Non-meiotic allele introgression

Abstract

Methods, uses, and compositions for manipulating genomic DNA. Some of the embodiments of the invention provide for making a founder animal that is completely free of all unplanned genetic modifications. Some embodiments are directed to removing genetic faults in established breeds without making other alterations to the genome. Other embodiments are directed to particular tools or processes such as TALENs or CRISPR with a preferred truncation. One embodiment involves introducing a targeted targeting endonuclease system and a HDR template into a cell (optionally with a mismatch in the binding of the targeting endonuclease and the targeted site). Another embodiment includes processes of making a genetically modified livestock animal comprising a genome that comprises inactivation of a neuroendocrine gene selective for sexual maturation, with the inactivation of the gene preventing the animal from becoming sexually mature. One embodiment includes compositions and methods for making livestock with a polled allele, including migrating a polled allele into a bovine species without changing other genes or chromosomal portions.

1 . A method for altering the genome of an animal cell, the method comprising:
 identifying a target DNA region within the animal cell, the target region comprising a target cleavage site;   contacting the animal cell with a targeted nuclease such that the nuclease cleaves the target DNA region at the target cleavage site, wherein the targeted nuclease comprises one or more binding domains that specifically bind to one or more sequences within the target DNA region.   
     
     
         2 . The method of  claim 1 , wherein the targeted nuclease is selected from the group consisting of a transcription-activator-like effector nuclease (TALEN), a CRISPR-based nuclease (e.g., CRISPR/Cas9), and a zinc finger nuclease. 
     
     
         3 .- 11 . (canceled) 
     
     
         12 . The method  claim 1 , wherein the method is performed without introducing into the animal cell (1) a fluorescent marker gene or (2) a reporter gene that, when incorporated into chromosomal DNA of the cell, confers a trait on the cell that permits isolation by one or more survival selection criteria (e.g., survival in the presence of a small molecule). 
     
     
         13 .- 18 . (canceled) 
     
     
         19 . The method of  claim 1 , wherein the animal cell is a primary somatic cell. 
     
     
         20 . The method of  claim 19 , further comprising:
 cloning the primary somatic cell to produce one or more embryos; and   implanting the one or more embryos into a surrogate mother.   
     
     
         21 . The method of  claim 20 , wherein cloning the primary somatic cell comprises somatic cell nuclear transfer or chromatin transfer. 
     
     
         22 . The method of  claim 21 , further comprising producing a gene-edited animal from the implanted embryo. 
     
     
         23 . The method of  claim 1 , wherein the animal cell is a totipotent or pluripotent cell. 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 23 , further comprising implanting the embryo into a surrogate mother. 
     
     
         26 . The method of  claim 25 , further comprising producing a gene-edited animal from the implanted embryo. 
     
     
         27 . The method of  claim 1 , wherein the targeted nuclease cleaves the target DNA region at or adjacent to a neuroendocrine gene involved in sexual maturation. 
     
     
         28 . The method of  claim 27 , wherein the neuroendocrine gene is selected from the group consisting of gpr54, kiss1, and gnrh11. 
     
     
         29 . The method of  claim 28 , wherein the neuroendocrine gene of the resulting animal cell is inactivated. 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 29 , wherein inactivation of the neuroendocrine gene involves insertion of a stop codon in a sequence of the neuroendocrine gene. 
     
     
         32 . The method of  claim 31 , further comprising administering a rescue agent to an animal that comprises or is derived from the animal cell such that the animal proceeds to sexual maturity. 
     
     
         33 .- 55 . (canceled) 
     
     
         56 . A method of modifying a bovine cell, the method comprising:
 contacting the bovine cell with a targeted endonuclease that targets and cuts a gene encoding the prolactin receptor;   contacting the bovine cell with a homology-dependent repair template such that the template integrates into the genome of the bovine cell to encode a truncated prolactin receptor protein.   
     
     
         57 . The method of  claim 56 , wherein the truncated prolactin receptor protein is 461 amino acids in length. 
     
     
         58 . The method of  claim 56 , wherein the targeted endonuclease is selected from a zinc finger nuclease, a TAL effector nuclease (TALEN) and a CRISPR/Cas 9 nuclease. 
     
     
         59 .- 91 . (canceled)