西澳大利亚大学Ryan Lister和Brendan N. Kidd研究团队揭示了基于 CRISPRi 的植物基因表达控制回路。这一研究成果发表在2024年5月20日出版的国际学术期刊《自然—生物技术》上。

研究人员在植物中建立了基于CRISPR（簇状规则间隔短回文重复序列）干扰（CRISPRi）的可逆基因回路平台。研究人员创建了不同强度的工程可抑制启动子工具包，并在拟南芥原生质体中构建了NOT和NOR门。研究构建了利用RNA Pol II启动子表达单导RNA的最佳处理系统，以引入可编程的NOR门，从而与宿主调控序列连接。NOR门在稳定拟南芥植物转化株中的表现证明了，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Physcomitrium patens、Triticum aestivum和Brassica napus原生质体中的跨物种活性也得到了展示。将多个NOR逻辑门层叠在一起可以产生OR、NIMPLY 和AND逻辑功能，这突出了该系统的模块性。该CRISPRi系统具有正交、紧凑、可逆、可编程和模块化的特点，为研究植物基因表达的复杂时空调控提供了一个平台。

据介绍，由于缺乏正交和模块化部件，植物合成基因回路的构建一直受到限制。

附：英文原文

Title: CRISPRi-based circuits to control gene expression in plants

Author: 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

Issue&Volume: 2024-05-20

Abstract: 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. 

DOI: 10.1038/s41587-024-02236-w

Source: https://www.nature.com/articles/s41587-024-02236-w