Synthetic gRNA-mediated multiplex CRISPR enables the generation of translucent F0 Xenopus laevis for in vivo imaging

Synthetic gRNA-mediated multiplex CRISPR enables the generation of translucent F0 Xenopus laevis for in vivo imaging | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Synthetic gRNA-mediated multiplex CRISPR enables the generation of translucent F0 Xenopus laevis for in vivo imaging Albert Chesneau, Axel Benchetrit, Pierre Affaticati, Jean-Pierre Levraud, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8552006/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Transparent model organisms are invaluable for live imaging, yet generating them remains challenging. Here, we present a robust strategy to produce translucent Xenopus laevis , enabling non-invasive, deep-tissue imaging in intact organisms. Using CRISPR/Cas9 technology, we generated quadruple knockouts of slc2a7 , hps6 , and the two tyrosinase homeologs. Instead of in vitro -transcribed single guide RNAs, we employed chemically modified, commercially synthesized two-part guide RNAs, which enabled efficient multiplex genome editing. We produced a high proportion of translucent frogs directly in F0 founder tadpoles, eliminating the need for multi-generational breeding. We validated in vivo live imaging using two transgenic reporter lines with GFP expression in the eye, brain, and heart. Loss of both eumelanin and iridescent pigments in tyr ; hps6 knockouts markedly improved optical clarity and fluorescence visibility. Overall, this multiplexed genome-editing strategy enables the rapid generation of translucent transgenic X. laevis suitable for live imaging, while also providing a simple and efficient protocol for simultaneous multi-gene targeting in X. laevis . Biological sciences/Biological techniques Biological sciences/Biotechnology Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION In most vertebrates, the body’s opacity conceals internal organs, making them challenging to study without invasive procedures. This opacity is largely due to pigment cells (melanophores, xanthophores, and iridophores) [ 1 ]. Some fish species, such as zebrafish and medaka, naturally appear semi-transparent early in life due to their low number of pigment cells. However, as development proceeds, pigment cells progressively accumulate in the skin, peritoneum, and retinal pigment epithelium (RPE) resulting in the gradual loss of transparency. To address this limitation, transparent and translucent zebrafish strains have been generated by combining pigment cell-deficient mutations. For instance, the widely used casper strain, which carries mutations in mitfa and mpv17 , causing a lack of melanophores and iridophores [ 2 , 3 ]. The crystal strain, which additionally includes a mutation in slc45a2 , also lacks melanin in the RPE [ 4 ]. The mitfa gene (also known as nacre ) encodes a transcription factor required for melanophore differentiation [ 5 ], mpv17 encodes a mitochondrial protein essential for iridophore survival [ 6 ], and slc45a2 encodes a membrane-associated transporter required for melanin synthesis [ 7 ]. Together, these pigment-deficient mutants enable optical transparency that facilitates in vivo imaging in adult zebrafish. Similar approaches have been used to generate transparent or translucent strains in other teleosts. In medaka, the “see-through” or spooky strains were established by combining multiple pigment-deficient mutations [ 8 , 9 ]. Transparent phenotypes have also been described in the killifish [ 10 ], goldfish [ 11 ], or Nile tilapia [ 12 ]. These models have been developed through a combination of traditional breeding of natural color mutants, ENU mutagenesis, and targeted gene disruption via CRISPR/Cas9. Many retain transparency throughout life, offering great possibilities for real-time imaging. Efforts to generate translucent model organisms are not limited to fish. In the amphibian Xenopus tropicalis , translucency has been achieved by targeting multiple pigmentation genes that affect distinct chromatophore types. For instance, pigment-deficient frogs were produced by crossing slc2a7 single knockouts (KO) with tyrosinase/hps6 double KO, followed by intercrossing of the F1 progeny [ 13 ]. The gene slc2a7 is required for yellow pigment production in xanthophores [ 14 ]. The gene tyrosinase ( tyr ) encodes the enzyme catalyzing the first step of melanin biosynthesis, producing the dark brown to black eumelanin pigment responsible for the majority of skin and retinal pigmentation [ 15 ]. The gene hps6 contributes to the biogenesis of pigment-cell organelles in iridophores and melanophores, as shown in the X. tropicalis mutant no privacy ( nop ), which exhibits a reduction or complete loss of these chromatophores [ 16 ]. Another example of pigment-deficient X. tropicalis model is the mitf −/− mutant, which lacks both melanophores and xanthophores and consequently exhibit markedly transparent skin, allowing clear visualization of internal organs [ 17 ]. In contrast, Xenopus laevis , despite being the most widely used amphibian model in developmental biology, has seen far fewer genetic attempts at achieving transparency. Of the pigmentation-related genes mentioned above, only tyr has been knocked out to date, using either TALEN or CRISPR technologies [ 18 – 23 ]. The X. laevis genome contains multiple tyr -like genes distributed across chromosomes 2L, 2S, 6L, and 6S. However, only the two homeologs on chromosomes 2L and 2S encode the functional Tyrosinase enzyme required for eumelanin synthesis, as their simultaneous inactivation completely abolishes the production of brown/black melanin in melanophores and the RPE, resulting in albino individuals [ 23 ]. Here, to obtain a fully transparent X. laevis , we employed CRISPR/Cas9 to generate a quadruple knockout of slc2a7 , hps6 , and the two tyr2L and tyr2S homeologs. Importantly, rather than in vitro -transcribed single guide RNAs (sgRNAs), we used chemically modified, commercially synthesized two-part gRNAs (crRNA:tracrRNA), which exhibit enhanced stability and reduced cellular toxicity [ 24 , 25 ]. This strategy enabled efficient multiplex genome editing, yielding a high proportion of translucent X. laevis individuals directly in F0 embryos, eliminating the need for multi-generational breeding. Using two transgenic reporter lines, we demonstrated that these animals are suitable for in vivo visualization of fluorescent signals, even in the eye, where the combined loss of melanic and iridescent pigments provides greater optical clarity than that achieved in classical albino animals. RESULTS Simultaneous knockout of tyrosinase (2L, 2S) and hps6 genes enhances transparency in Xenopus laevis We sought to generate translucent X. laevis by disrupting pigment-related genes using CRISPR/Cas9. To achieve maximal transparency, we performed a triple knockout targeting both tyr genes on chromosomes 2L and 2S (using a gRNA that targets both genes), as well as hps6 (Fig. 1 A). Rather than in vitro -transcribed single guide RNAs (sgRNAs), we used chemically modified, commercially synthesized two-part guide RNAs (gRNAs), to maximize stability and knockout efficiency, which were co-injected with the Cas9 protein at the one-cell stage. We assessed pigmentation at stage 52–54, when iridescent pigments begin to markedly reduce tissue transparency (Fig. 1 B). Compared with wild-type, hps6 knockout (KO) tadpoles clearly lacked iridescent pigmentation over the gut and displayed reduced brown/black skin pigmentation, yet retained strong RPE pigmentation in the eye. In contrast, tyr double KOs exhibited a complete loss of brown/black pigmentation, including in the RPE, but retained iridescent pigments over the gut and eye. Triple KOs lacked both eumelanin (black or brown pigmentation) and iridescent pigments, resulting in a significant increase in transparency than tyr -only knockouts. This improvement was particularly striking in the eye, where loss of iridescent pigments in the triple KO abolished a major source of light scattering observed in tyr -only knockouts (Fig. 1 C). The effect was equally evident in froglets, where simultaneous loss of melanin and iridescent pigments markedly increased transparency compared with melanin loss alone, most notably across the entire ventral surface (Fig. 1 D). However, transparency becomes less apparent in adult frogs, due to the increased thickness of the skin. In terms of efficiency, injection of the two gRNAs yielded a mix of phenotypes: wild-type-like pigmentation, partial pigment loss (mosaicism), and complete pigment loss. The overall editing efficiency was high: 84.86% ±2.85 (SEM) of injected embryos exhibited complete loss of melanin pigmentation, indicating efficient knockout of tyr (based on 5 independent experiments, n = 429 embryos). To