Splicing-guaranteed artificial introns enable efficient homology-directed repair at internal exons and streamlined cell engineering
Year: 2026
Type: preprint
Abstract: Selection-marker-assisted homology-directed repair (HDR) efficiently edits coding sequence termini but is poorly suited for internal exons. Here, we developed artificial reporter intron-based HDR (ARI-HDR), which inserts a selectable intron into target exons to enable precise internal-exon editing. Although designed for scarless removal by splicing, ARIs frequently exhibit inaccurate splicing. Using a random mutagenesis reporter assay, we identified intron upstream sequences (IUS) as key determinants of splicing fidelity and screened degenerate IUS variants to obtain reliably spliced ARI donors. ARI-HDR successfully introduced site-specific mutations into five human genes. Combining long single-stranded DNA donors with conversion-tract optimization increased editing efficiency to over 80%. This efficiency enabled “Dirty-Editing,” a streamlined workflow that generates precisely edited mixed-cell populations without single-cell cloning. Together, ARI-HDR extends selection-based enrichment to internal exons across protein-coding genes, while our splicing-optimized intron design offers a broadly applicable solution for artificial intron-based technologies.
Institutions: Shenzhen University, Chinese Academy of Sciences, Shenzhen Institutes of Advanced Technology, Shenzhen Second People's Hospital, Shenzhen Institute of Synthetic Biology, Sun Yat-sen University, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou Institute of Systems Medicine
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7222761805