About one-third of cellular proteins in eukaryotic cells are localized to membrane-enclosed organelles in the endomembrane system. Trafficking of these membrane proteins (including soluble lumenal proteins) among the organelles is mediated by small sac-like vesicles. Vesicle-mediated membrane trafficking regulates a broad range of biological processes, many of which are still poorly understood at the molecular level. A powerful approach to dissect a vesicle-mediated membrane trafficking pathway is unbiased genome-wide genetic screening, which only recently became possible in mammalian cells with the isolation of haploid human cell lines and the development of CRISPR-Cas9 genome editing. Here, we describe a FACS-based method to select populations of live mutant cells based on the surface levels of endogenous proteins or engineered reporters. Collection of these mutant populations enables subsequent deep sequencing and bioinformatics analysis to identify genes that regulate the trafficking pathway. This method can be readily adapted to genetically dissect a broad range of mammalian membrane trafficking processes using haploid genetics or CRISPR-Cas9 screens. © 2018 by John Wiley & Sons, Inc.
Keywords: CRISPR-Cas9; flow cytometry; genome-wide genetic screen; membrane trafficking; vesicle transport.
© 2018 John Wiley & Sons, Inc.