CAMERS‐B: CRISPR/Cpf1 assisted multiple‐genes editing and regulation system for Bacillus subtilis

The clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have been widely used in genome editing and transcriptional regulation. In this study, by engineering the Francisella novicida U112 CRISPR/Cpf1 system, a powerful tool called CRISPR/Cpf1 assisted multiple-genes editing and regulation system for B. subtilis was constructed for engineering Bacillus subtilis, and a synthetic oligos mediated assembly of CRISPR RNA (crRNA) array method was created to build crRNA array. This system can achieve the double genes in-frame knocking out, multiple point mutations (up to six), or single gene insertion at a time with 100% efficiency. In addition, transcriptional regulation systems were also developed using the DNase deactivated Cas protein (dCpf1) and a transcription factor RemA, which can implement repression and activation on multiple-genes concurrently. Finally, as a proof-of-concept demonstration, the synthesis pathways of N-acetylglucosamine and acetoin in B. subtilis were engineered by using this system. Overall, we provide effective tools for genome editing and metabolic engineering of B. subtilis cell factories to produce various biochemicals.