Bofill-De Ros Xavier, Gu Shuo
Gene Regulation and Chromosome Biology Laboratory, Center For Cancer Research, National Cancer Institute, Frederick, MD, United States.
Gene Regulation and Chromosome Biology Laboratory, Center For Cancer Research, National Cancer Institute, Frederick, MD, United States.
Methods. 2016 Jul 1;103:157-66. doi: 10.1016/j.ymeth.2016.04.003. Epub 2016 Apr 12.
RNA interference (RNAi) is an extremely useful tool for inhibiting gene expression. It can be triggered by transfected synthetic small interfering RNA (siRNA) or by expressed small hairpin RNA (shRNA). The cellular machinery processes the latter into siRNA in vivo. shRNA is preferred or required in genetic screens and specific RNAi approaches in gene therapy settings. Despite its many successes, the field of shRNAs faces many challenges. Insufficient knockdowns and off-target effects become obstacles for shRNA usage in many applications. Numerous failures are triggered by pitfalls in shRNA design that is often associated with impoverished biogenesis. Here, based on current understanding of the miRNA maturation pathway, we discuss the principles of different shRNA design (pre-miRNA-like, pri-miRNA-like and Ago-shRNA) with an emphasis on the RNA structure. We also provide detailed instructions for an optimal design of pre-miRNA-like shRNA.
RNA干扰（RNAi）是抑制基因表达的一种极其有用的工具。它可以由转染的合成小干扰RNA（siRNA）或表达的小发夹RNA（shRNA）触发。细胞机制在体内将后者加工成siRNA。在基因治疗环境中的遗传筛选和特定RNAi方法中，shRNA是首选或必需的。尽管取得了许多成功，但shRNA领域仍面临许多挑战。敲低不足和脱靶效应成为shRNA在许多应用中使用的障碍。shRNA设计中的缺陷常常引发众多失败，这些缺陷通常与生物合成不足有关。在此，基于对miRNA成熟途径的当前理解，我们讨论不同shRNA设计（前体miRNA样、初级miRNA样和AGO-shRNA）的原则，重点是RNA结构。我们还提供了前体miRNA样shRNA最佳设计的详细说明。
