Abstract
Plant viruses can be engineered to carry sequences that direct silencing of target host genes, expression of heterologous proteins, or editing of host genes. A set of foxtail mosaic virus (FoMV) vectors was developed that can be used for transient gene expression and single guide RNA delivery for Cas9‐mediated gene editing in maize, Setaria viridis, and Nicotiana benthamiana. This was accomplished by duplicating the FoMV capsid protein subgenomic promoter, abolishing the unnecessary open reading frame 5A, and inserting a cloning site containing unique restriction endonuclease cleavage sites immediately after the duplicated promoter. The modified FoMV vectors transiently expressed green fluorescent protein (GFP) and bialaphos resistance (BAR) protein in leaves of systemically infected maize seedlings. GFP was detected in epidermal and mesophyll cells by epifluorescence microscopy, and expression was confirmed by Western blot analyses. Plants infected with FoMV carrying the bar gene were temporarily protected from a glufosinate herbicide, and expression was confirmed using a rapid antibody‐based BAR strip test. Expression of these proteins was stabilized by nucleotide substitutions in the sequence of the duplicated promoter region. Single guide RNAs expressed from the duplicated promoter mediated edits in the N. benthamiana Phytoene desaturase gene, the S. viridis Carbonic anhydrase 2 gene, and the maize HKT1 gene encoding a potassium transporter. The efficiency of editing was enhanced in the presence of synergistic viruses and a viral silencing suppressor. This work expands the utility of FoMV for virus‐induced gene silencing (VIGS), virus‐mediated overexpression (VOX), and virus‐enabled gene editing (VEdGE) in monocots.
Highlights
Plant viruses provide surprisingly versatile technology platforms enabling the expression of a wide array of coding and non-coding sequences in plants (Pasin, Menzel, & Daros, 2019)
A set of foxtail mosaic virus (FoMV) vectors was developed that can be used for transient gene expression and single guide RNA delivery for Cas9-mediated gene editing in maize, Setaria viridis, and Nicotiana benthamiana
It has been demonstrated that plant viruses or their derivatives can be used to deliver CRISPR-Cas reagents consisting of single guide RNAs, 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)
Summary
Plant viruses provide surprisingly versatile technology platforms enabling the expression of a wide array of coding and non-coding sequences in plants (Pasin, Menzel, & Daros, 2019). 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 Cas (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). Systemic gene editing was observed in leaves of all three species, and it was enhanced in the presence of an RNA silencing suppressor or a synergistic virus, demonstrating that FoMV can enable gene editing through the expression of functional gRNAs
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