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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.
FIGURE 1 Flowchart demonstrating our pipe-line for generation of GEXM by CRISPR/Cas9 (left) or TALEN techniques (right), converging
at the employed methods for assessing genome editing efficiencies in F0 mosaic GEXM (center). (Top left) sgRNA is designed using the
CRISPRScan online tool, and subsequently synthesized by a cloning-free method (Moreno-Mateos et al., 2015). For this, two DNA oligonucleotides
are annealed and extended in a PCR reaction and the resulting dsDNA template is used for in vitro transcription generating
injection-ready sgRNA, which is subsequently coinjected with Cas9 protein (Nakayama et al., 2014; Top right) TALEN pairs are designed
using TALE-NT 2.0 online tool, and are generated by Golden Gate cloning (Cermak et al., 2015; Doyle et al., 2012). Synthetic mRNAs
encoding obligate heterodimeric TALEN-KKR and TALEN-ELD pairs are subsequently co-injected. (Center) Independent of the choice of targeted
nuclease, HMA is used for qualitative genotyping of the F0 mosaic GEXM. If genome editing can be detected, qualitative genotyping
is performed by targeted deep next-generation sequencing and BATCH-GE analysis (Boel et al., 2016)
FIGURE 3 Rapid therapeutic target identification in genetically engineered Xenopus tropicalis tumor models. Multiplex genome engineering
by TALEN or CRISPR/Cas9 allows for rapid cotargeting of both tumor suppressor gene(s) and PTT gene within an F0 mosaic GEXM. A
subset of the tumors, by default characterized by biallelic tumor suppressor gene mutations, should exhibit biallelic mutations within the
PTT. Unless there is a selection system in place by which a tumor can never develop if the PTT is biallelically inactivated, making it an
interesting therapeutic target for drug exploration efforts
FIGURE 4 Hypothesis driven drug testing in genetically engineered Xenopus tropicalis tumor models. Straightforward delivery of targeted
nucleases (CRISPR/Cas9, TALEN) within the extrauterine developing Xenopus tropicalis embryo allows for generation of large cohorts (1100
animals) of tumor-bearing GEXM. Tumor-bearing GEXM can be directly subjected to drug treatment, by being reared in water with added
compound. Subsequently, the tumor responses can be analyzed in large cohorts adding power to statistical analyses
