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 targeted genomic modification comprises an insertion of a transgenic IgH locus comprising a human variable region comprising human VH, D and JH gene segments. 
     
     
         6 . The method of  claim 5 , wherein the transgenic IgH locus is targeted to the region between the J4 exon and the Cμ locus in the mouse genome IgH locus. 
     
     
         7 . The method of  claim 5 , wherein the transgenic IgH locus is targeted to the region between the J4 exon and the Cμ locus in the mouse genome IgH locus, and wherein the inserted transgenic human IgH locus comprises, in germline configuration, all of the V, D and J regions and intervening sequences from a human. 
     
     
         8 . The method of  claim 5 , wherein the transgenic IgH locus is targeted into mouse chromosome 12 between the end of the mouse J4 region and the Eμ region. 
     
     
         9 . The method of  claim 5 , wherein the 3′ end of the last human J sequence of the transgenic IgH locus is less than 2 kb from 3′ end of the inserted transgenic IgH locus. 
     
     
         10 . The method of  claim 5 , wherein the 3′ end of the last human J sequence of the transgenic IgH locus is less than 1 kb from 3′ end of the inserted transgenic IgH locus. 
     
     
         11 . The method of  claim 5 , wherein the inserted IgH locus is operatively connected upstream of (5′ of) a mouse constant region. 
     
     
         12 . The method of  claim 1 , wherein the targeted genomic modification comprises an insertion of a transgenic Igκ locus comprising a human variable region comprising human Vκ and Jκ gene segments. 
     
     
         13 . The method of  claim 12 , wherein the human light chain kappa V and J gene segments are targeted into mouse chromosome 6. 
     
     
         14 . The method of  claim 12 , wherein the human light chain kappa V and J gene segments are targeted into mouse chromosome 6, and wherein the inserted human kappa V and J gene segments comprise, in germline configuration, all of the V and J regions and intervening sequences from a human. 
     
     
         15 . The method of  claim 12 , wherein the inserted Igκ locus is operatively connected upstream of a mouse constant region. 
     
     
         16 . The method of  claim 1 , wherein the targeted genomic modification comprises an insertion of a transgenic Igλ locus comprising a human variable region comprising human Vλ and Jλ gene segments. 
     
     
         17 . The method of  claim 16 , wherein the human light chain lambda V and J gene segments are targeted into mouse chromosome 16. 
     
     
         18 . The method of  claim 16 , wherein the human light chain lambda V and J gene segments are targeted into mouse chromosome 16, and wherein the inserted human lambda V and J gene segments comprise, in germline configuration, all of the V and J regions and intervening sequences from a human. 
     
     
         19 . The method of  claim 16 , wherein the inserted Igλ locus is operatively connected upstream of a mouse constant region. 
     
     
         20 . The method of  claim 16 , wherein the transgenic Igλ locus further comprises at least one human Cλ region. 
     
     
         21 . The method of  claim 1  wherein the mouse ES cell is a wild-type 129, C57BL/6N, C57BL/6J, JMS, AB2.1, AB2.2, 129S5, 129S7 or 129Sv strain. 
     
     
         22 . The method of  claim 1 , wherein the mouse ES cell or its progeny is developed into a mouse. 
     
     
         23 . A mouse obtained by the method of  claim 1  or progeny thereof. 
     
     
         24 . A mouse obtained by the method of  claim 5  or progeny thereof. 
     
     
         25 . The mouse of  claim 24  which is heterozygous for the targeted genomic modification. 
     
     
         26 . The mouse of  claim 24  which is homozygous for the targeted genomic modification. 
     
     
         27 . The mouse of  claim 24 , which further comprises a targeted modification to insert one or more human antibody kappa light chain variable domains. 
     
     
         28 . The mouse of  claim 24 , which further comprises a homozygous targeted modification to insert one or more human antibody kappa light chain variable domains. 
     
     
         29 . An antibody produced by the mouse of  claim 23  or its clone or progeny. 
     
     
         30 . A method for producing an antibody comprising immunizing the mouse of  claim 23  or its clone or progeny with an antigen, and isolating an antibody produced by the mouse. 
     
     
         31 . The method of  claim 30  further comprising a step of making the mouse by modifying an ES cell according to  claim 1 .