Dr Yehwa Jin updates us on her genome editing work as part of the AquaLeap project. 3/12/2021 The AquaLeap consortium project is comprised of five teams, which combine university, government, and industry scientists. Each team pursues objectives related to advancing selective breeding in several UK aquaculture species. Since it began in January of 2019, some exciting progress has been made, and we are happy to provide some further updates. Dr Yehwa Jin, a Postdoctoral Research Fellow at the Roslin Institute, has been closely involved in AquaLeap since its inception. Following on from the report she provided for the project in 2020, she has written about her ongoing research into salmonids. Improving CRISPR genome editing in salmon cell lines and embryos Report by Yehwa Jin (University of Edinburgh) In the AquaLeap project, one of the goals of the University of Edinburgh team and their partners is to improve the efficiency of genome editing in Atlantic salmon, and to use these techniques to study and improve disease resistance. In the past year this has included two main developments: Improving the efficiency of in vivo genome editing in salmon The most widely used genome editing method in salmon is to inject Cas9 mRNA and guide RNA (gRNA) at 1 cell stage embryos. However, the editing frequency can be relatively low, with perhaps only 20-25% of injected animals carrying successful target edits. We have been working on optimising in vivo genome editing in salmon by injecting different molecular forms of Cas9 (mRNA and protein) at different cleavage stages to enhance both editing efficiency in individual injected animal and editing frequency in injected populations. We tested different injection time-points from 1 cell stage to 16 cell stage with Cas9 mRNA-gRNA. In addition, efficacy of Cas9 protein-gRNA ribonucleoprotein (Cas9 RNP) was compared to Cas9 mRNA-gRNA injection at cell stages 2 and 4. Interestingly, Injection of Cas9 RNP resulted in significantly higher editing frequency than Cas9 mRNA-gRNA at early cleavage stage, implying that poor translation of Cas9 mRNA at the early cleavage stage may be a limiting factor for the low editing frequency in salmon. Image With the optimised settings, we can achieve > 90% editing efficiency in > 80% of injected embryos, which is greatly improved compared to the previous editing methods used. We are currently investigating the timing of editing events in salmon embryos and the difference in the level of mosaicism by injection timing. Validation of functional genes underlying disease resistance in salmon To validate and identify functional variants underlying resistance to Infectious pancreatic necrosis virus (IPNV) in salmon we used the optimised in vitro and in vivo genome editing platforms. We successfully edited two high priority candidate gene targets in salmonid cell lines and investigated the impact of the targeted knockout on the viral infection in vitro. The results have been published in the journal Genomics. Based on the cell line results, we have tested editing efficiency of editing selected targets in vivo. You can access the Genomics paper detailing our study via this external link. In addition, we also have been testing primary embryonic cell culture using salmon hatchlings as a new tool to validate the impact of in vivo edits on disease resistance. In salmon primary embryonic cells, gene editing using Cas9 RNP electroporation was successful, showing > 90% editing efficiency, and the first trial of lentiviral delivery of antibiotic resistance genes worked well conferring the resistance to the antibiotics. The usage of primary embryonic cells may be beneficial as a model and reduce the number of animals used for disease challenges. More information about the AquaLeap team is available through this report on its 2021 annual meeting. Reports from earlier in the AquaLeap project are accessible via the AquaLeap 2020 Newsletter Readers can find recent news and updates on the AquaLeap consortium project through its Twitter account, available via this external link.
