通过自动成像和数字PCR筛选，使用CRISPR-CAS9基因组编辑和验证迅速生成纯合荧光敲入人类细胞
Rapid generation of homozygous fluorescent knock-in human cells using CRISPR-Cas9 genome editing and validation by automated imaging and digital PCR screening
影响因子:16.00000
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
摘要
我们先前介绍了一种针对哺乳动物培养细胞的基因组工程方案，该方案具有簇状的定期间隔短的短质体重复序列和相关的蛋白9（CRISPR-CAS9），以将荧光标签的纯合性敲入进入内源基因中。在这里，我们正在更新以前的协议，以反映有关效率和吞吐量的工作流程的重大改进。简而言之，我们通过结合了优化的CRISPR-CAS9试剂的高效率电穿孔，通过自动化的明亮场和荧光成像筛选单细胞衍生的克隆，快速评估标记等位基因的数量以及使用数字聚合酶链反应（PCR）迅速评估潜在的非目标，以及自动化的数据分析。与原始协议相比，我们的当前程序（1）大大提高了TAG集成的效率，（2）自动化了从单个细胞中识别具有标记蛋白质的正确亚细胞定位的克隆的识别，（3）提供了定量和高吞吐量的分析，以测量与数字PCR的target集成的数量。新程序的提高效率可以减少需要深入分析的克隆数量，而在单个基因组工程回合中，在多倍体癌细胞系中，在多倍体癌细胞系中的纯合子克隆的产量超过26％。总体而言，我们能够在整个〜10周的过程中从30 d降低到10 d，从而使一个人可以并行处理多达五个基因，假设有验证的试剂，例如PCR引物，数字PCR分析和Western Blot抗体，则可以并行处理五个基因。
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.