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

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

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

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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
Article number	35
Number of pages	9
Journal	BMC Biotechnology
Volume	20
DOIs	https://doi.org/10.1186/s12896-020-00626-x
Publication status	Published - 23 Jun 2020

Keywords

CRISPR
lentivirus
gene editing
CHSE
salmon
disease resistance
Disease resistance
Salmon
Gene editing
Lentivirus
VIRUS
TRANSDUCTION
FUTURE
GENES
VECTORS

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Gratacap_etal_BMC_Biotech_Efficient_CRISPR_VOR
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Final published version, 833 KBLicence: CC BY

Cite this

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

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, Disease resistance, Salmon, Gene editing, Lentivirus, VIRUS, TRANSDUCTION, FUTURE, GENES, VECTORS",

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

note = "The present study was funded by were funded by the Biotechnology and Biological Sciences Research Council (BB/R008612/1, BB/S004343/1 to RH and RG; grant BB/R008973/1 to SM and CD) and the Institute Strategic Programme Grants (BBS/E/D/20002172, BBS/E/D/30002275 and BBS/E/D/10002070, to RH and RG). The funders had no roles in the study design, data collection and analysis, decision to publish or preparation of the manuscript. ",

year = "2020",

month = jun,

day = "23",

doi = "10.1186/s12896-020-00626-x",

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T1 - Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

AU - Gratacap, Remi L

AU - Regan, Tim

AU - Dehler, Carola E

AU - Martin, Samuel A M

AU - Boudinot, Pierre

AU - Collet, Bertrand

AU - Houston, Ross D

N1 - The present study was funded by were funded by the Biotechnology and Biological Sciences Research Council (BB/R008612/1, BB/S004343/1 to RH and RG; grant BB/R008973/1 to SM and CD) and the Institute Strategic Programme Grants (BBS/E/D/20002172, BBS/E/D/30002275 and BBS/E/D/10002070, to RH and RG). The funders had no roles in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

PY - 2020/6/23

Y1 - 2020/6/23

N2 - 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 - 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

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KW - disease resistance

KW - Disease resistance

KW - Salmon

KW - Gene editing

KW - Lentivirus

KW - VIRUS

KW - TRANSDUCTION

KW - FUTURE

KW - GENES

KW - VECTORS

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U2 - 10.1186/s12896-020-00626-x

DO - 10.1186/s12896-020-00626-x

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JO - BMC Biotechnology

JF - BMC Biotechnology

SN - 1472-6750

M1 - 35

ER -

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

Abstract

Keywords

UN SDGs

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