Splicing-guaranteed artificial introns enable efficient homology-directed repair at internal exons and streamlined cell engineering
- 1The First Affiliated Hospital, Shenzhen University; Shenzhen Second People's Hospital
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.
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BibTeX
@misc{wang2026splicingguaranteed,
title = {{Splicing-guaranteed artificial introns enable efficient homology-directed repair at internal exons and streamlined cell engineering}},
author = {S. wang and X. Wang and M. Shang and M. Zhang and K. Wu and W. Huang},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.16.759525},
url = {https://www.biorxiv.org/content/10.64898/2026.10.16.759525v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Splicing-guaranteed artificial introns enable efficient homology-directed repair at internal exons and streamlined cell engineering
AU - S. wang
AU - X. Wang
AU - M. Shang
AU - M. Zhang
AU - K. Wu
AU - W. Huang
PY - 2026
DA - 2026/10/16
PB - bioRxiv
DO - 10.64898/2026.10.16.759525
UR - https://www.biorxiv.org/content/10.64898/2026.10.16.759525v1
AB - 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.
ER -