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bioRxiv · 10.1101/2025.03.05.640691

Step-by-step protocol for making a knock-in Xenopus laevis to visualize endogenous gene expression

Kagawa, N.·Umesono, Y.·Suzuki, K.-i. T.·Mochii, M.
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Abstract

We established a novel knock-in technique, New and Easy Xenopus Targeted integration (NEXTi), to recapitulate endogenous gene expression by reporter expression. NEXTi is a CRISPR-Cas9-based method to integrate a donor DNA containing a reporter gene (egfp) into target 5 untranslated region (UTR) of Xenopus laevis genome. It enables us to track eGFP expression under regulation of endogenous promoter/enhancer activities. We obtained about 2% to 13% of knock-in vector-injected embryos showing eGFP signal in a tissue-specific manner, targeting krt.12.2.L, myod1.S, sox2.L and bcan.S loci, as previously reported. In addition, F1 embryos which show stable eGFP signals were obtained by outcrossing the matured injected frogs with wild-type animals. Integrations of donor DNAs into target 5 UTRs were confirmed by PCR amplification and sequencing. Here, we describe the step-by-step protocol for preparation of donor DNA and single guide RNA, microinjection and genotyping of F1 animals for the NEXTi procedure. 1. HighlightO_LIIntended organism: Xenopus laevis C_LIO_LIPurpose of the protocol: Efficient knock-in for visualizing endogenous target gene expression using CRISPR-Cas9 system C_LIO_LIEssential equipment and materials: Microinjector, fluorescence microscopy, Cas9 protein, sgRNA, donor DNA C_LIO_LIFeatures: Expression of reporter genes depends on endogenous enhancer/promoter activities. C_LI

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Kagawa, N., Umesono, Y., Suzuki, K.-i. T., Mochii, M.. 2025-03-07. Step-by-step protocol for making a knock-in Xenopus laevis to visualize endogenous gene expression. https://doi.org/10.1101/2025.03.05.640691

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