title: Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.
abstract: The type II bacterial CRISPR/Cas9 system is rapidly becoming popular for genome-engineering due to its simplicity, flexibility, and high efficiency. Recently, targeted knock-in of a long DNA fragment via homology-independent DNA repair has been achieved in zebrafish using CRISPR/Cas9 system. This raised the possibility that knock-in transgenic zebrafish could be efficiently generated using CRISPR/Cas9. However, how widely this method can be applied for the targeting integration of foreign genes into endogenous genomic loci is unclear. Here, we report efficient generation of knock-in transgenic zebrafish that have cell-type specific Gal4 or reporter gene expression. A donor plasmid containing a heat-shock promoter was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, a sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. We have succeeded in establishing stable knock-in transgenic fish with several different constructs for 4 genetic loci at a frequency being exceeding 25%. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic zebrafish.
authors.affiliation: National Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Kimura
authors.affiliation: Laboratory for Cardiovascular Molecular Dynamics, RIKEN Quantitative Biology Center, Furuedai 6-2-3, Suita, Osaka, 565-0874, Japan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Hisano
authors.affiliation: 1] Laboratory for Cardiovascular Molecular Dynamics, RIKEN Quantitative Biology Center, Furuedai 6-2-3, Suita, Osaka, 565-0874, Japan [2] Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, Simigatou 1110, Chuo, Yamanashi, 409-3862, Japan.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kawahara
authors.affiliation: National Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan.
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Higashijima
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems; D055879:Gene Knock-In Techniques; D018390:Gene Targeting; D005818:Genetic Engineering; D016678:Genome; D030781:Organisms, Genetically Modified / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D015027:Zebrafish / Q000235:genetics*
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems