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 (e.g., 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 (e.g., 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.

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 20 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. 
   wherein the targeted genomic modification comprises insertion of:
 a. One or more human antibody heavy chain variable domains; 
 b. One or more human antibody kappa light chain variable domains; or 
 c. One or more human antibody lambda light chain variable domains; 
   
     
     
         2 . The method of  claim 1 , wherein the targeted genomic modification comprises deletion of one or more mouse antibody heavy chain variable domains and insertion of one or more human antibody heavy chain variable domains. 
     
     
         3 . The method of  claim 1 , wherein the targeted genomic modification comprises deletion of one or more mouse antibody kappa light chain variable domains and insertion of one or more human antibody kappa light chain variable domains. 
     
     
         4 . The method of  claim 1 , wherein the targeted genomic modification comprises deletion of one or more mouse antibody lambda light chain variable domains and insertion of one or more human antibody lambda light chain variable domains. 
     
     
         5 . The method of  claim 1 , wherein the mouse ES cell or progeny thereof is developed into a mouse. 
     
     
         6 . A mouse obtained by the method of  claim 1 . 
     
     
         7 . The mouse of  claim 6 , wherein the mouse is heterozygous for the targeted genomic modification. 
     
     
         8 . The mouse of  claim 6 , wherein the mouse is homozygous for the targeted genomic modification. 
     
     
         9 . The mouse of  claim 6 , wherein the mouse further comprises a targeted modification to insert one or more human antibody kappa light chain variable domains. 
     
     
         10 . The mouse of  claim 6 , wherein the mouse further comprises a homozygous targeted modification to insert one or more human antibody kappa light chain variable domains. 
     
     
         11 . An antibody produced by the mouse of  claim 6 . 
     
     
         12 . The antibody according to  claim 11 , wherein the mouse is heterozygous for the targeted genomic modification. 
     
     
         13 . The antibody according to  claim 11 , wherein the mouse is homozygous for the targeted genomic modification 
     
     
         14 . The antibody according to  claim 11 , wherein the mouse further comprises a targeted modification to insert one or more human antibody kappa light chain variable domains. 
     
     
         15 . The antibody according to  claim 11 , wherein the mouse further comprises a homozygous targeted modification to insert one or more human antibody kappa light chain variable domains. 
     
     
         16 . A method for producing an antibody comprising:
 producing a mouse with a modified genome from the ES cell modified by the method of  claim 1  or a progeny thereof,   immunizing the mouse with an antigen, and   isolating an antibody produced by the mouse.   
     
     
         17 . The method of  claim 16 , wherein the targeted genomic modification comprises deletion of one or more mouse antibody heavy chain variable domains and insertion of one or more human antibody heavy chain variable domains 
     
     
         18 . The method of  claim 16 , wherein the targeted genomic modification comprises deletion of one or more mouse antibody kappa light chain variable domains and insertion of one or more human antibody kappa light chain variable domains. 
     
     
         19 . The method of  claim 16 , wherein the targeted genomic modification comprises deletion of one or more mouse antibody lambda light chain variable domains and insertion of one or more human antibody lambda light chain variable domains.