PUBLICATION
Generation of Zebrafish Models of Human Retinitis Pigmentosa Diseases Using CRISPR/Cas9-Mediated Gene Editing System
- Authors
- Mirzaei, F., Eslahi, A., Karimi, S., Alizadeh, F., Salmaninejad, A., Rezaei, M., Mozaffari, S., Hamzehloei, T., Pasdar, A., Mojarrad, M.
- ID
- ZDB-PUB-231120-13
- Date
- 2023
- Source
- Molecular Biotechnology 66(10): 2909-2919 (Journal)
- Registered Authors
- Rezaei, Mohammad
- Keywords
- Animal model, CRISPR/Cas9, RPE65 gene, Retinitis pigmentosa, Zebrafish
- MeSH Terms
-
- Disease Models, Animal*
- CRISPR-Cas Systems*
- Gene Editing*/methods
- Retinitis Pigmentosa*/genetics
- Retinitis Pigmentosa*/therapy
- Zebrafish*/genetics
- Animals
- Gene Knockout Techniques
- RNA, Guide, CRISPR-Cas Systems*/genetics
- Humans
- cis-trans-Isomerases/genetics
- cis-trans-Isomerases/metabolism
- Zebrafish Proteins/genetics
- PubMed
- 37980693 Full text @ Mol. Biotechnol.
Citation
Mirzaei, F., Eslahi, A., Karimi, S., Alizadeh, F., Salmaninejad, A., Rezaei, M., Mozaffari, S., Hamzehloei, T., Pasdar, A., Mojarrad, M. (2023) Generation of Zebrafish Models of Human Retinitis Pigmentosa Diseases Using CRISPR/Cas9-Mediated Gene Editing System. Molecular Biotechnology. 66(10):2909-2919.
Abstract
Generating animal models can explore the role of new candidate genes in causing diseases and the pathogenicity of a specific mutation in the underlying genes. These animals can be used to identify new pharmaceutical or genetic therapeutic methods. In the present experiment, we developed a rpe65a knock out (KO) zebrafish as a retinitis pigmentosa (RP) disease model. Using the CRISPR/Cas9 system, the rpe65a gene was KO in zebrafish. Two specific single-guide RNAs (sgRNAs) were designed for the zebrafish rpe65a gene. SgRNAs were cloned into the DR274 plasmid and synthesized using in vitro transcription method. The efficiency of Ribonucleoprotein (synthesized sgRNA and recombinant Cas9) was evaluated by in vitro digestion experiment. Ribonucleoprotein complexes were microinjected into one to four-celled eggs of the TU zebrafish strain. The effectiveness of sgRNAs in KO the target gene was determined using the Heteroduplex mobility assay (HMA) and Sanger sequencing. Online software was used to determine the percent of mosaicism in the sequenced samples. By examining the sequences of the larvae that showed a mobility shift in the HMA method, the presence of indels in the binding region of sgRNAs was confirmed, so the zebrafish model for RP disease established. Zebrafish is an ideal animal model for the functional study of various diseases involving different genes and mutations and used for evaluating different therapeutic approaches in human diseases. This study presents the production of rpe65a gene KO zebrafish models using CRISPR/Cas9 technology. This model can be used in RP pathophysiology studies and preclinical gene therapy experiments.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping