Khan Muhammad Adil, Herring Gabrielle, Zhu Jia Yuan, Oliva Marina, Fourie Elliott, Johnston Benjamin, Zhang Zhining, Potter Jarred, Pineda Luke, Pflueger Jahnvi, Swain Tessa, Pflueger Christian, Lloyd James P B, Secco David, Small Ian, Kidd Brendan N, Lister Ryan
Australian Research Council Centre of Excellence in Plant Energy Biology, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia.
Australian Research Council Centre of Excellence in Plants for Space, School of Molecular Sciences, The University of Western Australia, Perth, Western Australia, Australia.
Nat Biotechnol. 2025 Mar;43(3):416-430. doi: 10.1038/s41587-024-02236-w. Epub 2024 May 20.
The construction of synthetic gene circuits in plants has been limited by a lack of orthogonal and modular parts. Here, we implement a CRISPR (clustered regularly interspaced short palindromic repeats) interference (CRISPRi)-based reversible gene circuit platform in plants. We create a toolkit of engineered repressible promoters of different strengths and construct NOT and NOR gates in Arabidopsis thaliana protoplasts. We determine the optimal processing system to express single guide RNAs from RNA Pol II promoters to introduce NOR gate programmability for interfacing with host regulatory sequences. The performance of a NOR gate in stably transformed Arabidopsis plants demonstrates the system's programmability and reversibility in a complex multicellular organism. Furthermore, cross-species activity of CRISPRi-based logic gates is shown in Physcomitrium patens, Triticum aestivum and Brassica napus protoplasts. Layering multiple NOR gates together creates OR, NIMPLY and AND logic functions, highlighting the modularity of our system. Our CRISPRi circuits are orthogonal, compact, reversible, programmable and modular and provide a platform for sophisticated spatiotemporal control of gene expression in plants.
植物中合成基因回路的构建一直受到缺乏正交和模块化元件的限制。在此，我们在植物中实现了一个基于CRISPR（成簇规律间隔短回文重复序列）干扰（CRISPRi）的可逆基因回路平台。我们创建了一套具有不同强度的工程化可抑制启动子的工具包，并在拟南芥原生质体中构建了非门和或非门。我们确定了从RNA聚合酶II启动子表达单向导RNA的最佳加工系统，以引入或非门可编程性，从而与宿主调控序列对接。或非门在稳定转化的拟南芥植株中的性能证明了该系统在复杂多细胞生物体中的可编程性和可逆性。此外，基于CRISPRi的逻辑门在小立碗藓、普通小麦和甘蓝型油菜原生质体中表现出跨物种活性。将多个或非门叠加在一起可创建或、蕴含非和与逻辑功能，突出了我们系统的模块化特性。我们的CRISPRi回路具有正交、紧凑、可逆、可编程和模块化的特点，为植物基因表达的复杂时空控制提供了一个平台。
