RNA干扰(RNAi)是抑制基因表达的非常有用的工具。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。细胞机器在体内将后者加工成siRNA。shRNA在基因筛选和基因治疗环境中的特定RNAi方法中是优选的或必需的。尽管取得了许多成功,shRNA领域面临许多挑战。击倒不足和脱靶效应成为许多应用中使用shRNA的障碍。许多失败是由shRNA设计中的陷阱引发的,这些陷阱通常与贫乏的生物发生有关。这里,基于目前对miRNA成熟途径的理解,我们讨论了不同shRNA设计的原理(pre-miRNA-like,pri-miRNA样和Ago-shRNA),重点是RNA结构。我们还提供了pre-miRNA样shRNA的最佳设计的详细说明。
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.