Exploiting heterologous and endogenous CRISPR-Cas systems for genome editing in the probiotic Clostridium butyricum

文献类型： 外文期刊

第一作者： Zhou, Xiuqing

作者： Zhou, Xiuqing;Wang, Xiaolu;Luo, Huiying;Wang, Yaru;Wang, Yuan;Tu, Tao;Qin, Xing;Su, Xiaoyun;Bai, Yingguo;Yao, Bin;Huang, Huoqing;Zhang, Jie

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关键词： Clostridium butyricum; CRISPR‐ Cas9; endogenous CRISPR‐ Cas system; genome editing; probiotic

期刊名称：BIOTECHNOLOGY AND BIOENGINEERING （ 影响因子：4.002； 五年影响因子：4.143 ）

ISSN： 0006-3592

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收录情况： SCI

摘要： Clostridium butyricum has been widely used as a probiotic for humans and food animals. However, the mechanisms of beneficial effects of C. butyricum on the host remain poorly understood, largely due to the lack of high-throughput genome engineering tools. Here, we report the exploitation of heterologous Type II CRISPR-Cas9 system and endogenous Type I-B CRISPR-Cas system in probiotic C. butyricum for seamless genome engineering. Although successful genome editing was achieved in C. butyricum when CRISPR-Cas9 system was employed, the expression of toxic cas9 gene result in really poor transformation, spurring us to develop an easy-applicable and high-efficient genome editing tool. Therefore, the endogenous Type I-B CRISPR-Cas machinery located on the megaplasmid of C. butyricum was co-opted for genome editing. In vivo plasmid interference assays identified that ACA and TAA were functional protospacer adjacent motif sequences needed for site-specific CRISPR attacking. Using the customized endogenous CRISPR-Cas system, we successfully deleted spo0A and aldh genes in C. butyricum, yielding an efficiency of up to 100%. Moreover, the conjugation efficiency of endogenous CRISPR-Cas system was dramatically enhanced due to the precluding expression of cas9. Altogether, the two approaches developed herein remarkably expand the existing genetic toolbox available for investigation of C. butyricum.

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