Potato virus X-delivered CRISPR activation programs lead to strong endogenous gene induction and transient metabolic reprogramming in Nicotiana benthamiana.

Programmable transcriptional regulators based on CRISPR architecture are promising tools for the induction of plant gene expression. In plants, CRISPR gene activation is effective with respect to modulating development processes, such as the flowering time or customizing biochemical composition. The most widely used method for delivering CRISPR components into the plant is Agrobacterium tumefaciens-mediated genetic transformation, either transient or stable. However, as a result of their versatility and their ability to move, virus-derived systems have emerged as an interesting alternative for supplying the CRISPR components to the plant, in particular guide RNA (gRNA), which represents the variable component in CRISPR strategies. In the present study, we describe a Potato virus X-derived vector that, upon agroinfection in Nicotiana benthamiana, serves as a vehicle for delivery of gRNAs, producing highly specific virus-induced gene activation. The system works in combination with a N. benthamiana transgenic line carrying the remaining complementary CRISPR gene activation components, specifically the dCasEV2.1 cassette, which has been shown previously to mediate strong programmable transcriptional activation in plants. Using an easily scalable, non-invasive spraying method, we show that gRNA-mediated activation programs move locally and systemically, generating a strong activation response in different target genes. Furthermore, by activating three different endogenous MYB transcription factors, we demonstrate that this Potato virus X-based virus-induced gene reprogramming strategy results in program-specific metabolic fingerprints in N. benthamiana leaves characterized by distinctive phenylpropanoid-enriched metabolite profiles.

Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector.

Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.

Strong and tunable anti-CRISPR/Cas activities in plants.

CRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti-CRISPR proteins as tools to prevent CRISPR/Cas-mediated gene editing and gene activation in plants has not been explored yet. This study describes the characterization of two anti-CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana. Our results demonstrate that AcrIIA4 prevents site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. In a similar way, AcrVA1 is able to prevent CRISPR/Cas12a-mediated gene editing. Moreover, using a N. benthamiana line constitutively expressing Cas9, we show that the viral delivery of AcrIIA4 using Tobacco etch virus is able to completely abolish the high editing levels obtained when the guide RNA is delivered with a virus, in this case Potato virus X. We also show that AcrIIA4 and AcrVA1 repress CRISPR/dCas-based transcriptional activation of reporter genes. In the case of AcrIIA4, this repression occurs in a highly efficient, dose-dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin-regulated activation of a downstream reporter gene. The strong anti-Cas activity of AcrIIA4 and AcrVA1 reported here opens new possibilities for customized control of gene editing and gene expression in plants.

RNA virus-mediated gene editing for tomato trait breeding.

Virus-induced genome editing (VIGE) leverages viral vectors to deliver CRISPR-Cas components into plants for robust and flexible trait engineering. We describe here a VIGE approach applying an RNA viral vector based on potato virus X (PVX) for genome editing of tomato, a mayor horticultural crop. Viral delivery of single-guide RNA into Cas9-expressing lines resulted in efficient somatic editing with indel frequencies up to 58%. By proof-of-concept VIGE of PHYTOENE DESATURASE (PDS) and plant regeneration from edited somatic tissue, we recovered loss-of-function pds mutant progeny displaying an albino phenotype. VIGE of STAYGREEN 1 (SGR1), a gene involved in fruit color variation, generated sgr1 mutant lines with recolored red-brown fruits and high chlorophyll levels. The obtained editing events were heritable, overall confirming the successful breeding of fruit color. Altogether, our VIGE approach offers great potential for accelerated functional genomics of tomato variation, as well as for precision breeding of novel tomato traits.

Tools and targets: The dual role of plant viruses in CRISPR-Cas genome editing.

The recent emergence of tools based on the clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have revolutionized targeted genome editing, thus holding great promise to both basic plant science and precision crop breeding. Conventional approaches for the delivery of editing components rely on transformation technologies or transient delivery to protoplasts, both of which are time-consuming, laborious, and can raise legal concerns. Alternatively, plant RNA viruses can be used as transient delivery vectors of CRISPR-Cas reaction components, following the so-called virus-induced genome editing (VIGE). During the last years, researchers have been able to engineer viral vectors for the delivery of CRISPR guide RNAs and Cas nucleases. Considering that each viral vector is limited to its molecular biology properties and a specific host range, here we review recent advances for improving the VIGE toolbox with a special focus on strategies to achieve tissue-culture-free editing in plants. We also explore the utility of CRISPR-Cas technology to enhance biotic resistance with a special focus on plant virus diseases. This can be achieved by either targeting the viral genome or modifying essential host susceptibility genes that mediate in the infection process. Finally, we discuss the challenges and potential that VIGE holds in future breeding technologies.

Heritable CRISPR-Cas9 editing of plant genomes using RNA virus vectors.

Viral vectors hold enormous potential for genome editing in plants as transient delivery vehicles of CRISPR-Cas components. Here, we describe a protocol to assemble plant viral vectors for single-guide RNA (sgRNA) delivery. The obtained viral constructs are based on compact T-DNA binary vectors of the pLX series and are delivered into Cas9-expressing plants through agroinoculation. This approach allows rapidly assessing sgRNA design for plant genome targeting, as well as the recovery of progeny with heritable mutations at targeted loci. For complete details on the use and execution of this protocol, please refer to Uranga et al. (2021)1 and Aragonés et al. (2022).2.

CRISPR-Cas12a Genome Editing at the Whole-Plant Level Using Two Compatible RNA Virus Vectors.

The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial CRISPR components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-Cas nuclease. Here, we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors derived from tobacco etch virus (TEV; genus Potyvirus) and potato virus X (PVX; genus Potexvirus), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.