PUBLICATION

Highly efficient generation of knock-in transgenic medaka by CRISPR/Cas9-mediated genome engineering

Authors
Watakabe, I., Hashimoto, H., Kimura, Y., Yokoi, S., Naruse, K., Higashijima, S.I.
ID
ZDB-PUB-180223-18
Date
2018
Source
Zoological letters   4: 3 (Journal)
Registered Authors
Hashimoto, Hisashi, Higashijima, Shin-ichi, Naruse, Kiyoshi
Keywords
CRISPR/Cas9, Knock-in, Medaka, Transgenic
MeSH Terms
none
PubMed
29445519 Full text @ Zoological Lett
Abstract
Medaka (Oryzias latipes) is a popular animal model used in vertebrate genetic analysis. Recently, an efficient (~ 30%) knock-in system via non-homologous end joining (NHEJ) was established in zebrafish using the CRISPR/Cas9 system. If the same technique were applicable in medaka, it would greatly expand the usefulness of this model organism. The question of the applicability of CRISPR/Cas9 in medaka, however, has yet to be addressed.
We report the highly efficient generation of knock-in transgenic medaka via non-homologous end joining (NHEJ). Donor plasmid containing a heat-shock promoter and a reporter gene was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, an sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. Broad transgene expression in the expression domain of a target gene was observed in approximately 25% of injected embryos. By raising these animals, we established stable knock-in transgenic fish with several different constructs for five genetic loci, obtaining transgenic founders at efficiencies of > 50% for all five loci. Further, we show that the method is useful for obtaining mutant alleles. In the experiments where transgene integrations were targeted between the transcription start site and the initiation methionine, the resultant transgenic fish became mutant alleles.
With its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in via NHEJ will become a standard method for the generation of transgenic and mutant medaka.
Genes / Markers
Figures
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Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping