ArticleNucleic acids research2014
High-efficiency genome editing via 2A-coupled co-expression of fluorescent proteins and zinc finger nucleases or CRISPR/Cas9 nickase pairs.
Article in Nucleic acids research, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 39 papers.
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Who cites it
39 citing papers in PubMed.
- Prime editing for the investigation of aberrant splicing defect associated with a pathogenicMolecular therapy. Nucleic acids · 2025Article
- Dynamic movement of the Golgi unit and its glycosylation enzyme zones.Nature communications · 2024Article
- Improving and Streamlining Gene Editing inJournal of fungi (Basel, Switzerland) · 2024Article
- An efficient and cost-effective method for disrupting genes in RAW264.7 macrophages using CRISPR-Cas9.PloS one · 2024Article
- Enrichment strategies to enhance genome editing.Journal of biomedical science · 2023Review
- Amelioration of cold-induced sweetening in potato by RNAi mediated silencing ofFrontiers in plant science · 2023Article
- HideRNAs protect against CRISPR-Cas9 re-cutting after successful single base-pair gene editing.Scientific reports · 2022Article
- Co-delivery of genes can be confounded by bicistronic vector design.MRS communications · 2022Article
- Synthetic polycistronic sequences in eukaryotes.Synthetic and systems biotechnology · 2021Review
- Fast but not furious: a streamlined selection method for genome-edited cells.Life science alliance · 2021Article
- O-glycan initiation directs distinct biological pathways and controls epithelial differentiation.EMBO reports · 2020Article
- Genetic tool development in marine protists: emerging model organisms for experimental cell biology.Nature methods · 2020Article
- Cellular sensing platform with enhanced sensitivity based on optogenetic modulation of cell homeostasis.Biosensors & bioelectronics · 2020Article
- Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin-glycan specificities in a natural context.The Journal of biological chemistry · 2018Article
- Probing the contribution of individual polypeptide GalNAc-transferase isoforms to theThe Journal of biological chemistry · 2018Article
- How to create state-of-the-art genetic model systems: strategies for optimal CRISPR-mediated genome editing.Nucleic acids research · 2018Review
- Site-specificThe Journal of biological chemistry · 2018Article
- Article
- Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing.Nature communications · 2017Article
- Precise integration of inducible transcriptional elements (PrIITE) enables absolute control of gene expression.Nucleic acids research · 2017Article
Corrections and comments
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Authors and funding
13 authors.
Funding
Abstract
Targeted endonucleases including zinc finger nucleases (ZFNs) and clustered regularly interspaced short palindromic repeats (CRISPRs)/Cas9 are increasingly being used for genome editing in higher species. We therefore devised a broadly applicable and versatile method for increasing editing efficiencies by these tools. Briefly, 2A peptide-coupled co-expression of fluorescent protein and nuclease was combined with fluorescence-activated cell sorting (FACS) to allow for efficient isolation of cell populations with increasingly higher nuclease expression levels, which translated into increasingly higher genome editing rates. For ZFNs, this approach, combined with delivery of donors as single-stranded oligodeoxynucleotides and nucleases as messenger ribonucleic acid, enabled high knockin efficiencies in demanding applications, including biallelic codon conversion frequencies reaching 30-70% at high transfection efficiencies and ∼ 2% at low transfection efficiencies, simultaneous homozygous knockin mutation of two genes with ∼ 1.5% efficiency as well as generation of cell pools with almost complete codon conversion via three consecutive targeting and FACS events. Observed off-target effects were minimal, and when occurring, our data suggest that they may be counteracted by selecting intermediate nuclease levels where off-target mutagenesis is low, but on-target mutagenesis remains relatively high. The method was also applicable to the CRISPR/Cas9 system, including CRISPR/Cas9 mutant nickase pairs, which exhibit low off-target mutagenesis compared to wild-type Cas9.
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