Extended Data Figure 5. Mono-allelic knockout of POLR2A sensitizes HCT116 cells to POLR2A inhibition.

a, Schematic illustration of the Cas9/sgRNA-targeting sites in the POLR2A gene. Two single-guide RNA(sgRNA)-targeting sequences are shown and the protospacer-adjacent motif (PAM) sequences are highlighted in red. b, Efficiency of the Cas9-mediated cleavage of POLR2A in HCT116 cells measured by the Surveyor assay. c, Sequences of mutant POLR2A alleles in the cell colonies #14 and #5. PAM sequences are highlighted in red. Small deletions in the targeted region led to open reading frame shift, producing only a short stretch of the N-terminal peptide without any functional domains of POLR2A. d, Protein levels of POLR2A in POLR2A^neutral and POLR2A^loss HCT116 cells. e, Growth curves of POLR2A^neutral and POLR2A^loss HCT116 cells. f, Relative proliferation of POLR2A^neutral and POLR2A^loss cells treated with actinomycin D. g, Effect of POLR2A knockdown on the POLR2A^neutral and POLR2A^loss HCT116 cells. Experiments were performed as described in Extended Data Fig. 3c. **p < 0.01, ns: not significant. h, Dox-induced partial suppression of POLR2A inhibited the growth of POLR2A^loss HCT116 cells, but not of parental POLR2A^neutral HCT116 cells. Data are mean and s.d. of three independent experiments in the figure.