EditID: an open-access workflow for the multiplexed, semi-automated identification of CRISPR edited cell clones.

The growing catalogue of genetic variants associated with human disease offers an unprecedented opportunity to harness CRISPR/Cas9-based gene editing to illuminate genotype-phenotype relationships. However, identifying and isolating appropriately edited cells among thousands of targeted cells remains a considerable challenge, especially if gene editing is performed in parallel at multiple loci. Here we describe a complete open-access workflow for sample tracking, deep sequencing of barcoded PCR amplicons, high-throughput protein analysis, and intuitive data visualisation that facilitates the rapid identification (ID) of cell clones carrying desired edits (GenEditID). To validate GenEditID, we first targeted STAT3 in the human breast cancer cell line MCF7, and found that loss of STAT3 protein correlated well with the total burden of frameshift mutations as determined by both Sanger and Illumina sequencing. To test its scalability, we targeted multiple genes in the FTO (fat mass and obesity associated gene) locus in human pluripotent stem cells (hPSCs), and demonstrate the rapid, parallel identification and prioritisation of knockout (KO) clones for each targeted gene. We further show that these tools can be used to rapidly optimise conditions for less efficient gene editing events, such as the targeted insertion of single-base changes by homology directed repair. Overall, GenEditID enables non-specialist groups to accelerate their gene targeting efforts, facilitating the modelling of genetically complex human disease.

与人类疾病相关的遗传变异目录日益丰富，为利用基于CRISPR/Cas9的基因编辑技术阐明基因型-表型（genotype-phenotype）关联提供了前所未有的机遇。然而，在数千个经靶向处理的细胞中鉴定并分离出经过精准编辑的细胞仍是一项重大挑战，尤其是当在多个基因座上并行开展基因编辑时。本文介绍一套完整的开源工作流程，涵盖样本追踪、带条形码的PCR扩增子（barcoded PCR amplicons）深度测序、高通量蛋白质分析以及直观的数据可视化，可助力快速鉴定携带预期编辑结果的细胞克隆，该整套流程被命名为GenEditID。为验证GenEditID的有效性，我们首先在人乳腺癌细胞系MCF7中靶向编辑STAT3基因，结果发现STAT3蛋白的表达缺失与移码突变的总负荷呈现显著相关性，该结果经桑格测序（Sanger sequencing）与Illumina测序（Illumina sequencing）确认。为测试该流程的可扩展性，我们在人多能干细胞（hPSCs）的FTO（脂肪量与肥胖相关基因）基因座上靶向编辑多个基因，并证明可对每个靶向基因的敲除（KO）克隆实现快速并行鉴定与优先级排序。我们进一步证明，该工具可用于快速优化低效率基因编辑事件的实验条件，例如通过同源定向修复（homology directed repair）实现单碱基改变的靶向插入。总体而言，GenEditID可帮助非专业研究团队加快基因靶向研究进度，助力构建遗传复杂性人类疾病的模型。