Multiplexed Gene Editing and Protein Overexpression Using a Tobacco mosaic virus Viral Vector

Abstract

Development of CRISPR/Cas9 transient gene editing screening tools in plant biology has been hindered by difficulty of delivering high quantities of biologically active single guide RNAs (sgRNAs). Furthermore, it has been largely accepted that in vivo generated sgRNAs need to be devoid of extraneous nucleotides, which has limited sgRNA expression by delivery vectors. Here, we increased cellular concentrations of sgRNA by transiently delivering sgRNAs using a -derived vector (TRBO) designed with 5' and 3' sgRNA proximal nucleotide-processing capabilities. To demonstrate proof-of-principle, we used the TRBO-sgRNA delivery platform to target GFP in (16c) plants, and gene editing was accompanied by loss of GFP expression. Surprisingly, indel (insertions and deletions) percentages averaged nearly 70% within 7 d postinoculation using the TRBO-sgRNA constructs, which retained 5' nucleotide overhangs. In contrast, and in accordance with current models, in vitro Cas9 cleavage assays only edited DNA when 5' sgRNA nucleotide overhangs were removed, suggesting a novel processing mechanism is occurring in planta. Since the Cas9/TRBO-sgRNA platform demonstrated sgRNA flexibility, we targeted the paralogs with one sgRNA and also multiplexed two sgRNAs using a single TRBO construct, resulting in indels in three genes. TRBO-mediated expression of an RNA transcript consisting of an sgRNA adjoining a GFP protein coding region produced indels and viral-based GFP overexpression. In conclusion, multiplexed delivery of sgRNAs using the TRBO system offers flexibility for gene expression and editing and uncovered novel aspects of CRISPR/Cas9 biology.

Multiplex gene editing in rice with simplified CRISPR‐Cpf1 and CRISPR‐Cas9 systems

“…In previous reports of STU systems for Cas9 in plants, special processing devices, such as the hammerhead ribozyme (Tang et al ) or tRNA element (Ding et al ), was used to release individual sgRNAs. Recently, it was shown that an RNA transcript, consisting of a sgRNA adjoining a GFP protein coding region in the Tobacco mosaic virus , caused target indels and viral‐based GFP overexpression, simultaneously (Cody et al ). Inspired by this finding, we speculated that the Cas9 protein and its sgRNAs may be assembled into a SSTU system, without any additional processing machinery.…”

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

“…Two tobraviruses, TRV and PEBV, can be used to edit N. benthamiana and Arabidopsis genes of interest (Ali et al, 2015(Ali et al, , 2018. 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, 2017). Very recently, BNYVV has also been used for gRNA delivery in N. benthamiana (Jiang et al, 2019).…”

“…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 ., ).…”

CRISPR-based tools for plant genome engineering

“…Both Tobacco Rattle Virus (TRV) [29] and Cabbage Leaf Curl Virus (CaLCuV) [30] have been adapted for this purpose. More recently, Cody et al [31] demonstrated targeted mutagenesis induced by transient expression of Cas9 simultaneous with delivery of sgRNAs from a tobacco mosaic virus-derived vector.…”