Genome modification using guide polynucleotide/cas endonuclease systems and methods of use

Abstract

Compositions and methods are provided for genome modification of a target sequence in the genome of a cell. The methods and compositions employ a guide polynucleotide/Cas endonuclease system to provide an effective system for modifying or altering target sites within the genome of a cell or organism. Once a genomic target site is identified, a variety of methods can be employed to further modify the target sites such that they contain a variety of polynucleotides of interest. Compositions and methods are also provided for editing a nucleotide sequence in the genome of a cell. Breeding methods and methods for selecting plants utilizing a two component RNA polynucleotide and Cas endonuclease system are also disclosed.

1 .- 43 . (canceled) 
     
     
         44 . A method for modifying a target site in a eukaryotic cell, the method comprising:
 providing the eukaryotic cell with a guide polynucleotide that complexes with a Cas endonuclease, the guide polynucleotide comprising:
 a) a Variable Targeting (VT) domain that is complementary to a polynucleotide sequence of the target site, wherein the VT domain is at least 12 nucleotides in length and comprises deoxyribonucleic acid; and 
 b) a Cas Endonuclease Recognition (CER) domain that interacts with the Cas endonuclease, 
   introducing a double strand break in the polynucleotide sequence of the target site upon complexing of the guide polynucleotide and the Cas endonuclease.   
     
     
         45 . The method of  claim 44 , further comprising:
 identifying eukaryotic cells comprising a modified target site, the modified target site comprising:
 i. a substitution of at least one nucleotide; 
 ii. a deletion of at least one nucleotide; 
 iii. an insertion of at least one nucleotide; or 
 iv. a combination of i-iii. 
   
     
     
         46 . The method of  claim 44 , further comprising:
 providing the eukaryotic cell with a donor DNA comprising a polynucleotide of interest; and   identifying eukaryotic cells comprising the polynucleotide of interest integrated at the target site.   
     
     
         47 . The method of  claim 44 , further comprising:
 providing the eukaryotic cell with the Cas endonuclease.   
     
     
         48 . The method of  claim 44 , wherein the VT domain and the CER domain are located on a single molecule. 
     
     
         49 . The method of  claim 44 , wherein the guide polynucleotide provided to the eukaryotic cell is less than 1 μg. 
     
     
         50 . The method of  claim 44 , wherein the guide polynucleotide provided to the eukaryotic cell is between, and inclusive of, 50 ng and 100 ng. 
     
     
         51 . The method of  claim 44 , wherein the guide polynucleotide provided to the eukaryotic cell is at least 50 ng and less than 1 ug. 
     
     
         52 . The method of  claim 44 , wherein the guide polynucleotide is provided to the eukaryotic cell by particle bombardment. 
     
     
         53 . The method of  claim 44 , wherein the guide polynucleotide is provided to the eukaryotic cell by  Agrobacterium -mediated transformation. 
     
     
         54 . The method of  claim 44 , wherein the target site is chromosomal DNA. 
     
     
         55 . The method of  claim 44 , wherein the eukaryotic cell is a plant cell. 
     
     
         56 . The method of  claim 55 , wherein the eukaryotic cell is a monocot plant cell. 
     
     
         57 . The method of  claim 55 , wherein providing the eukaryotic cell with the guide polynucleotide comprises delivering the guide polynucleotide to a plant embryo. 
     
     
         58 . The method of  claim 57 , wherein the guide polynucleotide is provided to the plant embryo by particle bombardment. 
     
     
         59 . The method of  claim 57 , wherein the guide polynucleotide is provided to the plant embryo by  Agrobacterium -mediated transformation.