confirm that both tyr homeologs ( tyr2L and tyr2S ) were successfully mutated, we performed PCR analysis on individuals showing a complete absence of eumelanin. In all cases ( n = 4), the crispant individuals carried only mutant alleles for both tyr genes, with no wild-type sequences detected (Supplementary Fig. 1). Furthermore, the presence of only 2 to 7 distinct mutations per individual suggests that genome editing occurred at an early developmental stage, likely before the fourth cleavage. Among these melanin-deficient tadpoles, 40.63% (n = 128) also lacked any detectable iridescent pigmentation. This proportion increased to 68.76% when mosaic individuals were included. Together, these results support efficient simultaneous disruption of pigment-associated loci in F0 animals, as assessed by phenotype and genotyping. Concurrent removal of melanin and iridescent pigments enhances live imaging of fluorescent reporters Pigmentation in Xenopus reduces optical transparency, particularly as development progresses, substantially impairing fluorescence imaging in transgenic lines and thereby restricting the feasibility of live imaging in tadpoles and froglets. The use of natural albino lines (arising from a spontaneous mutation in hsp4 gene [ 26 ]) already provides a practical way to mitigate this issue. For example, we recently demonstrated that the dynamics of retinal photoreceptor degeneration and regeneration can be monitored in vivo by live imaging in an albino Tg(rho:eGFP-NTR) transgenic line [ 27 ]. In this line, GFP is specifically expressed in rod photoreceptors under the rhodopsin promoter, and the NTR enzyme enables their selective ablation upon exposure to the prodrug metronidazole [ 28 , 29 ]. Here, we sought to determine whether removing both melanin and iridescent pigments could improve GFP fluorescence visualization in transgenic tadpoles. To this end, we crossed pigmented Tg(rho:eGFP-NTR) with wild-type individuals and injected the one-cell stage progeny with gRNAs targeting both tyr genes and hps6 (Fig. 2 A). The simultaneous loss of eumelanin and iridescent pigments in triple KO markedly improved fluorescence visibility in the eye compared with tyr -only KO (Fig. 2 B). In addition to reducing RPE transparency, we showed above that iridescent pigments also contribute to opacifying the entire ventral region. We therefore evaluated the benefit of the triple KO in a transgenic line expressing eGFP in the heart. The Tg(her4:eGFP, cmlc2:eGFP) line expresses GFP in Müller glial cells in the retina via the her4 promoter and in differentiated cardiomyocytes via the cardiac myosin light chain 2 ( cmlc2 ) promoter [ 30 ]. First, in this context as well, the visualization of the fluorescence in the retina is improved in tyr ; hps6 KO compared to tyr -only KO (Fig. 2 A, C). In addition, simultaneous removal of eumelanin and iridescent pigments also enhanced GFP visibility in the heart, compared with eumelanin removal alone (Fig. 2 C). Collectively, these findings demonstrate that combining transgenesis with targeted pigment-gene editing is a powerful approach for improving live fluorescence imaging across multiple tissues in X. laevis . Generation of translucent and white X. laevis by simultaneous removal of melanic, iridescent, and yellow pigmentation via CRISPR-mediated quadruple gene KO Yellow pigmentation is generally sparse throughout most larval stages. However, some individuals exhibit increased yellow pigment at later stages, which may compromise overall transparency. To address this, we extended our CRISPR-based strategy to include slc2a7 , a gene involved in yellow pigment production, in addition to tyr genes and hps6 , thereby generating quadruple KO individuals (Fig. 3 A). As expected, quadruple KO tadpoles lacking eumelanin, iridescent, and yellow pigments appeared noticeably whiter than triple tyr;hps6 KOs (Fig. 3 B). Because some individuals could naturally exhibit a whiter phenotype than usual, we confirmed by PCR genotyping that indeed it resulted specifically from slc2a7 disruption (Supplementary Fig. 2). As tadpoles, froglets lacking all three pigment types also exhibited white skin, in contrast to the yellow-toned skin of frogs in which only eumelanin and iridescent pigments were removed (Fig. 3 C). This phenotype persisted into adulthood: adult frogs displayed a progressive spectrum of skin coloration, from darkly pigmented to white, depending on whether one, two, or all three pigment types are disrupted (Fig. 3 D). White KO individuals were highly transparent up to the froglet stage (Fig. 3 E) while this transparency became less pronounced in adults (Fig. 3 D). Importantly, quadruple KO individuals generated in this study developed normally, indicating that neither full pigment loss nor the use of synthetic gRNAs induce detectable adverse effects during development. Moreover, they are fertile and capable of producing viable, morphologically normal F1 offspring exhibiting the same white and transparent phenotype as the parents (Fig. 3 F). We thus next asked whether removal of yellow pigments in addition to eumelanin and iridescent pigments could further enhance the visualization of fluorescent proteins. In the Tg(her4-GFP, clmc-GFP) line, GFP signal intensity in the brain (likely originating from radial glial cells, as described in the zebrafish brain [ 31 ]) was comparable between quadruple mutants and tyr;hps6 triple KOs (Fig. 3 G). These findings suggest that eliminating yellow pigmentation does not significantly improve fluorescence visibility. DISCUSSION The intense dark pigmentation of melanophores and strong light scattering produced by iridophores have long hindered live fluorescence imaging in X. laevis . Compared with X. tropicalis , where transparent lines were generated through sequential intercrossing of single-gene knockouts [ 13 ], similar strategies are considerably less practical in X. laevis because of its long reproductive cycle and allotetraploid genome. In this study, we show that translucent and even “white” X. laevis can instead be produced rapidly in a single step using multiplex CRISPR/Cas9-mediated disruption of key pigment-related genes. By simultaneously targeting tyr , hps6 , and slc2a7 , we eliminated eumelanin-, iridophore-, and xanthophore-derived pigmentation, generating live animals substantially more transparent than naturally albino or single-pigment mutants. This multiplex genome-editing approach bypasses the need for laborious multi-generational breeding and enables the direct production of translucent individuals in the F0 generation. Efficient multiplex genome editing has previously been demonstrated in Xenopus species: for example, by co-targeting tyr homeologs in X. laevis [ 23 ]. The Vleminckx laboratory also developed robust multi-gene disruption strategies in X. tropicalis [ 31 , 32 ]. More recently, Cas12a-based approaches have also been used to inactivate duplicated genes in X. laevis [ 33 ]. These studies collectively demonstrate the feasibility of multiplex genome editing in amphibians. In all of these studies however, in vitro -transcribed sgRNAs were used. Our work extends this multiplex genome editing by using synthetic gRNAs to achieve high-efficiency gene editing in X. laevis , enabling generation of F0 animals at high penetrance. We indeed employed chemically stabilized two-part synthetic gRNAs, which are commercially available from Integrated DNA Technologies (IDT). This company provides several gRNA formats. In this study, we selected the most cost-effective option, which nevertheless yielded excellent editing efficiencies and minimal cytotoxicity. These synthetic RNAs simplify the experimental workflow and offer superior stability compared to in vitro -transcribed sgRNAs [ 24 , 25 ], making them particularly well suited for simultaneous multi-gene targeting. Although we did not compare alternative formats, it is possible that other chemically modified variants could further extend gRNA half-life and improve even further editing efficiency. The limited number of distinct mutations per individual indicates that editing occurs at early cleavage stages, allowing reliable functional analyses directly in the F0 generation. The viability and fertility of quadruple knockout adults further confirm that this strategy does not induce detectable developmental defects, validating the use of synthetic gRNAs for multiplex genome editing in amphibians. The goal of generating depigmented X. laevis lines is to improve optical access for live imaging of fluorescent reporters. Our results demonstrate that removal of both eumelanin and iridescent pigments qualitatively enhances fluorescence visibility, in particular in the retina and heart, across multiple transgenic contexts. Notably, the elimination of iridescent pigments reduced light scattering in the ventral region and ocular tissues. On the other hand, our work suggests that eliminating yellow pigmentation does not significantly improve fluorescence visibility. We thus favor to target solely tyr and hps6 genes if the aim is to monitor fluorescence in live animals. Considering the green autofluorescence of yellow pteridine pigments observed in zebrafish [ 34 ], additional targeting of slc2a7 in Xenopus may be beneficial in challenging imaging conditions with weak GFP transgenes. Despite complete disruption of slc2a7 , some individuals retained faint traces of yellow pigmentation. This observation suggests that slc2a7 is not the sole contributor to xanthophore pigment synthesis in X. laevis . A similar situation has been described in X. tropicalis , where multiple genes contribute to xanthophore development. Indeed, slc2a7 knockout eliminates xanthophore-specific pterinosomes [ 14 ], and mitf null mutants also lack xanthophores [ 17 ]. It is also possible that X. laevis synthesizes additional pigment types, such as pheomelanin (a yellow to reddish melanin variant) although this has not been documented in this species. Pheomelanin has been reported in Hymenochirus boettgeri , another member of the Pipidae family [ 35 ], suggesting that the enzymatic machinery for pheomelanin biosynthesis may be conserved in some pipids. Furthermore, the existence of multiple tyrosinase paralogs in X. laevis , including those on chromosomes 6L and 6S, raises the possibility that these enzymes could contribute to alternative pigment pathways, potentially linked to pheomelanin-like compounds. While this remains speculative, the multiplex approach employed in the present study provides a powerful tool for investigating amphibian pigment evolution and diversity in future research. METHODS Ethics statement All animal experiments have been carried out in accordance with the European Community Council Directive of 22 September 2010 (2010/63/EEC) and in accordance to ARRIVE guidelines. Animal care and experimentations were conducted in accordance with institutional guidelines, under the institutional license C 91-471-102. The study protocols were approved by the Animal Welfare Structure (SBEA) under the number HC 01-02-2025. Animals X. laevis adults were maintained and bred under standard laboratory conditions. Embryos were obtained by natural mating or in vitro fertilization, dejellied prior to injection, and reared according to established procedures [36].Tadpoles were staged according to [37]. Tg (rho:eGFP-NTR) and Tg(her4:eGFP; cmlc2:eGFP) animals have been generated previously [29,30] using the simplified REMI method [38,39]. Wild type X. laevis animals and transgenic lines are maintained at the TEFOR Paris-Saclay Zootechnics Service, which houses the French Xenopus Resource Center. Two-part guide RNAs Guide RNAs (gRNAs) targeting the tyr , hps6 and slc2a7 genes were designed using the CRISPOR web tool (http://crispor.tefor.net) to maximize on-target efficiency and minimize potential off-target effects. All gRNAs were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). For each gene, target sites were selected within early exons to maximize the likelihood of generating loss-of-function alleles. We employed the two-part gRNA format consisting of an Alt-R™ CRISPR/Cas9 CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) (IDT, #1072533). The crRNAs contain proprietary chemical modifications that enhance stability by protecting the RNA from endogenous nuclease degradation. When annealed with tracrRNA, they form a functional duplex compatible with standard Cas9-mediated genome editing. For tyr , the gRNA was adapted from [20], with two additional nucleotides (CA) introduced at the 5′ end to improve cleavage efficiency. The crRNA sequence used was 5′-CATGAGATTCAGAAGCTCAC-3′. This gRNA is designed to target both tyr2S and tyr2L homeologs. For hps6 and slc2a7 , the crRNA sequences were 5′-GCAGAGTGTTCCGTACGGCT-3′ and 5′-CTGGGACTCTCCGGAAACCA-3′, respectively. Notably, both genes are present as single-copy and do not have homeologs. Each crRNA (2 nmol) was resuspended in 20 µL of Nuclease-Free Duplex Buffer (IDT) to yield a 100 µM stock solution. Working solutions of 3 µM duplex gRNA were prepared by mixing 3 µL of 100 µM crRNA, 3 µL of 100 µM tracrRNA, and 94 µL of Nuclease-Free Duplex Buffer. The mixture was heated at 95°C for 5 minutes and then allowed to cool to room temperature. The duplexed gRNAs were stored at -20°C until further use. Cas9 ribonucleoprotein complex The Cas9 protein used was Alt-R™ Streptococcus pyogenes HiFi Cas9 Nuclease V3 (IDT, #1081060), a high-fidelity variant engineered to minimize off-target cleavage while maintaining high on-target efficiency [40]. To assemble a 20 µL Cas9 ribonucleoprotein (RNP) complex, pre-annealed gRNA duplexes and Cas9 protein were combined immediately prior to injection. When one gRNA was used, the RNP mixture contained 16 µL of 3 µM gRNA ( tyr or hps6 ), 0.8 µL of Cas9 protein (10 µg/µL), and 3.2 µL of nuclease-free water. When two gRNAs were used, the RNP mixture contained 8 µL of 3 µM tyr gRNA, 8 µL of 3 µM hps6 gRNA, 0.8 µL of Cas9 protein (10 µg/µL), and 3.2 µL of nuclease-free water. When three gRNAs were used, the RNP mixture contained 5.3 µL of 3 µM tyr gRNA, 5.3 µL of 3 µM hps6 gRNA, 5.3 µL of 3 µM slc2a7 gRNA, 0.8 µL of Cas9 protein (10 µg/µL), and 3.2 µL of nuclease-free water. The mixture was incubated at 37°C for 10 minutes to facilitate RNP complex formation and then cooled to room temperature prior to use. Microinjection procedure and conditions Microinjections were performed at the one-cell stage in wild-type or transgenic one-cell stage embryos using a micromanipulator and pulled glass capillaries connected to a Picospritzer III pressure injector (Parker Hannifin). Approximately 10 nL of the CRISPR RNP complex was injected into the animal hemisphere. Given that 1 µg of Cas9 corresponds to approximately 6.1 pmol, and assuming a 1:1 molar ratio between Cas9 and the total amount of gRNAs, injection of 10 nL of RNP complex delivers approximately 24 femtomoles of Cas9 and an equimolar quantity of gRNAs. Injections were completed at room temperature within 30 minutes post-dejellying to maximize editing efficiency and minimize mosaicism. When handling a large number of one-cell embryos, the onset of the first cleavage can be delayed by maintaining the embryos at 14 °C prior to microinjection, allowing sufficient time for processing. After injections, embryos were raised under standard conditions until the desired stage. Genotyping Tadpoles were sacrificed in 0.01% benzocaine before genomic DNA extraction: they were lysed individually in 100 μL of Viagen DirectPCR™ lysis buffer , supplemented with 1 μL of 20 mg/mL proteinase K , and incubated overnight at 55°C . This was followed by a 1-hour incubation at 95°C to inactivate the enzyme. A volume of 3 μL of the lysate was used directly as template for PCR amplification. Primers were designed using the Primer-BLAST tool (NCBI), targeting genomic regions flanking each CRISPR guide RNA site (Supplementary Table 1). Amplicons were purified using PCR clean-up kit (Macherey-Nagel) and sequenced directly for genotyping. We then used the DECODR website (https://decodr.org/) to analyze gene editing efficiency and identify insertions and deletions (indels). Microscopy Fluorescence and brightfield images were obtained with a stereomicroscope Olympus SZX12 and a Zeiss AxioCam ICc5 camera. They were then processed using Zen (Zeiss, Germany), and Photoshop CS5 (Adobe) softwares. For each transgenic line, fluorescence analyses were performed on at least 10 tadpoles per condition and replicated in at least three independent experiments. Prior to microscopy analysis, animals were anesthetized for 10 minutes in 0.005% benzocaine (tadpoles) or 0.01% benzocaine (froglets and adults). Declarations ACKNOWLEDGMENTS This work has benefited from the I2BC sequencing facility (supported by IBiSA, Région Île de France, Plan Cancer, CNRS and Paris-Saclay University), and the TEFOR Paris-Saclay’s zootechnics service for the maintenance of Xenopus . Some figures were created with icons from BioRender.com and with Xenopus illustrations from Xenbase (www.xenbase.org RRID:SCR_003280) and © Natalya Zahn (2022) [37]. FUNDING DECLARATION This research was supported by grants to M.P. from Retina France association. AUTHOR CONTRIBUTIONS AC conducted all the analyses and drafted the manuscript. AC and MP analyzed data and prepared all figures. MP supervised the study and wrote the manuscript. All authors contributed to the conceptualization and design of the project and revised and approved the manuscript. DATA AVAILABILITY Data are available from the corresponding author upon request. COMPETING INTERESTS The authors declare no competing interests. References Rawls, J. 