Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

Remi Gratacap; Tim Regan; Carola E.  Dehler; Samuel A.M.  Martin; Pierre  Boudinot; Bertrand Collet; Ross Houston

doi:10.1186/s12896-020-00626-x

Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

Remi Gratacap, Tim Regan, Carola E. Dehler , Samuel A.M. Martin, Pierre Boudinot, Bertrand Collet, Ross Houston

Royal (Dick) School of Veterinary Studies

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Journal	BMC Biotechnology
Early online date	20 Jun 2020
DOIs	https://doi.org/10.1186/s12896-020-00626-x
Publication status	E-pub ahead of print - 20 Jun 2020

Keywords / Materials (for Non-textual outputs)

CRISPR
Lentivirus
Gene editing
CHSE
Salmon
Disease resistance

Access to Document

10.1186/s12896-020-00626-x

s12896-020-00626-x
© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder ...
Final published version, 833 KBLicence: Creative Commons: Attribution (CC-BY)

AquaLeap: Innovation in Genetics and Breeding to Advance UK Aquaculture Production
Houston, R. (Principal Investigator), Bean, T. (Co-investigator), Hickey, J. (Co-investigator) & Whitelaw, B. (Co-investigator)
Biotechnology and Biological Sciences Research Council
1/01/19 → 31/12/22
Project: Research
Improving resistance to infectious salmon anaemia using genome editing: Novel approaches to tackling viral disease in aquaculture
Houston, R. (Principal Investigator)
Biotechnology and Biological Sciences Research Council
1/04/18 → 31/03/22
Project: Research

Cite this

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title = "Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system",

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.",

keywords = "CRISPR, Lentivirus, Gene editing, CHSE, Salmon, Disease resistance",

author = "Remi Gratacap and Tim Regan and Dehler, \{Carola E.\} and Martin, \{Samuel A.M.\} and Pierre Boudinot and Bertrand Collet and Ross Houston",

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AU - Gratacap, Remi

AU - Regan, Tim

AU - Dehler , Carola E.

AU - Martin, Samuel A.M.

AU - Boudinot, Pierre

AU - Collet, Bertrand

AU - Houston, Ross

PY - 2020/6/20

Y1 - 2020/6/20

N2 - 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.

AB - 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.

KW - CRISPR

KW - Lentivirus

KW - Gene editing

KW - CHSE

KW - Salmon

KW - Disease resistance

U2 - 10.1186/s12896-020-00626-x

DO - 10.1186/s12896-020-00626-x

M3 - Article

SN - 1472-6750

JO - BMC Biotechnology

JF - BMC Biotechnology

ER -

Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

Abstract

Keywords / Materials (for Non-textual outputs)

Access to Document

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Projects

AquaLeap: Innovation in Genetics and Breeding to Advance UK Aquaculture Production

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

Cite this