In parallel with the expansion of RNA interference (RNAi) techniques, accumulating evidence indicates that RNAi analyses might be seriously biased due to the off-target effects of gene-specific short hairpin RNAs (shRNAs). Our findings indicated that off-target effects of non-targeting shRNA comprise another source of misinterpreted shRNA-based data. We found that SHC016, which is one of two non-targeting shRNA controls for the MISSION (commercialized TRC) library, exerts deleterious effects that lead to elimination of the shRNA-coding cassette from the genomes of cultured murine and human cells. Here, we used a lentiviral vector with inducible SHC016 expression to confirm that this shRNA induces apoptosis in murine cells and senescence or mitotic catastrophe depending on the p53 status in human tumor cells. We identified the core spliceosomal protein, small nuclear ribonucleoprotein Sm D3 (SNRPD3), as a major SHC016 target in several cell lines and confirmed that CRISPRi knockdown of SNRPD3 mimics the effects of SHC016 expression in A549 and U251 cells. The overexpression of SNRPD3 rescued U251 cells from SHC016-induced mitotic catastrophe. Our findings disqualified non-targeting SHC016 shRNA and added a new premise to the discussion about the sources of uncertainty in RNAi results.

随着 RNA 干扰 (RNAi) 技术的扩展，越来越多的证据表明，由于基因特异性短发夹 RNA (shRNA) 的脱靶效应，RNAi 分析可能存在严重偏差。我们的研究结果表明，非靶向 shRNA 的脱靶效应是另一个误解的基于 shRNA 的数据来源。我们发现 SHC016 是 MISSION（商业化 TRC）文库的两个非靶向 shRNA 对照之一，它发挥有害作用，导致从培养的小鼠和人类细胞的基因组中消除 shRNA 编码盒。在这里，我们使用具有可诱导 SHC016 表达的慢病毒载体来确认该 shRNA 诱导鼠细胞凋亡和衰老或有丝分裂灾难，具体取决于人类肿瘤细胞中的 p53 状态。SNRPD3模拟 A549 和 U251 细胞中 SHC016 表达的影响。SNRPD3 的过度表达将 U251 细胞从 SHC016 诱导的有丝分裂灾难中拯救出来。我们的发现取消了非靶向 SHC016 shRNA 的资格，并为关于 RNAi 结果不确定性来源的讨论增加了一个新前提。