Targeted non-viral dna insertions

Abstract

Provided herein are methods and compositions for editing the genome of a cell. In some embodiments, a nucleotide sequence of at least 200 nucleotides in length is inserted into a target region in the genome of a cell.

What is claimed is: 
     
         1 . A method of editing the genome of a cell, the method comprising:
 a) providing a Cas9 ribonucleoprotein complex (RNP)-DNA template complex comprising:
 (i) the RNP, wherein the RNP comprises a Cas9 nuclease domain and a guide RNA, wherein the guide RNA specifically hybridizes to a target region of the genome of the cell, and wherein the Cas9 nuclease domain cleaves the target region to create an insertion site in the genome of the cell; and 
 (ii) a double-stranded or single-stranded DNA template, wherein the size of the DNA template is greater than about 200 nucleotides, wherein the 5′ and 3′ ends of the DNA template comprise nucleotide sequences that are homologous to genomic sequences flanking the insertion site, and wherein the molar ratio of RNP to DNA template in the complex is from about 3:1 to about 100:1; and 
   b) introducing the RNP-DNA template complex into the cell.   
     
     
         2 . The method of  claim 1 , wherein the RNP-DNA template complex is formed by incubating the RNP with the DNA template for less than about one minute to about thirty minutes, at a temperature of about 20° to 25° C. 
     
     
         3 . The method of  claim 1  or  2 , wherein the DNA template is a linear DNA template. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the DNA template is a single-stranded DNA template 
     
     
         5 . The method of any one of  claims 1 - 3 , wherein the DNA template is a pure single-stranded DNA template. 
     
     
         6 . The method of any one of  claims 1 - 5 , wherein the RNP-DNA template complex and the cell are mixed prior to introducing the RNP-DNA template complex into the cell. 
     
     
         7 . The method of any one of  claims 1 - 6 , wherein the RNP comprises a Cas9 nuclease. 
     
     
         8 . The method of any one of  claims 1 - 6 , wherein the RNP comprises a Cas9 nickase. 
     
     
         9 . The method of any of  claims 1 - 8 , wherein the RNP-DNA template complex comprises at least two structurally different RNP complexes. 
     
     
         10 . The method of  claim 9 , wherein the at least two structurally different RNP complexes contain structurally different guide RNAs. 
     
     
         11 . The method of  claim 10 , wherein each of the structurally different RNP complexes comprises a Cas9 nickase, and wherein the structurally different guide RNAs hybridize to opposite strands of the target region. 
     
     
         12 . The method of  claim 9 , wherein the at least two structurally different RNP complexes contain structurally different Cas9 nuclease domains. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein the introducing comprises electroporation. 
     
     
         14 . The method of any one of  claims 1 - 13 , wherein the molar ratio of RNP to DNA template is from about 5:1 to about 15:1. 
     
     
         15 . The method of any one of  claims 1 - 14 , wherein the molar ratio of RNP to DNA template is from about 5:1 to about 10:1. 
     
     
         16 . The method of any one of  claims 1 - 15 , wherein the molar ratio of RNP to DNA template is from about 8:1 to about 12:1. 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the size of the DNA template is greater than about 1 kb. 
     
     
         18 . The method of any one of  claims 1 - 17 , wherein the DNA template is at a concentration of about 2.5 pM to about 25 pM. 
     
     
         19 . The method of any one of  claims 1 - 18 , wherein the amount of DNA template is about 1 μg to about 10 μg. 
     
     
         20 . The method of any one of  claims 1 - 19 , wherein the cell is a primary hematopoietic cell or a primary hematopoietic stem cell. 
     
     
         21 . The method of any one of  claims 1 - 20 , wherein the RNP-DNA template complex is introduced into about 1×10 5  to about 2×10 6  cells. 
     
     
         22 . The method of any one of  claims 1 - 21 , wherein the cell is a primary hematopoietic cell. 
     
     
         23 . The method of  claim 22 , wherein the primary hematopoietic cell is an immune cell. 
     
     
         24 . The method of  claim 23 , wherein the immune cell is a T cell. 
     
     
         25 . The method of  claim 24 , wherein the T cell is a regulatory T cell, an effector T cell, or a naïve T cell. 
     
     
         26 . The method of  claim 25 , wherein the regulatory T cell, effector T cell, or naïve T cell is a CD4 +  T cell. 
     
     
         27 . The method of  claim 24 , wherein the T cell is a CD8 +  T cell. 
     
     
         28 . The method of  claim 24 , wherein the T cell is a CD4 + CD8 +  T cell.