Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Republic of Korea.
Department of Biomedical Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Republic of Korea.
Commun Biol. 2020 Apr 2;3(1):154. doi: 10.1038/s42003-020-0888-2.
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.
基于 CRISPR 的筛选方法结合单细胞 RNA 测序 (scRNA-seq) 技术，可全面分析敲除突变引起的基因扰动。然而，目前使用 scRNA-seq 评估取代突变还存在限制。我们结合 CRISPR RNA 引导的脱氨酶和 scRNA-seq 技术，开发了一种可在多个基因中引入突变并评估突变相关特征的平台。利用该平台，我们构建了一个包含 420 个 sgRNA 的文库，进行了 sgRNA 追踪分析，并评估了在人黑色素瘤细胞系中引入突变对 vemurafenib 反应的效应大小，该细胞系已通过基于敲除的dropout 筛选进行了深入研究。然而，尚未应用取代突变文库筛选，也未评估作用机制的转录信息。我们的平台通过整合 sgRNA 候选物和基因表达读数，可以区分几种候选突变，这些候选突变与其他突变的作用方式不同。我们预计，我们的平台将能够高通量分析与各种生物学事件相关的机制。
