title: Efficient nonmeiotic allele introgression in livestock using custom endonucleases.
abstract: We have expanded the livestock gene editing toolbox to include transcription activator-like (TAL) effector nuclease (TALEN)- and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-stimulated homology-directed repair (HDR) using plasmid, rAAV, and oligonucleotide templates. Toward the genetic dehorning of dairy cattle, we introgressed a bovine POLLED allele into horned bull fibroblasts. Single nucleotide alterations or small indels were introduced into 14 additional genes in pig, goat, and cattle fibroblasts using TALEN mRNA and oligonucleotide transfection with efficiencies of 10-50% in populations. Several of the chosen edits mimic naturally occurring performance-enhancing or disease- resistance alleles, including alteration of single base pairs. Up to 70% of the fibroblast colonies propagated without selection harbored the intended edits, of which more than one-half were homozygous. Edited fibroblasts were used to generate pigs with knockout alleles in the DAZL and APC genes to model infertility and colon cancer. Our methods enable unprecedented meiosis-free intraspecific and interspecific introgression of select alleles in livestock for agricultural and biomedical applications.
authors.affiliation: Department of Animal Science, University of Minnesota, Saint Paul, MN 55108.
authors.forename: Wenfang
authors.initials: W
authors.lastname: Tan
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Carlson
authors.forename: Cheryl A
authors.initials: CA
authors.lastname: Lancto
authors.forename: John R
authors.initials: JR
authors.lastname: Garbe
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Webster
authors.forename: Perry B
authors.initials: PB
authors.lastname: Hackett
authors.forename: Scott C
authors.initials: SC
authors.lastname: Fahrenkrug
mesh_terms: D000818:Animals; D001947:Breeding / Q000379:methods*; D004252:DNA Mutational Analysis; D003851:Deoxyribonucleases / Q000378:metabolism*; D018014:Gene Transfer Techniques; D014644:Genetic Variation; D005828:Genetics, Population; D055029:Inverted Repeat Sequences / Q000235:genetics; D058751:Livestock / Q000235:genetics*; D016296:Mutagenesis; D059645:Mutation Rate; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics
chemical_list: D009841:Oligonucleotides; D003851:Deoxyribonucleases