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. Epub 2025 Feb 20.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apolipoproteins E / genetics
  • Apolipoproteins E / metabolism
  • CRISPR-Cas Systems* / genetics
  • Cell Differentiation
  • Humans
  • Induced Pluripotent Stem Cells* / cytology
  • Induced Pluripotent Stem Cells* / metabolism
  • Lipid Metabolism
  • Mechanistic Target of Rapamycin Complex 1 / metabolism
  • Microglia* / cytology
  • Microglia* / metabolism
  • Models, Biological*
  • Myeloid Cells* / cytology
  • Myeloid Cells* / metabolism
  • Signal Transduction

Substances

  • Mechanistic Target of Rapamycin Complex 1
  • Apolipoproteins E