Compositions and methods for improving homogeneity of dna generated using a crispr/cas9 cleavage system

Abstract

The invention relates to the unexpected discovery of a system and methods for precise homology directed repair after CRISPR/Cas9 cleavage. The invention includes a DNA cleavage and repair system comprising a CRISPR/Cas9 system and an oligonucleotide 100% complementary to cleaved DNA to promote homology directed DNA repair. The invention further includes methods for inducing homology directed repair of cleaved DNA and repairing a CRISPR/Cas9 cleavage.

1 .- 12 . (canceled) 
     
     
         13 . A method of exchanging a single nucleotide in a DNA sequence using a CRISPR/Cas9 system in a synchronized and released population of cells in vitro or ex vivo, the method comprising:
 cleaving a first DNA within 20 bases of a single nucleotide target site in the synchronized and released population of cells using at least one CRISPR/Cas9 system comprising a ribonucleoprotein (RNP) complex, thus generating a first cleaved DNA comprising a first cleavage end;   exchanging a single nucleotide at the single nucleotide target site by (a) annealing the first cleaved DNA with a single-stranded deoxyoligonucleotide about 72 nucleotides in length comprising (i) a first region 100% complementary to the first cleavage end and at least one deoxynucleotide adjacent to the first cleavage end of the first cleaved DNA, (ii) a second region 100% complementary to a second cleavage end of a second DNA and at least one deoxynucleotide adjacent to the second cleavage end of the second cleaved DNA, and (iii) a single mismatched nucleotide as compared to the single nucleotide target site of the first DNA and (b) fusing the first and second cleavage ends of the cleaved DNA   with no insertion and deletion of one or more unwanted deoxynucleotides at the junction of the first cleavage end of the cleaved first DNA and the second cleavage end of the second DNA to generate a repaired DNA at the single nucleotide target site comprising a repaired nucleotide complementary to the single mismatched nucleotide of the single-stranded deoxyoligonucleotide.   
     
     
         14 .- 16 . (canceled) 
     
     
         17 . The method of  claim 13 , wherein the first region of the single-stranded deoxyoligonucleotide is directly linked to the second region of the single-stranded deoxyoligonucleotide. 
     
     
         18 . The method of  claim 13 , wherein no collateral on-site or off-site DNA mutagenesis is produced. 
     
     
         19 . A method of exchanging a single nucleotide in a first DNA cleaved by at least one CRISPR/Cas9 system a synchronized and released population of cells in vitro or ex vivo and a second cleavage end of a second DNA, the method comprising:
 exchanging a single nucleotide at a single nucleotide target site that is within 20 bases of the CRISPR/Cas9 cleavage site without introducing an insertion or deletion of one or more unwanted deoxynucleotides by (a) annealing a single-stranded oligonucleotide about 72 nucleotides in length to the first DNA cleaved in one or more sites using at least one CRISPR/Cas9 system comprising a ribonucleoprotein (RNP) complex in the synchronized and released population of cells and (b) fusing a first cleavage end of the first DNA with the second cleavage end of the second DNA with no insertion and deletion of one or more unwanted deoxynucleotides at the junction of the first cleavage end of the first DNA and the second cleavage end of the second DNA to generate a repaired DNA comprising a repaired nucleotide complementary to the single mismatched nucleotide of the single-stranded deoxyoligonucleotide,   wherein the single-stranded oligonucleotide comprises (i) a first region 100% complementary to the first cleavage end of the first DNA and at least one deoxynucleotide adjacent to the first cleavage end of the first DNA, (ii) a second region 100% complementary to the second cleavage end of the second DNA and at least one deoxynucleotide adjacent to the second cleavage end of the second DNA, and (iii) a single mismatched nucleotide as compared to the single nucleotide target site of the DNA.   
     
     
         20 .- 22 . (canceled) 
     
     
         23 . The method of  claim 19 , wherein the first region of the single-stranded deoxyoligonucleotide is directly linked to the second region of the single-stranded deoxyoligonucleotide. 
     
     
         24 . The method of  claim 19 , wherein no collateral on-site or off-site DNA mutagenesis is produced. 
     
     
         25 .- 26 . (canceled)