Highly efficient disruption of tyrb gene using CRISPR/Cas9 in grass carp (Ctenopharyngodon idella)

Abstract

Grass carp (Ctenopharyngodon idella) is the most important economic freshwater fish species in China. The stable production of high-quality grass carp depends significantly on excellent germplasm. In recent years, the generation of new germplasm based on genome editing has been applied to various cultured fish species. However, until now, there has been very few reports on the application of genome editing technology in grass carp. In this study, one-cell-stage embryos of grass carp were acquired through hormone-induced artificial spawning, thereby enabling the performance of genome editing in this species. The tyrb gene was isolated and chosen as the target of CRISPR/Cas9, because of its easily observable phenotype in F0 mutants. RT-PCR results indicated a high expression level of the tyrb gene in both skin and fin tissues. Subsequently, after the microinjection of the guide RNA (gRNA) and Cas9 protein mixture, targeted mutations were successfully identified through Sanger sequencing. Phenotypic analysis of the F0 mutants revealed that the disruption of tyrb led to a distinct golden phenotype, accompanied by a reduction or even absence of melanophores. Moreover, our data demonstrated that the combined utilization of two or three gRNAs caused large DNA fragment loss and a higher mutation rate in the F0 generation. Overall, this represents an application of CRISPR/Cas9 genome-editing technology in grass carp and may hold great significance for the future generation of new golden grass carp germplasm.