Pea early-browning virus-mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis

Abstract

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system has enabled efficient genome engineering in diverse plant species. However, delivery of genome engineering reagents, such as the single guide RNA (sgRNA), into plant cells remains challenging. Here, we report the engineering of Tobacco rattle virus (TRV) and Pea early browning virus (PEBV) to deliver one or multiple sgRNAs into Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants that overexpress a nuclear localization signal containing Cas9. Our data showed that TRV and PEBV can deliver sgRNAs into inoculated and systemic leaves, and this resulted in mutagenesis of the targeted genomic loci. Moreover, in N. benthamiana, PEBV-based sgRNA delivery resulted in more targeted mutations than TRV-based delivery. Our data indicate that TRV and PEBV can facilitate plant genome engineering and can be used to produce targeted mutations for functional analysis and other biotechnological applications across diverse plant species. Key message: Delivery of genome engineering reagents into plant cells is challenging and inefficient and this limit the applications of this technology in many plant species. RNA viruses such as TRV and PEBV provide an efficient tool to systemically deliver sgRNAs for targeted genome modification.

Protein expression and gene editing in monocots using foxtail mosaic virus vectors

“…Viruses that carry fragments of plant genes in sense and antisense orientation cause virus-induced gene silencing (VIGS) of the targeted sequence, or microRNA inserts can initiate silencing with high specificity. Most recently, it has been demonstrated that plant viruses or their derivatives can be used to deliver CRISPR-Cas reagents consisting of single guide RNAs (gRNAs), DNA repair templates, and site-specific nucleases such as Cas9 (Ali et al, 2015;Ali, Eid, Ali, & Mahfouz, 2018;Butler, Baltes, Voytas, & Douches, 2016;Cody, Scholthof, & Mirkov, 2017;Dahan-Meir et al, 2018;Gao et al, 2019;Gil-Humanes et al, 2017;Jiang et al, 2019;Mahas, Ali, Tashkandi, & Mahfouz, 2019;Wang et al, 2017). These capabilities show that plant viruses can be useful biotechnological tools for gene function studies in plants, and they can have practical applications as well (Pasin et al, 2019;Zaidi & Mansoor, 2017).…”

A barley stripe mosaic virus‐based guide RNA delivery system for targeted mutagenesis in wheat and maize

“…Currently, four RNA viruses have been developed as VIGE vectors. Two tobraviruses, TRV and PEBV, can be used to edit N. benthamiana and Arabidopsis genes of interest (Ali et al ., , ). TMV was also reported to be able to mediate target gene editing in the local leaves by partially substituting the CP ORF with a gRNA (Cody et al ., ).…”

“…Recent studies indicate that viruses can be developed to deliver gRNAs for targeted mutagenesis in plants (Ali et al ., , ; Cody et al ., ; Gil‐Humanes et al ., ; Jiang et al ., ; Yin et al ., ). Compared to the traditional gRNA delivery methods via Agrobacterium transformation, plant virus‐mediated gRNA delivery systems have several advantages: (1) the gRNAs can accumulate to high levels owing to viral replication and systemic spread in plants and may contribute to a higher genome editing efficiency, (2) multiple functional gRNAs can be expressed from a single viral genome, which provides the potential for multi‐targeted genome editing, (3) phenotypic alterations may appear in infected plants in a relatively short period of time after gene targeting by virus‐induced genome editing (VIGE), and (4) transformation and regeneration of agriculturally important crops such as wheat is laborious and time‐consuming, but VIGE may shorten this period and simplify operation and editing processes of a target gene in the specific tissues.…”

“…Compared to the traditional gRNA delivery methods via Agrobacterium transformation, plant virus‐mediated gRNA delivery systems have several advantages: (1) the gRNAs can accumulate to high levels owing to viral replication and systemic spread in plants and may contribute to a higher genome editing efficiency, (2) multiple functional gRNAs can be expressed from a single viral genome, which provides the potential for multi‐targeted genome editing, (3) phenotypic alterations may appear in infected plants in a relatively short period of time after gene targeting by virus‐induced genome editing (VIGE), and (4) transformation and regeneration of agriculturally important crops such as wheat is laborious and time‐consuming, but VIGE may shorten this period and simplify operation and editing processes of a target gene in the specific tissues. Several plant RNA viruses including tobacco rattle virus (TRV) (Ali et al ., ), pea early‐browning virus (PEBV) (Ali et al ., ), tobacco mosaic virus (TMV) (Cody et al ., ) and beet necrotic yellow vein virus (BNYVV) (Jiang et al ., ) have been reported to enable targeted genome editing in the model plants Nicotiana benthamiana and Arabidopsis thaliana or both. The DNA virus cabbage leaf curl virus (CaLCuV) has also been engineered for plant genome editing in tobacco (Yin et al ., ).…”

Development of Beet necrotic yellow vein virus‐based vectors for multiple‐gene expression and guide RNA delivery in plant genome editing

“…We also demonstrated that BNYVV‐based vectors can be used to deliver gRNA for CRISPR/Cas9 plant genome editing (Figure ). Compared with currently virus‐based gRNA delivery systems (Ali et al ., ,, ; Yin et al ., ), the efficiency of NbPDS3 editing by our system is up to 85% (Figure d), suggesting BNYVV‐based vectors can be used as efficient gRNA delivery tools for genome editing of sugar beet plants. Thus, the engineering and application of BNYVV‐based expression systems in Beta species will provide a valuable platform for insight into functional characterization of sugar beet genes and to promote future sugar beet genome research.…”