RNA-coupled CRISPR screens reveal ZNF207 as a regulator of LMNA aberrant splicing in progeria
Amit K. Behera, Jeongjin J. Kim, Shreya Kordale, Filip Pekovic, Arun Prasath Damodaran, Bandana Kumari, Sandra Vidak, Ethan Dickson, Mei‐Sheng Xiao, Gerard Duncan, Thorkell Andresson, Tom Misteli, Eugene Valkov, Thomas Gonatopoulos-Pournatzis
Abstract
Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-seq (CRISPR-based identification of regulators of alternative splicing with phenotypic sequencing), a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitatively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-seq identified both known and untested regulators, enriched for proteins involved in RNA splicing and metabolism. As a proof-of-concept, CRASP-seq analysis of the 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 protein levels in patient-derived cells. We further show that ZNF207’s zinc-finger domain broadly impacts alternative splicing through direct interactions with U1 small nuclear ribonucleoprotein (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. • CRASP-seq: RNA-coupled CRISPR screen quantifying gene and domain impact on splicing • Profiling of five events identified 370 genes influencing alternative splicing • ZNF207 regulates splicing by interacting with U1 snRNP via its zinc-finger domains • Depletion of ZNF207 corrects the aberrant LMNA splicing underlying progeria This study introduces CRASP-seq, an RNA-coupled CRISPR screening platform designed to systematically identify regulatory factors and domains of alternative pre-mRNA splicing. Using this technology, ZNF207 is identified as a bona fide splicing regulator through its interaction with U1 snRNP components. ZNF207 modulates aberrant LMNA splicing, a critical event linked to progeria.