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Genome editing simplifies the generation of new animal models for congenital disorders. However, the detailed and unbiased phenotypic assessment of altered embryonic development remains a challenge. Here, we explore how deep learning (U-Net) can automate segmentation tasks in various imaging modalities, and we quantify phenotypes of altered renal, neural and craniofacial development in Xenopus embryos in comparison with normal variability. We demonstrate the utility of this approach in embryos with polycystic kidneys (pkd1 and pkd2) and craniofacial dysmorphia (six1). We highlight how in toto light-sheet microscopy facilitates accurate reconstruction of brain and craniofacial structures within X. tropicalis embryos upon dyrk1a and six1 loss of function or treatment with retinoic acid inhibitors. These tools increase the sensitivity and throughput of evaluating developmental malformations caused by chemical or genetic disruption. Furthermore, we provide a library of pre-trained networks and detailed instructions for applying deep learning to the reader's own datasets. We demonstrate the versatility, precision and scalability of deep neural network phenotyping on embryonic disease models. By combining light-sheet microscopy and deep learning, we provide a framework for higher-throughput characterization of embryonic model organisms. This article has an associated 'The people behind the papers' interview.
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Aprendizado Profundo , Desenvolvimento Embrionário/genética , Fenótipo , Animais , Anormalidades Craniofaciais/embriologia , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , Modelos Animais de Doenças , Processamento de Imagem Assistida por Computador , Camundongos , Microscopia , Mutação , Redes Neurais de Computação , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Doenças Renais Policísticas/embriologia , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
In this Letter, the surname of author Lena Vlaminck was misspelled 'Vlaeminck'. In addition, author Kris Vleminckx should have been associated with affiliation 16 (Center for Medical Genetics, Ghent University, Ghent, Belgium). These have been corrected online.
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The four R-spondin secreted ligands (RSPO1-RSPO4) act via their cognate LGR4, LGR5 and LGR6 receptors to amplify WNT signalling1-3. Here we report an allelic series of recessive RSPO2 mutations in humans that cause tetra-amelia syndrome, which is characterized by lung aplasia and a total absence of the four limbs. Functional studies revealed impaired binding to the LGR4/5/6 receptors and the RNF43 and ZNRF3 transmembrane ligases, and reduced WNT potentiation, which correlated with allele severity. Unexpectedly, however, the triple and ubiquitous knockout of Lgr4, Lgr5 and Lgr6 in mice did not recapitulate the known Rspo2 or Rspo3 loss-of-function phenotypes. Moreover, endogenous depletion or addition of exogenous RSPO2 or RSPO3 in triple-knockout Lgr4/5/6 cells could still affect WNT responsiveness. Instead, we found that the concurrent deletion of rnf43 and znrf3 in Xenopus embryos was sufficient to trigger the outgrowth of supernumerary limbs. Our results establish that RSPO2, without the LGR4/5/6 receptors, serves as a direct antagonistic ligand to RNF43 and ZNRF3, which together constitute a master switch that governs limb specification. These findings have direct implications for regenerative medicine and WNT-associated cancers.
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Proteínas de Ligação a DNA/antagonistas & inibidores , Extremidades/embriologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Deformidades Congênitas dos Membros/genética , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Fibroblastos , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Proteínas Oncogênicas/antagonistas & inibidores , Proteínas Oncogênicas/metabolismo , Fenótipo , Receptores Acoplados a Proteínas G/deficiência , Ubiquitina-Proteína Ligases/metabolismo , Xenopus/genéticaRESUMO
Cancer precision medicine implies identification of tumor-specific vulnerabilities associated with defined oncogenic pathways. Desmoid tumors are soft-tissue neoplasms strictly driven by Wnt signaling network hyperactivation. Despite this clearly defined genetic etiology and the strict and unique implication of the Wnt/ß-catenin pathway, no specific molecular targets for these tumors have been identified. To address this caveat, we developed fast, efficient, and penetrant genetic Xenopus tropicalis desmoid tumor models to identify and characterize drug targets. We used multiplexed CRISPR/Cas9 genome editing in these models to simultaneously target a tumor suppressor gene (apc) and candidate dependency genes. Our methodology CRISPR/Cas9 selection-mediated identification of dependencies (CRISPR-SID) uses calculated deviations between experimentally observed gene editing outcomes and deep-learning-predicted double-strand break repair patterns to identify genes under negative selection during tumorigenesis. This revealed EZH2 and SUZ12, both encoding polycomb repressive complex 2 components, and the transcription factor CREB3L1 as genetic dependencies for desmoid tumors. In vivo EZH2 inhibition by Tazemetostat induced partial regression of established autochthonous tumors. In vitro models of patient desmoid tumor cells revealed a direct effect of Tazemetostat on Wnt pathway activity. CRISPR-SID represents a potent approach for in vivo mapping of tumor vulnerabilities and drug target identification.
