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Dev Biol 2024 Feb 01;506:42-51. doi: 10.1016/j.ydbio.2023.11.010.
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A CRISPR-Cas9-mediated versatile method for targeted integration of a fluorescent protein gene to visualize endogenous gene expression in Xenopus laevis.

Mochii M , Akizuki K , Ossaka H , Kagawa N , Umesono Y , Suzuki KT .

Xenopus laevis is a widely used model organism in developmental and regeneration studies. Despite several reports regarding targeted integration techniques in Xenopus, there is still room for improvement of them, especially in creating reporter lines that rely on endogenous regulatory enhancers/promoters. We developed a CRISPR-Cas9-based simple method to efficiently introduce a fluorescent protein gene into 5' untranslated regions (5'UTRs) of target genes in Xenopus laevis. A donor plasmid DNA encoding an enhanced green fluorescent protein (eGFP) flanked by a genomic fragment ranging from 66 bp to 878 bp including target 5'UTR was co-injected into fertilized eggs with a single guide RNA and Cas9 protein. Injections for krt12.2.L, myod1.S, sox2.L or brevican.S resulted in embryos expressing eGFP fluorescence in a tissue-specific manner, recapitulating endogenous expression of target genes. Integrations of the donor DNA into the target regions were examined by genotyping PCR for the eGFP-expressing embryos. The rate of embryos expressing the specific eGFP varied from 2.1% to 13.2% depending on the target locus and length of the genomic fragment in the donor plasmids. Germline transmission of an integrated DNA was observed. This simple method provides a powerful tool for exploring gene expression and function in developmental and regeneration research in X. laevis.

PubMed ID: 38052295
Article link: Dev Biol

Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: bcan krt12.2 myod1 sox2
GO keywords: embryo development
gRNAs referenced: bcan gRNA1 krt12.2 gRNA1 myod1 gRNA1 sox2 gRNA4

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