Student Theses and Dissertations
Date of Award
2025
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Luciano Marraffini
Keywords
CRISPR-Cas, type III CRISPR systems, cyclic oligoadenylate (cOA), CARF domain, Cam1/Cad1/Cat1 effectors, phage immunity
Abstract
Bacteria are under constant threat by the viruses that infect them called bacteriophages or phages for short. It is predicted that phages outnumber bacteria by an order of magnitude, producing a tremendous pressure for resistance mechanisms against this threat. One of the most abundant immune systems bacteria have evolved to combat this are CRISPR-Cas systems, which have been studied extensively due to their fascinating biology and usefulness in biotechnology and therapeutics. My thesis focuses on the discovery and characterization of three new CRISPR-Cas effector proteins that provide immunity in bacteria in response to phage challenge with highly diverse activities. CRISPR-Cas systems are the only known adaptive immune system found in bacteria. Immunity by these systems is achieved in two main steps: (1) immunization, which is the process of acquisition of small sequences of nucleic acids from the invader into CRISPR arrays as ‘spacers’ on the host chromosome as immunological memories of pathogens and (2) interference, which is the process of the transcription and maturation of CRISPR arrays into crRNAs containing spacer sequences that are subsequently loaded onto a CRISPR effector protein or protein complex that survey nucleic acids for sequences complementary to transcribed spacer sequences (protospacers). Upon base pairing between crRNAs and protospacers, the Cas machinery mediates destruction of invader nucleic acids or initiates a different immune response. My thesis work has focused on a specific type of CRISPR systems called type III CRISPR systems. These CRISPR systems are unique in that they recognize invader RNA transcripts rather than DNA (which is more common) and upon recognition two activities are unleashed for immunity. The first is destruction of ssDNA by the Cas10-Csm complex, specifically via the catalytic activity of the HD domain within the Cas10 protein. Second, and exclusive to type III CRISPR, is the synthesis of cyclic oligoadenylate molecules (cOAs) of various sizes from ATP substrates by the catalytic activity of the palm domain. cOAs go on to activate additional accessory immune effectors encoded within type III CRISPR loci that provide immunity primarily through the activation of some toxic activity via their effector domains. The most common protein domain among these effectors is the CARF domain. This protein domain is responsible for receiving the cOA signals that Cas10- Csm makes upon sensing an infection in the cell. In my doctoral studies I have studied three proteins containing CARF domains fused to various other domains that are activated upon binding cOAs. The first protein I characterized, called cyclic oligoadenylate-activated membrane protein 1 (Cam1) contains a CARF domain fused to single a transmembrane helix. This protein was first identified as active in our model CRISPR system in a screen by a former member of the Marraffini laboratory, Jakob Rostøl. I determined that it mediates membrane depolarization by forming a tetrameric pore in the membrane of cells that likely opens when it binds a cOA. After this, I identified two additional proteins that were active in our CRISPR system, a CARF-adenosine deaminase protein fusion called CRISPR-associated adenosine deaminase 1 (Cad1), which deaminates ATP to ITP upon cOA binding and a CARFTIR protein fusion called CRISPR-associated TIR protein 1 (Cat1), which hydorlyzes NAD+ molecules upon cOA binding. I was able to identify these proteins by leveraging new bioinformatic tools developed during my PhD and characterize their molecular mechanisms. At the start of my doctoral studies, only type III CRISPR accessory proteins containing nuclease domains were characterized. My work has extensively expanded the mechanistic diversity in effector activities within the CRISPR-Cas immune response in bacteria. Additionally, these proteins offer potential uses in diagnostic tools, as their enzymatic responses can be stimulated by the presence of a RNA molecule of interest coupled with a crRNA-loaded Cas10-Csm complex and read out with relatively simple assays.
License and Reuse Information
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Recommended Citation
Baca, Christian Fredrick, "Identification and Characterization of Novel CRISPR-Cas Immune Effectors" (2025). Student Theses and Dissertations. 792.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/792
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy