Single-cell analysis of a mutant library generated using CRISPR-guided deaminase

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 simultaneously assessing the mutation-associated perturbations and signatures in a high-throughput manner. 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 melanoma A375 cell line, which has been well-studied via GeCKO but not transcriptome analysis. A convenient and efficient workflow not possible with abundance-based assays enabled the characterization of surviving cells. Our platform permits discrimination of several hit-mutations within a large-scale library by integrating sgRNA hits and gene expression readout. We anticipate that our platform will enable high-throughput analyses of the mechanisms related to a variety of biological events.