Poor Splice-Site Recognition in a Humanized Zebrafish Knockin Model for the Recurrent Deep-Intronic c.7595-2144A>G...

Area of Science:

• Genetics
• Molecular Biology
• Ophthalmology

Background:

• A common deep-intronic mutation (c.7595-2144A>G) in the USH2A gene creates a pseudoexon (PE40), leading to premature termination of usherin protein.
• This aberrant splicing is implicated in Usher syndrome, a genetic disorder affecting vision and hearing.
• Previous studies demonstrated that antisense oligonucleotides could correct USH2A splicing in patient fibroblasts.

Purpose of the Study:

• To investigate the functional impact of the c.7595-2144A>G mutation and USH2A splice redirection on retinal function.
• To establish and analyze a humanized zebrafish model for studying USH2A splicing defects.

Main Methods:

• Generated a humanized zebrafish model by replacing a segment of zebrafish ush2a intron 40 with the human sequence using CRISPR/Cas9.
• Analyzed USH2A transcript incorporation of the human pseudoexon (PE40) in adult zebrafish retinas.
• Performed immunohistochemical analyses to assess usherin expression and localization.
• Utilized antisense morpholinos to target and correct aberrant USH2A splicing.

Main Results:

• In homozygous humanized zebrafish retinas, only 7.4% of ush2a transcripts incorporated the human PE40 sequence.
• No significant differences in usherin expression or localization were observed between humanized and wild-type zebrafish larvae retinas.
• Partial correction of aberrant ush2a splicing was achieved using a PE40-targeting antisense morpholino.

Conclusions:

• Significant differences exist between human and zebrafish splicing machinery in recognizing splice sites.
• The humanized zebrafish model showed limited efficacy in recapitulating the splicing defect caused by the human mutation.
• A zebrafish-specific cell-based splice assay is proposed as a preliminary step before generating humanized zebrafish models to study splice-modulating mutations.