Abstract
BackgroundGenome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques.ResultsIn the current study, we developed an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214). As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated.ConclusionsThe optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.
Highlights
Genome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation
Efficient transduction of Chinook salmon cells with lentivirus To improve Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) delivery and genome editing efficiency, lentiviral transduction was optimised for use in salmonid cell lines
No editing was detected in either of the regions (FigS3). These results demonstrate that endogenous genes in the chinook salmon CHSE cell line can be targeted for genome editing using lentivirus. These results show that the lentivirus delivery strategy described together with the Cas9 expressing CHSE cell line (CHSE-EC) can rapidly and efficiently be used to edit the genome of salmonid cells, and suggests that the method could be applied to the new generation of CRISPR/Cas9 platforms such as base editors and CRISPR activation/inhibition
Summary
Genome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. Selective breeding for improved disease resistance is a promising avenue to tackle these diseases, and has Genome editing using reprogrammed CRISPR/Cas systems has emerged as a revolutionary tool to make specific and targeted changes to genomes of species’ of interest. CRISPR/Cas can be applied to test targeted perturbation of candidate genes and variants within QTL regions, to assess the consequence on the trait of interest. This knowledge raises the possibility of enhancing genomic selection accuracy via increased weighting on functional variants. Genome editing can potentially by applied to create de novo variation, or to introduce favourable alleles segregating in closely related strains or species [6]
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