GeneWeld: Efficient targeted integration directed by short homology in Zebrafish

Jordan M. Welker, Wesley A. Wierson, Maira P. Almeida, Carla M. Mann, Melanie E. Torrie, Zhitao Ming, Stephen C. Ekker, Karl J Clark, Drena L. Dobbs, Jeffrey J. Essner, Maura McGrail

Research output: Contribution to journalArticlepeer-review

Abstract

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for germline recovery of integration alleles. The GeneWeld protocol and pGTag (plasmids for Gene Tagging) vector series provide a set of resources to streamline precision gene targeting in zebrafish. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at a CRISPR/Cas induced DNA double-strand break. The pGTag vectors contain reporters flanked by a universal CRISPR sgRNA sequence to liberate the targeting cassette in vivo and expose homology arms for homology-driven integration. Germline transmission rates for precision-targeted integration alleles range 22-100%. Our system provides a streamlined, straightforward, and cost-effective approach for high-efficiency gene targeting applications in zebrafish.

Original languageEnglish (US)
Article numbere4100
JournalBio-protocol
Volume11
Issue number14
DOIs
StatePublished - Jul 20 2021

Keywords

  • CRISPR/Cas9
  • Homology mediated-end joining
  • Knock-in
  • Targeted integration
  • Zebrafish

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)
  • Plant Science

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