Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish.
Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish.
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DOI：
10.1038/srep08841
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发表时间：
2015-03-05
影响因子：
4.6
通讯作者：
Kawahara A
中科院分区：
文献类型：
--
作者：
Hisano Y;Sakuma T;Nakade S;Ohga R;Ota S;Okamoto H;Yamamoto T;Kawahara A
CRISPR/Cas9 系统为包括斑马鱼在内的各种模型生物体的基因组编辑提供了强大的工具。通过 CRISPR/Cas9 介导的基因组修饰，可以轻松实现靶向基因破坏的斑马鱼（敲除）的建立。最近，报道了外源DNA通过同源无关的DNA修复整合到斑马鱼基因组中，但这种整合在基因组和整合DNA的连接处包含各种突变。因此，对目标基因组基因座的精确基因组修饰仍有待实现。在这里，我们描述了使用基因组靶基因座侧翼含有短同源序列 (10–40bp) 的供体载体进行高效、精确的 CRISPR/Cas9 介导的整合。我们成功地将外源 mCherry 或 eGFP 基因高效整合到框架内的目标基因（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无需骨架载体序列即可精确框内整合。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进行精确的靶向基因敲入。
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.
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影响因子：
7
作者：
Auer TO;Duroure K;De Cian A;Concordet JP;Del Bene F
通讯作者：
Del Bene F
DOI：
10.1016/j.tig.2008.08.007
发表时间：
2008-11
期刊：
Trends in genetics : TIG
影响因子：
--
作者：
McVey M;Lee SE
通讯作者：
Lee SE
影响因子：
4.6
作者：
Sakuma T;Nishikawa A;Kume S;Chayama K;Yamamoto T
通讯作者：
Yamamoto T
影响因子：
2.4
作者：
Ansai S;Kinoshita M
通讯作者：
Kinoshita M
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F