Protocol for pooled FACS-based CRISPR knockout screening in human iPSC-derived microglia

Sam J Washer; Elena Navarro-Guerrero; Sally A Cowley; Daniel V Ebner; Andrew R Bassett

doi:10.1016/j.xpro.2025.104111

Protocol for pooled FACS-based CRISPR knockout screening in human iPSC-derived microglia

STAR Protoc. 2025 Sep 20;6(4):104111. doi: 10.1016/j.xpro.2025.104111. Online ahead of print.

Authors

Sam J Washer¹, Elena Navarro-Guerrero², Sally A Cowley³, Daniel V Ebner², Andrew R Bassett⁴

Affiliations

¹ Wellcome 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. Electronic address: sam.washer@cmd.ox.ac.uk.
² Target Discovery Institute, Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Old Road Campus, Oxford OX3 7FZ, UK.
³ 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.
⁴ Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton CB10 1SA, UK; Open Targets, Wellcome Genome Campus, Hinxton CB10 1SA, UK.

Abstract

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.

Keywords: CRISPR; cell culture; high-throughput screening; sequencing; stem cells.