CN111979262A - plant modification - Google Patents

Abstract

The present invention generally relates to methods and materials for gene or genome editing in plants. The present invention provides an expression system for expressing a complete genome editing (GE) system (such as CRISPR Cas9) for editing target sequences in plants, the expression system comprising: at least one nucleotide sequence, the at least one nucleoside The acid sequence includes: (i) a promoter, (ii) a FoMV cDNA, wherein the promoter is operably linked to a FoMV cDNA encoding one or more recombinant Foxtail Mosaic Virus (FoMV) viral vectors, so The one or more recombinant foxtail mosaic virus vectors include at least one subgenomic promoter (SGP) operably linked to a heterologous nucleic acid encoding the GE system. The present invention also provides FoMV RNA transcripts suitable for editing the target.

Description

plant modification

technical field

The present invention generally relates to methods and materials for gene or genome editing in plants.

Background technique

Gene targeting and genome editing (GE) systems hold great promise for specific gene targeting or precise genome editing, whether for functional characterization of plant genes or genetic improvement of crops.

GE systems generally involve the use of engineered nuclease systems. Nucleases can induce double-strand breaks (DSBs) or nicks in target DNA sequences such that repair of the breaks by non-homologous end joining or homology-directed repair can result in knockout of a gene and/or insertion of a sequence of interest (target). to integration). Regulation and/or cleavage of endogenous genes can be achieved through the use of proteins and systems such as the transcriptional activation of zinc finger protein transcription factors (ZFP-TFs), zinc finger nucleases (ZFNs), such as effector transcription factors (TALE-TFs) CRISPR/Cas transcription factors (see e.g. Perez-Pinera et al. (2013) Nature Methods 10:973-976), transcriptional activators such as effector nucleases (TALENs), Ttago nucleases or combining the CRISPR/Cas system with Engineered crRNA/tracr RNA ("single guide RNA") is used in combination to direct specific cleavage.

In plants, one of the key challenges in inducing GE is the delivery of the entire system (nuclease and guide) into cells for efficient expression. This is usually achieved by time-consuming and laborious plant transformation of the whole or part of the system, but not for species that are difficult or stubbornly difficult to transform.

Transient toolboxes such as virus-induced gene silencing (VIGS) ^5,6 , gene complementation (VIGC) ⁷ and flowering (VIF) ^8,9 have been widely used in plants, and several RNA/DNA viruses have been used previously to Expression of GE-only sgRNA components in Cas9-transgenic plants ^10-12 .

WO2014194190 relates to plant gene targeting and genome editing methods in the field of molecular biology and genetic engineering of DNA molecules introduced on specific types of vectors.

Nonetheless, it can be seen that general non-transgenic strategies that can be used to utilize GE technology in plant functional genomics and crop improvement will contribute to the field.

SUMMARY OF THE INVENTION

The inventors have provided a non-transgenic ViGE system using Foxtail Mosaic Virus (FoMV), a positive single-stranded RNA mosaic virus capable of infecting both monocotyledonous and dicotyledonous plants, for simultaneous transient expression with Cas9, sgRNA and The GE system represented by the RNAi suppressor p19 is used to edit target genes in plants.

Although Cas9 transgenic plants have been used in the art as described above (see Kaya et al., Plant and CellPhysiology 58.4(2017):643-649), the present invention is believed to be the first to express Cas9 from a plant viral vector as a complete transient GE expression system a part of.

Accordingly, in one aspect, there is provided a plant expression system for expressing a complete genome editing (GE) system for editing a target sequence in a plant, the expression system comprising at least one nucleotide sequence, the at least one nuclear The nucleotide sequence includes:

(i) a plant-active promoter operably linked to the FoMV cDNA;

(ii) a FoMV cDNA encoding one or more recombinant foxtail mosaic virus (FoMV) viral vectors comprising at least one subgenomic promoter (SGP) ), the at least one subgenomic promoter is operably linked to a heterologous nucleic acid encoding the GE system, and a terminator sequence;

(iii) Terminator sequences.

The present invention also provides a process for producing the expression system, a method for editing a target sequence in plant tissue, an RNA transcript from the expression system, a virus or virus particle that encapsulates the RNA transcript, including the Kits of expression systems, and plant host cells, tissues or whole plants comprising the expression systems.

Preferably, the FoMV vector includes all sequences required for replication and movement in the plant. The cDNA may be within an expression cassette, wherein the cDNA encodes the entire FoMV RNA genome (see eg, Figure 1A), but includes a subgenomic promoter operably linked to the heterologous nucleic acid.

Therefore, the FoMV vector of the expression system is preferably fully functional. Once infiltrated into plant cells, they replicate and form viral particles that move from cell to cell and spread systemically, creating systemic infections even in non-infiltrated leaf tissue.

However, the present invention is based on the use of transient system representations and thus does not employ stable integration of one or more elements of the GE system as the prior art does. Thus, the system can be used to generate "transgenic-free" plant tissue.

In another aspect, the system uses RNA transcripts that have been expressed in vitro directly, eg, extracted from suitable plasmids or other nucleic acids in vitro. Accordingly, the present invention additionally provides an in vitro expression system for expressing one or more RNA transcripts that collectively encode a complete genome editing (GE) system for editing target sequences in plants, wherein the expression system comprises at least one A nucleotide sequence, the at least one nucleotide sequence comprising:

(i) a promoter operably linked to the FoMV cDNA;

(ii) a FoMV cDNA encoding one or more recombinant foxtail mosaic virus (FoMV) viral vectors comprising at least one subgenomic promoter (SGP) ), the at least one subgenomic promoter is operably linked to a heterologous nucleic acid encoding the GE system.

In another aspect, the system can use RNA transcripts that have been expressed outside the plant, eg, RNA isolated in vivo from a suitable plasmid or other encoding nucleic acid in a suitable microorganism or other isolated cell transcript. Accordingly, the present invention additionally provides an in vivo expression system for expressing one or more RNA transcripts that collectively encode a complete genome editing (GE) system for editing target sequences in plants, wherein the expression system comprises at least one A nucleotide sequence, the at least one nucleotide sequence comprising:

(i) a promoter suitable for use in microorganisms or isolated cells operably linked to the FoMV cDNA;

(ii) a FoMV cDNA encoding one or more recombinant foxtail mosaic virus (FoMV) viral vectors comprising at least one subgenomic promoter (SGP) ), the at least one subgenomic promoter is operably linked to a heterologous nucleic acid encoding the GE system, and a terminator sequence;

(iii) Terminator sequences.

In another aspect, one or more isolated RNA transcripts (eg, produced from the expression systems described herein) are provided that collectively encode for editing a target sequence in a plant Complete genome editing (GE) system with RNA transcripts encoding one or more recombinant foxtail mosaic virus (FoMV) viral vectors containing at least one subgenomic promoter (SGP) operably linked to the at least one subgenomic promoter to a heterologous nucleic acid encoding the GE system.

Preferably, the heterologous nucleic acid further comprises an RNAi inhibitor, eg, p19 or a derivative thereof.

Preferably, the GE system consists of (a) Cas9, and (b) a small guide RNA (sgRNA) for targeting the GE system to a target sequence. The sgRNA targets the target sequence, and the Cas9 nuclease cleaves the DNA molecule.

Thus, the expression systems of the present invention may themselves comprise vectors or plasmids suitable for expression in plants or in vitro. They may consist of first and second expression vectors comprising or encoding first and second FoMV viral vectors, each of said first and second FoMV viral vectors comprising a heterologous nucleic acid encoding the GE system, eg , the first vector expresses a nuclease such as Cas9 and the second vector expresses the sgRNA and optionally the repressor.

FoMV is a member of the genus Potexvirus and possesses a broad host range that includes 56 species of grasses and at least 35 species of dicots (Paulsen and Niblett, 1977; Short and Davies, 1987; Petty et al., 1989). ).

FoMV consists of a sense and antisense single-stranded (ss) RNA genome with a 5'-methylguanosine cap, 3'-polyA tail and five major open reading frames (ORFs) and a unique 5A gene (Robertson et al., 2000). The five major ORFs all encode a functional protein (Robertson et al., 2000).

"Plant-active promoter" refers to a nucleotide sequence of transcription initiation of DNA operably linked downstream (ie, in the 3' direction on the sense strand of double-stranded DNA).

"Operably linked" means linked as part of the same nucleic acid molecule in a position and orientation suitable for initiating transcription from a promoter (or subgenomic or other promoter). A nucleic acid operably linked to a promoter is "under transcription initiation regulation" of the promoter.

