Date of Award
2026
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Luciano Marraffini
Keywords
viruses, bacteriophages, immune system, CRISPR-Cas, gene editing, antiviral defense
Abstract
All domains of life can be infected by viruses. The simplest forms of life, bacteria, are outnumbered by their viruses, called bacteriophage or phage, by 10 to 1. It is estimated there are more phages on Earth than stars in the sky. Phage act as parasites to bacteria by ejecting their DNA into the cell and hijacking host machinery to produce more viral particles. Once new phage particles are assembled inside the cell, the bacterium will lyse, leading to not only to the death of the bacterium but also the proliferation of more viruses. Bacteria encode a range of strategies to mitigate viral infection. One such example is an adaptive immune system encoded in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) loci. DNA repeats are separated by short sequences, called spacers, which are acquired from invading genetic elements and incorporated into the CRISPR array by CRISPR-associated (Cas) proteins Cas1 and Cas2.The CRISPR locus also encodes either a multi-protein complex or single protein with nuclease activity. Upon infection via phage or plasmid, the host will transcribe the CRISPR array, generating guide RNA sequences called crRNA. These crRNA will associate with a CRISPR nuclease to create an RNA-guided nuclease that mediates site-specific degradation of invader DNA or RNA that is complementary to the crRNA. The type III CRISPR-Cas system is also able to synthesize a cyclic small molecule from ATP through the cyclase domain of Cas10. This molecule, termed cyclicoligoadenylate (cOA), typically contains 3, 4, or 6 cyclized AMP molecules to produce cA3, cA4, or cA6, respectively. This signaling molecule can, in turn, activate an accessory protein of type III CRISPR. Several accessory proteins have been characterized to date. They often contain a CARF domain to sense cOA and an additional effector domain. Binding of cOA leads to a conformational change in the effector domain, which then provides immunity on a community level by inducing a growth arrest of individual infected cells. In my doctoral studies, I have characterized novel CARF effectors and also investigated the role of canonical CARF effector, Csm6, in mediating immunity against mutant targets. The first proteins I characterized are closely related homologs Chp1 and Chp2 (CRISPR-associated HAD phosphatase). These proteins were first identified in a deep search bioinformatics study.1I determined that these proteins function by inducing a growth arrest in the cell through depletion of essential NTPs. Next, I characterized a novel CARF effector containing a PIN domain, which was bioinformatically identified by another graduate student in the lab, Christian Baca. I demonstrated that the PIN-CARF effector depletes RNA upon activation and alone is sufficient to provide antiviral immunity. Lastly, I studied the role of canonical CARF effector Csm6. I genetically dissected the nuclease activity of Cas10 and the RNase activity of Csm6 in the type III CRISPR response and demonstrated that Csm6 is more often required for immunity when a target sequence contains mismatches against the crRNA. From my work, I have contributed to the growing arsenal of diverse CARF effectors and demonstrated, in the case of Csm6, an advantage accessory proteins yield by leading to efficient targeting of mutated target sequences.
License and Reuse Information
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Recommended Citation
Stella, Gianna Marie, "Investigation of Diverse CARF Effectors in Type III CRISPR - Cas Immunity" (2026). Student Theses and Dissertations. 846.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/846
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy