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Cells 2024 Jan 29;133:. doi: 10.3390/cells13030247.
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Simplifying Genotyping of Mutants from Genome Editing with a Parallel qPCR-Based iGenotype Index.

Fu L , Wang S , Liu L , Shibata Y , Okada M , Luu N , Shi YB .

Targeted genome editing is a powerful tool in reverse genetic studies of gene function in many aspects of biological and pathological processes. The CRISPR/Cas system or engineered endonucleases such as ZFNs and TALENs are the most widely used genome editing tools that are introduced into cells or fertilized eggs to generate double-strand DNA breaks within the targeted region, triggering cellular DNA repair through either homologous recombination or non-homologous end joining (NHEJ). DNA repair through the NHEJ mechanism is usually error-prone, leading to point mutations or indels (insertions and deletions) within the targeted region. Some of the mutations in embryos are germline transmissible, thus providing an effective way to generate model organisms with targeted gene mutations. However, point mutations and short indels are difficult to be effectively genotyped, often requiring time-consuming and costly DNA sequencing to obtain reliable results. Here, we developed a parallel qPCR assay in combination with an iGenotype index to allow simple and reliable genotyping. The genotype-associated iGenotype indexes converged to three simple genotype-specific constant values (1, 0, -1) regardless of allele-specific primers used in the parallel qPCR assays or gene mutations at wide ranges of PCR template concentrations, thus resulting in clear genotype-specific cutoffs, established through statistical analysis, for genotype identification. While we established such a genotyping assay in the Xenopus tropicalis model, the approach should be applicable to genotyping of any organism or cells and can be potentially used for large-scale, automated genotyping.

PubMed ID: 38334640
PMC ID: PMC10854663
Article link: Cells
Grant support: [+]

Species referenced: Xenopus tropicalis
Genes referenced: amdhd1 ctrl mbd3 slc22a18 wt1

Article Images: [+] show captions
References [+] :
Bell, A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing. 2014, Pubmed