ArticleBMC genomics2016
A simplified method for identifying early CRISPR-induced indels in zebrafish embryos using High Resolution Melting analysis.
Article in BMC genomics, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 37 papers.
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Who cites it
37 citing papers in PubMed, 80 citations in OpenAlex.
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- Loss of carbohydrate sulfotransferase 6 function leads to macular corneal dystrophy phenotypes and skeletal defects in zebrafish.The FEBS journal · 2025Article
- Naa80 is required for actin N-terminal acetylation and normal hearing in zebrafish.Life science alliance · 2024Article
- Neural progenitor-derived Apelin controls tip cell behavior and vascular patterning.Science advances · 2024Article
- scRNA-seq reveals the diversity of the developing cardiac cell lineage and molecular players in heart rhythm regulation.iScience · 2024Article
- Biallelic BORCS8 variants cause an infantile-onset neurodegenerative disorder with altered lysosome dynamics.Brain : a journal of neurology · 2024Article
- Dysregulated lysosomal exocytosis drives protease-mediated cartilage pathogenesis in multiple lysosomal disorders.iScience · 2024Article
- Zebrafish raptor mutation inhibits the activity of mTORC1, inducing craniofacial defects due to autophagy-induced neural crest cell death.Development (Cambridge, England) · 2024Article
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- DNA extraction from recently fertilised Atlantic salmon embryos for use in microsatellite validation of triploidy.PloS one · 2023Article
- Parental mutations influence wild-type offspring via transcriptional adaptation.Science advances · 2022Article
- Review
- Principles and Applications of CRISPR Toolkit in Virus Manipulation, Diagnosis, and Virus-Host Interactions.Cells · 2022Review
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
backgroundThe CRISPR/Cas9 system has become a regularly used tool for editing the genome of many model organisms at specific sites. However, two limiting steps arise in the process of validating guide RNA target sites in larvae and adults: the time required to identify indels and the cost associated with identifying potential mutant animals.
resultsHere we have combined and optimized the HotSHOT genomic DNA extraction technique with a two-steps Evagreen PCR, followed by a high-resolution melting (HRM) assay, which facilitates rapid identification of CRISPR-induced indels. With this technique, we were able to genotype adult zebrafish using genomic DNA extracted from fin-clips in less than 2 h. We were also able to obtain a reliable and early read-out of the effectiveness of guide RNAs only 4 h after the embryos were injected with the constructs for the CRISPR/Cas9 mutagenic system. Furthermore, through mutagenesis kinetic assay, we identified that the 2-cell stage is the earliest time point at which indels can be observed.
conclusionsBy combining an inexpensive and rapid genomic DNA extraction method with an HRM-based assay, our approach allows for high-throughput genotyping of adult zebrafish and embryos, and is more sensitive than standard PCR approaches, permitting early identification of CRISPR-induced indels and with applications for other model organisms as well.
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