Prosjektnummer
901631
Utnytte artsuavhengig variasjon i resistens mot lakselus / Harnessing cross-species variation in sea lice resistance (CrispResist)
Prosjektet samler forskere fra Norge,
Storbritannia, USA, Canada, Sverige og Australia for å finne de underliggende mekanismene for artsuavhengig variasjon i vertsresistens mot lakselus Lepeophtheirus salmonis,
og bruke denne kunnskapen til å styrke resistens hos atlantisk laks. Det er allerede fastslått at bestemte arter
stillehavslaks er motstandsdyktige mot lakselus og kan forsvare seg mot lus i et tidlig
stadium av parasitteringen, i motsetning til atlantisk laks som er svært sårbar overfor lus. Årsaken til forskjellen er først og fremst variasjon i effektivitet i tidlig immunrespons, men lusens immunmodulering ser også ut til å spille en nøkkelrolle. En svært lovende, men lite utnyttet tilnærming til lakseluskontroll er forbedring og utvikling av den medfødte genetiske resistensen hos den atlantiske laksen.
Background
This project draws together a leading team of researchers from Norway, UK, USA, Canada, Sweden and Australia to discover the mechanisms underlying cross-species variation in host resistance to sea lice Lepeophtheirus salmonis and apply this knowledge to boost Atlantic salmon resistance. It is well established that certain Pacific salmon species are resistant to sea lice and are able to kill lice in the early stages of parasitisation, whereas Atlantic salmon are highly susceptible. This difference is primarily due to variation in the effectiveness of the early-stage immune response, but immunomodulation by the lice is likely to play a key role. Improving the innate genetic resistance of the Atlantic salmon host to the lice is a highly promising but underexploited approach to sea lice control.
Background
This project draws together a leading team of researchers from Norway, UK, USA, Canada, Sweden and Australia to discover the mechanisms underlying cross-species variation in host resistance to sea lice Lepeophtheirus salmonis and apply this knowledge to boost Atlantic salmon resistance. It is well established that certain Pacific salmon species are resistant to sea lice and are able to kill lice in the early stages of parasitisation, whereas Atlantic salmon are highly susceptible. This difference is primarily due to variation in the effectiveness of the early-stage immune response, but immunomodulation by the lice is likely to play a key role. Improving the innate genetic resistance of the Atlantic salmon host to the lice is a highly promising but underexploited approach to sea lice control.
Hovedmål
Å utforske og dokumentere potensialet som ligger i utnytting av genetiske egenskaper og mekanismer for lakselusresistens i stillehavslaks som verktøy for å oppnå høy eller full lakselusresistens hos atlantisk laks.
Delmål
• Å identifisere og dokumentere gener og mekanismer som ligger til grunn for forskjellen i lakselusresistens mellom lakseartene.
• Å utforske og dokumentere potensialet som ligger i identifiserte genetiske egenskaper og mekanismer for lakselusresistens som verktøy for å oppnå høy eller full lakselusresistens.
• Å gjennomføre en risikovurdering av mulighetene for og konsekvensene av at lakselus tilpasser seg atlantisk laks med lakselusresistens.
Objectives
Main objective
To elaborate and document the potential for utilising genetic traits and mechanisms of salmon lice resistance in Pacific salmon as tools to achieve an Atlantic salmon with high or full salmon lice (L. salmonis) resistance.
Sub-objectives
• To identify and document genes and mechanisms responsible for the difference in salmon lice resistance between salmonid species.
• To elaborate and document the potential for utilising the identified genetic traits and mechanisms of salmon lice resistance as tools to achieve an Atlantic salmon with high or full salmon lice resistance.
• To conduct a risk evaluation on the possibilities for, and consequences of salmon lice adapting to Atlantic salmon with salmon lice resistance.
Å utforske og dokumentere potensialet som ligger i utnytting av genetiske egenskaper og mekanismer for lakselusresistens i stillehavslaks som verktøy for å oppnå høy eller full lakselusresistens hos atlantisk laks.
Delmål
• Å identifisere og dokumentere gener og mekanismer som ligger til grunn for forskjellen i lakselusresistens mellom lakseartene.
• Å utforske og dokumentere potensialet som ligger i identifiserte genetiske egenskaper og mekanismer for lakselusresistens som verktøy for å oppnå høy eller full lakselusresistens.
• Å gjennomføre en risikovurdering av mulighetene for og konsekvensene av at lakselus tilpasser seg atlantisk laks med lakselusresistens.
Objectives
Main objective
To elaborate and document the potential for utilising genetic traits and mechanisms of salmon lice resistance in Pacific salmon as tools to achieve an Atlantic salmon with high or full salmon lice (L. salmonis) resistance.
Sub-objectives
• To identify and document genes and mechanisms responsible for the difference in salmon lice resistance between salmonid species.
• To elaborate and document the potential for utilising the identified genetic traits and mechanisms of salmon lice resistance as tools to achieve an Atlantic salmon with high or full salmon lice resistance.
• To conduct a risk evaluation on the possibilities for, and consequences of salmon lice adapting to Atlantic salmon with salmon lice resistance.
Nofima
har beregnet at luseparasittisme koster norsk laksenæring minst 5 milliarder
kroner i året. I tillegg til økonomiske kostnader representerer lakselus
alvorlige dyrevelferdsbekymringer, både patologisk tilknyttet parasittering og
stress som påføres ved kontrolltiltak. Andre kontrolltiltak har alvorlige
ulemper og ingen tilnærmingsmåte er fullt ut effektiv. Vellykket implementering
av løsningene som skal utvikles i dette prosjektet vil kunne forandre norsk lakseoppdrett gjennom årlige besparelser på mer enn 5 milliarder kroner årlig og betydelig forbedring av dyrevelferden (en samlet verdi på minst 100 ganger
prosjektkostnadene).
Expected project impact
Nofima has estimated that lice parasitism costs the Norwegian salmon sector at least 5 billion Norwegian kroner per year. In addition to the economic cost, sea lice present a serious animal welfare concern, both due to the pathology associated with parasitisation, and the stress associated with control measures. Other control measures have serious downsides and no single approach is fully effective. Successful implementation of the approach developed by this project will transform salmon aquaculture in Norway by saving the sector more than 5 billion kroner per annum and by greatly improving animal welfare (a total value of at least 100 times project costs).
