Optimization of the CRISPR/Cas9 knock-in technology and application in salmon and trout

The primary aim of the project is to optimize the CRISPR/Cas9 knock-in technology in zebrafish and establish the protocol for salmon and trout. The secondary aim is to utilize these tools to explain mechanisms behind some key biological features in fish such as time of maturity, pigmentation, immune functions, sex determination, reproduction and egg quality. The main objectives to accomplish the aim will be 1.) Improvement of the knock-in protocol in zebrafish (primarily) and Medaka by testing different designs of donor DNA constructs and Cas9 proteins 2.) Implementing the best practices for knock-in from zebrafish and Medaka to salmon and trout 3.) Utlizing knock-in of putative important genetic variants in salmon and trout. Our hypothesis is that the enhanced CRISPR methodology will be transferable from the model species to farmed species, enabling functional studies of key life history traits, which will ultimately lead to genetically improved aquaculture fish. Our results show that it is fully possible to achieve efficient knock-in in salmon using protocols based on work in zebrafish and medaka. We achieve higher efficiency than anyone else has shown in fish and our analyzes provide information on how CRISPR knock-in experiments can also be optimized in other species. Results from this will generate a larger knowledge base for aquaculture and also explore whether some of these traits may be transferable to farming of salmonids.

Resultatene i dette prosjektet har klart en svært stor innvirkning på flere forskningsfelt. Vår data viser at man kan gjøre knock-in med høy effektivitet og ikke bare knock-out. For det første åpner denne utvidete verktøykassen for genredigering som vi har utviklet for laks mange muligheter som ikke til stede før dette prosjektet. For grunnforskning kan dette brukes til å studere sykdom, kjønnsmodning og annet. I tillegg kan våre metoder brukes i andre arter. Også for næringsliv og samfunnet kan dette få stor verdi. Muliggjøring av funksjonelle studier av viktige livshistorietrekk vil kunne gi forståelse som er viktig for oppdrett, men dette viser også at det er mulig bruke genredigering direkte for å oppnå fisk med endrete egenskaper som kan gi både mer bærekraftig og lønnsom oppdrettsfisk.

Recent biotechnological innovations currently allow the development of new approaches to apply genetic engineering to non-model organisms, including economically important salmonid species. This has been mediated by the introduction of the highly efficient and specific CRISPR-Cas9 methodology. In recent years several studies have revealed that single SNPs in the genomes of salmonids can explain important traits such as time of maturity and disease resistance. Based on these findings further studies need to aim at elucidating how single nucleotide exchanges can alter important traits for aquaculture such as growth, reproduction and disease resistance. Hence, there is a need to develop technologies that can precisely alter single nucleotides in the genome. This can be obtained by knock in- by a combination of gene editing and homology-directed repair as previously done in zebrafish. So far knock out by gene editing has been established in both rainbow trout and Atlantic salmon. Both species of fish have a long generation time, therefore it will be necessary to perform double allelic knock in by homologous recombination already in the F0. We have successfully established a methodology using pigmentation as a tracer for double allelic mutations in Atlantic salmon, this methodology can be further explored for knocking in traits. The project will therefore focus on establishing an efficient knock in technology in salmon and rainbow trout. This will be done in combination with exploring the technology further in zebrafish and medaka since testing out technologies is much faster in these model fish species. By doing so, we will focus our technology development on genes essential for, sex determination, reproduction and egg quality since our groups have been exploring these fields for a long time and results produced can in addition to providing technological improvements explain mechanisms behind some key biological features in fish and other species.