GE Systems

GE systems generally involve the use of engineered nuclease systems. In particular, nucleases can induce double-strand breaks (DSBs) or nicks in target DNA sequences such that repair of breaks by non-homologous end joining (NHEJ) or homology-directed repair (HDR) can lead to gene knockout and/or insertion of target sequences (targeted integration). Regulation and/or cleavage of endogenous genes can be achieved through the use of proteins and systems such as the transcriptional activation of zinc finger protein transcription factors (ZFP-TFs), zinc finger nucleases (ZFNs), such as effector transcription factors (TALE-TFs) (2013) Nature Methods 10:973-976), transcriptional activators such as effector nucleases (TALENs), Ttago nucleases, or combining the CRISPR/Cas system with Engineered crRNA/tracr RNA ("single guide RNA") is used in combination to direct specific cleavage.

Since the specificity of the CRISPR/Cas system is based on nucleotide pairing rather than protein-DNA interactions, this approach may be simpler, more specific, more efficient, and applicable to plant genomes compared to existing ZFN and TALEN systems edit. WO2014194190…

suppressor

Gene silencing suppressors useful in the present invention are known in the art and described in WO/2007/135480. Preferably, the suppressor is tomato bush dwarf virus P19 or a mutant thereof.

Nucleic Acids and Vectors

Nucleic acids of the invention may be isolated and/or purified, in essentially pure or homogeneous form, or free or substantially free of other nucleic acids. The term "isolated" covers all of these possibilities.

The vector used in the present invention is a "ViGE vector", which means a vector suitable for delivering gene editing systems to plants by replicating viral transcripts in the plant. These can be delivered through expression systems introduced into plants or directly through RNA transcripts. In general, given the present disclosure, one skilled in the art will be able to construct vectors according to the present invention, whether for plants or microbial cells or other isolated cells or expression systems. In addition to the promoter, terminator, the vector may include other regulatory sequences, eg, to define an expression cassette consisting of the modified recombinant FoMV cDNA and the heterologous nucleotide sequence. For details, see, for example, Molecular Cloning: a Laboratory Manual: Second Edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulating nucleic acids, such as preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis, are detailed in Protocols in Molecular Biology, Second Edition, Ausubel et al. eds ., described in John Wiley & Sons, 1992. Specific procedures and vectors that have previously been widely successful in plants are described in Bevan, Nucl. Acids Res. (1984) 12, 8711-8721) and Guerineau and Mullineaux (1993) Plant transformation and expression vectors. See: PlantMolecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp121-148.

In one aspect of the invention, a microbial expression vector is based on a plasmid such as pUC or pBluescript that can be used to transform E. coli expressing T7 RNA polymerase.

In one aspect of the invention, a plant expression vector is based on a plant binary transformation vector, such as pYL44, a pBIN19-based T vector that contains repeated CaMV 35S promoter and NOS terminator (Liu et al., 2002b; also Materials and methods below).

transfer sequence

In one embodiment, the plant expression system further comprises a border sequence that allows transfer of the nucleotide sequence into the plant genome at the entry site, eg, a border sequence extending from Agrobacterium tumefaciens. In these embodiments, the nucleotide sequences are located between border sequences and are capable of insertion into the plant genome under appropriate conditions. Typically, this is achieved by using so-called "Agrobacterium infiltration", which uses Agrobacterium-mediated transient transformation. Briefly, the technology is based on the property of A. tumefaciens to transfer a portion of its DNA ("T-DNA") into a host cell where it may integrate into nuclear DNA. T-DNA is defined by about 25 nucleotides in length around-border sequences. In the present invention, the border sequence is included around the "transfer nucleotide sequence" (T-DNA), wherein the entire vector is introduced into the plant by agro-infiltration, also in the form of a binary transformation vector.

However, such border sequences are not essential for the application of the present invention, which relies on the replication and movement of FoMV viral nucleic acids in plants. Therefore, such boundary sequences may not exist.

plant promoter

Suitable plant-active promoters are well known to those skilled in the art and include the cauliflower mosaic virus 35S (CaMV 35S) gene promoter which is expressed at high levels in virtually all plant tissues. The promoter may in principle be an inducible promoter, such as the maize glutathione-S-transferase isoform II (GST-II-27) gene promoter, which is responsive to the release of an exogenous safener. Activated by administration (WO93/01294, ICI Ltd). The GST-II-27 gene promoter has been shown to be inducible by certain compounds useful in growing plants. Another suitable promoter may be the DEX promoter (Plant Journal (1997) 11:605-612).

In vitro synthesis of RNA transcripts

As an alternative to introducing an expression system into plants, the present invention can be practiced using in vitro or in vivo synthesis of single-stranded FoMV RNA molecules.

In vitro synthesis of single-stranded RNA molecules is a routine laboratory procedure. Modern commercially available multipurpose cloning vectors typically contain a multiple cloning site (MCS) flanked on each side by multiple promoters for different polymerases (e.g. SP6, T7 and T3 phage RNA polymerases, e.g. from ThermoFisher Scientific).

Plasmid templates are typically linearized with restriction enzymes to allow the lost RNA transcripts to have a defined end for synthesis.

Most eukaryotic mRNA molecules have 5'7-methylguanosine residues or cap structures, both of which play a role in the initiation of protein synthesis, protecting mRNA from intracellular nuclease digestion. Capped in vitro transcripts can be synthesized by replacing a portion of GTP with a cap analog (m7G(5')ppp(5')G) in the transcription reaction.

Thus, the in vitro and in vivo FoMV GE expression systems and the FoMV GE RNA transcripts constitute the invention of the present invention.

Subgenomic Promoters (SGPs)

According to the ViGE vector of the present invention, the heterologous GE system nucleic acid is operably linked to a subgenomic promoter recognized by an efficient replicase, resulting in transcription of subgenomic RNA corresponding to all or part of the GE system.

In one embodiment, the SGP is a FoMV SGP, such as the FoMV SGP of coat protein. However, SGPs of other potato panviruses such as Potato X (PVX) (5'-gaacggttaagtttccattgatactcgaaaga-3') can be used in place of the FoMV SGP to control the expression of heterologous nucleic acids encoding the GE system.

The SGP linked to the heterologous nucleic acid encoding the GE system is usually an additional SGP introduced into the FoMV sequence, or a repeating (second) SGP based on the FoMV native SGP. The first subgenomic promoter operably linked to the heterologous sequence is preferably the same sequence as the second subgenomic promoter. In this embodiment, the first and second SGPs may both be from the same FoMVORF, eg, they may both be coat protein (CP) SGPs or both may be triple gene block (TGB) SGPs.

Therefore, some aspects and embodiments herein take as an example an incoming replica (replica) of the FoMV CP SGP, which is 5' away from the local CP SGP and ORF. However, given the present disclosure, one of skill in the art will appreciate that other subgenomic promoters may be used, eg, those from the Tri-Gene Block (TGB) of FoMV.

For example, the two subgenomic promoters include the native FoMV coat protein subgenomic promoter. This can be derived from the 170-bp sequence shown in 5202 to 5371 of SEQ ID NO: 1, although it will be appreciated that slightly longer or shorter versions can also be used. Typically, promoters are 100 to 500 nucleotides in length.

Some specific preferred embodiments will now be discussed:

CRISPR/CAS9

A preferred GE system in the context of this document is the "Clustered Regularly Interspaced Short Palindromic Repeats" (CRISPR)/CRISPR-associated protein (Cas) system ¹ . This system is an adaptive immune mechanism found in bacterial and archaeal species that allows the host to resist pathogens, such as bacteriophages (Barrangou et al., Science 315, 1709-1712 (2007); Marraffini, LA & Sontheimer, EJScience 322, 1843- 1845 (2008); Bhaya, D., Davison, M. & Barrangou, R. Annual review of genetics 45, 273-297 (2011); Garneau, JE et al. Nature 468, 67-71 (2010)). A phage-derived 30 bp DNA fragment is inserted into the CRISPR locus of the host cell and transcribed into CRISPR RNA (crRNA). These form complexes with trans-coding RNA (tracrRNA) and CRISPR-associated (Cas) proteins, and this complex introduces site-specific cleavage at DNA sites that match the crRNA sequence. This mechanism has been used for specific and multiplex GE in eukaryotes ^1-4 .

CRISPR-Cas9 is a type II CRISPR-Cas system. The CRISPR-Cas9 system of Streptococcus pyogenes is used in the art as a simple and versatile tool for RNA-guided genome editing (RGE) in different organisms. In Cas9-mediated RGE, single or dual short RNA molecules (short guide RNAs or sgRNAs) direct Cas9 to target desired DNA sites for genome modification or transcriptional control. sgRNA-Cas9 recognizes target DNA by sgRNA-DNA pairing between the 5'-terminal leader sequence of the sgRNA (called the sgRNA spacer) and one DNA strand (the complementary strand of the protospacer). Cas9 also requires the presence of a protospacer-adjacent motif (PAM) in the target site following the sgRNA-DNA pairing region.

In the expression systems described herein, the Cas9 sequence can include FLAG and/or nuclear localization signals. A non-limiting example of a Cas9 sequence is shown in SEQ ID No: 3, although variants of this Cas9 sequence as well as other GE systems may also be used within the invention described herein.

