CRISPR/Cas9-mediated ablation of elovl2 in Atlantic salmon (Salmo salar L.) inhibits elongation of polyunsaturated fatty acids and induces Srebp-1 and target genes

Abstract

Atlantic salmon can synthesize polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (20:5n-3), arachidonic acid (20:4n-6) and docosahexaenoic acid (22:6n-3) via activities of very long chain fatty acyl elongases (Elovls) and fatty acyl desaturases (Fads), albeit to a limited degree. Understanding molecular mechanisms of PUFA biosynthesis and regulation is a pre-requisite for sustainable use of vegetable oils in aquafeeds as current sources of fish oils are unable to meet increasing demands for omega-3 PUFAs. By generating CRISPR-mediated elovl2 partial knockout (KO), we have shown that elovl2 is crucial for multi-tissue synthesis of 22:6n-3 in vivo and that endogenously synthesized PUFAs are important for transcriptional regulation of lipogenic genes in Atlantic salmon. The elovl2 -KOs showed reduced levels of 22:6n-3 and accumulation of 20:5n-3 and docosapentaenoic acid (22:5n-3) in the liver, brain and white muscle, suggesting inhibition of elongation. Additionally, elovl2 -KO salmon showed accumulation of 20:4n-6 in brain and white muscle. The impaired synthesis of 22:6n-3 induced hepatic expression of sterol regulatory element binding protein-1 ( srebp-1 ), fatty acid synthase-b, Δ6fad-a , Δ5fad and elovl5 . Our study demonstrates key roles of elovl2 at two penultimate steps of PUFA synthesis in vivo and suggests Srebp-1 as a main regulator of endogenous PUFA synthesis in Atlantic salmon.

A perspective of the future value and challenges of genetic engineering in aquaculture

“…Gene editing technology utilizing CRISPR/Cas9 has been successfully performed in several aquaculture species, including Atlantic salmon, rainbow trout, catfish, tilapia, and carp (Chakrapani et al, 2016;Cleveland, Yamaguchi, Radler, & Shimizu, 2018;Datsomor et al, 2019;Khalil et al, 2017;Li et al, 2014;Osmond & Colombo, 2019;Wargelius et al, 2016;Zhong et al, 2016). Traits known to be significantly regulated by a single gene present themselves as obvious targets for gene editing as some production traits can be predictably improved by simply targeting/disrupting one gene.…”

Targeted mutagenesis of Δ5 and Δ6 fatty acyl desaturases induce multiplex-mutagenesis and lipogenesis in Atlantic salmon (Salmo salar)

“…It is difficult to conclude the reason for the differential expression of lipid metabolism genes since plant oils are devoid of cholesterol and LC-HUFA and contains high amount of C 18 PUFA precursors and phytosterols compared to fish oil [14–16]. In a recent study we disrupted LC-HUFA synthesis pathway in salmon by mutating elovl2 gene using CRISPR/Cas9 technology [17]. In addition to the decreased DHA content in mutant fish, we have identified up-regulation of fads2 genes as well as several genes involved in fatty acid biosynthesis and lipogenesis [17].…”

“…In a recent study we disrupted LC-HUFA synthesis pathway in salmon by mutating elovl2 gene using CRISPR/Cas9 technology [17]. In addition to the decreased DHA content in mutant fish, we have identified up-regulation of fads2 genes as well as several genes involved in fatty acid biosynthesis and lipogenesis [17]. This suggests a systemic change of lipid metabolism regulation in response to the disruption of LC-HUFA synthesis in salmon.…”

“…CRISPR/Cas9 technology has recently been used in salmon to edit genes and generate mutants [17–20]. Both guide RNA (gRNA) and Cas9 mRNA are injected to one-cell stage salmon embryos to induce a targeted double-strand break, followed by non-homologous end joining (NHEJ) which generates random insertions and deletions (indels) at the target sites that can leads to a truncated protein.…”

“…The studies of CRISPR/Cas9 edited salmon have so far been done on the F0 generation because the fish has a long maturation period of 2-4 years. As a mosaic pattern of mutations forms in F0 crispant salmon, which translates into multiple alleles in the fish [17].…”