Loss of MAR1 Function is a Marker for Co-Selection of CRISPR-Induced Mutations in Plants
In this study, we describe the establishment of the knockout marker gene MAR1 for selection of CRISPR/Cas9-edited Arabidopsis seedlings and tomato explants in tissue culture. MAR1 encodes a transporter that is located in mitochondria and chloroplasts and is involved in iron homeostasis. It also oppo...
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2021-08-01
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Online Access: | https://www.frontiersin.org/articles/10.3389/fgeed.2021.723384/full |
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doaj-8cfab7a7e86347b2b030b2d4d2f39d3f2021-08-09T06:15:46ZengFrontiers Media S.A.Frontiers in Genome Editing2673-34392021-08-01310.3389/fgeed.2021.723384723384Loss of MAR1 Function is a Marker for Co-Selection of CRISPR-Induced Mutations in PlantsJannis RinneClaus-Peter WitteMarco HerdeIn this study, we describe the establishment of the knockout marker gene MAR1 for selection of CRISPR/Cas9-edited Arabidopsis seedlings and tomato explants in tissue culture. MAR1 encodes a transporter that is located in mitochondria and chloroplasts and is involved in iron homeostasis. It also opportunistically transports aminoglycoside antibiotics into these organelles and defects of the gene render plants insensitive to those compounds. Here, we show that mutations of MAR1 induced by the CRISPR system confer kanamycin-resistance to Arabidopsis plants and tomato tissues. MAR1 is single-copy in a variety of plant species and the corresponding proteins form a distinct phylogenetic clade allowing easy identification of MAR1 orthologs in different plants. We demonstrate that in multiplexing approaches, where Arabidopsis seedlings were selected via a CRISPR/Cas9-induced kanamycin resistance mediated by MAR1 mutation, a mutation in a second target gene was observed with higher frequency than in a control population only selected for the presence of the transgene. This so called co-selection has not been shown before to occur in plants. The technique can be employed to select for edited plants, which might be particularly useful if editing events are rare.https://www.frontiersin.org/articles/10.3389/fgeed.2021.723384/fullCRISPRArabidopsistomatoknockout markerMAR1phylogeny |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jannis Rinne Claus-Peter Witte Marco Herde |
spellingShingle |
Jannis Rinne Claus-Peter Witte Marco Herde Loss of MAR1 Function is a Marker for Co-Selection of CRISPR-Induced Mutations in Plants Frontiers in Genome Editing CRISPR Arabidopsis tomato knockout marker MAR1 phylogeny |
author_facet |
Jannis Rinne Claus-Peter Witte Marco Herde |
author_sort |
Jannis Rinne |
title |
Loss of MAR1 Function is a Marker for Co-Selection of CRISPR-Induced Mutations in Plants |
title_short |
Loss of MAR1 Function is a Marker for Co-Selection of CRISPR-Induced Mutations in Plants |
title_full |
Loss of MAR1 Function is a Marker for Co-Selection of CRISPR-Induced Mutations in Plants |
title_fullStr |
Loss of MAR1 Function is a Marker for Co-Selection of CRISPR-Induced Mutations in Plants |
title_full_unstemmed |
Loss of MAR1 Function is a Marker for Co-Selection of CRISPR-Induced Mutations in Plants |
title_sort |
loss of mar1 function is a marker for co-selection of crispr-induced mutations in plants |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Genome Editing |
issn |
2673-3439 |
publishDate |
2021-08-01 |
description |
In this study, we describe the establishment of the knockout marker gene MAR1 for selection of CRISPR/Cas9-edited Arabidopsis seedlings and tomato explants in tissue culture. MAR1 encodes a transporter that is located in mitochondria and chloroplasts and is involved in iron homeostasis. It also opportunistically transports aminoglycoside antibiotics into these organelles and defects of the gene render plants insensitive to those compounds. Here, we show that mutations of MAR1 induced by the CRISPR system confer kanamycin-resistance to Arabidopsis plants and tomato tissues. MAR1 is single-copy in a variety of plant species and the corresponding proteins form a distinct phylogenetic clade allowing easy identification of MAR1 orthologs in different plants. We demonstrate that in multiplexing approaches, where Arabidopsis seedlings were selected via a CRISPR/Cas9-induced kanamycin resistance mediated by MAR1 mutation, a mutation in a second target gene was observed with higher frequency than in a control population only selected for the presence of the transgene. This so called co-selection has not been shown before to occur in plants. The technique can be employed to select for edited plants, which might be particularly useful if editing events are rare. |
topic |
CRISPR Arabidopsis tomato knockout marker MAR1 phylogeny |
url |
https://www.frontiersin.org/articles/10.3389/fgeed.2021.723384/full |
work_keys_str_mv |
AT jannisrinne lossofmar1functionisamarkerforcoselectionofcrisprinducedmutationsinplants AT clauspeterwitte lossofmar1functionisamarkerforcoselectionofcrisprinducedmutationsinplants AT marcoherde lossofmar1functionisamarkerforcoselectionofcrisprinducedmutationsinplants |
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