Center for Synthetic Microbiology, Philipps-Universität Marburg, Marburg, Germany.
Department of Biology, Philipps-Universität Marburg, Marburg, Germany.
Methods Mol Biol. 2025;2850:365-375. doi: 10.1007/978-1-0716-4220-7_20.
Vibrio natriegens is a gram-negative bacterium, which has received increasing attention due to its very fast growth with a doubling time of under 10 min under optimal conditions. To enable a wide range of projects spanning from basic research to biotechnological applications, we developed NT-CRISPR as a new method for genome engineering. This book chapter provides a step-by-step protocol for the use of this previously published tool. NT-CRISPR combines natural transformation with counterselection through CRISPR-Cas9. Thereby, genomic regions can be deleted, foreign sequences can be integrated, and point mutations can be introduced. Furthermore, up to three simultaneous modifications are possible.
嗜水气单胞菌是一种革兰氏阴性菌，由于其在最佳条件下的倍增时间不到 10 分钟，生长速度非常快，因此受到越来越多的关注。为了能够开展从基础研究到生物技术应用等广泛项目，我们开发了 NT-CRISPR 作为一种新的基因组工程方法。本章提供了使用此先前发表的工具的逐步方案。NT-CRISPR 将自然转化与 CRISPR-Cas9 反选择相结合。由此，可以删除基因组区域、整合外源序列和引入点突变。此外，还可以同时进行多达三个修饰。
