State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No.1 Beichen West Road, Beijing, 100101, China.
Key Laboratory of Development and Application of Rural Renewable Energy, Biogas Institute of Ministry of Agriculture and Rural Affairs, Chengdu, 610041, China.
Microb Cell Fact. 2024 Sep 4;23(1):239. doi: 10.1186/s12934-024-02492-0.
The type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CObased biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory.
This study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5'end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes.
Therefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.
基于 II 型的 CRISPR-Cas 系统在古菌中受到限制，古菌是与细菌和真核生物并列的生命领域的一个特征。Methanococcus maripaludis 以快速生长和遗传可操作性而闻名，是研究古菌生物学和探索基于 CObased 的生物技术应用的理想模型。然而，用于控制基因调控的工具仍然不足，并且 CRISPR-Cas 工具在这个古菌中仍需要改进，限制了它作为古菌模型细胞工厂的应用。
本研究不仅改进了 CRISPR-Cas9 系统以优化多重基因组编辑和 CRISPR 质粒构建效率，而且还开创了一种有效的 CRISPR 干扰（CRISPRi）系统，用于控制 M. maripaludis 中的基因调控。我们开发了两种用于平衡表达多个 sgRNA 的新策略，促进了高效的多重基因组编辑。我们还构建了一个基因组表达 Cas9 的菌株，简化了 CRISPR 质粒的构建并促进了更有效的基因组修饰，包括无标记和无痕基因敲入。重要的是，我们使用无催化活性的 dCas9 建立了 CRISPRi 系统，实现了靶基因高达 100 倍的抑制。在这里，靶向转录起始位点和 5'端 ORF 的近侧和下游区域的 sgRNA 达到了最高的抑制效果。此外，我们开发了基于 TetR/tetO 平台的诱导型 CRISPRi-dCas9 系统。这促进了基因的诱导性抑制，特别是对必需基因。
因此，这些进展不仅扩展了遗传操作工具包，而且还弥合了 M. maripaludis 中控制基因调控的方法学差距，特别是对必需基因。在这里开发的强大工具包为将 M. maripaludis 用作重要的模型古菌细胞工厂铺平了道路，促进了古菌的基础生物学研究和应用生物技术的发展。
