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An Optimized microRNA Backbone for Effective Single-Copy RNAi

2013; Cell Press; Volume: 5; Issue: 6 Linguagem: Inglês

10.1016/j.celrep.2013.11.020

2639-1856

Christof Fellmann, Thomas Hoffmann, Vaishali Sridhar, Barbara Hopfgartner, Matthias Muhar, Mareike Roth, Dan Yu Lai, Inês Amorim Monteiro Barbosa, Jung Shick Kwon, Yuanzhe Guan, Nishi Sinha, Johannes Zuber,

Advanced biosensing and bioanalysis techniques

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.