Highly efficient heritable genome editing in wheat using an RNA virus and bypassing tissue culture

Li, Tingdong; Hu, Jiajia; Sun, Yu; Li, Boshu; Zhang, Dingliang; Li, Wenli; Liu, Jin-Xing; Li, Dawei; Gao, Caixia; Zhang, Yongliang; Wang, Yanpeng

doi:10.1016/j.molp.2021.07.010

Cited by 102 publications

(140 citation statements)

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“…This strategy has also been implemented in monocot crops such as wheat with surprising results. Cas9 transgenic wheat lines were infected with BSMV expressing either unaugmented gRNAs or augmented gRNAs with wheat FT ( Li et al, 2021 ). Unexpectedly, gene edits were present in almost all M1 progeny from plants infected with unaugmented gRNA whereas, the progeny of plants infected with BSMV- FT augmented gRNA were rarely edited.…”

Section: Virus Induced Genome Editingmentioning

confidence: 99%

“…To quickly remove the Cas9 transgene, the authors used anthers from plants infected with BSMV-gRNA for pollination of wild type plants. The progeny was self-pollinated to obtain edited plants lacking the Cas9 transgene ( Li et al, 2021 ). This BSMV VIGE system has the potential to circumvent the need for tissue culture in genome editing pipelines involving monocots such as maize and barley which BSMV infects.…”

Section: Virus Induced Genome Editingmentioning

confidence: 99%

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Non-GM Genome Editing Approaches in Crops

2021

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CRISPR/Cas-based genome editing technologies have the potential to fast-track large-scale crop breeding programs. However, the rigid cell wall limits the delivery of CRISPR/Cas components into plant cells, decreasing genome editing efficiency. Established methods, such as Agrobacterium tumefaciens-mediated or biolistic transformation have been used to integrate genetic cassettes containing CRISPR components into the plant genome. Although efficient, these methods pose several problems, including 1) The transformation process requires laborious and time-consuming tissue culture and regeneration steps; 2) many crop species and elite varieties are recalcitrant to transformation; 3) The segregation of transgenes in vegetatively propagated or highly heterozygous crops, such as pineapple, is either difficult or impossible; and 4) The production of a genetically modified first generation can lead to public controversy and onerous government regulations. The development of transgene-free genome editing technologies can address many problems associated with transgenic-based approaches. Transgene-free genome editing have been achieved through the delivery of preassembled CRISPR/Cas ribonucleoproteins, although its application is limited. The use of viral vectors for delivery of CRISPR/Cas components has recently emerged as a powerful alternative but it requires further exploration. In this review, we discuss the different strategies, principles, applications, and future directions of transgene-free genome editing methods.

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Section: Virus Induced Genome Editingmentioning

confidence: 99%

Section: Virus Induced Genome Editingmentioning

confidence: 99%

Non-GM Genome Editing Approaches in Crops

2021

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“…To generate gene-edited plants without tissue culture, plant viral vectors have been engineered; these vectors express SpCas9 proteins and/or gRNA in plants ( Ali et al, 2015 ; Ariga et al, 2020 ; Ellison et al, 2020 ; Lei et al, 2021 ; Li et al, 2021 ; Wang et al, 2017 ; Zhang et al, 2020 ). This system is called the virus-induced genome editing (VIGE) system ( Oh et al, 2021a ).…”

Section: Introductionmentioning

confidence: 99%

“…Rather than focus on the delivery of both SpCas9 and gRNA, scientists have focused on the delivery of gRNA into SpCas9-expressing meristematic cells. This approach results in the production of gene-edited seeds ( Ellison et al, 2020 ; Lei et al, 2021 ; Li et al, 2021 ). Guide RNA fused with the mobile Flowering Locus T ( FT ) mRNA sequence enabled the acquisition of heritable mutants with a mutation frequency of 65%-100% from the TRV-infected Nicotiana benthamiana ( Ellison et al, 2020 ), which expresses a SpCas9 gene under the control of the 35S PPDK promoter (the 35S promoter fused to the maize C4PPDK basal promoter; Baltes et al, 2015 ; Li et al, 2013 ; Yoo et al, 2007 ) .…”

Section: Introductionmentioning

confidence: 99%

“…FT-fused gRNA induced the heritable mutation with a frequency of 4%-9% from the cotton leaf crumple virus-infected Arabidopsis ( Lei et al, 2021 ), which expressed SpCas9 under the control of YAO and 35S promoter. A barley stripe mosaic virus-derived gRNA also generated heritable mutations (13%-100%) in the transgenic wheat, which expresses SpCas9 under the control of maize ubiquitin promoter ( Li et al, 2021 ). However, whether the promoter used for SpCas9 expression is critical to induce heritable gene editing with virus-delivered gRNA remains unknown.…”

Section: Introductionmentioning

confidence: 99%

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RPS5A Promoter-Driven Cas9 Produces Heritable Virus-Induced Genome Editing in Nicotiana attenuata

Kim

2021

Molecules and Cells

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The virus-induced genome editing (VIGE) system aims to induce targeted mutations in seeds without requiring any tissue culture. Here, we show that tobacco rattle virus (TRV) harboring guide RNA (gRNA) edits germ cells in a wild tobacco, Nicotiana attenuata, that expresses Streptococcus pyogenes Cas9 (SpCas9). We first generated N. attenuata transgenic plants expressing SpCas9 under the control of 35S promoter and infected rosette leaves with TRV carrying gRNA. Gene-edited seeds were not found in the progeny of the infected N. attenuata. Next, the N. attenuata ribosomal protein S5 A (RPS5A) promoter fused to SpCas9 was employed to induce the heritable gene editing with TRV. The RPS5A promoter-driven SpCas9 successfully produced monoallelic mutations at three target genes in N. attenuata seeds with TRV-delivered guide RNA. These monoallelic mutations were found in 2%-6% seeds among M 1 progenies. This editing method provides an alternative way to increase the heritable editing efficacy of VIGE.

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