Prime editing in zebrafish and Atlantic salmon : exploring a new CRISPR technique in non-mammalian species

• Main Author: Bryhni, Jenny Heyerdahl
• Other Authors: Sandvik, Guro Katrine, Young, Darshan
• Format: Master Thesis
• Language: English
• Published: Norwegian University of Life Sciences, Ås 2022
• Subjects: Kjølvannet; Atlantic salmon; Salmo salar
• Online Access: https://hdl.handle.net/11250/3028110

The CRISPR-Cas system has enabled precise genome editing in a multitude of organisms, and gene knockout experiments can now be done with relative ease due to the simplicity of the system. However, unintended genomic side-effects of such editing have surfaced in the last couple of years. In this thesis, the CRISPR-based genome editing system prime editing was explored as an alternative gene knockout approach to avoid the unintended editing outcomes seen in the wake of regular CRISPR-Cas editing. Prime editing was applied to zebrafish (Danio rerio) by microinjection of RNP complexes and to Atlantic salmon (Salmo salar) cells by electroporation of plasmids and RNP complexes. No successful prime editing outcomes was observed in zebrafish. Low survival rate of both microinjected and uninjected embryos remained an obstacle throughout the project, and should be addressed before continuing zebrafish prime editing experiments. Prime editing of Atlantic salmon cells using plasmids did not produce any positive results. Prime editing with RNP complexes in Atlantic salmon cells showed signs of successful editing, but results remains inconclusive as further analysis is needed to properly validate the editing outcomes from this experiment. CRISPR-Cas har muliggjort presis redigering av genomer i en rekke organsimer, og kan brukes til å relativt enkelt slå ut gener for ulike formål. Til tross for at det er en presis og effektiv metode for genomredigering, har utilsiktede konsekvenser av bruk av CRISPRCas9 kommet til overflaten de siste årene. I denne oppgaven ble det CRISPR-baserte genomredigeringsverktøyet prime editing utforsket, for å se om det kan fungere som en alternativ metode for å slå ut gener og unngå de utilsiktede konsevensene som kan oppstå i kjølvannet av vanlig CRISPR-Casredigering. Prime editing ble brukt i zebrafisk (Danio rerio) ved mikroinjesering av RNP-komplekser og i lakseceller (Salmo salar), ved elektroporering av plasmider og RNP-komplekser. Bruk av prime editing i zebrafisk ga ingen positive ...