期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 109, 期 43, 页码 17382-17387出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1211446109
关键词
Tal-effector nuclease; biotechnology; gene-editing
资金
- Small Business Technology Transfer program [1R41HL108440-01]
- Biotechnology and Biological Sciences Research Council Institute Strategic Programme [BBS/E/D/07731442]
- Genus plc
- BBSRC [BBS/E/D/05251443, BBS/E/D/05251444, BBS/E/D/20221658, BBS/E/D/05251442, BBS/E/D/05251445] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BBS/E/D/05251442, BBS/E/D/05251445, BBS/E/D/05251443, BBS/E/D/20221658, BBS/E/D/05251444] Funding Source: researchfish
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. Coselection 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.
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