Establishing Combinatorial Screening Platform Coupled to Single-cell Analysis

Division Of Basic Sciences - Nci

Investigators

Abstract

By optimizing the hgRNA scaffold sequence and refining library preparation protocols, we have significantly enhanced the CHyMErA exon deletion screening platform for single-cell experiments. This advanced tool enables efficient combinatorial genetic perturbations and precise gene segment deletions, while uniquely supporting the simultaneous capture of Cas9 and Cas12a guide RNAs alongside polyadenylated transcripts at single-cell resolution. We have designated this platform single-cell CHyMErA sequencing (scCHyMErA-Seq). To validate scCHyMErA-Seq, we performed a screen focused on the deletion of alternative exons. Alternative splicing is a pervasive gene regulatory mechanism and plays a critical role in expanding proteomic diversity in human cells. Our platform enables efficient exon deletion while simultaneously capturing guide RNA identity and transcriptomic output from individual cells. Applying scCHyMErA-Seq to high-throughput profiling of alternative cassette exons, we identified numerous exons with strong regulatory effects on gene expression. Importantly, the gene expression profiles generated using scCHyMErA-Seq closely recapitulate findings from traditional, labor-intensive orthogonal approaches, while offering substantially greater scalability and efficiency. Overall, scCHyMErA-Seq represents a robust and versatile platform for systematically dissecting the functional consequences of alternative splicing, enabling direct linkage of splicing variants to transcriptional phenotypes at single-cell resolution.