President's Showcase

Katie Jo Gelasco

Poster Presentation, Ballroom D
Katie Jo Gelasco headshot.jpg

Phi Eta Sigma Undergraduate Research Award

Engineering ThermoCas9 for Improved Gene Editing Outcomes
Supervising Professor: Dr. Hong Li
Katie Jo Gelasco is a senior dual degree pursuant in Biochemistry and Music. She has served as a research assistant for Dr. Hong Li since August 2022. In her lab, she engineers CRISPR-Cas9 proteins to learn more about their mechanism and function. She is a member of Florida State’s chapter of Alpha Chi Sigma, the national chemistry fraternity, and serves on the executive board as the Alumni Secretary, where she organizes professional events and writes a quarterly newsletter. Apart from science, Katie Jo is a member and cellist in the prestigious, internationally renowned University Symphony Orchestra, and has previously been a principal player of the University Philharmonic. She plans to earn her PhD after graduation in the biomedical sciences, and would like to work in a national lab and continue a career in research.


CRISPR-associated (Cas) proteins are a heritable immune response found in bacteria that cleave foreign DNA from phage infection. These endonucleases have profound specificity and are easily programmable by altering the guide-RNA. The protospacer adjacent motif, or PAM sequence is also an important prerequisite for target cleavage. ThermoCas9 is a thermophilic Cas9 found in Geobacillus thermodenitrificans that is sensitive to cytosine methylation in its PAM sequence, a rare characteristic of Cas proteins. We are interested in improving the gene editing efficiency of ThermoCas9. To do so, we created a library of ThermoCas9 proteins that have mutations in the hinge region and transformed them into E. coli cells that have a truncated protospacer and a CG PAM sequence. With these non-ideal conditions for catalysis, survivors would therefore be more catalytically enhanced than the wildtype ThermoCas9. We discovered a double mutant E655G/N696I of ThermoCas9 improves its catalytic efficiency. From there, we inserted point mutations into the PAM-interacting domain region that directly interacts with the methylated cytosine to identify interactions with the substrate. With the double mutant, we intend
to characterize the methylation sensitivity in vivo by performing a triple selection assay in E. coli cells. This will demonstrate that ThermoCas9 can selectively cleave a plasmid whether it is methylated or non-methylated using HaeIII methyltransferase. This investigation allows us to explore the molecular mechanisms of ThermoCas9’s sensitivity to cytosine methylation.

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