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

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, product...

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Published inSTAR protocols Vol. 6; no. 4; p. 104111
Main Authors Washer, Sam J., Navarro-Guerrero, Elena, Cowley, Sally A., Ebner, Daniel V., Bassett, Andrew R.
Format Journal Article
LanguageEnglish
Published United States 19.12.2025
Subjects
CRISPR
stem cells
cell culture
high-throughput screening
sequencing
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ISSN2666-1667
2666-1667
DOI10.1016/j.xpro.2025.104111

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Summary: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. .
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ISSN:2666-1667
2666-1667
DOI:10.1016/j.xpro.2025.104111