Eschstruth Alexis, Schneider-Maunoury Sylvie, Giudicelli François
Sorbonne Université, CNRS UMR7622, Inserm U1156, Institut de Biologie Paris-Seine (IBPS) - Laboratoire de Biologie du développement, Paris, France.
Genesis. 2020 Jan;58(1):e23340. doi: 10.1002/dvg.23340. Epub 2019 Sep 30.
CRISPR/Cas9-based strategies are widely used for genome editing in many organisms, including zebrafish. Although most applications consist in introducing double strand break (DSB)-induced mutations, it is also possible to use CRISPR/Cas9 to enhance homology directed repair (HDR) at a chosen genomic location to create knock-ins with optimally controlled precision. Here, we describe the use of CRISPR/Cas9-targeted DSB followed by HDR to generate zebrafish transgenic lines where exogenous coding sequences are added in the nefma gene, in frame with the endogenous coding sequence. The resulting knock-in embryos express the added gene (fluorescent reporter or KalTA4 transactivator) specifically in the populations of neurons that express nefma, making them convenient tools for research on these populations.
基于CRISPR/Cas9的策略被广泛应用于包括斑马鱼在内的许多生物体的基因组编辑。尽管大多数应用是引入双链断裂（DSB）诱导的突变，但也可以使用CRISPR/Cas9增强选定基因组位置的同源定向修复（HDR），以创建具有最佳控制精度的敲入。在这里，我们描述了使用CRISPR/Cas9靶向的DSB，随后进行HDR，以生成斑马鱼转基因品系，其中外源编码序列被添加到nefma基因中，与内源编码序列读框一致。由此产生的敲入胚胎在表达nefma的神经元群体中特异性表达添加的基因（荧光报告基因或KalTA4反式激活因子），使其成为研究这些群体的便利工具。
