The Effects of a POLQ Deletion in MCM10-Deficient HCT116 Cells
Discipline: Biological Sciences
Subcategory: Genetics
Session: 2
Room: Woodley Park
Aliyyah Roberson - University of Maryland Baltimore County
Co-Author(s): Brian Ruis, University of Virginia, Charlottesville,Virginia; Moriam Munni, University of Virginia, Charlottesville, Virginia; Anja Bielinsky, University of Virginia, Charlottesville, Virginia.
MCM10 is an essential gene that ensures complete DNA replication. In humans, MCM10 deficiency results in genome instability syndromes because mutant cells under-replicate their DNA and are prone to double stranded breaks (DSBs). Cells repair DSBs by utilizing one of three DSB repair pathways: homologous recombination, classic nonhomologous end-joining, and alternative nonhomologous end-joining. POLQ plays an important role specifically in alternative end-joining (A-EJ). Our research aimed to determine whether POLQ becomes essential in HCT116 MCM10-deficient cells. We used CRISPR-Cas9 genome editing to delete POLQ in heterozygous MCM10+/- cells. Subsequent TIDE analysis of the Cas9 cleavage site in exon 2 of POLQ suggested that editing occurred at a rate of over 80%. Based on these results, we conclude that POLQ is likely not essential in MCM10+/- mutants. Our results suggest the MCM10-deficient cells do not solely rely on A-EJ for DSB repair. Although our findings suggest that POLQ is likely not essential in MCM10+/- mutants, it is still possible that a POLQ deletion significantly alters the proliferation rate of MCM10+/- mutants.
Funder Acknowledgement(s): Supported by NIH grant R35GM141805 to AKB
Faculty Advisor: Anja Bielinsky, azu3jn@virginia.edu
Role: I deleted the POLQ gene using CRISPR-Cas9 gene editing. I purified DNA extracted from edited cells to send for sequencing. I used TIDE analysis to analyze the edited gene sequence and confirm editing and determine efficiency of editing.