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Fellmann, Christof; Hoffmann, Thomas; Sridhar, Vaishali; Hopfgartner, Barbara; Muhar, Matthias; Roth, Mareike; Lai, Dan Yu; Barbosa, Inês A.M.; Kwon, Jung Shick; Guan, Yuanzhe; Sinha, Nishi; Zuber, Johannes
Cell reports, 12/2013, Volume: 5, Issue: 6Journal Article
Short hairpin RNA (shRNA) technology enables stable and regulated gene repression. For establishing experimentally versatile RNAi tools and minimizing toxicities, synthetic shRNAs can be embedded into endogenous microRNA contexts. However, due to our incomplete understanding of microRNA biogenesis, such “shRNAmirs” often fail to trigger potent knockdown, especially when expressed from a single genomic copy. Following recent advances in design of synthetic shRNAmir stems, here we take a systematic approach to optimize the experimental miR-30 backbone. Among several favorable features, we identify a conserved element 3′ of the basal stem as critically required for optimal shRNAmir processing and implement it in an optimized backbone termed “miR-E”, which strongly increases mature shRNA levels and knockdown efficacy. Existing miR-30 reagents can be easily converted to miR-E, and its combination with up-to-date design rules establishes a validated and accessible platform for generating effective single-copy shRNA libraries that will facilitate the functional annotation of the genome. Display omitted •The optimized “miR-E” backbone strongly and generally improves knockdown potency•A conserved ACNNC motif is critical for efficient biogenesis of miR-30-based shRNAs•miR-E enhances pri-miRNA processing and boosts mature miRNA levels up to 30-fold•Combining “Sensor rules” and miR-E primarily yields effective single-copy shRNAs Fellmann, Zuber, and colleagues identify an optimized shRNAmir backbone termed miR-E that strongly enhances target knockdown. A critical ACNNC sequence motif in the 3′ flank leads to better processing of synthetic pri-miRNAs and a 10-fold increase in mature small RNA levels. Existing miR-30 reagents can be easily converted to miR-E, and combining Sensor-based shRNA predictions and miR-E can lead to potent target knockdown at single copy.
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