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. Two experimental groups. First group has CRISPR knockout of SF3B5, SF3B6, AQR, or FLUC(no-targeting). Second group has CRISPR knockout of GCN1 or FLUC, followed by harringtonine treatment for 6hrs (0hrs is no treatment). The first group does not have replicates. The second group has two replicates per sample and two different sgRNA pairs for GCN1 (1_2 and 3_4). All CRISPR knockouts use paired sgRNAs integrated at the AAVS1 locus and are induced through doxycline-mediated induction of Cas9. See manuscript for further details.