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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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. .
AbstractList 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.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.
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. .
ArticleNumber 104111
Author Washer, Sam J.
Cowley, Sally A.
Navarro-Guerrero, Elena
Bassett, Andrew R.
Ebner, Daniel V.
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Cites_doi 10.1007/978-1-4939-7514-3_13
10.1038/s41598-022-23477-2
10.1016/j.cell.2019.08.053
10.1016/j.stem.2022.11.004
10.1016/j.xpro.2024.102903
10.1186/s13059-014-0554-4
10.1038/s41596-018-0113-7
10.1038/s41588-020-00776-w
10.1016/j.celrep.2016.09.079
10.1093/brain/awaa309
10.1038/nmeth.3047
10.1038/s41588-020-0610-9
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stem cells
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References Bellenguez (10.1016/j.xpro.2025.104111_bib3) 2022; 1
Burgold (10.1016/j.xpro.2025.104111_bib11) 2024
Wang (10.1016/j.xpro.2025.104111_bib13) 2019; 14
Pimentel (10.1016/j.xpro.2025.104111_bib15) 2024
Navarro-Guerrero (10.1016/j.xpro.2025.104111_bib8) 2024; 5
Schwartzentruber (10.1016/j.xpro.2025.104111_bib4) 2021; 53
Prinz (10.1016/j.xpro.2025.104111_bib2) 2019; 179
Clarke (10.1016/j.xpro.2025.104111_bib6) 2020; 143
Tzelepis (10.1016/j.xpro.2025.104111_bib10) 2016; 17
Pantazis (10.1016/j.xpro.2025.104111_bib14) 2022; 29
Yau (10.1016/j.xpro.2025.104111_bib16) 2018; 1712
Perez-Alcantara (10.1016/j.xpro.2025.104111_bib1) 2025
Bryois (10.1016/j.xpro.2025.104111_bib5) 2020; 52
Washer (10.1016/j.xpro.2025.104111_bib7) 2022; 12
Li (10.1016/j.xpro.2025.104111_bib12) 2014; 15
Sanjana (10.1016/j.xpro.2025.104111_bib9) 2014; 11
References_xml – volume: 1712
  start-page: 203
  year: 2018
  ident: 10.1016/j.xpro.2025.104111_bib16
  article-title: Next-Generation Sequencing of Genome-Wide CRISPR Screens
  publication-title: Methods Mol. Biol.
  doi: 10.1007/978-1-4939-7514-3_13
– volume: 12
  start-page: 1
  year: 2022
  ident: 10.1016/j.xpro.2025.104111_bib7
  article-title: Single-cell transcriptomics defines an improved, validated monoculture protocol for differentiation of human iPSCs to microglia
  publication-title: Sci. Rep.
  doi: 10.1038/s41598-022-23477-2
– volume: 179
  start-page: 292
  year: 2019
  ident: 10.1016/j.xpro.2025.104111_bib2
  article-title: Microglia Biology: One Century of Evolving Concepts
  publication-title: Cell
  doi: 10.1016/j.cell.2019.08.053
– volume: 29
  start-page: 1685
  year: 2022
  ident: 10.1016/j.xpro.2025.104111_bib14
  article-title: A reference human induced pluripotent stem cell line for large-scale collaborative studies
  publication-title: Cell Stem Cell
  doi: 10.1016/j.stem.2022.11.004
– volume: 5
  year: 2024
  ident: 10.1016/j.xpro.2025.104111_bib8
  article-title: Optimized protocol for CRISPR knockout of human iPSC-derived macrophages
  publication-title: STAR Protoc.
  doi: 10.1016/j.xpro.2024.102903
– volume: 15
  start-page: 554
  year: 2014
  ident: 10.1016/j.xpro.2025.104111_bib12
  article-title: MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens
  publication-title: Genome Biol.
  doi: 10.1186/s13059-014-0554-4
– volume: 14
  start-page: 756
  year: 2019
  ident: 10.1016/j.xpro.2025.104111_bib13
  article-title: Integrative analysis of pooled CRISPR genetic screens using MAGeCKFlute
  publication-title: Nat. Protoc.
  doi: 10.1038/s41596-018-0113-7
– volume: 53
  start-page: 392
  year: 2021
  ident: 10.1016/j.xpro.2025.104111_bib4
  article-title: Genome-wide meta-analysis, fine-mapping and integrative prioritization implicate new Alzheimer’s disease risk genes
  publication-title: Nat. Genet.
  doi: 10.1038/s41588-020-00776-w
– volume: 17
  start-page: 1193
  year: 2016
  ident: 10.1016/j.xpro.2025.104111_bib10
  article-title: A CRISPR Dropout Screen Identifies Genetic Vulnerabilities and Therapeutic Targets in Acute Myeloid Leukemia
  publication-title: Cell Rep.
  doi: 10.1016/j.celrep.2016.09.079
– volume: 143
  start-page: 3526
  year: 2020
  ident: 10.1016/j.xpro.2025.104111_bib6
  article-title: The microglial component of amyotrophic lateral sclerosis
  publication-title: Brain
  doi: 10.1093/brain/awaa309
– year: 2024
  ident: 10.1016/j.xpro.2025.104111_bib15
  article-title: A model for accurate quantification of CRISPR effects in pooled FACS screens
  publication-title: bioRxiv
– volume: 1
  start-page: 1
  year: 2022
  ident: 10.1016/j.xpro.2025.104111_bib3
  article-title: New insights into the genetic etiology of Alzheimer’s disease and related dementias
  publication-title: Nat. Genet.
– volume: 11
  start-page: 783
  year: 2014
  ident: 10.1016/j.xpro.2025.104111_bib9
  article-title: Improved vectors and genome-wide libraries for CRISPR screening
  publication-title: Nat. Methods
  doi: 10.1038/nmeth.3047
– year: 2024
  ident: 10.1016/j.xpro.2025.104111_bib11
  article-title: Genetic interaction library screening with a next-generation dual guide CRISPR system
  publication-title: bioRxiv
– year: 2025
  ident: 10.1016/j.xpro.2025.104111_bib1
  article-title: Integrated QTL mapping and CRISPR screening in pooled iPSC-derived microglia reveals genetic drivers of neurodegenerative risk
  publication-title: bioRxiv
– volume: 52
  start-page: 482
  year: 2020
  ident: 10.1016/j.xpro.2025.104111_bib5
  article-title: Genetic Identification of Cell Types Underlying Brain Complex Traits Yields Novel Insights Into the Etiology of Parkinson’s Disease
  publication-title: Nat. Genet.
  doi: 10.1038/s41588-020-0610-9
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