Hisano Yu, Sakuma Tetsushi, Nakade Shota, Ohga Rie, Ota Satoshi, Okamoto Hitoshi, Yamamoto Takashi, Kawahara Atsuo
Laboratory for Developmental Gene Regulation, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
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.
Sci Rep. 2015 Mar 5;5:8841. doi: 10.1038/srep08841.
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.
CRISPR/Cas9系统为包括斑马鱼在内的各种模式生物的基因组编辑提供了一个强大的工具。通过CRISPR/Cas9介导的基因组修饰，很容易实现靶向基因敲除斑马鱼（基因敲除）的构建。最近，有报道称通过不依赖同源性的DNA修复将外源DNA整合到斑马鱼基因组中，但这种整合在基因组DNA和整合DNA的连接处包含各种突变。因此，仍有待实现对靶向基因组位点的精确基因组修饰。在这里，我们描述了一种高效、精确的CRISPR/Cas9介导的整合方法，该方法使用一个供体载体，该载体在基因组靶位点两侧带有短同源序列（10 - 40 bp）。我们成功地将外源mCherry或eGFP基因高效地框内整合到靶向基因（酪氨酸酶和krtt1c19e）中。当在左同源臂、eGFP序列和右同源臂两侧引入Cas9切割位点时，我们发现外源DNA能够精确地框内整合，且没有骨架载体序列。此外，我们证实这种精确的基因组修饰是可遗传的。这种简单的方法能够在斑马鱼中实现精确的靶向基因敲入。
