Homology-directed repair (HDR) is the cellular pathway that enables precision genome engineering. When a double-strand break occurs, cells can repair the damage using a homologous DNA sequence as a template, faithfully copying the template information into the break site. In genome editing, researchers exploit this pathway by providing an exogenous donor template containing desired modifications flanked by sequences homologous to the target locus, directing the cell to incorporate specific changes during the repair process.
A major challenge with HDR-based editing is its limited efficiency, particularly in post-mitotic cells. HDR is primarily active during the S and G2 phases of the cell cycle, making it difficult to achieve high knock-in rates in non-dividing cells such as neurons and cardiomyocytes. This cell-cycle restriction has driven significant research into improving HDR efficiency through chemical inhibition of the competing NHEJ pathway, cell-cycle synchronization, and engineering of Cas9 variants that recruit HDR machinery. Companies like Tessera Therapeutics have developed novel writing approaches that bypass the requirement for double-strand breaks entirely, potentially overcoming HDR limitations.
The design of donor templates significantly impacts HDR outcomes. Single-stranded oligodeoxynucleotide (ssODN) donors work well for small edits but are limited in the size of insertions they can deliver. Longer double-stranded DNA donors and adeno-associated virus (AAV) vectors enable larger knock-ins but can be subject to off-target integration. Emerging approaches using long single-stranded DNA donors produced enzymatically or through asymmetric PCR offer improved efficiency and reduced toxicity. The optimization of donor template design remains an active area of research critical for therapeutic gene correction and precision cell engineering.