. In some embodiments, the RT domain initiates TPRT when 3nts within the target site immediately upstream of the first-strand nick, for example, the genomic DNA priming the RNA template, have at least 66% or 100% complementarity to 3nts of homology in the RNA template. In some embodiments, the RT domain initiates TPRT when less than 5nts of mismatch exist between the homology of the template RNA and the target DNA-priming reverse transcription (e.g., less than 1, 2, 3, 4, or 5nts of mismatch). In some embodiments, the RT domain is modified to increase stringency in priming mismatches in the TPRT reaction, for example, the RT domain either does not tolerate any mismatches within the priming region or tolerates fewer mismatches compared to a wild-type (e.g., unmodified) RT domain. In some embodiments, the RT domain includes an HIV-1 RT domain. In several embodiments, the HIV-1 RT domain initiates synthesis at a lower level, even with three nucleotide mismatches, compared to alternative RT domains (e.g., as described by Jamburthugoda and Eickbush J Mol Biol 407(5):661-672 (2011) (the entire text of which is incorporated herein by reference)).

[0552] In some embodiments, the RT domain forms a dimer (e.g., a heterodimer or a homodimer). In some embodiments, the RT domain is a monomer. In some embodiments, the RT domain, for example, a retroviral RT domain, functions naturally as a monomer or a dimer (e.g., a heterodimer or a homodimer). In some embodiments, the RT domain functions naturally as a monomer, for example, derived from a monomeric virus. Exemplary monomeric RT domains, their viral sources, and their associated RT signatures can be found in Table 30, along with descriptions of domain signatures in Table 32. In some embodiments, the RT domain of the system described herein includes the amino acid sequence in Table 30, or a functional fragment or variant thereof, or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity thereto.In several embodiments, the RT domain is 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), 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 The RT domains are selected from the following: P14350), simian foamy virus (SFV) (e.g., UniProt P23074), bovine foamy / syncytial virus (BFV / BSV) (e.g., UniProt O41894), or functional fragments or variants thereof (e.g., amino acid sequences having at least 70%, 80%, 90%, 95%, or 99% identity thereto). In some embodiments, the RT domain is dimerized in its innate functionality. Exemplary dimeric RT domains, their viral sources, and associated RT signatures can be found in Table 31, along with descriptions of domain signatures in Table 32. In some embodiments, the RT domains of the systems described herein include the amino acid sequences in Table 31, or their functional fragments or variants, or sequences having at least 70%, 80%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain is derived from a virus that functions as a dimer.In several embodiments, the RT domain is derived 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...). The RT domain is selected from 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 with it). In nature, heterodimeric RT domains may also function as homodimers in some embodiments. In some embodiments, the dimeric RT domain is expressed as a fusion protein, for example, as a homodimeric fusion protein or a heterodimeric fusion protein. In some embodiments, the RT function of the system is satisfied by multiple RT domains (e.g., as described herein). In further embodiments, the multiple RT domains may be fused or separated and may reside, for example, on the same polypeptide or on different polypeptides.

[0553] In some embodiments, the GeneWriter described herein includes an integrase domain, for example, the integrase domain may be part of the RT domain. In some embodiments, the RT domain (for example, as described herein) includes an integrase domain. In some embodiments, the RT domain (for example, as described herein) lacks an integrase domain or includes an integrase domain that has been inactivated by mutation or deletion. In some embodiments, the GeneWriter described herein includes a ribonuclease H domain, for example, the ribonuclease H domain may be part of the RT domain. In some embodiments, the RT domain (for example, as described herein) includes a ribonuclease H domain, for example, an endogenous ribonuclease H domain or a heterologous ribonuclease H domain. In some embodiments, the RT domain (for example, as described herein) lacks a ribonuclease H domain. In some embodiments, the RT domain (for example, as described herein) includes a ribonuclease H domain that has been added to, deleted from, mutated, or replaced with a heterologous ribonuclease H domain. In some embodiments, mutations in the ribonuclease H domain are, for example, as described in Kotewicz et al., Nucleic Acids Res As measured by the method in 16(1):265-277(1988) (which is incorporated herein by reference in its entirety), for example, polypeptides exhibiting lower ribonuclease activity are produced, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower compared to other similar domains that do not have mutations. In some embodiments, ribonuclease H activity is lost.

[0554] In some embodiments, the RT domain undergoes mutation, resulting in increased fidelity compared to other similar domains that do not have mutations. For example, in some embodiments, the YADD (SEQ ID NO: 1539) or YMDD (SEQ ID NO: 1540) motif within the RT domain (e.g., in reverse transcriptase) is substituted with YVDD (SEQ ID NO: 1541). In several embodiments, the substitution of YADD (SEQ ID NO: 1539), YMDD (SEQ ID NO: 1540), or YVDD (SEQ ID NO: 1541) results in higher fidelity in retroviral reverse transcriptase activity (e.g., described in Jamburthugoda and Eickbush J Mol Biol 2011; the entire work is incorporated herein by reference).

[0555] In some embodiments, the reverse transcriptase domain is selected from the elements in Table 1 or Table 3.

[0556] [Table 192]

[0557] [Table 193]

[0558] [Table 194]

[0559] [Table 195]

[0560] [Table 196]

[0561] [Table 197]

[0562] Table 198

[0563] Table 199

[0564] Table 200

[0565] Table 201

[0566] Table 202

[0567] Table 203

[0568] Table 204

[0569] Table 205

[0570] Table 206

[0571] Table 207

[0572] [Table 208]

[0573] [Table 209]

[0574] [Table 210]

[0575] Table 3 (below) shows exemplary Gene Writer® proteins and associated sequences from various retrotransposases identified using data mining. Column 1 indicates the family to which the retrotransposon belongs. Column 2 lists the element names. Column 3 indicates the acceptance number, if present. Column 4 lists the organisms in which the retrotransposase is found. Column 5 lists the predicted 5' untranslated region, and Column 6 lists the predicted 3' untranslated region; both are predicted sequences that enable the template RNA to bind to the retrotransposase in Column 7. (It is understood that columns 5-6 represent DNA sequences, and RNA sequences following either of columns 5-6 may typically contain uracil rather than thymidine.) Column 7 lists the predicted retrotransposase amino acid sequences.

