Telomeres are regions of repetitive DNA at the ends of linear eukaryotic chromosomes. While telomeres play important roles in protecting the ends of chromosomes, they cannot be fully copied by the DNA replication machinery. To overcome this end-replication problem, an enzyme called telomerase adds sequences to the 3’ end of the chromosome. Without telomerase activity, telomere lengths decrease and may be a leading cause of aging; while up-regulation of telomerase activity is implicated in >85% of cancers. Interestingly, prokaryotes have circular chromosomes that lack telomeres and these organisms have no need for telomerase. So, why did linear chromosomes, and thus the need for telomeres and telomerase, evolve? To begin to address this broad question, I am genetically engineering a circularized version of Chromosome XVI in the yeast Saccharomyces cerevisiae. Our method involves inserting DNA cassettes containing selectable markers at the ends of both arms of Chromosome XVI. The selectable markers contain homologous sections, so that once the cassettes have been integrated into both arms we can select for a recombination event between the cassettes to cause circularization. Thus far, I have successfully confirmed integration of the left arm cassette in several candidates and have potential double integration candidates. In an effort to increase the efficiency of the integration of DNA cassettes in Chromosome XVI, we are designing new CRISPR gRNAs to targere double strand breaks in the desired location. Once yeast with a circular chromosome XVI are confirmed, we will assess their relative fitness. Our experimental approach will ultimatley shed light on the evolution of linear chromosomes in eukaryotes.
Richardson, Nadia and Mefford, Melissa, "Are Telomeres Required? Genetically Engineering A Eukaryote With Circular Chromosomes" (2021). 2021 Celebration of Student Scholarship - Oral Presentations. 66.