An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)

Yu Murakami, Satoshi Ansai, Akari Yonemura, Masato Kinoshita

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

Abstract

Background The CRISPR/Cas system is a powerful genome editing tool that enables targeted genome modifications in various organisms. In medaka (Oryzias latipes), targeted mutagenesis with small insertions and deletions using this system have become a robust technique and are now widely used. However, to date there have been only a small number of reports on targeted gene integration using this system. We thus sought in the present study to identify factors that enhance the efficiency of targeted gene integration events in medaka. Results We show that longer homology arms (ca. 500 bp) and linearization of circular donor plasmids by cleavage with bait sequences enhances the efficiency of targeted integration of plasmids in embryos. A new bait sequence, BaitD, which we designed and selected by in silico screening, achieved the highest efficiency of the targeted gene integration in vivo. Using this system, donor plasmids integrated precisely at target sites and were efficiently transmitted to progeny. We also report that the genotype of F 2 siblings, obtained by mating of individuals harboring two different colors of fluorescent protein genes (e.g. GFP and RFP) in the same locus, can be easily and rapidly determined non-invasively by visual observations alone. Conclusion We report that the efficiency of targeted gene integration can be enhanced by using donor vectors with longer homologous arms and linearization using a highly active bait system in medaka. These findings may contribute to the establishment of more efficient systems for targeted gene integration in medaka and other fish species.

Original languageEnglish
Article number10
JournalZoological Letters
Volume3
Issue number1
DOIs
Publication statusPublished - 2017
Externally publishedYes

Keywords

  • Bait sequence
  • CRISPR/Cas9
  • Homology-directed repair
  • Medaka
  • Targeted gene integration

ASJC Scopus subject areas

  • Animal Science and Zoology

Fingerprint

Dive into the research topics of 'An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)'. Together they form a unique fingerprint.

Cite this