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Major: Plant Science

Mentors: Dr. Sarwan Dhir and Dr. Michael Terns, University of Georgia

Title: An In-Vitro Investigation of Cas1 and Cas2 Proteins of the Streptococcus thermophilus CRISPR-Cas Immune SystemAbstract: The clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) proteins are the basis of bacterial and archaeal adaptive immune systems. This immune system helps protect them from phages and plasmids. The CRISPR is an array of short repeated sequences separated by spacers derived from invading nucleic acid. By adding new spacers, the invader can be recognized if it returns. The mechanism of action of CRISPR–Cas systems can be divided into three stages: adaptation, CRISPR RNA biogenesis, and invader silencing. In the adaptation stage, Cas proteins capture new spacers and integrate them into the CRISPR locus, to form an immunological memory. The remaining stages involve transcribing RNAs from the CRISPR locus that guide Cas proteins to silence the invader by degradation of the nucleic acid. Streptococcus thermophilus (Sth), a bacterium widely used in the dairy industry, contains four different CRISPR- Cas systems. In this study, the integrase function of the adaptation proteins, Cas1 and Cas2, were studied by placing them into an assay to detect whether spacer integration took place into a plasmid containing a CRISPR locus. Sth proteins were successfully expressed and purified from E. coli, and in vitro activity assays were used to test function. The results from the integration assays support the hypothesis that Cas1 and Cas2 adaptation proteins, in the presence of MnCl2, are sufficient for spacer integration into a CRISPR locus.