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

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.