Efficient nonmeiotic allele introgression in livestock using custom endonucleases

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. Towar...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2013-10, Vol.110 (41), p.16526-16531
Main Authors: Tan, Wenfang, Carlson, Daniel F., Lancto, Cheryl A., Garbe, John R., Webster, Dennis A., Hackett, Perry B., Fahrenkrug, Scott C.
Format: Article
Language:English
Subjects:
Alleles
Animals
Biological Sciences
Breeding - methods
colorectal neoplasms
dairy cattle
dehorning
Deoxyribonucleases - metabolism
DNA
DNA Mutational Analysis
Fibroblasts
Gene Transfer Techniques
Genetic loci
Genetic mutation
Genetic Variation
Genetics, Population
Genomes
goats
homozygosity
introgression
Inverted Repeat Sequences - genetics
Livestock
Livestock - genetics
Messenger RNA
Mutagenesis
Mutation Rate
oligonucleotides
Oligonucleotides - genetics
Plasmids
Plasmids - genetics
Stem cells
swine
transfection
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1310478110