Organisms homozygous for targeted modification

Abstract

Disclosed herein are homozygously modified organisms and methods of making and using these organisms.

1 . An organism that is homozygous for a modification at a gene locus having at least first and second alleles, wherein
 (i) the first and second alleles of the gene locus comprise one or more deletions such that the gene locus is inactivated and further wherein the first and second alleles do not comprise exogenous sequences; or   (ii) the first and second alleles of the gene locus comprise one or more exogenous sequences, wherein the one or more exogenous sequences do not encode a reporter or selectable marker.   
     
     
         2 . The organism of  claim 1 , wherein the first and second alleles comprise one or more deletions such that the gene locus is inactivated. 
     
     
         3 . The organism of  claim 1 , wherein the first and second alleles comprise one or more exogenous sequences not encoding a reporter or a selectable marker. 
     
     
         4 . The organism of  claim 1 , wherein the organism is a plant or an animal. 
     
     
         5 . The organism of  claim 4 , wherein the plant is selected from the group consisting of maize, rice, wheat, potato, soybean, tomato, tobacco, members of the Brassica family, and  Arabidopsis.    
     
     
         6 . A seed produced by the plant of  claim 5 . 
     
     
         7 . The organism of  claim 4 , wherein the animal is a mammal. 
     
     
         8 . A method of generating a homozygous organism according to  claim 1 , wherein the homozygous organism lacks exogenous sequences at a selected target locus, the method comprising,
 (a) introducing an exogenous sequence into a cell using a nuclease that mediates targeted integration of the exogenous sequence into a selected locus of the genome of the organism, the locus comprising at least first and second alleles;   (b) identifying cells comprising (i) the exogenous sequence in the first allele of the target locus and (ii) a non-homologous end joining (NHEJ) modification in the second allele of the selected target locus;   (c) allowing the cells identified in step (b) to develop into reproductively mature organisms;   (d) crossing the reproductively mature organisms to each other; and   (e) identifying progeny that exhibit NHEJ modifications at the first and second alleles of the selected target locus, thereby generating a homozygous organism lacking exogenous sequences at the selected target locus.   
     
     
         9 . A method of generating an organism according to  claim 1 , wherein the organism is homozygous for one or more exogenous sequences at a selected target locus of the genome, wherein the exogenous sequences do not comprise a reporter or selectable marker at the selected target locus, the method comprising,
 (a) introducing a reporter or selectable marker sequence into a cell of the organism using a nuclease that mediates targeted integration of the reporter into the selected target locus of the genome, the selected target locus comprising at least first and second alleles;   (b) introducing the one or more exogenous sequence(s) into the cell, wherein the nuclease mediates targeted integration of the exogenous sequence into the selected target locus of the genome,   (c) identifying cells comprising (i) the reporter or selectable marker in the first allele of the selected target locus and (ii) the one or more exogenous sequences in the second allele;   (d) allowing the cells identified in step (c) to develop to reproductively mature organisms;   (e) crossing the reproductively mature organisms to each other and;   (f) identifying progeny of the cross of step (e) that comprise the one or more exogenous sequences in the first and second alleles, thereby generating an organism that is homozygous for the exogenous sequences and lacking reporter or selectable marker sequences at the selected target locus.   
     
     
         10 . The method of  claim 8 , wherein the nuclease is selected from the group consisting of one or more zinc finger nucleases (ZFNs); one or more meganucleases and one or more TAL-effector domain nucleases. 
     
     
         11 . The method of  claim 9 , wherein the nuclease is selected from the group consisting of one or more zinc finger nucleases (ZFNs); one or more meganucleases and one or more TAL-effector domain nucleases. 
     
     
         12 . The method of  claim 9 , wherein the reporter or selectable marker sequence and the one or more exogenous sequences are introduced concurrently with the one or more nucleases. 
     
     
         13 . The method of  claim 9 , wherein the reporter or selectable marker sequence and the one or more exogenous sequences are introduced sequentially with the one or more nucleases. 
     
     
         14 . The method of  claim 8 , wherein the reporter sequence comprises a selectable or screening marker. 
     
     
         15 . The method of  claim 8 , wherein the one or more nucleases are introduced as a polynucleotide. 
     
     
         16 . The method of  claim 9 , wherein the one or more nucleases are introduced as a polynucleotide. 
     
     
         17 . A kit for generating an organism according to  claim 1 , the kit comprising:
 (a) one or more nucleases that bind to a target site in the selected target locus;   (b) one or more exogenous sequence for targeted integration into the selected target locus; and   (c) instructions for:   (i) introducing the nucleases and exogenous sequence into cells;   (ii) identifying cells comprising one or more exogenous sequences are inserted into the first allele at the selected target locus;   (iii) identifying cells of (ii) comprising a modification at the second allele of the selected target locus;   (iv) growing the cells of (iii) into reproductively mature organisms;   (v) crossing the organisms of (iv); and   (vi) identifying progeny of the crosses of (v) that a homozygous for the targeted gene modification.   
     
     
         18 . The kit of  claim 17 , wherein the nucleases are supplied as polynucleotides encoding the nucleases. 
     
     
         19 . The kit of  claim 17 , further comprising an optional donor transgene comprising sequences homologous to the target site, wherein the donor transgene does not comprise a reporter gene.