Improving resistance to infectious salmon anaemia using genome editing: Novel approaches to tackling viral disease in aquaculture

Lead Research Organisation: University of Aberdeen
Department Name: Inst of Biological and Environmental Sci

Abstract

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Technical Summary

Salmon aquaculture is worth approximately £1 billion per annum to the UK economy and is the primary source of employment in many rural and coastal communities. Infectious disease epidemics constrain sustainability and expansion, and cause major negative economic and animal welfare impact. Infectious Salmon Anaemia Virus (ISAV) is a notifiable disease that requires culling of stocks for control. Improving genetic resistance of salmon stocks is a critical component of tackling ISAV. Genome editing raises the possibility of creating targeted and informed de novo disease resistance alleles (e.g. CD163 deletions for PRRS virus in pigs), and to test the causality of naturally-occurring variants, and potentially increase genetic gain in breeding programs. ISAV is a close relative of influenza, and knowledge of the conserved function of viral binding, internalisation and replication across orthomyxoviridae is one route to identifying target genes for editing.

This proposal will use forward and reverse genetics to identify genome editing targets for resistance to ISAV in Atlantic salmon. Harnessing large scale disease challenge and pedigree data provided by the industrial partner Benchmark, the team will map and characterise genes underpinning host resistance. Genome editing will be applied in cell culture to screen putative functional resistance candidate genes, with targets including 'naturally-occurring' variants, and de novo targets with a predicted key role in the ISAV host-cell interaction. These approaches will deliver high priority candidates for in vivo genome editing of salmon embryos, followed by an ISAV challenge to assess the resistance of the edited fish. The outcomes will include fundamental knowledge of mechanisms of genetic resistance to ISAV, and potential novel strategies to combat viral diseases impacting on salmon aquaculture. As such, it is aligned to the BBSRC priorities on 'Sustainably enhancing agricultural production' and 'Animal health'.

Planned Impact

This proposal is applying novel and innovative methods with potential to develop an Atlantic salmon with resistance to ISAV as a result of genome editing. This outcome, and the research that leads to it, is likely to have high impact for academic, industry, government and societal groups.

It is likely that the major impact of the proposal will be medium and long term, due to uncertainty over the regulatory landscape and public appetite for genome editing technologies. However, high profile success stories are key, and this proposal has high potential to deliver such success stories. Use of genome editing to produce a disease resistant food fish has animal welfare, environmental and economic benefits.

It should also be noted that intermediate impacts of the research will include improved methods of genomic selection for disease resistance in aquaculture breeding, improved knowledge of host-pathogen interaction for ISAV, and optimised methods of using CRISPR-Cas9 for animal bioscience research. The following groups can expect positive impact from the proposal:

(i) UK and global aquaculture industry: ISAV has a major negative impact on aquaculture production, and is a notifiable disease in the UK. This proposal has potential for developing disease-resistant strains of salmon within a relatively short timeframe, and major breeding companies such as Benchmark view genome editing as a high priority R&D activity. While the regulatory landscape for genome editing is uncertain at present, achievement of the aims of this proposal is likely to be a driver to incorporate this technology to benefit food production.

(ii) Animal breeding industry: The improved genomic selection arising from the identification of functional genomic variants will have short term impact on selective breeding programmes. In the longer term, aquaculture species such as salmon are amenable to genome editing due to their high fecundity and easily visible embryos, and may be a good model for other species.

(iii) UK economy: This project has potential for long term impact for the UK economy via improved sustainable production of a high quality food product with reduced environmental impact. There will be direct contribution to the UK treasury via improved market share for the project partner Benchmark, and downstream impacts on fish farming companies, and the communities that depend on these industries.

(iv) Academic community: The impact for academic groups will include improved knowledge of the genetic basis of resistance to ISAV in salmon, specific genes and mutations impacting resistance, and improvement in methods of effective genome editing of cell lines and embryos.

(v) UK science capacity. This project will enable capacity and expertise for application of genome editing technology to address (applied) biological questions via research programs in academia and industry. This should lead to the UK taking a leading global position in the application of this technology for animal research and production.

(vi) Political and regulatory bodies. The regulatory landscape of genome editing currently precludes its use for food production in the short term. Successful achievement of the objectives of this project will influence ethical and regulatory frameworks to encourage full exploitation of genome editing in an informed and responsible manner.

(vii) General public and society. This project has potential to influence societal attitudes to genome editing in aquaculture and, more broadly, in food production animals. In the longer term, there will be direct benefits to society via improved economic stability and reduced environmental impact of the aquaculture industry.
 
