[CRISPR/Cas9-based knockout of GPR43 gene in RAW264.7 cells inhibits their phagocytosis to Klebsiella pneumoniae].

Objective To construct cell line RAW264.7 with stable knockout of GPR43 gene using CRISPR/Cas9 system, and explore the role and mechanism of GPR43 gene in Klebsiella pneumoniae infection. Methods Three pairs of small-guide RNA (sgRNA) targeting the GPR43 gene were designed and inserted into plasmid pLenticrisprV2. The recombinant plasmid pLenticrisprV2 containing sgRNA was packaged using a lentivirus packaging system. RAW264.7 cells were transfected with viruses, and monoclonal cells were screened using puromycin. The genomic DNA was extracted from the amplified monoclonal cells. The GPR43 gene-related sequences were sequenced and compared with the wild-type GPR43 gene to confirm the cell line with successful knockout (GPR43 ^-/- RAW264.7 cells). The expression of GPR43 protein was detected by Western blotting. After GPR43 ^-/- RAW264.7 cells were transfected with Klebsiella pneumoniae, the changes in the expression of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in the cells were detected using real-time quantitative PCR. Additionally, the phagocytic capacity of RAW264.7 cells after GPR43 knockout was observed. Results Western blotting confirmed that GPR43 protein was not expressed in the selected monoclonal cells, and DNA sequencing showed that 34 bases were missing at the insertion site of sgRNA, which proved that GPR43 gene was successfully knocked out. After GPR43 ^-/- RAW264.7 cells were transfected with Klebsiella pneumoniae, the levels of IL-1β, IL-6 and TNF-α expression in the cells were all lower than those in the control group, and the phagocytic capacity of GPR43 ^-/- RAW264.7 cells to Klebsiella pneumoniae decreased. Conclusion CRISPR/Cas9-based knockout of GPR43 gene in RAW264.7 cells can inhibit their phagocytosis for Klebsiella pneumoniae and production of inflammatory cytokines.