Efficient TALEN-mediated gene knockout in livestock

Daniel F. Carlson, Wenfang Tan, Simon G. Lillico, Dana Stverakova, Chris Proudfoot, Michelle Christian, Daniel F. Voytas, Charles R. Long, C. Bruce A. Whitelaw, Scott C. Fahrenkrug

Research output: Contribution to journalArticlepeer-review

453 Scopus citations


Transcription activator-like effector nucleases (TALENs) are programmable nucleases that join FokI endonuclease with the modular DNA-binding domain of TALEs. Although zinc-finger nucleases enable a variety of genome modifications, their application to genetic engineering of livestock has been slowed by technical limitations of embryo-injection, culture of primary cells, and difficulty in producing reliable reagents with a limited budget. In contrast, we found that TALENs could easily be manufactured and that over half (23/36, 64%) demonstrate high activity in primary cells. Cytoplasmic injections of TALEN mRNAs into livestock zygotes were capable of inducing gene KO in up to 75% of embryos analyzed, a portion of which harbored biallelic modification. We also developed a simple transposon coselection strategy for TALEN-mediated gene modification in primary fibroblasts that enabled both enrichment for modified cells and efficient isolation of modified colonies. Coselection after treatment with a single TALEN-pair enabled isolation of colonies with mono- and biallelic modification in up to 54% and 17% of colonies, respectively. Co-selection after treatment with two TALEN-pairs directed against the same chromosome enabled the isolation of colonies harboring large chromosomal deletions and inversions (10% and 4% of colonies, respectively). TALEN-modified Ossabaw swine fetal fibroblasts were effective nuclear donors for cloning, resulting in the creation of miniature swine containing mono- and biallelic mutations of the LDL receptor gene as models of familial hypercholesterolemia. TALENs thus appear to represent a highly facile platform for the modification of livestock genomes for both biomedical and agricultural applications.

Original languageEnglish (US)
Pages (from-to)17382-17387
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number43
StatePublished - Oct 23 2012


  • Biotechnology
  • Gene-editing
  • Tal-effector nuclease


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