Title Additional file 1 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 1 Figure S1. Puromycin can be used to select for resistant cells. CHSE-EC cells were treated for 7 days with different concentrations of Puromycin and the survival was calculated by CellTiter-Glo. A concentration of 0.25 µg/mL of puromycin was found to be the minimal concentration to efficiently kill all non-antibiotic-resistant cells. 
Type Of Art Film/Video/Animation 
Year Produced 2020 
URL https://springernature.figshare.com/articles/Additional_file_1_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Additional file 1 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 1 Figure S1. Puromycin can be used to select for resistant cells. CHSE-EC cells were treated for 7 days with different concentrations of Puromycin and the survival was calculated by CellTiter-Glo. A concentration of 0.25 µg/mL of puromycin was found to be the minimal concentration to efficiently kill all non-antibiotic-resistant cells. 
Type Of Art Film/Video/Animation 
Year Produced 2020 
URL https://springernature.figshare.com/articles/Additional_file_1_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Additional file 2 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 2 Figure S2. Editing efficiency estimation, The analysis of the editing of pooled cell population samples using ICE online software. a. The chromatograms (.ab1 file) from the control (non-edited) and edited samples, along with the gRNA sequence are uploaded on ice.sythego.com. b. The platform verifies that the cut site corresponds to the start of the mixed population chromatogram and deconvolutes the picks to original sequences + or - a few bases. c. The results are presented as the percentage of each edited sample present in the pooled population contributing to the mixed chromatogram. 
Type Of Art Film/Video/Animation 
Year Produced 2020 
URL https://springernature.figshare.com/articles/Additional_file_2_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Additional file 2 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 2 Figure S2. Editing efficiency estimation, The analysis of the editing of pooled cell population samples using ICE online software. a. The chromatograms (.ab1 file) from the control (non-edited) and edited samples, along with the gRNA sequence are uploaded on ice.sythego.com. b. The platform verifies that the cut site corresponds to the start of the mixed population chromatogram and deconvolutes the picks to original sequences + or - a few bases. c. The results are presented as the percentage of each edited sample present in the pooled population contributing to the mixed chromatogram. 
Type Of Art Film/Video/Animation 
Year Produced 2020 
URL https://springernature.figshare.com/articles/Additional_file_2_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Additional file 3 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 3 Figure S3. Off-target editing. Off-target evaluation of RIG-I editing. a: The sequence of the top 2 off-target sites are represented along the gRNA sequence used for targeting RIG-I (red box in the middle). Blue letters indicate differences with the original sequence. An additional nucleotide was sequenced in the CHSE-EC cell line, not present in the published sequence (Otsh_v1.0, green box). b: Diagram representing the editing efficiency in the off-target regions (OffT1: ch7:73101728-73,102,398 and OffT2: ch14:42341999-42,342,859). No off-target was detected by Sanger sequencing in either sample (Puro- and Puro+; all sequences, including CHSE-EC (wt) were compared to CHSEwt). c: Representative chromatogram from the sequencing of off-target region 1 (OffT1) in CHSEwt (Control sample, bottom track) and CHSE-EC-RIG-I Puro + (Edited Sample, top track). 
Type Of Art Film/Video/Animation 
Year Produced 2020 
URL https://springernature.figshare.com/articles/Additional_file_3_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Additional file 3 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 3 Figure S3. Off-target editing. Off-target evaluation of RIG-I editing. a: The sequence of the top 2 off-target sites are represented along the gRNA sequence used for targeting RIG-I (red box in the middle). Blue letters indicate differences with the original sequence. An additional nucleotide was sequenced in the CHSE-EC cell line, not present in the published sequence (Otsh_v1.0, green box). b: Diagram representing the editing efficiency in the off-target regions (OffT1: ch7:73101728-73,102,398 and OffT2: ch14:42341999-42,342,859). No off-target was detected by Sanger sequencing in either sample (Puro- and Puro+; all sequences, including CHSE-EC (wt) were compared to CHSEwt). c: Representative chromatogram from the sequencing of off-target region 1 (OffT1) in CHSEwt (Control sample, bottom track) and CHSE-EC-RIG-I Puro + (Edited Sample, top track). 
Type Of Art Film/Video/Animation 
Year Produced 2020 
URL https://springernature.figshare.com/articles/Additional_file_3_of_Efficient_CRISPR_Cas9_genome_editi...
 
