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- W3137906344 abstract "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." @default.
- W3137906344 created "2021-03-29" @default.
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- W3137906344 date "2021-04-14" @default.
- W3137906344 modified "2023-10-05" @default.
- W3137906344 title "Exploiting heterologous and endogenous CRISPR‐Cas systems for genome editing in the probiotic <i>Clostridium butyricum</i>" @default.
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- W3137906344 doi "https://doi.org/10.1002/bit.27753" @default.
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