CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy
CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy
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使用无缝集成策略进行基于 CRISPR/Cas9 的斑马鱼基因组工程
DOI：
10.1096/fj.201800077rr
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发表时间：
2018-09-01
期刊：
影响因子：
4.8
通讯作者：
Pei, De-Sheng
中科院分区：
文献类型：
--
作者：
Luo, Juan-Juan;Bian, Wan-Ping;Pei, De-Sheng
已经开发了许多将外源DNA序列的大片段插入斑马鱼基因组的可行方法，以及使用可编程核酸酶的基因组编辑技术。然而，靶内源基因的编码序列被破坏，插入的外源基因的表达模式不能完全重现内源基因的表达模式。在这里，我们描述了一种新的策略的建立内源性启动子驱动和微同源介导的末端连接依赖性整合的供体载体使用成簇的规则间隔短回文重复序列（CRISPR）/CRISPR相关（Cas）9。我们成功地将mCherry整合到目标基因的最终编码序列中，以高效率产生无缝转基因斑马鱼品系。这种新的无缝转基因技术既保持了内源基因的完整性，又不破坏目的基因的功能。因此，我们的微同源介导的末端连接介导的转基因策略可能在基因治疗中有更广泛的应用。此外，这种新的无缝基因编辑策略在斑马鱼中提供了一种有价值的新转基因技术，该技术由内源性启动子和体内动物报告模式驱动，用于转化医学。它有望成为斑马鱼领域的标准基因编辑技术，为早期胚胎发生研究带来一些重要突破。J.，Bian，W.- P.的人，Liu，Y.，黄鸿是的，尹，Q.杨X- J.，Pei，D.- S.使用无缝整合策略的基于CRISPR/Cas9的斑马鱼基因组工程。
Numerous feasible methods for inserting large fragments of exogenous DNA sequences into the zebrafish genome have been developed, as has genome editing technology using programmable nucleases. However, the coding sequences of targeted endogenous genes are disrupted, and the expression patterns of inserted exogenous genes cannot completely recapitulate those of endogenous genes. Here we describe the establishment of a novel strategy for endogenous promoter-driven and microhomology-mediated end-joining-dependent integration of a donor vector using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9. We successfully integrated mCherry into the final coding sequence of targeted genes to generate seamless transgenic zebrafish lines with high efficiency. This novel seamless transgenesis technique not only maintained the integrity of the endogenous gene but also did not disrupt the function of targeted gene. Therefore, our microhomology-mediated end-joining-mediated transgenesis strategy may have broader applications in gene therapy. Moreover, this novel seamless gene-editing strategy in zebrafish provides a valuable new transgenesis technique, which was driven by endogenous promoters and in vivo animal reporter modes for translational medicine. It is expected to be a standard gene-editing technique in the field of zebrafish, leading to some important breakthroughs for studies in early embryogenesis.Luo, J.-J., Bian, W.-P., Liu, Y., Huang, H.-Y., Yin, Q., Yang, X.-J., Pei, D.-S. CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.