Mapping Non-Crossover Repair Events in CRISPR/Cas9-Mediated DNA Double Strand Breaks in Drosophila melanogaster

Homologous recombination (HR) is an essential DNA repair mechanism that helps restore damaged DNA. Although crossover (CO) HR events are rare in mitotic cells, non-crossover (NCO) HR events are more common and typically occur through mechanisms like synthesis-dependent strand annealing (SDSA). By avoiding the exchange of large segments of homologous chromosomes that result from COs, NCO pathways play a crucial role in minimizing loss of heterozygosity (LOH), which can lead to deleterious consequences for the organism. Research in this field has primarily focused on CO repair products; we sought to investigate the mechanisms of NCO repair in <em>Drosophila melanogaster </em>to further understand this conservative process. Using CRISPR/Cas9 to create targeted double strand breaks (DSBs) in the <em>rosy</em> gene, we studied repair products via single nucleotide polymorphism (SNP) tracking and used mismatch repair gene knockout to preserve heteroduplex DNA for analysis. Results showed predominant NCO repair (58.2%) over CO (6.2%), indicating a cellular preference for conservative repair mechanisms like SDSA. Theta-mediated end joining (TMEJ), a backup repair pathway, was also found to have a significant role in repairing DSBs, particularly in situations where canonical pathways failed. We found an unforeseen 119 bp insertion of a foreign fly transcript in one of the sequences, suggesting unexpected genetic exchanges during repair. These findings provide critical insights into the framework of repair pathways in maintaining genomic stability and inform the refinement of CRISPR/Cas9 gene editing technologies.