Compositions and methods for making and using CRISPR-Cas systems are described in US Patent No. 8,697,359, entitled "CRISPR-CAS SYSTEMS AND METHODS FOR ALTERING EXPRESSION OF GENE PRODUCTS," which is incorporated herein by reference in its entirety.

CRISPR-cas9 plasmids for use in plants are commercially available, eg from addgene, see: www.addgene.org/crispr/plant/.

U6 promoter & sgRNA

sgRNA production is typically driven by promoters of small nuclear RNAs (snRNAs) such as U6 and U3 snRNAs (see, e.g., Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X , Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cassystems. Science. 2013; 339(6121):819–823).

SEQ ID No. 4 shows a non-limiting example of a U6 promoter scaffold suitable for use in the present invention. This scaffold also encodes tracrRNA (transactivating crRNA) that plays a role in the maturation of sgRNAs.

The sgRNA typically contains a targeting sequence that corresponds to the target sequence in the target gene and contains a PAM.

suppressor

Gene silencing suppressors useful in the present invention are known in the art and described in WO/2007/135480. They include HcPro from Potatovirus Y, He-Pro from TEV, P19 from TBSV, rgsCam, B2 protein from FHV, small coat protein from CPMV and coat protein from TCV. Preferably, the suppressor is P19 or a mutant thereof.

Preferred mutants are mutated P19 RNAi inhibitors with potent RNAi inhibitory activity but lacking pathogenic function 15 . A non-limiting example is shown in SEQ ID NO:2. Other modified P19 mutants such as Shi et al. (2009) "Suppression of local RNA silencing is not sufficient to promote cell-to-cellmovement of Turnip crinkle virus in Nicotiana benthamiana" Plant Signaling & Behavior 4:1, 15-22 or as in US20100269220 As stated by Scholthof.

Recombinant FoMV cDNA

Any suitable strain of FoMV that produces replicating, infectious FoMV viral transcripts can be used in the present invention.

Within the FoMV sequence, it is preferred to retain all native FoMV ORFs, ie the FoMV RNA-dependent RNA polymerase, triple gene block and coat protein.

However, one or more sequences can be deleted if the nucleic acid can still be used to generate replicating infectious transcripts.

Preferably, the elements in the nucleotide sequence are in the 5' to 3' sequence listed.

Preferably, the vector system is based on FoMV/P19: sgRNA (targeting the desired endogenous gene) and FoMV/Cas9, as described herein, eg with reference to Figure 1 .

For example, the expression system can include FoMV/P19:sgRNApds, in which the PDS sequence is replaced with a different target gene sequence, including the appropriate PAM. The FoMV vector backbone shown in SEQ ID No: 1 contains HpaI/AscI sites to facilitate the cloning of different target gene sequences.

Also included within the scope of the invention are substantially homologous variants of this sequence. In particular, vectors derived from these FoMV vectors and having the characteristics of those vectors (described herein) are also included.

target gene

For ViGE vectors, the GE system will include a "targeting gene", usually within a guide RNA or analog. This corresponds to the sequence in the plant to be edited.

Typically, targeting sequences can be derived from endogenous plant nuclear genes or transgenes.

The methods of the present invention can include identifying PAMs in the complementary strand of a target gene of interest. The methods of the invention can include engineering a sgRNA to be complementary to a selected target, wherein the 5' end of the engineered sgRNA is adjacent to the PAM.

A non-limiting example of a U6 promoter scaffold encoding an sgRNA (in this case, a plant PDS gene) is shown in SEQ ID No. 5.

ViGE is particularly suitable for studying gene function because it can be used to make precise changes to a specific gene, which can provide information about how that gene functions in the body. In such cases, the targeting sequence may not be known, but the methods of the invention can be used to identify its specific phenotype.

An "endogenous" gene is a gene that is present in a specific cell at a specific developmental stage under specific environmental conditions. For example, endogenous nucleic acid may comprise the genome of chromosomes, mitochondria, chloroplasts or other organelles, or naturally occurring episomal nucleic acids. Other endogenous molecules can include proteins such as transcription factors and enzymes.

For the purposes of the present invention, "gene" includes regions of DNA that encode the gene product (see below), as well as all regions of DNA that regulate the production of the gene product, whether or not these regulatory sequences are adjacent to the coding and/or transcription sequences. Thus, genes include, but are not limited to, promoter sequences, terminators, translation regulatory sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, border elements, origins of replication, substrates Attachment sites and locus control regions.

Targeted genes include those that confer traits on plants and thus may require editing using ViGE. For example, specific genes in ripe tomatoes to improve processing and handling characteristics of harvested fruit; genes involved in pollen formation that allow breeders to regenerate F1 hybrids to produce male sterile plants; genes involved in lignin biosynthesis , which improves the quality of pulp made from plant vegetative tissues; genes involved in anthocyanin production, which produce new flower colors; genes involved in regulatory pathways that control developmental or environmental responses, which produce new growth habits Or, for example, disease resistant plants; elimination of toxic secondary metabolites by gene silencing of genes required for toxin production.

Plants to which the present invention has utility

The present invention is particularly applicable to plants as natural hosts (compatible with FoMV).

"Compatible" means able to operate with other components of the system, in which case the FoMV must be able to replicate in the plant in question. These include not only the plants used in the examples below, but also other known hosts in grasses, other monocotyledonous and dicotyledonous plants ¹³ .

The plants may be dicotyledonous or monocotyledonous.

Foxtail millet is a genus of Poaceae and is a model system for understanding functional genomics and photosynthesis of cereal crops and _C4 plants. In preferred embodiments, the plant may thus be a Poaceae and/or C4 plant.

Non-limiting examples of preferred plants include wheat, millet, barley, corn, soybean, rice, cotton, canola (canola, OSR), sugar cane, sugar beet, potato, sorghum, and sunflower.

Other aspects of the invention

One aspect of the present invention is a process for producing a vector as described above substantially as described in the Examples below, eg, introducing the GE System nucleic acid into a cDNA encoding the FoMV genome, optionally with An additional CP subgenomic promoter (SGP) was introduced together.

One aspect of the present invention includes a method of editing a target sequence or gene using ViGE in a plant or plant tissue, the method comprising the steps of introducing into the plant a vector as described above, wherein the vector comprises as A GE sequence of a targeting sequence such that the GE system is expressed in cells in the plant or tissue. The editing may alter the sequence or expression of the gene product of the target sequence, wherein the target sequence is the target gene.

"Plant tissue" is any tissue of a plant within a plant or in culture, including whole plant organs, cuttings, or any group of plant cells organized into structural and functional units.

The plant tissue may be part of an early plant, eg two or three plant stages.

The method is preferably useful for causing non-lethal ViGE of the target gene in plants, eg only associated with mosaic symptoms of mild FoMV infection.

As mentioned above, for introduction into the plant, the vector may be in the form of an Agrobacterium binary vector. The vector is introduced into the plant cell by Agrobacterium-mediated T-DNA transfer, and the transfer sequence can be transiently integrated into the plant (cell) genome and subsequently transcribed into RNA from the plant promoter.

Further provided is a process comprising introducing the ViGE vector into a plant.

Another aspect of the present invention provides a method comprising causing or allowing transcription within the genome of a plant cell to produce viral particles from one or more of the ViGE vectors disclosed herein.

The present invention also discloses a method for characterizing a target gene, the method comprising the following steps:

(a) using the FoMV ViGE system as described above to edit the target gene in a part of the plant or at a certain growth stage;

(b) Observe the phenotype of the part of the plant where the target gene is edited.

Typically, observations will be compared to plants expressing the gene of interest to characterize that gene (ie, establish one or more phenotypic traits).

In another aspect, a method of altering the phenotype of a plant is disclosed, the method comprising using the editing method described above. Traits that may require altering their phenotype include: plant growth status, herbicide tolerance, drought resistance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, modified Seed yield, percent improved oil, percent improved protein, and resistance to bacterial, fungal, or viral diseases.

In another aspect of the present invention, the present invention discloses a host cell comprising an expression system (vector or encoded virus or viral particle). These may be plant cells, or may be microbial (especially bacterial, especially Agrobacterium) cells.

In another aspect, a plant or plant tissue is disclosed, including a plant or plant tissue transiently transformed by one or more vectors of the present invention.

The invention also provides plants comprising such host cells or tissues.

The present disclosure also encompasses plant seeds that have been transiently infected, wherein in the seeds, the target genes have been edited as described above. The seeds themselves may not include the expression system. The invention also includes progeny, clones, cell lines or cells of the edited plants described above, wherein the progeny, clones, cell lines or cells have the edited gene, but optionally may not include the expression system itself.

In another aspect of the present invention, RNA transcripts from the above-described expression systems are provided.

In another aspect of the invention, viruses or virus particles are provided that encapsulate RNA transcripts from an expression system as described above.

