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Efficient nonmeiotic allele introgression in livestock using custom endonucleases.

Wenfang Tan1, Daniel F Carlson, Cheryl A Lancto

  • 1Department of Animal Science, University of Minnesota, Saint Paul, MN 55108.

Proceedings of the National Academy of Sciences of the United States of America
|September 10, 2013
PubMed
Summary

Researchers expanded livestock gene editing using transcription activator-like effector nuclease (TALEN) and CRISPR/Cas9. These methods enable efficient gene editing for agricultural and biomedical applications in livestock.

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Area of Science:

  • Agricultural Science
  • Molecular Biology
  • Genetics

Background:

  • Gene editing technologies are crucial for livestock improvement.
  • Existing methods have limitations in efficiency and application scope.

Purpose of the Study:

  • To expand the livestock gene editing toolbox with TALEN and CRISPR/Cas9-stimulated homology-directed repair (HDR).
  • To demonstrate the utility of these methods for agricultural and biomedical applications.

Main Methods:

  • Utilized TALEN and CRISPR/Cas9-stimulated HDR with plasmid, rAAV, and oligonucleotide templates.
  • Introduced single nucleotide alterations and small insertions/deletions (indels) into 14 genes across cattle, pig, and goat fibroblasts.
  • Generated gene knockout pigs for DAZL and APC genes.

Main Results:

  • Achieved gene editing efficiencies of 10-50% in fibroblast populations.
  • Successfully introgressed a bovine POLLED allele for genetic dehorning in cattle.
  • Demonstrated high editing success rates (up to 70% of colonies edited, >50% homozygous) without selection.
  • Created pigs modeling infertility and colon cancer.

Conclusions:

  • Developed novel gene editing tools for livestock, including TALEN and CRISPR/Cas9-stimulated HDR.
  • These methods facilitate efficient, meiosis-free allele introgression in livestock.
  • The technology holds significant potential for advancing livestock breeding and disease modeling.