High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells

Synthetic riboswitches mediating ligand-dependent RNA cleavage or splicing-modulation represent elegant tools to control gene expression in various applications, including next-generation gene therapy. However, due to the limited understanding of context-dependent structure-function relationships, the identification of functional riboswitches requires large-scale-screening of aptamer-effector-domain designs, which is hampered by the lack of suitable cellular high-throughput methods. Here we describe a fast and broadly applicable method to functionally screen complex riboswitch libraries (~1.8x104 constructs) by cDNA-amplicon-sequencing in transiently transfected and stimulated human cells. The self-barcoding nature of each construct enables quantification of differential mRNA levels without additional pre-selection or cDNA-manipulation steps. We apply this method to engineer tetracycline- and guanine-responsive ON- and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices. In summary, our method enables fast and efficient high-throughput riboswitch identification, thereby overcoming a major hurdle in the development cascade for therapeutically applicable gene switches. DOI:10.1038/s41467-020-14491-x Identification of tetracycline- and guanine-responsive ON- and OFF-switches based on hammerhead, hepatitis-delta-virus and Twister ribozymes as well as U1-snRNP polyadenylation-dependent RNA devices from plasmid libraries containing up to 1.8x10^4 constructs by tetracycline or guanine stimulation at increasing dosages and subsequent cDNA amplicon sequencing.

可介导配体依赖型RNA切割或剪接调控的人工合成核糖开关（riboswitch），是一类在包括下一代基因治疗在内的诸多应用场景中用于精准调控基因表达的精巧工具。然而，由于对上下文依赖的结构-功能关系认知不足，功能性核糖开关的筛选需针对适配体-效应结构域设计开展大规模筛选；但当前缺乏适配的细胞内高通量检测方法，这一需求因此受到极大限制。本文报道一种快速且普适性优异的方法，可在经瞬时转染并接受刺激的人源细胞中，通过cDNA扩增子测序（cDNA-amplicon-sequencing）对复杂度高达约1.8×10^4个构建体的核糖开关文库开展功能筛选。每个构建体自带的条码化特性，无需额外开展预筛选或cDNA操作步骤，即可实现差异mRNA水平的精准定量。我们利用该方法对基于锤头核酶（hammerhead ribozyme）、丁型肝炎病毒核酶、Twister核酶以及U1小核核糖核蛋白（U1-snRNP）依赖型多聚腺苷酸化RNA元件的四环素、鸟嘌呤响应型开启/关闭型核糖开关进行了工程化改造与验证。综上，本方法可快速高效地完成高通量核糖开关的筛选与鉴定，从而攻克治疗级基因开关开发流程中的一大核心技术障碍。DOI:10.1038/s41467-020-14491-x 本研究通过梯度浓度四环素或鸟嘌呤刺激含多达1.8×10^4个构建体的质粒文库，结合后续cDNA扩增子测序，成功从该文库中筛选得到基于锤头核酶、丁型肝炎病毒核酶、Twister核酶以及U1小核核糖核蛋白依赖型多聚腺苷酸化RNA元件的四环素、鸟嘌呤响应型开启/关闭型核糖开关。