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Generation of Zebrafish Models of Human Retinitis Pigmentosa Diseases Using CRISPR/Cas9-Mediated Gene Editing System

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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.

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Data availability

The datasets generated and analyzed during the current study are not publicly available but are available from the corresponding author upon request.

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Funding

Funding was provided by Mashhad University of Medical Sciences (Grant No. 991444).

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Authors and Affiliations

  1. Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

    Farzaneh Mirzaei, Atiyeh Eslahi, Farzaneh Alizadeh, Sina Mozaffari, Tayebeh Hamzehloei, Alireza Pasdar & Majid Mojarrad

  2. Department of Anatomy and Cell Biology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

    Sareh Karimi

  3. Regenerative Medicine, Organ Procurement and Transplantation Multi-Disciplinary Center, Razi Hospital, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran

    Arash Salmaninejad

  4. Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

    Mohammad Rezaei

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  1. Farzaneh Mirzaei
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Correspondence to Majid Mojarrad.

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Mirzaei, F., Eslahi, A., Karimi, S. et al. Generation of Zebrafish Models of Human Retinitis Pigmentosa Diseases Using CRISPR/Cas9-Mediated Gene Editing System. Mol Biotechnol (2023). https://doi.org/10.1007/s12033-023-00907-8

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