Presage Biosciences, Seattle, WA 98109, USA.
RNA. 2010 May;16(5):879-84. doi: 10.1261/rna.1977810. Epub 2010 Mar 26.
Along with silencing intended target genes, transfected siRNAs regulate numerous unintended transcripts through a mechanism in which the equivalent of a microRNA-like seed region in the siRNA recognizes complementary sequences in transcript 3' UTRs. Amelioration of this off-target silencing would lead to more accurate interpretation of RNA interference (RNAi) experiments and thus greatly enhance their value. We tested whether lentivirus-mediated delivery of shRNA is prone to the sequence-based off-target activity prevalent in siRNA experiments. We compared target gene silencing and overall impact on global gene expression caused by multiple sequences delivered as both transfected siRNAs and lentivirus vector-expressed shRNAs. At equivalent levels of target gene silencing, signatures induced by shRNAs were significantly smaller than those induced by cognate siRNAs and arose less frequently from seed region activity. Interestingly, the low level of seed region-based off-target activity exhibited by shRNAs resulted in down-regulation of transcripts that were largely distinct from those regulated by siRNAs. On the basis of these observations, we recommend lentivirus-mediated RNAi for pathway profiling experiments that measure whole genome transcriptional readouts as well as for large-scale screens when resources for extensive follow up are limited.
除了沉默预期的靶基因外，转染的 siRNA 还通过一种机制调节许多非预期的转录本，其中 siRNA 中的 miRNA 样种子区域识别转录本 3'UTR 中的互补序列。减轻这种脱靶沉默将导致更准确地解释 RNA 干扰 (RNAi) 实验，从而极大地提高它们的价值。我们测试了慢病毒介导的 shRNA 传递是否容易受到 siRNA 实验中普遍存在的基于序列的脱靶活性的影响。我们比较了作为转染的 siRNA 和慢病毒载体表达的 shRNA 传递的多种序列引起的靶基因沉默和对全局基因表达的整体影响。在等效的靶基因沉默水平下，shRNA 诱导的特征明显小于同源 siRNA 诱导的特征，并且较少由种子区域活性引起。有趣的是，shRNA 表现出的低水平的基于种子区域的脱靶活性导致了与 siRNA 调节的转录物截然不同的转录物的下调。基于这些观察结果，我们建议将慢病毒介导的 RNAi 用于测量全基因组转录读数的途径分析实验，以及在资源有限时进行大规模筛选。
