METHODS AND COMPOSITIONS FOR MODULATING A GENOME
Methods and compositions for modulating a target genome are disclosed.
1 - 108 . (canceled)
109 . A polypeptide or a 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, wherein the RT domain has a sequence of Table 30 or 44, or a sequence having at least 90% identity thereto.
110 . A system for modifying DNA comprising:
(a) the polypeptide or a nucleic acid encoding the polypeptide of claim 109 ; and
(b) a template RNA (or DNA encoding the template RNA) comprising, from 5′ to 3′, (i) optionally a sequence that binds a target site, (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain.
111 . The system of claim 110 , wherein the DBD comprises a CRISPR/Cas domain.
112 . The system of claim 110 , wherein the endonuclease domain is a nickase domain.
113 . The system of claim 110 , wherein the RT domain comprises one or more mutations relative to a naturally occurring RT domain or an RT domain or functional fragment chosen from Table 30 or 44.
114 . The system of claim 112 , wherein the RT domain is positioned C-terminal of the nickase domain in the polypeptide.
115 . The system of claim 110 , wherein the polypeptide comprises a linker positioned between the RT domain and the DBD.
116 . The system of claim 110 , wherein the linker is between 2-50 amino acids in length.
117 . The system of claim 110 , wherein the 3′ target homology domain is complementary to a sequence adjacent to a site to be modified by the system.
118 . The system of claim 110 , wherein the heterologous object sequence is complementary to a site to be modified by the system except at a position or positions to be modified.
119 . The system of claim 110 , wherein the heterologous object sequence inserts at least 1 nucleotide into the target site.
120 . The system of claim 110 , wherein the sequence that binds the polypeptide comprises a gRNA scaffold.
121 . The system of claim 110 , wherein the sequence that binds a target site is at least 18 nucleotides long.
122 . The system of claim 110 , wherein the sequence that binds a target site is complementary to the second strand of the target site.
123 . The system of claim 110 , further comprising a second strand-targeting gRNA that directs the endonuclease domain to nick the second strand.
124 . The system of claim 123 , wherein the template RNA further comprises the second strand-targeting gRNA.
125 . The system of claim 123 , wherein the second strand-targeting gRNA is disposed on a separate nucleic acid from the template RNA.
126 . The system of claim 110 , wherein the template RNA comprises a 2′-O-methyl modified nucleotide.
127 . The system of claim 110 , wherein the nucleic acid encoding the polypeptide comprises 5-methoxyuridine.
128 . The system of claim 110 , wherein at least 80% of the template RNA present contains one or more modified nucleotides.
129 . The system of claim 110 , wherein the polypeptide and/or template RNA are packaged into a virus, viral-like particle, virosome, liposome, vesicle, exosome, or LNP.
130 . The system of claim 110 , which is substantially free of pyrogen, virus, fungus, bacterial pathogen, and/or host cell protein contamination.
131 . A method for modifying a target site in genomic DNA in a cell, the method comprising contacting the cell with a system of claim 110 , thereby modifying the target site in genomic DNA in a cell.
132 . A method for treating a subject having a disease or condition associated with a genetic defect, the method comprising administering to the subject the system of claim 110 , thereby treating the subject having a disease or condition associated with a genetic defect.