PUBLICATION
Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human genetic cardiovascular disorders
- Authors
- Tessadori, F., Roessler, H.I., Savelberg, S.M.C., Chocron, S., Kamel, S.M., Duran, K.J., van Haelst, M.M., van Haaften, G., Bakkers, J.
- ID
- ZDB-PUB-181026-16
- Date
- 2018
- Source
- Disease models & mechanisms 11(10): (Journal)
- Registered Authors
- Bakkers, Jeroen, Chocron, Sonja
- Keywords
- ABCC9, CRISPR/Cas9, Cantú syndrome, Genome editing, KCNJ8, Point mutation, Zebrafish
- MeSH Terms
-
- Animals
- Base Sequence
- CRISPR-Associated Protein 9/metabolism*
- CRISPR-Cas Systems/genetics*
- Cardiovascular Diseases/genetics*
- Disease Models, Animal
- Gene Editing*
- Gene Knock-In Techniques
- Genetic Testing
- Heterozygote
- Humans
- Mutation/genetics
- Nucleotides/genetics*
- Zebrafish/genetics*
- PubMed
- 30355756 Full text @ Dis. Model. Mech.
Citation
Tessadori, F., Roessler, H.I., Savelberg, S.M.C., Chocron, S., Kamel, S.M., Duran, K.J., van Haelst, M.M., van Haaften, G., Bakkers, J. (2018) Effective CRISPR/Cas9-based nucleotide editing in zebrafish to model human genetic cardiovascular disorders. Disease models & mechanisms. 11(10):.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping