Optimized gene editing utilizing a recombinant endonuclease system

Abstract

Described herein are methods and compositions for genomic editing. Endonucleases for genomic editing involve inducing breaks in double stranded DNA, for which knock-ins are notoriously inefficient for relying on random integration of homologous DNA sequences into the break site by repair proteins. To address these issues, described herein are novel recombinant fusion proteins that actively recruit linear DNA inserts in closer proximity to the genomic cleavage site, increasing integration efficiency of large DNA fragments into the genome. Such improvements to genomic editing technology allow one to use lower linear DNA concentrations without sacrificing efficiency and can be further combined with other features, such as fluorescent protein reporting systems.

1 . A composition comprising a vector encoding a fusion protein comprising at least one endonuclease and a DNA binding moiety. 
     
     
         2 . The composition of  claim 1 , wherein the fusion protein comprises at least one endonuclease selected from the group consisting of: cas regularly interspaced short palindromic (CRISPR) protein, a zinc finger nuclease (ZFNs) and transcription activator-like effector nucleases (TALENs). 
     
     
         3 . The composition of  claim 2 , wherein the CRISPR protein comprises cas9. 
     
     
         4 . The composition of  claim 1 , wherein the DNA binding moiety comprises a zinc finger protein. 
     
     
         5 . The composition of  claim 4 , wherein the zinc finger comprises a left handed CCR5 binding protein. 
     
     
         6 . The composition of  claim 1 , wherein the at least one endonuclease and DNA binding moiety are joined by a linker comprising two, three, four, five, six, seven, eight, nine, ten or more amino acids. 
     
     
         7 . The composition of  claim 1 , wherein the fusion protein comprises a fluorescent labeled protein. 
     
     
         8 . The composition of  claim 7 , wherein the fluorescent labeled protein comprises one or more proteins selected from the group consisting of: green fluorescent protein (GFP), enhanced (eGFP), red fluorescent protein (RFP) and mCherry. 
     
     
         9 . The composition of  claim 1 , wherein the fusion protein comprises a nuclear localization signal (NLS). 
     
     
         10 . The composition of  claim 9 , wherein the NLS is SV40 NLS. 
     
     
         11 - 18 . (canceled) 
     
     
         19 . A kit for genomic editing comprising:
 one or more vectors encoding a fusion protein comprising at least one endonuclease and a DNA binding moiety; and   template DNA comprising at least one expression cassette, two flanking sequences, and a DNA binding moiety sequence.   
     
     
         20 . The kit of  claim 19 , further comprising one or more guide RNAs (gRNAs). 
     
     
         21 . The kit of  claim 19 , wherein the fusion protein comprises at least one endonuclease CRISPR protein, a DNA binding moiety that is a zinc finger protein. 
     
     
         22 . The kit of  claim 21 , further comprising a fluorescent labeled protein. 
     
     
         23 . The kit of  claim 21 , further comprising a nuclear localization signal (NLS).