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
  • Gene Knock-In Techniques
  • Zebrafish/genetics*
  • Cardiovascular Diseases/genetics*
  • Animals
  • Nucleotides/genetics*
  • Genetic Testing
  • CRISPR-Associated Protein 9/metabolism*
  • Mutation/genetics
  • CRISPR-Cas Systems/genetics*
  • Base Sequence
  • Disease Models, Animal
  • Gene Editing*
  • Heterozygote
  • Humans
(all 14)
PubMed
30355756 Full text @ Dis. Model. Mech.
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
Figures
Figure Gallery (6 images)
Show all Figures
Expression
Gene Antibody Fish Conditions Stage Qualifier Anatomy Assay Figure
GFPkcnj8hu11872/+; la116Tgstandard conditionsDay 5IFL
GFPla116Tgstandard conditionsDay 5IFL
1 - 2 of 2
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Phenotype
Fish Conditions Stage Phenotype Figure
abcc9hu11838/+standard conditionsDay 5
abcc9hu11838/+standard conditionsDay 5
abcc9hu11838/hu11838standard conditionsDay 5
abcc9hu11838/hu11838standard conditionsDay 5
abcc9hu11859/+; la116Tgstandard conditionsDay 5
abcc9hu11859/+; la116Tgstandard conditionsDay 5
abcc9hu11859/+; la116Tgstandard conditionsDay 5
abcc9hu11859/+; la116Tgstandard conditionsDay 5
kcnj8hu11872/+standard conditionsDay 5
kcnj8hu11872/+standard conditionsDay 5
1 - 10 of 24
Show
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
hu10742
    		Small Deletion
    hu11838
      		Complex
      hu11859
        		Complex
        hu11872
          		Point Mutation
          la116TgTransgenic Insertion
            1 - 5 of 5
            Show
            Human Disease / Model
            Human Disease Fish Conditions Evidence
            hypertrichotic osteochondrodysplasia Cantu typeabcc9hu11838/+standard conditionsTAS
            hypertrichotic osteochondrodysplasia Cantu typekcnj8hu11872/+standard conditionsTAS
            1 - 2 of 2
            Show
            Sequence Targeting Reagents
            Target Reagent Reagent Type
            abcc9CRISPR1-abcc9CRISPR
            abcc9CRISPR2-abcc9CRISPR
            kcnj8CRISPR1-kcnj8CRISPR
            pln1CRISPR1-pln1CRISPR
            1 - 4 of 4
            Show
            Fish
            Fish
            abcc9hu11838/hu11838
            abcc9hu11838/+
            abcc9hu11859/hu11859; la116Tg
            abcc9hu11859/+; la116Tg
            kcnj8hu11872/hu11872
            kcnj8hu11872/+
            kcnj8hu11872/+; la116Tg
            la116Tg
            pln1hu10742/+
            1 - 9 of 9
            Show
            Antibodies
            No data available
            Orthology
            No data available
            Engineered Foreign Genes
            Marker Marker Type Name
            GFPEFGGFP
            1 - 1 of 1
            Show
            Mapping
            No data available
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            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):.