LAUSR

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.