Reduced seed region-based off-target activity with lentivirus-mediated RNAi

In addition to 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. The ability to limit such off-target effects would substantially facilitate 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 seed region-based off-target activity prevalent in siRNA experiments. We compared target gene silencing and overall impact on global gene expression caused by multiple 21-mer duplex sequences delivered as both transfected siRNA and lentivirus vector-expressed shRNA. 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 RNA interference 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. To assess the extent to which lentivirus-mediated RNAi is prone to off-target effects, we compared gene expression profiles induced by siRNAs and shRNAs that encode the exact same 19-mer RNA duplex sequence in HeLa cells. siRNA/shRNA pairs targeting 4 distinct genes (cdkn1a, e2f1, ezh2, and fdxr) were analyzed. We also generated HCT-116 colon carcinoma cells (wild type for TP53) harboring a stable tetracycline-regulatable expression construct encoding an shRNA designed to silence TP53. We compared transcript regulation induced by doxycycline-mediated expression of a TP53-targeting shRNA to that induced by transfection of the same cells with a TP53 siRNA with the same 19-mer core sequence.