ArticleJournal of biomedical science2024
Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration.
Article in Journal of biomedical science, 2024. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
6 citing papers in PubMed.
- Dual-line Genome-scale CRISPR Screening Enables Robust Target Gene Discovery.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2026Article
- Stepwise Evaluation of Plasmid- and Adeno-Associated Virus-Based Knock-In Using A Triple-Reporter Platform.Genes to cells : devoted to molecular & cellular mechanisms · 2026Article
- Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain.Scientific reports · 2026Article
- Human Genome Safe Harbor Sites: A Comprehensive Review of Criteria, Discovery, Features, and Applications.Cells · 2026Review
- Application of Gene Editing Technology Based on Targeted Delivery Materials in TNBC.ACS omega · 2025Review
- High-efficiency homology-directed insertion into the genome using the engineered homing endonuclease ARCUS.Nucleic acids research · 2025Article
Corrections and comments
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Authors and funding
8 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundThe field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR-Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency.
methodHere we report the development of a multicolour fluorescence assay for studying CRISPR-Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR-Cas9 strategies.
resultWe found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration.
conclusionOur results highlight the need for a more stringent assessment of CRISPR-Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.
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