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

Abstract

Microglia are emerging as key drivers of neurological diseases. However, we lack a systematic understanding of the underlying mechanisms. Here, we present a screening platform to systematically elucidate functional consequences of genetic perturbations in human induced pluripotent stem cell-derived microglia. We developed an efficient 8-day protocol for the generation of microglia-like cells based on the inducible expression of six transcription factors. We established inducible CRISPR interference and activation in this system and conducted three screens targeting the ‘druggable genome’. These screens uncovered genes controlling microglia survival, activation and phagocytosis, including neurodegeneration-associated genes. A screen with single-cell RNA sequencing as the readout revealed that these microglia adopt a spectrum of states mirroring those observed in human brains and identified regulators of these states. A disease-associated state characterized by osteopontin (SPP1) expression was selectively depleted by colony-stimulating factor-1 (CSF1R) inhibition. Thus, our platform can systematically uncover regulators of microglial states, enabling their functional characterization and therapeutic targeting.

Functional regulatory variants implicate distinct transcriptional networks in dementia

“…For the CROP-seq (pooled CRISPRi) screen, two iPSC lines were maintained at the University of California, San Francisco. For microglial differentiation, a transcription factor-based protocol (iTF-microglia) was used as described by Dräger et al (33). Similarly, neurons for the CROP-seq screen were generated using the transcription factor-based i 3 N approach as described by Tian et al (32).…”

Cerebral Organoids as an Experimental Platform for Human Neurogenomics

“…Disruption of nuclear architecture of progenitor cells has been observed [ 230 ]. The advent of CRISPR interference technology, which enables flexible targeting of genes upregulated in disease states [ 231 , 232 , 233 ], can be applied to target and limit abnormal activation of pathways discovered using in vitro models [ 234 ].…”

Pluripotent stem cell strategies for rebuilding the human brain

“…One study has shown that overexpression of transcription factors CEBPA and PU.1 coupled with CNS-patterning molecules described above can generate Microglia-like cells from human iPSC ( Chen et al, 2021 ) with a second one showing improved efficiency by overexpressing PU.1 from primitive hematopoietic progenitors ( Sonn et al, 2022 ). A recent study has defined a set of six transcription factors for the generation of microglia-like cells at a scale sufficient for genetic screening ( Drager et al, 2022 ). Following the progress in the derivation of specific neuronal populations, it is plausible that newer approaches might find that just a few transcriptional factors could be sufficient, when coupled with small molecules, for the generation of this cell type.…”