Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics.

Alternative splicing is a pervasive gene regulatory mechanism critical for diversifying the human proteome. To systematically investigate its role in cell fate determination, we develop scCHyMErA-Seq, a scalable CRISPR-based exon deletion screening platform integrated with 10x Genomics single-cell transcriptomic readouts. This tool enables efficient exon deletion while simultaneously capturing Cas9/Cas12a guides and polyadenylated transcripts at single-cell resolution. Applying scCHyMErA-Seq to high-throughput profiling of alternative cassette exons, we identify numerous exons with pronounced regulatory effects on gene expression and cell cycle progression. Analysis of the alternative NRF1 exon-7 demonstrates that its inclusion modulates NRF1's regulatory function by influencing its recruitment to the promoters of target genes. Importantly, gene expression profiles generated using scCHyMErA-Seq accurately recapitulate findings from traditional, labor-intensive orthogonal methods, while offering enhanced scalability and efficiency. Overall, scCHyMErA-Seq represents a versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.