Identifying Cleaved and Noncleaved Targets of Small Interfering RNAs and MicroRNAs in Mammalian Cells by SpyCLIP.

Recently, the US Food and Drug Administration (FDA) approved the first small interfering RNA (siRNA) drug, marking a significant milestone in the therapeutic use of RNA interference (RNAi) technology. However, off-target gene silencing by siRNA remains one of the major obstacles in siRNA therapy. Although siRNA off-target effects caused by a mechanism known for microRNA (miRNA)-mediated gene repression have been extensively discussed, whether RNAi can cause unintended cleavage through the effector protein AGO2 at sites harboring partially complementary sequences to the siRNA remains unknown. Here, we report a strategy to establish a comprehensive picture of siRNA cleaved and noncleaved off-targets by performing SpyCLIP using wild-type and catalytically inactive AGO2 mutants in parallel. Additionally, we investigated naturally occurring cleavage events mediated by endogenous miRNAs using the same strategy. Our results demonstrated that AGO2 SpyCLIP is a powerful method to identify both the cleaved and noncleaved targets of siRNAs, providing valuable information for improving siRNA design rules. Approval of patisiran and givosiran by the FDA heralds the arrival age of siRNA medicines. However, off-target effect remains a challenge in developing siRNA therapy. Herein, corresponding authors and colleagues report an effective strategy to identify siRNA cleaved and noncleaved off-targets, providing valuable information for improving siRNA design rules.

近期，美国食品药品监督管理局（FDA）批准了首款小干扰RNA（siRNA）药物，这标志着RNA干扰（RNAi）技术在治疗应用方面取得了重大里程碑。然而，siRNA的脱靶基因沉默现象仍是siRNA疗法的主要障碍之一。尽管由一种已知的微小RNA（miRNA）介导的基因抑制机制所导致的siRNA脱靶效应已被广泛探讨，但RNAi是否会通过效应蛋白AGO2在与siRNA具有部分互补序列的位点引发非预期的切割，目前仍不清楚。在此，我们报告了一种策略，即通过同时使用野生型和催化失活的AGO2突变体进行SpyCLIP技术，来全面描绘siRNA切割和未切割的脱靶情况。此外，我们运用相同策略研究了内源性miRNA介导的自然发生的切割事件。我们的研究结果表明，AGO2 SpyCLIP是一种强大的方法，可识别siRNA的切割和未切割靶标，为完善siRNA设计规则提供了有价值的信息。 FDA对帕替西韦（patisiran）和吉沃西韦（givosiran）的批准预示着siRNA药物时代的到来。然而，脱靶效应在开发siRNA疗法过程中仍是一项挑战。在此，通讯作者及其同事报告了一种有效的策略，用于识别siRNA切割和未切割的脱靶情况，为完善siRNA设计规则提供了有价值的信息。