Supporting data for "A Precise shRNA Backbone for siRNA Target Screening and Quantitative microRNA Biology"
Over the past two decades, short hairpin RNA (shRNA) has emerged as a powerful tool for loss-of-function studies, enabling researchers to effectively silence gene expression and investigate gene function. Current shRNA backbones, however, were designed without consideration for small RNA homogeneity, resulting in imprecise processing by endogenous microRNA biogenesis machinery. This inherent limitation leads to the production of unintended small RNAs, which can be loaded onto Argonaute (AGO) proteins and yield functional small RNAs, introducing unintended gene regulation beyond the intended gene knockdown and restricting broader applications.
To address this limitation, we engineered a novel shRNA backbone, named shRNAoneV4, which allows for precise processing and significantly reduces the production of unintended guides. shRNAoneV4 demonstrates enhanced small RNA homogeneity of the intended guide while expressing a library of guides with varying thermostability at both ends, making it a robust tool for precise small RNA expression in RNA interference (RNAi) screening.
Furthermore, we propose that shRNA screening could replace siRNA screening, as both strategies leverage the endogenous RNAi pathway. As a proof-of-concept experiment, we applied shRNAoneV4 in an siRNA target tiling screen, utilizing a library of shRNAoneV4 targeting tiling sites on TTR transcripts. We identified two regions on TTR transcripts that contained efficient shRNA target sites, suggesting that these regions are more effective for RNAi. The efficiencies of these shRNA target sites were validated with siRNAs, underscoring the feasibility of replacing the labour-intensive siRNA screening approach with a simpler pooled shRNA screening method.
Additionally, we demonstrated the utility of shRNAoneV4 in studying microRNA functions. By mimicking endogenous microRNAs with the shRNAoneV4 library, we successfully identified microRNAs that regulate sorafenib resistance in hepatocellular carcinoma (HCC). This highlights the potential of exploiting shRNAoneV4 to investigate microRNA functions in various biological contexts—an endeavour that was hindered by previous shRNA backbones due to noise introduced by imprecisely processed small RNAs. Moreover, libraries encoding microRNA variants revealed microRNAs that rely on non-seed regions for target recognition, facilitating studies on the principles governing non-seed contributions to defining target sites.
In conclusion, we present a new gold standard shRNA backbone to the research community worldwide, opening a reliable approach for RNAi screening. Our findings demonstrate the feasibility of employing this RNAi screening strategy for the large-scale identification of microRNA functions, thereby accelerating the research community’s understanding of microRNAs’ roles in various biological processes.