ArticleDisease models & mechanisms2018
CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments.
Article in Disease models & mechanisms, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 58 papers.
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58 citing papers in PubMed, 101 citations in OpenAlex.
- Interfering with DNA repair pathways to enhance CRISPR-Cas9-mediated homology-directed repair in a chelicerate genetic model.iScience · 2026Article
- Efficient Endogenous Tagging in the Sea Urchin,bioRxiv : the preprint server for biology · 2026Article
- Precise, predictable genome integrations by deep-learning-assisted design of microhomology-based templates.Nature biotechnology · 2026Article
- Universal conditional knockout approach to multiple species, from fish to human induced pluripotent stem cells.Nucleic acids research · 2026Article
- Strategies and considerations for the generation of ssDNA-Based HDR templates for CRISPR-based genome editing.BMC genomics · 2026Review
- Enhancing CRISPR Homology Directed Repair in IAL-PiD2 Insect Cells via Reagent Delivery Optimization and Cell Synchronization.Biochemical engineering journal · 2026Article
- Early death and neuronal abnormalities inbioRxiv : the preprint server for biology · 2025Article
- Transferable approaches to CRISPR-Cas9 induced genome editing in non-model insects: a brief guide.Frontiers in zoology · 2025Review
- Genetically encoded affinity reagents are a toolkit for visualizing and manipulating endogenous protein function in vivo.Nature communications · 2025Article
- Engineering precision zebrafish alleles of human disease.bioRxiv : the preprint server for biology · 2025Article
- Modeling thoracic aortic genetic variants in the zebrafish: useful for predicting clinical pathogenicity?Frontiers in cardiovascular medicine · 2025Review
- Efficient DNA-free co-targeting of nuclear genes in Chlamydomonas reinhardtii.Biology direct · 2024Article
- Instantaneous visual genotyping and facile site-specific transgenesis via CRISPR-Cas9 and phiC31 integrase.Biology open · 2024Article
- A cis-regulatory element regulates ERAP2 expression through autoimmune disease risk SNPs.Cell genomics · 2024Article
- Mutation Knock-in Methods Using Single-Stranded DNA and Gene Editing Tools in Zebrafish.Methods in molecular biology (Clifton, N.J.) · 2024Review
- High-fidelity large-diversity monoclonal mammalian cell libraries by cell cycle arrested recombinase-mediated cassette exchange.Nucleic acids research · 2023Article
- Retained chromosomal integrity following CRISPR-Cas9-based mutational correction in human embryos.Molecular therapy : the journal of the American Society of Gene Therapy · 2023Article
- Fast, precise and cloning-free knock-in of reporter sequences in vivo with high efficiency.Development (Cambridge, England) · 2023Article
- Enrichment of transgene integrations by transient CRISPR activation of a silent reporter gene.Molecular therapy. Methods & clinical development · 2023Article
- Activated PI3K delta syndrome 1 mutations cause neutrophilia in zebrafish larvae.Disease models & mechanisms · 2023Article
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Authors and funding
7 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Targeted genome editing by CRISPR/Cas9 is extremely well fitted to generate gene disruptions, although precise sequence replacement by CRISPR/Cas9-mediated homology-directed repair (HDR) suffers from low efficiency, impeding its use for high-throughput knock-in disease modeling. In this study, we used next-generation sequencing (NGS) analysis to determine the efficiency and reliability of CRISPR/Cas9-mediated HDR using several types of single-stranded oligodeoxynucleotide (ssODN) repair templates for the introduction of disease-relevant point mutations in the zebrafish genome. Our results suggest that HDR rates are strongly determined by repair-template composition, with the most influential factor being homology-arm length. However, we found that repair using ssODNs does not only lead to precise sequence replacement but also induces integration of repair-template fragments at the Cas9 cut site. We observed that error-free repair occurs at a relatively constant rate of 1-4% when using different repair templates, which was sufficient for transmission of point mutations to the F1 generation. On the other hand, erroneous repair mainly accounts for the variability in repair rate between the different repair templates. To further improve error-free HDR rates, elucidating the mechanism behind this erroneous repair is essential. We show that the error-prone nature of ssODN-mediated repair, believed to act via synthesis-dependent strand annealing (SDSA), is most likely due to DNA synthesis errors. In conclusion, caution is warranted when using ssODNs for the generation of knock-in models or for therapeutic applications. We recommend the application of in-depth NGS analysis to examine both the efficiency and error-free nature of HDR events.This article has an associated First Person interview with the first author of the paper.
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