Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish.
The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10-40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.
Laboratory for Developmental Gene Regulation, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Yu
Y
Hisano
Molecular Genetics Laboratory, Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
Tetsushi
T
Sakuma
Molecular Genetics Laboratory, Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
Shota
S
Nakade
Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan.
Rie
R
Ohga
Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan.
Satoshi
S
Ota
Laboratory for Developmental Gene Regulation, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Hitoshi
H
Okamoto
Molecular Genetics Laboratory, Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
Takashi
T
Yamamoto
Laboratory for Developmental Biology, Center for Medical Education and Sciences, Graduate School of Medical Science, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi, 409-3898, Japan.
Atsuo
A
Kawahara
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D055879:Gene Knock-In Techniques; D018390:Gene Targeting / Q000379:methods; D017930:Genes, Reporter; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome; D059765:Homologous Recombination; D008969:Molecular Sequence Data; D015027:Zebrafish / Q000235:genetics*