Ryan O'Hanrahan

Telomeres function as protective ends of eukaryotic chromosomes and have a role in the aging process in the cell. In addition, it is thought that telomeres interact with regions of chromatin to regulate gene expression. The Barthel Lab has developed a method for identifying these telomere-chromatin interactions and found interactions between telomeres and promoters as well as non-coding regions. Results from the screen showed telomeres interacting with repetitive elements, including Interstitial Telomeric Sequences (ITS). In order to understand if this interaction is biologically relevant, we used CRISPR to attempt to disrupt the interaction in a fibroblast cell line.      

A CRISPR CAS-9 strategy was used to excise the ITS regions on Chromosomes 18 and 19 using both electroporation and lipofection to introduce the CRISPR reagents. To validate CRISPR excision, we utilized PCR amplification and agarose gel electrophoresis. PCR products were sequenced as a final check to confirm the knockout.        Comparing transfection methods, electroporation is shown to be over two times more effective at introducing a knockout than lipofection. Moreover, successful PCR screening of clones varied by polymerase and annealing temperature used. PCR screening of clones shows that the CRISPR strategy successfully knocks out the ITS region on chromosomes 18 and 19 with frequencies of 55% and 38% respectively. Lastly, DNA sequence analysis of PCR products reveals that the excision is occurring in the correct location. These cell lines can now be assessed for phenotypes.