[0576] [Table 211]

[0577] [Table 212]

[0578] [Table 213]

[0579] Table 214

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[0625] Table 260

[0626] Table 261

[0627] Table 262

[0628] Table 263

[0629] Table 264

[0630] Table 265

[0631] Table 266

[0632] Table 267

[0633] Table 268

[0634] Table 269

[0635] Table 270

[0636] Table 271

[0637] Table 272

[0638] Table 273

[0639] Table 274

[0640] Table 275

[0641] Table 276

[0642] Table 277

[0643] Table 278

[0644] Table 279

[0645] Table 280

[0646] Table 281

[0647] Table 282

[0648] Table 283

[0649] Table 284

[0650] Table 285

[0651] Table 286

[0652] Table 287

[0653] Table 288

[0654] Table 289

[0655] Table 290

[0656] Table 291

[0657] Table 292

[0658] Table 293

[0659] Table 294

[0660] Table 295

[0661] Table 296

[0662] Table 297

[0663] Table 298

[0664] Table 299

[0665] Table 300

[0666] Table 301

[0667] Table 302

[0668] Table 303

[0669] Table 304

[0670] Table 305

[0671] Table 306

[0672] Table 307

[0673] Table 308

[0674] Table 309

[0675] Table 310

[0676] Table 311

[0677] Table 312

[0678] Table 313

[0679] Table 314

[0680] Table 315

[0681] Table 316

[0682] Table 317

[0683] Table 318

[0684] Table 319

[0685] Table 320

[0686] Table 321

[0687] Table 322

[0688] Table 323

[0689] Table 324

[0690] Table 325

[0691] Table 326

[0692] Table 327

[0693] Table 328

[0694] Table 329

[0695] Table 330

[0696] Table 331

[0697] Table 332

[0698] Table 333

[0699] Table 334

[0700] Table 335

[0701] Table 336

[0702] Table 337

[0703] Table 338

[0704] Table 339

[0705] Table 340

[0706] Table 341

[0707] Table 342

[0708] Table 343

[0709] Table 344

[0710] Table 345

[0711] Table 346

[0712] Table 347

[0713] Table 348

[0714] Table 349

[0715] Table 350

[0716] Table 351

[0717] Table 352

[0718] Table 353

[0719] Table 354

[0720] Table 355

[0721] Table 356

[0722] Table 357

[0723] Table 358

[0724] Table 359

[0725] Table 360

[0726] Table 361

[0727] Table 362

[0728] Table 363

[0729] Table 364

[0730] Table 365

[0731] Table 366

[0732] Table 367

[0733] Table 368

[0734] Table 369

[0735] Table 370

[0736] Table 371

[0737] Table 372

[0738] Table 373

[0739] Table 374

[0740] Table 375

[0741] Table 376

[0742] Table 377

[0743] Table 378

[0744] Table 379

[0745] Table 380

[0746] Table 381

[0747] Table 382

[0748] Table 383

[0749] Table 41 presents a list of retrotransposase proteins and their associated retrotransposons 5'UTR and 3'UTR for use in novel Gene Writing systems. The reverse transcriptase domains in the proteins described herein were identified using conserved RT signatures and annotated to indicate the presence and location of the RT domains within the polypeptide sequences. In some embodiments, the systems or methods described herein include polypeptides or functional fragments thereof having amino acid sequences listed in Table 41 or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the systems or methods described herein include domains (e.g., reverse transcriptase domains) or functional fragments thereof having amino acid sequences following the domains in Table 41 (e.g., reverse transcriptase domains) or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the systems or methods described herein include a template RNA or a functional fragment thereof containing a sequence that conforms to one or both of the predicted 5'UTR and the predicted 3'UTR of Table 41, or a sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0750] [Table 384]

[0751] [Table 385]

[0752] [Table 386]

[0753] [Table 387]

