ArticleNucleic acids research2017
CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.
Article in Nucleic acids research, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 47 papers.
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Who cites it
47 citing papers in PubMed.
- CRISPR-HAWK: haplotype- and variant-aware guide design toolkit for CRISPR-Cas.Bioinformatics (Oxford, England) · 2026Article
- Sensitizing carbapenem-resistant Klebsiella pneumoniae using a compact Cascade-Cas3 system.International microbiology : the official journal of the Spanish Society for Microbiology · 2026Article
- Compound delivery of eVLPs enhances prime editing for targeted genome engineering and high-throughput screening.Cell genomics · 2026Article
- Targeted multiplex gene knockouts in Lemna minor using CRISPR/Cas9.Transgenic research · 2026Article
- Essentiality and dynamic expression of the human tRNA pool during viral infection.Molecular systems biology · 2026Article
- CRISPR-HAWK: Haplotype- and Variant-aware guide design toolkit for CRISPR-Cas.bioRxiv : the preprint server for biology · 2025Article
- Targeted S100A8 PP-Cas9@PLGA-apt microparticles reduced cartilage degradation and subchondral bone isomerism in osteoarthritis treatment through JAK/STAT3 pathway modulation.Materials today. Bio · 2025Article
- Development of CRISPR-Cas9-based genome editing tools for non-model microorganismSynthetic and systems biotechnology · 2025Article
- The impact of the ATP-binding cassette (ABC) transporter family on multidrug resistance in head and neck tumors.Molecular biology reports · 2025Review
- Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening.Nature communications · 2025Article
- A long journey towards genome editing technologies in plants: a technical and critical review of genome editing technologies.Frontiers in genome editing · 2025Review
- Artificial dynamic structure ensemble-guided rational design of a universal RNA aptamer-based sensing tag.Proceedings of the National Academy of Sciences of the United States of America · 2024Article
- A platform to deliver single and bi-specific Cas9/guide RNA to perturb genes in vitro and in vivo.Molecular therapy : the journal of the American Society of Gene Therapy · 2024Article
- Improvement in Tol2 transposon for efficient large-cargo capacity transgene applications in cultured cells and zebrafish (Zoological research · 2024Article
- Expanding the CRISPR Toolbox for Engineering Lycopene Biosynthesis inMicroorganisms · 2024Article
- Quantum measurement enables single biomarker sensitivity in flow cytometry.Scientific reports · 2024Article
- Extracellular vesicles associated microRNAs: Their biology and clinical significance as biomarkers in gastrointestinal cancers.Seminars in cancer biology · 2024Review
- The Current Situation and Development Prospect of Whole-Genome Screening.International journal of molecular sciences · 2024Review
- Modulation of EZH2 Activity Induces an Antitumoral Effect and Cell Redifferentiation in Anaplastic Thyroid Cancer.International journal of molecular sciences · 2023Article
- A Polymeric Nanoparticle Formulation for Targeted mRNA Delivery to Fibroblasts.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2023Article
Corrections and comments
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Authors and funding
7 authors.
Funding
Abstract
CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform.
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Registered trials
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