Competitive PCR with dual fluorescent primers enhances the specificity and reproducibility of genotyping animals generated from genome editing

Targeted genome editing is a powerful tool for studying gene function in almost every aspect of biological and pathological processes. The most widely used genome editing approach is to introduce engineered endonucleases or CRISPR/Cas system into cells or fertilized eggs to generate double-strand DN...

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Bibliographic Details
Published inCell & bioscience Vol. 13; no. 1; p. 83
Main Authors Fu, Liezhen, Ma, Emily, Okada, Morihiro, Shibata, Yuki, Shi, Yun-Bo
Format Journal Article
LanguageEnglish
Published England BioMed Central Ltd 11.05.2023
BioMed Central
BMC
Subjects
Analysis
Animal genetic engineering
Competition
CRISPR
Deoxyribonucleic acid
Design
DNA
DNA damage
DNA repair
Dyes
Embryos
Fluorescent PCR
Gene mutations
Genes
Genetic research
Genome editing
Genomes
Genomics
Genotype & phenotype
Genotyping
Homologous recombination
Letter to the Editor
Mutation
Non-homologous end joining
Polymerase chain reaction
Proteins
Xenopus tropicalis
Genome editing
Genotyping
Xenopus tropicalis
Fluorescent PCR
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Summary:Targeted genome editing is a powerful tool for studying gene function in almost every aspect of biological and pathological processes. The most widely used genome editing approach is to introduce engineered endonucleases or CRISPR/Cas system into cells or fertilized eggs to generate double-strand DNA breaks within the targeted region, leading to DNA repair through homologous recombination or non-homologous end joining (NHEJ). DNA repair through NHEJ mechanism is an error-prone process that often results in point mutations or stretches of indels (insertions and deletions) within the targeted region. Such mutations in embryos are germline transmissible, thus providing an easy means to generate organisms with gene mutations. However, point mutations and short indels present difficulty for genotyping, often requiring labor intensive sequencing to obtain reliable results. Here, we developed a single-tube competitive PCR assay with dual fluorescent primers that allowed simple and reliable genotyping. While we used Xenopus tropicalis as a model organism, the approach should be applicable to genotyping of any organisms.
Bibliography:SourceType-Other Sources-1
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ObjectType-Correspondence-1
ISSN:2045-3701
2045-3701
DOI:10.1186/s13578-023-01042-2