Expected project impact
Nofima has estimated that lice parasitism costs the Norwegian salmon sector at least 5 billion Norwegian kroner per year. In addition to the economic cost, sea lice present a serious animal welfare concern, both due to the pathology associated with parasitisation, and the stress associated with control measures. Other control measures have serious downsides and no single approach is fully effective. Successful implementation of the approach developed by this project will transform salmon aquaculture in Norway by saving the sector more than 5 billion kroner per annum and by greatly improving animal welfare (a total value of at least 100 times project costs).
Text in English below. Links to some presentations at the bottom of this section.
Prosjektet er organisert i tre arbeidspakker (AP-er):
AP1: Mekanismer i motstandsdyktighet mot lakselus
Målet er å få en detaljert funksjonell forståelse av resistens og mottakelighet, rettet mot molekylære prosesser og gennettverk som kan endres gjennom genomredigering for å overføre resistens hos stillehavslaks til atlantisk laks. Genaktivitet, proteinprofiler og semiokjemiske profiler av to resistente (coho og pink) og to mottakelige (atlantisk og keta) laksearter sammenlignes og alle de forskjellige komponentene i resistens vurderes: Immunitetsrespons og tiltrekningskraft hos vert og lusens immunmodulering.
A. Prøvetaking
Oppgave 1.1. Kontrollerte smitteforsøk med lus
De fire lakseartene blir utfordret gjennom kontrollerte forsøk med lakselus. Det vil innhentes prøver for teknisk analyse som beskrevet nedenfor.
B. Motstandsdyktighet etter kontakt med lus
Oppgave 1.2.1. Genaktivitet i enkelt-cellekjerner
For å forstå de forskjellige lakseartenes respons på lakselus, inkludert de impliserte cellepopulasjonene, vil det utføres genaktivitetsanalyse på enkelt-cellekjerner i fiskens skinn på forskjellige tidspunkt før smitte og etter at lusen fester seg. Artsuavhengig sammenligning av enkelt-cellekjerner vil avdekke differensiert genaktivitet innen ulike celletyper.
Oppgave 1.2.2. Lokale genetiske signaler som driver immuncelleinfiltrasjon
Her vil det kartlegges genaktivitet i to dimensjoner, som vil komplementere genaktivitetsanalyse på enkelt-cellekjerner ved å muliggjøre spesifikke målinger av genetiske signaler som driver immuncelleinfiltrasjon og respons fra cellene som ligger i grenseflaten mellom laks og lus. Ved å benytte både analyse av enkelt-cellekjerner og romlig fordeling kan en avdekke lokale forskjeller i genaktivitet mellom atlantisk laks og resistent stillehavslaks tidlig i smitteforløpet.
Oppgave 1.2.3. Interaksjoner mellom vertsceller og immunmodulerende proteiner hos lus
For å gjenkjenne potensielle immunmodulerende interaksjoner mellom lus og vertsceller, vil det identifiseres forskjeller mellom lakseartenes sammensetning av membranproteiner på steder der lusen sitter fast. Dette muliggjør identifisering av immunmodulerende proteiner hos lus og deres respektive proteinmål på verten, som igjen vil avdekke forskjeller mellom lakseartene. Dette vil kunne fastslå hvordan vertens og lusens proteiner påvirker hverandre, og gjøre det mulig med målrettet genredigering for å unngå lusens immunmodulering. Universitetet i Bergen/ Sea Lice Research Center (SLRC) ble inkludert i prosjektet fra 2022 for å styrke dette arbeidet.
C. Motstandsdyktighet før kontakt med lus
Oppgave 1.3.1. Kjemiske stoffer utskilt fra resistente og mottakelige laksearter
Målet er å finne kjemiske stoffer som utskilles fra mottakelig atlantisk laks og ketalaks, men ikke fra resistent coholaks og pinklaks, og vurdere hvordan disse komponentene påvirker aktiviteten hos lus etter at de har festet seg på verten.
Oppgave 1.3.2. Atferdstest for tiltrekning av lus: Identifisering av semiokjemiske stoffer
Kjemiske stoffer som er spesifikke for mottakelige arter eller som finnes i høyere nivå hos mottakelige og ikke resistente arter (oppgave 1.3.1) vil undersøkes gjennom en totrinns prosess for å identifisere semiokjemiske feromoner hos lakselus. Man vil måle atferdsrespons som økning i lakselusens svømmehastighet, hoppefrekvens og distanse som tilbakelegges som respons på testforbindelser. Forbindelser som er antatt attraktive for lus testes ytterligere for å måle distansen fra en punktkilde på laksen der forbindelsene aktiverer frittsvømmende lus.
Oppgave 1.3.3. Genrespons hos lus på identifiserte semiokjemiske stoffer
Det vil testes om eksponering for semiokjemiske stoffer utløser differensiell genaktivitet hos lakselus (f.eks. induserer produksjon av immunmodulerende forbindelser). Karakterisering av genaktivitetsprofiler fra behandlet og ubehandlet lus vil gi informasjon om hvorvidt luseproteiner som gjenkjennes av verten (identifisert i oppgave 1.2.3) er påvirket av semiokjemiske stoffer utskilt fra resistente eller mottakelige laks.
Oppgave 1.3.4. Mekanismer som blokkerer produksjon av semiokjemiske stoffer fra atlantisk laks for genredigering
For å finne målgener for blokkering av produksjon av luseferomoner vil det fokuseres på aktivitetsprofilene til gener som koder for enzymer for sekundærmetabolitter, som finnes hos mottakelige laksearter, er nedregulerte eller fraværende i resistente laksearter og er fraværende i andre marine arter.
D. Metaanalyse
Oppgave 1.4. Integrert analyse av “omiske” og semiokjemiske datasett
Gener og synteseveier som skiller resistente og mottakelige laksearter vil bli identifisert slik at viktige målgener kan testes for overføring av resistens til atlantisk laks.
AP2: Mulighet for å innføre lakselusresistens loci i bestander av atlantisk laks ved genredigering
Genredigering av kandidatgener utvalgt på grunnlag av resultatene fra AP1 vil bli brukt til å teste effektiviteten i overføring av lakselusresistens fra arter av stillehavslaks til atlantisk laks. I denne arbeidsplanen vil det også undersøkes hvordan oppdrettsteknologi kan brukes så effektiv som mulig til å implementere antatte resistens loci.
