An efficient, non-viral arrayed CRISPR screening platform for iPSC-derived myeloid and microglia models

  • Stem Cell Reports. 2025 Mar 11;20(3):102420. doi: 10.1016/j.stemcr.2025.102420.
Sonja Meier  1 ,  Anne Sofie Gry Larsen  2 ,  Florian Wanke  1 ,  Nicolas Mercado  3 ,  Arianna Mei  4 ,  Livia Takacs  1 ,  Eva Suszanna Mracsko  1 ,  Ludovic Collin  1 ,  Martin Kampmann  5 ,  Filip Roudnicky  6 ,  Ravi Jagasia  1
Affiliations
  • 1. Pharma Research and Early Development, Neuroscience and Rare Diseases, F. Hoffmann-La Roche Ltd, 4070 Basel, Switzerland.
  • 2. Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, England; Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, England.
  • 3. Pharma Research and Early Development, Immunology and Inflammation, F. Hoffmann-La Roche Ltd, 4070 Basel, Switzerland.
  • 4. Pharma Research and Early Development, Therapeutic Modalities, F. Hoffmann-La Roche Ltd, 4070 Basel, Switzerland.
  • 5. Department of Biochemistry and Biophysics, Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, CA 94158, USA.
  • 6. Pharma Research and Early Development, Therapeutic Modalities, F. Hoffmann-La Roche Ltd, 4070 Basel, Switzerland. Electronic address: [email protected].
PMID: 39983727 DOI: 10.1016/j.stemcr.2025.102420
Abstract

Here, we developed a CRISPR-Cas9 arrayed screen to investigate lipid handling pathways in human induced pluripotent stem cell (iPSC)-derived microglia. We established a robust method for the nucleofection of CRISPR-Cas9 ribonucleoprotein complexes into iPSC-derived myeloid cells, enabling genetic perturbations. Using this approach, we performed a targeted screen to identify key regulators of lipid droplet formation dependent on Apolipoprotein E (apoE). We identify the Mammalian Target of Rapamycin Complex 1 (mTORC1) signaling pathway as a critical modulator of lipid storage in both APOE3 and apoE knockout microglia. This study is a proof of concept underscoring the utility of CRISPR-Cas9 technology in elucidating the molecular pathways of lipid dysregulation associated with Alzheimer's disease and neuroinflammation.

Keywords
APOE; CRISPR-Cas9 gene editing; arrayed genetic screening; iPSC-derived microglia; lipid accumulation; lipid droplet screen; lipid metabolism; lipid regulation; lysosome; mTORC1.
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