Livestock with genetically modified prolactin receptor

Abstract

Methods, uses, and animals for introgression of alleles between animals, including SNPs. One embodiment involves introducing a targeted targeting endonuclease system and a HDR template into a cell with a mismatch in the binding of the targeting endonuclease and the targeted site.

1 . A genetically modified livestock animal from a first breed comprising an exogenous allele of a gene from another species or a second breed, said exogenous allele replacing a native allele of the gene, wherein a genome of the first breed is free of meiotic recombination with a genome of the other species or the second breed and the exogenous allele is selected from the group consisting of prolactin receptor (PRLR), and pleiomorphic adenoma gene 1 (Plag1). 
     
     
         2 . The animal of  claim 1  wherein the animal of the first breed is free of genetically engineered changes other than the replacement of the native allele by the exogenous allele. 
     
     
         3 . The animal of  claim 1  wherein the livestock animal is an artiodactyl. 
     
     
         4 . The animal of  claim 1  being homozygous for the exogenous allele. 
     
     
         5 . The animal of  claim 1  wherein the exogenous allele differs from the native allele by a number of bases, with the number ranging from 1 to 20. 
     
     
         6 . The animal of  claim 1  wherein a difference between the exogenous allele and the native allele differs comprises a single nucleotide polymorphism (SNP). 
     
     
         7 . The animal of  claim 1  wherein the exogenous allele is prolactin receptor Trunc461. 
     
     
         8 . A livestock cell or embryo from a first breed comprising an exogenous allele of a gene from another species or a second breed, said exogenous allele replacing a native allele of the gene, wherein a genome of the first breed is free of meiotic recombination with a genome of the other species or the second breed and the exogenous allele is selected from the group consisting of prolactin receptor (PRLR), and pleiomorphic adenoma gene 1 (Plag1). 
     
     
         9 . The cell of  claim 8  being selected from the group consisting of a primary cell, a primary somatic cell, a zygote, a germ cell, a stem cell, an oocyte, a sperm, and an embryo. 
     
     
         10 . A method of making an improved livestock animal comprising introgression of an exogenous allele into a native genome to replace the native allele, with the allele being prolactin receptor (PRLR) or pleiomorphic adenoma gene 1 (Plag1). 
     
     
         11 . The method of  claim 10  wherein the native allele of a cell is replaced with the exogenous allele and the cell is used to make the livestock animal, wherein the cell is selected from the group consisting of a primary cell, a primary somatic cell, a zygote, a germ cell, a stem cell, an oocyte, a sperm, and an embryo. 
     
     
         12 . The method of  claim 10  comprising introducing a targeted endonuclease system and a HDR template into a cell, with the targeted nuclease system comprising a DNA-binding member for specifically binding an endogenous cognate sequence in the chromosomal DNA, wherein the targeted nuclease system and the HDR template operate to alter the chromosomal DNA to have identity to the HDR template sequence to knockout the target gene, wherein the target gene is selected from the group consisting of prolactin receptor (PRLR) and pleiomorphic adenoma gene 1 (Plag1). 
     
     
         13 . The method of  claim 12  wherein the targeted endonuclease system and/or HDR template comprising a feature to reduce specific binding of the targeting endonuclease system to DNA. 
     
     
         14 . The method of  claim 12  wherein the targeted endonuclease system comprises
 a plurality of TAL effector repeat sequences that are fused to a nuclease (TALEN) or 
 a Cas9 nuclease and a guide RNA, with the mismatch being in the gRNA sequence relative to the endogenous cognate sequence. 
 
     
     
         15 . The method of  claim 10  wherein, upon introgression of the exogenous allele, the animal of the first breed is free of genetically engineered changes other than the replacement of the native allele by the exogenous allele. 
     
     
         16 . The method of  claim 10  wherein the livestock animal is an artiodactyl. 
     
     
         17 . The method of  claim 10 , with the animal being homozygous for the exogenous allele. 
     
     
         18 . The method of  claim 10  wherein the exogenous allele differs from the native allele by a number of bases, with the number ranging from 1 to 20. 
     
     
         19 . The method of  claim 10  wherein a difference between the exogenous allele and the native allele differs comprises a single nucleotide polymorphism (SNP). 
     
     
         20 . The method of  claim 10  wherein the exogenous allele is prolactin receptor Trunc461. 
     
     
         21 . The method of  claim 10  wherein the targeted endonuclease system is introduced into the cell as an mRNA. 
     
     
         22 . A genetically modified livestock animal comprising a knockout of a beta-lactoglobulin gene. 
     
     
         23 . The animal of  claim 22  wherein the livestock animal is an artiodactyl. 
     
     
         24 . The animal of  claim 22  being homozygous for the knockout of the gene. 
     
     
         25 . The animal of  claim 22  wherein the knockout gene is knocked out by deleting, inserting, or changing a number of nucleic acids in a nucleic acid sequence of the target gene, with the number ranging from 1 to 4. 
     
     
         26 . The animal of  claim 25  wherein the change to the gene comprises a single nucleotide polymorphism (SNP). 
     
     
         27 . A livestock cell or embryo comprising a knockout of a beta-lactoglobulin gene. 
     
     
         28 . The cell of  claim 27  being selected from the group consisting of a primary cell, a primary somatic cell, a zygote, a germ cell, a stem cell, an oocyte, a sperm, and an embryo. 
     
     
         29 . A method of making an improved livestock animal comprising knocking out a beta-lactoglobulin gene. 
     
     
         30 . The method of  claim 29  comprising introducing a targeted endonuclease system and a HDR template into the cell, with the targeted nuclease system comprising a DNA-binding member for specifically binding an endogenous cognate sequence in the chromosomal DNA, wherein the targeted nuclease system and the HDR template operate to alter the chromosomal DNA to have identity to the HDR template sequence to knockout the beta-lactoglobulin gene. 
     
     
         31 . The method of  claim 30  wherein the targeted endonuclease system and/or HDR template comprising a feature to reduce specific binding of the targeting endonuclease system to DNA. 
     
     
         32 . The method of  claim 30  wherein the targeted endonuclease system comprises
 a plurality of TAL effector repeat sequences that are fused to a nuclease (TALEN) or 
 a Cas9 nuclease and a guide RNA, with the mismatch being in the gRNA sequence relative to the endogenous cognate sequence. 
 
     
     
         33 . The method of  claim 30  wherein the targeted endonuclease system is introduced into the cell as an mRNA. 
     
     
         34 . The method of  claim 29  comprising knocking out the beta-lactoglobulin gene with an insertion, a deletion, or a substitution of a nucleic acid base. 
     
     
         35 . The method of  claim 34  wherein the insertion, deletion, or substitution has a length from 1 to 20 residues. 
     
     
         36 . The method of  claim 29  comprising altering the beta-lactoglobulin gene at only a single nucleic acid base (SNP). 
     
     
         37 . The method of  claim 29  comprising altering the beta-lactoglobulin gene with a plurality of single nucleotide polymorphisms (SNP). 
     
     
         38 . The method of  claim 29  wherein the native allele of a cell is replaced with the exogenous allele and the cell is used to make the livestock animal, wherein the cell is selected from the group consisting of a primary cell, a primary somatic cell, a zygote, a germ cell, a stem cell, an oocyte, a sperm, and an embryo.