Matt Arbrust

Biological Engineering

Mentor: Dr. Ryan Jackson

Molecular Phylogeny of Two Distinct Type IV CRISPR-Associated (Cas) Proteins

CRISPR-Cas systems are prokaryotic adaptive immune systems. CRISPR systems are diverse (Type I-VI) and include many different subtypes. Currently, there is little information detailing Type IV CRISPR system function. By analyzing the role of Type IV CRISPR proteins in prokaryotic adaptive immunity, we gain a better understanding of Type IV CRISPR system mechanisms and basic prokaryote biology. I determined phylogenetic relationships for two distinct Type-IV Cas proteins, Cas6 and DinG, to begin to elucidate their function. These proteins likely have a critical function in Type IV CRISPR systems, as Cas6 is necessary for immunity in several well-characterized CRISPR systems and DinG is predicted to interact with DNA. To perform phylogenetic analysis, each signature Cas6 sequence was analyzed using the Basic Local Alignment Search Tool (BLAST) and returned sequences were collected for a multiple sequence alignment using Clustal Omega. DinG protein sequences were handled in a similar manner, using a database of known Type-IV sequences instead of BLAST. The results indicate that most Type-IV Cas6 proteins do not belong to an already defined group of Cas6 ribonucleases, though some may have been recruited from other CRISPR systems as “orphan” proteins. Also, Type-IV DinG proteins are more closely related to each other than DinG proteins not belonging to CRISPR systems, indicating they may function distinctly from chromosomal DinG. The data I show here will be used to guide our biochemical and structural studies of Cas6 and DinG and will likely result in a basic understanding of Type IV CRISPR system function.