The large yellow croaker (Larimichthys crocea) is an economically important marine species with the highest annual production among the farmed marine fishes in China. However, the aquaculture industry of this species ...The large yellow croaker (Larimichthys crocea) is an economically important marine species with the highest annual production among the farmed marine fishes in China. However, the aquaculture industry of this species is suffering from severe problems that include weakened disease resistance, decreased growth rate, and reduced meat quality due to frequent inbreeding. Genome editing, which has a huge potential for solving those problems by introducing favorable genetic changes, is not yet available for the large yellow croaker. Here, we pioneered the techniques of embryo microinjection and genome editing using the CRISPR/Cas9 system in this species. Recombinant plasmids encoding green fluorescent protein (GFP) were introduced into the fertilized eggs of L. crocea by microinjection before the chorion had hardened. A high survival rate (40%) and GFP-positive larvae rate (81.8%) were achieved, indicating that the microinjection technique in L. crocea was successfully established. On this basis, Cas9 mRNA and sgRNA targeting the tyrosinase a gene in L. crocea (Lc-tyra) were co-injected into fertilized eggs of L. crocea. Mutant individuals with insertion and deletion mutations of Lc-tyra were detected. These results indicated that the CRISPR/Cas9-based genome editing technology established herein could efficiently introduce mutations at a specific site in the L. crocea genome. This method provides the potential for genetic improvement and functional genomic study in this species. This is the first report on effective CRISPR/Cas9-based genome editing in L. crocea.展开更多
基金The work was supported by grants from the National Key R&D Program of China(2018YFD0900505)National Natural Science Foundation of China(U1905204 and 31802337)+1 种基金China Agricultural Research System(CARS-47)Marine Economic Development Subsidy Fund of Fujian Province(FJHJF-L-2019-2).
文摘The large yellow croaker (Larimichthys crocea) is an economically important marine species with the highest annual production among the farmed marine fishes in China. However, the aquaculture industry of this species is suffering from severe problems that include weakened disease resistance, decreased growth rate, and reduced meat quality due to frequent inbreeding. Genome editing, which has a huge potential for solving those problems by introducing favorable genetic changes, is not yet available for the large yellow croaker. Here, we pioneered the techniques of embryo microinjection and genome editing using the CRISPR/Cas9 system in this species. Recombinant plasmids encoding green fluorescent protein (GFP) were introduced into the fertilized eggs of L. crocea by microinjection before the chorion had hardened. A high survival rate (40%) and GFP-positive larvae rate (81.8%) were achieved, indicating that the microinjection technique in L. crocea was successfully established. On this basis, Cas9 mRNA and sgRNA targeting the tyrosinase a gene in L. crocea (Lc-tyra) were co-injected into fertilized eggs of L. crocea. Mutant individuals with insertion and deletion mutations of Lc-tyra were detected. These results indicated that the CRISPR/Cas9-based genome editing technology established herein could efficiently introduce mutations at a specific site in the L. crocea genome. This method provides the potential for genetic improvement and functional genomic study in this species. This is the first report on effective CRISPR/Cas9-based genome editing in L. crocea.
