Abstract
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.
Highlights
Introduction and enrichment of resistant mutationTo assess the efficiency of our system, we investigated the rate of conversion of the C base targeted in a single locus
The editing efficiency of BE3 is lower than Cas[94] (>90%), we believe this efficiency is high enough compared to homologous recombination-based substitution mutationgenerating methods[3]
We investigated whether transcriptional changes in the clusters composed primarily of #176 sgRNA targeting close to E203 in MAP2K1 were related to vemurafenib resistance by identifying marker genes
Summary
Introduction and enrichment of resistant mutationTo assess the efficiency of our system, we investigated the rate of conversion of the C base targeted in a single locus. After treatment with vemurafenib for 25 days, the substitution efficiency increased to 41.11–77.17%, whereas DMSO vehicle treatment yielded an efficiency of only 9.67–12.08% These data suggest that continuous engineering occurred during culture and that the mutation confers resistance to vemurafenib. The same analysis of DMSO-treated cells revealed that 12.06% of the viable cells were E203K clones These results confirmed that enrichment of the mutation occurred with vemurafenib treatment. The results of our singleplex experiments suggest that the mutations artificially introduced via base editing functioned well and that mutants can be enriched using drugs in a manner similar to naturally acquired mutations in patients These data demonstrate the possibility of using this system to measure the effect sizes of various mutations in terms of drug resistance
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