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Sci Rep. 2014 Oct 8;4:6545. doi: 10.1038/srep06545.

Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering.

Author information

1
National Institutes of Natural Sciences, Okazaki Institute for Integrative Bioscience, National Institute for Physiological Sciences, Okazaki, Aichi 444-8787, Japan.
2
Laboratory for Cardiovascular Molecular Dynamics, RIKEN Quantitative Biology Center, Furuedai 6-2-3, Suita, Osaka, 565-0874, Japan.
3
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.

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.

PMID:
25293390
PMCID:
PMC4189020
DOI:
10.1038/srep06545
[Indexed for MEDLINE]
Free PMC Article

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