title: Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide.
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.
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan.
authors.forename: Sivasundaram
authors.initials: S
authors.lastname: Karnan
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan.
authors.forename: Akinobu
authors.initials: A
authors.lastname: Ota
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Konishi
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan.
authors.forename: Md
authors.initials: M
authors.lastname: Wahiduzzaman
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Hosokawa
authors.affiliation: Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan hkonishi@aichi-med-u.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Konishi
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D005091:Exons; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D000067556:Internal Ribosome Entry Sites; D007438:Introns; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D016662:Virus Integration
chemical_list: D000067556:Internal Ribosome Entry Sites; D008565:Membrane Proteins; D010455:Peptides; C080086:phosphatidylinositol glycan-class A protein; D049452:Green Fluorescent Proteins