RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria [CRASP-Seq]

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. Overall design: CRASP-Seq assays of six different splicing minigene reporters (FAS exon-6, EZH2 exon-14, SRSF7 poison exon, PKM mutually exclusive exons, LMNA HGPS, LMNA WT) were performed in three different cell lines (HAP1, RPE1, and HepG2) at two different time points (T1 and T4).