Department of Biosystems Engineering, Auburn University, Auburn, AL, USA.
Department of Biosystems Engineering, Auburn University, Auburn, AL, USA; Key Laboratory of Endemic and Ethnic Diseases (Guizhou Medical University), Ministry of Education, Guiyang, People's Republic of China.
Clin Microbiol Infect. 2018 Oct;24(10):1095-1099. doi: 10.1016/j.cmi.2018.03.026. Epub 2018 Mar 29.
Clostridium difficile is a notorious pathogenic species that can cause severe gastrointestinal infections in humans and animals. C. difficile infection (CDI) results in thousands of deaths worldwide every year. The elucidation of related mechanisms of CDI and exploration of potential therapeutic strategies are largely delayed due to the lack of efficient genetic engineering tools for C. difficile strains.
Plasmids carrying the CRISPR-Cas9 system were constructed and transformed into C. difficile through conjugation. Mutants were identified using colony PCR with primers annealing to the regions flanking the target gene deletion/integration locus. Heat-survival assay was used to compare the sporulation frequency between the mutant with spo0A deletion and the wild type strain. The fluorescence in the mutant with the insertion of the green fluorescent protein (GFP) gene was inspected under a fluorescent microscope.
An efficient genome editing tool was developed for C. difficile based on the CRISPR-Cas9 system. With this tool, spo0A was deleted with a 100% mutation efficiency. Conversely, an anaerobic GFP gene was successfully inserted into the C. difficile chromosome (with a mutation efficiency of 80%).
The developed CRISPR-Cas9-based genome engineering tool will facilitate functional genomic studies in C. difficile as well as the elucidation of mechanisms related to host-bacteria interaction and pathogenesis of CDI. This will be highly beneficial for the development of innovative strategies for CDI diagnostics and therapies.
艰难梭菌是一种臭名昭著的致病物种，可导致人类和动物严重的胃肠道感染。每年，艰难梭菌感染（CDI）在全球导致数千人死亡。由于缺乏有效的艰难梭菌菌株遗传工程工具，相关 CDI 机制的阐明和潜在治疗策略的探索在很大程度上被推迟。
构建携带 CRISPR-Cas9 系统的质粒，并通过接合将其转化为艰难梭菌。使用退火到靶基因缺失/整合位点侧翼区域的引物通过菌落 PCR 鉴定突变体。使用热存活试验比较spo0A 缺失突变体和野生型菌株的孢子形成频率。在插入绿色荧光蛋白（GFP）基因的突变体中，通过荧光显微镜检查荧光。
基于 CRISPR-Cas9 系统，为艰难梭菌开发了一种有效的基因组编辑工具。使用该工具，spo0A 的缺失突变效率达到 100%。相反，成功地将厌氧 GFP 基因插入艰难梭菌染色体（突变效率为 80%）。
开发的基于 CRISPR-Cas9 的基因组工程工具将促进艰难梭菌的功能基因组学研究，以及阐明与宿主-细菌相互作用和 CDI 发病机制相关的机制。这将非常有利于开发用于 CDI 诊断和治疗的创新策略。
