UC San Diego

CRISPR-based genome editing holds great promise for many therapeutic applications; however, conventional Cas9 introduces double-strand breaks (DSBs) that can lead to large deletions and mutagenic off-target effects. In contrast, Cas9-derived nickases generate single-strand breaks (SSBs) and are associated with fewer mutagenic outcomes. These breaks are repaired either by error-prone non-homologous end joining (NHEJ) or precisely by homology-directed repair(HDR). An alternative repair mechanism utilizes the homologous chromosome as a repair template by a process referred to as homologous chromosome-templated repair (HTR) or inter-chromosomal gene conversion (IGC). Using a genetic reporter system at the white locus in Drosophila melanogaster, we previously showed that HTR can be induced more efficiently by nickases than by Cas9 with a strong gRNA. An RNAi screen identified components of the Fanconi anemia (FA) pathway as key regulators of HTR, revealing opposing roles in Cas9- and nickase-induced repair. We are currently extending this analysis to additional genomic loci, including pale and Notch to assess whether this effect is generalizable, and to mammalian systems to determine whether the underlying mechanism is conserved. These studies aim to better understand how repair pathway choice is regulated during genome editing and how nickase-induced HTR may provide a less disruptive approach for precise genetic modifications.