High-throughput approaches to understand and engineer bacteriophages

Bacteriophage research has been vital to fundamental aspects of modern biology. Advances in metagenomics have revealed treasure troves of new and uncharacterized bacteriophages ('phages') that are not yet understood. However, our ability to find new phages has outpaced our understanding of how sequence encodes function in phages. Traditional approaches for characterizing phages are limited in scale and face hurdles in determining how changes in sequence drive function. We describe powerful emerging technologies that can be used to clarify sequence–function relationships in phages through high-throughput genome engineering. Using these approaches, up to 105 variants can be characterized through pooled selection experiments and deep sequencing. We describe caveats when using these tools and provide examples of basic science and engineering goals that are pursuable using these approaches. High-throughput approaches can generate large libraries of phages that can be screened simultaneously in pooled selection experiments and scored using deep sequencing.Approaches for creating phage libraries frequently have a tradeoff between creating mutations throughout larger areas of a phage genome (breadth) versus our ability to specify mutations (programmability).Untargeted mutagenesis is a simple and effective approach for introducing random mutations and is a useful tool for high-throughput reverse genetics.Targeted mutagenesis can characterize phage–host interactions at a high resolution by accurately mapping key functional regions in a phage genome.Phage deletion libraries can be used to study gene essentiality under different environmental and host conditions, while insertion libraries can be used to characterize metagenomic gene function.