Research Article

Disruption of HPV16-E7 by CRISPR/Cas System Induces Apoptosis and Growth Inhibition in HPV16 Positive Human Cervical Cancer Cells

Figure 2

Evaluation of gRNA/Cas9-mediated HPV16-E7 DNA cleavage by the SSA assay. (a) Schematic representation of luciferase assay. Inactive luciferase gene is separated by left homology arm (LH), 20 nt gRNA recognition site, corresponding PAM sequence, a stop codon, and right homology arm (RH). When the CRISPR gRNA/Cas9 system causes DSBs at specific recognition sites, an active luciferase will form due to SSA repair pathway. Cas9 plasmid, site-specific gRNA, pSSA Rep-gRNA, and renilla plasmid were cotransfected into HEK293 cells. At 48 h after transfection, the firefly luciferase (b) and renilla luciferase (c) activity were measured by a microplate reader. Cells transfected with GZF3-L3, GZF1-R3, and pSSA Rep3-1 plasmids were used as positive control and cells treated with only Cas9 plasmid were applied as negative control. Cells treated with Con-gRNA (HPV16E6-gRNA-1) which had been proved to be inactive preexperimentally and Cas9 enzyme together were used as a control. RLU represents relative light units ( compared to the negative test, , per Student’s -test).
612823.fig.002a
(a)
612823.fig.002b
(b)
612823.fig.002c
(c)