利用CRISPR-Cas9基因编辑技术快速产生纯合荧光基因敲入人类细胞并通过自动成像和数字PCR筛选进行验证

Rapid generation of homozygous fluorescent knock-in human cells using CRISPR-Cas9 genome editing and validation by automated imaging and digital PCR screening

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Nature Protocols

影响因子:16

分区:生物学2区 Top / 生化研究方法1区

发表日期:2025 Jan

作者: Andrea Callegari, Moritz Kueblbeck, Natalia Rosalía Morero, Beatriz Serrano-Solano, Jan Ellenberg

DOI: 10.1038/s41596-024-01043-6

摘要

我们之前描述了一种利用簇状规律间隔短回文重复序列（CRISPR-Cas9）进行哺乳动物培养细胞基因组工程的方案，以在内源基因中生成荧光标签的纯合敲入。本文对该方案进行了更新，反映出工作流程在效率和通量方面的主要改进。简而言之，我们通过结合高效电穿孔优化的CRISPR-Cas9试剂、自动明场和荧光成像筛选单细胞克隆、快速评估标记等位基因数及潜在离靶位点的数字PCR（PCR）和自动数据分析，显著提高了效率。与原始方案相比，目前的流程（1）大幅提高了标签整合的效率，（2）实现了自动识别具有正确亚细胞定位的单细胞克隆，以及（3）提供了利用数字PCR进行定量和高通量测定在靶向和非靶向整合数目的方法。新流程的效率提升使得需要深入分析的克隆数减少十倍以上，并在一次基因组工程操作中在多倍体癌细胞系中获得超过26%的纯合克隆。总体而言，我们将整体操作时间从30天大幅缩短至10天（整个约10周的流程），使单人可同时处理多达五个基因，前提是有验证用试剂（如PCR引物、数字PCR试剂盒和蛋白免疫检测抗体）可用。

Abstract

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.