Student Theses and Dissertations


Robert Heler

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Marraffini Laboratory


Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral attack in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5′-NGG-3′ protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. Before my graduate work, it was not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. My work revealed that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminates or changes PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. This was a newly identified function of Cas9 in the genesis of prokaryotic immunological memory. To further explore the link between Cas9 and spacer acquisition, I performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. I identified a mutation, I473F, which increases the rate of spacer acquisition by more than two orders of magnitude. This patented variant of Cas9 highlights the enzyme’s role during CRISPR immunization, provides a useful tool to study this otherwise rare process, and holds promise to be developed into a biotechnological application. Researching Cas9 and spacer acquisition involved many rounds of high-throughput sequencing of millions of spacers acquired by bacteria during phage infection. These experiments revealed that the abundance of each spacer in the surviving population was highly uneven. Since the molecular mechanisms underlying this bias were not known, I decided to look into the factors that affect the distribution of individual spacer sequences during phage infection of cells harboring the CRISPR system from Streptococcus pyogenes. My work has shown that spacer patterns are established early during infection and correlate with spacer acquisition rates, but not with spacer targeting efficiency. The data suggests that the rate of spacer acquisition depends on unique sequence elements within the spacers and therefore determines the abundance of different spacers within the adapted population. These results elucidate a fundamental mechanism behind the generation of immunological diversity during the type II CRISPR-Cas response.


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

Included in

Life Sciences Commons