期刊论文详细信息
Disease Models & Mechanisms
Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human genetic cardiovascular disorders
Karen J. Duran1  Mieke M. van Haelst1  Helen I. Roessler1  Gijs van Haaften1  Sanne M. C. Savelberg1  Sarah M. Kamel2  Jeroen Bakkers2  Sonja Chocron2  Federico Tessadori2 
[1] Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, 3584 CX Utrecht, the Netherlands;Hubrecht Institute-KNAW and UMC Utrecht, 3584 CT Utrecht, the Netherlands;
关键词: CRISPR/Cas9;    Zebrafish;    Genome editing;    Point mutation;    Cantú syndrome;    KCNJ8;    ABCC9;   
DOI  :  10.1242/dmm.035469
来源: DOAJ
【 摘 要 】

The zebrafish (Danio rerio) has become a popular vertebrate model organism to study organ formation and function due to its optical clarity and rapid embryonic development. The use of genetically modified zebrafish has also allowed identification of new putative therapeutic drugs. So far, most studies have relied on broad overexpression of transgenes harboring patient-derived mutations or loss-of-function mutants, which incompletely model the human disease allele in terms of expression levels or cell-type specificity of the endogenous gene of interest. Most human genetically inherited conditions are caused by alleles carrying single nucleotide changes resulting in altered gene function. Introduction of such point mutations in the zebrafish genome would be a prerequisite to recapitulate human disease but remains challenging to this day. We present an effective approach to introduce small nucleotide changes in the zebrafish genome. We generated four different knock-in lines carrying distinct human cardiovascular-disorder-causing missense mutations in their zebrafish orthologous genes by combining CRISPR/Cas9 with a short template oligonucleotide. Three of these lines carry gain-of-function mutations in genes encoding the pore-forming (Kir6.1, KCNJ8) and regulatory (SUR2, ABCC9) subunits of an ATP-sensitive potassium channel (KATP) linked to Cantú syndrome (CS). Our heterozygous zebrafish knock-in lines display significantly enlarged ventricles with enhanced cardiac output and contractile function, and distinct cerebral vasodilation, demonstrating the causality of the introduced mutations for CS. These results demonstrate that introducing patient alleles in their zebrafish orthologs promises a broad application for modeling human genetic diseases, paving the way for new therapeutic strategies using this model organism.

【 授权许可】

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