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Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/isolamento & purificação , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Edição de Genes/métodos , Neoplasias Abdominais/genética , Polipose Adenomatosa do Colo/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Fibromatose Agressiva/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso , Oncogenes , Complexo Repressor Polycomb 2/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt , Xenopus , beta CateninaRESUMO
SIGNIFICANCE STATEMENT: Mutations in hepatocyte nuclear factor-1 ß ( HNF1B ) are the most common monogenic causes of congenital renal malformations. HNF1B is necessary to directly reprogram fibroblasts to induced renal tubule epithelial cells (iRECs) and, as we demonstrate, can induce ectopic pronephric tissue in Xenopus ectodermal organoids. Using these two systems, we analyzed the effect of HNF1B mutations found in patients with cystic dysplastic kidney disease. We found cross-species conserved targets of HNF1B, identified transcripts that are differentially regulated by the patient-specific mutant protein, and functionally validated novel HNF1B targets in vivo . These results highlight evolutionarily conserved transcriptional mechanisms and provide insights into the genetic circuitry of nephrogenesis. BACKGROUND: Hepatocyte nuclear factor-1 ß (HNF1B) is an essential transcription factor during embryogenesis. Mutations in HNF1B are the most common monogenic causes of congenital cystic dysplastic renal malformations. The direct functional consequences of mutations in HNF1B on its transcriptional activity are unknown. METHODS: Direct reprogramming of mouse fibroblasts to induced renal tubular epithelial cells was conducted both with wild-type HNF1B and with patient mutations. HNF1B was expressed in Xenopus ectodermal explants. Transcriptomic analysis by bulk RNA-Seq identified conserved targets with differentially regulated expression by the wild-type or R295C mutant. CRISPR/Cas9 genome editing in Xenopus embryos evaluated transcriptional targets in vivo . RESULTS: HNF1B is essential for reprogramming mouse fibroblasts to induced renal tubular epithelial cells and induces development of ectopic renal organoids from pluripotent Xenopus cells. The mutation R295C retains reprogramming and inductive capacity but alters the expression of specific sets of downstream target genes instead of diminishing overall transcriptional activity of HNF1B. Surprisingly, targets associated with polycystic kidney disease were less affected than genes affected in congenital renal anomalies. Cross-species-conserved transcriptional targets were dysregulated in hnf1b CRISPR-depleted Xenopus embryos, confirming their dependence on hnf1b . CONCLUSIONS: HNF1B activates an evolutionarily conserved program of target genes that disease-causing mutations selectively disrupt. These findings provide insights into the renal transcriptional network that controls nephrogenesis.
