CRISPR-Cas9 assisted non-homologous end joining genome editing system of Halomonas bluephagenesis for large DNA fragment deletion

作者：Chunyan Liu, Ya-Xin Yue, Yanfen Xue, Cheng Zhou, Yanhe Ma · 发表于：Microbial Cell Factories · 年份：2023 · DOI：10.1186/s12934-023-02214-y · 被引用次数：15 · 研究领域：CRISPR and Genetic Engineering、Bacterial Genetics and Biotechnology、Microbial Metabolic Engineering and Bioproduction

Abstract Background Halophiles possess several unique properties and have broad biotechnological applications including industrial biotechnology production. Halomonas spp., especially Halomonas bluephagenesis , have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), some proteins, small molecular compounds, organic acids, and has the potential to become a chassis cell for the next-generation of industrial biotechnology (NGIB) owing to its simple culture, fast growth, contamination-resistant, low production cost, and high production value. An efficient genome editing system is the key for its engineering and application. However, the efficiency of the established CRISPR-Cas-homologous recombination (HR) gene editing tool for large DNA fragments was still relatively low. In this study, we firstly report a CRISPR-Cas9 gene editing system combined with a non-homologous end joining (NHEJ) repair system for efficient large DNA fragment deletion in Halomonas bluephagenesis . Results Three different NHEJ repair systems were selected and functionally identified in Halomonas bluephagenesis TD01. The NHEJ system from M. tuberculosis H37Rv (Mt-NHEJ) can functionally work in H. bluephagenesis TD01, resulting in base deletion of different lengths for different genes and some random base insertions. Factors affecting knockout efficiencies, such as the number and position of sgRNAs on the DNA double-strands, the Cas9 protein promoter, and the interaction be...