ArticleNucleic acids research2016
Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide.
Article in Nucleic acids research, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed, 18 citations in OpenAlex.
- Fine-Tuning Homology-Directed Repair (HDR) for Precision Genome Editing: Current Strategies and Future Directions.International journal of molecular sciences · 2025Review
- Targeted knock-in ofMolecular therapy. Nucleic acids · 2024Article
- CAMK2D: a novel molecular target for BAP1-deficient malignant mesothelioma.Cell death discovery · 2023Article
- Flow cytometry-based quantification of genome editing efficiency in human cell lines using the L1CAM gene.PloS one · 2023Article
- Flow cytometry-based quantification of targeted knock-in events in human cell lines using a GPI-anchor biosynthesis gene PIGP.Bioscience reports · 2021Article
- Generation of scalable cancer models by combining AAV-intron-trap, CRISPR/Cas9, and inducible Cre-recombinase.Communications biology · 2021Article
- Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins.Nature communications · 2020Article
- WDFY2 restrains matrix metalloproteinase secretion and cell invasion by controlling VAMP3-dependent recycling.Nature communications · 2019Article
- Review
- The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing.Frontiers in molecular neuroscience · 2016Article
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
6 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The adeno-associated virus (AAV)-based targeting vector has been one of the tools commonly used for genome modification in human cell lines. It allows for relatively efficient gene targeting associated with 1-4-log higher ratios of homologous-to-random integration of targeting vectors (H/R ratios) than plasmid-based targeting vectors, without actively introducing DNA double-strand breaks. In this study, we sought to improve the efficiency of AAV-mediated gene targeting by introducing a 2A-based promoter-trap system into targeting constructs. We generated three distinct AAV-based targeting vectors carrying 2A for promoter trapping, each targeting a GFP-based reporter module incorporated into the genome, PIGA exon 6 or PIGA intron 5. The absolute gene targeting efficiencies and H/R ratios attained using these vectors were assessed in multiple human cell lines and compared with those attained using targeting vectors carrying internal ribosome entry site (IRES) for promoter trapping. We found that the use of 2A for promoter trapping increased absolute gene targeting efficiencies by 3.4-28-fold and H/R ratios by 2-5-fold compared to values obtained with IRES. In CRISPR-Cas9-assisted gene targeting using plasmid-based targeting vectors, the use of 2A did not enhance the H/R ratios but did upregulate the absolute gene targeting efficiencies compared to the use of IRES.
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