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Nat Med 24 , 1216–1224 (2018). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFile.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 19 Feb, 2026 Reviews received at journal 19 Feb, 2026 Reviews received at journal 01 Feb, 2026 Reviewers agreed at journal 28 Jan, 2026 Reviews received at journal 28 Jan, 2026 Reviewers agreed at journal 22 Jan, 2026 Reviewers agreed at journal 20 Jan, 2026 Reviewers invited by journal 20 Jan, 2026 Editor assigned by journal 20 Jan, 2026 Editor invited by journal 19 Jan, 2026 Submission checks completed at journal 16 Jan, 2026 First submitted to journal 16 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":92169,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of translucent \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eX. laevis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e by simultaneous knockout of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003etyr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehps6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of the procedure used in (B-D) to generate \u003cem\u003etyr;hps6\u003c/em\u003e KO \u003cem\u003eX. laevis\u003c/em\u003e. Two duplex gRNAs, each composed of a crRNA targeting \u003cem\u003etyr\u003c/em\u003e or \u003cem\u003ehps6\u003c/em\u003eand a tracrRNA, were co-injected with Cas9 protein into one-cell-stage wild-type embryos. Injected embryos were raised as tadpole (stage 52–54) or froglet (stage 66) for phenotypic analysis. \u003cstrong\u003e(B)\u003c/strong\u003e Bright-field images showing dorsal (top) and ventral (bottom) views of tadpoles of the indicated genotypes: wild type, \u003cem\u003ehps6\u003c/em\u003e single KO, \u003cem\u003etyr\u003c/em\u003e double KO (2S and 2L), and \u003cem\u003etyr;hps6\u003c/em\u003e triple KO. \u003cstrong\u003e(C)\u003c/strong\u003e Higher-magnification dorsal views of the eyes of \u003cem\u003etyr\u003c/em\u003e KO and \u003cem\u003etyr;hps6\u003c/em\u003e KO tadpoles. \u003cstrong\u003e(D)\u003c/strong\u003eBright-field ventral views of froglets of \u003cem\u003etyr\u003c/em\u003e KO and \u003cem\u003etyr;hps6\u003c/em\u003e KO genotypes. \u003cem\u003eXenopus\u003c/em\u003e illustrations © Natalya Zahn (2022).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8552006/v1/74bf921dd820a065ca8029a2.jpg"},{"id":100929955,"identity":"830686a3-1c42-4599-9bb0-f7f244af5405","added_by":"auto","created_at":"2026-01-23 00:39:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":83400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of translucent \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTg(rho:eGFP-NTR)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTg(her4:eGFP; cmlc2:eGFP)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transgenic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eX. laevis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e by simultaneous knockout of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003etyr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehps6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A)\u003c/strong\u003e Schematic representation of the procedure used in (B, C) to generate \u003cem\u003etyr;hps6\u003c/em\u003eKO transgenic \u003cem\u003eX. laevis\u003c/em\u003e. Adult \u003cem\u003eTg(rho:eGFP-NTR)\u003c/em\u003e or \u003cem\u003eTg(her4;eGFP; cmlc2:eGFP)\u003c/em\u003e frogs were crossed with wild-type animals, and embryos at the one-cell stage were co-injected with Cas9 protein and two duplex gRNAs, each composed of a crRNA targeting \u003cem\u003etyr\u003c/em\u003e or \u003cem\u003ehps6\u003c/em\u003e and a tracrRNA. Injected embryos were raised to the tadpole (stages 52–54) stages for phenotypic analysis. \u003cstrong\u003e(B)\u003c/strong\u003e Fluorescence images showing lateral views of the eye region in \u003cem\u003eTg(rho:eGFP-NTR)\u003c/em\u003e tadpoles of the indicated genotypes: wild type, \u003cem\u003etyr\u003c/em\u003e double KO (2S and 2L), and \u003cem\u003etyr;hps6\u003c/em\u003e triple KO. \u003cstrong\u003e(C)\u003c/strong\u003eFluorescence images of \u003cem\u003eTg(her4:eGFP; cmlc2:eGFP)\u003c/em\u003e tadpoles showing lateral views of the eye (top) and ventral views of the heart region (bottom) in wild type, \u003cem\u003etyr\u003c/em\u003e KO, and \u003cem\u003etyr;hps6\u003c/em\u003e KO animals.\u003cem\u003e Xenopus\u003c/em\u003eillustrations © Natalya Zahn (2022) and BioRender.com.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8552006/v1/e0836fc617f4b77e99891992.jpg"},{"id":100930052,"identity":"4141d5cf-0549-409a-8d17-52f3f15dd9c0","added_by":"auto","created_at":"2026-01-23 00:39:27","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":98621,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration of translucent and white \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eX. laevis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eby simultaneous knockout of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003etyr\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehps6\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eslc2a7\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of the procedure used in (B-G)\u003cem\u003e \u003c/em\u003eto generate \u003cem\u003etyr;hps6;slc2a7\u003c/em\u003e KO \u003cem\u003eX. laevis\u003c/em\u003e. Three duplex gRNAs, each composed of a crRNA targeting \u003cem\u003etyr\u003c/em\u003e, \u003cem\u003ehps6,\u003c/em\u003e or \u003cem\u003eslc2a7,\u003c/em\u003e and a tracrRNA, were co-injected with Cas9 protein into one-cell-stage embryos, wild-type or \u003cem\u003eTg(her4:eGFP; cmlc2:eGFP)\u003c/em\u003e. Injected embryos were raised to the tadpole, froglet, or adult stages for phenotypic analysis. \u003cstrong\u003e(B,C)\u003c/strong\u003e Bright-field images showing dorsal views of stage 59-60 F0 tadpoles (B) and F0 3-month-old froglets (C) of the indicated genotypes: \u003cem\u003etyr;hps6\u003c/em\u003e KO and \u003cem\u003etyr;hps6;slc2a7\u003c/em\u003eKO. \u003cstrong\u003e(D) \u003c/strong\u003eBright-field dorsal views of F0 adult frogs of the indicated genotypes: wild type, \u003cem\u003ehps6\u003c/em\u003e KO, \u003cem\u003etyr\u003c/em\u003e KO, \u003cem\u003etyr;hps6\u003c/em\u003e KO and \u003cem\u003etyr;hps6;slc2a7\u003c/em\u003eKO. \u003cstrong\u003e(E)\u003c/strong\u003e Bright-field dorsal view of an F0\u003cem\u003e tyr;hps6;slc2a7\u003c/em\u003e KO 1-month-old froglet. \u003cstrong\u003e(F) \u003c/strong\u003eDorsal and ventral bright-field views of an F₁\u003cem\u003etyr;hps6;slc2a7\u003c/em\u003e KO tadpole. \u003cstrong\u003e(G)\u003c/strong\u003e Bright field (top) and fluorescence (bottom) dorsal views of \u003cem\u003eTg(her4:eGFP; cmlc2:eGFP)\u003c/em\u003e tadpoles (stage 54-55) with the indicated genotypes:\u003cem\u003e tyr;hps6\u003c/em\u003e KO and\u003cem\u003e tyr;hps6;slc2a7\u003c/em\u003e KO. \u003cem\u003eXenopus\u003c/em\u003eillustrations © Natalya Zahn (2022) and BioRender.com.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8552006/v1/6ecb3e77bc81a2aa87f412a5.jpg"},{"id":100929881,"identity":"daab38ce-2edd-4eff-8806-28ad220438e7","added_by":"auto","created_at":"2026-01-23 00:39:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56610,"visible":true,"origin":"","legend":"","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8552006/v1/6549675a156341d432a71230.jpg"},{"id":100953603,"identity":"cd40870c-8013-45bc-9e1f-459759dfb12f","added_by":"auto","created_at":"2026-01-23 07:22:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1316962,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8552006/v1/518dffb1-48d3-4bde-a315-6d477264a262.pdf"},{"id":100929836,"identity":"73f1f535-61df-48ee-989e-a51b2f801525","added_by":"auto","created_at":"2026-01-23 00:39:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":799783,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.docx","url":"https://assets-eu.researchsquare.com/files/rs-8552006/v1/6a84c1d585cdb528a4dcb158.