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 pronounced regulatory effects on gene expression and cell cycle progression. Detailed analysis of the alternative NRF1 exon-7 demonstrated 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 robust and versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.

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 pronounced regulatory effects on gene expression and cell cycle progression. Detailed analysis of the alternative NRF1 exon-7 demonstrated 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 robust and versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.

Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-Seq, a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-Seq identified both known and novel regulators, enriched for proteins involved in RNA splicing and metabolism. As proof-of-concept, CRASP-Seq analysis of an LMNA cryptic splicing event linked to progeria uncovered ZNF207, primarily known for mitotic spindle assembly, as a regulator of progerin splicing. ZNF207 depletion enhances canonical LMNA splicing and decreases progerin levels in patient-derived cells. We further show that ZNF207’s zinc finger domain broadly impacts alternative splicing through direct interactions with U1 snRNP components. These findings position ZNF207 as a U1 snRNP auxiliary factor and demonstrate the power of CRASP-Seq to uncover key regulators and domains of alternative splicing.

Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-Seq, a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-Seq identified both known and novel regulators, enriched for proteins involved in RNA splicing and metabolism. As proof-of-concept, CRASP-Seq analysis of an LMNA cryptic splicing event linked to progeria uncovered ZNF207, primarily known for mitotic spindle assembly, as a regulator of progerin splicing. ZNF207 depletion enhances canonical LMNA splicing and decreases progerin levels in patient-derived cells. We further show that ZNF207’s zinc finger domain broadly impacts alternative splicing through direct interactions with U1 snRNP components. These findings position ZNF207 as a U1 snRNP auxiliary factor and demonstrate the power of CRASP-Seq to uncover key regulators and domains of alternative splicing.