Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P. R. China.
CAS Key Laboratory of Green Process and Engineering, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P. R. China.
ACS Synth Biol. 2023 Apr 21;12(4):1204-1215. doi: 10.1021/acssynbio.2c00676. Epub 2023 Apr 5.
Haloalkaliphilic , a dominant species for sulfide removal, has attracted increasing attention. However, research on is limited by the lack of genetic manipulation tools. In this work, we developed a CRISPR/AsCas12a-mediated system in for an efficient and implementable genome editing workflow. Compared to the CRISPR/Cas9-mediated system, the CRISPR/AsCas12a system exhibited enhanced editing efficiency. Additionally, as Cas12a is capable of processing the crRNA maturation independently, the CRISPR/AsCas12a system allowed multiplex gene editing and large-fragment DNA knockout by expressing more than one crRNA under the control of one promoter. Using the CRISPR/AsCas12a system, five key genes of the elemental sulfur oxidation pathway were knocked out. Simultaneous deletion of the and genes disrupted the ability of to metabolize elemental sulfur, resulting in a 24.7% increase in elemental sulfur generation and a 15.2% reduction in sulfate production. This genome engineering strategy significantly improved our understanding of sulfur metabolism in spp.
耐碱嗜盐菌是一种主要的硫化物去除菌，受到越来越多的关注。然而，由于缺乏遗传操作工具，对耐碱嗜盐菌的研究受到限制。在这项工作中，我们在耐碱嗜盐菌中开发了一种 CRISPR/AsCas12a 介导的系统，用于高效和可实现的基因组编辑工作流程。与 CRISPR/Cas9 介导的系统相比，CRISPR/AsCas12a 系统表现出更高的编辑效率。此外，由于 AsCas12a 能够独立地处理 crRNA 的成熟，CRISPR/AsCas12a 系统允许通过在一个启动子的控制下表达多个 crRNA 进行多重基因编辑和大片段 DNA 敲除。利用 CRISPR/AsCas12a 系统，敲除了元素硫氧化途径的五个关键基因。同时敲除 和 基因破坏了 代谢元素硫的能力，导致元素硫生成增加 24.7%，硫酸盐生成减少 15.2%。这项基因组工程策略显著提高了我们对耐碱嗜盐菌属中硫代谢的理解。