Prosjektet er organisert i tre arbeidspakker (AP-er):
AP1: Mekanismer i motstandsdyktighet mot lakselus
Målet er å få en detaljert funksjonell forståelse av resistens og mottakelighet, rettet mot molekylære prosesser og gennettverk som kan endres gjennom genomredigering for å overføre resistens hos stillehavslaks til atlantisk laks. Genaktivitet, proteinprofiler og semiokjemiske profiler av to resistente (coho og pink) og to mottakelige (atlantisk og keta) laksearter sammenlignes og alle de forskjellige komponentene i resistens vurderes: Immunitetsrespons og tiltrekningskraft hos vert og lusens immunmodulering.
A. Prøvetaking
Oppgave 1.1. Kontrollerte smitteforsøk med lus
De fire lakseartene blir utfordret gjennom kontrollerte forsøk med lakselus. Det vil innhentes prøver for teknisk analyse som beskrevet nedenfor.
B. Motstandsdyktighet etter kontakt med lus
Oppgave 1.2.1. Genaktivitet i enkelt-cellekjerner
For å forstå de forskjellige lakseartenes respons på lakselus, inkludert de impliserte cellepopulasjonene, vil det utføres genaktivitetsanalyse på enkelt-cellekjerner i fiskens skinn på forskjellige tidspunkt før smitte og etter at lusen fester seg. Artsuavhengig sammenligning av enkelt-cellekjerner vil avdekke differensiert genaktivitet innen ulike celletyper.
Oppgave 1.2.2. Lokale genetiske signaler som driver immuncelleinfiltrasjon
Her vil det kartlegges genaktivitet i to dimensjoner, som vil komplementere genaktivitetsanalyse på enkelt-cellekjerner ved å muliggjøre spesifikke målinger av genetiske signaler som driver immuncelleinfiltrasjon og respons fra cellene som ligger i grenseflaten mellom laks og lus. Ved å benytte både analyse av enkelt-cellekjerner og romlig fordeling kan en avdekke lokale forskjeller i genaktivitet mellom atlantisk laks og resistent stillehavslaks tidlig i smitteforløpet.
Oppgave 1.2.3. Interaksjoner mellom vertsceller og immunmodulerende proteiner hos lus
For å gjenkjenne potensielle immunmodulerende interaksjoner mellom lus og vertsceller, vil det identifiseres forskjeller mellom lakseartenes sammensetning av membranproteiner på steder der lusen sitter fast. Dette muliggjør identifisering av immunmodulerende proteiner hos lus og deres respektive proteinmål på verten, som igjen vil avdekke forskjeller mellom lakseartene. Dette vil kunne fastslå hvordan vertens og lusens proteiner påvirker hverandre, og gjøre det mulig med målrettet genredigering for å unngå lusens immunmodulering. Universitetet i Bergen/ Sea Lice Research Center (SLRC) ble inkludert i prosjektet fra 2022 for å styrke dette arbeidet.
C. Motstandsdyktighet før kontakt med lus
Oppgave 1.3.1. Kjemiske stoffer utskilt fra resistente og mottakelige laksearter
Målet er å finne kjemiske stoffer som utskilles fra mottakelig atlantisk laks og ketalaks, men ikke fra resistent coholaks og pinklaks, og vurdere hvordan disse komponentene påvirker aktiviteten hos lus etter at de har festet seg på verten.
Oppgave 1.3.2. Atferdstest for tiltrekning av lus: Identifisering av semiokjemiske stoffer
Kjemiske stoffer som er spesifikke for mottakelige arter eller som finnes i høyere nivå hos mottakelige og ikke resistente arter (oppgave 1.3.1) vil undersøkes gjennom en totrinns prosess for å identifisere semiokjemiske feromoner hos lakselus. Man vil måle atferdsrespons som økning i lakselusens svømmehastighet, hoppefrekvens og distanse som tilbakelegges som respons på testforbindelser. Forbindelser som er antatt attraktive for lus testes ytterligere for å måle distansen fra en punktkilde på laksen der forbindelsene aktiverer frittsvømmende lus.
Oppgave 1.3.3. Genrespons hos lus på identifiserte semiokjemiske stoffer
Det vil testes om eksponering for semiokjemiske stoffer utløser differensiell genaktivitet hos lakselus (f.eks. induserer produksjon av immunmodulerende forbindelser). Karakterisering av genaktivitetsprofiler fra behandlet og ubehandlet lus vil gi informasjon om hvorvidt luseproteiner som gjenkjennes av verten (identifisert i oppgave 1.2.3) er påvirket av semiokjemiske stoffer utskilt fra resistente eller mottakelige laks.
Oppgave 1.3.4. Mekanismer som blokkerer produksjon av semiokjemiske stoffer fra atlantisk laks for genredigering
For å finne målgener for blokkering av produksjon av luseferomoner vil det fokuseres på aktivitetsprofilene til gener som koder for enzymer for sekundærmetabolitter, som finnes hos mottakelige laksearter, er nedregulerte eller fraværende i resistente laksearter og er fraværende i andre marine arter.
D. Metaanalyse
Oppgave 1.4. Integrert analyse av “omiske” og semiokjemiske datasett
Gener og synteseveier som skiller resistente og mottakelige laksearter vil bli identifisert slik at viktige målgener kan testes for overføring av resistens til atlantisk laks.
AP2: Mulighet for å innføre lakselusresistens loci i bestander av atlantisk laks ved genredigering
Genredigering av kandidatgener utvalgt på grunnlag av resultatene fra AP1 vil bli brukt til å teste effektiviteten i overføring av lakselusresistens fra arter av stillehavslaks til atlantisk laks. I denne arbeidsplanen vil det også undersøkes hvordan oppdrettsteknologi kan brukes så effektiv som mulig til å implementere antatte resistens loci.
Prosjektutvidelse primo 2023 (in English only):
ODN guided knock-out (KO). In order to minimize the occurrence of in-frame mutations in F0, the knockout will be guided towards loss-of-function alleles using oligos. To facilitate effective screening, an albino knockout will be utilized as a visual tracer for other CRISPR targets in the edited salmon.
Oppgave 2.1. Målrettet redigering av kandidater for lakselusresistens i atlantiske laksembryoer
Her skal det fremstilles genredigerte embryoer fra atlantisk laks. Etiske, sosiale og juridiske implikasjoner ved å bruke genredigering til å forske på funksjonene hos kandidatgener i laks skal evalueres gjennom en indikatorkartlegging for ansvarlig forskning og innovasjon.
Prosjektutvidelse, primo 2023 (in English only):
KO lice trial. In the 4th quarter of 2024, editing success will be assessed and a recommendation/proposal made to FHF about extending the project and budget to perform this task depending on whether the project has conclusive and potentially valuable results. When fish are one year old and if FHF approve extension of the project at that time, the project will expose edited and control fish in common garden to salmon lice infestation (copepodits). Salmon lice will be counted 2 and 8 weeks after infestation. Additionally, skin samples will be obtained from the infection site, and head kidney samples will be collected from both control and edited fish to monitor potential changes in the response to infection. Immunohistochemistry will be employed to identify potential macrophages, T-cells, and B-cells in the infected salmon skin. Edit modifications that lead to increased or decreased infection intensity of salmon lice, or exhibit modified immune response to lice infection, will undergo closer monitoring. For fish with altered immune response, skin tissue from the lice infection site will be sampled. Based on the snRNAseq study, it is possible to observe which cell types are expected to be affected and determine whether these cells are affected using qPCR. This approach will result in Atlantic salmon knockouts that can be utilized to evaluate potential effects on host resistance.
KO lice trial. In the 4th quarter of 2024, editing success will be assessed and a recommendation/proposal made to FHF about extending the project and budget to perform this task depending on whether the project has conclusive and potentially valuable results. When fish are one year old and if FHF approve extension of the project at that time, the project will expose edited and control fish in common garden to salmon lice infestation (copepodits). Salmon lice will be counted 2 and 8 weeks after infestation. Additionally, skin samples will be obtained from the infection site, and head kidney samples will be collected from both control and edited fish to monitor potential changes in the response to infection. Immunohistochemistry will be employed to identify potential macrophages, T-cells, and B-cells in the infected salmon skin. Edit modifications that lead to increased or decreased infection intensity of salmon lice, or exhibit modified immune response to lice infection, will undergo closer monitoring. For fish with altered immune response, skin tissue from the lice infection site will be sampled. Based on the snRNAseq study, it is possible to observe which cell types are expected to be affected and determine whether these cells are affected using qPCR. This approach will result in Atlantic salmon knockouts that can be utilized to evaluate potential effects on host resistance.
Oppgave 2.2 Sammenligne resistens mot lakselus hos redigert og ikke-redigert atlantisk laks
Genredigert laks fra oppgave 2.1 vil bli smittet med lakselus sammen med uredigerte og mock-redigerte kontrollfisk fra de samme familiene. Vertsresistens mot lakselus evalueres ved å måle og sammenligne antall lakselus på redigert laks og kontroll-laks.
Prosjektutvidelse, primo 2023 (in English only):
Knock-in (KI) –Use of fine-tuned editing to up- or down-regulate expression in Atlantic salmon. Resistance candidates will be targeted with the goal of modulating their expression. This can be achieved by impacting mRNA integrity, incorporating promoter elements, or introducing additional copies of genes into desired promoters within the salmon genome. Protocols for inserting larger fragments (unpublished data from NRC project TUNESAL (2020–2024, led by IMR)), performing single SNP exchanges, and inserting smaller fragments have already been developed. These established techniques will be employed to upregulate or downregulate genes identified in the ongoing CRISPresist project. Additionally, multiple precise edits will be attempted in a single fish to more accurately represent a lice-resistant salmon genotype. This approach will enable the creation of knock-in Atlantic salmon strains that can be utilized to evaluate potential effects on host resistance.
KI lice trial – Breeding knock-in Atlantic salmon and lice trial on heterozygous KI. Again, in the fourth quarter of 2024, the success of the editing will be evaluated, and a recommendation/proposal will be submitted to FHF regarding the potential extension of the project and budget for this task. The decision will depend on the presence of conclusive and potentially valuable results. The efficiency of knock-in's can be high using shorter oligos (below 100nt), enabling some functional studies to be performed in F0 Crispants. However, because larger inserts are integrated at a much lower frequency than regular mutations caused by CRISPR, it will be necessary when making large insertions to cross-out F0 Crispants to create an F1 generation of heterozygous fish, in which at least one allele carries the desired insertion. Different from knockout models, insertion into one of the two alleles may confer a clear phenotype. To carry out these modifications in salmon, the editing process will be conducted in F0 individuals, and positive fish will be identified through PCR and miSeq analysis of the targeted F0 fish. F0 fish with detected edits will be kept up to one year of age. At one year of age fish will be stimulated to enter early maturation, 6 months after stimulation males can be crossed out to wild type (wt) eggs.
Knock-in (KI) –Use of fine-tuned editing to up- or down-regulate expression in Atlantic salmon. Resistance candidates will be targeted with the goal of modulating their expression. This can be achieved by impacting mRNA integrity, incorporating promoter elements, or introducing additional copies of genes into desired promoters within the salmon genome. Protocols for inserting larger fragments (unpublished data from NRC project TUNESAL (2020–2024, led by IMR)), performing single SNP exchanges, and inserting smaller fragments have already been developed. These established techniques will be employed to upregulate or downregulate genes identified in the ongoing CRISPresist project. Additionally, multiple precise edits will be attempted in a single fish to more accurately represent a lice-resistant salmon genotype. This approach will enable the creation of knock-in Atlantic salmon strains that can be utilized to evaluate potential effects on host resistance.
KI lice trial – Breeding knock-in Atlantic salmon and lice trial on heterozygous KI. Again, in the fourth quarter of 2024, the success of the editing will be evaluated, and a recommendation/proposal will be submitted to FHF regarding the potential extension of the project and budget for this task. The decision will depend on the presence of conclusive and potentially valuable results. The efficiency of knock-in's can be high using shorter oligos (below 100nt), enabling some functional studies to be performed in F0 Crispants. However, because larger inserts are integrated at a much lower frequency than regular mutations caused by CRISPR, it will be necessary when making large insertions to cross-out F0 Crispants to create an F1 generation of heterozygous fish, in which at least one allele carries the desired insertion. Different from knockout models, insertion into one of the two alleles may confer a clear phenotype. To carry out these modifications in salmon, the editing process will be conducted in F0 individuals, and positive fish will be identified through PCR and miSeq analysis of the targeted F0 fish. F0 fish with detected edits will be kept up to one year of age. At one year of age fish will be stimulated to enter early maturation, 6 months after stimulation males can be crossed out to wild type (wt) eggs.
