CRISPR–Cas9 Based Bacteriophage Genome Editing

Bacteriophages are the most abundant entities in the biosphere, and many genomes of rare and novel bacteriophages have been sequenced to date. However, bacteriophage functional genomics has been limited by a lack of effective research methods. Clustered regularly interspaced short palindromic repeat/CRISPR-associated gene (CRISPR-Cas) systems provide bacteriophages with a new mechanism for attacking host bacteria as well as new tools for study bacteriophage functional genomics. It has been reported that bacteriophages are not only the driving elements of the evolution of prokaryote CRISPR arrays but also the targets of CRISPR-Cas systems. In this study, a phage genome editing platform based on the heterologous CRISPR-Cas9 system was theoretically designed, and a Vibrio natriegens phage TT4P2 genome editing experiment was carried out in vivo in the host bacterium Vibrio natriegens TT4 to achieve phage gene deletion and replacement. The construction of this phage genome editing platform is expected to solve the problem of insufficient research on phage gene diversity, promote the development of phage synthetic biology and nanotechnology, and even accelerate the discovery of new molecular biology tools. IMPORTANCE Bacteriophages are the most numerous organisms on earth and are known for their diverse lifestyles. Since the discovery of bacteriophages, our knowledge of the wider biological world has undergone immense and unforeseen changes. A variety of V. natriegens phages have been detected, but few have been well characterized. CRISPR was first documented in Escherichia coli in 1987. It has been reported that the CRISPR-Cas system can target and cleave invaders, including bacteriophages, in a sequence-specific manner. Here, we show that the construction of a phage genome editing platform based on the heterologous CRISPR-Cas9 system can achieve V. natriegens phage TT4P2 gene editing and can also improve the efficiency and accuracy of phage TT4P2 gene editing.