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Fator 1-beta Nuclear de Hepatócito , Doenças Renais Císticas , Animais , Camundongos , Fator 1-beta Nuclear de Hepatócito/genética , Rim/metabolismo , Doenças Renais Císticas/genética , Mutação , Xenopus laevisRESUMO
BACKGROUND: About 40 disease genes have been described to date for isolated CAKUT, the most common cause of childhood CKD. However, these genes account for only 20% of cases. ARHGEF6, a guanine nucleotide exchange factor that is implicated in biologic processes such as cell migration and focal adhesion, acts downstream of integrin-linked kinase (ILK) and parvin proteins. A genetic variant of ILK that causes murine renal agenesis abrogates the interaction of ILK with a murine focal adhesion protein encoded by Parva , leading to CAKUT in mice with this variant. METHODS: To identify novel genes that, when mutated, result in CAKUT, we performed exome sequencing in an international cohort of 1265 families with CAKUT. We also assessed the effects in vitro of wild-type and mutant ARHGEF6 proteins, and the effects of Arhgef6 deficiency in mouse and frog models. RESULTS: We detected six different hemizygous variants in the gene ARHGEF6 (which is located on the X chromosome in humans) in eight individuals from six families with CAKUT. In kidney cells, overexpression of wild-type ARHGEF6 -but not proband-derived mutant ARHGEF6 -increased active levels of CDC42/RAC1, induced lamellipodia formation, and stimulated PARVA-dependent cell spreading. ARHGEF6-mutant proteins showed loss of interaction with PARVA. Three-dimensional Madin-Darby canine kidney cell cultures expressing ARHGEF6-mutant proteins exhibited reduced lumen formation and polarity defects. Arhgef6 deficiency in mouse and frog models recapitulated features of human CAKUT. CONCLUSIONS: Deleterious variants in ARHGEF6 may cause dysregulation of integrin-parvin-RAC1/CDC42 signaling, thereby leading to X-linked CAKUT.
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Sistema Urinário , Anormalidades Urogenitais , Humanos , Camundongos , Animais , Cães , Anormalidades Urogenitais/genética , Rim/anormalidades , Sistema Urinário/anormalidades , Integrinas/metabolismo , Proteínas Mutantes/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/genéticaRESUMO
The development of hindlimbs in tetrapod species relies specifically on the transcription factor TBX4. In humans, heterozygous loss-of-function TBX4 mutations cause dominant small patella syndrome (SPS) due to haploinsufficiency. Here, we characterize a striking clinical entity in four fetuses with complete posterior amelia with pelvis and pulmonary hypoplasia (PAPPA). Through exome sequencing, we find that PAPPA syndrome is caused by homozygous TBX4 inactivating mutations during embryogenesis in humans. In two consanguineous couples, we uncover distinct germline TBX4 coding mutations, p.Tyr113∗ and p.Tyr127Asn, that segregated with SPS in heterozygous parents and with posterior amelia with pelvis and pulmonary hypoplasia syndrome (PAPPAS) in one available homozygous fetus. A complete absence of TBX4 transcripts in this proband with biallelic p.Tyr113∗ stop-gain mutations revealed nonsense-mediated decay of the endogenous mRNA. CRISPR/Cas9-mediated TBX4 deletion in Xenopus embryos confirmed its restricted role during leg development. We conclude that SPS and PAPPAS are allelic diseases of TBX4 deficiency and that TBX4 is an essential transcription factor for organogenesis of the lungs, pelvis, and hindlimbs in humans.
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Anormalidades Múltiplas/etiologia , Doenças do Desenvolvimento Ósseo/etiologia , Ectromelia/etiologia , Quadril/anormalidades , Homozigoto , Ísquio/anormalidades , Mutação com Perda de Função , Pneumopatias/etiologia , Pulmão/anormalidades , Patela/anormalidades , Pelve/anormalidades , Proteínas com Domínio T/genética , Anormalidades Múltiplas/patologia , Adolescente , Doenças do Desenvolvimento Ósseo/patologia , Criança , Ectromelia/patologia , Feminino , Quadril/patologia , Humanos , Ísquio/patologia , Pulmão/patologia , Pneumopatias/patologia , Masculino , Patela/patologia , Linhagem , Pelve/patologia , PrognósticoRESUMO
The speed by which clinical genomics is currently identifying novel potentially pathogenic variants is outperforming the speed by which these can be functionally (genotype-phenotype) annotated in animal disease models. However, over the past few years the emergence of CRISPR/Cas9 as a straight-forward genome editing technology has revolutionized disease modeling in vertebrate non-mammalian model organisms such as zebrafish, medaka and Xenopus. It is now finally possible, by CRISPR/Cas9, to rapidly establish clinically relevant disease models in these organisms. Interestingly, these can provide both cost-effective genotype-phenotype correlations for gene-(variants) and genomic rearrangements obtained from clinical practice, as well as be exploited to perform translational research to improve prospects of disease afflicted patients. In this review, we show an extensive overview of these new CRISPR/Cas9-mediated disease models and provide future prospects that will allow increasingly accurate modeling of human disease in zebrafish, medaka and Xenopus.
