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
Related in: MedlinePlus
Mentions: To validate our findings in a relevant preclinical setting, we created nine shRNAs against the hepatitis C virus (HCV) genome. All were designed as a 21-mer stem–loop structure with fixed ends as described previously, leaving only positions 2–18 as guide strand variables. Three were designed by the new scheme: GC-rich sequences in the central and AU-rich sequences in both seed and 3′ regions. Three shRNAs with balanced GC content (group I) and another three containing an AU-rich seed but overall balanced GC content (group II) were selected as controls (Figure 3A). As expected, all anti-HCV shRNAs had marginal passenger strand-mediated off-target effects (Supplementary Figure S3A) and relatively potent on-target activity (Figure 3B). In contrast, the guide-strand-mediated miRNA-like repression (off-targeting parameter) varied (Figure 3B). The degree of off-targeting did not correlate with the amount of guide strand (Supplementary Figure S3B), but rather with the GC content distribution profile. Off-targeting was observed in both control groups, but not with shRNAs containing AU-rich sequences in both seed and 3′ regions (Figure 3B). Similar observations were made in MEF cells (Supplementary Figure S3C). Together, our results demonstrate that potent anti-HCV shRNAs with reduced off-targeting could be achieved by following the new design scheme.
Affiliation: Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.