Genome‐wide CRISPR knockout screening to identify key regulators of phagocytosis in induced pluripotent stem cell derived microglia

Abstract

AbstractBackgroundA whole genome knockout CRISPR screen in human induced pluripotent stem cell (hiPSC) derived microglia was undertaken to identify regulators of phagocytosis. The secondary objective was to cross reference these genes to Alzheimer’s Disease genome‐wide association studies to provide potential therapeutic targets for drug development.MethodHiPSC‐derived microglia precursor cells were transduced with the TKOv3 genome‐wide CRISPR/Cas9 knockout library and differentiated for two weeks to microglia in our recently published media. hiPSC‐microglia were then fed dead GFP/mCherry fluorescent labelled SH‐SY5Y neuronal cells as phagocytic meal. Phagocytosis proceeded for 6 hours before washing away non‐phagocytosed SH‐SY5Y, harvesting and fixing the hiPSC‐microglia. hiPSC‐microglia were sorted into high and low levels of phagocytosis by fluorescent activated cell sorting (FACS) for single mCherry‐positive cells as GFP is quenched due to a low pH environment. Genomic DNA was extracted from sorted cell populations and the integrated guide RNA were sequenced by Illumina Novoseq.ResultMicroglia were successfully transduced at a multiplicity of infection at 0.7 to maximize single guide integration. Successful phagocytosis was observed via FACS, with over 60% of hiPSC‐microglia phagocytosing dead neurons after 6 hours. Four populations of varying levels of phagocytosis were harvested and genomic DNA extracted and sequenced.ConclusionWe have developed a pipeline for the first successful genome‐wide CRISPR knockout screening in hiPSC‐microglia and have used this to identify regulators of phagocytosis.