Study on the Application of CRISPR/Cas9 Technology in Editing of Genes Related to Sex and Muscle Development in Olive Flounder (Paralichthys olivaceus)
WANG Ling1,2,3, TAN Xun-Gang1,2,*, WU Zhi-Hao1,2, WANG Li-Juan1,2, YOU Feng1,2,*
1 Institute of Oceanology/Center for Ocean Mega-Science/Key Laboratory of Experimental Marine Biology, Chinese Academy of Sciences, Qingdao 266071, China; 2 Pilot National Laboratory for Marine Science and Technology (Qingdao)/Laboratory for Marine Biology and Biotechnology, Qingdao 266237, China; 3 University of Chinese Academy of Sciences, Beijing 100049, China
Abstract CRISPR/Cas9 is a gene editing technology for specific DNA modification of targeted genes. Based on the simplicity of design, low cost, and high efficiency, CRISPR/Cas9 system has been extensively used in mammal for gene function study. It has been well developed in model fish and freshwater fish for gene function analysis through gene knockout method, but there were few studies on marine fish species, especially on flatfish. Olive flounder (Paralichthys olivaceus) is an important maricultured fish in China with high economic value. In this study, low microinjection concentrations of Cas9 message RNA (Cas9 mRNA) (250 ng/μL) and guide RNA (gRNA) (100 ng/μL) were firstly administrated to edit genes related to gonadal and muscle development, including myogenic differentiation (myod), adaptor-related protein complex 3 (ap3), R-spondin 1 (rspo1), and dynein axonemal light intermediate chain 1 (dnali1) (dnali1-part A and dnali1-part B) in olive flounder, respectively. Two gRNAs for dnali1-part A and dnali1-part B, and one gRNA for the other genes were designed to be used for the editing, respectively. The results showed that no mutants were detected in the newly hatched larvae of the rspo1, myod and dnali1-part A gene editing groups, and the mutation rates of the ap3 and dnali1-part B groups were no more than 14.3% and 42.9%~45%, respectively. And then, the higher injection concentrations of Cas9 mRNA (1 500 ng/μL) and gRNA (1 000 ng/μL) were used for the genes editing. Under the condition, the mutation rate of dnali1-part B increased to 65%, and the mutation rate of of individual detection increased from 10%~20% to 80%~90%. The marker gene dead end (dnd) of primordial germ cells, doublesex and mab-3 related transcription factor 1 (dmrt1), and hydroxysteroid (17-beta) dehydrogenase 1 (17β-Hsd1) genes with 2 gRNAs were further edited by using higher concentrations of Cas9 mRNA and gRNA, and the mutation rates of these genes were 60%~80% at hatched stage. The results also showed that the editing efficiency at low concentration condition with one gRNA site (14.3%~45%) was lower than those at high concentration condition with 2 gRNA sites (60%~80%). This study established a CRISPR/Cas9 gene editing method in olive flounder primarily, and would provide a reference for its application in the flatfish.
WANG Ling,TAN Xun-Gang,WU Zhi-Hao, et al. Study on the Application of CRISPR/Cas9 Technology in Editing of Genes Related to Sex and Muscle Development in Olive Flounder (Paralichthys olivaceus)[J]. 农业生物技术学报, 2021, 29(9): 1774-1784.
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