Knockout of human anxa6 gene in Caco-2 cells by CRISPR/Cas9 system

Abstract:

[Objective] To study the interaction between Escherichia coli O157:H7 effector protein EspF and host ANXA6 protein and its pathogenic mechanism, we constructed a stable Caco-2 cell line with the knockout of anxa6 using CRISPR/Cas9 system.[Methods] We designed and synthesized three small guide RNA (sgRNA) which can specifically recognize anxa6 gene. We constructed Lenticrisprv2-sgRNA recombinant plasmid and transfected it into 293T cells to prepare sgRNA-Cas9 lentivirus. Then we infected Caco-2 cells with lentivirus, and applied puromycin to screen the positive cells. We isolated the monoclonal cells by limiting dilution and sequenced the cells to evaluate the knock-out of gene anxa6 and the off-target effect. Western blotting was employed to detect the expression level of ANXA6, cell counting kit 8 (CCK8) assay to determine cell proliferation, and immunofluorescence to detect the distribution of tight junction protein ZO-1. [Results] The anxa6 gene in Caco-2 cell line was knocked out, and no off-target effect in the 10 predicted sites was found. The knockout of anxa6 had no significant effect on cell proliferation. ZO-1 of Caco-2 and Caco-2^anxa6‒/‒ cells displayed continuous distribution along the cell membrane, with complete structure. After transfection with EspF plasmids, the distribution of tight junction was incomplete with clear gaps and crack-like appearance. [Conclusion] We successfully constructed the Caco-2 cell line with the knock-out of anxa6. The cell line was used to preliminarily explore the role of ANXA6 protein in distribution of tight junction protein. This study provides an effective tool for exploiting the molecular mechanism of O157:H7 in mediating intestinal barrier injury through EspF-ANXA6 interaction.

Key words:CRISPR/Cas9 system;anxa6;EspF proteins;Caco-2 cells;tight junction