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Article
Efficient targeted integration directed by short homology in zebrafish and mammalian cells
eLife
  • Wesley A. Wierson, Iowa State University
  • Jordan M. Welker, Iowa State University
  • Maira P. Almeida, Iowa State University
  • Carla M. Mann, Iowa State University
  • Melanie E. Torrie, Iowa State University
  • Trevor J. Weiss, Iowa State University
  • Sekhar Kambakam, Iowa State University
  • Merrina Lan, Iowa State University
  • Kenna C. McKeighan, Iowa State University
  • Jacklyn Levey, Iowa State University
  • Zhitao Ming, Iowa State University
  • Alec Wehmeier, Iowa State University
  • Christopher S. Mikelson, Iowa State University
  • Jeffrey A. Haltom, Iowa State University
  • Drena L. Dobbs, Iowa State University
  • Maura McGrail, Iowa State University
  • Jeffrey Essner, Iowa State University
  • et al., et al.
Document Type
Article
Publication Version
Published Version
Publication Date
1-1-2020
DOI
10.7554/eLife.53968
Abstract

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.

Comments

This article is published as Wierson, Wesley A., Jordan M. Welker, Maira P. Almeida, Carla M. Mann, Dennis A. Webster, Melanie E. Torrie, Trevor J. Weiss et al. "Efficient targeted integration directed by short homology in zebrafish and mammalian cells." Elife 9 (2020): e53968. doi: 10.7554/eLife.53968.

Creative Commons License
Creative Commons Attribution 4.0
Copyright Owner
Wierson et al.
Language
en
File Format
application/pdf
Citation Information
Wesley A. Wierson, Jordan M. Welker, Maira P. Almeida, Carla M. Mann, et al.. "Efficient targeted integration directed by short homology in zebrafish and mammalian cells" eLife Vol. 9 (2020) p. e53968
Available at: http://works.bepress.com/maura-mcgrail/20/