Student Theses and Dissertations

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Marraffini Laboratory


Prokaryotes have developed numerous defense strategies to combat the constant threat of viruses (bacteriophages) that endanger them. Clustered, regularly interspaced short palindromic repeats (CRISPR) loci provide archaea and bacteria with adaptive immune systems that allow them to counteract these rapidly evolving genetic parasites. These diverse systems all generally contain two components: a set of CRISPR-associated (cas) genes and a series of repetitive DNA elements intercalated with variable sequences known as spacers. Following viral infection, these sequences are acquired from the viral genome and integrated in the CRISPR array as new spacers. Spacers are then transcribed into CRISPR RNAs (crRNAs) that direct the Cas nucleases to destroy the invader following sequence-specific recognition of either DNA or RNA. Thus, spacers function as a form of immunologic memory that can be called upon again and again to defend the cell from reinfection. In type II CRISPR-Cas systems, spacer sequences direct the Cas9 nuclease to target infecting bacteriophages and cleave their double-stranded (ds)DNA genomes. Whether and how pre-exiting anti-viral spacers in type II systems affect memory generation and the acquisition of new spacers is unknown. Here, in my thesis work, I demonstrate that previously acquired spacers promote additional spacer capture from the vicinity of the Cas9 cut site at an enhanced rate. I go on to show that Cas9-mediated dsDNA break (DSB) formation is required for spacer-mediated spacer acquisition and that the rate of spacer acquisition is correlated with the efficiency of Cas9 cleavage. As a result of this mechanism, cells with preexisting viral immunity can utilize their spacerderived crRNAs to direct the acquisition of additional spacers in a new phase of immune response known as primed spacer acquisition or priming. A consequence of priming is that immune cells can acquire additional spacers as Cas9 destroys the infecting virus. I go on to show that spacers acquired during Cas9- mediated priming endow potent benefits to bacterial communities faced with virulent bacteriophages. In particular, priming suppresses the emergence of CRISPR escaper and related viruses that emerge during Cas9 targeting. I show that this anti-viral immunity is achieved in three ways. Firstly, priming expands the hosts immune repertoire, thereby improving the existing anti-phage immunity. In addition, I show that primed spacer acquisition allows the host to contain the propagation escapers that have mutations in their target sequence that abrogate Cas9 targeting. Finally, by preemptively immunizing the host with additional spacers during the initial Cas9 targeting response, priming allows the host to anticipate secondary infections by escaper and related viruses. This “prophylactic” immunity is a unique feature in CRISPR systems that allows type II systems to overcome future threats from viruses that would have overcome the defense provided by the initial anti-viral spacer. CRISPR-Cas immune systems allow their host to rapidly adapt to the viruses that challenge them. Collectively, my thesis work has revealed a new phases of the type II-A CRISPR-Cas9 immune response that is fundamental to how these systems defend their hosts against bacteriophages.


A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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