利用 CRISPR-Cpf1 系统对谷氨酸棒杆菌进行基因组编辑。

Genome Editing of Corynebacterium glutamicum Using CRISPR-Cpf1 System.

机构信息

School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, China.

Shanghai Research and Development Center of Industrial Biotechnology, Shanghai, China.

出版信息

Methods Mol Biol. 2022;2479:189-206. doi: 10.1007/978-1-0716-2233-9_13.

DOI:10.1007/978-1-0716-2233-9_13

PMID:35583740

Abstract

Corynebacterium glutamicum, as an important microbial chassis, has great potential in industrial application. However, complicated genetic modification is severely slowed by lack of efficient genome editing tools. The Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been verified as a very powerful tool for mediating genome alteration in many microorganisms but cannot work well in C. glutamicum. We recently developed two Francisella novicida (Fn) CRISPR-Cpf1 assisted systems for genome editing via homologous recombination in C. glutamicum. Here, we describe the protocols and demonstrated that N iterative rounds of genome editing can be achieved in 3 N + 4 or 3 N + 2 days, respectively.

摘要

谷氨酸棒杆菌作为一种重要的微生物底盘，在工业应用中具有巨大的潜力。然而，由于缺乏有效的基因组编辑工具，复杂的遗传修饰严重受阻。酿脓链球菌（Sp）CRISPR-Cas9 系统已被证明是一种非常强大的工具，可用于调节许多微生物中的基因组改变，但在谷氨酸棒杆菌中不能很好地工作。我们最近开发了两种弗朗西斯氏菌（Fn）CRISPR-Cpf1 辅助系统，用于通过同源重组在谷氨酸棒杆菌中进行基因组编辑。在这里，我们描述了这些方案，并证明了可以在 3N+4 或 3N+2 天内分别实现 N 轮迭代的基因组编辑。