Genome-scale top-down reduction of phages to generate viable minimal phage genomes

Abstract Reduction of tailed-phage genomes to generate viable minimal genome phages is important for expanding our understanding of phage biology, providing insights for phage synthetic biology. Many efforts have been made to minimize living cells, but such work remains a challenge for phages due to the extraordinary genomic diversity and lack of genome-scale editing techniques. Here, we developed a CRISPR/Cas9-based iterative phage genome reduction (CiPGr) approach to detect the nonessential gene set of phages and minimize phage genomes. By CiPGr, inactivated genes accumulated on the phage genome, and mutant progeny with robust growth gradually arose, eventually becoming predominant in the populations. CiPGr was applied to four distinct tailed phages (model phages T7 and T4; wild-type phages seszw and selz), resulting in mutants of these phages with deletion of 8–20% (3.3–33 kbp) sequences, and leading to minimal genomes. Metagenomic sequencing of the mutant phage populations generated showed that 46.7 to 65.4% of genes of these phages were removed. Loss of some genes (39.6%-50%) in the removable gene sets was likely severely detrimental to phage growth. This made the corresponding mutant progenies recede in the populations, leading to the failure of detection of these genes in the genomes of the isolated mutants. In summary, our results for these four distinct tailed phages demonstrated that CiPGr is a generic yet effective approach suitable for use in novel phages without prior knowledge.