[0754] Table 388

[0755] Table 389

[0756] Table 390

[0757] Table 391

[0758] Table 392

[0759] Table 393

[0760] Table 394

[0761] Table 395

[0762] Table 396

[0763] Table 397

[0764] Table 398

[0765] Table 399

[0766] Table 400

[0767] Table 401

[0768] Table 402

[0769] Table 403

[0770] Table 404

[0771] Table 405

[0772] Table 406

[0773] Table 407

[0774] Table 408

[0775] Table 409

[0776] Table 410

[0777] Table 411

[0778] Table 412

[0779] Table 413

[0780] Table 414

[0781] Table 415

[0782] Table 416

[0783] Table 417

[0784] Table 418

[0785] Table 419

[0786] Table 420

[0787] Table 421

[0788] Table 422

[0789] Table 423

[0790] Table 424

[0791] Table 425

[0792] Table 426

[0793] Table 427

[0794] Table 428

[0795] Table 429

[0796] Table 430

[0797] Table 431

[0798] Table 432

[0799] Table 433

[0800] Table 434

[0801] Table 435

[0802] Table 436

[0803] Table 437

[0804] Table 438

[0805] Table 439

[0806] Table 440

[0807] Table 441

[0808] Table 442

[0809] Table 443

[0810] Table 444

[0811] Table 445

[0812] Table 446

[0813] Table 447

[0814] Table 448

[0815] Table 449

[0816] Table 450

[0817] Table 451

[0818] Table 452

[0819] Table 453

[0820] Table 454

[0821] Table 455

[0822] Table 456

[0823] Table 457

[0824] Table 458

[0825] Table 459

[0826] Table 460

[0827] Table 461

[0828] Table 462

[0829] Table 463

[0830] Table 464

[0831] Table 465

[0832] Table 466

[0833] Table 467

[0834] Table 468

[0835] Table 469

[0836] Table 470

[0837] Table 471

[0838] Table 472

[0839] Table 473

[0840] Table 474

[0841] Table 475

[0842] Table 476

[0843] Table 477

[0844] Table 478

[0845] Table 479

[0846] Table 480

[0847] Table 481

[0848] Table 482

[0849] Table 483

[0850] Table 484

[0851] Table 485

[0852] Table 486

[0853] Table 487

[0854] Table 488

[0855] Table 489

[0856] Table 490

[0857] Table 491

[0858] Table 492

[0859] Table 493

[0860] Table 494

[0861] Table 495

[0862] Table 496

[0863] Table 497

[0864] Table 498

[0865] Table 499

[0866] Table 500

[0867] Table 501

[0868] Table 502

[0869] Table 503

[0870] Table 504

[0871] Table 505

[0872] Table 506

[0873] Table 507

[0874] Table 508

[0875] Table 509

[0876] Table 510

[0877] Table 511

[0878] Table 512

[0879] Table 513

[0880] Table 514

[0881] Table 515

[0882] Table 516

[0883] Table 517

[0884] Table 518

[0885] Table 519

[0886] Table 520

[0887] Table 521

[0888] Table 522

[0889] Table 523

[0890] Table 524

[0891] Table 525

[0892] Table 526

[0893] Table 527

[0894] Table 528

[0895] Table 529

[0896] Table 530

[0897] Table 531

[0898] Table 532

[0899] Table 533

[0900] Table 534

[0901] Table 535

[0902] Table 536

[0903] Table 537

[0904] Table 538

[0905] Table 539

[0906] Table 540

[0907] Table 541

[0908] Table 542

[0909] Table 543

[0910] Table 544

[0911] Table 545

[0912] Table 546

[0913] Table 547

[0914] Table 548

[0915] Table 549

[0916] Table 550

[0917] Table 551

[0918] Table 552

[0919] Table 553

[0920] Table 554

[0921] Table 555

[0922] Table 556

[0923] Table 557

[0924] Table 558

[0925] Table 559

[0926] Table 560

[0927] Table 561

[0928] Table 562

[0929] Table 563

[0930] Table 564

[0931] Table 565

[0932] Table 566

[0933] Table 567

[0934] Table 568

[0935] Table 569

[0936] Table 570

[0937] Table 571

[0938] Table 572

[0939] Table 573

[0940] Table 574

[0941] Table 575

[0942] Table 576

[0943] Table 577

[0944] Table 578

[0945] Table 579

[0946] Table 580

[0947] Table 581

[0948] Table 582

[0949] Table 583

[0950] Table 584

[0951] Table 585

[0952] Table 586

[0953] Table 587

[0954] Table 588

[0955] Table 589

[0956] Table 590

[0957] Table 591

[0958] Table 592

[0959] Table 593

[0960] Table 594

[0961] Table 595

[0962] Table 596

[0963] Table 597

[0964] Table 598

[0965] Table 599

[0966] Table 600

[0967] Table 601

[0968] Table 602

[0969] Table 603

[0970] Table 604

[0971] Table 605

[0972] Table 606

[0973] Table 607

[0974] Table 608

[0975] Table 609

[0976] Table 610

[0977] Table 611

[0978] Table 612

[0979] Table 613

[0980] Table 614

[0981] Table 615

[0982] Table 616

[0983] Table 617

[0984] Table 618

[0985] Table 619

[0986] Table 620

[0987] Table 621

[0988] Table 622

[0989] Table 623

[0990] Table 624

[0991] Table 625

[0992] Table 626

[0993] Table 627

[0994] Table 628

[0995] Table 629

[0996] Table 630

[0997] Table 631

[0998] Table 632

[0999] Table 633

[1000] Table 634

[1001] Table 635

[1002] Table 636

[1003] Table 637

[1004] Table 638

[1005] Table 639

[1006] Table 640

[1007] Table 641

[1008] Table 642

[1009] Table 643

[1010] Table 644

[1011] Table 645

[1012] Table 646

[1013] Table 647

[1014] Table 648

[1015] Table 649

[1016] Table 650

[1017] Table 651

[1018] Table 652

[1019] Table 653

[1020] Table 654

[1021] Table 655

[1022] Table 656

[1023] Table 657

[1024] Table 658

[1025] Table 659

[1026] Table 660

[1027] Table 661

[1028] Table 662

[1029] Table 663

[1030] Table 664

[1031] Table 665

[1032] Table 666

[1033] Table 667

[1034] Table 668

[1035] Table 669

[1036] Table 670

[1037] Table 671

[1038] Table 672

[1039] Table 673

[1040] Table 674

[1041] Table 675

[1042] Table 676

[1043] Table 677

[1044] Table 678

