CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines

Finfish production has seen over three-fold increase in the past 30 years (1990–2020), and Atlantic salmon (A. salmon; salmo salar ) accounted for approximately 32.6% of the total marine and coastal aquaculture of all finfish species in the year 2020, making it one of the most profitable farmed fish...

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Published in:International Journal of Molecular Sciences
Main Authors: Trygve A. H. Strømsnes, Sebastian E. Schmidke, Mitra Azad, Øyvind Singstad, Idun M. Grønsberg, Roy A. Dalmo, Arinze S. Okoli
Format: Article in Journal/Newspaper
Language:English
Published: MDPI AG 2022
Subjects:
Online Access:https://doi.org/10.3390/ijms232416218
https://doaj.org/article/666b71bca78743528a4f64f23ab39dbc
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spelling ftdoajarticles:oai:doaj.org/article:666b71bca78743528a4f64f23ab39dbc 2023-05-15T15:32:50+02:00 CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines Trygve A. H. Strømsnes Sebastian E. Schmidke Mitra Azad Øyvind Singstad Idun M. Grønsberg Roy A. Dalmo Arinze S. Okoli 2022-12-01T00:00:00Z https://doi.org/10.3390/ijms232416218 https://doaj.org/article/666b71bca78743528a4f64f23ab39dbc EN eng MDPI AG https://www.mdpi.com/1422-0067/23/24/16218 https://doaj.org/toc/1661-6596 https://doaj.org/toc/1422-0067 doi:10.3390/ijms232416218 1422-0067 1661-6596 https://doaj.org/article/666b71bca78743528a4f64f23ab39dbc International Journal of Molecular Sciences, Vol 23, Iss 16218, p 16218 (2022) CRISPR/Cas9 A. salmon gene editing ASK-1 SHK-1 CHSE-214 Biology (General) QH301-705.5 Chemistry QD1-999 article 2022 ftdoajarticles https://doi.org/10.3390/ijms232416218 2022-12-30T19:31:35Z Finfish production has seen over three-fold increase in the past 30 years (1990–2020), and Atlantic salmon (A. salmon; salmo salar ) accounted for approximately 32.6% of the total marine and coastal aquaculture of all finfish species in the year 2020, making it one of the most profitable farmed fish species globally. This growth in production is, however, threatened by a number of problems which can be solved using the CRISPR/Cas technology. In vitro applications of CRISPR/Cas using cell lines can complement its in vivo applications, but salmonids-derived cell lines are difficult to gene edit because they grow slowly, are difficult to transfect and isolate single clones of gene-edited cells. While clonal isolation of the gene-edited Chinook salmon cell line (CHSE-214) has successfully been performed, there is no report of successful clonal isolation of the gene-edited A. salmon ASK-1 and SHK-1cell lines. In the current study, two gene loci— cr2 and mmp9 of A. salmon—were efficiently edited using the ribonucleoprotein (RNP) and plasmid CRISPR/Cas9 strategies. Edited cells were enriched using flow cytometer-activated cell sorting (FACS), followed by clonal isolation and expansion of edited cells. The study both confirms the recent report of the highly efficient editing of these widely used model cell lines, as well as extends the frontline in the single-cell cloning of gene-edited salmonids cells. The report also highlights the pitfalls and future directions in the application of CRISPR/Cas9 in these cells. Article in Journal/Newspaper Atlantic salmon Salmo salar Directory of Open Access Journals: DOAJ Articles International Journal of Molecular Sciences 23 24 16218
institution Open Polar
collection Directory of Open Access Journals: DOAJ Articles
op_collection_id ftdoajarticles
language English
topic CRISPR/Cas9
A. salmon
gene editing
ASK-1
SHK-1
CHSE-214
Biology (General)
QH301-705.5
Chemistry
QD1-999
spellingShingle CRISPR/Cas9
A. salmon
gene editing
ASK-1
SHK-1
CHSE-214
Biology (General)
QH301-705.5
Chemistry
QD1-999
Trygve A. H. Strømsnes
Sebastian E. Schmidke
Mitra Azad
Øyvind Singstad
Idun M. Grønsberg
Roy A. Dalmo
Arinze S. Okoli
CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines
topic_facet CRISPR/Cas9
A. salmon
gene editing
ASK-1
SHK-1
CHSE-214
Biology (General)
QH301-705.5
Chemistry
QD1-999
description Finfish production has seen over three-fold increase in the past 30 years (1990–2020), and Atlantic salmon (A. salmon; salmo salar ) accounted for approximately 32.6% of the total marine and coastal aquaculture of all finfish species in the year 2020, making it one of the most profitable farmed fish species globally. This growth in production is, however, threatened by a number of problems which can be solved using the CRISPR/Cas technology. In vitro applications of CRISPR/Cas using cell lines can complement its in vivo applications, but salmonids-derived cell lines are difficult to gene edit because they grow slowly, are difficult to transfect and isolate single clones of gene-edited cells. While clonal isolation of the gene-edited Chinook salmon cell line (CHSE-214) has successfully been performed, there is no report of successful clonal isolation of the gene-edited A. salmon ASK-1 and SHK-1cell lines. In the current study, two gene loci— cr2 and mmp9 of A. salmon—were efficiently edited using the ribonucleoprotein (RNP) and plasmid CRISPR/Cas9 strategies. Edited cells were enriched using flow cytometer-activated cell sorting (FACS), followed by clonal isolation and expansion of edited cells. The study both confirms the recent report of the highly efficient editing of these widely used model cell lines, as well as extends the frontline in the single-cell cloning of gene-edited salmonids cells. The report also highlights the pitfalls and future directions in the application of CRISPR/Cas9 in these cells.
format Article in Journal/Newspaper
author Trygve A. H. Strømsnes
Sebastian E. Schmidke
Mitra Azad
Øyvind Singstad
Idun M. Grønsberg
Roy A. Dalmo
Arinze S. Okoli
author_facet Trygve A. H. Strømsnes
Sebastian E. Schmidke
Mitra Azad
Øyvind Singstad
Idun M. Grønsberg
Roy A. Dalmo
Arinze S. Okoli
author_sort Trygve A. H. Strømsnes
title CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines
title_short CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines
title_full CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines
title_fullStr CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines
title_full_unstemmed CRISPR/Cas9-Mediated Gene Editing in Salmonids Cells and Efficient Establishment of Edited Clonal Cell Lines
title_sort crispr/cas9-mediated gene editing in salmonids cells and efficient establishment of edited clonal cell lines
publisher MDPI AG
publishDate 2022
url https://doi.org/10.3390/ijms232416218
https://doaj.org/article/666b71bca78743528a4f64f23ab39dbc
genre Atlantic salmon
Salmo salar
genre_facet Atlantic salmon
Salmo salar
op_source International Journal of Molecular Sciences, Vol 23, Iss 16218, p 16218 (2022)
op_relation https://www.mdpi.com/1422-0067/23/24/16218
https://doaj.org/toc/1661-6596
https://doaj.org/toc/1422-0067
doi:10.3390/ijms232416218
1422-0067
1661-6596
https://doaj.org/article/666b71bca78743528a4f64f23ab39dbc
op_doi https://doi.org/10.3390/ijms232416218
container_title International Journal of Molecular Sciences
container_volume 23
container_issue 24
container_start_page 16218
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