In another aspect of the present invention there is provided a kit comprising the vector as described above.

It will be appreciated by those of skill in the art that a nucleotide sequence defined or recited herein, whether derived from or heterologous to FoMV, need not be "wild-type", but may optionally be a variant (eg, a mutant or other variants, or substantially homologous derivatives), as long as their function is not reversed. For example, the function of the FoMV coat protein is to wrap the FoMV genome and allow it to move. The various ORFs of FoMV and their functions will be discussed later. Likewise, p19 derivatives maintain the function of suppressing gene silencing.

"Substantially homologous" means that the sequence in question is at least about 70% or 80% identical to a reference sequence, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% identical identity. The identity can be at the level of the nucleotide sequence and/or the encoded amino acid sequence. Preferably, sequence comparisons are performed using FASTA and FASTP (see Pearson & Lipman, 1988. Methods in Enzymology 183:63-98). It is best to use the default matrix to set the parameters as follows: Gapopen (penalty for first residue in gap): -12 for protein / -16 for DNA; Gapext (penalty for other residues in gap): -2 for protein/-4 for DNA; KTUP wordlength: 2 for protein/6 for DNA

Any subheadings contained herein are for convenience only and should not be construed as limiting the disclosure in any way.

The invention will be further described with reference to the following non-limiting figures and examples. In view of these, other embodiments of the invention will occur to those skilled in the art.

The disclosures of all references cited herein are expressly incorporated herein by cross-reference, as those skilled in the art can use to practice the present invention.

Description of drawings

Figure 1A shows PDS gene editing induced by viral delivery of Cas9 and sgRNApds.

(a) FoMV-based expression vectors for Cas9, sgRNApds and p19. FoMV/Cas9, FoMV/sgRNApds, and FoMV/sgRNAnon were used to express Cas9 labeled with 3xFLAG and NLS tags at the N-terminus and C-terminus, respectively (FLAG:NLS:Cas9:NLS), sgRNAs targeting PDS genes, and empty sgRNA scaffolds . FoMV/P19: sgRNApds are expected to co-express the p19RNAi suppressor and sgRNApds. The sgRNApds sequence, PDS target region and protospacer adjacent motif (PAM) and FoMV genome and regulatory elements (left and right borders LB and RB, cloning site, dual 35S promoter 2x35S and NOS terminator) are shown. Arrows indicate native and repetitive coat protein (CP) subgenomic RNA promoters.

(b) Cas9 mRNA was detected in young leaf tissues of 3 Nb plants infected with FoMV/Cas9+FoMV/P19:sgRNApds, but not in 3 mock-inoculated controls. The position and size of the DNA ladder (M) are shown, along with the position of detected Cas9 mRNA.

(c, d) FoMV-mediated whole-body ViGE was detected in young leaf tissues. Comparison of wild-type (WT) and 17 edited PDS sequences (P1 to P17) revealed various point mutations and 2nt deletions in the sgRNApds target region (box, c). A representation of the chromaticity diagram shows the sgRNApds targeting sequences (P1, P4, P13 and P16) with ViGE-mediated substitutions (asterisk) and deletions (2 triangles). (d).

(e) Summary of the various types and occurrences of ViGE events in PDS genes targeted by sgRNApds. The PAM is highlighted (c, d), the position number of the nucleotide upstream of the PAM (c, e).

Figure IB shows an in vitro system for producing RNA transcripts encoding FoMV vectors for delivery of Cas9 and sgRNApds.

Figure 2 shows efficient ViGE by co-delivery of Cas9, sgRNApds and p19 from FoMV.

(a-d) ViGE induction. The experimental strategy (a) is outlined. Broken seed coat (SC) seeds were infiltrated with mixed Agrobacterium carrying FoMV/Cas9 and FoMV/P19:sgRNApds under vacuum pressure (b). Systemic viral symptoms appear in Nb plants grown from agroinfiltrated seeds (c, d). Plants were photographed on day 2 seeds (b) spread on water-soaked filter paper and on days 28 (c) and 60 (d) after seed Agrobacterium infiltration.

(e) Analysis of p19 mRNA. Expression of p19 was detected in virus-infected Nbs (lanes 1 and 2) but not in mock plants (lane 3). The position and size of the DNA ladder (lane M) are shown.

(f) Delivery of p19 from FoMV enhances viral co-expression of Cas9 protein in plants. In Nb young leaf tissue infected with FoMV/Cas9+FoMV/P19: sgRNApds (lane 5) and in p19 transgenic young leaf tissue infected with FoMV/Cas9+FoMV/sgRNApds (lane 6), Western blotting was used to detect 160 -KDa FLAG-tagged Cas9 (asterisk), but not detected in Nb young leaf tissue infected with FoMC/Cas9+FoMC/sgRNA pds (lanes 1 and 2) or mock controls (lanes 3 and 4). The location and size of protein markers (lane M) are shown.

(g, h) FoMV-mediated ViGE in young leaf tissues of the system. A comparison of wild type (WT) and 10 edited PDS sequences (P1 to P10) is shown (g). Representation of chromaticity diagrams (P1, P3, P9 and P10) showing nucleotide substitutions (asterisks) in the sgRNApds target sequence (h). PAM highlighted (g, h).

Figure 3 shows transient expression of eGFP from FoMV/eGFP.

(a) FoMV-based eGFP expression vector. The organization of the FoMV genome is shown, and arrows indicate native and repetitive coat protein (CP) subgenomic RNA promoters.

(b-c) Confocal microscopy analysis of eGFP expression in Agrobacterium-infiltrated Nb leaves by FoMV/eGFP. Brightfield image of Nb epidermal cells (b), eGFP expression in green lane (c) and merged image of panels b and c (d). (c) Test strip = 100 microns.

Figure 4 shows ViGE in Nb.

(a-d) Nb infection with recombinant FoMV. Plants infiltrated with FoMV/sgRNAnon (a), FoMV/sgRNAnon+FoMV/Cas9 (b), FoMV/sgRNApds (c) or FoMV/sgRNApds+FoMV/Cas9 (d) had no apparent viral symptoms. Photographs were taken 21 days after the leaves were infiltrated by Agrobacterium.

(e, f) Genomic PCR/MlyI analysis of ViGE. In two separate experiments, Expt I (e) and Expt II (f), agarose gel analysis of 6 representative samples (Nb infected with FoMV/sgRNApds + FoMV/Cas9) showed that the sgRNApds target PCR products Fully sensitive to MlyI cleavage, indicating that ViGE did not occur in FoMV-infected plants. Repeated experiments yielded similar results.

(g, h) Cloning/sequencing analysis of ViGE. The MlyI digested genomic PCR product from one randomly selected (RS) sample of Expts I and II was isolated from the gel section (box, g) and cloned into the T vector. Sanger sequencing plasmid DNA extracted from randomly selected colonies further confirmed that no mutation (h) was introduced in the sgRNApds target PDS sequence. The positions and sizes of DNA ladders are shown (e-g). The corresponding regions of the wild-type (WT) PDS gene and the sgRNApds target PDS sequence in these expected ViGE samples (P1 to P9) were identical (h). The MlyI site is marked and the sgRNApds target PDS sequence region is underlined. PAM(h) is highlighted in the figure.

Figure 5 shows potential FoMV infection in N. benthamiana

(a) Mock-seeded Nb. (b) FoMV-infected Nb. (c) RT-PCR detection of FoMV. FoMV was detected in whole-body young (ie not Agro-infiltrated) leaf tissue of three representative plants (P1, P2 and P3) infiltrated with FoMV, but not in mock controls (P1 and P2). All plants remained healthy and did not show mosaic disease, chlorosis, or leaf curling that is characteristic of viral infections. Plants were photographed 21 days after leaf infiltration with Agrobacterium (a, b). The position and size of the DNA ladder are shown (c).

Figure 6 shows whole body ViGE in p19 transgenic Nb.

(a) p19-transgene expression cassette in binary vector ^pEAQ -HT15. Nbs were transformed with Agrobacterium tumefaciens LBA4404 carrying the pEAQ-HT vector. Five single-copy homozygous lines were obtained.

(b) Representative of healthy p19 transgenes. (c) Healthy Nb.

(d, e) Two independent p19 transgenic lines infected with FoMV/Cas9+FoMV/sgRNApds showed severe mosaicism, chlorosis and curling in plants. Photographs were taken 21 days after the leaves were infiltrated by Agrobacterium.

