Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan.
Front Immunol. 2023 Jul 7;14:1169725. doi: 10.3389/fimmu.2023.1169725. eCollection 2023.
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 (Aβ) and glioblastoma phagocytosis in microglia. We showed that CRISPR genome editing could enhance the phagocytic activities of HMC3.
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 by fluorescent Aβ and glioblastoma phagocytosis assays.
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 and 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 Aβ aggregates in the acidified lysosomes. HMC3 mutants also changed the phagocytic characteristic toward apoptotic glioblastoma cells.
CRISPR 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.
小胶质细胞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存在挑战，我们对小胶质细胞生物学的理解有限。HMC3 是人小胶质细胞研究的重要细胞系，因为它易于获得，并且在标准实验室中易于维持。尽管 HMC3 被广泛用于小胶质细胞研究，但尚未描述强大的遗传方法。在这里，我们报告了一种 CRISPR 基因组编辑平台，通过电穿孔 Cas9 核糖核蛋白（Cas9 RNP）和合成 DNA 修复模板，实现 HMC3 的快速和精确遗传修饰。作为概念验证演示，我们针对调节小胶质细胞中淀粉样蛋白 β（Aβ）和神经胶质瘤吞噬作用的基因进行了靶向。我们表明，CRISPR 基因组编辑可以增强 HMC3 的吞噬活性。
我们通过电穿孔预先组装的 Cas9 RNP 在 HMC3 中进行 CRISPR 基因敲除（KO）。共导入 DNA 修复模板可实现特定位置的基因敲入（KI），包括表位标签、合成启动子和荧光报告基因。通过 DNA 测序进行基因型编辑效率测定，通过免疫荧光染色和流式细胞术进行表型编辑效率测定。通过荧光 Aβ 和神经胶质瘤吞噬测定来检查基因编辑的 HMC3 细胞。
我们的平台通过高通量 DNA 测序实现了强大的单（>90%）和双（>70%）KO，而没有检测到脱靶编辑。我们还插入了一个合成的 SFFV 启动子，以有效地上调与小胶质细胞吞噬作用相关的内源性基因和基因的表达。CRISPR 编辑的 HMC3 显示出稳定的表型，并增强了对荧光标记的 Aβ1-42 肽的吞噬作用。共聚焦显微镜进一步证实了 Aβ 聚集体在酸化溶酶体中的定位。HMC3 突变体还改变了对凋亡神经胶质瘤细胞的吞噬特性。
Cas9 RNP 电穿孔的 CRISPR 基因组编辑是一种强大的方法，可以对 HMC3 进行遗传修饰，用于功能研究，如 Aβ 和肿瘤吞噬作用的研究，并且易于采用来研究小胶质细胞生物学的其他方面。
Zotero 插件
