Generation of golden goldfish Carassius auratus via tyrosinase gene targeting by CRISPR/Cas9

Gene editing technology based on CRISPR/cas9 system has a pivotal application prospect in promoting the genetic improvement of economic traits of aquaculture animals, and has been widely used in the genetic improvement of various aquatic animals. The captivating coloration and intricate color patterns in ornamental fish are vital phenotypic traits that directly influence their economic and aesthetic value. Goldfish (Carassius auratus), regarded as one of the world's earliest ornamental fish, has garnered significant attention from researchers due to its diverse range of color patterns and unique morphological variations. Tyrosinase (tyr) serves as the rate-limiting enzyme in the enzymatic cascade responsible for melanin biosynthesis. In our study, we have successfully developed a highly efficient and precise genome editing technology for Carassius auratus tyrosinase (tyr), resulting in the creation of a strikingly golden goldfish. The duplicated tyr genes (tyrA and tyrB) were first identified in C. auratus, and the CRISPR/Cas9 was used to disrupt both tyr genes. The edited albino mutants displayed a complete absence of melanocytes in both their eyes and body surface, whereas mosaic mutants exhibited varying degrees of melanin reduction. Notably, disrupting only tyrA or tyrB failed to yield a reduction in melanin content. The whole-genome resequencing was employed to comprehensively screen the off-target sites in the mutant individuals at the genome-wide scale. The results revealed that the absence of off-target mutations in the predicted location, which exhibited sequence similarity to the target sites. Our findings underscored the indispensable role of tyr genes in melanin synthesis within goldfish, while also demonstrating the remarkable efficiency and accuracy of the CRISPR/Cas9 editing system in generating novel phenotypes in fish. This bright golden goldfish represented a captivating addition to the realm of ornamental fish strains and provided valuable insights into the regulation of color patterning and the potential for gene-editing breeding in fish for future research.