In the F1 generation, edit-positive embryo batches will be screened using an e-DNA screen. Positive egg-batches (n=50 embryos) will be identified and kept. Positive fish will then at one-year of age be studied as described in subtask 2.1 KO.
Prosjektutvidelse, primo 2023 (in English only):
Oppgave 2.3.
Coho gene editing at UoPEI in Canada.
For five genes that exhibit higher expression levels in coho salmon compared to Atlantic salmon following lice infection, attempts will be made to generate coho knockouts. The objective is to investigate whether suppressing the immune response of coho salmon against lice is possible. The need for this investigation was not initially anticipated and exceeds the budget of the original application. Through this approach, coho knockouts will be generated, enabling the assessment of potential effects on host resistance.
Oppgave 2.4. Implementering og formidling
Mulige planer for overlevering av lusresistens fra avlsprogrammene i Norge til kommersielt produksjonsnivå vil simuleres og evalueres i samråd med oppdrettsnæringen. Formidlingsplanen som utvikles vil følges i henhold til retningslinjene for ansvarlig forskning og innovasjon ved bruk av genredigering.
AP3: Risikoanalyse og modellering av potensial for utvikling av lakselus som kan motstå genetisk resistens
En merkesak i implementeringsplanen vil være å redusere risikoen for tilpasning hos lus.
Oppgave 3.1. Modellering av evolusjonsmessig potensial for tilpasning av lakselus til resistent atlantisk laks
Modellen vil beskrive demografi hos lus (i grupper av villaks og oppdrettslaks, genflyt mellom oppdrettsanlegg) og redegjøre for flokkimmunitet. Modellen vil brukes til å vurdere styringsstrategier for laksestammer for å redusere den adaptive responsen hos lus. Resultater brukes som et filter for utvikling av den endelige genredigeringsstrategien.
Oppgave 3.2. Risikoanalyse
Her vil det vurderes hvordan ulike strategier for å utnytte resistent laks, i kombinasjon med andre forebyggende smittemekanismer, kan minske risiko for at lusen utvikler strategier for å motstå økende lakseresistens. Formidlingsplanen (AP2) og risikoanalysen (AP3) resulterer i en overordnet plan for implementering i oppdrettsnæringen.
Prosjektorganisering
Nofima AS Norway (hovedorganisasjon og ansvarlig for oppgaver 1.3.1, 1.3.2, 1.3.4, 1.4, 2.1, 2.2 og 2.3)
Roslin Institute of the University of Edinburgh UK (ansvarlig for oppgaver 1.2.1, 1.2.2 og 1.2.3)
Universitetet i Bergen / SLRC med fra 2022 (tillegg spesielt for oppgave 1.2.3)
Institute of Aquaculture of Sterling University UK (ansvarlig for oppgave 1.3.3)
University of Melbourne Australia (ansvarlig for oppgaver 3.1 og 3.2) - fra 2023 overtatt av Deakin University i Australia
University of Prince Edward Island Canada (ansvarlig for oppgave 1.1)
Rothamsted Research UK (AP1)
Institute of Marine Research Norway (AP1 og utvidet ved tillegg til også AP2)
University of Gothenburg Sweden (AP1) og
Bigelow Laboratory for Ocean Sciences USA (AP1).
Benchmark, Salmar og Mowi har viktige konsultasjons- og ledelsesroller.
Benchmark og Salmar donerer fisken.
Prosjektet vil bli fulgt av et internasjonalt sammensatt rådgivende vitenskapelig panel.
Project design and implementation
The project is organised as three work packages (WPs):
WP1: Mechanisms of resistance to sea lice
The goal is to gain a detailed functional understanding of resistance and susceptibility, pointing to molecular processes and gene networks that can be modified by genome editing to transfer Pacific salmon resistance to Atlantic salmon. Gene activity, protein and semiochemical profiles of two resistant (coho and pink) and two susceptible (Atlantic and chum) salmon species will be compared, assessing all the different components of resistance: host immune responses, host attractiveness and lice immunomodulation.
A. Sampling
Task 1.1. Challenge test
The four salmon species will be experimentally challenged with infectious lice copepodids. Samples will be collected for technical analysis described below.
B. Resistance post-contact
Task 1.2.1. Single-nuclei gene activity
To understand the detailed response of the different salmon species to sea lice, including the cellular populations involved, single-nuclei gene activity analysis of the skin will be performed at different time points before infection and post-attachment. Cross-species comparison of single-nuclei will reveal differential gene activity within cell types.
Task 1.2.2. Capturing local genetic signals driving immune cell infiltration
Gene activity will be mapped in two dimensions which will complement single-nuclei gene activity analysis by enabling specific measurement of genetic signals driving immune cell infiltration and response from the cells situated at the attachment boundary between the host and louse. Using both single-nuclei and spatial analysis will allow to detect localized differences in gene activity between Atlantic and resistant Pacific salmon during early infection.
Task 1.2.3. Interactions between host cells and lice immunomodulatory proteins
To identify potential immunomodulatory interactions between lice and host cells the project group will identify differences in membrane protein composition at lice attachment sites between salmonid species which will enable the identification of lice immunomodulatory proteins and their host protein targets, highlighting differences between salmonid species. Further, this will identify host protein sequences interacting with lice proteins, enabling targeted gene editing to evade lice immunomodulation. The University of Bergen/ Sea Lice Research Center (SLRC) was included in the project from 2022 to strengthen this task.
C. Resistance pre-contact
Task 1.3.1. Chemical analysis of water conditioned with resistant and susceptible species
The goal is to find compounds that are released by the susceptible Atlantic and chum salmon, but not by the resistant pink and coho salmon, and to assess how these compounds affect the activity of the lice post-attachment.
