Figure 2

Experimental validation of the therapeutic strategy with essential gene RRP9 in human colorectal cancer cell lines. (a) Genomic DNA sequences of wild-type RRP9 and RRP9 of the SNUC4 cell line, and sgRNA with the protospacer adjacent motif (PAM) sequence targeting passenger single-nucleotide variant (SNV) of SNUC4 RRP9. The SNV is represented in red and the PAM sequence in blue. (b) Frequency of insertions and deletions (InDels) and wild-type reads from RRP9-targeted next-generation sequencing in sgRRP9-SNV-transduced SNUC4 and SW620 cells. (c) The effect of sgRNA targeting the RRP9 SNV of SNUC4 on RRP9 protein levels was assessed via immunoblotting whole-cell lysates from SNUC4 and SW620 cell lines. sgNT, non-targeting sgRNA. (d) Effect of sgRNA targeting RRP9 SNV of SNUC4 on colony formation of SNUC4 and SW620 cell lines. (Left) Representative images from the colony formation assay and (right) quantification of crystal violet-stained colonies. The statistical significance of the difference in colony numbers between groups was assessed using a two-sided Wilcoxon rank-sum test. Data are presented as mean ± standard deviation (s.d.). NS, not significant. (e) Effect of sgRNA targeting the RRP9 SNV of SNUC4 on (left) SNUC4 and (right) SW620 tumor growth. The statistical significance of the difference in tumor growth between groups was determined using two-way ANOVA. Data are presented as mean ± standard deviation (s.d.). Images show the dissected tumors after being sacrificed. NS, not significant.