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Dis Model Mech. 2018 Oct 18;11(10). pii: dmm035469. doi: 10.1242/dmm.035469.

Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human genetic cardiovascular disorders.

Author information

1
Hubrecht Institute-KNAW and UMC Utrecht, 3584 CT Utrecht, the Netherlands.
2
Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, 3584 CX Utrecht, the Netherlands.
3
Department of Clinical Genetics, Amsterdam Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands.
4
Department of Clinical Genetics, Free University Medical Center, 1018 HV Amsterdam, the Netherlands.
5
Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, 3584 CX Utrecht, the Netherlands G.vanHaaften@umcutrecht.nl j.bakkers@hubrecht.eu.
6
Hubrecht Institute-KNAW and UMC Utrecht, 3584 CT Utrecht, the Netherlands G.vanHaaften@umcutrecht.nl j.bakkers@hubrecht.eu.
7
Department of Medical Physiology, Division of Heart and Lungs, University Medical Center Utrecht, 3584 CX Utrecht, the Netherlands.

Abstract

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.

KEYWORDS:

ABCC9; CRISPR/Cas9; Cantú syndrome; Genome editing; KCNJ8; Point mutation; Zebrafish

PMID:
30355756
PMCID:
PMC6215435
DOI:
10.1242/dmm.035469
[Indexed for MEDLINE]
Free PMC Article

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