A Pipeline for Precise and Efficient Genome Editing by sgRNA-Cas9 RNPs in <i>Drosophila</i>

Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of <i>Drosophila melanogaster</i> as well as to other <i>Drosophila</i> species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against <i>eyes absent</i>. This allows for screening based on eye morphology rather than color. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.

借助同源定向修复（homology-directed repair, HDR）进行基因组编辑，已实现对基因序列的精准可控修饰。其中，CRISPR/Cas9介导的同源定向修复是实现该操作的最简方案。然而，当前仍存在技术瓶颈：亟待提升编辑效率，并拓展其在黑腹果蝇（Drosophila melanogaster）及其他果蝇属物种的任意遗传背景中的应用范围。为解决上述问题，我们开发了一种双阶段标记辅助策略：先向胚胎注射核糖核蛋白复合物（ribonucleoprotein complexes, RNPs），并通过T7核酸内切酶I（T7EI）进行预筛选；随后使用与重组Cas9蛋白结合的单向导RNA（single guide RNA, sgRNA），检测各sgRNA的基因组切割效率。接着，我们利用切割效率优异的sgRNA开展同源定向修复，并使用靶向无眼基因（eyes absent）的RNAi转化标记，该标记可基于眼形态而非眼色完成筛选。本研究开发的新型工具，既可用于目标区域内的单点突变或一系列等位基因替换，也可用于构建平衡染色体等其他遗传工具。