CRISPR-mediated knock-in editing in Xenopus

Versatile Xenopus tropicalis model with targeted integration of human BRAFV600E.

Ran R, Li L, Chen P, Li S, Wang P, Zhu Z, Wang X, Chen Y, Hang J, Liang W.

Proc Natl Acad Sci U S A. 2025 Sep 30;122(39):e2426981122. doi: 10.1073/pnas.2426981122. Epub 2025 Sep 26.

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Abstract

Targeting exogenous gene integrations in animals often exhibits low efficiency, limiting the development of gene knock-in models. Theoretically, by screening founder generation individuals based on the cell phenotypes resulting from gene knock-ins and leveraging the high fecundity of animals, heritable descendants with targeted knock-ins can be efficiently generated. Therefore, we utilized the high fecundity of Xenopus tropicalis and easily observable pigment phenotypes to construct a BRAFV600E-targeted mitf locus knock-in model. Results indicated that this approach enabled efficient generation of BRAFV600E knock-in X. tropicalis and produced a versatile frog model. The BRAFV600E knock-in induced the transdifferentiation of RPE cells into retinal cells, resulting in a symmetric retinal structure in the eyes of these frogs. The transformation of RPE cells ultimately leads to these frogs becoming eyeless frogs, which serve as a tool for retinal regeneration research. Additionally, in eyeless frogs the BRAFV600E knock-in led to the abnormal proliferation of both melanocytes and xanthophores into melanocytic and xanthocytic nevi respectively. Consequently, eyeless frogs provide a model for studying abnormal pigment cell proliferation, offering a platform for investigating pigment cell nevus formation. Furthermore, the cdkn2b-knockout eyeless frogs serve as a valuable xanthophoroma model for tumor biology research. Overall, the BRAFV600E-targeted knock-in X. tropicalis not only represents a strategy for constructing gene knock-in animal models but also serves as a versatile tool for research in retinal regeneration and tumor biology.

Figure 1. Generation of BRAFV600E knock-in X. tropicalis. (A) Strategy for generating the BRAFV600E knock-in line in X. tropicalis. (B) Dorsal (DS) and ventral (VS) views of wild-type tadpoles (n ≥ 10 tadpoles were examined), showing consistent pigmentation patterns. (C) Abnormal yellow and black pigmentation observed in the head and tail of eight G0 BRAFV600E knock-in tadpoles out of >600 examined. Arrows indicate regions of abnormal black pigmentation. (D) Summary of pancreas-specific EGFP-positive tadpoles at stage 52 from independent injection batches (N1–N3) and mitf–/– rescue injections (mitf-R). Pancreatic EGFP expression indicates genomic integration of the knock-in plasmid. (E) Statistics of BRAF+ tadpoles (those with abnormal pigmentation) at stage 52 across different injection batches for both BRAFV600E and mitf–/– rescue groups. In (D and E), EGFP refers to pancreas-specific expression. (F) Representative images of mitf knock-in line 1 (F1 generation) and wild-type tadpoles. Red arrows indicate magnified views of the eye. (n ≥100 tadpoles were examined per genotype). (G) Genotyping of mitf knock-in line 1. (Ma, Mb) and (Mc, Md) indicate genotypes at the upstream (UI) and downstream (DI) integration sites, respectively; (Me, Mf) target the UI site. A single copy of the plasmid was integrated, with a 6 bp deletion at UI and a 21 bp deletion at DI. Genotyping was performed using genomic DNA from a single heterozygous F1 tadpole and validated by Sanger sequencing (n = 40). PCR with primers Me/Mf detected only three outcomes: WT, –6 bp, and –21 bp, representing the unedited mitf allele and integration-associated indels, respectively (SI Appendix, Fig. S3). (Scale bars in panels C–F, 1 mm.)

