A CRISPRi/a platform in human iPSC-derived microglia uncovers regulators of disease states

Microglia are critical for brain health, yet understanding how specific genetic variants influence their diverse functional states in neurodegenerative diseases like Alzheimer's remains a major challenge. The authors sought to create a scalable, reliable platform to systematically map the functional consequences of genetic perturbations in human microglia, enabling both discovery and therapeutic screening.

To overcome the limitations of lengthy differentiation protocols—which often create population bottlenecks—the authors engineered human iPSCs to inducibly express six key transcription factors (PU.1, MAFB, CEBPα, CEBPβ, IRF5, and IRF8). This allowed for the efficient production of microglia-like cells (iTF-Microglia) in just 8 days. By integrating inducible CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) machinery into these cells, the researchers conducted pooled screens targeting the 'druggable genome' to identify modifiers of survival, inflammatory activation (using CD38 as a marker), and phagocytosis of rat brain synaptosomes.

The platform successfully identified several microglia-specific regulators of key cellular processes. Specifically, the authors found that CSF1R is essential for microglial survival, while knocking down genes such as MED1 and CDK12 increased inflammatory activation markers. Furthermore, through single-cell RNA sequencing (CROP-seq), they uncovered nine distinct transcriptional states within the iTF-Microglia, including a SPP1-expressing state that closely resembles microglia seen in human Alzheimer's disease brains. Crucially, they demonstrated that the abundance of these SPP1+ microglia is highly sensitive to genetic and pharmacological modulation of the MAPK14 and CSF1R pathways.

This platform bridges the gap between high-throughput genetic screening and human-relevant cellular models. By enabling the functional characterization of diverse microglial states, it provides a powerful tool for identifying novel therapeutic targets that could potentially reset diseased microglial populations to a more homeostatic state, rather than simply depleting them. The data are openly available for the research community to explore on the CRISPRbrain platform.