Weak base pairing in both seed and 3′ regions reduces RNAi off-targets and enhances si/shRNA designs. Issue 19 (30th September 2014)

• Record Type: Journal Article
• Authors: Gu, Shuo; Zhang, Yue; Jin, Lan; Huang, Yong; Zhang, Feijie; Bassik, Michael C.; Kampmann, Martin; Kay, Mark A.
• Is Part Of: Nucleic acids research. Volume 42:Issue 19(2014)
• Page Start: 12169
• Page End: 12176
• Publication Date: 2014-09-30
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gku854
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: The use of RNA interference is becoming routine in scientific discovery and treatment of human disease. However, its applications are hampered by unwanted effects, particularly off-targeting through miRNA-like pathways. Recent studies suggest that the efficacy of such off-targeting might be dependent on binding stability. Here, by testing shRNAs and siRNAs of various GC content in different guide strand segments with reporter assays, we establish that weak base pairing in both seed and 3′ regions is required to achieve minimal off-targeting while maintaining the intended on-target activity. The reduced off-targeting was confirmed by RNA-Seq analyses from mouse liver RNAs expressing various anti-HCV shRNAs. Finally, our protocol was validated on a large scale by analyzing results of a genome-wide shRNA screen. 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.