Chimeric genome engineering molecules and methods

Abstract

The present disclosure provides compositions and methods for increasing mutation efficiency and homologous recombination rates of site-specific endonucleases. The compositions and methods comprise a chimeric polypeptide comprising a site-specific endonuclease or a domain thereof and a functional moiety. The current inventions relate to functional enhancement of the CRISPR-Cas enzymes. Disclosed herein include possible variants and their intended improvements.

1 . A polypeptide comprising a programmable endonuclease fused in frame to a DNA modifying enzyme comprising at least 70% sequence identity to RecE, RecJ, RecBCD, Mungbean, ExoIII, or ExoVII, wherein the polypeptide comprises an increased mutagenesis activity at a locus contacted with the programmable endonuclease compared to a mutagenesis activity of a wild type programmable endonuclease. 
     
     
         2 . The polypeptide of  claim 1 , wherein the programmable endonuclease comprises at least 70% identity to Cas9 or Cpf1. 
     
     
         3 . The polypeptide of  claim 1 , wherein the DNA modifying enzyme generates 3′ OH overhang, 
     
     
         4 . The polypeptide of  claim 1 , wherein the DNA modifying enzyme exposes a recessed 3′ OH. 
     
     
         5 . The polypeptide of  claim 1 , wherein the DNA modifying enzyme comprises cleaved end resection activity. 
     
     
         6 . The polypeptide of  claim 1 , wherein the increased mutagenesis activity comprises an increased mutation efficiency or an increased homologous recombination efficiency relative to a mutagenesis activity exerted by the wild type programmable endonuclease. 
     
     
         7 . The polypeptide of  claim 6 , wherein the increased mutation efficiency or the increased homologous recombination efficiency relative to a wild type Cas9. 
     
     
         8 . The polypeptide of  claim 6 , wherein the increased mutation efficiency or increased homologous recombination efficiency is measured in a human cell or a plant cell. 
     
     
         9 . The polypeptide of  claim 1 , wherein the increased mutagenesis activity comprises increased deletion rate at the locus contacted with the polypeptide comprising the programmable endonuclease compared to a deletion rate exerted by the wild type programmable endonuclease. 
     
     
         10 . The polypeptide of  claim 1 , wherein the increased mutagenesis activity comprises increased insertion rate at the locus contacted with the polypeptide comprising the programmable endonuclease compared to an insertion rate exerted by the wild type programmable endonuclease. 
     
     
         11 . The polypeptide of  claim 1 , wherein the increased mutagenesis activity comprises increased homologous recombination rate at the locus contacted with the polypeptide comprising the programmable endonuclease compared to a homologous recombination rate exerted by the wild type programmable endonuclease. 
     
     
         12 . The polypeptide of  claim 1 , wherein the polypeptide is substantially free of bacterial cellular contaminant. 
     
     
         13 . The polypeptide of  claim 1 , wherein the polypeptide does not have an animal glycosylation pattern. 
     
     
         14 . The polypeptide of  claim 1 , wherein the polypeptide does not have a bacterial glycosylation pattern. 
     
     
         15 . The polypeptide of  claim 1 , wherein the polypeptide does not have a fungal glycosylation pattern. 
     
     
         16 . A nucleic acid encoding the polypeptide of  claim 1 . 
     
     
         17 . The nucleic acid of  claim 16 , wherein the nucleic acid comprises an open reading frame that is at least partially codon optimized for expression of the polypeptide in a plant cell, a bacterium, or an animal cell. 
     
     
         18 . The nucleic acid of  claim 16 , wherein the nucleic acid comprises a 5′ UTR that is at least partially codon optimized for expression of the polypeptide in a plant cell, in a bacterium, or in an animal cell. 
     
     
         19 . The nucleic acid of  claim 16 , wherein the nucleic acid comprises a 3′ UTR that is at least partially codon optimized for expression of the polypeptide in a plant cell, in a bacterium, or in an animal cell. 
     
     
         20 . The nucleic acid of  claim 16 , wherein the nucleic acid is a vector.