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CRISPR/Cas9 has become the favorite method for gene knockouts in a range of vertebrate model organisms due to its ease of use and versatility. Gene-specific guide RNAs can be designed to a unique genomic sequence and used to target the Cas9 endonuclease, which causes a double-stranded break at the desired locus. Repair of the breaks through non-homologous end joining often results in the deletion or insertion of several nucleotides, which frequently result in nonsense mutations. Xenopus frogs have long been an excellent model organism in which to study gene function, and they have proven to be useful in gene-editing experiments, especially the diploid species, X. tropicalis. In this chapter, we present our protocols for gene disruption in Xenopus, which we regularly use to investigate developmental processes and model human genetic disease.
Fig. 1
ssDNA and dsDNA templates visualized using the intercalating reagent Ethidium Bromide
Fig. 2
sgRNA constructs visualized by agarose gel electrophoresis
Fig. 3
Genotype analysis of F0 mosaic tadpoles. The target locus is amplified from genomic DNA preparations of injected (crispant) and uninjected (control) tadpoles. Amplicons are digested with T7 Endonuclease I, which reveals a second band unique to crispant tadpoles that correspond to the location of the CRISPR target site (A). Sanger sequencing is used to confirm the presence of indels in these crispant samples. Analysis of the Sanger sequencing trace files by Synthego ICE revealed the editing efficiency of the target domain is around 50% (B)
Fig. 4
Detailed structural differences are examined in Xenopus tadpoles using high-resolution Micro Computed Tomography. The imaged volumes show a whole tadpole with a 3D reconstruction of the cardiovascular system and gut visualized using TXM3DViewer (Carl Zeiss Microscopy) that can be exported as cross-section high-resolution TIFF image files
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