Targeted disruption of the endogenous zebrafish

Area of Science:

• Genetics
• Molecular Biology
• Ophthalmology

Background:

• Retinitis pigmentosa (RP) is a group of genetic disorders causing photoreceptor degeneration and blindness.
• Mutations in the rhodopsin gene (RHO) are a frequent cause of RP.
• Zebrafish offer a valuable model system for studying genetic eye diseases.

Purpose of the Study:

• To generate novel zebrafish models of photoreceptor degeneration by inducing mutations in the rh1-1 gene.
• To utilize clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology for precise gene editing in zebrafish.
• To establish germline transmission of induced mutations for creating disease models.

Main Methods:

• Microinjection of zebrafish embryos with Cas9 mRNA and single guide RNA (gRNA) targeting the rh1-1 locus.
• Detection of mutations using restriction fragment length polymorphism (RFLP) and DNA sequencing.
• Histological and cellular analysis using rod- and cone-specific antibodies and Rho-specific immunoblotting.

Main Results:

• Generated dominant and recessive alleles in the rh1-1 gene, leading to rapid rod photoreceptor degeneration.
• Identified mutations mimicking disease-associated alleles, including a null allele (T17*) and alleles disrupting glycosylation (N15) and phosphorylation (S339) sites.
• Observed rod degeneration in both homozygous and heterozygous states for specific alleles, with no impact on cone cells.
• Demonstrated Rho expression is essential for rod survival and altered Rho localization in mutated cells.

Conclusions:

• CRISPR/Cas9 efficiently generates zebrafish alleles that phenocopy RP.
• These novel zebrafish lines serve as in-vivo models for photoreceptor degeneration.
• The models will facilitate high-throughput screening for therapeutic compounds and protective genes for RP.