CRISPR-Based Screening for Stress Response Factors in Mammalian Cells

In the presence of different physiological and environmental stresses, cells rapidly initiate stress responses to re-establish cellular homeostasis. Stress responses usually orchestrate both transcriptional and translational programs via distinct mechanisms. With the advance of transcriptomics and proteomics technologies, transcriptional and translational outputs to a particular stress condition have become easier to measure; however, these technologies lack the ability to reveal the upstream regulatory pathways. Unbiased genetic screens based on a transcriptional or translational reporter are powerful approaches to identify regulatory factors of a specific stress response. CRISPR/Cas-based technologies, together with next-generation sequencing, enable genome-scale pooled screens to systematically elucidate gene function in mammalian cells, with a significant reduction in the rate of off-target effects compared to the previously used RNAi technology. Here, we describe our fluorescence-activated cell sorting (FACS)-based CRISPR interference (CRISPRi) screening platform using a translational reporter to identify novel genetic factors of the mitochondrial stress response in mammalian cells. This protocol provides a general framework for scientists who wish to establish a reporter-based CRISPRi screening platform to address questions in their area of research.