Abstract
Homology-directed repair (HDR) enables precise genome editing; however, its application in mammalian cells is limited by low efficiency owing to competition from error-prone repair pathways and intrinsically restricted HDR activity. Existing HDR-enhancement strategies, including small-molecule treatments and marker-based selection, are constrained by cytotoxicity, genomic scarring, and inconsistent performance. Here, we present essential-gene-supported scarless HDR (ESS-HDR), a robust, drug- and marker-free platform that selectively enriches HDR-proficient cells. By leveraging essential-gene coediting, ESS-HDR enables precise and scarless genome modification with enhanced efficiency. CRISPR–Cas9 induces double-strand breaks at both the target locus and an essential gene, accompanied by two donor templates: one introducing the desired edit and the other restoring essential-gene function. Only cells that undergo accurate HDR at the essential locus survive, providing endogenous selection without exogenous markers. Single-cell clone analysis confirms that enrichment of HDR-proficient cells enhances editing at the target locus. Using ssODN donors carrying a 1 nucleotide substitution or a 10 nucleotide insertion, ESS-HDR increases HDR efficiencies by sevenfold to 16-fold in HEK293 cells and 41-fold in primary epidermal keratinocytes compared with conventional single-site HDR. With plasmid donors targeting TUBA1B, LMNB1, or ACTB, ESS-HDR improves knock-in efficiencies by sixfold to 34-fold across HEK293, U2OS, and HeLa cells. ESS-HDR also outperforms chemical enhancers including RS-1, SCR7, nocodazole, and AZD7648. Together, these findings establish ESS-HDR as a broadly applicable strategy for efficient, scarless genome editing without external selection markers.