我们之前描述了一种哺乳动物培养细胞基因组工程方案，具有成簇的规则间隔短回文重复序列和相关蛋白 9 (CRISPR-Cas9)，以将荧光标签纯合敲入到内源基因中。在这里，我们更新了之前的协议，以反映工作流程在效率和吞吐量方面的重大改进。简而言之，我们通过结合优化 CRISPR-Cas9 试剂的高效电穿孔、通过自动明场和荧光成像筛选单细胞衍生克隆、使用快速评估标记等位基因的数量和潜在脱靶来改进我们的方法。数字聚合酶链反应 (PCR) 和自动数据分析。与原始方案相比，我们当前的程序（1）大大提高了标签整合的效率，（2）自动识别来自单细胞的克隆，并正确定位标签蛋白的亚细胞定位，（3）提供定量和高通量通过数字 PCR 测量脱靶整合次数的测定。新程序效率的提高将需要深入分析的克隆数量减少了十倍以上，并在单轮基因组工程中产生多倍体癌细胞系中超过 26% 的纯合克隆。总体而言，在整个约 10 周的程序中，我们能够将实际操作时间从 30 天大幅减少到 10 天，允许一个人并行处理多达 5 个基因，前提是使用经过验证的试剂（例如 PCR 引物） 、数字 PCR 检测和蛋白质印迹抗体可供使用。© 2024。Springer Nature Limited。

We previously described a protocol for genome engineering of mammalian cultured cells with clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR-Cas9) to generate homozygous knock-ins of fluorescent tags into endogenous genes. Here we are updating this former protocol to reflect major improvements in the workflow regarding efficiency and throughput. In brief, we have improved our method by combining high-efficiency electroporation of optimized CRISPR-Cas9 reagents, screening of single cell-derived clones by automated bright-field and fluorescence imaging, rapidly assessing the number of tagged alleles and potential off-targets using digital polymerase chain reaction (PCR) and automated data analysis. Compared with the original protocol, our current procedure (1) substantially increases the efficiency of tag integration, (2) automates the identification of clones derived from single cells with correct subcellular localization of the tagged protein and (3) provides a quantitative and high throughput assay to measure the number of on- and off-target integrations with digital PCR. The increased efficiency of the new procedure reduces the number of clones that need to be analyzed in-depth by more than tenfold and yields to more than 26% of homozygous clones in polyploid cancer cell lines in a single genome engineering round. Overall, we were able to dramatically reduce the hands-on time from 30 d to 10 d during the overall ~10 week procedure, allowing a single person to process up to five genes in parallel, assuming that validated reagents-for example, PCR primers, digital PCR assays and western blot antibodies-are available.© 2024. Springer Nature Limited.