[1045] Table 679

[1046] Table 680

[1047] Table 681

[1048] Table 682

[1049] Table 683

[1050] Table 684

[1051] Table 685

[1052] Table 686

[1053] Table 687

[1054] Table 688

[1055] Table 689

[1056] Table 690

[1057] Table 691

[1058] Table 692

[1059] Table 693

[1060] Table 694

[1061] Table 695

[1062] Table 696

[1063] Table 697

[1064] Table 698

[1065] Table 699

[1066] Table 700

[1067] Table 701

[1068] Table 702

[1069] Table 703

[1070] Table 704

[1071] Table 705

[1072] Table 706

[1073] Table 707

[1074] Table 708

[1075] Table 709

[1076] Table 710

[1077] Table 711

[1078] Table 712

[1079] Table 713

[1080] Table 714

[1081] Table 715

[1082] Table 716

[1083] Table 717

[1084] Table 718

[1085] Table 719

[1086] Table 720

[1087] Table 721

[1088] Table 722

[1089] Table 723

[1090] Table 724

[1091] Table 725

[1092] Table 726

[1093] Table 727

[1094] Table 728

[1095] Table 729

[1096] Table 730

[1097] Table 731

[1098] Table 732

[1099] Table 733

[1100] Table 734

[1101] Table 735

[1102] Table 736

[1103] Table 737

[1104] Table 738

[1105] Table 739

[1106] Table 740

[1107] Table 741

[1108] Table 742

[1109] Table 743

[1110] Table 744

[1111] Table 745

[1112] Table 746

[1113] Table 747

[1114] Table 748

[1115] Table 749

[1116] Table 750

[1117] Table 751

[1118] Table 752

[1119] Table 753

[1120] Table 754

[1121] Table 755

[1122] Table 756

[1123] Table 757

[1124] Table 758

[1125] Table 759

[1126] Table 760

[1127] Table 761

[1128] Table 762

[1129] Table 763

[1130] Table 764

[1131] Table 765

[1132] Table 766

[1133] Table 767

[1134] Table 768

[1135] Table 769

[1136] Table 770

[1137] Table 771

[1138] Table 772

[1139] Table 773

[1140] Table 774

[1141] Table 775

[1142] Table 776

[1143] Table 777

[1144] Table 778

[1145] Table 779

[1146] Table 780

[1147] Table 781

[1148] Table 782

[1149] Table 783

[1150] Table 784

[1151] Table 785

[1152] Table 786

[1153] Table 787

[1154] Table 788

[1155] Table 789

[1156] Table 790

[1157] Table 791

[1158] Table 792

[1159] Table 793

[1160] Table 794

[1161] Table 795

[1162] Table 796

[1163] Table 797

[1164] Table 798

[1165] Table 799

[1166] Table 800

[1167] Table 801

[1168] Table 802

[1169] Table 803

[1170] Table 804

[1171] Table 805

[1172] Table 806

[1173] Table 807

[1174] Table 808

[1175] Table 809

[1176] Table 810

[1177] Table 811

[1178] Table 812

[1179] Table 813

[1180] Table 814

[1181] Table 815

[1182] Table 816

[1183] Table 817

[1184] Table 818

[1185] Table 819

[1186] Table 820

[1187] Table 821

[1188] Table 822

[1189] Table 823

[1190] Table 824

[1191] Table 825

[1192] Table 826

[1193] Table 827

[1194] Table 828

[1195] Table 829

[1196] Table 830

[1197] Table 831

[1198] Table 832

[1199] Table 833

[1200] Table 834

[1201] Table 835

[1202] Table 836

[1203] Table 837

[1204] Table 838

[1205] Table 839

[1206] Table 840

[1207] Table 841

[1208] Table 842

[1209] Table 843

[1210] Table 844

[1211] Table 845

[1212] Table 846

[1213] Table 847

[1214] Table 848

[1215] Table 849

[1216] Table 850

[1217] Table 851

[1218] Table 852

[1219] Table 853

[1220] Table 854

[1221] Table 855

[1222] Table 856

[1223] Table 857

[1224] Table 858

[1225] Table 859

[1226] Table 860

[1227] Table 861

[1228] Table 862

[1229] Table 863

[1230] Table 864

[1231] Table 865

[1232] Table 866

[1233] Table 867

[1234] Table 868

[1235] Table 869

[1236] Table 870

[1237] Table 871

[1238] Table 872

[1239] Table 873

[1240] Table 874

[1241] Table 875

[1242] Table 876

[1243] Table 877

[1244] Table 878

[1245] Table 879

[1246] Table 880

[1247] Table 881

[1248] Table 882

[1249] Table 883

[1250] Table 884

[1251] Table 885

[1252] Table 886

[1253] Table 887

[1254] Table 888

[1255] Table 889

[1256] Table 890

[1257] Table 891

[1258] Table 892

[1259] Table 893

[1260] Table 894

[1261] Table 895

[1262] Table 896

[1263] Table 897

[1264] Table 898

[1265] Table 899

[1266] Table 900

[1267] Table 901

[1268] Table 902

[1269] Table 903

[1270] Table 904

[1271] Table 905

[1272] Table 906

[1273] Table 907

[1274] Table 908

[1275] Table 909

[1276] Table 910

[1277] Table 911

[1278] Table 912

[1279] Table 913

[1280] Table 914

[1281] Table 915

[1282] Table 916

[1283] Table 917

[1284] Table 918

[1285] Table 919

[1286] Table 920

[1287] Table 921

[1288] Table 922

[1289] Table 923

[1290] Table 924

[1291] Table 925

[1292] Table 926

[1293] Table 927

[1294] Table 928

[1295] Table 929

[1296] Table 930

[1297] Table 931

[1298] Table 932

[1299] Table 933

[1300] Table 934

[1301] Table 935

[1302] Table 936

[1303] Table 937

[1304] Table 938

[1305] Table 939

[1306] Table 940

[1307] Table 941

[1308] Table 942

[1309] [Table 943]

[1310] [Table 944]

[1311] [Table 945]

[1312] [Table 946]

[1313] Table 44 provides retroviral reverse transcriptase domains for use in Gene Writer polypeptides. The wild-type reverse transcriptase enzymes were collected and prioritized according to the description herein (see Example 33). The type column indicates whether the sequence corresponds to the wild-type sequence ("root") or contains a mutation ("derivative") that may improve the enzyme's activity.

