Decoding RNA metabolism by RNA-linked CRISPR screening in human cells

RNAs undergo a complex choreography of metabolic processes that are regulated by thousands of RNA-associated proteins. Here we present a massively parallel RNA-linked CRISPR (ReLiC) platform to measure the responses of diverse RNA metabolic events to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC relies on a pooled, virus-free, serine recombinase-based strategy to integrate DNA libraries encoding Cas9, multiple sgRNAs, and barcoded reporters into a defined genomic locus. Combining ReLiC with polysome fractionation reveals distinct effects of perturbing translation and proteostasis machineries on ribosome occupancy. Isoform-specific ReLiC captures differential regulation of intron retention and exon skipping by SF3b complex subunits. Chemogenomic screens using ReLiC decipher translational regulators upstream of mRNA decay and uncover a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a versatile platform for discovering and dissecting regulatory principles of human RNA metabolism.