Single-Cell Exon Deletion Profiling Reveals Splicing Events That Shape Gene Expression and Cell State Dynamics [RNA-seq_Rescue_exp]
Alternative splicing is a pervasive gene regulatory mechanism critical for diversifying the human proteome. To systematically investigate its role in cell fate determination, we developed 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 identified numerous exons with strong regulatory effects on gene expression. Analysis of NRF1 alternative exon-7 revealed that this exon modulates NRF1 transcriptional activity by regulating 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 robust and versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes. Overall design: RNA-Sequencing was performed in HEK293 Flp-in cells depleted from endogenous NRF1 with RNAi (non-targeting siRNAs were used as control) while expressing either full-length NRF1 (NRF1-FL) or the NRF1 isoform lacking exon-7(NRF1-?E7) at endogenous levels using dox-inducible HEK293 Flp-In cell lines to systematically explore if NRF1 exon-7 regulates gene expression.