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthetic gRNA-mediated multiplex CRISPR enables the generation of translucent F0 Xenopus laevis for in vivo imaging","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIn most vertebrates, the body\u0026rsquo;s opacity conceals internal organs, making them challenging to study without invasive procedures. This opacity is largely due to pigment cells (melanophores, xanthophores, and iridophores) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Some fish species, such as zebrafish and medaka, naturally appear semi-transparent early in life due to their low number of pigment cells. However, as development proceeds, pigment cells progressively accumulate in the skin, peritoneum, and retinal pigment epithelium (RPE) resulting in the gradual loss of transparency. To address this limitation, transparent and translucent zebrafish strains have been generated by combining pigment cell-deficient mutations. For instance, the widely used \u003cem\u003ecasper\u003c/em\u003e strain, which carries mutations in \u003cem\u003emitfa\u003c/em\u003e and \u003cem\u003empv17\u003c/em\u003e, causing a lack of melanophores and iridophores [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The \u003cem\u003ecrystal\u003c/em\u003e strain, which additionally includes a mutation in \u003cem\u003eslc45a2\u003c/em\u003e, also lacks melanin in the RPE [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The \u003cem\u003emitfa\u003c/em\u003e gene (also known as \u003cem\u003enacre\u003c/em\u003e) encodes a transcription factor required for melanophore differentiation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], \u003cem\u003empv17\u003c/em\u003e encodes a mitochondrial protein essential for iridophore survival [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and \u003cem\u003eslc45a2\u003c/em\u003e encodes a membrane-associated transporter required for melanin synthesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Together, these pigment-deficient mutants enable optical transparency that facilitates \u003cem\u003ein vivo\u003c/em\u003e imaging in adult zebrafish.\u003c/p\u003e \u003cp\u003eSimilar approaches have been used to generate transparent or translucent strains in other teleosts. In medaka, the \u0026ldquo;see-through\u0026rdquo; or \u003cem\u003espooky\u003c/em\u003e strains were established by combining multiple pigment-deficient mutations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Transparent phenotypes have also been described in the killifish [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], goldfish [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], or Nile tilapia [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These models have been developed through a combination of traditional breeding of natural color mutants, ENU mutagenesis, and targeted gene disruption via CRISPR/Cas9. Many retain transparency throughout life, offering great possibilities for real-time imaging.\u003c/p\u003e \u003cp\u003eEfforts to generate translucent model organisms are not limited to fish. In the amphibian \u003cem\u003eXenopus tropicalis\u003c/em\u003e, translucency has been achieved by targeting multiple pigmentation genes that affect distinct chromatophore types. For instance, pigment-deficient frogs were produced by crossing \u003cem\u003eslc2a7\u003c/em\u003e single knockouts (KO) with \u003cem\u003etyrosinase/hps6\u003c/em\u003e double KO, followed by intercrossing of the F1 progeny [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The gene \u003cem\u003eslc2a7\u003c/em\u003e is required for yellow pigment production in xanthophores [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The gene \u003cem\u003etyrosinase\u003c/em\u003e (\u003cem\u003etyr\u003c/em\u003e) encodes the enzyme catalyzing the first step of melanin biosynthesis, producing the dark brown to black eumelanin pigment responsible for the majority of skin and retinal pigmentation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The gene \u003cem\u003ehps6\u003c/em\u003e contributes to the biogenesis of pigment-cell organelles in iridophores and melanophores, as shown in the \u003cem\u003eX. tropicalis\u003c/em\u003e mutant \u003cem\u003eno privacy\u003c/em\u003e (\u003cem\u003enop\u003c/em\u003e), which exhibits a reduction or complete loss of these chromatophores [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Another example of pigment-deficient \u003cem\u003eX. tropicalis\u003c/em\u003e model is the \u003cem\u003emitf\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mutant, which lacks both melanophores and xanthophores and consequently exhibit markedly transparent skin, allowing clear visualization of internal organs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast, \u003cem\u003eXenopus laevis\u003c/em\u003e, despite being the most widely used amphibian model in developmental biology, has seen far fewer genetic attempts at achieving transparency. Of the pigmentation-related genes mentioned above, only \u003cem\u003etyr\u003c/em\u003e has been knocked out to date, using either TALEN or CRISPR technologies [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The \u003cem\u003eX. laevis\u003c/em\u003e genome contains multiple \u003cem\u003etyr\u003c/em\u003e-like genes distributed across chromosomes 2L, 2S, 6L, and 6S. However, only the two homeologs on chromosomes 2L and 2S encode the functional Tyrosinase enzyme required for eumelanin synthesis, as their simultaneous inactivation completely abolishes the production of brown/black melanin in melanophores and the RPE, resulting in albino individuals [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Here, to obtain a fully transparent \u003cem\u003eX. laevis\u003c/em\u003e, we employed CRISPR/Cas9 to generate a quadruple knockout of \u003cem\u003eslc2a7\u003c/em\u003e, \u003cem\u003ehps6\u003c/em\u003e, and the two \u003cem\u003etyr2L and tyr2S\u003c/em\u003e homeologs. Importantly, rather than \u003cem\u003ein vitro\u003c/em\u003e-transcribed single guide RNAs (sgRNAs), we used chemically modified, commercially synthesized two-part gRNAs (crRNA:tracrRNA), which exhibit enhanced stability and reduced cellular toxicity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This strategy enabled efficient multiplex genome editing, yielding a high proportion of translucent \u003cem\u003eX. laevis\u003c/em\u003e individuals directly in F0 embryos, eliminating the need for multi-generational breeding. Using two transgenic reporter lines, we demonstrated that these animals are suitable for \u003cem\u003ein vivo\u003c/em\u003e visualization of fluorescent signals, even in the eye, where the combined loss of melanic and iridescent pigments provides greater optical clarity than that achieved in classical albino animals.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eSimultaneous knockout of\u003c/b\u003e \u003cb\u003etyrosinase\u003c/b\u003e \u003cb\u003e(2L, 2S) and\u003c/b\u003e \u003cb\u003ehps6\u003c/b\u003e \u003cb\u003egenes enhances transparency in\u003c/b\u003e \u003cb\u003eXenopus laevis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe sought to generate translucent \u003cem\u003eX. laevis\u003c/em\u003e by disrupting pigment-related genes using CRISPR/Cas9. To achieve maximal transparency, we performed a triple knockout targeting both \u003cem\u003etyr\u003c/em\u003e genes on chromosomes 2L and 2S (using a gRNA that targets both genes), as well as \u003cem\u003ehps6\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Rather than \u003cem\u003ein vitro\u003c/em\u003e-transcribed single guide RNAs (sgRNAs), we used chemically modified, commercially synthesized two-part guide RNAs (gRNAs), to maximize stability and knockout efficiency, which were co-injected with the Cas9 protein at the one-cell stage. We assessed pigmentation at stage 52\u0026ndash;54, when iridescent pigments begin to markedly reduce tissue transparency (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Compared with wild-type, \u003cem\u003ehps6\u003c/em\u003e knockout (KO) tadpoles clearly lacked iridescent pigmentation over the gut and displayed reduced brown/black skin pigmentation, yet retained strong RPE pigmentation in the eye. In contrast, \u003cem\u003etyr\u003c/em\u003e double KOs exhibited a complete loss of brown/black pigmentation, including in the RPE, but retained iridescent pigments over the gut and eye. Triple KOs lacked both eumelanin (black or brown pigmentation) and iridescent pigments, resulting in a significant increase in transparency than \u003cem\u003etyr\u003c/em\u003e-only knockouts. This improvement was particularly striking in the eye, where loss of iridescent pigments in the triple KO abolished a major source of light scattering observed in \u003cem\u003etyr\u003c/em\u003e-only knockouts (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The effect was equally evident in froglets, where simultaneous loss of melanin and iridescent pigments markedly increased transparency compared with melanin loss alone, most notably across the entire ventral surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). However, transparency becomes less apparent in adult frogs, due to the increased thickness of the skin. In terms of efficiency, injection of the two gRNAs yielded a mix of phenotypes: wild-type-like pigmentation, partial pigment loss (mosaicism), and complete pigment loss. The overall editing efficiency was high: 84.86% \u0026plusmn;2.85 (SEM) of injected embryos exhibited complete loss of melanin pigmentation, indicating efficient knockout of \u003cem\u003etyr\u003c/em\u003e (based on 5 independent experiments, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;429 embryos). To confirm that both \u003cem\u003etyr\u003c/em\u003e homeologs (\u003cem\u003etyr2L\u003c/em\u003e and \u003cem\u003etyr2S\u003c/em\u003e) were successfully mutated, we performed PCR analysis on individuals showing a complete absence of eumelanin. In all cases (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4), the crispant individuals carried only mutant alleles for both \u003cem\u003etyr\u003c/em\u003e genes, with no wild-type sequences detected (Supplementary Fig.