(f) Genomic PCR/MlyI analysis of whole body ViGE. An experimental outline for the genomic PCR/MlyI analysis of ViGE in p19 transgenic Nb is shown. Genomic PCR was performed using primers PDS_MLY ID-F3 and PDS_Mly_ID-R (Supplementary Table 1), and the resulting product (407 bp) was treated with MlyI. Partial or complete digestion indicated that ViGE occurred in p19 transgenic Nbs, but not in normal Nbs infected with FoMV/Cas9+FoMV/sgRNApds. The MlyI-insensitive PCR product (e.g. triangle (f) here) was purified from agarose gel, cloned into a T vector and sequenced. The position and size of the DNA ladders are shown (f). It should be noted that in these ViGE plants there were no any albinism or dwarf phenotypes (d, e and Fig. 2c, d), probably due to the fact that most of the mutations were point mutations (Fig. 2g, h), which did not cause The amino acid sequence of the PDS protein changes.

Figure 7 shows systemic ViGE by FoMV-mediated co-delivery of Cas9, sgRNApds and p19.

(a) Overview of the second method of ViGE detection. Genomic PCR was performed using primers PDS_MLY ID-F3 and PDS_Mly_ID-R (Supplementary Table 1), and the resulting PCR products were cloned directly into the T vector. After colony PCR screening and MlyI treatment of colony PCR products, Sanger sequencing was performed on the miniprep plasmid DNA of MlyI-insensitive samples.

(b, c) MlyI-treated agarose gel analysis of colony PCR products. In two separate ViGE experiments, 63

(b) and 26(c) Colony PCR products were treated (shown with diagonal lines) or without MlyI. Those sequences of the expected size (407 bp) and fully resistant to MlyI digestion (asterisk) were selected for sequencing (Fig. 2g,h).

Detailed ways

Example 1 ViGE using FoMV vector without gene silencing

To test ViGE, we utilized a FoMV vector originally designed for VIGS ¹³ , but recently modified for VIF ¹⁴ and transient gene expression (Figure 3). We cloned the coding sequence of 3xFLAG and the nuclear localization signal (NLS)-tagged Cas9, phytoene desaturase (PDS)-targeted sgRNApds, or sgRNA lacking any targeting sequence (sgRNAnon) into FoMV and Generated in FoMV/Cas9, FoMV/sgRNApds and FoMV/sgRNAnon.

Nicotiana benthamiana (Nb) plants infected with either FoMV/Cas9 combined with FoMV/sgRNAnon or FoMV/Cas9+FoMV/sgRNApds by co-Agrobacterium infiltration of leaves did not develop any symptoms (Fig. 4a–d), which was consistent with detection by RT-PCR Potential FoMV infection by viral RNA was consistent (Fig. 5a-c). Then, we extracted genomic DNA from whole-body leaf tissue and amplified the sgRNApds target PDS genes. Complete MlyI digestion of the resulting PCR product and subsequent sequencing analysis indicated that ViGE did not occur (Fig. 4e-h). We suspect that the initial failure of ViGE may be due to the low efficacy of FoMV infection and insufficient viral expression of Cas9 and sgRNApds in plants.

Example 2 ViGE using FoMV vector with transiently expressed p19 suppressor

To enhance FoMV infectivity to increase the levels of Cas9 and sgRNApds, we co-expressed tomato bush dwarf virus p19, a mutant RNAi suppressor with potent RNAi inhibitory activity but no pathogenesis in plants15. We generated a single-copy homozygous Nb line transformed with the p19 expression cassette (Fig. 6a). Transgenic plants infected with FoMV/sgRNApds and FoMV/Cas9 exhibited mosaic, chlorosis, and leaf curling (Fig. 6b-e), and viral expression of Cas9 mRNA was easily detected. Genomic PCR-MlyI screening of 24 infected p19 transgenes in 4 different experiments revealed that sgRNApds targets were only cleaved by MlyI, indicating that plant whole-body ViGE occurred (Fig. 6f). Further cloning and sequencing of PCR products against MlyI identified 104 mutations, including nucleotide deletions and substitutions in the target region. These findings are supported by the finding of increased GE content in RNAi pathway-deficient Arabidopsis ¹⁶ .

Example 3 Versatile Transient Whole Body ViGE System for Plants

Then, we generated a new vector FoMV/P19:sgRNApds to express p19 and sgRNApds. Following agro-infiltration of germinated Nb seeds with FoMV/Cas9 and FoMV/P19:sgRNApds, plants grew and developed obvious systemic virus symptoms (Fig. 2a-d). Viral delivery of p19 significantly enhanced Cas9 protein levels in young leaf tissue (Fig. 2e, f) and resulted in systemic ViGE that is potent against PDS targets (Fig. 2g, h and Fig. 7).

Example 4 Use of the ViGE System in Monocotyledonous Plants

Examples 1 to 3 illustrate how ViGE can be used effectively in dicots. The host range of the FoMV virus includes both monocotyledonous and dicotyledonous plants, so the present invention is equally applicable to monocotyledonous plants.

Known techniques for transient expression were used to introduce the vector systems described in Example 2 (FoMV/Cas9 and FoMV/P19: sgRNAxxx) from maize (Zea maysL) into tissues, with the sgRNA targeting the desired endogenous gene. Alternatively, RNA transcripts prepared by in vitro transcription were introduced into maize tissue by bombardment (see e.g., Yadava P, Abhishek A, Singh R et al Advances in Maize Transformation Technologies and Development of Transgenic Maize. Front Plant Sci. 2017;7: 1949. Published January 6, 2017, doi:10.3389/fpls.2016.01949). Once infiltrated into plant cells, the transcript replicates and forms viral particles, which move between cells and spread systemically within the plant, establishing a systemic infection.

Example conclusion

Combining our data shows that simultaneous delivery of Cas9, sgRNA and the RNAi suppressor p19 from FoMV results in ViGE production in plants. The expression of large proteins (eg Cas9) over 160 kD in size from FoMV is also unprecedented for any plant virus-based gene expression system ¹⁷ . This fast and efficient method involves neither plant transformation nor transgenic expression of Cas9 or sgRNA. Considering the breadth of its host ^species13,14 , FoMV-based ViGE can be applied to both dicotyledonous and monocotyledonous plants, including important cereal crops.

Methods of Examples 1 to 3

Construct FoMV-based expression vector. Using high-fidelity KOD-Plus-Neo DNA polymerase (Toyobo), a plasmid templated with FLAG:NLS:Cas9:NLS ⁴ and a set of primers Cas9-3X-NLS-Hpa-MLU-F and Cas9-3X- NLS-Xhol-ASC-R, amplified 3xFLAG and the coding sequence of Cas9 (designated as FLAG:NLS:Cas9:NLS) tagged with a nuclear localization signal (NLS) (Supplementary Table 1). The resulting PCR product, approximately 4.2 Kb in length, was then treated with HpaI and AscI and cloned into the HpaI/AscI site of the binary vector pCambia2300-FoMV ¹³ to generate FoMV/Cas9. For the production of FoMV/sgRNAnon and FoMV/sgRNApds, high fidelity KOD-Plus-Neo DNA polymerase, pT-U6p-scaffold-U6t ¹¹ or pCVA-gRNA::NbPDS plasmid DNA ¹¹ was used as template and primer AUT-Hpa-F3 was used and U6T-ASC-R2 (Supplementary Table 1), digested with HpaI/AscI, and then cloned into the HpaI/AscI site of the binary vector pCambia2300-FoMV ¹³ to amplify the corresponding DNA fragments. To generate FoMV/P19:sgRNApds, high-fidelity KOD-Plus-Neo DNA polymerase, pEAQ-HT plasmid templated with p19 coding sequence ¹⁵ and a set of primers P19-ORF-F and P19-ORF-R (Supplementary Table 1), and cloned into the Hpal site of FoMV/sgRNApds to amplify the p19 gene. Similarly, using primers eGFP-ORF-F and eGFP-ORF-R (Supplementary Table 1), plasmid pEGFP (Clontech) as template, and cloned into the Hpal site of pCambia2300-FoMV to produce FoMV/eGFP (Fig. 3a), The eGFP gene was amplified. The insertion of FLAG:NLS:Cas9:NLS, sgRNAnon, sgRNApds or P19:sgRNApds in these FoMV vectors was verified by PCR using a pair of primers Fomv seq_6830_5K-F and Fomv Seq_7260_SUBP-R (Supplementary Table 1) and further sequenced by Sanger to confirm. All FoMV constructs and binary vector pEAQ-HT ¹⁵ were individually transformed into A. tumefaciens LBA4404 by electroporation ¹⁸ prior to use in subsequent agricultural infiltration assays and plant transformations and were micro-prepared by The plasmids were sequenced to confirm.

Plant Transformation A number of primary p19-transgenic lines were generated by leaf disc transformation of N. benthamiana (Nb) with A. tumefaciens LBA4404 containing pEAQ-HT as described in ¹⁹ . Specific primers P19-ORF-F and P19-ORF-R (Supplementary Table 1) were used to verify transformation by PCR amplification of the integrated p19 transgene. After self-fertilization, T1 and T2 progeny were tested for antibiotic susceptibility by germinating seeds on 0.5 mg/ml kanamycin. Five independent single-copy homozygous Nb lines transformed by the p19 transgene were obtained, as evidenced by a Mendelian 3:1 segregation ratio between kanamycin-resistant and susceptible seedlings. As with WT Nb, all p19 transgenic plants grew properly and were well developed.

