F1 transgenic channel catfish, Ictalurus punctatus, containing the common carp beta-actin promoter-masu salmon, Oncorhynchus masou, Δ5-desaturase like gene (D5D) showed a 33% increase in the relative proportion of n-3 fatty acids coupled with a 15% decrease in n-6 fatty acids and a 17% decrease in n-9 fatty acids when compared to non-transgenic full-siblings (P < 0.01). However, while the relative proportion of n-3 fatty acids was achieved, the total amount of fatty acids in the transgenic fish decreased resulting in a reduction of all fatty acids. Insertion of the D5D transgene into channel catfish also had large effects on metabolism, physiology, body composition, and growth of channel catfish. Transgenic channel catfish grew faster, were more disease resistant, and had higher protein percentage than full-sib controls. There were sex effects as performance changes were more dramatic in males. The D5D transgenic channel catfish were also more uniform in their fatty acid composition, growth and other traits.
Masu salmon elongase (elovl2) was substituted for D5D in the transgene and transferred 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%) detected and expressed in twelve different tissues of P1 transgenic fish including relatively high expression in liver and muscle, 13.4 and 9.2 fold-change, respectively. 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 that the β-actin-elovl2 transgene improved biosynthesis of LC-PUFA in channel catfish as a whole. Potential off-target gene editing effects were likely the major factor responsible for morphological deformities detected found in positive and negative siblings compared to sham-injected controls.
The evol2 gene was also inserted targeting a non-coding region of chromosome 1 or randomly inserted without CRISPR/Cas9. Integration rates were similar in this experiment and in both cases DHA was increased by about 20%. The benefit of the transgene is affected by diet causing genotype-environment interactions.
A variety of approaches were used to knock in 2-3 omega-3 fatty acid biosynthesis genes, masu salmon elovl2, rabbitfish (Siganus canaliculatus) Δ4 and Δ6 (Δ5) desaturase genes, simultaneously. In some cases (7%), all 3 genes were transferred by microinjecting a cocktail targeting multiple long repeat sequences (LRS) in the genome. For another strategy, 2 or 3 gene tandem arrays driven by a single promoter targeted either LRS or lh gene. This technique gave slightly higher and more consistent transformation with no target effect and double the integration with 2 gene constructs compared to 3 gene constructs. The last strategy involved knockin of C. elegans Δ12 desaturase and Δ15 desaturase transgenes utilizing mRNA and Cas9/gRNA RNP coupled with homology directed repair, microhomology-mediated end-joining non-homologous end joining with preliminary results indicating possible integration rates as high as 70%.