\u0026nbsp;1). Furthermore, the presence of only 2 to 7 distinct mutations per individual suggests that genome editing occurred at an early developmental stage, likely before the fourth cleavage. Among these melanin-deficient tadpoles, 40.63% (n\u0026thinsp;=\u0026thinsp;128) also lacked any detectable iridescent pigmentation. This proportion increased to 68.76% when mosaic individuals were included. Together, these results support efficient simultaneous disruption of pigment-associated loci in F0 animals, as assessed by phenotype and genotyping.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConcurrent removal of melanin and iridescent pigments enhances live imaging of fluorescent reporters\u003c/h2\u003e \u003cp\u003ePigmentation in \u003cem\u003eXenopus\u003c/em\u003e reduces optical transparency, particularly as development progresses, substantially impairing fluorescence imaging in transgenic lines and thereby restricting the feasibility of live imaging in tadpoles and froglets. The use of natural albino lines (arising from a spontaneous mutation in \u003cem\u003ehsp4\u003c/em\u003e gene [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]) already provides a practical way to mitigate this issue. For example, we recently demonstrated that the dynamics of retinal photoreceptor degeneration and regeneration can be monitored \u003cem\u003ein vivo\u003c/em\u003e by live imaging in an albino \u003cem\u003eTg(rho:eGFP-NTR)\u003c/em\u003e transgenic line [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this line, GFP is specifically expressed in rod photoreceptors under the \u003cem\u003erhodopsin\u003c/em\u003e promoter, and the NTR enzyme enables their selective ablation upon exposure to the prodrug metronidazole [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Here, we sought to determine whether removing both melanin and iridescent pigments could improve GFP fluorescence visualization in transgenic tadpoles. To this end, we crossed pigmented \u003cem\u003eTg(rho:eGFP-NTR)\u003c/em\u003e with wild-type individuals and injected the one-cell stage progeny with gRNAs targeting both \u003cem\u003etyr\u003c/em\u003e genes and \u003cem\u003ehps6\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The simultaneous loss of eumelanin and iridescent pigments in triple KO markedly improved fluorescence visibility in the eye compared with \u003cem\u003etyr\u003c/em\u003e-only KO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition to reducing RPE transparency, we showed above that iridescent pigments also contribute to opacifying the entire ventral region. We therefore evaluated the benefit of the triple KO in a transgenic line expressing eGFP in the heart. The \u003cem\u003eTg(her4:eGFP, cmlc2:eGFP)\u003c/em\u003e line expresses GFP in M\u0026uuml;ller glial cells in the retina via the \u003cem\u003eher4\u003c/em\u003e promoter and in differentiated cardiomyocytes via the \u003cem\u003ecardiac myosin light chain 2\u003c/em\u003e (\u003cem\u003ecmlc2\u003c/em\u003e) promoter [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. First, in this context as well, the visualization of the fluorescence in the retina is improved in \u003cem\u003etyr\u003c/em\u003e;\u003cem\u003ehps6\u003c/em\u003e KO compared to \u003cem\u003etyr\u003c/em\u003e-only KO (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C). In addition, simultaneous removal of eumelanin and iridescent pigments also enhanced GFP visibility in the heart, compared with eumelanin removal alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Collectively, these findings demonstrate that combining transgenesis with targeted pigment-gene editing is a powerful approach for improving live fluorescence imaging across multiple tissues in \u003cem\u003eX. laevis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneration of translucent and white\u003c/b\u003e \u003cb\u003eX. laevis\u003c/b\u003e \u003cb\u003eby simultaneous removal of melanic, iridescent, and yellow pigmentation via CRISPR-mediated quadruple gene KO\u003c/b\u003e\u003c/p\u003e \u003cp\u003eYellow pigmentation is generally sparse throughout most larval stages. However, some individuals exhibit increased yellow pigment at later stages, which may compromise overall transparency. To address this, we extended our CRISPR-based strategy to include \u003cem\u003eslc2a7\u003c/em\u003e, a gene involved in yellow pigment production, in addition to \u003cem\u003etyr\u003c/em\u003e genes and \u003cem\u003ehps6\u003c/em\u003e, thereby generating quadruple KO individuals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). As expected, quadruple KO tadpoles lacking eumelanin, iridescent, and yellow pigments appeared noticeably whiter than triple \u003cem\u003etyr;hps6\u003c/em\u003e KOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Because some individuals could naturally exhibit a whiter phenotype than usual, we confirmed by PCR genotyping that indeed it resulted specifically from \u003cem\u003eslc2a7\u003c/em\u003e disruption (Supplementary Fig.\u0026nbsp;2). As tadpoles, froglets lacking all three pigment types also exhibited white skin, in contrast to the yellow-toned skin of frogs in which only eumelanin and iridescent pigments were removed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This phenotype persisted into adulthood: adult frogs displayed a progressive spectrum of skin coloration, from darkly pigmented to white, depending on whether one, two, or all three pigment types are disrupted (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). White KO individuals were highly transparent up to the froglet stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) while this transparency became less pronounced in adults (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Importantly, quadruple KO individuals generated in this study developed normally, indicating that neither full pigment loss nor the use of synthetic gRNAs induce detectable adverse effects during development. Moreover, they are fertile and capable of producing viable, morphologically normal F1 offspring exhibiting the same white and transparent phenotype as the parents (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). We thus next asked whether removal of yellow pigments in addition to eumelanin and iridescent pigments could further enhance the visualization of fluorescent proteins. In the \u003cem\u003eTg(her4-GFP, clmc-GFP)\u003c/em\u003e line, GFP signal intensity in the brain (likely originating from radial glial cells, as described in the zebrafish brain [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]) was comparable between quadruple mutants and \u003cem\u003etyr;hps6\u003c/em\u003e triple KOs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). These findings suggest that eliminating yellow pigmentation does not significantly improve fluorescence visibility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe intense dark pigmentation of melanophores and strong light scattering produced by iridophores have long hindered live fluorescence imaging in \u003cem\u003eX. laevis\u003c/em\u003e. Compared with \u003cem\u003eX. tropicalis\u003c/em\u003e, where transparent lines were generated through sequential intercrossing of single-gene knockouts [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], similar strategies are considerably less practical in \u003cem\u003eX. laevis\u003c/em\u003e because of its long reproductive cycle and allotetraploid genome. In this study, we show that translucent and even \u0026ldquo;white\u0026rdquo; \u003cem\u003eX. laevis\u003c/em\u003e can instead be produced rapidly in a single step using multiplex CRISPR/Cas9-mediated disruption of key pigment-related genes. By simultaneously targeting \u003cem\u003etyr\u003c/em\u003e, \u003cem\u003ehps6\u003c/em\u003e, and \u003cem\u003eslc2a7\u003c/em\u003e, we eliminated eumelanin-, iridophore-, and xanthophore-derived pigmentation, generating live animals substantially more transparent than naturally albino or single-pigment mutants. This multiplex genome-editing approach bypasses the need for laborious multi-generational breeding and enables the direct production of translucent individuals in the F0 generation.