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.

中文翻译：

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基于 EcCas6e 的反义 crRNA，用于微生物中的基因抑制和 RNA 编辑


精确的基因调控和可编程的RNA编辑是重要的RNA水平调控机制。以小非编码 RNA、microRNA 和 CRISPR-dCas 蛋白为基础的基因抑制工具，以及以作用于 RNA 的腺苷脱氨酶 (ADAR) 为基础的 RNA 编辑工具，在分子生物学和细胞工程中得到了广泛的应用。在这里，我们介绍了一种新方法，其中我们开发了 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 具有固有的 RNA 退火能力，归因于二次带正电荷的裂口，增强了 crRNA-mRNA 杂交和稳定性。基于此，我们证明 EcCas6e 及其同源 crRNA 重复序列（包含与目标 mRNA 核糖体结合位点的互补区域）可有效抑制基因表达高达 25 倍。此外，我们证明了多个 crRNA 可以轻松组装，并且可以同时靶向多达 13 个基因。最后，EcCas6e-crRNA 系统通过与 ADAR2 脱氨酶结构域融合而被开发为 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。