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

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. Highlight Intended organism: Xenopus laevis Purpose of the protocol: Efficient knock-in for visualizing endogenous target gene expression using CRISPR-Cas9 system Essential equipment and materials: Microinjector, fluorescence microscopy, Cas9 protein, sgRNA, donor DNA Features: Expression of reporter genes depends on endogenous enhancer/promoter activities. Competing Interest Statement The authors have declared no competing interest. Footnotes Advantages, limitation, Tip21 and Fig.S1were revised.