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| Construction of Transgenic Zebrafish (Danio rerio) Expressing Common Carp (Cyprinus carpio) Elovl5 Using CRISPR/Cas9 System |
| WANG Ya-Xin1,2, YANG Chen-Ru1,2, ZHAO Yu-Jie1,2, SUN Xiao-Qing1,2, LI Shang-Qi2, ZHANG Yan2, LIU Ying-Jie2, LI Jiong-Tang2,* |
1 National Experimental Teaching and Demonstration Center of Fisheries Science, Shanghai Ocean University, Shanghai 201306, China;
2 Biotechnology Research Center, Chinese Academy of Fishery Sciences/Key Laboratory of Aquatic Genomes, Ministry of Agriculture and Rural Affairs/Beijing Key Laboratory of Aquatic Biotechnology, Beijing 100141, China |
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Abstract CRISPR/cas9 technology is widely used in gene knockout. It is helpful for early screening of experimental animals to knock in fragments containing fluorescent protein and target gene. Elongation of very long chain fatty acids 5 (Elovl5) is one of key enzymes to regulate the biosynthesis of fish highly-unsaturated fatty acids (HUFAs). Constructing a transgenic animal model expressing Elovl5 will provide a new strategy to study the in vivo function of this enzyme. Common carp (Cyprinus carpio) is one of the most widely cultured freshwater fishes in our country. Although common carp Elovl5 was identified and its tissue expressional pattern and correlation to growth were studied, its elongase activity has not been investigated. To study the in vivo function of common carp Elovl5 in zebrafish (Danio rerio), a vector including Tol2 sequence, zebrafish myosin light chain 2 (Mylz2) promoter, red fluorescence gene (DsRed2), zebrafish Elovl5 promoter, common carp Elovl5 gene, 2A cleavage peptide, and green fluorescence gene, was constructed. A fragment consisting of these promoters and genes with a length of 7.5 kb was amplified from this vector and then knocked into zebrafish genome using the CRISPR/Cas9 method. From 72 h after fertilization, the red fluorescence was observed. For the transgenic zebrafish screened using the red fluorescence, PCR amplification of DsRed2 confirmed the insertion of this fragment into the genome. The result indicated that using fluorescence to screen transgenic animals was reliable, simple and practicable. Among all the surviving zebrafish, the knock-in rate was 28.7%. The study also knocked this fragment into the genome by using Tol2 system with a rate of 32.8%, which suggested that the knock-in efficiency of CRISPR/Cas9 method was lower than the Tol2 system. Compared to the control group, common carp Elovl5 was expressed only in the CRISPR/Cas9 experimental group and the Tol2 experimental group. Furthermore, its expression level was comparable between these two groups, suggesting that the inserted gene expression efficiency of the CRISPR/Cas9 method was equivalent to that of the Tol2 method. The contents of Elovl5 substrates in the CRISPR/Cas9 experimental group decreased by 18.6%. The contents of Elovl5 products had an increase of 31.8% and the C20:4n-3 fatty acid content was 2.4 times as high as that in the control group. These results indicated that common carp Elovl5 could in vivo convert the substrates into HUFAs. In this study, CRISPR/Cas9 technology was used to construct transgenic zebrafish expressing common carp Elovl5. It demonstrated that the CRISPR/Cas9 method could effectively insert the long fragments into genomes. It will be applied into the simultaneous knock-in of multiple genes from one pathway or regulating multiple traits. The HUFA contents increased in the transgenic zebrafish, demonstrating the elongase activity of common carp Elovl5. The transgenic zebrafish will be useful to study the biosynthesis mechanism of common carp HUFAs and the regulation mechanism of Elovl5.
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Received: 16 January 2020
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Corresponding Authors:
* lijt@cafs.ac.cn
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