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Abstract
Abstract
Background: 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. 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.
Results: In 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.
Conclusions: The 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.
Background: 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. 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.
Results: In 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.
Conclusions: The 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.
| Original language | English |
|---|---|
| Journal | BMC Biotechnology |
| Early online date | 20 Jun 2020 |
| DOIs | |
| Publication status | E-pub ahead of print - 20 Jun 2020 |
Keywords
- CRISPR
- Lentivirus
- Gene editing
- CHSE
- Salmon
- Disease resistance
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AquaLeap: Innovation in Genetics and Breeding to Advance UK Aquaculture Production
Houston, R., Bean, T., Hickey, J. & Whitelaw, B.
1/01/19 → 31/12/22
Project: Research
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Improving resistance to infectious salmon anaemia using genome editing: Novel approaches to tackling viral disease in aquaculture
Houston, R.
1/04/18 → 31/03/22
Project: Research