Targeted modification of rat genome

Abstract

Compositions and methods are provided for modifying a rat genomic locus of interest using a large targeting vector (LTVEC) comprising various endogenous or exogenous nucleic acid sequences as described herein. Compositions and methods for generating a genetically modified rat comprising one or more targeted genetic modifications in their germline are also provided. Compositions and methods are provided which comprise a genetically modified rat or rat cell comprising a targeted genetic modification in the rat interleukin-2 receptor gamma locus, the rat ApoE locus, the rat Rag2 locus, the rat Rag1 locus and/or the rat Rag2/Rag1 locus. The various methods and compositions provided herein allows for these modified loci to be transmitted through the germline.

We claim: 
     
         1 . A method for modifying a genomic locus of interest in a non-human pluripotent cell, comprising
 (a) introducing into the non-human pluripotent cell:
 (i) a large targeting vector (LTVEC) comprising a first nucleic acid flanked with a 5′ homology arm and a 3′ homology arm, wherein the sum total of the 5′ and 3′ homology arms of the LTVEC is at least 10 kb; 
 (ii) a first expression construct comprising a first promoter operably linked to a second nucleic acid encoding a Cas protein, 
 (iii) a second expression construct comprising a second promoter operably linked to a third nucleic acid encoding a guide RNA (gRNA) comprising a nucleotide sequence that hybridizes to a target sequence and a trans-activating CRISPR RNA (tracrRNA), wherein the first and the second promoters are active in the non-human pluripotent cell; and 
 (b) identifying a modified non-human pluripotent cell comprising a targeted genetic modification at the genomic locus of interest. 
   
     
     
         2 . The method of  claim 1 , wherein the LTVEC is from about 20 kb to about 30 kb, from about 30 kb to about 40 kb, from about 40 kb to about 50 kb, from about 50 kb to about 75 kb, or from about 75 kb to about 100 kb. 
     
     
         3 . The method of  claim 1 , wherein the LTVEC is from about 100 kb to about 125 kb, from about 125 kb to about 150 kb, from about 150 kb to about 175 kb, or from about 175 kb to about 200 kb. 
     
     
         4 . The method of  claim 1 , wherein the targeted genetic modification is a biallelic genetic modification. 
     
     
         5 . The method of  claim 1 , wherein the non-human pluripotent cell is a rodent pluripotent cell. 
     
     
         6 . The method of  claim 5 , wherein the rodent pluripotent cell is derived from a mouse, a rat, or a hamster. 
     
     
         7 . The method of  claim 5 , wherein the rodent pluripotent cell is a mouse or a rat embryonic stem (ES) cell. 
     
     
         8 . The method of  claim 5 , wherein the rodent pluripotent cell is a rat ES cell derived from a DA strain or an ACI strain. 
     
     
         9 . The method of  claim 1 , wherein the Cas protein is Cas9. 
     
     
         10 . The method of  claim 1 , wherein the target sequence is immediately flanked on the 3′ end by a Protospacer Adjacent Motif (PAM) sequence. 
     
     
         11 . The method of  claim 1 , wherein the sum total of the 5′ and the 3′ homology arms is from about 10 kb to about 150 kb. 
     
     
         12 . The method of  claim 1 , wherein the sum total of the 5′ and the 3′ homology arms of the LTVEC is from about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100 kb to about 120 kb, or from about 120 kb to 150 kb. 
     
     
         13 . The method of  claim 1 , wherein the targeted genetic modification comprises:
 (a) a replacement of an endogenous nucleic acid sequence with a homologous or an orthologous nucleic acid sequence;   (b) a deletion of an endogenous nucleic acid sequence;   (c) a deletion of an endogenous nucleic acid sequence,   wherein the deletion ranges from about 5 kb to about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100 kb to about 150 kb, or from about 150 kb to about 200 kb, from about 200 kb to about 300 kb, from about 300 kb to about 400 kb, from about 400 kb to about 500 kb, from about 500 kb to about 1 Mb, from about 1 Mb to about 1.5 Mb, from about 1.5 Mb to about 2 Mb, from about 2 Mb to about 2.5 Mb, or from about 2.5 Mb to about 3 Mb;   (d) insertion of an exogenous nucleic acid sequence;   (e) insertion of an exogenous nucleic acid sequence ranging from about 5 kb to about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about 40 kb, from about 40 kb to about 60 kb, from about 60 kb to about 80 kb, from about 80 kb to about 100 kb, from about 100 kb to about 150 kb, from about 150 kb to about 200 kb, from about 200 kb to about 250 kb, from about 250 kb to about 300 kb, from about 300 kb to about 350 kb, or from about 350 kb to about 400 kb;   (f) insertion of an exogenous nucleic acid sequence comprising a homologous or an orthologous nucleic acid sequence;   (g) insertion of a chimeric nucleic acid sequence comprising a human and a non-human nucleic acid sequence;   (h) insertion of a conditional allele flanked with site-specific recombinase target sequences;   (i) insertion of a selectable marker or a reporter gene operably linked to a third promoter active in the pluripotent cell; or   (j) a combination thereof.   
     
     
         14 . The method of  claim 1 , wherein the genomic locus of interest comprises (i) a 5′ target sequence that is homologous to the 5′ homology arm; and (ii) a 3′ target sequence that is homologous to the 3′ homology arm. 
     
     
         15 . The method of  claim 14 , wherein the 5′ target sequence and the 3′ target sequence is separated by at least 5 kb but less than 3 Mb. 
     
     
         16 . The method of  claim 14 , wherein the 5′ target sequence and the 3′ target sequence is separated by at least 5 kb but less than 10 kb, at least 10 kb but less than 20 kb, at least 20 kb but less than 40 kb, at least 40 kb but less than 60 kb, at least 60 kb but less than 80 kb, at least about 80 kb but less than 100 kb, at least 100 kb but less than 150 kb, or at least 150 kb but less than 200 kb, at least about 200 kb but less than about 300 kb, at least about 300 kb but less than about 400 kb, at least about 400 kb but less than about 500 kb, at least about 500 kb but less than about 1 Mb, at least about 1 Mb but less than about 1.5 Mb, at least about 1.5 Mb but less than about 2 Mb, at least about 2 Mb but less than about 2.5 Mb, or at least about 2.5 Mb but less than about 3 Mb. 
     
     
         17 . The method of  claim 1 , wherein the genomic locus of interest comprises the Interleukin-2 receptor gamma locus, the ApoE locus, the Rag1 locus, the Rag2 locus, or both of the Rag1 and the Rag2 loci. 
     
     
         18 . The method of  claim 1 , wherein the first and the second expression constructs are on a single nucleic acid molecule. 
     
     
         19 . A method for targeted genetic modification of a non-human animal, comprising:
 (a) modifying a genomic locus of interest in a non-human pluripotent cell according to the method of  claim 1 , thereby producing a genetically modified non-human pluripotent cell comprising a targeted genetic modification;   (b) introducing the modified non-human pluripotent cell of (a) into a non-human host embryo; and   (c) gestating the non-human host embryo comprising the modified pluripotent cell in a surrogate mother, wherein the surrogate mother produces F0 progeny comprising the targeted genetic modification,
 wherein the targeted genetic modification is capable of being transmitted through the germline. 
   
     
     
         20 . The method of  claim 19 , wherein the genomic locus of interest comprises the Interleukin-2 receptor gamma locus, the ApoE locus, the Rag1 locus, the Rag2 locus, or both of the Rag1 and the Rag2 loci.