In order to further explore the interaction of elongase 5 (elovl5), elongase 2 (elovl2) and desaturase 2 (fads2) in the synthesis of highly unsaturated fatty acids (HUFA) in freshwater fish, we successfully constructed four zebrafish mutations including elovl5-/- (E5-/-), elovl2-/-*elovl5-/- (E2-/-*E5-/-), elovl5-/-*fads2-/- (E5-/-*F2-/-) and elovl2-/-*elovl5-/-*fads2-/- (E2-/-*E5-/-*F2-/-) using CRISPR/Cas9 technology, and soybean oil (SO) and canola oil (CO) feeding experiments were carried out on four mutants. The responses of zebrafish elovl5, elovl2 and fads2 to different fat sources in the feed during the HUFA synthesis process were studied. The results showed that in the E5-/- and E2-/-*E5-/- group, the extension of C18PUFA to C20PUFA still existed, while C18:3n-3 (ALA) and C18:2n-6 (LNA) showed significant accumulation in the E5-/-*F2-/- and E2-/-*E5-/-*F2-/- group. The expression of elovl4a and elovl4b in the liver were induced after mutation of elovl2, elovl5 and fads2. The results of fatty acid analysis showed that the DHA content of E5-/- group was not significantly different from that of WT, and the DHA content of E5-/-*F2-/- group was significantly lower than that of WT after fed with SO feed. However, the DHA content of E5-/- group was significantly higher than that of WT, and there was no significant difference between E5-/-*F2-/- and WT after CO feed was fed. Zebrafish desaturation was significantly affected without fads2, indicating that fads2 is an essential enzyme in the process of HUFA synthesis. The missing phenotype of elovl2 and elovl5 may be compensated by other HUFA synthesis pathways. This experiment further verified that elovl2 and elovl5 had gene interactions in HUFA synthesis, and fads2 was essential in HUFA synthesis as a desaturase. This study provides a theoretical basis for improving the HUFA synthesis ability of freshwater fish.