Human microRNA-30a (MIR30A)-based shRNAmir backbone (miR-30)

Experimental shRNAmir backbone derived from human MIR30A in which a synthetic 22-nucleotide guide/passenger stem replaces the miR-30a stem, so an shRNA is expressed inside a microRNA context. Placed in Pol II transcripts, including the 3′ UTR of a TRE-driven fluorescent reporter, it is processed by Drosha/DGCR8 and Dicer into a mature small RNA that silences the target gene.

Length: 110 nt

RNA type: mirna

Origin: Human MIR30A with synthetic stem and XhoI/EcoRI sites

Characteristics

Shuttles shRNA stems as a roughly 110-bp XhoI–EcoRI fragment amplified from a 97-mer oligonucleotide. Relative to endogenous MIR30A, the experimental backbone has no stem bulge, has UA/UA instead of CU/GG flanking the loop, and has a cloning-site mutation in a conserved region 3′ of the basal stem. The endogenous MIR30A guide is miR-30a-5p; the backbone places the intended guide on the opposite, 3′ arm. For three Pten shRNAs, Dicer cleaves at the canonical site in 82–100% of reads and Drosha in 25–70%. Single-copy miR30 shRNAs do not disturb processing of endogenous miRNAs in cultured cells.

Applications: Constitutive and Tet-regulated shRNA expression from retroviral (LMP, TRMPV), lentiviral and Col1a1-targeted vectors. Pooled in vivo negative-selection screening of 824 shRNAs in MLL-AF9;NrasG12D leukemia, Rpa3 knockdown that cleared established AML in mice, and single-copy shRNA transgenic mice made by Flp-mediated cassette exchange into Col1a1 in KH2 ES cells.

Limitations: Potent single-copy knockdown is uncommon: only about 3% of possible shRNAs are potent, the intermediate shRNA Pten.1524 reaches only 62% protein knockdown, and only two of 11 new Dnmt3a designs matched the control Ren.713. Mature small RNA output is 7- to 32-fold lower than the same stems in miR-E. Rpa3 shRNAs are silenced in vivo, especially in bone marrow and intestine.

References

  1. Lagos-Quintana et al. (2002). Identification of tissue-specific microRNAs from mouse. Curr Biol - Lagos-Quintana 2002 Tissue miRNAs
  2. Zuber et al. (2010). Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi. Nat Biotechnol - Zuber 2010 TRMPV Tet RNAi
  3. Dow et al. (2012). A pipeline for the generation of shRNA transgenic mice. Nat Protoc - Dow 2012 shRNA Transgenic Mice
  4. Fellmann et al. (2013). An optimized microRNA backbone for effective single-copy RNAi. Cell Rep - Fellmann 2013 miR-E Backbone