A simple and efficient strategy to produce transgene-free gene edited plants in one generation using paraquat resistant 1 as a selection marker

Kong, Xiangjiu; Pan, Wenbo; Zhang, Tingyu; Liu, Lijing; Zhang, Huawei

doi:10.3389/fpls.2022.1051991

Cited by 4 publications

(11 citation statements)

References 41 publications

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“…Notably, loss-offunction mutations in the two close paralogs OsLAT1/OsPUT1 and OsLAT7/OsPUT2 did not convey resistance under these conditions. An independent study performed in parallel con rmed that loss-offunction mutations in AtPAR1 generated by CRIPSR/Cas9 can be used to identify edited lines (15). A similar approach that targeted the same three paralogs in rice as targeted here using a single construct showed that the triple oslat5/osput3/ospar1, oslat1/osput1, oslat7/osput2 mutants were also resistant to MV (30).…”

Section: Introductionmentioning

confidence: 75%

Loss-of-function mutation in the polyamine transporter gene OsLAT5 as a potential selectable marker for genome editing

Schenstnyi,

Zhang,

Liu

et al. 2024

Preprint

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Genome editing by TALENs and CRISPR/Cas has become routine tools. During stable plant transformation, genes coding for editing enzymes, e.g., Cas9, guide RNAs (gRNA), and selectable markers are integrated into the nuclear genome. Identification of successful transformants relies on selectable or screenable markers, typically genes providing resistance to herbicides or antibiotics. Selectable markers use a substantial portion of the T-DNA, hence reducing transfer efficiency by limiting the effective number of TALENs or guide/pegRNAs that can be used. Marker genes are frequently subject to gene silencing. Here, we generated loss-of-function mutations in PUT/LAT-type polyamine transporter family genes to confer resistance to methylviologen (MV). As proof of concept, CRISPR/Cas9 constructs with gRNAs were constructed to target three close homologs OsLAT1, OsLAT5, and OsLAT7. Loss of OsLAT5(also known as OsPUT3 or OsPAR1) function was sufficient to confer resistance to MV in rice seeds, seedlings and calli, validating the editing approach of OsLAT5 to obtain a selectable marker. We discuss use of a gRNA cassette (OsLAT5) as selectable marker and reporter for successful genome editing for optimizing editing protocols.

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Section: Introductionmentioning

confidence: 75%

Loss-of-function mutation in the polyamine transporter gene OsLAT5 as a potential selectable marker for genome editing

Schenstnyi,

Zhang,

Liu

et al. 2024

Preprint

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show abstract

“…While GMO concerns associated with genome editing could be resolved by segregating out the transgenic components through conventional crosses, a significant challenge remains when there is a desire to add muscat flavour to an existing elite grape for which the genetic makeup should otherwise be entirely maintained. There is extensive ongoing effort in developing transgene‐free editing systems, and one such system is to enrich transiently edited target genes using co‐editing genes such as ALS , MAR1 , and PAR1 as selection markers (Huang et al ., 2023; Kong et al ., 2023; Rinne et al ., 2021). Because most transgene‐free editing systems rely on transient editing, further optimization of various editing components for enhancing editing efficiencies continues to be a key area for future research.…”

Section: Discussionmentioning

confidence: 99%

Editing VvDXS1 for the creation of muscat flavour in Vitis vinifera cv. Scarlet Royal

Yang,

Wheatley,

Meakem

et al. 2024

Plant Biotechnology Journal

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SummaryMuscat flavour represents a group of unique aromatic attributes in some grape varieties. Biochemically, grape berries with muscat flavour produce high levels of monoterpenes. Monoterpene biosynthesis is mainly through the DOXP/MEP pathway, and VvDXS1 encodes the first enzyme in this plastidial pathway of terpene biosynthesis in grapevine. A single‐point mutation resulting in the substitution of a lysine with an asparagine at position 284 in the VvDXS1 protein has previously been identified as the major cause for producing muscat flavour in grapes. In this study, the same substitution in the VvDXS1 protein was successfully created through prime editing in the table grape Vitis vinifera cv. ‘Scarlet Royal’. The targeted point mutation was detected in most of the transgenic vines, with varying editing efficiencies. No unintended mutations were detected in the edited alleles, either by PCR Sanger sequencing or by amplicon sequencing. More than a dozen edited vines were identified with an editing efficiency of more than 50%, indicating that these vines were likely derived from single cells in which one allele was edited. These vines had much higher levels of monoterpenes in their leaves than the control, similar to what was found in leaf samples between field‐grown muscat and non‐muscat grapes.

