Methods and compositions for modulating a genome

Abstract

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.