Viral infection (ViGE), plant growth and maintenance. To prepare FoMV and recombinant FoMV inoculum, Agrobacterium tumefaciens LBA4404 with different FoMV constructs was grown overnight in LB medium containing 0.5 mg/ml streptomycin and 0.5 mg/ml kanamycin (Fig. 3a) , brought to a density of 1.0 _OD600 at 28C, then collected by centrifugation at 3000 rpm for 10 minutes and resuspended in sterile water to a final density of 0.5 _OD600 . For Agrobacterium-infiltrated leaves, infuse Agrobacterium into young leaves of wild-type or transgenic Nb plants at the six-leaf stage via a 0.5-mL needleless syringe. Alternatively, germinated seeds were agriculturally infiltrated to shorten the time of viral infection. Briefly, dozens of Nb seeds were spread on 3MM Whatman filter paper pre-soaked in sterile water and placed in a Petri dish (10 cm diameter). The dishes were kept in a growth chamber under constant light at 25 °C. After two days in these conditions, the seeds began to husk and germinate. At this stage, seeds were collected and mixed with 5 ml of 0.5OD ₆₀₀ Agrobacterium in a 50 mL-Falcon ^™ centrifuge tube. Agrobacterium infiltration of the seeds was achieved using a vacuum pump at a pressure of 0.085 MPa for 10 minutes. Then, the Agrobacterium-infiltrated seeds were transferred to compost and left in the dark for 24 hours. Subsequently, plants were grown in an insect-free growth chamber at 25°C under 16h light/8h dark conditions, regularly checked for local and systemic infections, and photographically recorded using a D7000 Sony NEX-5R camera.

Confocal microscope. To examine eGFP expression from FoMV/eGFP, Nb leaves were collected on day 4 after Agrobacterium infiltration and examined by Nikon A1 confocal microscopy at 488 nm excitation to excite GFP and monitor green fluorescence emission (510 nm) ²⁰ . The Nb epidermis was also photographed in brightfield following the manufacturer's instructions. Confocal images were processed using Nikon A1 Nis-Elements software.

RNA extraction and RT-PCR. RNA was extracted from whole body Nb young leaf tissue using RNAprep Pure Plant Kit (TIANGEN). First-strand cDNA was synthesized from DNase I-treated RNA (2 μg) by M-MLV reverse transcriptase using the FastQuant RT kit (TIANGEN) according to the manufacturer's instructions. PCR to detect FoMV genomic RNA was performed using cDNA as template with virally expressed Cas9 or p19 mRNA along with primers for each target (Supplementary Table 1) and analyzed by 1.2% agarose gel electrophoresis.

Western blot analysis. Total protein was extracted from whole body young Nb leaf tissue as described in ¹⁹ . After electrophoresis at 100V for 2 hours, protein aliquots (20 μg) were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad). Western blot analysis was performed using 1:2000 mouse anti-FLAG (Sigma-Aldrich) antibody, 1:5000 goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Abeam) and SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific). The chemiluminescent signal was detected using the ChemiDoc XRS+ Imaging System (Bio-Rad) following the manufacturer's instructions.

Genomic DNA extraction and molecular characterization of ViGE. DNA was isolated from whole body Nb young leaf tissue using the DNeasy Plant MiniKit (Qiagen) following the manufacturer's instructions. Genomic PCR amplification of the sgRNA target PDS gene (407 bp) was performed using high-fidelity KOD-Plus-Neo DNA polymerase, 10-100 ng DNA as template and primers PDS_MLY ID-F3 and PDS_Mly_ID-R (Supplementary Table 1). Subsequently, ViGE was characterized using two methods. In method I, genomic PCR products (approximately 400 ng) were treated with MlyI (NEB) for 6 hours at 37°C and analyzed by 1.5% agarose gel electrophoresis. Any undigested PCR fragments were purified from the gel and cloned into the pEASY-Blunt3 cloning vector (TransGenBiotech) for Sanger sequencing. In Method II, the genomic PCR product was cloned directly into the pEASY-Blunt3 cloning vector. Plasmid DNA was miniprepared for sequencing after PCR/MlyI digestion screening of high-preservation fungi colonies. It should be noted that the high fidelity KOD-Plus-Neo DNA polymerase and detection of deletions and substitutions of various nucleotides including A→T and T→A ensured that these mutations we identified were not the result of PCR errors, Instead, it is produced by the plant ViGE.

Supplementary Table 1

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SEQ ID NO: 1: FoMV vector sequence (6386nt, HpaI (5375) and AscI (5389) highlighted)

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gggtgacgtgctttggctcgcgtacaagcgggcgatgatgctcccagatgagcccatcaaatttaacccagagctctggtgggcatgtgcagatgaggtgcaaaagacctacc tctccaagcccatacacgcgctcaagaacggaattcttcggcaatcacccgactttgactggaacaaactgcagattttcc tcaagtcacagtgggttaagaaaattgacaaaatcgggaaaattgacgtcaacgctggacagacgattgccgccttttacc aaccaaccgttatgctgtttggaaccatggcgagatacatgcgccgcatccgcgacacttatcaacccggcgaaatactca tcaattgcgagaagaaccagaagcacatttcgaagtgggtcgagagcaattggaaccaccgcctacccgcttacaccaatg acttcactgcttacgaccaaagccaggacggggctatgttacagtttgaggtactgaaagccctgcaccatgatatccctc atgaggttgtggaagcctacgtagcccttaaactcaactcaaaaatgtttctgggcacactggcgattatgagattaactg gtgagggacccaccttcgacgctaacacagagtgcaacattgcttacacacacgcccggttcgagatcccaaagaacgtgg cgcaaatgtacgcgggtgacgactgtgcgctcaactgcaggcccgttgaacggcagtccttcttgcctcttgtggagaaat tcaccctgaaatcaaaacccaaagtatttgagcaaaaagttgggtcatggcctgagttctgcggcaatctgatcaccccac ggggctacctcaaggatcccatgaagctacaacactgcctgcaactggcacagaggaagaaaccatccgaacctgggtcgc tcaaagacgttgctgagaactacgctatggacttgctacccacatacgagctaggtgatgcactct acgaaatcttcgacg agagacaaatgaacgcgcactatcagtcggtcaggacgcttatcacatgcgcccacaccaaagtcctccgagtggcacaggcacttcaggaagactgcaccttctttagctccatctaacaggttttgagttaggctaactccactgacgaattaaataaca atggatagtgaaatagttgaacgactaacaaagcttggtttcgtcaagacttcacacacgcacatcgctggcgagcccctc gtgattcacgccgttgctggggccggtaaaaccaccctccttcggtccttacttgaattaccgggcgtggaagtcttcaca ggcggggagcacgatcctccaaatttgtcagggaaatatatccgctgcgctgcaccccctgtggccggtgcatacaacatt ctcgacgagtaccccgcgtacccaaattggcgatcgcaaccctggaacgtcctaatcgccgacaacctacaatacaaagaa cccacagctcgcgcccactacacatgcaatcgcactcaccgcctggggcagctcactgtcgacgctttgcgtagggttggt ttcgacatcacctttgccggcacgcagactgaagactacggattccaggaaggccatctctacaccagtcaattttacgga caggtcatttcacttgacacgcaggcccataagatcgctgtgcgccacggacttgcacccctgtccgctttagaaacccgg gggctggaatttgatgagaccactgtgataacgactaaaacctcgctggaggaagtgaaggataggcacatggtctatgtc gctctcacacggcacaggcgcacctgccatctctacaccgctcactttgcgccctccgcctgacaacacgaaagccatact aactatagctataggaatagccgcctccctcgtctttttcatgctcacacgcaacaatctgccacacgtcggtgataacat c cactcactaccccacggaggaagttacattgacggtaccaagtccatcaactaccgcccacctgcgtcacgctacccctc atctaacttactcgctttcgctccaccaatactcgccgcagtgctctttttcctcacacagccatatctagctaccagacgatccaggtgcgttcggtgcttcgttgtccacggcgcatgcacgaatcacacctagttgttatattagcgctgttactttta gctctgtggtgtcttagcactcgacccgttcaaccatcgtgccatgtcgaaatcaacggccactccatcatcgtcaccgga aactgctggcactccactcaacgaccgcattgagggtgttagggtaaccaacatcagtgaagagaaacaacccacctcgag tgtgacctcatcatttcaggacaca gttaacgcgtctcgaggcgcgc cactcgacccgttcaaccatcgtgccatgtcgaa atcaacggccactccatcatcgtcaccggaaactgctggcactccactcaacgaccgcattgagggtgttagggtaaccaa catcagtgaagagaaacaacccacctcgagtgtgacctcatcatttcaggacacaatggcaccacaagatgccgacgtcac tgatgcgacggactacaagaaaccgcctgctgaaactgagcagaaggcactcaccattcaaccacggtcaaacaaggcgcc cagtgacgaggagttggtacgcatcatcaacgcggcgcagaagcgaggcctcacacccgcggcctttgttcaagcagctat agtcttcaccatggaatccatggacaagggcgccaccgactccacgattttcacgggaaaatacaacactttcccaatgaa aagtctggcgctagcttgcaaagatgctggcgtgcccgtgcacaaactttgctacttctataccaagccggcttacgcgaa ccgtagggtcgccaac cagccgcctgctcgctggaccaacgagaatgtgcccaaagctaacaagtgggcggctttcgacac cttcgacgcacttctcgacccatacgtagtcccatcctctgtaccgtacgatgagcccacgccagaggatcgccaagtcaa tgagattttcaagaaggacaatttgagtcaggcagcatccagaaaccaactccgcgccctaggaacgcaagcctccatcacgcgcgggagactcaacggcgcaccagcactaccaaacaacgggcagtacttcatcgaggcacctcagtgatcagtagtatg ataccaataaataaatcgggcgaatccgcgcctcctgactatgggcaggtttacggaccaagctgtatcgagatacgacct aacagtaacgcagctaaggggtgaatgcacacatcgcttataaaaaaaaaaaaaaaaaaaaaaaaaaa

