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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
Abu-Daya,
Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6.
2009, Pubmed,
Xenbase
Abu-Daya,
Absence of heartbeat in the Xenopus tropicalis mutation muzak is caused by a nonsense mutation in cardiac myosin myh6.
2009,
Pubmed
,
Xenbase
Abu-Daya,
The secreted integrin ligand nephronectin is necessary for forelimb formation in Xenopus tropicalis.
2011,
Pubmed
,
Xenbase
Abu-Daya,
The hitchhiker's guide to Xenopus genetics.
2012,
Pubmed
,
Xenbase
Bhattacharya,
CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus.
2015,
Pubmed
,
Xenbase
Bibikova,
Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.
2002,
Pubmed
Blum,
Xenopus: An Undervalued Model Organism to Study and Model Human Genetic Disease.
2018,
Pubmed
,
Xenbase
Brinkman,
Easy quantitative assessment of genome editing by sequence trace decomposition.
2014,
Pubmed
Carroll,
Genome engineering with zinc-finger nucleases.
2011,
Pubmed
Goda,
Genetic screens for mutations affecting development of Xenopus tropicalis.
2006,
Pubmed
,
Xenbase
Hassnain Waqas,
Adipose tissue macrophages develop from bone marrow-independent progenitors in Xenopus laevis and mouse.
2017,
Pubmed
,
Xenbase
Hwang,
Xenopus: Driving the Discovery of Novel Genes in Patient Disease and Their Underlying Pathological Mechanisms Relevant for Organogenesis.
2019,
Pubmed
,
Xenbase
Ishibashi,
Highly efficient bi-allelic mutation rates using TALENs in Xenopus tropicalis.
2012,
Pubmed
,
Xenbase
McQueen,
An analysis of MyoD-dependent transcription using CRISPR/Cas9 gene targeting in Xenopus tropicalis embryos.
2017,
Pubmed
,
Xenbase
Metscher,
MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues.
2009,
Pubmed
Miller,
A TALE nuclease architecture for efficient genome editing.
2011,
Pubmed
Moreno-Mateos,
CRISPRscan: designing highly efficient sgRNAs for CRISPR-Cas9 targeting in vivo.
2015,
Pubmed
,
Xenbase
Naert,
CRISPR/Cas9 disease models in zebrafish and Xenopus: The genetic renaissance of fish and frogs.
2018,
Pubmed
,
Xenbase
Naert,
Maximizing CRISPR/Cas9 phenotype penetrance applying predictive modeling of editing outcomes in Xenopus and zebrafish embryos.
2020,
Pubmed
,
Xenbase
Nakajima,
Generation of albino Xenopus tropicalis using zinc-finger nucleases.
2012,
Pubmed
,
Xenbase
Nakayama,
Simple and efficient CRISPR/Cas9-mediated targeted mutagenesis in Xenopus tropicalis.
2013,
Pubmed
,
Xenbase
Session,
Genome evolution in the allotetraploid frog Xenopus laevis.
2016,
Pubmed
,
Xenbase
Shalem,
Genome-scale CRISPR-Cas9 knockout screening in human cells.
2014,
Pubmed
