A CRISPR-based technical strategy for gene tagging in lung cancer cells

Objective To establish a clustered regularly-interspaced short palindromic repeats (CRISPR)-based technical strategy for gene tagging in lung cancer cells. Methods Cas9, DCAF12small guide RNA (sgRNA) and double cut donor DNA plasmids were constructed by a PCR-based seamless cloning method. These plasmids were co-transfected into A549 cells and the stable expression clones were selected with puromycin. The puromycin-resistant cells were transfected with sall interfering RNA (siRNA) against DCAF12 for 72 h. The expression and knockdown efficiency of DCAF12 were then analyzed by Western blot with an anti-HA antibody to confirm the correct tagging. The floxed puromycin gene in the validated cells was removed by adenovirus expressing Cre recombinase which was further confirmed by PCR and Western blot. Cancer cell growth was determined by proliferation and clonogenic survival assays. Co-immunoprecipitation by anti-HA antibody was used to test whether DCAF12 protein is capable of binding to cullin-4-RING E3 ligase (CRL4) complex associated proteins. Results PCR and Western blot confirmed that the HA tag was successfully in frame fused to the N-terminal of DCAF12 protein and excision of puromycin resistant marker significantly increased HA-DCAF12 expression in cells. Cancer cell growth seemed not to be influenced by HA-DCAF12 expression. Functionally, HA-DCAF12 could efficiently pull down cullin-4A and DDB1, the key components of CRL4 complexes, indicating that the HA tagging did not affect DCAF12 biological functions. Conclusions By tagging DCAF12 gene in lung cancer cells, we successfully establish a simple and rapid method for gene tagging.