Elovl2 but not Elovl5 is essential for the biosynthesis of docosahexaenoic acid (DHA) in zebrafish: insight from a comparative gene knockout study

Teleost fish can synthesize one of the major omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFAs), docosahexaenoic acid (DHA, 22:6n-3), from dietary α-linolenic acid (ALA; 18:3n-3), via elongase of very long chain fatty acid (Elovl) and fatty acid desaturase (Fads). However, it remains unclear which elongase is responsible for the endogenous synthesis of DHA. Here in this study, the knockout models of the two major elongases, Elovl2 and Elovl5, were generated by CRISPR/Cas9 approach in zebrafish and comparatively analyzed. The homozygous mutants were validated by Sanger sequencing, mutation-mediated PCR and whole-mount in situ hybridization analysis of the endogenous target genes. Compared with wildtype (WT) counterparts, the content of DHA was significantly reduced by 67.1% (p<0.05) in the adult liver and by 91.7% (p<0.01) in the embryo at 3 day-post-fertilization (dpf) of the elovl2 mutant, but not of the elovl5 mutant. Further study revealed that elovl2 and fads2 was upregulated by 9.9-fold (p<0.01) and 9.7-fold (p<0.01) in the elovl5 mutant, and elovl5 and fads2 was upregulated by 15.1-fold (p<0.01) and 21.5-fold (p<0.01) in the elovl2 mutant. Our study indicates that although both Elovl2 and Elovl5 have the elongase activity toward C20, the upregulation of elovl2 could completely replace the genetic depletion of elovl5 , but upregulation of elovl5 could not compensate the endogenous deficiency of elovl2 in mediating DHA synthesis. In conclusion, the endogenous synthesis of DHA in is mediated by Elovl2 but not Elovl5 in teleost, and a DHA-deficient genetic model of zebrafish has been generated.