Single-cell analysis of a mutant library generated using CRISPR-guided deaminase in human melanoma cells

• Published in: Communications biology 2020-04, Vol.3 (1), p.154-154, Article 154
• Main Authors: Jun, Soyeong, Lim, Hyeonseob, Chun, Honggu, Lee, Ji Hyun, Bang, Duhee
• Format: Article
• Language: English
• Subjects: 38; 38/23; 38/39; 38/47; 38/70; 38/91; 42; 42/62; 45; 59; 631/61/212; 631/61/338; 64; Amino acid substitution; Antineoplastic Agents - pharmacology; Biology; Biomarkers, Tumor - genetics; Biomedical and Life Sciences; Cell Line, Tumor; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CRISPR-Cas Systems; Cytidine Deaminase - genetics; Cytidine Deaminase - metabolism; Female; Gene Editing; Gene expression; Gene Library; HEK293 Cells; Humans; Libraries; Life Sciences; Melanoma; Melanoma - drug therapy; Melanoma - genetics; Melanoma - metabolism; Melanoma - pathology; Mutants; Mutation; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems - genetics; RNA-Seq; Single-Cell Analysis; Skin Neoplasms - drug therapy; Skin Neoplasms - genetics; Skin Neoplasms - metabolism; Skin Neoplasms - pathology; Transcription; Vemurafenib - pharmacology
• ISSN: 2399-3642; 2399-3642
• 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. Jun, Lim et al. combined CRISPR RNA-guided deaminase and single-cell RNA sequencing technology to functionally discriminate the target mutations from off-target mutations. This study provides a platform that allows researchers to generate multiple mutations and to screen the cells that contain the target mutations in a high-throughput manner.