Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors
Viral vectors provide a quick and effective way to express exogenous sequences in eukaryotic cells and to engineer eukaryotic genomes through the delivery of CRISPR/Cas components. Here, we present JoinTRV, an improved vector system based on tobacco rattle virus (TRV) that simplifies gene silencing...
| Autores principales: | , , , |
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| Formato: | Artículo |
| Lenguaje: | Inglés |
| Publicado: |
Wiley
2022
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| Acceso en línea: | https://hdl.handle.net/20.500.12955/1803 https://doi.org/10.1002/biot.202100504 |
| _version_ | 1855490173616259072 |
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| author | Aragonés, Verónica Aliaga, Flavio Pasin, Fabio Daròs, José Antonio |
| author_browse | Aliaga, Flavio Aragonés, Verónica Daròs, José Antonio Pasin, Fabio |
| author_facet | Aragonés, Verónica Aliaga, Flavio Pasin, Fabio Daròs, José Antonio |
| author_sort | Aragonés, Verónica |
| collection | Repositorio INIA |
| description | Viral vectors provide a quick and effective way to express exogenous sequences in eukaryotic cells and to engineer eukaryotic genomes through the delivery of CRISPR/Cas components. Here, we present JoinTRV, an improved vector system based on tobacco rattle virus (TRV) that simplifies gene silencing and genome editing logistics. Our system consists of two mini T-DNA vectors from which TRV RNA1 (pLX-TRV1) and an engineered version of TRV RNA2 (pLX-TRV2) are expressed. The two vectors have compatible origins that allow their cotransformation and maintenance into a single Agrobacterium cell, as well as their simultaneous delivery to plants by a one-Agrobacterium/two-vector approach. The JoinTRV vectors are substantially smaller than those of any known TRV vector system, and pLX-TRV2 can be easily customized to express desired sequences by one-step digestion-ligation and homology-based cloning. The system was successfully used in Nicotiana benthamiana for launching TRV infection, for recombinant protein production, as well as for robust virus-induced gene silencing (VIGS) of endogenous transcripts using bacterial suspensions at low optical densities. JoinTRV-mediated delivery of single-guide RNAs in a Cas9 transgenic host allowed somatic cell editing efficiencies of ≈90%; editing events were heritable and >50% of the progeny seedlings showed mutations at the targeted loci. |
| format | Artículo |
| id | INIA1803 |
| institution | Institucional Nacional de Innovación Agraria |
| language | Inglés |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Wiley |
| publisherStr | Wiley |
| record_format | dspace |
| spelling | INIA18032022-11-21T20:02:06Z Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors Aragonés, Verónica Aliaga, Flavio Pasin, Fabio Daròs, José Antonio CRISPR/Cas9 Heritable gene editing PLX binary vector multiplexing Tobacco rattle virus Virus-induced gene silencing (VIGS) Virus-induced genome editing (VIGE) https://purl.org/pe-repo/ocde/ford#4.04.00 Viral vectors provide a quick and effective way to express exogenous sequences in eukaryotic cells and to engineer eukaryotic genomes through the delivery of CRISPR/Cas components. Here, we present JoinTRV, an improved vector system based on tobacco rattle virus (TRV) that simplifies gene silencing and genome editing logistics. Our system consists of two mini T-DNA vectors from which TRV RNA1 (pLX-TRV1) and an engineered version of TRV RNA2 (pLX-TRV2) are expressed. The two vectors have compatible origins that allow their cotransformation and maintenance into a single Agrobacterium cell, as well as their simultaneous delivery to plants by a one-Agrobacterium/two-vector approach. The JoinTRV vectors are substantially smaller than those of any known TRV vector system, and pLX-TRV2 can be easily customized to express desired sequences by one-step digestion-ligation and homology-based cloning. The system was successfully used in Nicotiana benthamiana for launching TRV infection, for recombinant protein production, as well as for robust virus-induced gene silencing (VIGS) of endogenous transcripts using bacterial suspensions at low optical densities. JoinTRV-mediated delivery of single-guide RNAs in a Cas9 transgenic host allowed somatic cell editing efficiencies of ≈90%; editing events were heritable and >50% of the progeny seedlings showed mutations at the targeted loci. Abstract. 1. Introduction. 2. Experimental section. 3. Results. 4. Discussion. References. 2022-08-03T13:17:43Z 2022-08-03T13:17:43Z 2022-07-14 info:eu-repo/semantics/article Aragonés, V.; Aliaga, F.; Pasin, F. & Darós, J. (2022). Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors. Biotechnology Journal Volume 17, Issue 7, 2100504. doi: 10.1002/biot.202100504 https://hdl.handle.net/20.500.12955/1803 Biotechnology Journal https://doi.org/10.1002/biot.202100504 eng https://doi.org/10.1002/biot.202100504 info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by/4.0/ application/pdf application/pdf Wiley Instituto Nacional de Innovación Agraria Repositorio Institucional - INIA |
| spellingShingle | CRISPR/Cas9 Heritable gene editing PLX binary vector multiplexing Tobacco rattle virus Virus-induced gene silencing (VIGS) Virus-induced genome editing (VIGE) https://purl.org/pe-repo/ocde/ford#4.04.00 Aragonés, Verónica Aliaga, Flavio Pasin, Fabio Daròs, José Antonio Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors |
| title | Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors |
| title_full | Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors |
| title_fullStr | Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors |
| title_full_unstemmed | Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors |
| title_short | Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors |
| title_sort | simplifying plant gene silencing and genome editing logistics by a one agrobacterium system for simultaneous delivery of multipartite virus vectors |
| topic | CRISPR/Cas9 Heritable gene editing PLX binary vector multiplexing Tobacco rattle virus Virus-induced gene silencing (VIGS) Virus-induced genome editing (VIGE) https://purl.org/pe-repo/ocde/ford#4.04.00 |
| url | https://hdl.handle.net/20.500.12955/1803 https://doi.org/10.1002/biot.202100504 |
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