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Sistemas CRISPR-Cas , Modelos Animais de Doenças , Edição de Genes , Animais , Marcação de Genes , Terapia Genética/métodos , Genômica , Mutação , Xenopus , Peixe-ZebraRESUMO
The targeted nuclease revolution (TALENs, CRISPR/Cas9) now allows Xenopus researchers to rapidly generate custom on-demand genetic knockout models. These novel methods to perform reverse genetics are unprecedented and are fueling a wide array of human disease models within the aquatic diploid model organism Xenopus tropicalis (X. tropicalis). This emerging technology review focuses on the tools to rapidly generate genetically engineered X. tropicalis models (GEXM), with a focus on establishment of genuine genetic and clinically relevant cancer models. We believe that due to particular advantageous characteristics, outlined within this review, GEXM will become a valuable alternative animal model for modeling human cancer. Furthermore, we provide perspectives of how GEXM will be used as a platform for elucidation of novel therapeutic targets and for preclinical drug validation. Finally, we also discuss some future prospects on how the recent expansions and adaptations of the CRISPR/Cas9 toolbox might influence and push forward X. tropicalis cancer research.
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Sistemas CRISPR-Cas/genética , Engenharia Genética , Neoplasias/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Animais , Modelos Animais de Doenças , Marcação de Genes , Humanos , Neoplasias/patologia , Xenopus/genéticaRESUMO
Imaging large, cleared samples requires microscope objectives that combine a large field of view (FOV) with a long working distance (WD) and a high numerical aperture (NA). Ideally, such objectives should be compatible with a wide range of immersion media, which is challenging to achieve with conventional lens-based objective designs. Here we introduce the multi-immersion 'Schmidt objective' consisting of a spherical mirror and an aspherical correction plate as a solution to this problem. We demonstrate that a multi-photon variant of the Schmidt objective is compatible with all homogeneous immersion media and achieves an NA of 1.08 at a refractive index of 1.56, 1.1-mm FOV and 11-mm WD. We highlight its versatility by imaging cleared samples in various media ranging from air and water to benzyl alcohol/benzyl benzoate, dibenzyl ether and ethyl cinnamate and by imaging of neuronal activity in larval zebrafish in vivo. In principle, the concept can be extended to any imaging modality, including wide-field, confocal and light-sheet microscopy.
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Telescópios , Animais , Imersão , Microscopia/métodos , Peixe-ZebraRESUMO
In 2015, we launched the mesoSPIM initiative, an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of such microscopes. Here, we introduce the next-generation mesoSPIM ("Benchtop") with a significantly increased field of view, improved resolution, higher throughput, more affordable cost, and simpler assembly compared to the original version. We develop an optical method for testing detection objectives that enables us to select objectives optimal for light-sheet imaging with large-sensor cameras. The improved mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, magnification up to 20×, and supports sample sizes ranging from sub-mm up to several centimeters while being compatible with multiple clearing techniques. The microscope serves a broad range of applications in neuroscience, developmental biology, pathology, and even physics.
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Microscopia , Neurociências , Microscopia/métodosRESUMO
CRISPR-mediated simultaneous targeting of candidate tumor suppressor genes in Xenopus tropicalis allows fast functional assessment of co-driver genes for various solid tumors. Genotyping of tumors that emerge in the mosaic mutant animals rapidly exposes the gene mutations under positive selection for tumor establishment. However, applying this simple approach to the blood lineage has not been attempted. Multiple hematologic malignancies have mutations in EZH2, encoding the catalytic subunit of the Polycomb Repressive Complex 2. Interestingly, EZH2 can act as an oncogene or a tumor suppressor, depending on cellular context and disease stage. We show here that mosaic CRISPR/Cas9 mediated ezh2 disruption in the blood lineage resulted in early and penetrant acute myeloid leukemia (AML) induction. While animals were co-targeted with an sgRNA that induces notch1 gain-of-function mutations, sequencing of leukemias revealed positive selection towards biallelic ezh2 mutations regardless of notch1 mutational status. Co-targeting dnm2, recurrently mutated in T/ETP-ALL, induced a switch from myeloid towards acute T-cell leukemia. Both myeloid and T-cell leukemias engrafted in immunocompromised hosts. These data underline the potential of Xenopus tropicalis for modeling human leukemia, where mosaic gene disruption, combined with deep amplicon sequencing of the targeted genomic regions, can rapidly and efficiently expose co-operating driver gene mutations.