SEQ ID NO: 2 mutated P19 sequence (mutated nucleotides are underlined)

SEQ ID NO: 3-3xFlag-Cas9-NLS (bold sequence: 3xFlag; underlined sequence: NLS; oblique Body marked sequence: Cas9)

SEQ ID NO: 4-AtU6-Scaffold (AtU6 promoter: bold; tracrRNA, underlined; terminator, oblique body)

SEQ ID NO: 5-AtU6-sgRNApds-Scaffold (AtU6 promoter: bold; sgRNApds: uppercase; tracrRNA, underlined; terminator T, italics)

sequence listing

<110> Hangzhou Normal University

<120> Plant modification

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6386

<212> DNA

<213> FoMV vector (FoMV)

<400> 1

gaaaactctt ccgaaaccga aactgactga aactacctcg accgacctta gaacccaaga 60

acccaacggg tgcggccact atgtctatcg aggcagtttt cgaccaggtt acagacccat 120

cgctccgcgc tgtgattcag gaggaagcgc acaaacagat caaagatttg tttaaggaaa 180

cgacgcgctg caatccctac tccataccgc aagctgggcg caaggttttg gaaaagtacg 240

ccatccccta caacccgtac tctctcaaac tacaccctca cgcagcctca aaagcgtttg 300

aagtgtcgct ctacgaggct gcgtctaact acctcccctc cacctcctca actcctgtca 360

cattcatgtt cacaaaaccg ggcaagctca gattctttag gcgccgaggt cacgtggaca 420

aattcgttaa tgctgacata gttccaagag acttggctag atacccacgc gacacagtct 480

acagttatct gcccgagatc accaccacac acgctttcat tggcgacacc ctacaccact 540

tcggtgagga ctttctcgtc gaggttttct ccaggtcacc gaaactagaa gtgcttctag 600

ccaccatggt attaccaccc gaggcctttt acagaatgga gtcccttcat ccctcggttt 660

acactctcct ctacagggac gaccgattcc tatacctgcc tggtggcctg cctggcggtg 720

agtacgaaca tcgctataag gacctaaact ggctaacatt tggcacagtt acgcacggcg 780

ggatcactat cacaggagaa cgcattgaga ctaaggccgc gaatcatctt ttcctcttca 840

gacgagggcg actagagaca ccaaaattcc gctcattcga catgcccgag cctatggtcc 900

tgcttcccaa ggttttccgc cccgcaaagt acaatgtaca gaagccaatt ccccgggaga 960

aagcaaacaa atggttgatg tacgttaaat ccatcggcaa tgccaccatt cgtgacgtat 1020

gggctaagct gaggcaaacc atagccaatg cagacattgg actcttctcg cccactgagc 1080

tcgtgcatct cacgaattac ttcctgctcc tgggccggct tgactcacac aactccttcg 1140

accaagtact ggccgacagt gtgctgaaag catggttcag accaatggtc gcaaagcttc 1200

aggagatcaa gcacaaactc atggggcaga cccaattcat gcaactctgc caagcgctag 1260

agatgacgga ggtggacctc gtttttgagg tccgggactc caagactccc cacaaacaag 1320

ctgtgccgtt ggaccgtgaa attgaaaacg ttttgttgga aggagtctca tcggagccaa 1380

cttacacgga aaccgaaggc gttgctgata gtccacttcc ccccccaatg caaactgcag 1440

ccgagccgtc cgcgacctca gacgagcccg agagctctag ctcgcgtgaa attgagcacc 1500

aaccggcgcc tgagatcacg cttgacgagg aagaacctca gcgagacgat ctgccttggg 1560

acgcttggag aacacaatta agggcgcttg gctttgaggc ctctgaaagg cagtatgacc 1620

cggacggtga actgatctct cctatcctga gcacccgaag gttacctaag actcccatag 1680

acacaacact ctacgccacg ctagacaaga ttgcacgctg cccaactttc tacaagcctg 1740

acacagatcg cgcgcagact tacgctcgcg atgtcatggc ggggaaaacc ggtgccattc 1800

tcaagcaaca accctttgag tggaaaacca cgctcaaacg caagactaaa gaggaaccga 1860

aggaaattca ccttgcggtg ttgcatggtg cgggcgggtc gggcaaatcc tacgcactgc 1920

aggaatttat gcggaacaac tctgacacac cgattacggt catcctgccg actaacgagc 1980

tcagggccga ctggaagaaa aaattgcccg cccacgacaa agacacattt atgacatacg 2040

aaaacgcgct cttgtgccct cgtggagaca tcttcattat ggacgattac acaaaattgc 2100

ccaggggcta cattgaggct ttcgtgcaga atgcacctgc cctctcactt ctgatactca 2160

ccggtgaccc caaccaagcc gaacactttg agaccactga ggacaatgaa attaacagcc 2220

tcgcccccgc ctcagtggtc ttcggcaagt tctctaggta ccacataaat gccacacacc 2280

gcaaccccag aaacttggca aacgccctcg gtgtttactc cgagacgccc ggggaggtta 2340

aagtgcttta cacgaggaac atcaagaccg gttatcacaa tctcgtgccc tcacaaatga 2400

agatgagaaa ctacgcctca ctcgggcagc gagcgtccac ctatgcgggt tgtcagggga 2460

tcactgcgcc ccgcgttcaa atcatcctag actccgacac accccggtgc accaggcaag 2520

tcatgtacac tgcgctttca agggccacga cggaagtggt gctctgcaac acgatgccgg 2580

atgagaaaag ctttttccag aaggttgaag caacaccgta cctcaaagcc atcctcaacc 2640

tcaacaaaga gattaaagtc actgagggtg acttgacaga agaaccgccg agggagcccg 2700

ctcctcccac cacacacctg cctgttgaaa acagaattat tcttaatgag gccctagtcg 2760

aaccgctgcc cgacaaacat gaccgcgaga tctactccaa ctccactggc ttttcaaact 2820

gcatacagac tcaagacccg tacatccaag ccttccaaca tcagcaagcc aaggacgaga 2880

cattgttctg ggcaaccgtc gagaagaggc tcgcagcatc cacgccgaag gacaactgga 2940

cagaattcaa gaccaagaga cctctgggtg acgtgctttg gctcgcgtac aagcgggcga 3000

tgatgctccc agatgagccc atcaaattta acccagagct ctggtgggca tgtgcagatg 3060

aggtgcaaaa gacctacctc tccaagccca tacacgcgct caagaacgga attcttcggc 3120

aatcacccga ctttgactgg aacaaactgc agattttcct caagtcacag tgggttaaga 3180

aaattgacaa aatcgggaaa attgacgtca acgctggaca gacgattgcc gccttttacc 3240

aaccaaccgt tatgctgttt ggaaccatgg cgagatacat gcgccgcatc cgcgacactt 3300

atcaacccgg cgaaatactc atcaattgcg agaagaacca gaagcacatt tcgaagtggg 3360

tcgagagcaa ttggaaccac cgcctacccg cttacaccaa tgacttcact gcttacgacc 3420

aaagccagga cggggctatg ttacagtttg aggtactgaa agccctgcac catgatatcc 3480

ctcatgaggt tgtggaagcc tacgtagccc ttaaactcaa ctcaaaaatg tttctgggca 3540

cactggcgat tatgagatta actggtgagg gacccacctt cgacgctaac acagagtgca 3600

acattgctta cacacacgcc cggttcgaga tcccaaagaa cgtggcgcaa atgtacgcgg 3660

gtgacgactg tgcgctcaac tgcaggcccg ttgaacggca gtccttcttg cctcttgtgg 3720

agaaattcac cctgaaatca aaacccaaag tatttgagca aaaagttggg tcatggcctg 3780

agttctgcgg caatctgatc accccacggg gctacctcaa ggatcccatg aagctacaac 3840

actgcctgca actggcacag aggaagaaac catccgaacc tgggtcgctc aaagacgttg 3900

ctgagaacta cgctatggac ttgctaccca catacgagct aggtgatgca ctctacgaaa 3960

tcttcgacga gagacaaatg aacgcgcact atcagtcggt caggacgctt atcacatgcg 4020

cccacaccaa agtcctccga gtggcacagg cacttcagga agactgcacc ttctttagct 4080

ccatctaaca ggttttgagt taggctaact ccactgacga attaaataac aatggatagt 4140

gaaatagttg aacgactaac aaagcttggt ttcgtcaaga cttcacacac gcacatcgct 4200

ggcgagcccc tcgtgattca cgccgttgct ggggccggta aaaccaccct ccttcggtcc 4260

ttacttgaat taccgggcgt ggaagtcttc acaggcgggg agcacgatcc tccaaatttg 4320

tcagggaaat atatccgctg cgctgcaccc cctgtggccg gtgcatacaa cattctcgac 4380

gagtaccccg cgtacccaaa ttggcgatcg caaccctgga acgtcctaat cgccgacaac 4440

ctacaataca aagaacccac agctcgcgcc cactacacat gcaatcgcac tcaccgcctg 4500

gggcagctca ctgtcgacgc tttgcgtagg gttggtttcg acatcacctt tgccggcacg 4560

cagactgaag actacggatt ccaggaaggc catctctaca ccagtcaatt ttacggacag 4620

gtcatttcac ttgacacgca ggcccataag atcgctgtgc gccacggact tgcacccctg 4680

tccgctttag aaacccgggg gctggaattt gatgagacca ctgtgataac gactaaaacc 4740

tcgctggagg aagtgaagga taggcacatg gtctatgtcg ctctcacacg gcacaggcgc 4800

acctgccatc tctacaccgc tcactttgcg ccctccgcct gacaacacga aagccatact 4860

aactatagct ataggaatag ccgcctccct cgtctttttc atgctcacac gcaacaatct 4920

gccacacgtc ggtgataaca tccactcact accccacgga ggaagttaca ttgacggtac 4980

caagtccatc aactaccgcc cacctgcgtc acgctacccc tcatctaact tactcgcttt 5040

cgctccacca atactcgccg cagtgctctt tttcctcaca cagccatatc tagctaccag 5100

acgatccagg tgcgttcggt gcttcgttgt ccacggcgca tgcacgaatc acacctagtt 5160

gttatattag cgctgttact tttagctctg tggtgtctta gcactcgacc cgttcaacca 5220

tcgtgccatg tcgaaatcaa cggccactcc atcatcgtca ccggaaactg ctggcactcc 5280

actcaacgac cgcattgagg gtgttagggt aaccaacatc agtgaagaga aacaacccac 5340

ctcgagtgtg acctcatcat ttcaggacac agttaacgcg tctcgaggcg cgccactcga 5400

cccgttcaac catcgtgcca tgtcgaaatc aacggccact ccatcatcgt caccggaaac 5460

tgctggcact ccactcaacg accgcattga gggtgttagg gtaaccaaca tcagtgaaga 5520

