siRNAs with decreased off-target effect facilitate the identification of essential genes in cancer cells

Since the essential genes are crucial to the proliferation and survival of cancer cells, the interference of these genes is promising to be an option for cancer therapy to overcome heterogeneity. However, the essential genes are highly overestimated by RNA interference (RNAi) screenings, which is mainly caused by the pervasive off-target effect of small interference RNA (siRNA) and short hairpin RNA (shRNA). In the present study, we designed Match-Mismatch paired siRNAs to discriminate the on-target effect from off-target effect of siRNAs on cell viability. Only one of the 7 potential essential genes was validated as essential to cell viability, which demonstrates the high false positive rate in RNAi screenings. We modified the siRNA by introducing random nucleotides (N) into the guide strand to mitigate the offtarget effect, without significantly compromising the on-target effect. The whole transcriptome profile analysis of cells transfected with siRNAs with or without N indicates that siRNA-dN (with Ns on both the 2^nd and the 18^th bases of the guide strand) weakens the off-target effect by decreasing the unintended targets. The optimized siRNAs can be applied in the characterization of essential genes in cancer cells.