Table_1_Interrogation of human microglial phagocytosis by CRISPR genome editing.xlsx

BackgroundMicroglia 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 (Aβ) and glioblastoma phagocytosis in microglia. We showed that CRISPR genome editing could enhance the phagocytic activities of HMC3.
MethodsWe 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 Aβ and glioblastoma phagocytosis assays.
ResultsOur 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 Aβ1-42 peptides. Confocal microscopy further confirmed the localization of Aβ1-42 aggregates in the acidified lysosomes. HMC3 mutants also changed the phagocytic characteristic toward apoptotic glioblastoma cells.
ConclusionCRISPR genome editing by Cas9 RNP electroporation is a robust approach to genetically modify HMC3 for functional studies such as the interrogation of Aβ and tumor phagocytosis, and is readily adoptable to investigate other aspects of microglial biology.

【背景】小胶质细胞（microglia）是中枢神经系统的重要组成部分，但由于获取和培养原代人小胶质细胞（primary human microglia）存在技术难题，目前学界对小胶质细胞生物学特性的认知仍较为有限。HMC3是研究人源小胶质细胞的重要细胞系（cell line），因其易于获取且可在常规实验室中稳定培养。尽管HMC3已广泛应用于小胶质细胞相关研究，但目前尚未报道成熟可靠的遗传操作方法。本研究构建了一套基于Cas9核糖核蛋白（Cas9 ribonucleoproteins, Cas9 RNP）电穿孔与合成DNA修复模板（synthetic DNA repair templates）的CRISPR基因组编辑（CRISPR genome editing）平台，可实现对HMC3细胞的快速精准遗传修饰（genetic modifications）。为验证该平台的可行性，我们靶向调控小胶质细胞中β淀粉样蛋白（amyloid beta, Aβ）与胶质母细胞瘤（glioblastoma）吞噬作用（phagocytosis）的相关基因进行编辑，结果显示CRISPR基因组编辑可增强HMC3细胞的吞噬活性。
【方法】本研究通过电穿孔预组装的Cas9 RNP，在HMC3细胞中实现CRISPR基因敲除（gene knockout, KO）。同步导入DNA修复模板可实现表位标签（epitope tag）、合成启动子（synthetic promoter）与荧光报告基因（fluorescent reporter gene）的位点特异性敲入（site-specific knock-in, KI）。编辑效率通过DNA测序从基因型水平进行验证，并通过免疫荧光染色（immunofluorescent staining）与流式细胞术（flow cytometry）从表型水平进行检测。我们采用荧光标记Aβ与胶质母细胞瘤吞噬实验，对基因编辑后的HMC3细胞进行体外功能评估。
【结果】本平台可实现高效的单基因（敲除效率>90%）与双基因（敲除效率>70%）敲除，且经高通量DNA测序（high throughput DNA sequencing）未检测到脱靶编辑。我们还通过插入合成SFFV启动子，有效上调了与小胶质细胞吞噬功能相关的内源性CD14与TREM2基因的表达。经CRISPR编辑的HMC3细胞表型稳定，且对荧光标记的Aβ1-42肽段的吞噬能力显著增强。共聚焦显微镜（confocal microscopy）进一步证实，Aβ1-42聚集物定位于酸化溶酶体（acidified lysosomes）中。HMC3基因突变细胞对凋亡胶质母细胞瘤细胞的吞噬特性也发生了改变。
【结论】通过Cas9 RNP电穿孔实现的CRISPR基因组编辑是一种可靠的遗传修饰方法，可用于HMC3细胞的功能研究（如解析Aβ与肿瘤吞噬机制），且易于推广应用于小胶质细胞生物学其他方面的研究。