Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish

被引:176
作者
Hisano, Yu [1 ]
Sakuma, Tetsushi [2 ]
Nakade, Shota [2 ]
Ohga, Rie [3 ]
Ota, Satoshi [3 ]
Okamoto, Hitoshi [1 ]
Yamamoto, Takashi [2 ]
Kawahara, Atsuo [3 ]
机构
[1] RIKEN, Brain Sci Inst, Lab Dev Gene Regulat, Wako, Saitama 3510198, Japan
[2] Hiroshima Univ, Grad Sch Sci, Dept Math & Life Sci, Mol Genet Lab, Higashihiroshima, Hiroshima 7398526, Japan
[3] Univ Yamanashi, Grad Sch Med Sci, Ctr Med Educ & Sci, Lab Dev Biol,Chuo Ku, Yamanashi 4093898, Japan
基金
日本学术振兴会;
关键词
GENOME MODIFICATIONS; CAS9; NUCLEASE; KNOCK-IN; RECOMBINATION; GENERATION; TALENS; GENES; CELLS;
D O I
10.1038/srep08841
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页数:7
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