Methods for improving crispr/cas-mediated genome-editing

Abstract

This application provides improved methods of editing the genome of a target cell. Cas9 molecules can be used to create a break in a genomic region of interest. To increase the likelihood that the break is repaired by homology-directed repair (HDR), the cell can be contacted with an HDR-enhancer. The cell may be, e.g., a human cell, a non-human animal cell, a bacterial cell, or a plant cell.

1 . A Cas9 system comprising:
 an HDR-enhancer molecule, wherein the enhancer molecule is not an inhibitor of DNA-PK or an inhibitor of Ligase IV,   a Cas9 molecule, and   a gRNA molecule, wherein the gRNA molecule is capable of targeting the Cas9 molecule to a target nucleic acid.   
     
     
         2 . The Cas9 system of  claim 1 , wherein the HDR-enhancer molecule is an HDR-enhancing gRNA, an siRNA, a peptide, an antibody, an miRNA, an antiMiR, or a small molecule. 
     
     
         3 . The Cas9 system of  claim 1 , further comprising a second gRNA molecule, wherein the second gRNA molecule is capable of targeting the Cas9 molecule to the target nucleic acid. 
     
     
         4 . A Cas9 system comprising:
 a Cas9 nickase molecule,   a gRNA molecule, wherein the gRNA molecule is capable of targeting the Cas9 nickase molecule to a target nucleic acid, and   a second gRNA molecule, wherein the second gRNA molecule is capable of targeting the Cas9 nickase molecule to the target nucleic acid,   wherein the gRNA molecule and the second gRNA molecule are designed to be oriented on the target nucleic acid such that protospacer adjacent motifs (PAMs) are facing out,   wherein the gRNA molecule will position the Cas9 nickase molecule to make a single-strand break in the target nucleic acid which results a 5′ overhang in the target nucleic acid.   
     
     
         5 . The Cas9 system of  claim 1 , further comprising a template nucleic acid. 
     
     
         6 .- 9 . (canceled) 
     
     
         10 . The Cas9 system of  claim 1 , wherein the HDR-enhancer is a down-regulator of HR, a down-regulator of canonical NHEJ, a down-regulator of alt-NHEJ, a down-regulator of an antirecombinant factor, a down-regulator of SSA, a down-regulator of SSBR, a down-regulator of MMR, a chromatin modification agent, a cell cycle arrest compound, an agent capable of promoting resection at a double strand break, a down-regulator of SD-MMEJ, an up-regulator of HDR, or a down-regulator of blunt EJ. 
     
     
         11 .- 62 . (canceled) 
     
     
         63 . The Cas9 system of  claim 1 , which further comprises at least one additional HDR-enhancer molecule. 
     
     
         64 .- 66 . (canceled) 
     
     
         67 . The Cas9 system of  claim 1 , wherein the Cas9 molecule is fused to a transcription activator or a transcription repressor. 
     
     
         68 . The Cas9 system of  claim 1 , wherein the Cas9 molecule is an enzymatically inactive Cas9 molecule (eiCas9) or an enzymatically active Cas9 (eaCas9) molecule. 
     
     
         69 .- 74 . (canceled) 
     
     
         75 . The Cas9 system of  claim 1 , further comprising a second Cas9 molecule. 
     
     
         76 .- 93 . (canceled) 
     
     
         94 . The Cas9 system of  claim 1  further comprising a cell cycle arrest compound. 
     
     
         95 .- 96 . (canceled) 
     
     
         97 . A Cas9 system comprising:
 a Cas9 nickase molecule,   a gRNA molecule, wherein the gRNA molecule is capable of targeting the Cas9 nickase molecule to a target nucleic acid, and   a second gRNA molecule, wherein the second gRNA molecule is capable of targeting the Cas9 nickase molecule to the target nucleic acid,   wherein the gRNA molecule and the second gRNA molecule are designed to be oriented on the target nucleic acid such that protospacer adjacent motifs (PAMs) are facing out,   wherein the gRNA molecule will position the Cas9 nickase molecule to make a single-strand break in the target nucleic acid which results a 3′ overhang in the target nucleic acid.   
     
     
         98 . A cell comprising the Cas9 system of  claim 1 . 
     
     
         99 . A composition comprising the Cas9 system of  claim 1 . 
     
     
         100 . A pharmaceutical composition comprising the composition of  claim 99  and a pharmaceutically acceptable carrier. 
     
     
         101 . A kit comprising the Cas9 system of  claim 1 . 
     
     
         102 . (canceled) 
     
     
         103 . A vector comprising the Cas9 system of  claim 1 . 
     
     
         104 .- 105 . (canceled) 
     
     
         106 . A reaction mixture comprising the cell of  claim 98 , and a solution. 
     
     
         107 . (canceled) 
     
     
         108 . A method of altering the structure of a cell comprising contacting the cell with:
 a Cas9 system of  claim 1 ,   under conditions that allow for alteration of the structure of the cell, thereby altering the structure of the cell.   
     
     
         109 . (canceled) 
     
     
         110 . A method of treating a subject by altering the structure of a cell in the subject, comprising contacting the cell with:
 a Cas9 system of  claim 1     under conditions that allow for alteration of the structure of the cell, thereby treating the subject by altering the structure of the cell in the subject.   
     
     
         111 . A method of promoting DNA repair of a break in a nucleic acid in a cell via an HDR pathway, the method comprising contacting the cell with:
 a Cas9 system of  claim 1  under conditions that allow for repair of the break in the nucleic acid in the cell via an HDR pathway.   
     
     
         112 . A method of promoting DNA repair of a double strand break or two single strand breaks in a target nucleic acid in a cell by an HDR pathway, the method comprising contacting the cell with a gRNA molecule, a Cas9 molecule, and a second gRNA molecule,
 wherein the gRNA molecule and the second gRNA molecule are oriented on the target nucleic acid such that protospacer adjacent motifs (PAMs) are facing out,   wherein the Cas9 nickase molecule cuts the target nucleic acid, resulting in a first 5′ overhang and a second 5′ overhang,   thereby promoting DNA repair of the double strand break or the two single strand breaks in the target nucleic acid in the cell via an HDR pathway.   
     
     
         113 .- 128 . (canceled) 
     
     
         129 . The method of  claim 110 , wherein the subject has a disorder that is caused by a mutation in the target nucleic acid. 
     
     
         130 .- 136 . (canceled) 
     
     
         137 . A method of producing the Cas9 system of  claim 1 , the method comprising providing and admixing the gRNA molecule, the Cas9 molecule, and the HDR-enhancer molecule. 
     
     
         138 . (canceled)