Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. Here we describe a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2–3 months and can be applied to other polyploid cell lines or high-copy-number genes.

精确高效的基因组修饰为生物学研究提供了强大的工具。以前的细胞系中的 CRISPR 基因敲除方法依赖于所有等位基因中随机插入/删除引起的移码。然而，这种方法对于由于多倍体或基因扩增而具有高拷贝数的基因效率低下，因为所有等位基因中的移码可能难以生成和检测。在这里，我们描述了一种在多倍体果蝇S2R+ 细胞系中敲除基因的同源定向插入方法。该方案允许使用插入盒生成纯合突变细胞系，该插入盒自动催化在所有等位基因中生成插入突变。使用这种方法产生的敲除细胞可以直接通过 PCR 进行鉴定，无需进行 DNA 测序。该方案需要 2-3 个月，可应用于其他多倍体细胞系或高拷贝数基因。