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Leucemia Mieloide Aguda , RNA Guia de Sistemas CRISPR-Cas , Animais , Humanos , Histona Metiltransferases/genética , Xenopus/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , MutaçãoRESUMO
In 2015, we launched the mesoSPIM initiative (www.mesospim.org), an open-source project for making light-sheet microscopy of large cleared tissues more accessible. Meanwhile, the demand for imaging larger samples at higher speed and resolution has increased, requiring major improvements in the capabilities of light-sheet microscopy. Here, we introduce the next-generation mesoSPIM ("Benchtop") with significantly increased field of view, improved resolution, higher throughput, more affordable cost and simpler assembly compared to the original version. We developed a new method for testing objectives, enabling us to select detection objectives optimal for light-sheet imaging with large-sensor sCMOS cameras. The new mesoSPIM achieves high spatial resolution (1.5 µm laterally, 3.3 µm axially) across the entire field of view, a magnification up to 20x, and supports sample sizes ranging from sub-mm up to several centimetres, while being compatible with multiple clearing techniques. The new microscope serves a broad range of applications in neuroscience, developmental biology, and even physics.
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Alterations of the retinoblastoma and/or the p53 signaling network are associated with specific cancers such as high-grade astrocytoma/glioblastoma, small-cell lung cancer (SCLC), choroid plexus tumors, and small-cell pancreatic neuroendocrine carcinoma (SC-PaNEC). However, the intricate functional redundancy between RB1 and the related pocket proteins RBL1/p107 and RBL2/p130 in suppressing tumorigenesis remains poorly understood. Here we performed lineage-restricted parallel inactivation of rb1 and rbl1 by multiplex CRISPR/Cas9 genome editing in the true diploid Xenopus tropicalis to gain insight into this in vivo redundancy. We show that while rb1 inactivation is sufficient to induce choroid plexus papilloma, combined rb1 and rbl1 inactivation is required and sufficient to drive SC-PaNEC, retinoblastoma and astrocytoma. Further, using a novel Li-Fraumeni syndrome-mimicking tp53 mutant X. tropicalis line, we demonstrate increased malignancy of rb1/rbl1-mutant glioma towards glioblastoma upon concomitant inactivation of tp53. Interestingly, although clinical SC-PaNEC samples are characterized by abnormal p53 expression or localization, in the current experimental models, the tp53 status had little effect on the establishment and growth of SC-PaNEC, but may rather be essential for maintaining chromosomal stability. SCLC was only rarely observed in our experimental setup, indicating requirement of additional or alternative oncogenic insults. In conclusion, we used CRISPR/Cas9 to delineate the tumor suppressor properties of Rbl1, generating new insights in the functional redundancy within the retinoblastoma protein family in suppressing neuroendocrine pancreatic cancer and glioma/glioblastoma.
