View Program - 63rd Annual Drosophila Research Conference

Genome stability is key to longevity of multicellular organisms and avoidance of disease. Despite daily challenges from numerous sources of DNA damage threatening this stability, cells regularly repair DNA to maintain genomic resilience. Improper or misregulated repair causes accumulation of “scars” in the form of detrimental mutations within the genome, eventually leading to genome instability, cancer, and other genetic disease. Homology directed repair (HDR) of DNA double strand breaks is one DNA repair pathway that, if improperly regulated, leads to accumulation of mutations via mitotic (non-meiotic) crossovers and loss of heterozygosity. Therefore, better understanding of mitotic crossover mechanisms and regulators is critical to prevention of cancer and other genetic disease. CRISPR/Cas9 has also become increasingly reliant on accurate HDR to integrate desired mutations or corrections in genome editing, but the precise CRISPR/Cas9 HDR mechanisms remain elusive. I have begun to test mitotic crossover mechanisms using both CRISPR/Cas9 induced double strand breaks and mutation of Bloom Syndrome Helicase (Blm), a key HDR regulator that prevents mitotic crossovers. Through combination with total mismatch repair (MMR) knockout only possible in Drosophila (through both Msh6 canonical and backup Xpc short-patch pathways), it is now possible to analyze resulting mitotic crossover products using Sanger sequencing for CRISPR/Cas9 induced double strand breaks and whole genome sequencing for Blm mutants. Using these tools, I have begun to accurately define mitotic crossover mechanisms for the first time in a multicellular organism, with an unligated double Holliday junction resolution model indicated as the primary mechanism. Unexpectedly, combination of Blm and MMR knockout also appears to substantially increase the rate of mitotic crossovers compared to knockout of either alone, indicating a previously uncharacterized genetic interaction between Blm and MMR components in HDR and prevention of mitotic crossovers. This work has begun to enhance understanding of how DNA is repaired in both CRISPR and Blm mutant contexts, expanding knowledge of how mitotic crossovers lead to genome instability, identifying previously uncharacterized interactions in HDR and mitotic crossover regulation, and providing better understanding of how to beneficially utilize mitotic crossover mechanisms in genome editing.

Defining Mitotic Crossover Mechanisms Using CRISPR/Cas9 and Bloom Syndrome Helicase