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.

中文翻译：

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CAMERS-B：CRISPR/Cpf1 辅助枯草芽孢杆菌的多基因编辑和调控系统。

成簇的规则间隔短回文重复序列和 CRISPR 相关蛋白 (CRISPR-Cas) 系统已广泛用于基因组编辑和转录调控。本研究通过对新生弗朗西斯菌 U112 CRISPR/Cpf1 系统进行工程改造，构建了一个名为 CRISPR/Cpf1 的强大工具辅助枯草芽孢杆菌多基因编辑和调控系统，用于工程化枯草芽孢杆菌，以及合成寡核苷酸介导的 CRISPR RNA 组装(crRNA) 阵列方法被创建来构建 crRNA 阵列。该系统可实现双基因框内敲除、多点突变（最多6个）或单基因插入，效率100%。此外，还使用 ​​DNase 失活的 Cas 蛋白 (dCpf1) 和转录因子 RemA 开发了转录调控系统，可以同时对多个基因进行抑制和激活。最后，作为概念验证演示，使用该系统设计了枯草芽孢杆菌中 N-乙酰氨基葡萄糖和乙偶姻的合成途径。总的来说，我们为枯草芽孢杆菌细胞工厂的基因组编辑和代谢工程提供了有效的工具，以生产各种生化产品。