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.

中文翻译：

---

通过Cas9介导的同源重组在nefma基因中创建斑马鱼敲入报告基因系。

基于CRISPR / Cas9的策略已在许多生物中广泛用于基因组编辑，包括斑马鱼。尽管大多数应用包括引入双链断裂（DSB）诱导的突变，但也可以使用CRISPR / Cas9在选定的基因组位置上增强同源性定向修复（HDR），从而以最佳控制的精度创建敲入基因。在这里，我们描述了使用CRISPR / Cas9靶向DSB和HDR来生成斑马鱼转基因品系，其中将内源编码序列与内源编码序列一起添加到nefma基因中。产生的敲入胚胎在表达nefma的神经元群体中表达添加的基因（荧光报告基因或KalTA4反式激活子），使其成为针对这些人群进行研究的便捷工具。