Kaadt Erik, Alsing Sidsel, Cecchi Claudia R, Damgaard Christian Kroun, Corydon Thomas J, Aagaard Lars
Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.
Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
Mol Ther Nucleic Acids. 2019 Mar 1;14:318-328. doi: 10.1016/j.omtn.2018.11.013. Epub 2018 Dec 1.
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.
短发夹RNA（shRNA）的表达可能会因共加工的过客链而产生不必要的活性。最近的研究表明，缩短传统shRNA的茎干可消除过客链的释放。这些由RNA聚合酶III（Pol III）启动子表达的不依赖Dicer的shRNA，类似于miR-451，依赖Ago2进行加工。使用链特异性报告基因，我们测试了两种设计，结果支持过客链活性的丧失。我们证明，由Pol II启动子表达的人工初级微小RNA（pri-miRNA）转录本可以有效地沉默目标基因。在测试的六种不同支架中，miR-324和miR-451很容易被重新靶向，以从CMV或U1 snRNA启动子直接进行有效的敲低。重要的是，miR-shRNA没有过客链活性，并且在Dicer基因敲除细胞中仍保持活性。我们的载体设计简单，因为我们用在碱基处具有不成对A-C核苷酸的模仿miR-451的不依赖Dicer的shRNA替换了pre-miR-324或-451序列。使用Pol II启动子可实现可控表达，而包含pri-miRNA序列可能需要Drosha加工，因此模仿了微小RNA的生物合成过程。由于这种改进的、可调节的系统绕过了对Dicer活性的需求，并完全消除了过客链活性，因此在通用性和增强安全性方面，它可能在研究和治疗应用中都具有优势。
