Efficient non-meiotic allele introgression in livestock

A method for making a non-human animal, the method comprising:

• (a) introducing into a cell isolated from a non-human animal line
• (i) a CRISPR/Cas9 endonuclease;
• (ii) a guide RNA (gRNA) comprising a spacer RNA sequence that interacts with a target sequence in the chromosomal DNA of the cell;
• (iii) a homology-directed repair (HDR) template sequence encoding an allele or a gene flanked by sequences homologous to the target sequence;
• (b) introducing the cell that results from (a) into an enucleated oocyte of the same species as the non-human animal line to produce an embryo;
• and
• (c) implanting the embryo in a surrogate mother to produce the non-human animal,
• wherein said introducing step (a) results in cleavage of the chromosomal DNA at the target sequence in the cell and insertion of the HDR template sequence into the cleavage site to cause the chromosomal DNA of the cell to have identity with the HDR template sequence at the target sequence in the chromosomal DNA, thereby introgressing the allele or the gene into the chromosomal DNA of the cell, wherein the HDR template sequence also comprises a DNA sequence encoding a mismatch in the target sequence that alters the interaction with the RNA spacer sequence of the gRNA, wherein the mismatch is introduced into the chromosomal DNA of the cell and creates a sequence in the chromosomal DNA of the animal that is not found in the non-human animal line, wherein the non-human animal has a phenotypic difference relative to the non-human animal line,
• wherein the non-human animal line is selected from the group consisting of a swine line, a cattle line, a sheep line, a non-human primate line, a mouse line, and a rat line.