Student Theses and Dissertations

Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Marraffini Laboratory


Viruses that infect prokaryotes, called bacteriophages or phages, are thought to outnumber bacteria by a ratio of 10:1 and provide a constant threat to their hosts. In response, bacteria have evolved numerous defense mechanisms, attacking all major steps of the phage infection and replication cycle. In turn, phages have evolved their own counter defenses. One phage defense system, only recently characterized, has proved revolutionary for our understanding of prokaryote-phage dynamics and our ability to edit genomes of nearly any cell type. CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes) functions as an adaptive immune system by identifying and degrading specific sequences in invading phage nucleic acids. CRISPR loci consist of repeat sequences interspersed with short fragments of phage DNA (spacers). The cas genes code for proteins that regulate 1) the acquisition of new spacers during phage infection (“immunization”) and 2) the use of spacer transcripts to identify and degrade complementary foreign DNA sequences (protospacers) to prevent infection (“immunity”). In the Streptococcus pyogenes type II-A CRISPR-Cas system, the protospacer targets on the viral genome are followed by a conserved “N-G-G” DNA motif, known as the protospacer adjacent motif (PAM). The PAM identifies the phage DNA as “foreign” and triggers the Cas nuclease, Cas9, to cleave the target phage DNA. Most of the spacers present in bacterial populations that survive phage infection target protospacers flanked by N-G-G PAMs. As a consequence, the great majority of acquired spacers target such sequences. However, there is a small fraction of acquired spacers that target non- canonical PAMs. Whether these “non-canonical” spacers originate through accidental acquisition of phage sequences and/or provide efficient defense has been unknown. In the present body of work, we found that many of the identified non-canonical spacers match phage target regions flanked by a PAM of N-A-G-G. Despite being scarcely present in bacterial populations, these “NAGG spacers” provide substantial immunity in vivo and generate RNA guides that support robust DNA cleavage by Cas9 in vitro. Moreover, during phage infection competition assays, bacteria harboring NAGG spacers are similarly fit as, and sometimes even outcompete, those organisms carrying spacers targeting canonical AGG sequences. In contrast, when we tested the acquisition efficiency of NAGG spacers, we found that they are infrequently incorporated into the CRISPR array, several orders of magnitude less often than AGG spacers. We therefore conclude that, while NAGG PAMs facilitate robust targeting of phage DNA, these particular non-canonical sequences are selected against during spacer acquisition. Thus, they seem to operate under different mechanisms during each stage of phage defense. Our findings demonstrate that NAGG PAMs can mediate efficient immunity against invading phages, and at times, result in immune levels previously only seen with canonical PAMs. This indicates a lesser degree of stringency in PAM recognition by Cas9 than is commonly thought. Additionally, our results reveal unexpected, and hitherto unappreciated, differences in PAM recognition during the spacer acquisition versus targeting stages of the type II-A CRISPR-Cas immune response, suggesting a different role of Cas9-PAM interactions in these stages. Together, this work furthers our understanding of how bacteria generate, store, and use “memories” of interactions with foreign DNA, thereby driving the co-evolution of phage, bacteria, and in some cases, human hosts. Moreover, because of CRISPR-Cas9’s many biotechnological applications, deciphering the biological relevance of non-canonical PAMs to Cas9 also has wide-ranging implications, from modulating the specificity and efficiency of gene editing, to tracking the recording of cell memory, to developing potentially life-saving phage therapies that combat antibiotic resistance.


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

Available for download on Friday, March 22, 2024

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