CRISPR-based screening methods using single-cell RNA sequencing (scRNA-seq) technology enable comprehensive profiling of gene perturbations from knock-out mutations. However, evaluating substitution mutations using scRNA-seq is currently limited. We combined CRISPR RNA-guided deaminase and scRNA-seq technology to develop a platform for introducing mutations in multiple genes and assessing the mutation-associated signatures. Using this platform, we generated a library consisting of 420 sgRNAs, performed sgRNA tracking analysis, and assessed the effect size of the response to vemurafenib in the human melanoma cell line, which has been well-studied via knockout-based drop-out screens. However, a substitution mutation library screen has not been applied and transcriptional information for mechanisms of action was not assessed. Our platform permits discrimination of several candidate mutations that function differently from other mutations by integrating sgRNA candidates and gene expression readout. We anticipate that our platform will enable high-throughput analyses of the mechanisms related to a variety of biological events.

使用单细胞RNA测序（scRNA-seq）技术的基于CRISPR的筛选方法可对敲除突变产生的基因扰动进行全面分析。但是，使用scRNA-seq评估取代突变目前受到限制。我们结合了CRISPR RNA引导的脱氨酶和scRNA-seq技术，开发了一个平台，可在多个基因中引入突变并评估与突变相关的特征。使用该平台，我们生成了一个由420个sgRNA组成的文库，进行了sgRNA跟踪分析，并评估了人类黑素瘤细胞系中对vemurafenib的反应的效应大小，已通过基于基因剔除的筛选进行了深入研究。但是，尚未应用替代突变文库筛选，也未评估作用机理的转录信息。我们的平台通过整合sgRNA候选物和基因表达读数，可以区分几种功能与其他突变不同的候选物突变。我们预计，我们的平台将能够对与各种生物事件相关的机制进行高通量分析。