Targeted Mutagenesis in Atlantic Salmon (Salmo salar L.) Using the CRISPR/Cas9 System Induces Complete Knockout Individuals in the F0 Generation

Abstract

Understanding the biological function behind key proteins is of great concern in Atlantic salmon, both due to a high commercial importance and an interesting life history. Until recently, functional studies in salmonids appeared to be difficult. However, the recent discovery of targeted mutagenesis using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system enables performing functional studies in Atlantic salmon to a great extent. We used the CRISPR/Cas9 system to target two genes involved in pigmentation, tyrosinase (tyr) and solute carrier family 45, member 2 (slc45a2). Embryos were assayed for mutation rates at the 17 somite stage, where 40 and 22% of all injected embryos showed a high degree of mutation induction for slc45a2 and tyr, respectively. At hatching this mutation frequency was also visible for both targeted genes, displaying a graded phenotype ranging from complete lack of pigmentation to partial loss and normal pigmentation. CRISPRslc45a2/Cas9 injected embryos showing a complete lack of pigmentation or just a few spots of pigments also lacked wild type sequences when assaying more than 80 (slc45a2) sequence clones from whole embryos. This indicates that CRISPR/Cas9 can induce double-allelic knockout in the F0 generation. However, types and frequency of indels might affect the phenotype. Therefore, the variation of indels was assayed in the graded pigmentation phenotypes produced by CRISPR/Cas9-slc45a2. The results show a tendency for fewer types of indels formed in juveniles completely lacking pigmentation compared to juveniles displaying partial pigmentation. Another interesting observation was a high degree of the same indel type in different juveniles. This study shows for the first time successful use of the CRISPR/Cas9 technology in a marine cold water species. Targeted double-allelic mutations were obtained and, though the level of mosaicism has to be considered, we demonstrate that F0 fish can be used for functional studies in Atlantic salmon.

Approaches for delaying sexual maturation in salmon and their possible ecological and ethical implications

“…Detailed knowledge on the maturation process is continuously gained by researchers, and could in combination with technological advances (Edvardsen et al, 2014, Wargelius et al, 2016 lead to efficient ways for postponing or even blocking maturation in salmon.…”

“…DNA markers and genetic modification opens up vast possibilities for genetic changes in aquaculture species (Edvardsen et al, 2014, Fjalestad et al, 2003, Hayes and Andersen, 2005. Marker assisted selection (MAS) is a genotype based method for selecting brood stock.…”

“…This encompasses modifying growth rate, feed utilization, disease resistance, stress tolerance, and maturation processes, thereby enhancing the quality and cost effectiveness of the farmed salmon. It can also lead to developing new products through alterations of the salmon genome (Edvardsen et al, 2014, Melamed et al, 2002.…”

“…The possibility to alter or knockout the function of genes in salmon has been enabled through the establishment of targeted mutagenesis technology (such as zinc-finger, TALENs and CRISPR/Cas technologies) in fish (Edvardsen et al, 2014, Meng et al, 2008, Doyon et al, 2008, Huang et al, 2011, Hwang et al, 2013. This technology allows for small changes in the salmon genome and will be a valuable research tool for investigating important production traits, such as precocious maturation, disease resistance, and immune parameters, among others.…”

“…However, genetic engineering technology is advancing quickly. Increased understanding of processes and improved specificity could eventually produce fish with less off-target effects (Edvardsen et al, 2014). It is important when genetically modifying one trait, such as timing of maturation, that the overall performance and welfare of the fish is evaluated in case of unforeseen and negative effects.…”