Targeted genomic knock-ins are a valuable tool to probe gene function. However, knock-in methods involving homology-directed repair (HDR) can be laborious. Here, we adapt the mammalian CRISPaint [clustered regularly interspaced short palindromic repeat (CRISPR)-assisted insertion tagging] homology-independent knock-in method for Drosophila melanogaster, which uses CRISPR/Cas9 and nonhomologous end joining to insert "universal" donor plasmids into the genome. Using this method in cultured S2R+ cells, we efficiently tagged four endogenous proteins with the bright fluorescent protein mNeonGreen, thereby demonstrating that an existing collection of CRISPaint universal donor plasmids is compatible with insect cells. In addition, we inserted the transgenesis marker 3xP3-red fluorescent protein into seven genes in the fly germ line, producing heritable loss-of-function alleles that were isolated by simple fluorescence screening. Unlike in cultured cells, insertions/deletions always occurred at the genomic insertion site, which prevents predictably matching the insert coding frame to the target gene. Despite this effect, we were able to isolate T2A-Gal4 insertions in four genes that serve as in vivo expression reporters. Therefore, homology-independent insertion in Drosophila is a fast and simple alternative to HDR that will enable researchers to dissect gene function.

中文翻译：

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使用通用供体质粒的同源独立插入在果蝇中进行基因敲入。


靶向基因组敲入是探测基因功能的宝贵工具。然而，涉及同源定向修复（HDR）的敲入方法可能很费力。在这里，我们采用哺乳动物 CRISPaint [成簇规则间隔短回文重复 (CRISPR) 辅助插入标记]果蝇的同源独立敲入方法，该方法使用 CRISPR/Cas9 和非同源末端连接将“通用”供体质粒插入到基因组。在培养的 S2R+ 细胞中使用这种方法，我们用明亮的荧光蛋白 mNeonGreen 有效标记了四种内源蛋白，从而证明现有的 CRISPaint 通用供体质粒集合与昆虫细胞兼容。此外，我们将转基因标记3xP3-red 荧光蛋白插入果蝇种系的七个基因中，产生可遗传的功能丧失等位基因，并通过简单的荧光筛选分离出这些基因。与培养细胞不同，插入/缺失总是发生在基因组插入位点，这阻止了插入编码框与目标基因的可预测匹配。尽管存在这种影响，我们还是能够在四个基因中分离出T2A-Gal4插入，这些基因充当体内表达报告基因。因此，果蝇中的同源独立插入是 HDR 的一种快速而简单的替代方案，使研究人员能够剖析基因功能。