ArticleScientific reports2017
Disruption of diphthamide synthesis genes and resulting toxin resistance as a robust technology for quantifying and optimizing CRISPR/Cas9-mediated gene editing.
Article in Scientific reports, 2017. 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, 17 citations in OpenAlex.
- ShcD adaptor protein drives invasion of triple negative breast cancer cells by aberrant activation of EGFR signaling.Molecular oncology · 2025Article
- Improving the Efficiency of CRISPR Ribonucleoprotein-Mediated Precise Gene Editing by Small Molecules in Porcine Fibroblasts.Animals : an open access journal from MDPI · 2024Article
- CRISPR-Cas9-Mediated Bioluminescent Tagging of Endogenous Proteins by Fluorescent Protein-Assisted Cell Sorting.Methods in molecular biology (Clifton, N.J.) · 2024Article
- CRISPR/Cas9 Genome Editing for Tissue-Specific In Vivo Targeting: Nanomaterials and Translational Perspective.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2023Review
- Efficient cell death mediated by bioengineered killer extracellular vesicles.Scientific reports · 2023Article
- Approaches to Enhance Precise CRISPR/Cas9-Mediated Genome Editing.International journal of molecular sciences · 2021Review
- SCR7, a potent cancer therapeutic agent and a biochemical inhibitor of nonhomologous DNA end-joining.Cancer reports (Hoboken, N.J.) · 2021Review
- Diphthamide-deficiency syndrome: a novel human developmental disorder and ribosomopathy.European journal of human genetics : EJHG · 2020Article
- Modulation of DNA double-strand break repair as a strategy to improve precise genome editing.Oncogene · 2020Review
- Antibody-targeted chromatin enables effective intracellular delivery and functionality of CRISPR/Cas9 expression plasmids.Nucleic acids research · 2019Article
Corrections and comments
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivation was ~30-50-fold more frequent than cassette integration. Mono-allelic inactivation without integration occurred >100-fold more frequently than integration. Assessment of gRNA length confirmed 20mers to be most effective length for inactivation, while 16-18mers provided the highest overall integration efficacy. The overall efficacy was ~2-fold higher for CRISPR/Cas9 than for zinc-finger nuclease and was significantly increased upon modulation of non-homologous end joining or homology-directed repair. The frequencies and ratios of editing events were similar for two different DPH genes (independent of the target sequence or chromosomal location), which indicates that the optimization parameters identified with this method can be generalized.
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