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1. Application of genome editing 8:
Farm animals in aquatic systems
Anna Troedsson-Wargelius
Institute of Marine Research, Bergen, Norway

3. Aquaculture environments for fish and shellfish
Ottinger et al 2016

4. Ottinger et al 2016
Global aquaculture production

5. Norwegian aquaculture of Atlantic salmon
• Norway is the world’s largest producer of Atlantic salmon
• > 1.2 million metric tonnes in 2012- 2016
Norwegian farming of salmon, 1980-2015
What are the current challenges in salmon farming?
Rainbow trout
Atlantic salmon

6. -Environmental impact
Disease
Genetic introgression of
farmed fish into wild populations
-Feed resources
Sustainability issues

7. Atlantic Salmon genome
Lien et al 2016
Ayllon et al 2015
Kjærner- Semb et al 2016

8. Establishment of CRISPR/Cas9 methodology in
Atlantic salmon
Edvardsen et al., Plos One (2014)
Injection of guide RNA + Cas9 in newly fertilized eggs
Targeting a pigmentation gene: slc45a2 (albino)
Successful editing = albinosSuccessful editing = F0 albinos
Likewise the technology has been established in other aquaculture species.:
Tilapia (Wang et al 2014)
Cat fish (Zhong et al 2016)
Carp (Khalil et al 2017)

9. Generation time in salmon 3-4 years

10. CRISPR/Cas9
Research tool and possible industrial applications
in salmon aquaculture
- Genetic containment of wild populations
- Filet quality
- Disease resistance
- Move traits between strains

11. Environmental concerns
- Disease
- Genetic introgression of farmed escapees into wild populations
- Feed resources

12. Triploid sterile salmon
Genetic containment
- Produced by pressure treatment of eggs
- One extra set of chromosomes  sterile
- Sensitive to warmer temperatures and low oxygen
- More likely to develop skeletal deformities and cataract
- Males og through puberty
- Triplodization does not always result in 100% sterile fish batches
- Prone to get bacterial disease

13. Sterile salmon through germ cell ablation
Genetic containment
1) Broodstock/eggs:
Inhibit germ cell formation
2) Juveniles:
Induce apoptosis of germ cells

14. Sterile salmon through germ cell ablation
Genetic containment
χ

15. Germ cell-free (sterile) dnd-knockout
dnd + albino Knockout female Control female
Genetic containment Wargelius et al., Sci. Rep. (2016)

16. Germ cell-free salmon – what next?
Genetic containment Kleppe et al., Sci. Rep. (2017)
Ongoing: Long-term (2017-2020) growth experiment
~80 GCF and ~80 controls
-Common garden
-Induced early puberty
-Measured growth and sex steroid levels
-no puberty
-normal growth

17. Environmental concerns
- Disease
- Genetic introgression of farmed escapees into wild populations
- Feed resources

18. Omega-3 synthesis
Filet quality
EPA
DHA
Control
elovl2 + albino knockout
Datsmoor, Winge, in prep.

19. 0
5
10
15
20
25
30
35
40
C18:3n3 C18:2n6 C20:2n6 C20:4n6 C20:5n3 C22:5n3 C22:6n3
%offattyacidsfromphospholipidsinthe
liver
Wildtype
Elovl2 knockout
C20:5n3 C22:5n3 C22:6n3
p-values 0,0043 0,027 0,031
Filet quality Datsmoor, Winge, in prep.
Effects of elovl2 knockout on DHA synthesis in the
liver under standard diet

20. Environmental concerns
- Disease
- Genetic introgression of farmed escapees into wild populations
- Feed resources Number of wild salmon that
returns to rivers to breed

21. Natura Disease resistance in wild strains
Kjærner-Semb et al 2016, BMC genomicsMove traits between strains

22. Gene editing by CRISPR/Cas9
NHEJ=
Non-Homologous End Joining
HDR=
Homology Directed Recombination

23. Region under selection for time of maturation
Move traits between strains Barson et al., Nature (2015)
Ayllon et al., Plos Genetics (2015)

24. Ayllon et al., Plos Genetics (2015)Move traits between strains
SNPs explaining the trait in farmed fish

25. Straume, Edvardsen, Unpubl. res.
Move minor genetic elements between salmon
populations
Project:
AquaCrispr

26. Responsible Research Innovation (RRI)
- Interactions with public, presentations, media debates
- Ethics and integrity
-Local highschool integration
(Nordahl Grieg og Amalie Skram)

27. Summary – Gene editing in aquaculture
- Targeted mutagenesis using Crispr-Cas9 is established
in a number of farmed fish species
- F0 mutant studies are enabled
- Homologous recombination of single causative SNPs is being explored
(Disease resistance, control of reproduction, nutritional content, fish robustness)
- Mutants representing sustainable traits are being studied
(Germ cell-free fish, altered omega-3 metabolism, time of maturation etc.)

28. Reproduction and
Developmental biology group
at IMR
Anna Wargelius
Eva Andersson
Sven Leininger
Kai Ove Skaftnesmo
Geir Lasse Taranger
Erik Kjærner Semb
Tomasz Furmanek
Per Gunnar Fjelldal
Thomas Fraser
Tom Hansen
Birgitta Norberg
Hilal Güralp
Anne Hege Straume
Rolf Edvardsen
Population genetics group
at IMR
Kevin Glover
Fernando Ayllon
Vidar Wennevik
Calle Rubin
Dorothy Dankel
Alex Datsomoor
Per Winge
Nina Santi
Maren Mommens
Helge Tveiten
Øivind Andersen
Rüdiger W. Schulz
Hildre Sindre
Acknowledgements
Funding:
-Norwegian Research Council
BIOTEK2021 project SALMOSTERILE
HAVBRUK_BIOTEK2021 project SALMAT
HAVBRUK project STERWELL and MATGEN
-EU
AQUACRISPR

29. Thank your for the attention!