A CRISPR-Cas9-mediated versatile method for targeted integration of a fluorescent protein gene to visualize endogenous gene expression in Xenopus laevis

Xenopus laevis is a widely used model organism in developmental and regeneration studies. Despite several re-ports 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.