ZFIN ID: ZDB-PUB-181004-7
Poor Splice-Site Recognition in a Humanized Zebrafish Knockin Model for the Recurrent Deep-Intronic c.7595-2144A>G Mutation in USH2A
Slijkerman, R., Goloborodko, A., Broekman, S., de Vrieze, E., Hetterschijt, L., Peters, T., Gerits, M., Kremer, H., van Wijk, E.
Date: 2018
Source: Zebrafish   15(6): 597-609 (Journal)
Registered Authors: de Vrieze, Erik, Goloborodko, Alexander, Hetterschijt, Lisette, Kremer, Hannie, Slijkerman, Ralph, van Wijk, Erwin
Keywords: CRISPR/Cas9, USH2A, minigene splice assay, retinal degeneration, splicing, usher syndrome
MeSH Terms:
  • Animals
  • Extracellular Matrix Proteins/genetics*
  • Gene Expression Regulation*
  • Humans
  • Introns
  • Larva/growth & development
  • Larva/metabolism
  • Mutation
  • RNA Splice Sites
  • RNA Splicing
  • Zebrafish/genetics*
  • Zebrafish/growth & development
PubMed: 30281416 Full text @ Zebrafish
The frequent deep-intronic c.7595-2144A>G mutation in intron 40 of USH2A generates a high-quality splice donor site, resulting in the incorporation of a pseudoexon (PE40) into the mature transcript that is predicted to prematurely terminate usherin translation. Aberrant USH2A pre-mRNA splicing could be corrected in patient-derived fibroblasts using antisense oligonucleotides. With the aim to study the effect of the c.7595-2144A>G mutation and USH2A splice redirection on retinal function, a humanized zebrafish knockin model was generated, in which 670 basepairs of ush2a intron 40 were exchanged for 557 basepairs of the corresponding human sequence using an optimized CRISPR/Cas9-based protocol. However, in the retina of adult homozygous humanized zebrafish, only 7.4% ± 3.9% of ush2a transcripts contained the human PE40 sequence and immunohistochemical analyses revealed no differences in the usherin expression and localization between the retina of humanized and wild-type zebrafish larvae. Nevertheless, we were able to partially correct aberrant ush2a splicing using a PE40-targeting antisense morpholino. Our results indicate a clear difference in splice-site recognition by the human and zebrafish splicing machinery. Therefore, we propose a protocol in which the effect of human splice-modulating mutations is studied in a zebrafish-specific cell-based splice assay before the generation of a humanized zebrafish knockin model.