PUBLICATION

Targeted disruption of the endogenous zebrafish rhodopsin locus as models of rapid rod photoreceptor degeneration.

Authors
Zelinka, C.P., Sotolongo-Lopez, M., Fadool, J.M.
ID
ZDB-PUB-180914-5
Date
2018
Source
Molecular Vision   24: 587-602 (Journal)
Registered Authors
Fadool, James M.
Keywords
none
MeSH Terms
  • Animals, Genetically Modified
  • Animals
  • RNA, Messenger/genetics
  • Zebrafish Proteins/genetics*
  • Rhodopsin/genetics*
  • Frameshift Mutation/genetics
  • Retinal Rod Photoreceptor Cells/pathology*
  • Polymorphism, Restriction Fragment Length
  • Gene Targeting
  • Immunoblotting
  • Disease Models, Animal*
  • Retinal Degeneration/genetics*
  • Retinal Degeneration/pathology
  • Zebrafish/genetics*
  • Codon, Terminator/genetics
  • CRISPR-Cas Systems
(all 16)
PubMed
30210230
Abstract
Retinitis pigmentosa (RP) is a collection of genetic disorders that results in the degeneration of light-sensitive photoreceptor cells, leading to blindness. RP is associated with more than 70 loci that may display dominant or recessive modes of inheritance, but mutations in the gene encoding the visual pigment rhodopsin (RHO) are the most frequent cause. In an effort to develop precise mutations in zebrafish as novel models of photoreceptor degeneration, we describe the generation and germline transmission of a series of novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced insertion and deletion (indel) mutations in the major zebrafish rho locus, rh1-1.
One- or two-cell staged zebrafish embryos were microinjected with in vitro transcribed mRNA encoding Cas9 and a single guide RNA (gRNA). Mutations were detected by restriction fragment length polymorphism (RFLP) and DNA sequence analyses in injected embryos and offspring. Immunolabeling with rod- and cone-specific antibodies was used to test for histological and cellular changes.
Using gRNAs that targeted highly conserved regions of rh1-1, a series of dominant and recessive alleles were recovered that resulted in the rapid degeneration of rod photoreceptors. No effect on cones was observed. Targeting the 5'-coding sequence of rh1-1 led to the recovery of several indels similar to disease-associated alleles. A frame shift mutation leading to a premature stop codon (T17*) resulted in rod degeneration when brought to homozygosity. Immunoblot and fluorescence labeling with a Rho-specific antibody suggest that this is indeed a null allele, illustrating that the Rho expression is essential for rod survival. Two in-frame mutations were recovered that disrupted the highly conserved N-linked glycosylation consensus sequence at N15. Larvae heterozygous for either of the alleles demonstrated rapid rod degeneration. Targeting of the 3'-coding region of rh1-1 resulted in the recovery of an allele encoding a premature stop codon (S347*) upstream of the conserved VSPA sorting sequence and a second in-frame allele that disrupted the putative phosphorylation site at S339. Both alleles resulted in rod death in a dominant inheritance pattern. Following the loss of the targeting sequence, immunolabeling for Rho was no longer restricted to the rod outer segment, but it was also localized to the plasma membrane.
The efficiency of CRISPR/Cas9 for gene targeting, coupled with the large number of mutations associated with RP, provided a backdrop for the rapid isolation of novel alleles in zebrafish that phenocopy disease. These novel lines will provide much needed in-vivo models for high throughput screens of compounds or genes that protect from photoreceptor degeneration.
Genes / Markers
Figures
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Expression
Phenotype
Mutations / Transgenics
Allele Construct Type Affected Genomic Region
fl1TgTransgenic Insertion
    fl6
      Indel
      fl7
        Indel
        fl8
          Indel
          fl9
            Small Deletion
            fl10
              Small Deletion
              1 - 6 of 6
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              Human Disease / Model
              Sequence Targeting Reagents
              Target Reagent Reagent Type
              rhoCRISPR17-rhoCRISPR
              rhoCRISPR18-rhoCRISPR
              1 - 2 of 2
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              Fish
              Antibodies
              Orthology
              No data available
              Engineered Foreign Genes
              Marker Marker Type Name
              EGFPEFGEGFP
              1 - 1 of 1
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              Mapping
              No data available