ArticleInternational journal of molecular sciences2021
Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes.
Article in International journal of molecular sciences, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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Who cites it
19 citing papers in PubMed, 22 citations in OpenAlex.
- Review
- CRISPR Gene Tagging for Illuminating Endogenous Protein Dynamics.International journal of molecular sciences · 2026Review
- Zebrafish as a Model for Translational Immuno-Oncology.Journal of personalized medicine · 2025Review
- CRISPR-based functional genomics tools in vertebrate models.Experimental & molecular medicine · 2025Review
- Identifying optimal conditions for precise knock-in of exogenous DNA into the zebrafish genome.Development (Cambridge, England) · 2025Article
- Engineering precision zebrafish alleles of human disease.bioRxiv : the preprint server for biology · 2025Article
- A review on current advancement in zebrafish models to study chronic inflammatory diseases and their therapeutic targets.Heliyon · 2024Review
- Article
- Mutation Knock-in Methods Using Single-Stranded DNA and Gene Editing Tools in Zebrafish.Methods in molecular biology (Clifton, N.J.) · 2024Review
- Protein Tyrosine Phosphatase Studies in Zebrafish.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Article
- Fluorescent PCR-based Screening Methods for Precise Knock-in of Small DNA Fragments and Point Mutations in Zebrafish.Bio-protocol · 2023Article
- Modelling human lower urinary tract malformations in zebrafish.Molecular and cellular pediatrics · 2023Review
- Review
- Improved selection of zebrafish CRISPR editing by early next-generation sequencing based genotyping.Scientific reports · 2023Article
- A robust pipeline for efficient knock-in of point mutations and epitope tags in zebrafish using fluorescent PCR based screening.BMC genomics · 2022Article
- Scrutinizing pathogenicity of the USH2A c.2276 G > T; p.(Cys759Phe) variant.NPJ genomic medicine · 2022Article
- Zebrafish as a Model to Study Vascular Elastic Fibers and Associated Pathologies.International journal of molecular sciences · 2022Review
- Zebrafish, Medaka and Turquoise Killifish for Understanding Human Neurodegenerative/Neurodevelopmental Disorders.International journal of molecular sciences · 2022Review
Corrections and comments
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Authors and funding
8 authors at 1 institution in 2 countries.
Funding
Abstract
CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3-8% of alleles, and 30-45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models.
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Registered trials
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