Fig. 4: CRISPR interference targeting enhancers on hybrid ecDNA blocks HPV oncogene expression.

CRISPR interference, using dCas9-KRAB to target enhancers on the hybrid ecDNA of HMS001, was performed. gRNAs targeting S1: the long part (gRNA#1) and S2: the short part (gRNA#2) of the enhancer on hybrid ecDNA of HMS001 and nontarget controls were used (A). The expression of dCas9 after doxycycline induction was confirmed by qPCR (B) and Western blotting (C). Two biological replicates were used and the median with SD was shown in qPCR results (B). MYC and GAPDH were used as controls (C). Western blotting results of E6 and E7 of each CRISPRi condition indicate E6 and E7 expression were reduced by CRISPRi targeting the S2 enhancer (D). Proliferation assay results targeting the nontarget control (nonT), S1 enhancer, and S2 enhancer in HMS001, SCC154, and NOKSI were shown. Twelve biological replicates were used and median with SD were shown. Dox induction significantly inhibited the proliferation only in targeting S2 in HMS001 (**P = 0.004, two-tailed student’s t-test) (E), but not in SCC154 or NOKSI (F and G, two-tailed student’s t-test). Source Data is available for panels B and E–G.