Comparative analysis of RNA interference approaches for targeted gene silencing: Efficiency, durability, and off-target effects

Abstract

RNA interference (RNAi) has revolutionised functional genomics and emerged as a promising therapeutic modality, yet optimal strategies for achieving efficient and specific gene silencing remain incompletely defined. This research systematically compared small interfering RNA (siRNA) and short hairpin RNA (shRNA) approaches for silencing six therapeutically relevant oncogenes, evaluating knockdown efficiency, temporal dynamics, and off-target effects. The investigation was conducted at the State Key Laboratory of Molecular Genetics, Jiangnan Institute of Biotechnology, Suzhou, China, between January 2023 and October 2023. Human cancer cell lines (HeLa, A549, MCF-7) were transfected with chemically synthesised siRNAs or lentiviral-delivered shRNAs targeting VEGFA, BCL2, EGFR, MYC, KRAS, and TP53. Gene silencing was quantified by RT-qPCR and Western blotting at multiple time points over 168 hours. Off-target effects were assessed by transcriptome-wide RNA sequencing. siRNA transfection achieved superior initial knockdown efficiency compared to shRNA (mean 83.5% versus 69.8% mRNA reduction at 48 hours, p50% of control by 96 hours. shRNA-mediated silencing, while initially less potent, maintained stable suppression (4 weeks in stably transduced cells. Off-target analysis revealed that siRNA sequences with seed region complementarity to unintended transcripts caused measurable downregulation of 12-47 genes per siRNA, while shRNA showed similar off-target profiles. Importantly, optimised siRNA design incorporating 2'-O-methyl modifications at positions 1-2 of the sense strand reduced off-target effects by 67% without compromising on-target activity. These findings establish quantitative benchmarks for RNAi approach selection based on experimental requirements.