Efficient Knockdown and Lack of Passenger Strand Activity by Dicer-Independent shRNAs Expressed from Pol II-Driven MicroRNA Scaffolds

Abstract

The expression of short hairpin RNAs (shRNAs) may result in unwanted activity from the co-processed passenger strand. Recent studies have shown that shortening the stem of conventional shRNAs abolishes passenger strand release. These Dicer-independent shRNAs, expressed from RNA polymerase III (Pol III) promoters, rely on Ago2 processing in resemblance to miR-451. Using strand-specific reporters, we tested two designs, and our results support the loss of passenger strand activity. We demonstrate that artificial primary microRNA (pri-miRNA) transcripts, expressed from Pol II promoters, can potently silence a gene of choice. Among six different scaffolds tested, miR-324 and miR-451 were readily re-targeted to direct efficient knockdown from either a CMV or a U1 snRNA promoter. Importantly, the miR-shRNAs have no passenger strand activity and remain active in Dicer-knockout cells. Our vectors are straightforward to design, as we replace the pre-miR-324 or -451 sequences with a Dicer-independent shRNA mimicking miR-451 with unpaired A-C nucleotides at the base. The use of Pol II promoters allows for controlled expression, while the inclusion of pri-miRNA sequences likely requires Drosha processing and, as such, mimics microRNA biogenesis. Since this improved and tunable system bypasses the requirement for Dicer activity and abolishes passenger strand activity completely, it will likely prove favorable in both research and therapeutic applications in terms of versatility and enhanced safety.

circZNF827 nucleates a transcription inhibitory complex to balance neuronal differentiation

“…To ascertain whether the highly upregulated circRNAs might contribute to the process of neuronal differentiation, we next depleted a number of candidate circRNAs by RNA interference. We first tested knockdown efficiency of circZfp827 ( circZNF827 in humans) by lentivirally delivered dishRNAs ( Kaadt et al, 2019 ) targeting the backsplicing junction in either mESC, p19, SH-SY5Y or L-AN-5 cells, of which the latter three cell lines are well-established models of neuronal differentiation following retinoic acid treatment. Knockdown efficiency in mESC and p19 proved relatively poor (30–60% remaining circRNA) compared to the two human cell lines: SH-SY5Y (10% remaining) and L-AN-5, which displayed superior results (<8% remaining) ( Figure 2A and Figure 2—figure supplement 1A ).…”

A functional screen for optimization of short hairpin RNA biogenesis and RISC loading

“…Interestingly, asymmetric mutations in the stem that leads to a kinked structure has previously been reported to reduce accuracy of Dicer cleavage; however our data suggests that this is dependent on kink position and stem GC content (Starega-Roslan et al 2011). Interestingly, the Ago-2 slicing pathway has been successfully harnessed for use via Dicer-independent AgoshRNAs (Herrera-Carrillo et al 2014;Harwig et al 2015;Kaadt et al 2019). Ago2 cleavage of precursors is dependent on a perfect duplex stem, a feature that is absent in most endogenous miRNA precursors.…”

Short Hairpin RNAs for Strand-Specific Small Interfering RNA Production

“…The optimal shRNA for expressing a specific siRNA should be experimentally tested based on the principles described above. In addition, similar to a Dicer-dependent shRNA, Ago-shRNA could be engineered into a primary transcript that requires Drosha processing (Kaadt et al, 2019), but the principle of processing a primary transcript of Ago-shRNA is complicated. For example, recent research shows that although pri-miR-451 is dependent on Microprocessor, its short stem and small terminal loop render it an intrinsically weak Microprocessor substrate.…”