One of major problems facing sustainable aquaculture is infectious disease outbreaks, which can result in significant mortality and animal welfare issues. Disease resistance can be improved by selective breeding, but genetic gain can be relatively slow for species with long generation time, and is limited to the existing genetic variation in the population. Combining genomic analyses and genome editing tools such as CRISPR-Cas9 has potential to accelerate the process by identifying causative genes and variants, or by transferring disease resistance alleles between populations or even species1,2.
Our typical workflow begins with high-throughput genomics research leading to potential targets underlying intra- or inter-specific genetic variation in disease resistance. The function of target genes is then evaluated in cell lines using CRISPR-Cas9 mediate targeted genome editing, which has now been optimised in various salmonid cell lines3. These shortlisted candidates are evaluable in vivo by microinjecting CRISPR-Cas9 molecules into zygotes, followed by disease challenge experiments to assess phenotypes.
One recent example of combining genomic studies and genome editing to identify a causative gene is elucidation of NEDD-8 activating enzyme 1 (nae1) gene as a causative gene underlying the major QTL affecting resistance to IPNV in Atlantic salmon4. In this study, whole genome sequencing and functional annotation approaches characterised genes and variants in the QTL region. In addition, differential expression analysis between homozygous resistant and susceptible salmon fry challenged with IPNVs pointed to nae1 as a putative causative gene underlying the QTL effect. The function of nae1 in IPNV resistance was evaluated via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the Nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication, indicating that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon.
Our research is currently focussed on applying similar technologies to tackle sea lice and other infectious diseases affecting salmonids. The sea lice projects consist of cross-species genomics comparison approaches between salmonid species with differential susceptibility to sea lice to identify genomic features, followed by genome editing to precisely transfer the resistant alleles between species2. Other foci include development of CRISPR-Cas9 mediated genome-wide screens to identify de novo resistance alleles, and optimising methods of in vivo editing to improve editing efficiency and reduce mosaicism and routes to application, including via surrogate broodstock5. Depending on acceptable regulatory and public perceptions, genome editing technology holds the potential to develop disease-resistant fish strains with significant downstream positive impact on aquaculture sustainability and animal health.
1. Gratacap, R. L., Wargelius, A., Edvardsen, R. B. & Houston, R. D. Potential of Genome Editing to Improve Aquaculture Breeding and Production. Trends Genet. 35, 672–684 (2019).
2. Houston, R. D. et al. Harnessing genomics to fast-track genetic improvement in aquaculture. Nat. Rev. Genet. 21, 389–409 (2020).
3. Gratacap, R. L., Jin, Y. H., Mantsopoulou, M. & Houston, R. D. Efficient genome editing in multiple salmonid cell lines using ribonucleoprotein complexes. Mar. Biotechnol. 22, 717–724 (2020).
4. Pavelin, J. et al. The nedd-8 activating enzyme gene underlies genetic resistance to infectious pancreatic necrosis virus in Atlantic salmon. Genomics 113, 3842–3850 (2021).
5. Jin, Y. H., Robledo, D., Hickey, J. M., McGrew, M. J. & Houston, R. D. Surrogate broodstock to enhance biotechnology research and applications in aquaculture. Biotechnol. Adv. 49, 107756 (2021).