CRISPR/Cas9-Mediated Transgenesis of the Masu Salmon (Oncorhynchus masou) elovl2 Gene Improves n-3 Fatty Acid Content in Channel Catfish (Ictalurus punctatus)

摘要

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play a very important role in human health. Channel catfish (Ictalurus punctatus) is one of the leading freshwater aquaculture species in the USA, but has low levels of EPA and DHA compared to some fish such as salmon. To improve EPA and DHA content, a modification of the n-3 PUFA biosynthetic pathway was achieved through the insertion of an elovl2 transgene isolated from masu salmon (Oncorhynchus masou) driven by a carp beta-actin promoter using a two-hit by gRNA and two oligos with a targeting plasmid (2H2OP) CRISPR/Cas9 approach. Integration rate of the transgene was high (37.5%) and detected in twelve different tissues of P-1 transgenic fish with tissue-specific gene expression. Liver and muscle had relative high gene expression (13.4- and 9.2-fold change, respectively). Fatty acid analysis showed DHA content in the muscle from transgenic fish was 1.62-fold higher than in non-transgenic fish (P < 0.05). Additionally, total n-3 PUFAs and omega-6 polyunsaturated fatty acids (n-6 PUFAs) increased to 1.41-fold and 1.50-fold, respectively, suggesting the beta-actin-elovl2 transgene improved biosynthesis of PUFAs in channel catfish as a whole. The n-9 fatty acid level decreased in the transgenic fish compared to the control. Morphometric analysis showed that there were significant differences between injected fish with sgRNAs (including positive and negative fish) and sham-injected controls (P < 0.001). Potential off-target effects are likely the major factor responsible for morphological deformities. Optimization of sgRNA design to maximize activity and reduce off-target effects of CRISPR/Cas9 should be examined in future transgenic research, but this research shows a promising first step in the improvement of n-3 PUFAs in channel catfish.