This document summarizes a study that used CRISPR/Cas9 to knockout the elovl2 gene in Atlantic salmon in order to increase their omega-3 fatty acid content. The researchers found that elovl2 knockout inhibited the elongation of polyunsaturated fatty acids like EPA and DHA, leading to higher levels of shorter chain omega-3s. This inhibition also induced the expression of hepatic genes related to lipogenesis. The study demonstrates the key role of elovl2 in salmon fatty acid biosynthesis and suggests its potential as a selective breeding marker to increase the conversion of plant oils to EPA/DHA for aquaculture and human nutrition.

1. Animal Biotechnology: Case Study
Melvin Yong (0336533)
Jason Felix (0336302)
Azim (0336036)
Ashley Lai-Foenander (0335937)

3. Introduction
Fish oils are one of the most consumed dietary
supplements as it’s rich in beneficial omega-3 fatty acids.
Atlantic salmon contains a lot of long chain
polyunsaturated fatty acids (LC-PUFAs), which are a
prerequisite for fish oils, but farmers are struggling to
keep up due to the recent high in demand for fish oils.
Through the use of CRISPR we are going to knock-out
elovl2 genes inorder generate Atlantic salmon with
higher yield of LC-PUFAs

4. Advantages
● Can bind to more sites
● Simpler and easier to use
● Cheaper
● More precise
● Less time-needed
● Higher chance of success rates
● May Induce unwanted/off-target
effects mutation to the organism
● Laboratory testing is needed
● CRISPR/Cas9 approach makes the
identification of unwanted genomic
modifications at the target site very
challenging
Disadvantages

5. Methodology
Microinjection of
embryos
Preparation of gRNA and Cas9 mRNA
Fertilization of eggs in
freshwater
★ gRNA (elovl2 + slc45a2)
★ Cas9 mRNA
Genes of interest:
● fad
● elovl
● fasn
● srebp-1
Screening for
CRISPR-induced
mutations
Feeding trials Sampling of tissue
★ Standard
commercial
diet
★ Low PUFA
diet
★ White muscle,
liver, whole brain
★ Extraction
★ PCR
★ Subcloning
★ Sequencing
Analysis of lipids in tissue
Analysis of lipids in
liver cells
Transcriptional
analysis by RNAseq
Gene expression
analysis
★ Phospholipids
★ Triacylglycerol (TAG)
★ Assay of fatty acyl
desaturation/elongation

6. Results
GROWTH PERFORMANCE
No difference in growth and length
No mortality
CONFIRMATION AND IDENTIFICATION
Three types of KOs were found- Loss of Function (LOF), Splice Site(SS),
In Frame(IN). Of three genes, missplicing of exon 4 and 6 were found
INHIBITION OF PUFA ELONGATION BY KO ELOVL2 OF
SALMON
Increase of 20:5n-3, 22:5n-3 and reduction of 22:6n-3 in
brain, liver and white muscle cells of KO salmon.
Increased 20:4n-6 in phospholipids in brain and white
muscle of low PUFA fed diet.
Increase of 20:4n-6, 20:5n-3 and 22:5n-3 with reduction of
22:6n-3 in white muscle TAGs in KO salmon.
IMPAIRED ENDOGENOUS SYNTHESIS OF 22:6n-3 INDUCES HEPATIC
mRNA EXPRESSION OF SREBP-1 AND TARGET GENES.
Higher expression of fad genes in LC-PUFA pathway in KO salmon.
Higher expression of srebp-1 genes in liver of KO salmon with low
PUFA diet.
Expression of the srebp-1 genes of KO was affected less in standard diet
compared to WT.

7. Discussion
1. Atlantic salmon fed diet containing relatively high levels of 20:5n-3 (7.3% of total fatty acids, FAs) and 22:6n-3
(10.5% of total FAs) showed reduced hepatocyte fatty acyl desaturation and elongation.
1. Current study suggest that fish fed low PUFA and standard commercial diet had active fatty acyl desaturation and
elongation.
1. It is expected that the relatively high levels of 20:5n-3 and 22:6n-3 in the standard diet feedback inhibit desaturation
and elongation, the presence of appreciable levels of 18:2n-6 (10.8% of total FAs) and 18:3n-3 (4.8% of total FAs)
may have had stimulatory effects on PUFA synthesis.
1. Present study highlight in vivo functions of elovl2 in multiple tissues in Atlantic salmon LC-PUFA biosynthesis - key
roles of elovl2 in elongation of 20:4n-6 for synthesis of 22:5n-6 as well as 20:5n-3 and 22:5n-3 in 22:6n-3 synthesis in
vivo.
1. Extent highlights the crucial roles of endogenously synthesized PUFAs in the regulation of hepatic lipogenic genes,
most likely in a Srebp-1-dependent manner. The obvious changes in the levels of LC-PUFAs in our elovl2 KO salmon
coupled with the significant hepatic transcript response, and with the fact that Elovl2 catalyses two important
penultimate steps of PUFA synthesis suggests elovl2 as a potential selective Atlantic salmon breeding marker for
ensuring an increased conversion of C18 fatty acids present in vegetable oils to 20:5n-3 and 22:6n-3.

8. Applications AquacultureHuman health
Enhance
understanding
Molecular
mechanisms
Regulatory
pathways
Nutritional Transcriptional

9. Conclusion
● Key role of elovl2 in synthesis of LC-PUFA
● Endogenously synthesized PUFAs is crucial in the regulation of
hepatic lipogenic genes
● elovl2 will be effective as a selectable marker in Atlantic
salmon breeding to get higher conversion of vegetable oil into
LC-PUFA

10. References
❖ Datsomor, A., Zic, N., Li, K., Olsen, R., Jin, Y., Vik, J., Edvardsen, R., Grammes, F., Wargelius, A. and
Winge, P. (2019). 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. Scientific
Reports, 9(1).
❖ Taconic.com. (2019). CRISPR Genome Engineering: Advantages and Limitations. [online] Available
at: https://www.taconic.com/taconic-insights/model-generation-solutions/crispr-genome-
engineering-advantages-limitations.html [Accessed 15 Oct. 2019].