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“…Here, we developed a recessive marker that is caused by loss of function of an endogenous rice gene, i.e., LAT5 / PUT3 / PAR1 , to confer resistance to the phytotoxin methylviologen (MV) as a method of selection. Note that, others used the LAT homolog PAR1 from Arabidopsis to establish a similar selection system [14]. Also in parallel, Lyu et al .…”

Section: Discussionmentioning

confidence: 99%

“…Since 1 μM MV blocked callus induction from WT mature embryos (Fig 5C), MV-based selection of lat5 / put3 / par1 calli directly after transformation or transfection is possible. In the case of Arabidopsis , PAR1 null alleles generated by CRISPR-Cas9 were shown to confer resistance to 1 µM and 10µM MV [14]. The authors stated that heritable transgene-free mutations at target loci were identified in the T1 generation .…”

Section: Discussionmentioning

confidence: 99%

“…Notably, loss-of-function mutations in the two close paralogs OsLAT1/OsPUT1 and OsLAT7/OsPUT2 did not convey resistance under these conditions. An independent study performed in parallel confirmed that loss-of-function mutations in AtPAR1 generated by CRIPSR/Cas9 can be used to identify edited lines [14]. A similar approach that targeted the same three paralogs in rice as targeted here using a single construct showed that the triple oslat5/osput3/ospar1, oslat1/osput1, oslat7/osput2 mutants were also resistant to MV [29].…”

Section: Introductionmentioning

confidence: 88%

See 1 more Smart Citation

Loss-of-function mutation in the polyamine transporter geneOsLAT5as a selectable marker for genome editing

Schenstnyi,

Zhang,

Liu

et al. 2023

Preprint

View full text Add to dashboard Cite

Genome editing by TALENs, CRISPR/Cas, base or prime editing have become routine tools. During stable plant transformation, the gene coding for the editing enzyme, e.g.,Cas9,the guide RNAs (gRNAs), alongside a selectable marker are integrated into the nuclear genome. Identification of successful transformants relies on selectable or screenable markers, typically genes providing resistance to antibiotics or herbicides. Selectable markers use a substantial portion of the T-DNA, hence reducing transfer efficiency by limiting the effective number of TALENs or guide/pegRNAs that can be used in parallel. Moreover, marker genes are frequently subject to gene silencing. Here, we generated loss-of-function mutations in PUT/LAT-type polyamine transporter family genes to confer resistance to the phytotoxin methylviologen (MV) as a method for selection. As a proof of concept, CRISPR/Cas9 vectors with gRNAs were constructed to target three close homologsOsLAT1,OsLAT5, andOsLAT7. We show that loss ofOsLAT5(also known asOsPUT3orOsPAR1) function was sufficient to confer resistance to MV in rice seeds, seedlings and calli, validating the editing approach ofOsLAT5to obtain a selectable marker. We discuss the potential of incorporating a gRNA cassette (forOsLAT5) as a selectable marker and a reporter for successful genome editing for optimizing editing protocols.

show abstract

A simple and efficient strategy to produce transgene-free gene edited plants in one generation using paraquat resistant 1 as a selection marker

Cited by 4 publications

References 41 publications

Loss-of-function mutation in the polyamine transporter gene OsLAT5 as a potential selectable marker for genome editing

Loss-of-function mutation in the polyamine transporter gene OsLAT5 as a potential selectable marker for genome editing

Editing VvDXS1 for the creation of muscat flavour in Vitis vinifera cv. Scarlet Royal

Loss-of-function mutation in the polyamine transporter geneOsLAT5as a selectable marker for genome editing

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