Date of Award
Doctor of Philosophy (PhD)
CRISPR-Cas systems endow bacteria and archaea with adaptive immunity against foreign genetic threats, like phages and plasmids. These immune systems are comprised of CRISPR-associated (Cas) protein effectors and DNA-based storage of immunological memories in the CRISPR array. The CRISPR array is a series of direct repeats intercalated by variable spacer sequences (~30bp) of foreign origin. Upon infection, spacers are excised from the foreign genome and integrated into the array. The array is then transcribed and parsed into individual CRISPR RNAs, each containing a single spacer sequence, which are used by Cas nucleases to identify foreign nucleic acids for destruction. Thus, spacer sequences represent molecular memories that serve to define the specificity of the CRISPR immune response. New spacers are added invariably to the 5’ end of the array; therefore, the first spacer matches the most recent foreign invader. How this order is established and whether this highly polarized order of spacer insertion influences CRISPR-Cas immunity has not been explored. In my thesis work, I showed that conserved nucleotides within the leader, a sequence located immediately upstream of the CRISPR array, specify the site of new spacer integration with high fidelity. Mutation of this sequence results in erroneous incorporation of new spacers into the middle of the array. To interrogate the importance of polarized spacer addition, I compared the immune responses generated by CRISPR systems containing wild type and mutant leader sequences. I showed that spacers added through polarized acquisition give rise to more robust immunity than spacers added to the middle of the array. This demonstrated that the CRISPR-Cas system specifies the site of spacer integration to optimize the immune response against the latest and most immediate threat to the host. Because addition of new spacers pushes existing spacers further downstream, each spacer added to the CRISPR array weakens the immunity provided by already existing spacers within the array. How CRISPR systems address this conundrum had not been explored. In this thesis work, I showed that CRISPR systems exhibit significant natural variation in the rates of spacer acquisition and thereby can modulate the lifespan of existing spacers in the array. Fast-adapting systems can respond quickly to new invaders, but existing spacers rapidly lose their potency. In contrast, slow-adapting systems preserve potency of existing spacers at the cost of reduced rates of spacer acquisition. I showed that bacteria have overcome these tradeoffs by harboring multiple CRISPR systems that acquire new spacers at different rates. I also found that leader-repeat junctions serve as a means for spacer acquisition complexes to discriminate between related CRISPR arrays. I propose a model whereby bacteria can harbor two related CRISPR systems as a means to form both shortand long-term immunological memories against foreign invaders. Bacteria were once thought to possess only primitive forms of innate immunity, but this notion was turned on its head by the discovery of the CRISPRCas immune system. My thesis work has revealed a deeper complexity of bacterial immunity and evolution by demonstrating that CRISPR systems functionally organize molecular memories of past invaders as a means to confer optimal immunity to the host.
McGinn, Jon, "Functional Organization of Molecular Memories in the CRISPR-Cas Immune System" (2019). Student Theses and Dissertations. 508.