Discovery of novel phage-bacterial interactions

项目总结

PROJECT SUMMARY Interactions between bacteria and their viruses (phages) are among the most ubiquitous in nature and have yielded transformative tools for genetic engineering, such as restriction enzymes and CRISPR-Cas. More than three dozen new bacterial immune systems have recently been discovered across many bacterial species. Some of these immune systems, such as CRISPR, target phage for cleavage, while others sense phage infection and induce bacterial death to halt phage spread. In turn, phages express “anti-immune” proteins to disarm these bacterial defenses, including “anti-CRISPR” (Acr) proteins that inhibit Cas effector functions. Phage-derived interactors (either inhibitors or activators) have not yet been found for most of these immune systems, however. The long-term objective of this proposal is to identify phage proteins that interact with or trigger activation of these immune systems. These interactions will be identified using yeast two hybrid screens and validated using affinity purification-mass spectrometry analysis in bacteria. In the two-hybrid screen, the Gal4 transcription factor will be split into an activation domain and DNA-binding domain and fused to each phage “prey” protein and bacterial immune “bait” protein, respectively. Interaction between the prey protein and bait protein should reconstitute the full transcription factor and enable expression of a reporter gene that confers survival on selective media. Rationally selected phage proteins will be screened for interactions with CRISPR- Cas proteins as well as immune proteins that lack known interactors. This versatile platform will accelerate the discovery of phage-bacterial interactions, which have long transformed molecular biology and gene therapy. In parallel, the strategies that phage use to inactivate CRISPR-Cas systems in bacteria will be applied to gene therapy in human cells to reduce cytotoxicity and off-target effects. Phages that constitutively inactivate Cas9 and Cas12a in bacteria often block both targeting and expression, which is likely optimal for long-term Cas inactivation. Mammalian gene editing performed with Cas9 delivered on viral vectors often causes off-target mutations and cytotoxicity associated with long-term Cas9 expression. To mitigate these off-target effects, strategies to inactivate CRISPR-Cas complexes and reduce their expression (after on-target editing has occurred) will be combined and compared. This work will be performed at UCSF, which hosts world-class facilities and a highly intellectual and collaborative research community. It will also provide me with the expertise in protein-protein interaction screens and gene editing that I need to fulfill my postdoctoral training goals and pioneer an independent research program in bacterial-phage interactions.