citations

Interrogation of human microglial phagocytosis by CRISPR genome editing

Authors:

Jason Cheng-Yu Chang, Cheng-You Wang, Steven Lin

In:

Source: Frontiers in Immunology
Publication Date: (2023)
Issue: :

Research Area:

Neurobiology

Basic Research

Cells used in publication:

BV2: Species: mouse; Tissue Origin: brain
HMC3: Species: human; Tissue Origin: brain

Platform:

4D-Nucleofector® X-Unit

Experiment

Electroporation was performed in the Lonza 4D Nucleofector X-unit using the pulse codes listed in Table S3. The electroporation procedure was as described previously with modifications (29). Briefly, 2 × 10⁵ of HMC3 cells per electroporation reaction were collected by Accutase dissociation. The cells were washed once with DPBS and resuspended in 20 µL of P3 nucleofection buffer (Lonza). Plasmid pmaxGFP (encoding the turboGFP reporter gene, Lonza) was added at 0.4 µg to screen for the DNA electroporation condition. Cas9 RNP (targeting the CD40 gene) was added at 40 pmol to screen for the RNP electroporation condition. [...]

Electroporation was performed using the protocol for HMC3 and a set of BV-2-specific pulse codes listed in Table S3. Briefly, 2 × 10⁵ of BV-2 cells per electroporation reaction were collected by Accutase dissociation, washed once with DPBS, and resuspended in 20 µL of SF or P3 nucleofection buffer (Lonza). Cas9 RNP (in complex with sgRNA17 targeting the mouse CD40 gene) was added at 40 pmol to test the RNP electroporation condition. CM158 + SF is the condition suggested by Lonza and CA137 + P3 is our best condition for HMC3. The other conditions were recommended by Lonza’s optimization scheme. [...]

Table 3: Screening for optimal electroporation condition using plasmid pmaxGFP and CD40-targeting Cas9 RNP as cargos. Pulse codes for Lonza 4D nucleofector system are listed. The percentages of viability, GFP-positive and CD40-negative cells were determined three days after electroporation by flow cytometry. The top seven codes for plasmid electroporation were further tested for Cas9 RNP electroporation in HMC3 cells. The electroporation of BV-2 cells used a different set of pulse codes. CM158 + SF buffer is recommended by Lonza Nucleofection database, and CA137 + P3 buffer is the best HMC3 condition for cross comparison.

Abstract

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 Aß_1-42 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.

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