Single-Cell Exon Deletion Profiling Reveals Splicing Events That Shape Gene Expression and Cell State Dynamics [scRNA-seq]

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: HAP1 cells co-expressing spCas9 and opCas12a were transduced with a hybrid guide RNA (hgRNA) library designed to induce deletions in 224 exons and knockouts in 161 genes at a low multiplicity of infection (MOI = 0.1). Transduced cells underwent selection with puromycin for two days. Four days post-transduction, a total of 413,190 cells were processed using the 10x Genomics Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index), with protocol modifications to enable the specific amplification of Cas12a gRNA libraries. For sequencing, libraries corresponding to 1) gene expression, 2) CRISPR perturbations (Cas9 & Cas12a gRNAs using conventional 10x Genomics CRISPR libraries), and 3) Cas12a-specific sequencing (using a custom Cas12 gRNA-specific amplification protocol) were pooled at a 4:1:1 molar ratio. Sequencing was performed on an Illumina NovaSeq platform using a 100-cycle kit, following the standard 10x Genomics protocol: Read 1: 28 bp, Index i7: 10 bp, Index i5: 10 bp, Read 2: 90 bp.