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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.

STAR Protocols
|September 21, 2025
PubMed
Summary

This study details a CRISPR knockout screening protocol for human induced pluripotent stem cell-derived microglia (iMGL). The method uses lentiviral CRISPR-Cas9 and VPX virus-like particles (VPX-VLPs) for efficient genetic screening in microglia.

Keywords:
CRISPRcell culturehigh-throughput screeningsequencingstem cells

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Area of Science:

  • Neuroscience
  • Immunology
  • Genetics

Background:

  • Microglia play crucial roles in brain health and disease.
  • Studying microglia function is essential for understanding neurological disorders.
  • CRISPR screening offers a powerful tool for functional genomics in specific cell types.

Purpose of the Study:

  • To establish a robust protocol for CRISPR knockout screening in human induced pluripotent stem cell-derived microglia (iMGL).
  • To enable large-scale genetic analysis of microglial functions, such as phagocytosis.
  • To provide a comprehensive guide for researchers utilizing advanced gene-editing techniques in microglia.

Main Methods:

  • Large-scale production of iMGL from human induced pluripotent stem cells (hiPSCs).
  • Generation and titration of lentiviral CRISPR-Cas9 and VPX virus-like particle (VPX-VLP) libraries.
  • Execution of pooled CRISPR screens focusing on phagocytosis.
  • Computational analysis pipeline for CRISPR screening data.

Main Results:

  • Successful implementation of a CRISPR knockout screening protocol in iMGL.
  • Demonstration of efficient lentiviral delivery of CRISPR-Cas9 and VPX-VLPs for genetic manipulation.
  • Establishment of a comprehensive pipeline for analyzing CRISPR screening data in microglia.

Conclusions:

  • This protocol facilitates high-throughput genetic screening in human iMGL.
  • The developed method is valuable for dissecting microglial functions and identifying genes involved in neurological processes.
  • This work provides a foundation for future functional genomic studies in microglia using CRISPR technology.