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Dev Biol 2015 Dec 15;4082:196-204. doi: 10.1016/j.ydbio.2015.11.003.
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CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus.

Bhattacharya D , Marfo CA , Li D , Lane M , Khokha MK .

Congenital malformations are the major cause of infant mortality in the US and Europe. Due to rapid advances in human genomics, we can now efficiently identify sequence variants that may cause disease in these patients. However, establishing disease causality remains a challenge. Additionally, in the case of congenital heart disease, many of the identified candidate genes are either novel to embryonic development or have no known function. Therefore, there is a pressing need to develop inexpensive and efficient technologies to screen these candidate genes for disease phenocopy in model systems and to perform functional studies to uncover their role in development. For this purpose, we sought to test F0 CRISPR based gene editing as a loss of function strategy for disease phenocopy in the frog model organism, Xenopus tropicalis. We demonstrate that the CRISPR/Cas9 system can efficiently modify both alleles in the F0 generation within a few hours post fertilization, recapitulating even early disease phenotypes that are highly similar to knockdowns from morpholino oligos (MOs) in nearly all cases tested. We find that injecting Cas9 protein is dramatically more efficacious and less toxic than cas9 mRNA. We conclude that CRISPR based F0 gene modification in X. tropicalis is efficient and cost effective and readily recapitulates disease and MO phenotypes.

PubMed ID: 26546975
PMC ID: PMC4684459
Article link: Dev Biol
Grant support: [+]

Species referenced: Xenopus tropicalis
Genes referenced: ccdc40 dnah9 foxj1 foxj1.2 galnt11 kit pax2 pax8 tyr
Antibodies: Tuba4b Ab4 Tuba4b Ab5
Morpholinos: foxj1 MO1 pax8 MO3
gRNAs referenced: ccdc40 gRNA1 ccdc40 gRNA2 dnah9 gRNA1 dnah9 gRNA2 foxj1 gRNA5 galnt11 gRNA1 galnt11 gRNA2 pax8 gRNA1 tyr gRNA10 tyr gRNA11

Disease Ontology terms: visceral heterotaxy [+]
Phenotypes: Xtr Wt + Cas9 (fig.3.b) [+]

Article Images: [+] show captions
References [+] :
Bassett, CRISPR/Cas9 mediated genome engineering in Drosophila. 2014, Pubmed