Task 1.3.2. Behavioural test of lice attraction: identification of semiochemicals
Compounds specific to susceptible species or identified at higher levels in susceptible than resistant species (Task 1.3.1) will be examined using a two-step process to identify semiochemical attractants to sea lice. The project group will measure behavioural responses such as increase in copepodid swimming speed, hop frequency, and distance travelled in response to test compounds. Putative lice-attractive compounds will be further tested to estimate the distance from a point source of salmon at which the compounds would activate free-swimming lice.
Task 1.3.3. Gene response of the lice to salmon conditioned water and putative semiochemicals
The project group will test if exposure to semiochemicals provokes differential gene activity in sea lice (e.g. induces the production of immunomodulatory compounds). Gene activity profiling of treated and untreated lice will inform as to whether host recognised louse proteins identified in Task 1.2.3 are influenced by semiochemicals released by resistant or susceptible salmonid hosts.
Task 1.3.4. Mechanism(s) blocking semiochemical production from Atlantic salmon for gene editing
To identify target genes for blocking the production of lice attractants the project group shall focus on the activity profiles of genes coding for enzymes of secondary metabolism that are present in susceptible salmon species, down regulated or absent in resistant salmon species and absent in other marine species.
D. Meta-analysis
Task 1.4. Integrated analysis of ‘omic’ and semiochemical datasets
Genes and pathways that differentiate resistant and susceptible salmonid species will be identified allowing prioritization of key target genes to test for transferring resistance into Atlantic salmon.
WP2. Potential to introduce sea lice resistance loci into Atlantic salmon stocks using genome editing
Genome editing of candidate genes selected based on the results of WP1 will be used to test the effectiveness of transferring sea lice resistance from Pacific species to Atlantic salmon. This WP will also explore how breeding technologies can be most effectively used to disseminate putative resistance loci.
Project extension primo 2023:
Project design and implementation
The project is organised as three work packages (WPs):
WP1: Mechanisms of resistance to sea lice
The goal is to gain a detailed functional understanding of resistance and susceptibility, pointing to molecular processes and gene networks that can be modified by genome editing to transfer Pacific salmon resistance to Atlantic salmon. Gene activity, protein and semiochemical profiles of two resistant (coho and pink) and two susceptible (Atlantic and chum) salmon species will be compared, assessing all the different components of resistance: host immune responses, host attractiveness and lice immunomodulation.
A. Sampling
Task 1.1. Challenge test
The four salmon species will be experimentally challenged with infectious lice copepodids. Samples will be collected for technical analysis described below.
B. Resistance post-contact
Task 1.2.1. Single-nuclei gene activity
To understand the detailed response of the different salmon species to sea lice, including the cellular populations involved, single-nuclei gene activity analysis of the skin will be performed at different time points before infection and post-attachment. Cross-species comparison of single-nuclei will reveal differential gene activity within cell types.
Task 1.2.2. Capturing local genetic signals driving immune cell infiltration
Gene activity will be mapped in two dimensions which will complement single-nuclei gene activity analysis by enabling specific measurement of genetic signals driving immune cell infiltration and response from the cells situated at the attachment boundary between the host and louse. Using both single-nuclei and spatial analysis will allow to detect localized differences in gene activity between Atlantic and resistant Pacific salmon during early infection.
Task 1.2.3. Interactions between host cells and lice immunomodulatory proteins
To identify potential immunomodulatory interactions between lice and host cells the project group will identify differences in membrane protein composition at lice attachment sites between salmonid species which will enable the identification of lice immunomodulatory proteins and their host protein targets, highlighting differences between salmonid species. Further, this will identify host protein sequences interacting with lice proteins, enabling targeted gene editing to evade lice immunomodulation. The University of Bergen/ Sea Lice Research Center (SLRC) was included in the project from 2022 to strengthen this task.
C. Resistance pre-contact
Task 1.3.1. Chemical analysis of water conditioned with resistant and susceptible species
The goal is to find compounds that are released by the susceptible Atlantic and chum salmon, but not by the resistant pink and coho salmon, and to assess how these compounds affect the activity of the lice post-attachment.
Task 1.3.2. Behavioural test of lice attraction: identification of semiochemicals
Compounds specific to susceptible species or identified at higher levels in susceptible than resistant species (Task 1.3.1) will be examined using a two-step process to identify semiochemical attractants to sea lice. The project group will measure behavioural responses such as increase in copepodid swimming speed, hop frequency, and distance travelled in response to test compounds. Putative lice-attractive compounds will be further tested to estimate the distance from a point source of salmon at which the compounds would activate free-swimming lice.
Task 1.3.3. Gene response of the lice to salmon conditioned water and putative semiochemicals
The project group will test if exposure to semiochemicals provokes differential gene activity in sea lice (e.g. induces the production of immunomodulatory compounds). Gene activity profiling of treated and untreated lice will inform as to whether host recognised louse proteins identified in Task 1.2.3 are influenced by semiochemicals released by resistant or susceptible salmonid hosts.
Task 1.3.4. Mechanism(s) blocking semiochemical production from Atlantic salmon for gene editing
To identify target genes for blocking the production of lice attractants the project group shall focus on the activity profiles of genes coding for enzymes of secondary metabolism that are present in susceptible salmon species, down regulated or absent in resistant salmon species and absent in other marine species.
D. Meta-analysis
Task 1.4. Integrated analysis of ‘omic’ and semiochemical datasets
Genes and pathways that differentiate resistant and susceptible salmonid species will be identified allowing prioritization of key target genes to test for transferring resistance into Atlantic salmon.
WP2. Potential to introduce sea lice resistance loci into Atlantic salmon stocks using genome editing
Genome editing of candidate genes selected based on the results of WP1 will be used to test the effectiveness of transferring sea lice resistance from Pacific species to Atlantic salmon. This WP will also explore how breeding technologies can be most effectively used to disseminate putative resistance loci.
Project extension primo 2023:
ODN guided knock-out (KO). In order to minimize the occurrence of in-frame mutations in F0,
the knockout will be guided towards loss-of-function alleles using
oligos. To facilitate effective screening, an albino knockout will be
utilized as a visual tracer for other CRISPR targets in the edited
salmon.
Task 2.1. Targeted editing of sea lice resistance candidate in Atlantic salmon embryos
Genome edited Atlantic salmon embryos will be created. Ethical, social and legal implications of using gene editing to study the functions of candidate genes in salmon will be evaluated through a Responsible Research and Innovation multi-indicator mapping approach.
