Developing a Cas9-based tool to engineer native plasmids in Synechocystis sp. PCC 6803

Yi Xiao, Shaojie Wang, Sarah Rommelfanger, Andrea Balassy, Carlos Barba-Ostria, Pengfei Gu, Jonathan M. Galazka, Fuzhong Zhang

Producción científica: Contribución a una revistaArtículorevisión exhaustiva

27 Citas (Scopus)

Resumen

The oxygenic photosynthetic bacterium Synechocystis sp. PCC 6803 (S6803) is a model cyanobacterium widely used for fundamental research and biotechnology applications. Due to its polyploidy, existing methods for genome engineering of S6803 require multiple rounds of selection to modify all genome copies, which is time-consuming and inefficient. In this study, we engineered the Cas9 tool for one-step, segregation-free genome engineering. We further used our Cas9 tool to delete three of seven S6803 native plasmids. Our results show that all three small-size native plasmids, but not the large-size native plasmids, can be deleted with this tool. To further facilitate heterologous gene expression in S6803, a shuttle vector based on the native plasmid pCC5.2 was created. The shuttle vector can be introduced into Cas9-containing S6803 in one step without requiring segregation and can be stably maintained without antibiotic pressure for at least 30 days. Moreover, genes encoded on the shuttle vector remain functional after 30 days of continuous cultivation without selective pressure. Thus, this study provides a set of new tools for rapid modification of the S6803 genome and for stable expression of heterologous genes, potentially facilitating both fundamental research and biotechnology applications using S6803.

Idioma originalInglés
Páginas (desde-hasta)2305-2314
Número de páginas10
PublicaciónBiotechnology and Bioengineering
Volumen115
N.º9
DOI
EstadoPublicada - sep. 2018
Publicado de forma externa

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