CRISPR/Cas9 induced disruption of oca2 and tyr genes associated with pigmentation in Danio rerio

Abstract

Targeted disruption of genes has been successfully carried out in several model and non-model fish species. In ornamental fish industries, Danio rerio (D. rerio) represents the most significant fish species and is available in several color varieties. Recently, CRISPR/Cas9 has been used to disrupt pigmented genes in several fish species, including D. rerio . However, there have been few reports on the use of CRISPR/Cas9 for the disruption of pigmented genes tyrosinase (tyr) and oculocutaneous albinism II ( oca2 ) in D. rerio to date. In this study, guide RNA (gRNA) constructs specific to the tyr (T1) and oca2 (T2) genes were designed and synthesized, while Cas9 mRNA was synthesized through in vitro transcription. Subsequently, both the Cas9 mRNA and the gRNA constructs were validated using gel electrophoresis and sequencing. The validated Cas9 mRNA and gRNA mixture was then co-microinjected into D. rerio embryos at the 1–2 cell stage, which were obtained through natural spawning and later reared under controlled conditions. The results of gel electrophoresis and sequencing confirmed the successful synthesis and validation of the T1 and T2 targeting Cas9 mRNA and gRNA constructs. Melanophores were reduced when the T1 and T2 genes were disrupted, according to phenotypic studies of the Fo and F1 mutants, leading to visible pigmentation alterations. Furthermore, the validated gene editing constructs developed in this study may serve as useful tools for targeted pigmentation modification in other ornamental fish species, thereby supporting the development of high-value ornamental strains and advancing genetic improvement programs in aquaculture. However, further studies are required at the molecular level to confirm the mutation by genotyping.