ArticleScientific reports2019
CRISPR/Cas9-mediated one step bi-allelic change of genomic DNA in iPSCs and human RPE cells in vitro with dual antibiotic selection.
Article in Scientific reports, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed, 35 citations in OpenAlex.
- Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9.Cells · 2025Article
- Precise measurement of CRISPR genome editing outcomes through single-cell DNA sequencing.Molecular therapy. Methods & clinical development · 2025Article
- Engineered iPSC-derived natural killer cells: recent innovations in translational innate anti-cancer immunotherapy.Clinical & translational immunology · 2025Review
- Comparative analysis of regulations and studies on stem cell therapies: focusing on induced pluripotent stem cell (iPSC)-based treatments.Stem cell research & therapy · 2024Review
- Exploring the promising potential of induced pluripotent stem cells in cancer research and therapy.Molecular cancer · 2023Review
- Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells.Stem cell reviews and reports · 2023Review
- Article
- Rapid clonal identification of biallelic CRISPR/Cas9 knock-ins using SNEAK PEEC.Scientific reports · 2023Article
- Article
- Biallelic and gene-wide genomic substitution for endogenous intron and retroelement mutagenesis in human cells.Nature communications · 2022Article
- Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK.Scientific reports · 2021Article
- Targeted mutagenesis in human iPSCs using CRISPR genome-editing tools.Methods (San Diego, Calif.) · 2021Article
- CRISPR-based knock-in mutagenesis of the pioneer transcription factor FOXA1: optimization of strategies for multi-allelic proteins in cancer cells.FEBS open bio · 2021Article
- Identifying adaptive alleles in the human genome: from selection mapping to functional validation.Human genetics · 2021Review
- Increased Rate of Retinal Pigment Epithelial Cell Migration and Pro-Angiogenic Potential Ensuing From Reduced Cystatin C Expression.Investigative ophthalmology & visual science · 2020Article
- Bi-allelic Homology-Directed Repair with Helper-Dependent Adenoviruses.Molecular therapy. Methods & clinical development · 2019Article
- A transient reporter for editing enrichment (TREE) in human cells.Nucleic acids research · 2019Article
Corrections and comments
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Authors and funding
4 authors at 1 institution in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
CRISPR/Cas9 causes double-stranded DNA breaks that can undergo DNA repair either via non-homologous end joining (NHEJ) or, in the presence of a template, homology-directed repair (HDR). HDR is typically used to insert a specific genetic modification into the genome but has low efficiency compared to NHEJ, which is lowered even further when trying to create a homozygous change. In this study we devised a novel approach for homozygous single base editing based on utilising simultaneously two donor DNA templates cloned in plasmids with different antibiotic resistant genes. The donor templates were co-transfected alongside the CRISPR/Cas9 machinery into cells and a double antibiotic selection was optimised and allowed the isolation of viable desired clones. We applied the method for obtaining isogenic cells homozygous for variant B cystatin C, a recessive risk factor for age-related macular degeneration and Alzheimer's disease, in both induced Pluripotent Stem Cells (iPSCs) and a human RPE cell line. Bi-allelic gene edited clones were validated by sequencing, demonstrating that the double antibiotic templates approach worked efficiently for both iPSCs and human differentiated cells. We propose that this one step gene editing approach can be used to improve the specificity and frequency of introducing homozygous modifications in mammalian cells.
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