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 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 eGFPs signal were obtained by outcrossing multiple founders 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.