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Protocol for pooled FACS-based CRISPR knockout screening in human iPSC-derived microglia
Sam J Washer1, Elena Navarro-Guerrero2, Sally A Cowley3
1Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton CB10 1SA, UK; Open Targets, Wellcome Genome Campus, Hinxton CB10 1SA, UK; James and Lillian Martin Centre for Stem Cell Research, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK; Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Oxford OX3 7FZ, UK.
None:
Here, we present a protocol for CRISPR knockout screening in human induced pluripotent stem cell (hiPSC)-derived microglia (iMGL) using lentiviral delivery of CRISPR-Cas9 and co-transduction of VPX virus-like particles (VPX-VLPs). We first describe large-scale production of iMGL from hiPSCs, production of the lentiviral and VPX-VLP libraries, and titration. Next, we describe how to perform a pooled CRISPR screen for phagocytosis including the computational analysis pipeline of CRISPR screening data. For complete details on the use and execution of this protocol, please refer to Perez-Alcantara et al.1.