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Carcinoma Neuroendócrino/patologia , Carcinoma de Células Pequenas/patologia , Glioblastoma/patologia , Neoplasias Pancreáticas/patologia , Proteína p107 Retinoblastoma-Like/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Carcinoma Neuroendócrino/genética , Carcinoma de Células Pequenas/genética , Modelos Animais de Doenças , Edição de Genes , Glioblastoma/genética , Humanos , Neoplasias Pancreáticas/genética , Proteína p107 Retinoblastoma-Like/genética , Transdução de Sinais/genética , Xenopus , Proteínas de Xenopus/genética , Neoplasias PancreáticasRESUMO
CRISPR/Cas9 genome editing has revolutionized functional genomics in vertebrates. However, CRISPR/Cas9 edited F0 animals too often demonstrate variable phenotypic penetrance due to the mosaic nature of editing outcomes after double strand break (DSB) repair. Even with high efficiency levels of genome editing, phenotypes may be obscured by proportional presence of in-frame mutations that still produce functional protein. Recently, studies in cell culture systems have shown that the nature of CRISPR/Cas9-mediated mutations can be dependent on local sequence context and can be predicted by computational methods. Here, we demonstrate that similar approaches can be used to forecast CRISPR/Cas9 gene editing outcomes in Xenopus tropicalis, Xenopus laevis, and zebrafish. We show that a publicly available neural network previously trained in mouse embryonic stem cell cultures (InDelphi-mESC) is able to accurately predict CRISPR/Cas9 gene editing outcomes in early vertebrate embryos. Our observations can have direct implications for experiment design, allowing the selection of guide RNAs with predicted repair outcome signatures enriched towards frameshift mutations, allowing maximization of CRISPR/Cas9 phenotype penetrance in the F0 generation.
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Sistemas CRISPR-Cas , Edição de Genes/métodos , Penetrância , Xenopus laevis/embriologia , Xenopus laevis/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Proteína 9 Associada à CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Mutação da Fase de Leitura , Frequência do Gene , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , RNA Guia de Cinetoplastídeos/genéticaRESUMO
In this chapter, we convey a state-of-the art update to the 2014 Nakayama protocol for CRISPR/Cas9 genome engineering in Xenopus tropicalis (X. tropicalis). We discuss in depth, gRNA design software and rules, gRNA synthesis, and procedures for tissue- and tissue-specific CRISPR/Cas9 genome editing by targeted microinjection in X. tropicalis embryos. We demonstrate the methodology by which any standard equipped Xenopus researcher with microinjection experience can generate F0 CRISPR/Cas9 mediated mosaic mutants (crispants) within one to two work-week(s). The described methodology allows CRISPR/Cas9 efficiencies to be high enough to read out phenotypic consequences, and thus perform gene function analysis, in the F0 crispant. Additionally, we provide the framework for performing multiplex tissue-specific CRISPR/Cas9 experiments generating crispants mosaic mutant in up to four genes simultaneously, which can be of importance for Laevis researchers aiming to target by CRISPR/Cas9 both the S and L homeolog of a gene simultaneously. Finally, we discuss off-target concerns, how to minimize these and ways to rapidly bypass reviewer off-target critique by exploiting the advantages of X. tropicalis.
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Sistemas CRISPR-Cas/genética , Engenharia Genética/métodos , Genoma , Especificidade de Órgãos/genética , Xenopus/genética , Animais , Proteína 9 Associada à CRISPR/metabolismo , Edição de Genes , Microinjeções , Monofenol Mono-Oxigenase/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Xenopus/embriologiaRESUMO
The targeted nuclease revolution (ZFN, TALEN, and CRISPR/Cas9) has led to a myriad of reports describing genotyping methodologies for genome edited founders (F0-crispants) and their offspring (F1). As such, choosing a specific genotyping methodology for your Xenopus CRISPR/Cas9 experiments can be challenging. In this chapter we will discuss, with emphasis on Xenopus tropicalis (X. tropicalis), different methods for assessing genome editing efficiencies within F0 CRISPR/Cas9 founders and for identification of their hetero-, compound hetero-, and homozygous mutant F1 offspring. For F0 crispants, we will provide the protocols and the respective (dis)advantages of genotyping with heteroduplex mobility assay (HMA), subclone Sanger sequencing, and sequence trace decomposition. Furthermore, we provide a previously unpublished pipe-line for rapid genotyping of F1 offspring-high resolution melting analysis (HRMA) and sequence trace decomposition-procured from breeding with F0 crispants. As such, we report here the current state-of-the-art cost- and time-effective approaches to perform genotyping of CRISPR/Cas9 experiments for the Xenopus tropicalis researcher.
