Cas9 nickase-mediated gene editing enables precise large-fragment integration with minimal byproducts
Year: 2026
Type: preprint
Abstract: Precise installation of large DNA sequences remains constrained by the core logic of current genome-editing platforms. Canonical homology-directed repair (HDR) relies on double-strand breaks and long homology arms, whereas prime-editing-based approaches depend on pegRNA-encoded information and reverse transcription. Here we report Nickase-mediated Gene Editing (NKGE), a paired-nick strategy for precise genome writing that is mechanistically distinct from both paradigms. NKGE employs Cas9(H840A) nickases and paired sgRNAs to generate spatially defined target-site nicks, thereby creating localized single-stranded regions that support donor capture and seamless DNA installation using only ~17-bp terminal homology, without requiring double-strand breaks, long homology arms, reverse transcriptase, recombinases, or transposases. This design actively circumvents indel and off-target formation by leveraging the high-fidelity repair pathways associated with single-strand nicks. Across multiple genomic loci and human cell lines, NKGE supported precise insertion of DNA fragments up to 10 kb with minimal indels and negligible off-target integration. NKGE also retained substantial activity under cell-cycle arrest and consistently outperformed HDR, prime editing, Prime Assembly, and QuadPE for medium- to large-scale insertions under matched conditions. Together, these findings establish NKGE as a distinct route for precise genome writing and broaden the mechanistic and practical scope of therapeutic DNA installation.
Source: Research Square
Institution: Tsinghua University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7214959090