\u003c/p\u003e \u003cp\u003eEfficient multiplex genome editing has previously been demonstrated in \u003cem\u003eXenopus\u003c/em\u003e species: for example, by co-targeting \u003cem\u003etyr\u003c/em\u003e homeologs in \u003cem\u003eX. laevis\u003c/em\u003e [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The Vleminckx laboratory also developed robust multi-gene disruption strategies in \u003cem\u003eX. tropicalis\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. More recently, Cas12a-based approaches have also been used to inactivate duplicated genes in \u003cem\u003eX. laevis\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These studies collectively demonstrate the feasibility of multiplex genome editing in amphibians. In all of these studies however, \u003cem\u003ein vitro\u003c/em\u003e-transcribed sgRNAs were used. Our work extends this multiplex genome editing by using synthetic gRNAs to achieve high-efficiency gene editing in \u003cem\u003eX. laevis\u003c/em\u003e, enabling generation of F0 animals at high penetrance. We indeed employed chemically stabilized two-part synthetic gRNAs, which are commercially available from Integrated DNA Technologies (IDT). This company provides several gRNA formats. In this study, we selected the most cost-effective option, which nevertheless yielded excellent editing efficiencies and minimal cytotoxicity. These synthetic RNAs simplify the experimental workflow and offer superior stability compared to \u003cem\u003ein vitro\u003c/em\u003e-transcribed sgRNAs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], making them particularly well suited for simultaneous multi-gene targeting. Although we did not compare alternative formats, it is possible that other chemically modified variants could further extend gRNA half-life and improve even further editing efficiency. The limited number of distinct mutations per individual indicates that editing occurs at early cleavage stages, allowing reliable functional analyses directly in the F0 generation. The viability and fertility of quadruple knockout adults further confirm that this strategy does not induce detectable developmental defects, validating the use of synthetic gRNAs for multiplex genome editing in amphibians.\u003c/p\u003e \u003cp\u003eThe goal of generating depigmented \u003cem\u003eX. laevis\u003c/em\u003e lines is to improve optical access for live imaging of fluorescent reporters. Our results demonstrate that removal of both eumelanin and iridescent pigments qualitatively enhances fluorescence visibility, in particular in the retina and heart, across multiple transgenic contexts. Notably, the elimination of iridescent pigments reduced light scattering in the ventral region and ocular tissues. On the other hand, our work suggests that eliminating yellow pigmentation does not significantly improve fluorescence visibility. We thus favor to target solely \u003cem\u003etyr\u003c/em\u003e and \u003cem\u003ehps6\u003c/em\u003e genes if the aim is to monitor fluorescence in live animals. Considering the green autofluorescence of yellow pteridine pigments observed in zebrafish [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], additional targeting of \u003cem\u003eslc2a7\u003c/em\u003e in \u003cem\u003eXenopus\u003c/em\u003e may be beneficial in challenging imaging conditions with weak GFP transgenes.\u003c/p\u003e \u003cp\u003eDespite complete disruption of \u003cem\u003eslc2a7\u003c/em\u003e, some individuals retained faint traces of yellow pigmentation. This observation suggests that \u003cem\u003eslc2a7\u003c/em\u003e is not the sole contributor to xanthophore pigment synthesis in \u003cem\u003eX. laevis\u003c/em\u003e. A similar situation has been described in \u003cem\u003eX. tropicalis\u003c/em\u003e, where multiple genes contribute to xanthophore development. Indeed, \u003cem\u003eslc2a7\u003c/em\u003e knockout eliminates xanthophore-specific pterinosomes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and \u003cem\u003emitf\u003c/em\u003e null mutants also lack xanthophores [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It is also possible that \u003cem\u003eX. laevis\u003c/em\u003e synthesizes additional pigment types, such as pheomelanin (a yellow to reddish melanin variant) although this has not been documented in this species. Pheomelanin has been reported in \u003cem\u003eHymenochirus boettgeri\u003c/em\u003e, another member of the \u003cem\u003ePipidae\u003c/em\u003e family [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], suggesting that the enzymatic machinery for pheomelanin biosynthesis may be conserved in some pipids. Furthermore, the existence of multiple \u003cem\u003etyrosinase\u003c/em\u003e paralogs in \u003cem\u003eX. laevis\u003c/em\u003e, including those on chromosomes 6L and 6S, raises the possibility that these enzymes could contribute to alternative pigment pathways, potentially linked to pheomelanin-like compounds. While this remains speculative, the multiplex approach employed in the present study provides a powerful tool for investigating amphibian pigment evolution and diversity in future research.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments have been carried out in accordance with the European Community Council Directive of 22 September 2010 (2010/63/EEC) and in accordance to ARRIVE guidelines. Animal care and experimentations were conducted in accordance with institutional guidelines, under the institutional license C 91-471-102. The study protocols were approved by the Animal Welfare Structure (SBEA) under the number HC 01-02-2025.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eX. laevis\u003c/em\u003e adults were maintained and bred under standard laboratory conditions. Embryos were obtained by natural mating or \u003cem\u003ein vitro\u003c/em\u003e fertilization, dejellied prior to injection, and reared according to established procedures [36].Tadpoles were staged according to [37].\u0026nbsp;\u003cem\u003eTg (rho:eGFP-NTR)\u0026nbsp;\u003c/em\u003eand\u0026nbsp;\u003cem\u003eTg(her4:eGFP; cmlc2:eGFP)\u0026nbsp;\u003c/em\u003eanimals have been generated previously\u0026nbsp;[29,30]\u0026nbsp;using the\u0026nbsp;simplified REMI\u0026nbsp;method\u0026nbsp;[38,39]. Wild type\u0026nbsp;\u003cem\u003eX. laevis\u003c/em\u003e animals and transgenic\u0026nbsp;lines are maintained at the TEFOR Paris-Saclay Zootechnics Service, which houses the French \u003cem\u003eXenopus\u003c/em\u003e Resource Center.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTwo-part guide RNAs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGuide RNAs (gRNAs) targeting the \u003cem\u003etyr\u003c/em\u003e, \u003cem\u003ehps6\u003c/em\u003e and \u003cem\u003eslc2a7\u003c/em\u003e genes were designed using the CRISPOR web tool (http://crispor.tefor.net) to maximize on-target efficiency and minimize potential off-target effects. All gRNAs were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). For each gene, target sites were selected within early exons to maximize the likelihood of generating loss-of-function alleles. We employed the two-part gRNA format consisting of an Alt-R™ CRISPR/Cas9 CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) (IDT, #1072533). The crRNAs contain proprietary chemical modifications that enhance stability by protecting the RNA from endogenous nuclease degradation. When annealed with tracrRNA, they form a functional duplex compatible with standard Cas9-mediated genome editing. For \u003cem\u003etyr\u003c/em\u003e, the gRNA was adapted from [20], with two additional nucleotides (CA) introduced at the 5′ end to improve cleavage efficiency. The crRNA sequence used was 5′-CATGAGATTCAGAAGCTCAC-3′. This gRNA is designed to target both \u003cem\u003etyr2S\u003c/em\u003e and \u003cem\u003etyr2L\u003c/em\u003e homeologs. For \u003cem\u003ehps6\u003c/em\u003e and \u003cem\u003eslc2a7\u003c/em\u003e, the crRNA sequences were 5′-GCAGAGTGTTCCGTACGGCT-3′ and 5′-CTGGGACTCTCCGGAAACCA-3′, respectively. Notably, both genes are present as single-copy and do not have homeologs. Each crRNA (2 nmol) was resuspended in 20 µL of Nuclease-Free Duplex Buffer (IDT) to yield a 100 µM stock solution. Working solutions of 3 µM duplex gRNA were prepared by mixing 3 µL of 100 µM crRNA, 3 µL of 100 µM tracrRNA, and 94 µL of Nuclease-Free Duplex Buffer. The mixture was heated at 95°C for 5 minutes and then allowed to cool to room temperature. The duplexed gRNAs were stored at -20°C until further use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCas9 ribonucleoprotein complex\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Cas9 protein used was Alt-R™ \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e HiFi Cas9 Nuclease V3 (IDT, #1081060), a high-fidelity variant engineered to minimize off-target cleavage while maintaining high