Thomas Naert

TALENs and CRISPR/Cas9 fuel genetically engineered clinically relevant Xenopus tropicalis tumor models

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.

RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6

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.Independently of the LGR4/5/6 receptors, RSPO2 acts as a direct antagonistic ligand to RNF43 and ZNRF3 during embryogenesis, and specifies the position and number of limbs that an embryo should form.

CRISPR/Cas9-Mediated Knockout of Rb1 in Xenopus tropicalis

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.

Genotyping of CRISPR/Cas9 Genome Edited Xenopus tropicalis

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.

Cancer Models in Xenopus tropicalis by CRISPR/Cas9 Mediated Knockout of Tumor Suppressors

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.

Author Correction: RSPO2 inhibition of RNF43 and ZNRF3 governs limb development independently of LGR4/5/6

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.

CRISPR/Cas9 disease models in zebrafish and Xenopus: The genetic renaissance of fish and frogs

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.