Genome-wide CRISPRi Screen for Regulators of Endoplasmic Reticulum Autophagy (ER-phagy)

This is a genome-wide CRISPR-interference (CRISPRi - gene repression using catalytically dead Cas9 fused with the KRAB transcriptional repressor) screen to identify regulators of starvation-induced endoplasmic reticulum autophagy (ER-phagy). ER-phagy activity is measured by FACS to measure change in fluorescence intensity of mCherry versus eGFP of an recombinant ER surface protein, mCherry-eGFP-RAMP4 upon starvation. Cells with sgRNA-mediated knockdown that result in phenotypic change (enhanced or inhibited ER-phagy) are FACS sorted and subjected to next generation sequencing to identify the sgRNA sequence and its targeting gene.