[1314] [Table 947]

[1315] [Table 948]

[1316] [Table 949]

[1317] [Table 950]

[1318] Table 951

[1319] Table 952

[1320] Table 953

[1321] Table 954

[1322] Table 955

[1323] Table 956

[1324] Table 957

[1325] Table 958

[1326] Table 959

[1327] Table 960

[1328] Table 961

[1329] Table 962

[1330] Table 963

[1331] Table 964

[1332] Table 965

[1333] Table 966

[1334] Table 967

[1335] In some embodiments, the reverse transcriptase domain is modified, for example, by site-directed mutation. In some embodiments, the reverse transcriptase domain is modified to have improved properties, such as SuperScript IV (SSIV) reverse transcriptase derived from MMLV RT. In some embodiments, the reverse transcriptase domain may be modified to have a lower error rate, for example, as described in International Publication No. 2001068895, incorporated herein by reference. In some embodiments, the reverse transcriptase domain may be modified to have increased thermal stability. In some embodiments, the reverse transcriptase domain may be modified to have increased processability. In some embodiments, the reverse transcriptase domain may be modified to have resistance to inhibitors. In some embodiments, the reverse transcriptase domain may be modified to be faster. In some embodiments, the reverse transcriptase domain may be modified to have increased resistance to modified nucleotides in the RNA template. In some embodiments, the reverse transcriptase domain may be modified to insert modified DNA nucleotides. In some embodiments, the reverse transcriptase domain is modified to bind to template RNA. In some embodiments, one or more mutations are selected from D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K, or D653N within the RT domain of mouse leukemia virus reverse transcriptase, or from corresponding mutations at corresponding positions in other RT domains.In some embodiments, one or more mutations are present in the RT domain of the R2Bm retrotransposase (e.g., C952S, and / or C956S, and / or C952S, C956S (double mutant), and / or C969S, and / or H970Y, and / or R979Q, and / or R976Q, and / or R1071S, and / Alternatively, mutations of R328A, and / or R329A, and / or Q336A, and / or R328A, R329A, Q336A (triple mutant), and / or G426A, and / or D428A, and / or G426A, D428A (double mutant), and / or any combination thereof (as selected for the position relative to Sequence ID No. 52 in International Publication No. 2018089860A1), or as the corresponding mutation at the corresponding position in another RT domain.

[1336] In some embodiments, the RT domain possesses proofreading activity. In some embodiments, the RT domain evolves from DNA-dependent DNA polymerase to obtain RNA-dependent DNA polymerase activity. A synthetically evolved proofreading RT (RTX, Genbank: QFN49000.1), known as reverse transcription xenopolymerase, was previously constructed by obtaining DNA-dependent DNA polymerase (KOD, Genbank: ABN15964.1) and selecting for RNA-dependent DNA polymerase activity (Ellefson et al 2016). In some embodiments, the modified RT may include the signature of DNA-dependent DNA polymerase as a result of wild-type enzymes, e.g., IPR006134, PF00136, cd05536.

[1337] In some embodiments, the reverse transcription domain simply recognizes a specific template and reverse transcribes it. In some embodiments, the template includes a specific sequence. In some embodiments, the template includes the inclusion of a UTR (e.g., an untranslated region (UTR) from a retrotransposon, e.g., the 3' UTR of an R2 retrotransposon) that associates the nucleic acid with the reverse transcriptase domain.

[1338] The writing domain may also include DNA-dependent DNA polymerase activity, such as enzymatic activity capable of writing DNA to the genome from a template DNA sequence. In some embodiments, DNA-dependent DNA polymerase activity is provided by the DNA polymerase domain in the polypeptide. In some embodiments, DNA-dependent DNA polymerase activity is provided by a reverse transcriptase domain capable of DNA-dependent DNA polymerization, such as second-strand synthesis.

[1339] In some embodiments, the writing domain (e.g., the RT domain) includes, for example, an RNA-binding domain that specifically binds to an RNA sequence. In some embodiments, the template RNA includes an RNA sequence that is specifically bound by the RNA-binding domain of the writing domain.

[1340] In contrast to other types of reverse transcription mechanisms, such as retroviral RT and LTR retrotransposons, reverse transcription in non-LTR retrotransposons like R2 is carried out exclusively against an RNA template containing a specific recognition sequence. To initiate TPRT, the R2 retrotransposase requires its template to contain a minimum 3'UTR region (Luan and Eickbush Mol Cell Biol 15, 3882-91 (1995)). In some embodiments, the Gene Writer polypeptide is derived from a retrotransposase having the required binding motif, and the template RNA is designed to contain the said binding motif, thereby resulting in a specific retrotransposition only on the desired template. In some embodiments, the Gene Writer polypeptide is derived from a retrotransposon selected from Table 3, and the 3'UTR on the RNA template contains a 3'UTR from the same retrotransposon in Table 3.

[1341] Template nucleic acid binding domain: Gene Writer® polypeptides typically have a region capable of associating with a Gene Writer® template nucleic acid (e.g., template RNA). In some embodiments, the template nucleic acid binding domain is an RNA binding domain. In some embodiments, the RNA binding domain is a modular domain capable of associating with RNA molecules containing a specific signature, e.g., a structural motif, e.g., a secondary structure present in the 3'UTR of non-LTR retrotransposons. In other embodiments, the template nucleic acid binding domain (e.g., RNA binding domain) is contained within a reverse transcription domain, for example, a component derived from reverse transcriptase having a known signature for RNA preference, e.g., a secondary structure present in the 3'UTR of non-LTR retrotransposons. In other embodiments, the template nucleic acid binding domain (e.g., RNA binding domain) is contained within a DNA binding domain. For example, in some embodiments, the DNA binding domain is a CRISPR-associated protein that recognizes the structure of a template nucleic acid (e.g., template RNA) containing gRNA. In some embodiments, gRNA is a short synthetic RNA consisting of a scaffold sequence involved in the binding of CRISPR-related proteins and a user-defined target sequence of approximately 20 nucleotides for a genomic target. The structure of a complete gRNA is described in Nishimasu et al. Cell 156, pp. 935-949 (2014). The gRNA (also called sgRNA, corresponding to a single guide RNA) consists of crRNA-derived and tracrRNA-derived sequences linked by an artificial tetraloop. The crRNA sequence can be divided into a guide (20nt) and repeat (12nt) region, while the tracrRNA sequence can be divided into an anti-repeat (14nt) and three tracrRNA stem-loops (Nishimasu et al. Cell 156, pp. 935-949 (2014)). In practice, the guide RNA sequence is generally designed to have a length of between 17 and 24 nucleotides (e.g., 19, 20, or 21 nucleotides) and to be complementary to the target nucleic acid sequence. Custom gRNA generators and algorithms are commercially available for use in the design of effective guide RNAs.In some embodiments, the gRNA comprises two RNA components from the natural 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 sequences from both tracrRNA (for nuclease binding) and at least one crRNA (for guiding the nuclease to a targeted sequence for editing / binding). Chemically modified sgRNAs have also been proven effective for use with CRISPR-related proteins; see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991. In some embodiments, the gRNA comprises a nucleic acid sequence complementary to the DNA sequence associated with the target gene. In some embodiments, the polypeptide comprises a DNA-binding domain containing a CRISPR-related protein that associates with the gRNA, enabling the DNA-binding domain to bind to the target genomic DNA sequence. In some embodiments, the gRNA is contained within a template nucleic acid (e.g., template RNA), so the DNA-binding domain is also the template nucleic acid-binding domain. In some embodiments, the polypeptide has RNA-binding function within multiple domains, for example, it can bind to gRNA structures in a CRISPR-associated DNA-binding domain and to 3'UTR structures in reverse transcription domains derived from non-LTR retrotransposons.

