Weak base pairing in both seed and 3' regions reduces RNAi off-targets and enhances si/shRNA designs.
Bottom Line: The reduced off-targeting was confirmed by RNA-Seq analyses from mouse liver RNAs expressing various anti-HCV shRNAs.Compared with previously established work, the new algorithm was more effective in reducing off-targeting without jeopardizing on-target potency.These studies provide new rules that should significantly improve on siRNA/shRNA design.
Affiliation: Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.Show MeSH
Mentions: We next sought to reduce binding stability by maximizing AU content beyond the seed region. Indeed, off-target effects were almost undetectable when five shRNAs with extensive AU sequences were tested (Supplementary Figure S2A). However, the on-target knock-down efficiencies were correspondingly reduced (Supplementary Figure S2A). In vitro biochemical studies indicated that base paring in the central region was critical for on-target cleavage but less important for RISC–target association (7). Consistent with this idea, we found that on-target knock-down efficacy was positively correlated with the GC content in the central region when the rest of the guide strand was AU-rich (Figure 2A and Supplementary Figure S2B). Taken together, these results indicate that AU enrichment in both the seed and 3′ regions, but not the central region, could be an OD scheme. Indeed, five shRNAs designed using these criteria were shown to have potent on-target activity while having minimal off-target effects in both HEK293 cells (Figure 2B) and MEF cells (Supplementary Figure S2C).
Affiliation: Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.