Saturday, April 27, 2024
4/27/2024
Project Summary CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against their viruses (phages). The hallmark of the CRISPR-Cas immune response is the acquisition of a “memory” of infection in the form of a short DNA sequence from the invading phage genome. This sequence, known as “spacer”, is integrated into the CRISPR locus of the host and then transcribed and processed into a CRISPR RNA (crRNA) guide. CRISPR-associated (Cas) effectors use the crRNA to recognize the nucleic acids of the invading phage through base-pair complementarity and trigger different defense strategies. For Type III CRISPR systems, commonly present in the human pathogen Staphylococcus aureus, target recognition leads to the dormancy of the infected cell, an event that prevents viral replication and propagation. Here, we propose to investigate a central, yet unanswered, question about this mechanism: if there are spacers that trigger a growth arrest in the host, how are they maintained in the bacterial community after they are acquired? Our central hypothesis is that the degradation of the phage DNA eventually eliminates the viral genome from the host, enabling growth and the fixation of the spacer in the population. To investigate this, we will explore several aspects of the Csm6-mediated response required for the defense mediated by type III CRISPR spacers that match late- expressed viral genes. First, we will define whether dormant cells eventually die or are able to exit this state, survive infection and continue growing. Second, we will determine whether spacers that match late-expressed phage genes can provide a selective advantage to the cell that harbors them, even when they trigger host dormancy. Third, we will determine if these spacers are actually acquired during infection. In all these experiments we will test our central hypothesis by using mutant staphylococci lacking in the expression of several nucleases to determine if they are required for the fixation of dormancy-triggering spacers. Finally, we will use a transposon library of mutants to investigate, in an unbiased manner, the impact of host genes that could be involved in the exit from dormancy. Our proposed experiments, aimed at understanding how spacers from dormancy-inducing CRISPR systems are fixed in the host population, will fill in a fundamental knowledge gap in our understanding of the hallmark feature of CRISPR immunity: the generation of a memory of infection. In addition, by directly addressing a fundamental mechanism of phage defense of staphylococci, our proposal can facilitate the success of phage therapies for the treatment of staphylococcal disease. In a more indirect manner, the characterization of the molecular mechanisms of type III CRISPR systems can lead to avenues to repurpose these immune systems for gene editing, particularly for the development of gene therapies to treat genetic diseases.
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