Efficient targeted integration directed by short homology in zebrafish and mammalian cells.

Autor:	Wierson WA; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Welker JM; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Almeida MP; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Mann CM; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Webster DA; Recombinetics, Inc, St. Paul, United States., Torrie ME; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Weiss TJ; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Kambakam S; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Vollbrecht MK; Recombinetics, Inc, St. Paul, United States., Lan M; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., McKeighan KC; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Levey J; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Ming Z; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Wehmeier A; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Mikelson CS; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Haltom JA; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Kwan KM; Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, United States., Chien CB; Department of Neurobiology and Anatomy, University of Utah Medical Center, Salt Lake City, United States., Balciunas D; Department of Biology, Temple University, Philadelphia, United States., Ekker SC; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, United States., Clark KJ; Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, United States., Webber BR; Department of Pediatrics, Masonic Cancer Center, University of Minnesota, Minneapolis, United States., Moriarity BS; Department of Pediatrics, Masonic Cancer Center, University of Minnesota, Minneapolis, United States., Solin SL; Recombinetics, Inc, St. Paul, United States., Carlson DF; Recombinetics, Inc, St. Paul, United States., Dobbs DL; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., McGrail M; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States., Essner J; Department of Genetics, Development and Cell Biology, Iowa State University, Ames, United States.
Jazyk:	angličtina
Zdroj:	ELife [Elife] 2020 May 15; Vol. 9. Date of Electronic Publication: 2020 May 15.
DOI:	10.7554/eLife.53968
Abstrakt:	Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Here, we describe a set of resources to streamline reporter gene knock-ins in zebrafish and demonstrate the broader utility of the method in mammalian cells. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at high frequency at a double strand break in the targeted gene. Our vector series, pGTag (plasmids for Gene Tagging), contains reporters flanked by a universal CRISPR sgRNA sequence which enables in vivo liberation of the homology arms. We observed high rates of germline transmission (22-100%) for targeted knock-ins at eight zebrafish loci and efficient integration at safe harbor loci in porcine and human cells. Our system provides a straightforward and cost-effective approach for high efficiency gene targeting applications in CRISPR and TALEN compatible systems. Competing Interests: WW Interests in Lifengine and Lifengine Animal Health, JW, MA, CM, MT, TW, SK, MV, ML, KM, JL, ZM, AW, CM, JH, KK, CC, DB, BW, BM, DD, MM No competing interests declared, DW, SS, DC Shares in Recombinetics, Inc, SE Shares in Lifengine, and Lifengine Animal Health, KC Shares in Recombinetics, Inc, Lifengine and Lifengine Animal Health, JE JJE has a financial conflict of interest with Recombinetics, Inc; Immusoft, Inc; LifEngine and LifEngine Animal Technologies; (© 2020, Wierson et al.)
Databáze:	MEDLINE
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