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.近二十年来，短发夹RNA（short hairpin RNA，shRNA）已成为功能缺失研究的强大工具，助力研究人员有效沉默基因表达并探究基因功能。然而，当前的shRNA骨架在设计时未考虑小RNA的均一性，导致内源微RNA（microRNA）生成机器对其加工不够精确。这一固有局限会产生非靶向小RNA，这些非靶向小RNA可结合至Argonaute（AGO）蛋白并形成功能性小RNA，引发超出预期靶基因沉默之外的非靶向基因调控，进而限制了其更广泛的应用。为解决这一局限，我们设计了一种新型shRNA骨架，命名为shRNAoneV4，该骨架可实现精准加工，并显著减少非靶向向导RNA的生成。shRNAoneV4可提升靶向向导RNA的小RNA均一性，同时表达两端热稳定性各异的向导RNA文库，使其成为RNA干扰（RNA interference，RNAi）筛选中实现精准小RNA表达的可靠工具。此外，我们提出shRNA筛选可替代小干扰RNA（small interfering RNA，siRNA）筛选，因为二者均利用内源RNAi通路。作为概念验证实验，我们将shRNAoneV4应用于siRNA靶标平铺筛选，使用靶向运甲状腺素蛋白（TTR）转录本平铺位点的shRNAoneV4文库。我们在TTR转录本上鉴定出两个包含高效shRNA靶位点的区域，表明这些区域更适用于RNAi实验。我们通过siRNA验证了这些shRNA靶位点的敲低效率，证实了用更简便的混合shRNA筛选方法替代劳动密集型siRNA筛选方法的可行性。此外，我们还证实了shRNAoneV4在微RNA功能研究中的应用价值。通过用shRNAoneV4文库模拟内源微RNA，我们成功鉴定出了调控肝细胞癌（hepatocellular carcinoma，HCC）索拉非尼耐药性的微RNA。这凸显了利用shRNAoneV4探究不同生物学环境中微RNA功能的潜力——此前的shRNA骨架因加工不精确的小RNA引入噪声，阻碍了这类研究的开展。此外，编码微RNA变体的文库还揭示了依赖非种子区域进行靶标识别的微RNA，助力解析调控非种子区域对靶位点定义的作用机制。综上，我们向全球科研界推出了一款全新的金标准shRNA骨架，为RNAi筛选提供了可靠的技术路径。我们的研究结果证实，利用该RNAi筛选策略可大规模鉴定微RNA功能，从而加速科研界对微RNA在各类生物学过程中作用的认知。