[1342] Endonuclease domain: In some embodiments, the Gene Writer® polypeptide has the function of cleaving a target DNA site via an endonuclease domain. In some embodiments, the endonuclease domain is also a DNA-binding domain. In some embodiments, the endonuclease domain is also a template nucleic acid (e.g., template RNA)-binding domain. For example, in some embodiments, the polypeptide includes a CRISPR-related endonuclease domain that binds to a template RNA containing gRNA, binds to a target DNA sequence (e.g., having complementarity to a portion of the gRNA), and cleaves the target DNA sequence. In certain embodiments, the endonuclease / DNA-binding domain of an APE-type retrotransposon or the endonuclease domain of an RLE-type retrotransposon may be used in or modified (e.g., by insertion, deletion, or substitution of one or more residues) in the Gene Writer® system described herein. In some embodiments, the endonuclease domain or the endonuclease / DNA-binding domain is modified from its native sequence to have improved, modified codon use, for example, in human cells. In some embodiments, the endonuclease element is a heterologous endonuclease element, such as Fok1 nuclease, type II restriction I-like endonuclease (RLE-type nuclease), or another RLE-type endonuclease (also known as REL). In some embodiments, the heterologous endonuclease activity has nickase activity and does not form double-strand breaks. The amino acid sequence of the endonuclease domain of the Gene Writer® system described herein may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% identical to the amino acid sequence of the endonuclease domain of a retrotransposon whose DNA sequence is referenced in Table 1 or Table 3.Those with common skills in the art can use tools such as the Basic Local Alignment Search Tool (BLAST) to identify endonuclease domains based on homology to other known endonuclease domains. In certain embodiments, the heterologous endonuclease is Fok1 or a functional fragment thereof. In certain embodiments, the heterologous endonuclease is a Holliday junction resolverase or its homolog, e.g., Holliday junction cleavage enzyme (Ssol Hje) from Sulfolobus solfataricus (Govindaraju et al., Nucleic Acids Research 44:7, 2016). In certain embodiments, the heterologous endonuclease is an endonuclease of a large fragment of a spliceosome protein such as Prp8 (Mahbub et al., Mobile DNA 8:16, 2017). In certain embodiments, the heterologous endonucleases are derived from CRISPR-related proteins, such as Cas9. In certain embodiments, the heterologous endonucleases are modified to have only ssDNA cleavage activity, for example, only nickase activity, such as Cas9 nickase. For example, the Gene Writer® polypeptide described herein may include an endonuclease domain comprising a reverse transcriptase domain from an APE-type or RLE-type retrotransposon and Fok1 or a functional fragment thereof. In yet another embodiment, the homologous endonuclease domain is modified, for example, by site-directed mutation to alter its DNA endonuclease activity. In yet another embodiment, the endonuclease domain is modified to remove sequence specificity of any latent DNA.

[1343] In some embodiments, the endonuclease domain has nickase activity and does not form double-strand breaks. In some embodiments, the endonuclease domain forms single-strand breaks more frequently than double-strand breaks, for example, at least 90%, 95%, 96%, 97%, 98%, or 99% of the breaks are single-strand breaks, or less than 10%, 5%, 4%, 3%, 2%, or 1% of the breaks are double-strand breaks. In some embodiments, the endonuclease substantially does not form double-strand breaks. In some embodiments, the endonuclease does not form detectable levels of double-strand breaks.

[1344] In some embodiments, the endonuclease domain has nickase activity that cleaves the target site DNA on the first strand; for example, in some embodiments, the endonuclease domain cleaves the genomic DNA at the target site near the modification site on the strand that will be extended by the writing domain. In some embodiments, the endonuclease domain has nickase activity that cleaves the target site DNA on the first strand but not the target site DNA on the second strand. For example, when the polypeptide contains a CRISPR-related endonuclease domain that has nickase activity and does not form double-strand breaks, in some embodiments, the CRISPR-related endonuclease domain cleaves the target site DNA strand containing the PAM site (for example, but not the target site DNA strand that does not contain the PAM site). As a further example, when a polypeptide contains a CRISPR-related endonuclease domain that has nickase activity and does not form double-strand breaks, in some embodiments, the CRISPR-related endonuclease domain cleaves a target site DNA strand that does not contain a PAM site (for example, but does not cleave a target site DNA strand that contains a PAM site).

[1345] In some other embodiments, the endonuclease domain has nickase activity that cleaves the target site DNA of the first and second strands. While not intended to be bound by any particular theory, after the writing domain (e.g., RT domain) of the polypeptide described herein polymerizes (e.g., reverse transcribes) the heterologous target sequence of the template nucleic acid (e.g., template RNA), the cellular DNA repair mechanism must repair the nick on the first DNA strand. The target site DNA here comprises two distinct sequences relative to the first DNA strand: the first corresponding to the original genomic DNA and the second corresponding to the one polymerized from the heterologous target sequence. The two distinct sequences are thought to equilibrate with each other, with one hybridizing with the second strand first, then the other, where the incorporation of the cellular DNA repair mechanism into its repair target site is considered random. While not intended to be bound by any particular theory, the introduction of further nicks into the second strand may bias the cellular DNA repair mechanism to use the heterologous target sequence more frequently than the original genomic sequence. In some embodiments, further nicks are located at 5' or 3' of the target site modification (e.g., insertion, deletion, or substitution), or 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 relative to the nicks on the first strand.

