Construction Of Anxa6 Knockout Strain Based On CRISPR/Cas9 Technology And Its Mechanism In Cell Tight Junctions

Background and SingnificanceANXA6 is the largest member of the membrane association protein family and is highly expressed in most tissues.ANXA6 is mainly located in the plasma membrane and endosomal compartments and interacts with a variety of proteins and lipids to form membrane structural domains,signaling complexes,mediate transient membrane-actin interactions,and maintain intracellular cholesterol homeostasis.In several cellular and animal models,ANXA6 and several other membrane-linked proteins have been shown to be critical for membrane repair.Previously,our group screened and validated the interaction of host protein ANXA6 with E.coli O157:H7 EspF protein and found that both constitutive expression of EspF or ANXA6 and co-expression of EspF-ANXA6 reduced the levels of tight junction proteins ZO-1 and ocludin and disrupted the distribution of ZO-1.However,the role of ANXA6 protein in the disruption of tight junctions is unclear.In view of this,we intend to use the CRISPR/Cas9 system to construct a stable Caco-2 cell line with anxa6 gene knocked out,to study the proliferation and migration ability of cells with anxa6 gene deletion,and to explore how EspF protein and ANXA6 protein interaction affects the tight junctions of cells.Methodsa.According to the targeting principle of CRISPR/Cas9 system,three Sgmas targeting exons of anxa6 gene were designed,and LentiCRISPRv2-sgRNA recombinant plasmids were constructed.The plasmids were transformed into the receptor cell Stbl3,which was selected for expansion culture.The plasmids were extracted and sent for sequencing verification.b.Lentivirus package plasmids pVSVG and pCD were co-transfected with LentiCRISPRv2-sgRNA recombinant plasmids into 293T cells to synthesize lentivirus.The lentivirus was collected and infected with Caco-2 cells.The resistant cells were screened with purinomycin and monoclonal cells were produced by limited dilution.The deletion of anxa6 gene in Caco-2 cell line was detected by DNA sequencing,and the expression of ANXA6 protein was detected by WB.c.DNA fragment extraction and sequencing were performed for 10 potential off-target sites using Off-Spotter,an online off-target effect assessment tool,to verify the presence of off-target effect in Caco-2anxa6-/-cells.d.The proliferative activity of Caco-2anxa6-/-cells was measured by CCK-8 kit and the migration ability of Caco-2anxa6-/-cells was detected by scratch assay.e.The distribution of tight junction proteins and their expression levels after transfection of EspF plasmid into Caco-2-V2 cells and Caco-2anxa6-/-cells were detected by IF as well as WB.Resulta.LentiCRISPRv2-sgRNArecombinant expression plasmid was constructed.b.The lentivirus was successfully packaged and positive monoclonal cells were successfully screened with puromycin after lentivirus infection of Caco-2 cells,and a Caco-2 cell line with a 5-base deletion of anxa6 gene was successfully obtained.c.The results of off-target effect assessment showed no off-targeting at any of the predicted 10 off-target sites.d.The proliferation activity of Caco-2anxa6-/-cells was detected by CCK-8 kit,and the proliferation of Caco-2anxa6-/-cells was not significantly different from that of the original Caco-2.The migration of Caco-2anxa6-/-cells was determined by cell scratch assay,and the migration rate of Caco-2anxa6-/-cells decreased significantly compared with the original Caco-2 cells..e.After transfection of Caco-2-V2 cells and Caco-2anxa6-/-cells with EspF plasmid,immunofluorescence and WB techniques were used to detect the distribution and expression levels of tight junction proteins.Studies showed that,regardless of transfection of pEGFP-EspF plasmid or non-expression of ANXA6 protein,Proteins associated with tight junctions in cells are affected.ConclusionIn this paper,Caco-2 cell lines with stable knockout of anxa6 gene were constructed.The results showed that there was no off-target effect during the knockout process.Deletion of anxa6 gene affected cell migration.The role of ANXA6 protein in the distribution of tight junctions and the expression of related pathway proteins were initially explored,providing technical support for further research on the molecular mechanism of Escherichia coli O157:H7 mediated host intestinal barrier damage through EspF-ANXA6 interaction.