Genome-wide CRISPRi screen for myriocin sensitivity
To delineate cellular pathways underlying cell growth in the absence of sphingolipid syntehsis, we performed a genome-wide CRISPRi screen to identify genetic modifiers of myriocin sensitivity in human K562 cells. Overall design: Pooled CRISPR screening experiment. A library of constructs expressing sgRNAs were cloned and transduced as a pool in K562 cells. Cells were passaged in de-lipidated culture conditions and treated with 1 µM myriocin or DMSO. T0 ("time-zero", pre-treatment, 5-days post-infection) and myriocin/DMSO-treated endpoint samples (after 6 days of treatment) were harvested. Genomic DNA was processed and sequenced to obtain sgRNA counts in the cell population, and phenotypes were quantified by comparing representation at T0 and DMSO and myriocin-treated endpoints.