Interrogation of human microglial phagocytosis by CRISPR genome editing.

Background: Microglia are an integral part of central nervous system, but our understanding of microglial biology is limited due to the challenges in obtaining and culturing primary human microglia. HMC3 is an important cell line for studying human microglia because it is readily accessible and straightforward to maintain in standard laboratories. Although HMC3 is widely used for microglial research, a robust genetic method has not been described. Here, we report a CRISPR genome editing platform, by the electroporation of Cas9 ribonucleoproteins (Cas9 RNP) and synthetic DNA repair templates, to enable rapid and precise genetic modifications of HMC3. For proof-of-concept demonstrations, we targeted the genes implicated in the regulation of amyloid beta (Aß) and glioblastoma phagocytosis in microglia. We showed that CRISPR genome editing could enhance the phagocytic activities of HMC3. Methods: We performed CRISPR gene knockout (KO) in HMC3 by the electroporation of pre-assembled Cas9 RNP. Co-introduction of DNA repair templates allowed site-specific knock-in (KI) of an epitope tag, a synthetic promoter and a fluorescent reporter gene. The editing efficiencies were determined genotypically by DNA sequencing and phenotypically by immunofluorescent staining and flow cytometry. The gene-edited HMC3 cells were examined in vitro by fluorescent Aß and glioblastoma phagocytosis assays. Results: Our platform enabled robust single (>90%) and double (>70%) KO without detectable off-target editing by high throughput DNA sequencing. We also inserted a synthetic SFFV promoter to efficiently upregulate the expression of endogenous CD14 and TREM2 genes associated with microglial phagocytosis. The CRISPR-edited HMC3 showed stable phenotypes and enhanced phagocytosis of fluorescence-labeled Aß1-42 peptides. Confocal microscopy further confirmed the localization of Aß1-42 aggregates in the acidified lysosomes. HMC3 mutants also changed the phagocytic characteristic toward apoptotic glioblastoma cells. Conclusion: CRISPR genome editing by Cas9 RNP electroporation is a robust approach to genetically modify HMC3 for functional studies such as the interrogation of Aß and tumor phagocytosis, and is readily adoptable to investigate other aspects of microglial biology.