Description Background: Infectious Salmonid Anaemia Virus (ISAV) causes a notifiable disease that poses a large threat for Atlantic salmon (Salmo salar) aquaculture worldwide. There is no fully effective treatment or vaccine, and therefore selective breeding to increase resistance to ISAV is a promising avenue for disease prevention. Genomic selection and potentially genome editing can be applied to enhance host resistance, and these approaches benefit from improved knowledge of the genetic and functional basis of the target trait. The aim of this study was to characterise the genetic architecture of resistance to ISAV in a commercial Atlantic salmon population and study its underlying functional genomic basis using RNA Sequencing. To identify potential genes that infer resistance a challenge was carried out on 194 salmon families.
A genome-wide association analysis confirmed that resistance to ISAV was a polygenic trait, albeit a genomic region in chromosome 13 was significantly associated with resistance and explained 3% of the genetic variance. RNA sequencing of the heart of 16 infected (7 and 14 days post infection) and 8 control fish highlighted 4,927 and 2,437 differentially expressed genes at 7 and 14 days post infection respectively. The complement and coagulation pathway was down-regulated in infected fish, while several metabolic pathways were up-regulated. The interferon pathway showed no up-regulation at 7 days post infection and was mildly activated at 14 days, suggesting a crosstalk between host and virus. These genes are being used as candidates for gene editing. Other targets basded on viral literature have been identified and are in process of being edited in salmon cells.
Exploitation Route Tools for gene editing in salmonid fish and salmon cell lines are being further developed and will be then able to be used by other research groups and industry to use these techniques. Information is also leading to new knowledge about viral responses in fish and cell lines giving more information about how viruses can manipulate the host antiviral response.
Sectors Agriculture

Food and Drink

 
Description AQUA-FAANG Horizon 2020 Framework Programme
Amount € 6,000,000 (EUR)
Funding ID 817923 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 04/2019 
End 04/2023
 
Title RNAseq 
Description We have used RNAseq to examine the differences in gene expression during early life history changes in Atlantic salmon. We have preformed RNAseq on both pituitary and hypothalamus from salmon smolts. Analysis for this is still underway. 
Type Of Material Biological samples 
Provided To Others? No  
Impact We have been developing a comprehensive Atlantic salmon transcriptome which will be of use for others working in the field, but it is not fully completed. 
 
Title Additional file 4 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 4 Table S1. Predicted Off-target sites. List of all predicted off-targets from CRISPOR website for 354 bp in RIG-I exon 2 (Tab1). The selected gRNA is labeled 224 forw and the list of off-targets can be found lines 2127-2240. Tab2 summarises the predicted off-targets for gRNA 224 forw. The sum of the two off-target scores (MIT and CDF) were calculated to rank the results. The 2 targets selected are highlighted. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
URL https://springernature.figshare.com/articles/Additional_file_4_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Additional file 4 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 4 Table S1. Predicted Off-target sites. List of all predicted off-targets from CRISPOR website for 354 bp in RIG-I exon 2 (Tab1). The selected gRNA is labeled 224 forw and the list of off-targets can be found lines 2127-2240. Tab2 summarises the predicted off-targets for gRNA 224 forw. The sum of the two off-target scores (MIT and CDF) were calculated to rank the results. The 2 targets selected are highlighted. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
URL https://springernature.figshare.com/articles/Additional_file_4_of_Efficient_CRISPR_Cas9_genome_editi...
 
Description INRA, Fish Virology and Immunology. Dr Collet and Dr Boudinot 
Organisation French National Institute of Agricultural Research
Department INRA Versailles
Country France 
Sector Academic/University 
PI Contribution The Lab in INRA is also working on Gene editing in fish cell lines.
Collaborator Contribution Exchange of knowledge and approaches for gene editing
Impact None as yet
Start Year 2017
 
Description Salmon genomics 
Organisation University of Victoria
Department Department of Biology
Country Canada 
Sector Academic/University 
PI Contribution Prof Ben Koop is a leading fish genomics expert and is a partner on the BBSRC / Canada partnering award.
Collaborator Contribution Significant interactions in relation to transcriptomics and genomics in salmon
Impact The outputs have been a working white paper in BMC Genomics highlighting the next stages of functional annotation of the salmonid genomes.
Start Year 2017
 
Description Phylogeny and expression of the tetraspanin CD9 in salmonid cell lines in response to interferon stimulation. International Society for Fish and Shellfish Immunology, conference Spain, June 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Presentation at he International Society of Fish and Shellfish Immunology conference, las palmas, Spain. June 2019
Year(s) Of Engagement Activity 2019