[1346] Alternatively or additionally, and without intending to be bound by any particular theory, it is thought that further nicks to the second strand may facilitate second strand synthesis. In some embodiments, if Gene Writer™ inserts or substitutes a portion of the first strand, synthesis of a new sequence corresponding to the insertion / substitution in the second strand is required.

[1347] In some embodiments, the polypeptide comprises a single domain having endonuclease activity (e.g., a single endonuclease domain), the domain cleaving both the first and second strands. For example, in such embodiments, the endonuclease domain may be a CRISPR-associated endonuclease domain, and the template nucleic acid (e.g., template RNA) comprises a gRNA that leads to nicking of the first strand and a further gRNA that leads to nicking of the second strand. In some embodiments, the polypeptide comprises multiple domains having endonuclease activity, the first endonuclease domain cleaving the first strand and the second endonuclease domain cleaving the second strand (optionally, the first endonuclease domain may not cleaving the second strand (e.g., is unable to do so), and the second endonuclease domain may not cleaving the first strand (e.g., is unable to do so)).

[1348] In some embodiments, the endonuclease domain can puncture the first and second chains. In some embodiments, the first and second chain nicks occur at the same location in the target site but not on the reverse chain. In some embodiments, the second chain nick occurs at a twisted location, for example, upstream or downstream of the first nick. In some embodiments, the endonuclease domain generates a deletion at the target site if the second chain nick is upstream of the first chain nick. In some embodiments, the endonuclease domain generates a duplication of the target site if the second chain nick is downstream of the first chain nick. In some embodiments, the endonuclease domain does not generate duplication and / or deletion if the first and second chain nicks occur at the same location in the target site (for example, as described in Gladyshev and Arkhipova Gene 2009; the whole of which is incorporated herein by reference). In some embodiments, the endonuclease domain has modified activity depending on the protein structure or RNA binding state, for example (as described in Christensen et al. PNAS 2006; the whole thereof is incorporated herein by reference), which promotes the nicking of the first or second strand.

[1349] In some embodiments, the Gene Writer polypeptide includes modifications to the endonuclease domain compared to, for example, the wild-type polypeptide. In some embodiments, the endonuclease domain includes additions, deletions, substitutions, or modifications to the amino acid sequence of the original endonuclease domain. In some embodiments, the endonuclease domain is modified to include a heterologous functional domain that specifically binds to a target nucleic acid (e.g., DNA) sequence of interest and / or induces its endonuclease cleavage. In some embodiments, the endonuclease domain includes a Zn finger. In some embodiments, the endonuclease domain includes a Cas domain (e.g., Cas9 or its variants or variants). In several embodiments, the endonuclease domain including the Cas domain associates with a guide RNA (gRNA), for example, as described herein. In some embodiments, the endonuclease domain is modified to include a functional domain that does not target a specific target nucleic acid (e.g., DNA) sequence. In several embodiments, the endonuclease domain includes a Fok1 domain.

[1350] In some embodiments, the endonuclease domain includes a meganuclease or a functional fragment thereof. In some embodiments, the endonuclease domain includes a homing endonuclease or a functional fragment thereof. In some embodiments, the endonuclease domain includes a meganuclease or a functional fragment or variant thereof from the LAGLIDADG (SEQ ID NO: 1577), GIY-YIG, HNH, His-Cys Box, or PD-(D / E)XK family, for example, having a conserved amino acid motif as indicated by the family name. In some embodiments, the endonuclease domain includes a meganuclease or fragment thereof selected from, for example, I-SmaMI (Uniprot F7WD42), I-SceI (Uniprot P03882), I-AniI (Uniprot P03880), I-DmoI (Uniprot P21505), I-CreI (Uniprot P05725), I-TevI ​​(Uniprot P13299), I-OnuI (Uniprot Q4VWW5), or I-BmoI (Uniprot Q9ANR6). In some embodiments, the meganuclease is naturally present in its functional form as a monomer, e.g., I-SceI, I-TevI, or a dimer, e.g., I-CreI. For example, LAGLIDADG(SEQ ID NO: 1577) meganuclease having a single copy of the LAGLIDADG motif (SEQ ID NO: 1577) generally forms homodimers, while members having two copies of the LAGLIDADG motif (SEQ ID NO: 1577) are generally found as monomers. In some embodiments, meganucleases that normally form as dimers are expressed as fusions, for example, as an I-CreI dimer fusion, where the two subunits are optionally linked by a linker as a single ORF (Rodriguez-Fornes et al. Gene Therapy 2020; the whole is incorporated herein by reference).In some embodiments, the meganuclease, or functional fragment thereof, is modified to preferentially target nickase activity on one strand of a double-stranded DNA molecule, e.g., I-SceI (K122I and / or K223I) (Niu et al. J Mol Biol 2008), I-AniI (K227M) (McConnell Smith et al. PNAS 2009), I-DmoI (Q42A and / or K120M) (Molina et al. J Biol Chem 2015). In some embodiments, the meganuclease, or functional fragment thereof, having this preference for single-strand breaks, is used, for example, as an endonuclease domain having nickase activity. In some embodiments, the endonuclease domain includes a meganuclease, or functional fragment thereof, that naturally targets, or is modified to target, a safe port site, e.g., an SH6 site that targets I-CreI (Rodriguez-Fornes). (et al., above). In some embodiments, the endonuclease domain includes a meganuclease or a functional fragment thereof having a sequence-resistant catalytic domain, e.g., I-TevI ​​that recognizes the minimal motif CNNNG (Kleinstiver et al. PNAS 2012). In some embodiments, the target sequence-resistant catalytic domain is fused to a DNA-binding domain, and activity is induced, for example, by fusing I-TevI ​​to (i) a Zn finger for producing Tev-ZFE (Kleinstiver et al. PNAS 2012), (ii) another meganuclease for producing MegaTev (Wolfs et al. Nucleic Acids Res 2014), and / or (iii) Cas9 for producing TevCas9 (Wolfs et al. PNAS 2016).

