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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 the target 5' untranslated region (UTR) of the Xenopus laevis genome. It enables us to track eGFP expression under the 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.
FIGURE 1 | Overview of NEXTi. (A) Summary of the NEXTi procedure to make a knock- in founder and F1 offsprings. The NEXTi donor plasmid carrying the egfp is injected into fertilized Xenopus laevis eggs with Cas9 protein and a single guide RNA. Selected founder (F0) expressing tissue-specific eGFP is outcrossed with wild- type animal (wt) to obtain F1 animals. (B) Schematic representation of targeted integration by NEXTi. The Cas9 RNP binds and cleaves both the target sequences in the donor plasmid and the 5′ UTR of the target locus. The plasmid integrates into the target sites in the zygotic genome via DSB/NHEJ. Insertion and/or deletion (indel) is found in the upstream and downstream junctions.
FIGURE 2 | Expression of eGFP in knock- in vector injected embryos. Representative images of embryos targeted for myod1.S (A, A', NF stage 35/36), sox2.L (B, B′, NF stage 37/38) and krt12.2.L (C, C′, NF stage 41) loci. (A‑C), bright field images. (A'‑C′), fluorescence images of (A‑C). Clear eGFP signal was observed in somatic muscle (arrowhead in A'), brain and lens (arrowheads in B′), and fin (arrowhead in C′). Scale bar = 1 mm.
FIGURE 3 | Expression of eGFP in F1 siblings of sox2.L:Egfp. Representative images of F1 embryos (NF stage 33/34) obtained by crossing a female sox2.L:Egfp founder #2 (Table 3) and a wild- type male (A‑D). (A) bright field images. (A'), fluorescence image of (A). (B‑D) magnified view of each embryo in (A'). (B) specific eGFP signal is observed in brain (b), spinal cord (sc), optic vesicles (op), olfactory pit (ol), and otic vesicles (ot). (C) Ubiquitous eGFP signal at a low level is observed. (D) No eGFP signal is observed. Scale bar = 1 mm.
FIGURE 4 | Genotyping of F1 animals. (A) Scheme of an integrated target locus. PCR was performed with genomic DNAs obtained from F1 animals. Upstream and downstream sequences, including junctions between the donor and the target regions, were amplified using donor vector primers and gene- specific primers. Primer sequences are listed in Table S1. (B) Examples of PCR analysis for sox2.L:Egfp F1 embryos obtained from the founder #1 (1, 2) and founder #2 (3–6). PCR products for both upstream and downstream junction fragments were observed in DNAs from F1 embryos with specific eGFP expression (1–4) but not from embryos with ubiquitous eGFP (5, 6) or wild- type embryos (wt). Predicted sizes of the upstream and downstream PCR fragments are indicated as arrowheads. PCR products with larger or smaller sizes indicate insertion or deletion in the junctions. (C) Example of upstream and downstream junction sequences. The PCR products of sox2.L:Egfp F1 embryo (lane 3) were sequenced directly. Determined sequences are indicated with the wild- type partial sequence of the 5′ UTR. Underline indicates the sgRNA target sequence with PAM sequence (red underline).
