Efficient nonmeiotic allele introgression in livestock using custom endonucleases

Wenfang Tan, Daniel F. Carlson, Cheryl A. Lancto, John R. Garbe, Dennis A. Webster, Perry B. Hackett, Scott C. Fahrenkrug

Research output: Contribution to journalArticlepeer-review

201 Scopus citations

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 onehalf 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.

Original languageEnglish (US)
Pages (from-to)16526-16531
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number41
DOIs
StatePublished - Oct 8 2013

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