ArticlePloS one2014
A PCR based protocol for detecting indel mutations induced by TALENs and CRISPR/Cas9 in zebrafish.
Article in PloS one, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 60 papers.
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Who cites it
60 citing papers in PubMed.
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- Investigation of Peroxisome Proliferator-Activated Receptor Genes as Requirements for Visual Startle Response Hyperactivity in Larval Zebrafish Exposed to Structurally Similar Per- and Polyfluoroalkyl Substances (PFAS).Environmental health perspectives · 2024Article
- Temporal landscape of mitochondrial proteostasis governed by the UPRScience advances · 2023Article
- A Digital PCR Method Based on Highly Specific Taq for Detecting Gene Editing and Mutations.International journal of molecular sciences · 2023Article
- Deficiency of Adipose Triglyceride Lipase Induces Metabolic Syndrome and Cardiomyopathy in Zebrafish.International journal of molecular sciences · 2022Article
- Optimized In Vitro CRISPR/Cas9 Gene Editing Tool in the West Nile Virus Mosquito Vector,Insects · 2022Article
- An Optimized Thermal Feedback Methodology for Accurate Temperature Control and High Amplification Efficiency during Fluorescent qPCR.Bioengineering (Basel, Switzerland) · 2022Article
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- A Cysteine-Rich Protein, SpDIR1L, Implicated in S-RNase-Independent Pollen Rejection in the Tomato (International journal of molecular sciences · 2021Article
- Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis.International journal of molecular sciences · 2021Article
- Rapid visual genotyping method for germline mutants with small genomic fragment deletion by allele-specific PCR and lateral flow nucleic acid biosensor.Molecular biology reports · 2021Article
- A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes.eLife · 2021Article
- CRISPR-Cas-Led Revolution in Diagnosis and Management of Emerging Plant Viruses: New Avenues Toward Food and Nutritional Security.Frontiers in nutrition · 2021Review
- TMBIM6/BI-1 contributes to cancer progression through assembly with mTORC2 and AKT activation.Nature communications · 2020Article
- Effective identification of CRISPR/Cas9-induced and naturally occurred mutations in rice using a multiplex ligation-dependent probe amplification-based method.TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik · 2020Article
- Technologies and Computational Analysis Strategies for CRISPR Applications.Molecular cell · 2020Review
- Heat-shock-induced tyrosinase gene ablation with CRISPR in zebrafish.Molecular genetics and genomics : MGG · 2020Article
- cAMP signaling primes lung endothelial cells to activate caspase-1 duringAmerican journal of physiology. Lung cellular and molecular physiology · 2020Article
- MultiFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction.Scientific reports · 2020Article
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Authors and funding
4 authors.
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No grant is acknowledged in the PubMed record.
Abstract
Genome editing techniques such as the zinc-finger nucleases (ZFNs), transcription activator-like effecter nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system Cas9 can induce efficient DNA double strand breaks (DSBs) at the target genomic sequence and result in indel mutations by the error-prone non-homologous end joining (NHEJ) DNA repair system. Several methods including sequence specific endonuclease assay, T7E1 assay and high resolution melting curve assay (HRM) etc have been developed to detect the efficiency of the induced mutations. However, these assays have some limitations in that they either require specific sequences in the target sites or are unable to generate sequencing-ready mutant DNA fragments or unable to distinguish induced mutations from natural nucleotide polymorphism. Here, we developed a simple PCR-based protocol for detecting indel mutations induced by TALEN and Cas9 in zebrafish. We designed 2 pairs of primers for each target locus, with one putative amplicon extending beyond the putative indel site and the other overlapping it. With these primers, we performed a qPCR assay to efficiently detect the frequencies of newly induced mutations, which was accompanied with a T-vector-based colony analysis to generate single-copy mutant fragment clones for subsequent DNA sequencing. Thus, our work has provided a very simple, efficient and fast assay for detecting induced mutations, which we anticipate will be widely used in the area of genome editing.
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