Methods, Cells & Organisms

Abstract

The invention relates to an approach for introducing one or more desired insertions and/or deletions of known sizes into one or more predefined locations in a nucleic acid (eg, in a cell or organism genome). They developed techniques to do this either in a sequential fashion or by inserting a discrete DNA fragment of defined size into the genome precisely in a predefined location or carrying out a discrete deletion of a defined size at a precise location. The technique is based on the observation that DNA single-stranded breaks are preferentially repaired through the HDR pathway, and this reduces the chances of indels (eg, produced by NHEJ) in the present invention and thus is more efficient than prior art techniques. The invention also provides sequential insertion and/or deletions using single- or double-stranded DNA cutting.

We claim: 
     
         1 . An in vitro method for modifying a genome at a genomic locus of interest in a mouse ES cell, the method comprising:
 contacting the mouse ES cell with:
 a Cas9 protein; 
 a CRISPR RNA that hybridizes to a CRISPR target sequence at the genomic locus of interest; 
 a tracrRNA; and 
 an incoming nucleic acid sequence that is flanked by:
 (i) a 5′ homology arm that is homologous to a 5′ target sequence at the genomic locus of interest; and 
 (ii) a 3′ homolog arm that is homologous to a 3′ target sequence at the genomic locus of interest; 
 
   wherein the incoming nucleic acid sequence is at least 10 kb in size;   wherein following the contacting step, the genome of the mouse ES cell is modified to comprise a targeted genetic modification comprising:
 deletion of a region of the genomic locus of interest wherein the deletion is at least 20 kb; and/or 
 insertion of the insert nucleic acid at the genomic locus of interest wherein the insertion is at least 20 kb. 
   
     
     
         2 . The method of  claim 1 , wherein the incoming nucleic acid sequence is at least 20 kb in size. 
     
     
         3 . The method of  claim 1 , wherein the incoming nucleic acid sequence is at least 50 kb in size. 
     
     
         4 . The method of  claim 1 , wherein the incoming nucleic acid sequence is at least 100 kb in size. 
     
     
         5 . The method of  claim 1 , wherein the deletion and/or insertion is at least 50 kb in size. 
     
     
         6 . The method of  claim 1 , wherein the deletion and/or insertion is at least 100 kb in size. 
     
     
         7 . The method of  claim 1 , wherein the CRISPR RNA and the tracrRNA are introduced as a single nucleic acid molecule comprising the CRISPR RNA and the tracrRNA. 
     
     
         8 . The method of  claim 7 , wherein the single nucleic acid molecule comprises the CRISPR RNA and the tracrRNA fused together in the form of a single guide RNA (sgRNA). 
     
     
         9 . The method of  claim 1 , wherein the CRISPR RNA and the tracrRNA are introduced separately. 
     
     
         10 . The method of  claim 1 , wherein: (a) the Cas9 protein is introduced in the form of a protein, a messenger RNA (mRNA) encoding the Cas9 protein, or a DNA encoding the Cas9 protein; (b) the CRISPR RNA is introduced in the form of an RNA or a DNA encoding the CRISPR RNA; and (c) the tracrRNA is introduced in the form of an RNA or a DNA encoding the tracrRNA. 
     
     
         11 . The method of  claim 1 , wherein the targeted genetic modification comprises simultaneous deletion of an endogenous nucleic acid sequence at the genomic locus of interest and insertion of the insert nucleic acid at the genomic locus of interest. 
     
     
         12 . The method of  claim 1 , wherein the targeted genetic modification is a biallelic genetic modification. 
     
     
         13 . The method of  claim 1 , wherein the insert nucleic acid is at least 20 kb, at least 50 kb, or at least 100 kb; or wherein the targeted genetic modification comprises deletion of a region of the genomic locus of interest wherein the deletion is at least 20 kb, and the insert nucleic acid is at least 20 kb, at least 50 kb, or at least 100 kb. 
     
     
         14 . The method of  claim 1 , wherein the CRISPR target sequence is immediately flanked by a Protospacer Adjacent Motif (PAM) sequence. 
     
     
         15 . The method of  claim 1 , wherein the targeted genetic modification comprises: (a) replacement of an endogenous nucleic acid sequence with a homologous or an orthologous nucleic acid sequence; (b) deletion of an endogenous nucleic acid sequence; ((d) insertion of an exogenous nucleic acid sequence; (f) insertion of an exogenous nucleic acid sequence comprising a homologous or an orthologous nucleic acid sequence; (i) insertion of a selectable marker; or (j) a combination thereof. 
     
     
         16 . The method of  claim 1 , wherein the region of the genomic locus of interest deleted is at least 20 kb, and the inserted insert nucleic acid is at least 20 kb.