School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
Nucleic Acids Res. 2024 Aug 12;52(14):8628-8642. doi: 10.1093/nar/gkae612.
Precise gene regulation and programmable RNA editing are vital RNA-level regulatory mechanisms. Gene repression tools grounded in small non-coding RNAs, microRNAs, and CRISPR-dCas proteins, along with RNA editing tools anchored in Adenosine Deaminases acting on RNA (ADARs), have found extensive application in molecular biology and cellular engineering. Here, we introduced a novel approach wherein we developed an EcCas6e mediated crRNA-mRNA annealing system for gene repression in Escherichia coli and RNA editing in Saccharomyces cerevisiae. We found that EcCas6e possesses inherent RNA annealing ability attributed to a secondary positively charged cleft, enhancing crRNA-mRNA hybridization and stability. Based on this, we demonstrated that EcCas6e, along with its cognate crRNA repeat containing a complementary region to the ribosome binding site of a target mRNA, effectively represses gene expression up to 25-fold. Furthermore, we demonstrated that multiple crRNAs can be easily assembled and can simultaneously target up to 13 genes. Lastly, the EcCas6e-crRNA system was developed as an RNA editing tool by fusing it with the ADAR2 deaminase domain. The EcCas6e-crRNA mediated gene repression and RNA editing tools hold broad applications for research and biotechnology.
精确的基因调控和可编程的 RNA 编辑是至关重要的 RNA 水平调控机制。基于小非编码 RNA、microRNA 和 CRISPR-dCas 蛋白的基因抑制工具，以及基于腺苷脱氨酶作用于 RNA（ADAR）的 RNA 编辑工具，已在分子生物学和细胞工程中得到广泛应用。在这里，我们介绍了一种新方法，我们开发了一种 EcCas6e 介导的 crRNA-mRNA 退火系统，用于大肠杆菌中的基因抑制和酿酒酵母中的 RNA 编辑。我们发现 EcCas6e 具有内在的 RNA 退火能力，归因于一个带正电荷的二级裂缝，增强了 crRNA-mRNA 的杂交和稳定性。在此基础上，我们证明 EcCas6e 及其同源的 crRNA 重复序列包含与靶 mRNA 核糖体结合位点互补的区域，可有效抑制基因表达高达 25 倍。此外，我们证明可以轻松组装多个 crRNA，并可以同时靶向多达 13 个基因。最后，我们将 EcCas6e-crRNA 系统与 ADAR2 脱氨酶结构域融合，开发为一种 RNA 编辑工具。EcCas6e-crRNA 介导的基因抑制和 RNA 编辑工具在研究和生物技术方面具有广泛的应用。