Figure 2. The BRAFV600E knock-in mitf allele exhibits impaired protein function. (A) Strategy for verifying Mitf protein function using offspring from mitf−/− and mitf-BRAFV600E± X. tropicalis crosses, generating mitf-BRAFV600E+/mitf– individuals. “LOF” denotes loss of function, numbers 1–9 represent mitf exons. Irrelevant elements such as CreERT2 and EGFP are omitted from the schematic. (B) Representative stage 55 tadpoles from mitf−/− and mitf-BRAFV600E± crosses. Tadpoles with normal pigment cell development were genotyped as mitf±, while those with abnormal pigmentation were mitf-BRAFV600E+/mitf– (12 tadpoles with normal pigmentation and 6 tadpoles with abnormal pigmentation were identified.), confirmed via Sanger sequencing. mitf±and mitf-BRAFV600E+/mitf– genotyping were performed as described. mitf−/− stage 55 tadpoles served as controls lacking melanophores and xanthophores. Sanger sequencing results are shown in SI Appendix, Fig. S7. Red arrows: abnormal eyes. Enlarged views of regions adjacent to the red triangles are shown below the corresponding images. Black/yellow/blue arrows: melanophores/xanthophores/abnormal iridophores. (Scale bar, 1 mm.) (C) Quantification of normal vs. abnormal phenotypes from three independent mitf−/− X mitf-BRAFV600E± crosses (random subset, n indicated). Statistical analysis was performed using an unpaired t test; ns = not significant. (D) Representative western blot results of Mitf protein from WT, mitf-BRAFV600E± (annotated as BRAFV600E+/−), mitf−/−, and mitf-BRAFV600E+/mitf– tadpole skin samples (n = 3). Gapdh was used as a loading control. The relative expression level was expressed as the mean value of three ratios normalized to the internal reference protein. Statistical analysis was performed using one-way ANOVA. ** denotes P < 0.01. Unmarked intergroup comparisons showed no statistically significant differences.

Figure 3. Retinal Regeneration Model. (A)Early eye development in WT X. tropicalis and eyeless frogs. Representative images show right-eye development at stages 40 and 37–38 in three WT and three F2 mitf KI line 1 frogs. No differences were observed between the left and right eyes in eyeless frogs. After mating F1 mitf KI line 1 with WT frogs, 48 normally developing F2 embryos were selected at stage 12 and placed into two 24-well plates, one per well. Eye development was recorded at regular intervals. Of 44 embryos that developed normally to stage 49, 24 showed no abnormalities, while 20 displayed pigmentation and eye development issues. Eye abnormalities at stage 40 were traceable to stages 37–38 but were absent at stages 35–36. See SI Appendix, Fig. S9 for details. Arrows in the figure point to earlier stages of eye development in the same tadpoles. (B) Images show eye development in WT and mitf KI line 1 X. tropicalis at various stages (n ≥ 50 tadpoles/frogs per genotype were analyzed). Arrows indicate eyes. (C) H&E staining of WT and mitf KI line 1 eyes at stages 49 and 57 (For each genotype, 6 tadpoles were sampled and n ≥ 9 paraffin sections examined). Arrows point to regenerated retina from RPE transdifferentiation. (D and E) High-magnification H&E staining of stage 57 WT (D) and mitf KI line 1 (E) eyes (For each genotype, 6 tadpoles were sampled and n ≥ 9 paraffin sections examined). Structures of regenerative and primordial retina in stage 57 mitf KI line 1 tadpoles are labeled. cho, choroid; rpe, retinal pigmented epithelium; os, outer segments; onl, outer nuclear layer; opl, outer plexiform layer; inl, inner nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer. (Scale bars, 100 μm in A, 500 μm in B, 50 μm in C, and 10 μm in D and E.)

Adapted with permission from National Academy of Science on behalf of PNAS: Ran et al. (2025). Versatile Xenopus tropicalis model with targeted integration of human BRAFV600E. Proc Natl Acad Sci U S A. 2025 Sep 30;122(39):e2426981122. doi: 10.1073/pnas.2426981122. Epub 2025 Sep 26.

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Step-by-Step Protocol for Making a Knock-In Xenopus laevis to Visualize Endogenous Gene Expression.

Dev Growth Differ 2025 Jun 09;675:293-302. doi: 10.1111/dgd.70011.

Kagawa N, Umesono Y, Suzuki KT, 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 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.

Adapted with permission from John Wiley & Sons on behalf of Development, Growth & Differentiation: Kagawa et al. (2025).Step-by-Step Protocol for Making a Knock-In Xenopus laevis to Visualize Endogenous Gene Expression. Dev Growth Differ 2025 Jun 09;675:293-302. doi: 10.1111/dgd.70011.