Multiplexed CRISPR-Cpf1-Mediated Genome Editing in Clostridium difficile toward the Understanding of Pathogenesis of C. difficile Infection
Multiplexed CRISPR-Cpf1-Mediated Genome Editing in Clostridium difficile toward the Understanding of Pathogenesis of C. difficile Infection
复制标题
艰难梭菌中多重 CRISPR-Cpf1 介导的基因组编辑有助于了解艰难梭菌感染的发病机制
DOI：
10.1021/acssynbio.8b00087
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发表时间：
2018-06-01
影响因子：
4.7
通讯作者：
Wang, Yi
中科院分区：
文献类型：
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作者：
Hong, Wei;Zhang, Jie;Wang, Yi
艰难梭菌通常是医院内腹泻的主要原因，每年导致全世界数千人死亡。一个有效的基因组编辑工具的可用性C。difficile是了解其致病机制和生理行为的关键。尽管CRISPR-Cas9已被广泛用于各种生物体的基因组工程，但在基因工程工具不发达的微生物中，大基因缺失和多重基因组编辑仍然具有挑战性。在这里，我们描述了一个精简的基于CRISPR-Cpfl的工具包，用于在C中实现fur，tetM和ermB 1/2的精确缺失。高效率的困难。所有这些基因都与重要的表型（包括铁摄取、抗生素抗性和毒素产生）相关，这些表型与C.艰难梭菌感染（CDI）。此外，我们能够删除包含噬菌体基因组（phiCD 630 -2）的49.2-kb的极大基因座，并在一次接合中以高效率实现了多重基因组编辑（同时删除cwp 66和tcdA）。我们的工作突出了CRISPR-Cpf 1在C.艰难梭菌的致病机制，这两个关键的了解C。并制定战略来打击CDI。此外，对于DNA克隆，我们开发了一个一步组装协议沿着与基于Python的自动引物设计算法，缩短了质粒构建的时间到传统程序的一半。我们在此开发的方法容易且广泛地适用于其他微生物。我们的研究结果为在原核细胞中建立CRISPR-Cpf 1作为通用基因组工程工具提供了有价值的指导。
Clostridium difficile is often the primary cause of nosocomial diarrhea, leading to thousands of deaths annually worldwide. The availability of an efficient genome editing tool for C. difficile is essential to understanding its pathogenic mechanism and physiological behavior. Although CRISPR-Cas9 has been extensively employed for genome engineering in various organisms, large gene deletion and multiplex genome editing is still challenging in microorganisms with underdeveloped genetic engineering tools. Here, we describe a streamlined CRISPR-Cpfl-based toolkit to achieve precise deletions of fur, tetM, and ermB1/2 in C. difficile with high efficiencies. All of these genes are relevant to important phenotypes (including iron uptake, antibiotics resistance, and toxin production) as related to the pathogenesis of C. difficile infection (CDI). Furthermore, we were able to delete an extremely large locus of 49.2-kb comprising a phage genome (phiCD630-2) and realized multiplex genome editing in a single conjugation with high efficiencies (simultaneous deletion of cwp66 and tcdA). Our work highlighted the first application of CRISPR-Cpf1for multiplexed genome editing and extremely large gene deletion in C. difficile, which are both crucial for understanding the pathogenic mechanism of C. difficile and developing strategies to fight against CDI. In addition, for the DNA cloning, we developed a one-step-assembly protocol along with a Python-based algorithm for automatic primer design, shortening the time for plasmid construction to half that of conventional procedures. The approaches we developed herein are easily and broadly applicable to other microorganisms. Our results provide valuable guidance for establishing CRISPR-Cpf1 as a versatile genome engineering tool in prokaryotic cells.