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Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes , Técnicas de Genotipagem/métodos , Xenopus/genética , Animais , Cruzamento , Embrião não Mamífero/metabolismo , Homozigoto , Mutação/genética , Desnaturação de Ácido NucleicoRESUMO
The recent advent of CRISPR/Cas9 as a straightforward genome editing tool has allowed the establishment of the first bona fide genetic cancer models within the diploid aquatic model organism Xenopus tropicalis (X. tropicalis). Within this chapter, we demonstrate the methods for targeting tumor suppressors with the CRISPR/Cas9 system in the developing X. tropicalis embryo. We further illustrate genotyping and phenotyping of the resulting tumor-bearing F0 mosaic mutant animals (crispants). We focus in detail on the histopathological analysis of cancer neoplasms, the methodology to illustrate high proliferative index by proliferation marker immunofluorescence and how to isolate specific (tumor) cell populations by laser capture microdissection. As such, the described pipeline allows for rapid establishment of novel cancer models by CRISPR/Cas9 targeting of established tumor suppressor genes, or novel candidates obtained from clinical data. In conclusion, we thus provide the methodology for modeling human cancer with the highly efficient CRISPR/Cas9 system in F0 X. tropicalis.
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Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes/métodos , Genes Supressores de Tumor , Neoplasias/genética , Animais , Proliferação de Células , Modelos Animais de Doenças , Edição de Genes , Microdissecção e Captura a Laser , Mutação/genética , Neoplasias/patologia , Fenótipo , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Xenopus/genéticaRESUMO
At this time, no molecular targeted therapies exist for treatment of retinoblastoma. This can be, in part, attributed to the lack of animal models that allow for both rapid identification of novel therapeutic targets and hypothesis driven drug testing. Within this scope, we have recently reported the first genuine genetic nonmammalian retinoblastoma cancer model within the aquatic model organism Xenopus tropicalis (Naert et al., Sci Rep 6: 35263, 2016). Here we describe the methods to generate rb1 mosaic mutant Xenopus tropicalis by employing the CRISPR/Cas9 technology. In depth, we discuss short guide RNA (sgRNA) design parameters, generation, quality control, quantification, and delivery followed by several methods for assessing genome editing efficiencies. As such the reader should be capable, by minor changes to the methods described here, to (co-) target rb1 or any one or multiple gene(s) within the Xenopus tropicalis genome by multiplex CRISPR/Cas9 methodology.
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Sistemas CRISPR-Cas , Técnicas de Inativação de Genes/métodos , Genoma , Proteína do Retinoblastoma/antagonistas & inibidores , Xenopus/genética , Animais , Proteína do Retinoblastoma/genética , Xenopus/classificaçãoRESUMO
Retinoblastoma is a pediatric eye tumor in which bi-allelic inactivation of the Retinoblastoma 1 (RB1) gene is the initiating genetic lesion. Although recently curative rates of retinoblastoma have increased, there are at this time no molecular targeted therapies available. This is, in part, due to the lack of highly penetrant and rapid retinoblastoma animal models that facilitate rapid identification of targets that allow therapeutic intervention. Different mouse models are available, all based on genetic deactivation of both Rb1 and Retinoblastoma-like 1 (Rbl1), and each showing different kinetics of retinoblastoma development. Here, we show by CRISPR/Cas9 techniques that similar to the mouse, neither rb1 nor rbl1 single mosaic mutant Xenopus tropicalis develop tumors, whereas rb1/rbl1 double mosaic mutant tadpoles rapidly develop retinoblastoma. Moreover, occasionally presence of pinealoblastoma (trilateral retinoblastoma) was detected. We thus present the first CRISPR/Cas9 mediated cancer model in Xenopus tropicalis and the first genuine genetic non-mammalian retinoblastoma model. The rapid kinetics of our model paves the way for use as a pre-clinical model. Additionally, this retinoblastoma model provides unique possibilities for fast elucidation of novel drug targets by triple multiplex CRISPR/Cas9 gRNA injections (rb1 + rbl1 + modifier gene) in order to address the clinically unmet need of targeted retinoblastoma therapy.