title: Weak base pairing in both seed and 3' regions reduces RNAi off-targets and enhances si/shRNA designs.
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.
authors.affiliation: Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Shuo
authors.initials: S
authors.lastname: Gu
authors.affiliation: Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Lan
authors.initials: L
authors.lastname: Jin
authors.affiliation: Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Feijie
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Department of Genetics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Bassik
authors.affiliation: Department of Cellular and Molecular Pharmacology, Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, CA 94158, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Kampmann
authors.affiliation: Departments of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA markay@stanford.edu.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Kay
mesh_terms: D000465:Algorithms; D000818:Animals; D001482:Base Composition; D020029:Base Pairing; D002478:Cells, Cultured; D005260:Female; D057809:HEK293 Cells; D016174:Hepacivirus / Q000235:genetics; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry*
chemical_list: D034741:RNA, Small Interfering