To improve the stability and expand applications of genome editing in Bacillus subtilis, we propose a new concept of the Cre-Cas system, which combines Cre-lox72 and CRISPR-Cas9 into an effective and convenient method. Single homologous recombination is used to introduce the integration vector into the chromosome via appropriate guide DNA to inactivate and/or insert genes of interest. The Cre recombinase then removes the region of a selection marker that is no longer needed, and the Escherichia coli replicon between the lox66 and lox71 sites are recombined to a single lox72 site. The CRISPR-Cas9 system can then be applied to remove the inserted foreign gene by targeted cutting. After Cas9 cutting, B. subtilis self-repairs the broken region to its original state without the aid of additional DNA templates. To validate this system, we used T7 and keratinase expression cassettes; self-repair efficiency was evaluated based on the loss or maintenance of the antibiotic resistance gene, as analyzed on selective media. Our results demonstrated that the insertion position in the chromosome is a more critical factor than the insertion length of the gene for efficient selfrepair in the B. subtilis genome. This concept can provide the applicability of chromosomal editing in B. subtilis. (C) 2021, The Society for Biotechnology, Japan. All rights reserved.
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