A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based singlecell RNA-seq with a strategy for barcoding CRISPRmediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting similar to 100 hits to Perturb-seq enabled highprecision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to thesameperturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1 alpha. These studies provide insight intohowthe three sensors ofERhomeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.