Project extension primo 2023:
KO lice trial.
In the 4th quarter of 2024, editing success will be assessed and a
recommendation/proposal made to FHF about extending the project and
budget to perform this task depending on whether the project has
conclusive and potentially valuable results.
When fish are one year old and if FHF approve
extension of the project at that time, the project will expose edited
and control fish in common garden to salmon lice infestation
(copepodits). Salmon lice will be counted 2 and 8 weeks after infestation.
Additionally, skin samples will be obtained
from the infection site, and head kidney samples will be collected from
both control and edited fish to monitor potential changes in the
response to infection. Immunohistochemistry will be employed to identify
potential macrophages, T-cells, and B-cells in the infected salmon
skin. Edit modifications that lead to increased or decreased infection
intensity of salmon lice, or exhibit modified immune response to lice
infection, will undergo closer monitoring. For fish with altered immune
response, skin tissue from the lice infection site will be sampled.
Based on the snRNAseq study, it is possible to observe which cell types
are expected to be affected and determine whether these cells are
affected using qPCR. This approach will result in Atlantic salmon
knockouts that can be utilized to evaluate potential effects on host
resistance.
Task 2.2 Compare the resistance of edited and non-edited Atlantic salmon to sea lice
Genome edited salmon generated in Task 2.1 will be challenged with sea lice along with unedited and mock-edited control animals of the same families. Host resistance to sea lice will be evaluated by measuring and comparing counts of sea lice on edited and control salmon.
Genome edited salmon generated in Task 2.1 will be challenged with sea lice along with unedited and mock-edited control animals of the same families. Host resistance to sea lice will be evaluated by measuring and comparing counts of sea lice on edited and control salmon.
Project extension primo 2023:
Knock-in (KI) –Use of fine-tuned editing to up- or down-regulate expression in Atlantic salmon.
Resistance candidates will be targeted with the goal of modulating
their expression. This can be achieved by impacting mRNA integrity,
incorporating promoter elements, or introducing additional copies of
genes into desired promoters within the salmon genome. Protocols for
inserting larger fragments (unpublished data from NRC project TUNESAL
(2020–2024, led by IMR)), performing single SNP exchanges, and inserting
smaller fragments have already been developed. These established
techniques will be employed to upregulate or downregulate genes
identified in the ongoing CRISPresist project. Additionally, multiple
precise edits will be attempted in a single fish to more accurately
represent a lice-resistant salmon genotype. This approach will enable
the creation of knock-in Atlantic salmon strains that can be utilized to
evaluate potential effects on host resistance.
KI lice trial – Breeding knock-in Atlantic salmon and lice trial on heterozygous KI. Again, in the fourth quarter of 2024, the success of the editing will be evaluated, and a recommendation/proposal will be submitted to FHF regarding the potential extension of the project and budget for this task. The decision will depend on the presence of conclusive and potentially valuable results. The efficiency of knock-in's can be high using shorter oligos (below 100nt), enabling some functional studies to be performed in F0 Crispants. However, because larger inserts are integrated at a much lower frequency than regular mutations caused by CRISPR, it will be necessary when making large insertions to cross-out F0 Crispants to create an F1 generation of heterozygous fish, in which at least one allele carries the desired insertion. Different from knockout models, insertion into one of the two alleles may confer a clear phenotype. To carry out these modifications in salmon, the editing process will be conducted in F0 individuals, and positive fish will be identified through PCR and miSeq analysis of the targeted F0 fish. F0 fish with detected edits will be kept up to one year of age. At one year of age fish will be stimulated to enter early maturation, 6 months after stimulation males can be crossed out to wild type (wt) eggs.
KI lice trial – Breeding knock-in Atlantic salmon and lice trial on heterozygous KI. Again, in the fourth quarter of 2024, the success of the editing will be evaluated, and a recommendation/proposal will be submitted to FHF regarding the potential extension of the project and budget for this task. The decision will depend on the presence of conclusive and potentially valuable results. The efficiency of knock-in's can be high using shorter oligos (below 100nt), enabling some functional studies to be performed in F0 Crispants. However, because larger inserts are integrated at a much lower frequency than regular mutations caused by CRISPR, it will be necessary when making large insertions to cross-out F0 Crispants to create an F1 generation of heterozygous fish, in which at least one allele carries the desired insertion. Different from knockout models, insertion into one of the two alleles may confer a clear phenotype. To carry out these modifications in salmon, the editing process will be conducted in F0 individuals, and positive fish will be identified through PCR and miSeq analysis of the targeted F0 fish. F0 fish with detected edits will be kept up to one year of age. At one year of age fish will be stimulated to enter early maturation, 6 months after stimulation males can be crossed out to wild type (wt) eggs.
In the F1 generation, edit-positive embryo batches
will be screened using an e-DNA screen. Positive egg-batches (n=50
embryos) will be identified and kept. Positive fish will then at
one-year of age be studied as described in subtask 2.1 KO.
Project extension primo 2023:
Task 2.3.
Coho gene editing at UoPEI in Canada.
For five genes that exhibit higher expression levels in
coho salmon compared to Atlantic salmon following lice infection,
attempts will be made to generate coho knockouts. The objective is to
investigate whether suppressing the immune response of coho salmon
against lice is possible. The need for this investigation was not
initially anticipated and exceeds the budget of the original
application. Through this approach, coho knockouts will be generated,
enabling the assessment of potential effects on host resistance.
Task 2.4. Implementation and dissemination
Alternative schemes for delivering lice resistance from the breeding programs in Norway to the commercial production tier will be simulated and evaluated in consultation with industry. The dissemination plan produced will follow and expand on Responsible Research and Innovation guidelines for the use of gene editing.
WP3. Risk analysis and modelling of the potential for sea lice to evolve to overcome genetic resistance
A key plank of our implementation plan will be to minimise the risk of adaptation by lice.
Task 3.1. Model the evolutionary potential for the adaptation of sea lice to resistant Atlantic salmon
The model will describe lice demography (wild and farmed fish cohorts, gene flow between farms) and account for herd immunity. The project group will use the model to assess stock management strategies for potential to minimise the adaptive response by lice. Results will be used as another filter for developing our final gene editing strategy.
