Establishment of a CRISPR-Cas9-mediated keap1a knockout protocol in zebrafish (Danio rerio) for implementation in Vietnam

Abstract

This is the first study in Vietnam to generate Kelch-like ECH-associated protein 1a (keap1a) knockout zebrafish (Danio rerio) using clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) technology, aiming to investigate the Keap1-Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) system. This system plays a key role in protecting cells against oxidative stress zebrafish, possessing two keap1 paralogs (keap1a
and keap1b), serve as an ideal model for this investigation. The experimental procedure involved designing a specific guide RNA (gRNA) targeting the keap1a gene and microinjecting the gRNA/Cas9 complex into single-cell stage zebrafish embryos. The keap1a knockout fish exhibited normal development but demonstrated enhanced resistance to oxidative stress upon hydrogen peroxide (H₂O₂) exposure, as indicated by higher survival rates and elevated
expression of Nrf2 target genes (Glutathione S-transferase pi 1, gstp1 and Peroxiredoxin 1, prdx1). This study not only establishes an efficient gene knockout protocol in zebrafish but also opens new avenues for CRISPR-Cas9 applications in gene function research and aquaculture in Vietnam, while facilitating further investigations into the Keap1-Nrf2 system’s role in maintaining redox homeostasis and adapting to environmental stress in fish.