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

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 identifies 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 Z NF 207, 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. High-throughput mutagenesis further showed that ZNF207’s zinc finger domain broadly impacts alternative splicing through interactions with U1 snRNP factors. 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.