[1351] In some embodiments, the endonuclease domain includes a restriction enzyme, e.g., type IIS or type IIP restriction enzyme. In some embodiments, the endonuclease domain includes a type IIS restriction enzyme, e.g., FokI or a fragment or variant thereof. In some embodiments, the endonuclease domain includes a type IIP restriction enzyme, e.g., PvuII or a fragment or variant thereof. In some embodiments, the dimeric restriction enzyme is expressed as a fusion, e.g., a FokI dimer fusion, so that it functions as a single chain (Minczuk et al. Nucleic Acids Res 36(12):3926-3938(2008)).

[1352] Further uses of endonuclease domains are described, for example, in Guha and Edgell Int J Mol Sci 18(22):2565(2017) (which is incorporated herein by reference in its entirety).

[1353] In some embodiments, the endonuclease domain includes a CRISPR / Cas domain (also referred to herein as a CRISPR-related protein). In some embodiments, the DNA-binding domain includes a CRISPR / Cas domain. In some embodiments, the CRISPR / Cas domain includes a protein involved in clustered and regularly arranged short palindromic sequence repeats (CRISPR) systems, such as a Cas protein, which optionally binds to a guide RNA, such as a single guide RNA (sgRNA).

[1354] The CRISPR system is an adaptive defense system first discovered in bacteria and archaea. The CRISPR system uses RNA-guided nucleases, referred to as CRISPR-associated or "Cas" endonucleases (e.g., Cas9 or Cpf1), to cleave foreign DNA. For example, in a typical CRISPR / Cas system, the endonuclease is guided to a target nucleotide sequence (e.g., a site in the genome to be sequence-edited) by a sequence-specific, non-coding "guide RNA" that targets a single-stranded or double-stranded DNA sequence. Three classes (I-III) of CRISPR systems have been identified. Class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes type II Cas endonucleases such as Cas9, CRISPR RNA ("crRNA"), and transactivating crRNA ("tracrRNA"). crRNA typically contains a "guide RNA," which is an RNA sequence of approximately 20 nucleotides corresponding to the target DNA sequence. In the wild-type system, and in some modified systems, crRNA forms a partially double-stranded structure that binds to tracrRNA and is cleaved by ribonuclease III. cThis also includes regions that result in rRNA / tracrRNA hybrids. The crRNA / tracrRNA hybrid then guides the Cas9 endonuclease to recognize and cleave the target DNA sequence. The target DNA sequence is generally adjacent to a “protospacer facilitation motif” ("PAM") specific to a given Cas endonuclease, although PAM sequences are found throughout a given genome. CRISPR endonucleases identified from various prokaryotic species have their own specific PAM sequence requirements; examples of PAM sequences include 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningitidis). Some endonucleases, such as Cas9 endonuclease, associate with G-rich PAM sites, e.g., 5'-NGG, and perform blunt-end cleavage of target DNA at a position of three nucleotides upstream (from 5') of the PAM site. Another class II CRISPR system includes a smaller V-type endonuclease, Cpf1, than Cas9; examples include AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (from Lachnospiraceae sp.). Cpf1-associated CRISPR arrays do not require tracrRNA and are processed with mature crRNA; in other words, the Cpf1 system, in some embodiments, comprises exclusively the Cpf1 nuclease and crRNA to cleave the target DNA sequence. Cpf1 endonucleases typically associate with T-rich PAM sites, e.g., 5'-TTN. Cpf1 can also recognize the 5'-CTA PAM motif.Cpf1 typically cleaves target DNA by introducing offset or twisted double-strand breaks into the 5' overhangs of 4- or 5-nucleotides, for example, by cleaving target DNA with 5-nucleotide offset or twisted breaks located 18 nucleotides downstream (3' side) from the PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complementary strand; the 5-nucleotide overhangs resulting from these offset breaks enable more precise genome editing by DNA insertion via homologous recombination rather than insertion with blunt-end DNA. See, for example, Zetsche et al. (2015) Cell, 163:759-771.

[1355] Various CRISPR-related (Cas) genes or proteins can be used in the techniques provided by this disclosure, and the selection of Cas proteins will depend on the specific conditions of the method. Specific examples of Cas proteins include the Class II system comprising Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1, C2C1, or C2C3. In some embodiments, the Cas protein, e.g., the Cas9 protein, may be derived from any of various prokaryotic species. In some embodiments, a specific Cas protein, e.g., a specific Cas9 protein, is selected to recognize a specific protospacer-adjacent motif (PAM) sequence. In some embodiments, the DNA-binding domain or endonuclease domain includes a sequence-targeting polypeptide such as a Cas protein, e.g., Cas9. In certain embodiments, the Cas protein, e.g., the Cas9 protein, may be obtained from bacteria or archaea, or synthesized using known methods. In certain embodiments, the Cas protein may be derived from Gram-positive or Gram-negative bacteria. In certain embodiments, the Cas protein is found in streptococci (e.g., Streptococcus pyogenes or S. thermophilus), Francisella (e.g., F. nobicida), Staphylococcus (e.g., Staphylococcus aureus), and Acidaminococcus (e.g., Acidaminococcus species BV3L6). It may be derived from sp.BV3L6), Neisseria (e.g., Neisseria meningitidis), Cryptococcus, Corynebacterium, Haemophilus, Eubacterium, Pasteurella, Prevotella, Veillonella, or Marinobacter. 【13...

Claims

[Claim 1] The invention described in the specification.

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