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ZFIN ID: ZDB-PUB-141009-8
Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering
Kimura, Y., Hisano, Y., Kawahara, A., Higashijima, S.
Date: 2014
Source: Scientific Reports   4: 6545 (Journal)
Registered Authors: Higashijima, Shin-ichi, Kawahara, Atsuo
Keywords: none
MeSH Terms:
  • Animals
  • Animals, Genetically Modified
  • CRISPR-Cas Systems*
  • Gene Knock-In Techniques*
  • Gene Targeting
  • Genetic Engineering*
  • Genome
  • Organisms, Genetically Modified/genetics*
  • RNA, Guide/genetics
  • Zebrafish/genetics*
PubMed: 25293390 Full text @ Sci. Rep.
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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.
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