Reduced seed region-based off-target activity with lentivirus-mediated RNAi
Reduced seed region-based off-target activity with lentivirus-mediated RNAi
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DOI：
10.1261/rna.1977810
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发表时间：
2010-05-01
期刊：
影响因子：
4.5
通讯作者：
Cleary, Michele A.
中科院分区：
文献类型：
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作者：
Klinghoffer, Richard A.;Magnus, Jill;Cleary, Michele A.
除了沉默预期的靶基因外，转基因的siRNAs还通过一种机制调控许多意外的转录本，在这种机制中，siRNA中的类似microRNA的种子区识别转录本39UTRs中的互补序列。这种脱靶沉默的改进将导致对RNA干扰(RNAi)实验的更准确解释，从而大大提高其价值。我们测试了慢病毒介导的shRNA传递是否容易产生siRNA实验中普遍存在的基于序列的脱靶活性。我们比较了靶基因沉默和慢病毒载体表达的shRNAs所传递的多个序列对全局基因表达的整体影响。在相同的靶基因沉默水平下，shRNAs诱导的特征信号显著小于同源siRNAs诱导的特征信号，且较少由种子区活动引起。有趣的是，shRNAs表现出低水平的基于种子区域的非靶标活性，导致转录下调，这在很大程度上不同于siRNAs调控的转录。在这些观察的基础上，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路径图谱实验，以及在广泛后续资源有限的情况下进行大规模筛查。
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 39 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.