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）的表达可能导致协同处理的乘客链产生有害的活性。最近的研究表明，缩短传统shRNA的茎可以消除过客链的释放。从RNA聚合酶III（Pol III）启动子表达的这些独立于Dicer的shRNA依赖于Ago2加工，类似于miR-451。使用特定于链的报道子，我们测试了两种设计，我们的结果支持了乘客链活动的损失。我们证明了人工初级microRNA（pri-miRNA）转录物，从Pol II启动子表达，可以有效地沉默所选基因。在测试的六种不同支架中，miR-324和miR-451易于重新定向以直接从CMV或U1 snRNA启动子进行有效的敲低。重要的，miR-shRNA没有过客链活性，并在切丁酶敲除细胞中保持活性。我们的载体易于设计，因为我们用模仿miR-451的不切切酶的shRNA取代了pre-miR-324或-451序列，该miR-451的碱基带有未配对的AC核苷酸。Pol II启动子的使用可实现受控表达，而pri-miRNA序列的包含可能需要Drosha加工，因此可模拟microRNA的生物发生。由于此改进的可调系统绕开了Dicer活动的要求，并完全废除了乘客链活动，因此在多功能性和增强的安全性方面，在研究和治疗应用中都将被证明是有利的。因为我们用模仿DiR-451的不切丁酶的shRNA取代了miR-451或pre-miR-324序列，在碱基上有未配对的AC核苷酸。Pol II启动子的使用可实现受控表达，而pri-miRNA序列的包含可能需要Drosha加工，因此可模拟microRNA的生物发生。由于此改进的可调系统绕开了Dicer活动的要求，并完全废除了乘客链活动，因此在多功能性和增强的安全性方面，在研究和治疗应用中都将被证明是有利的。因为我们用模仿DiR-451的不切丁酶的shRNA取代了miR-451或pre-miR-324序列，在碱基上有未配对的AC核苷酸。Pol II启动子的使用可实现受控表达，而pri-miRNA序列的包含可能需要Drosha加工，因此可模拟microRNA的生物发生。由于此改进的可调系统绕开了Dicer活动的要求，并完全废除了乘客链活动，因此在多功能性和增强的安全性方面，在研究和治疗应用中都将被证明是有利的。