Task 3.2. Risk analysis
Here the project group will consider how different strategies for deploying resistant salmon, in combination with other preventative infection mechanisms, could mitigate the risk of lice evolving mechanisms to overcome the increased salmon resistance and consider risks associated with lice resistant salmon escaping and competing in the open ecosystem. The dissemination plan (WP2) and risk analysis (WP3) will result in an overall plan for industry implementation.
Project organization
Nofima AS Norway (lead organisation and leader of tasks 1.3.1, 1.3.2, 1.3.4, 1.4, 2.1, 2.2, and 2.3),
Roslin Institute of the University of Edinburgh UK (leader of tasks 1.2.1, 1.2.2 and 1.2.3)
University of Bergen / SLRC included from 2022 (regarding task 1.2.3),
Institute of Aquaculture of Sterling University UK (leader of task 1.3.3),
University of Melbourne Australia (leader of tasks 3.1 and 3.2),
University of Prince Edward Island Canada (leader of task 1.1),
Rothamsted Research UK (WP1),
Institute of Marine Research Norway (WP1 and with project extension also WP2),
University of Gothenburg Sweden (WP1), and
Bigelow Laboratory for Ocean Sciences USA (WP1).
Institute of Aquaculture of Sterling University UK (leader of task 1.3.3),
University of Melbourne Australia (leader of tasks 3.1 and 3.2),
University of Prince Edward Island Canada (leader of task 1.1),
Rothamsted Research UK (WP1),
Institute of Marine Research Norway (WP1 and with project extension also WP2),
University of Gothenburg Sweden (WP1), and
Bigelow Laboratory for Ocean Sciences USA (WP1).
Benchmark, Salmar and Mowi will play important consultative and steering roles.
Benchmark and Salmar will provide fish.
The project will be followed by an international scientific advisory panel.
Presentations on YouTube®
The International Sea Lice Conference (Sealice 2022) in the Faroe Islands:
– Nick Robinson: CrispResist with NoLice: Developing tools for boosting Atlantic salmon host resistance to sealice
– Andrew Coates: A metapopulation model reveals hotspots in the evolution of treatment resistance in salmon lice
– Nick Robinson: CrispResist with NoLice: Developing tools for boosting Atlantic salmon host resistance to sealice
– Andrew Coates: A metapopulation model reveals hotspots in the evolution of treatment resistance in salmon lice
For å sikre maksimal verdi for sektoren vil løpende formidling av
resultater skje via publikasjoner, pressemeldinger, åpne internettfora (i
forbindelse med prosjektmøter som legges ut som videoer på YouTube®), sosiale medier,
konferanser og bransjemesser på norsk og engelsk. Man vil etablere en nettside
som beskriver prosjektet og legge ut relevant material (formidlende
animasjonsfilmer, brosjyrer, informasjonsartikler). Pressemateriale deles med
partnere for distribusjon til mediekontakter i deres egne land. Genredigering
drøftes på årlige møter med myndighetene.
Outreach (åpne internettfora) vil bli avholdt årlig med avls- og
oppdrettsselskaper for å sette i gang toveisdialog og sikre høy sannsynlighet
for resultater. Interessentmøter for ansvarlig forskning og innovasjon
arrangeres årlig. Forskningsresultatene vil formidles i publikasjoner og på
nasjonale / internasjonale konferanser. Det planlegges å publisere mange
vitenskapelige artikler og innholdet i hver enkelt presenteres på nasjonale /
internasjonale konferanser.
Detaljerte tidsskjema for publikasjoner, konferansepresentasjoner og
pressemeldinger er planlagt.
Dissemination of project results
To ensure maximum value for the sector, ongoing dissemination of results will occur via publications, press releases, open on-line forums (in connection with project meetings recorded as YouTube® videos), social media, conferences and industry fairs in Norwegian and English. A webpage will be established describing the project and attach relevant material (dissemination animation videos, brochures, information articles). Press material will be shared with partners for distribution to their media contacts in their countries. Gene editing will be discussed at yearly meetings with government. Outreach (open on-line forums) will be held annually with breeding and farming companies to spark ongoing two-way dialogue and ensure higher probability of adoption of results. Responsible Research and Innovation stakeholder meetings held annually. Scientific results will be disseminated through publications and national / international conferences. The project group plans to publish many scientific articles and the content of each will be presented at international / national conferences. Detailed timetables for publications, conference presentations and media releases are planned.
Dissemination of project results
To ensure maximum value for the sector, ongoing dissemination of results will occur via publications, press releases, open on-line forums (in connection with project meetings recorded as YouTube® videos), social media, conferences and industry fairs in Norwegian and English. A webpage will be established describing the project and attach relevant material (dissemination animation videos, brochures, information articles). Press material will be shared with partners for distribution to their media contacts in their countries. Gene editing will be discussed at yearly meetings with government. Outreach (open on-line forums) will be held annually with breeding and farming companies to spark ongoing two-way dialogue and ensure higher probability of adoption of results. Responsible Research and Innovation stakeholder meetings held annually. Scientific results will be disseminated through publications and national / international conferences. The project group plans to publish many scientific articles and the content of each will be presented at international / national conferences. Detailed timetables for publications, conference presentations and media releases are planned.
Medieomtale
A metapopulation model reveals connectivity-driven hotspots in treatment resistance evolution in a marine parasite
ICES Journal of Marine Science
How are genetic technologies being applied to combat infectious diseases in aquaculture?
https://thefishsite.com/
Applying genetic technologies to combat infectious diseases in aquaculture
onlinelibrary.wiley.com
Nobel-Prize-winning tool CRISPR being explored in sea-lice fight
www.aquaculturenorthamerica.com
Omstridt genetisk metode brukes i Norge
Forskningsetikk.no
Ny kunnskap om avl for klimarobust laks
nofima.no
GENOMICS, GENE-EDITING AND THE BLUE REVOLUTION
pursuit
CRISPR-Cas9 may help battle sea lice in Atlantic salmon
https://www.feedstuffs.com/
Tecnología CRISPR-Cas9 para luchar contra el piojo de mar en el salmón atlántico
http://www.ipacuicultura.com/
Vil bruke styrken til stillehavslaksen for å nedkjempe lakselus
nofima.no
Gene editing project could help build Atlantic salmon’s resistance to lice
www.fishfarmermagazine.com