Gene silencing via short hairpin mediated RNAi (shRNA) is a valuable experimental tool and has promise as a therapeutic strategy. Several shRNA platforms make use of the loop and flanking sequences from the endogenous microRNA (miRNAs) miR-30a or other miRNAs to provide an RNA structure for efficient and accurate biogenesis of the RNA trigger. However, the stem regions of these shRNAs are typically designed as perfect duplex structures which is an uncommon feature for endogenous miRNA precursors. A limitation of these designs is that shRNAs with perfect duplex stems undergo extensive stem cleavage analogous to the Dicer independent miRNA miR-451, destroying the shRNA trigger sequence that is present in the 3P arm. We employed an unbiased screen of > 9000 shRNA structures to identify features that prevent stem cleavage and promote canonical biogenesis and loading into the effector complex RISC. We find that a central stem bulge or kink reduces central stem cleavage and improves accuracy of Dicer processing. Furthermore, 9 - 10 GC nucleotides in the guide strand improves shRNA efficiency. These design rules enable more effective shRNA tools and are compatible with existing sets of optimized guide/target sequences.

中文翻译：

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优化短发夹RNA生物发生和RISC加载的功能筛选

通过短发夹介导的RNAi（shRNA）进行基因沉默是一种有价值的实验工具，有望作为一种治疗策略。几个shRNA平台利用来自内源性microRNA（miRNA）miR-30a或其他miRNA的环和侧翼序列来提供RNA结构，以实现RNA触发的有效和准确的生物合成。然而，这些shRNA的茎区域通常被设计为完美的双链体结构，这对于内源性miRNA前体而言并不常见。这些设计的局限性在于，具有完美双链茎的shRNA会经历类似于Dicer独立的miRNA miR-451的广泛茎裂解，从而破坏了3P臂中存在的shRNA触发序列。我们采用了> 9000 shRNA结构可识别阻止茎裂解并促进经典生物发生并加载到效应复合物RISC中的特征。我们发现中央茎隆起或扭结减少了中央茎的分裂并提高了切丁机加工的准确性。此外，引导链中的9-10个GC核苷酸可提高shRNA效率。这些设计规则可启用更有效的shRNA工具，并与现有的优化指南/靶标序列集兼容。