An effective double gene knock-in strategy using small-molecule L755507 in the medaka fish (Oryzias latipes)
An effective double gene knock-in strategy using small-molecule L755507 in the medaka fish (Oryzias latipes)
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DOI：
10.1002/dvg.23465
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发表时间：
2022-01-24
期刊：
影响因子：
1.5
通讯作者：
Kobayashi，Toru
中科院分区：
文献类型：
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作者：
Murakami，Yu;Kobayashi，Toru
同源定向修复（HDR）介导的基因组编辑已成为以无缝且准确的方式改变染色体序列的强大方法。然而，大多数细胞中 HDR 的低效率阻碍了含有精确修饰基因组的所需菌株的建立。为了增强 HDR 介导的敲入事件，我们探索了两种方法，即低温孵育和显微注射后使用青鳉胚胎进行化学化合物施用。我们通过根据胚胎中荧光蛋白的表达区域计算敲入效率来验证每种方法的性能。体内测定表明，温度降低不会促进 HDR 事件，而在筛选的 9 种化合物中，小分子 L755507 可以增强 HDR 介导的报告基因盒的靶向整合。此外，基于L755507的方法允许将两个不同的DNA片段同时整合到两个目标基因座中，即双敲入。我们建立的结合L755507、供体质粒和CRISPR/Cas9切口酶系统的敲入系统可以减少转基因菌株生成的工作量，从而加速对生物现象的分子机制的研究。
Homology‐directed repair (HDR)‐mediated genome editing has become a powerful method for altering chromosomal sequences in a seamless and accurate manner. However, the low efficiency of HDR in most cells hinders the establishment of desired strains harboring accurately modified genomes. To enhance HDR‐mediated knock‐in events, we explored two approaches, namely low‐temperature incubation and chemical compound administration using medaka embryos after microinjection. We validated the performance of each method by calculating the knock‐in efficiencies according to the expression area of fluorescent protein in the embryos. The in vivo assay indicated that the reduction in temperature did not promote HDR events, whereas among the nine compounds screened, the small molecule L755507 could enhance the HDR‐mediated targeted integration of reporter cassettes. Additionally, the L755507‐based approach allowed for the simultaneous integration of two different DNA fragments into the two targeted loci, that is, double knock‐in. Our established knock‐in system combining L755507, donor plasmids, and the CRISPR/Cas9 nickase system can reduce the workload for genetically modified strain generation, thus accelerating studies on the molecular mechanisms of biological phenomena.