on-target efficiency [40]. To assemble a 20 µL Cas9 ribonucleoprotein (RNP) complex, pre-annealed gRNA duplexes and Cas9 protein were combined immediately prior to injection. When one gRNA was used, the RNP mixture contained 16 µL of 3 µM gRNA (\u003cem\u003etyr\u003c/em\u003e or \u003cem\u003ehps6\u003c/em\u003e), 0.8 µL of Cas9 protein (10 µg/µL), and 3.2 µL of nuclease-free water. When two gRNAs were used, the RNP mixture contained 8 µL of 3 µM \u003cem\u003etyr\u003c/em\u003e gRNA, 8 µL of 3 µM \u003cem\u003ehps6\u003c/em\u003e gRNA, 0.8 µL of Cas9 protein (10 µg/µL), and 3.2 µL of nuclease-free water. When three gRNAs were used, the RNP mixture contained 5.3 µL of 3 µM \u003cem\u003etyr\u003c/em\u003e gRNA, 5.3 µL of 3 µM \u003cem\u003ehps6\u003c/em\u003e gRNA, 5.3 µL of 3 µM \u003cem\u003eslc2a7\u003c/em\u003e gRNA, 0.8 µL of Cas9 protein (10 µg/µL), and 3.2 µL of nuclease-free water. The mixture was incubated at 37°C for 10 minutes to facilitate RNP complex formation and then cooled to room temperature prior to use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroinjection procedure and conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroinjections were performed at the one-cell stage in wild-type or transgenic one-cell stage embryos using a micromanipulator and pulled glass capillaries connected to a Picospritzer III pressure injector (Parker Hannifin). Approximately 10 nL of the CRISPR RNP complex was injected into the animal hemisphere. Given that 1 µg of Cas9 corresponds to approximately 6.1 pmol, and assuming a 1:1 molar ratio between Cas9 and the total amount of gRNAs, injection of 10 nL of RNP complex delivers approximately 24 femtomoles of Cas9 and an equimolar quantity of gRNAs. Injections were completed at room temperature within 30 minutes post-dejellying to maximize editing efficiency and minimize mosaicism. When handling a large number of one-cell embryos, the onset of the first cleavage can be delayed by maintaining the embryos at 14 °C prior to microinjection, allowing sufficient time for processing. After injections, embryos were raised under standard conditions until the desired stage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenotyping\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTadpoles were sacrificed in \u003cstrong\u003e0.01% benzocaine\u003c/strong\u003e before genomic DNA extraction: they were lysed individually in \u003cstrong\u003e100 μL of Viagen DirectPCR™ lysis buffer\u003c/strong\u003e, supplemented with 1\u003cstrong\u003e μL\u003c/strong\u003e of 20 mg/mL \u003cstrong\u003eproteinase K\u003c/strong\u003e, and incubated overnight at \u003cstrong\u003e55°C\u003c/strong\u003e. This was followed by a \u003cstrong\u003e1-hour incubation at 95°C\u003c/strong\u003e to inactivate the enzyme. A volume of \u003cstrong\u003e3 μL\u003c/strong\u003e of the lysate was used directly as template for PCR amplification. Primers were designed using the \u003cstrong\u003ePrimer-BLAST\u003c/strong\u003e tool (NCBI), targeting genomic regions flanking each CRISPR guide RNA site (Supplementary Table 1). Amplicons were purified using PCR clean-up kit (Macherey-Nagel) and sequenced directly for genotyping. We then used the DECODR website (https://decodr.org/) to analyze gene editing efficiency and identify insertions and deletions (indels).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescence and brightfield images were obtained with a stereomicroscope Olympus SZX12 and a Zeiss AxioCam ICc5 camera.\u0026nbsp;They were then processed using Zen (Zeiss, Germany), and Photoshop CS5 (Adobe) softwares. For each transgenic line, fluorescence analyses were performed on at least 10 tadpoles per condition and replicated in at least three independent experiments. Prior to microscopy analysis, animals were anesthetized for 10 minutes in 0.005% benzocaine (tadpoles) or 0.01% benzocaine (froglets and adults).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work has benefited from the I2BC sequencing facility (supported by IBiSA, Région Île de France, Plan Cancer, CNRS and Paris-Saclay University), and the TEFOR Paris-Saclay’s zootechnics service for the maintenance of \u003cem\u003eXenopus\u003c/em\u003e.\u0026nbsp;Some figures were created with icons from BioRender.com and with \u003cem\u003eXenopus\u003c/em\u003e illustrations from Xenbase (www.xenbase.org RRID:SCR_003280) and © Natalya Zahn (2022) [37].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING DECLARATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by grants to M.P. from Retina France association.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAC conducted all the analyses and drafted the manuscript. AC and MP analyzed data and prepared all figures. MP supervised the study and wrote the manuscript. All authors contributed to the conceptualization and design of the project and revised and approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRawls, J. F., Mellgren, E. M. \u0026amp; Johnson, S. L. How the Zebrafish Gets Its Stripes. \u003cem\u003eDevelopmental Biology\u003c/em\u003e \u003cstrong\u003e240\u003c/strong\u003e, 301\u0026ndash;314 (2001).\u003c/li\u003e\n\u003cli\u003eWhite, R. 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A. \u003cem\u003eet al.\u003c/em\u003e A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. \u003cem\u003eNat Med\u003c/em\u003e\u003cstrong\u003e24\u003c/strong\u003e, 1216\u0026ndash;1224 (2018).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8552006/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8552006/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTransparent model organisms are invaluable for live imaging, yet generating them remains challenging. Here, we present a robust strategy to produce translucent \u003cem\u003eXenopus laevis\u003c/em\u003e, enabling non-invasive, deep-tissue imaging in intact organisms. Using CRISPR/Cas9 technology, we generated quadruple knockouts of \u003cem\u003eslc2a7\u003c/em\u003e, \u003cem\u003ehps6\u003c/em\u003e, and the two \u003cem\u003etyrosinase\u003c/em\u003e homeologs. Instead of \u003cem\u003ein vitro\u003c/em\u003e-transcribed single guide RNAs, we employed chemically modified, commercially synthesized two-part guide RNAs, which enabled efficient multiplex genome editing. We produced a high proportion of translucent frogs directly in F0 founder tadpoles, eliminating the need for multi-generational breeding. We validated \u003cem\u003ein vivo\u003c/em\u003e live imaging using two transgenic reporter lines with GFP expression in the eye, brain, and heart. Loss of both eumelanin and iridescent pigments in \u003cem\u003etyr\u003c/em\u003e;\u003cem\u003ehps6\u003c/em\u003e knockouts markedly improved optical clarity and fluorescence visibility. Overall, this multiplexed genome-editing strategy enables the rapid generation of translucent transgenic \u003cem\u003eX. laevis\u003c/em\u003e suitable for live imaging, while also providing a simple and efficient protocol for simultaneous multi-gene targeting in \u003cem\u003eX. laevis\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Synthetic gRNA-mediated multiplex CRISPR enables the generation of translucent F0 Xenopus laevis for in vivo imaging","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-23 00:38:28","doi":"10.21203/rs.3.rs-8552006/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-19T18:49:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-19T15:43:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-01T23:21:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91202143427137337409057523242560710330","date":"2026-01-28T17:45:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T15:45:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258126733043141172462378880701803354616","date":"2026-01-22T09:25:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197416200544502935484793355839299978009","date":"2026-01-20T17:43:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-20T17:40:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-20T17:24:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-19T16:13:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-16T14:51:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-01-16T14:40:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bd634646-ddc2-46a3-ba5e-5a8041c5f951","owner":[],"postedDate":"January 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61488426,"name":"Biological sciences/Biological techniques"},{"id":61488427,"name":"Biological sciences/Biotechnology"}],"tags":[],"updatedAt":"2026-05-17T06:53:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-23 00:38:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8552006","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8552006","identity":"rs-8552006","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}