Methods for increasing cas9-mediated engineering efficiency

Abstract

Methods for use with Type II CRISPR-Cas9 systems for increasing Cas9-mediated genome engineering efficiency are disclosed. The methods can be used to decrease the number of off-target nucleic acid double-stranded breaks and/or to enhance homology-directed repair of a cleaved target nucleic acid.

1 - 68 . (canceled) 
     
     
         69 . A method for positioning a donor polynucleotide near a cleavage site, comprising:
 contacting a first complex with a first target nucleic acid comprising the cleavage site, wherein the first complex comprises a catalytically active Cas9 protein and a first guide polynucleotide that comprises a spacer that binds to the first target nucleic acid, and the first complex binds and cleaves the first target nucleic acid at the cleavage site; and   contacting a second complex with a second target nucleic acid, wherein the second complex comprises a catalytically inactive Cas9 protein (dCas9 protein), and a second guide polynucleotide that comprises a spacer that binds to the second target nucleic acid, wherein the second target nucleic acid is in proximity to the cleavage site, the second complex is associated with a first end of a donor polynucleotide, and the second complex binds but does not cleave the second target nucleic acid;   wherein binding of the second complex positions the donor polynucleotide near the cleavage site, and at least a portion of the donor polynucleotide is inserted into the first target nucleic acid.   
     
     
         70 . The method of  claim 69 , wherein the first guide polynucleotide is a single-guide RNA (sgRNA). 
     
     
         71 . The method of  claim 69 , wherein the second guide polynucleotide is a dual-guide RNA. 
     
     
         72 . The method of  claim 69 , further comprising:
 contacting a third complex with a third target nucleic acid, wherein the third complex comprises a dCas9 protein, and a third guide polynucleotide that comprises a spacer that binds to the third target nucleic acid, wherein the second target is located upstream of the cleavage site, the third target nucleic acid is located downstream of the cleavage site, the third complex is associated with a second end of the donor polynucleotide, and the third complex binds but does not cleave the third target nucleic acid.   
     
     
         73 . The method of  claim 72 , wherein the third guide polynucleotide is a sgRNA. 
     
     
         74 . The method of  claim 69 , wherein the catalytically active Cas9 protein is selected from the group consisting of a  Streptococcus pyogenes  Cas9 protein, a  Streptococcus thermophilus  Cas9 protein, a  Staphylococcus aureus  Cas9 protein, a  Neisseria meningitidis  Cas9 protein, and an orthologous Cas9 protein. 
     
     
         75 . The method of  claim 74 , wherein the catalytically active Cas9 protein is the  Streptococcus pyogenes  Cas9 protein. 
     
     
         76 . The method of  claim 69 , wherein the dCas9 protein is selected from the group consisting of a  Streptococcus pyogenes  dCas9 protein, a  Streptococcus thermophilus  dCas9 protein, a  Staphylococcus aureus  dCas9 protein, a  Neisseria meningitidis  dCas9 protein, and an orthologous dCas9 protein. 
     
     
         77 . The method of  claim 76 , wherein the dCas9 protein is the  Streptococcus pyogenes  dCas9 protein. 
     
     
         78 . The method of  claim 72 , wherein the dCas9 protein is selected from the group consisting of a  Streptococcus pyogenes  dCas9 protein, a  Streptococcus thermophilus  dCas9 protein, a  Staphylococcus aureus  dCas9 protein, a  Neisseria meningitidis  dCas9 protein, and an orthologous dCas9 protein. 
     
     
         79 . The method of  claim 78 , wherein the dCas9 protein is the  Streptococcus pyogenes  dCas9 protein. 
     
     
         80 . The method of  claim 69 , wherein the first target nucleic acid, the second target nucleic acid, and the third target nucleic acid comprise a double-stranded DNA. 
     
     
         81 . The method of  claim 69 , wherein the method is performed in vitro. 
     
     
         82 . The method of  claim 69 , wherein the donor polynucleotide is a single-stranded DNA. 
     
     
         83 . The method of  claim 69 , wherein the donor polynucleotide is a double-stranded DNA. 
     
     
         84 . A method for positioning a donor polynucleotide near a cleavage site in genomic DNA of a cell, comprising:
 introducing into the cell
 a first complex comprising a catalytically active Cas9 protein and a first guide polynucleotide that comprises a spacer that binds to a first target nucleic acid in the genomic DNA, the first target nucleic acid comprising the cleavage site, and 
 a second complex comprising a catalytically inactive Cas9 protein (dCas9 protein) and a second guide polynucleotide that comprises a spacer that binds to a second target nucleic acid in proximity to the cleavage site, and the second complex is associated with a first end of a donor polynucleotide; 
   wherein the first complex contacting the first target nucleic acid facilitates binding and cleaving the first target nucleic acid at the cleavage site; and   wherein the second complex contacting the second target nucleic acid facilitates binding to the second target nucleic acid, binding of the second complex positions the donor polynucleotide near the cleavage site, and at least a portion of the donor polynucleotide is inserted into the first target nucleic acid.   
     
     
         85 . The method of  claim 84 , wherein the genomic DNA is double-stranded DNA. 
     
     
         86 . The method of  claim 84 , wherein the first complex and the second complex are introduced into the cell by a method selected from the group consisting of transfection, transduction, electroporation, liposome delivery, lipid nanoparticles, and viral delivery. 
     
     
         87 . The method of  claim 84 , wherein the cell is a eukaryotic cell. 
     
     
         88 . The method of  claim 84 , wherein the cell is selected from the group consisting of a bacterial cell, a yeast cell, a mammalian cell, and a plant cell.