gaaacaaccc acctcgagtg tgacctcatc atttcaggac acaatggcac cacaagatgc 5580

cgacgtcact gatgcgacgg actacaagaa accgcctgct gaaactgagc agaaggcact 5640

caccattcaa ccacggtcaa acaaggcgcc cagtgacgag gagttggtac gcatcatcaa 5700

cgcggcgcag aagcgaggcc tcacacccgc ggcctttgtt caagcagcta tagtcttcac 5760

catggaatcc atggacaagg gcgccaccga ctccacgatt ttcacgggaa aatacaacac 5820

tttcccaatg aaaagtctgg cgctagcttg caaagatgct ggcgtgcccg tgcacaaact 5880

ttgctacttc tataccaagc cggcttacgc gaaccgtagg gtcgccaacc agccgcctgc 5940

tcgctggacc aacgagaatg tgcccaaagc taacaagtgg gcggctttcg acaccttcga 6000

cgcacttctc gacccatacg tagtcccatc ctctgtaccg tacgatgagc ccacgccaga 6060

ggatcgccaa gtcaatgaga ttttcaagaa ggacaatttg agtcaggcag catccagaaa 6120

ccaactccgc gccctaggaa cgcaagcctc catcacgcgc gggagactca acggcgcacc 6180

agcactacca aacaacgggc agtacttcat cgaggcacct cagtgatcag tagtatgata 6240

ccaataaata aatcgggcga atccgcgcct cctgactatg ggcaggttta cggaccaagc 6300

tgtatcgaga tacgacctaa cagtaacgca gctaaggggt gaatgcacac atcgcttata 6360

aaaaaaaaaa aaaaaaaaaa aaaaaa 6386

<210> 2

<211> 519

<212> DNA

<213> Mutated P19 (P19)

<400> 2

atggaacgag ctatacaagg aaacgacgct agggaacaag ctaacagtga acgttgggat 60

ggaggatcag gaggtaccac ttctcccttc aaacttcctg acgaaagtcc gagttggact 120

gagtggcggc tacataacga tgagacgaat tcgaatcaag ataatcccct tggtttcaag 180

gaaagctggg gtttcgggaa agttgtattt aagagatatc tcagatacga caggacggaa 240

gcttcactgc acagagtcct tggatcttgg acgggagatt cggttaacta tgcagcatct 300

cgatttttcg gtttcgacca gatcggatgt acctatagta ttcggtttcg aggagttagt 360

atcaccgttt ctggagggtc tcgaactctt cagcatctct gtgagatggc aattcggtct 420

aagcaagaac tgctacagct tgccccaatc gaagtggaaa gtaatgtatc aagaggatgc 480

cctgaaggta ctgagacctt cgaaaaagaa agcgagtaa 519

<210> 3

<211> 4269

<212> DNA

<213> Cas9 vector (Cas9)

<400> 3

atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat 60

gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc 120

gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 180

accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 240

agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 300

acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 360

ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 420

gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 480

atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 540

ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 600

atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 660

gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 720

aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 780

ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg 840

attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 900

gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 960

atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 1020

ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 1080

atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1140

cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1200

tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1260

aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1320

cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1380

attctgcggc ggcaggaaga ttttttaccca ttcctgaagg acaaccggga aaagatcgag 1440

aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1500

ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1560

gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1620

ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1680

aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc 1740

ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1800

aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1860

ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1920

aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1980

accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 2040

ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 2100

ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2160

ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2220

ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2280

gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2340

aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2400

gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2460

aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2520

gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2580

atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2640

gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac 2700

aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2760

tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2820

aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2880

gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2940

aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 3000

ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 3060

caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3120

cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3180

atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3240

atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3300

ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3360

accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3420

acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3480

agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3540

tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3600

gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt 3660

ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3720

tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3780

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3840

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3900

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3960

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 4020

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 4080

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4140

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4200

ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 4260

aagaaaaag 4269

<210> 4

<211> 527

<212> DNA

<213> U6 promoter (AtU6-Scaffold)

<400> 4

agcttcgttg aacaacggaa actcgacttg ccttccgcac aatacatcat ttcttcttag 60

ctttttttct tcttcttcgt tcatacagtt ttttttttgtt tatcagctta cattttcttg 120

aaccgtagct ttcgttttct tctttttaac tttccattcg gagtttttgt atcttgtttc 180

atagtttgtc ccaggattag aatgattagg catcgaacct tcaagaattt gattgaataa 240

aacatcttca ttcttaagat atgaagataa tcttcaaaag gcccctggga atctgaaaga 300

agagaagcag gcccatttat atgggaaaga acaatagtat ttcttatata ggcccattta 360

agttgaaaac aatcttcaaa agtcccacat cgcttagata agaaaacgaa gctgagttta 420

tatacagcta gagtcgaagt agtggtttta gagctagaaa tagcaagtta aaataaggct 480

agtccgttat caacttgaaa aagtggcacc gagtcggtgc ttttttt 527

<210> 5

<211> 546

<212> DNA

<213> U6 promoter (AtU6-sgRNApds-Scaffold)

<400> 5

agcttcgttg aacaacggaa actcgacttg ccttccgcac aatacatcat ttcttcttag 60

ctttttttct tcttcttcgt tcatacagtt ttttttttgtt tatcagctta cattttcttg 120

aaccgtagct ttcgttttct tctttttaac tttccattcg gagtttttgt atcttgtttc 180

atagtttgtc ccaggattag aatgattagg catcgaacct tcaagaattt gattgaataa 240

aacatcttca ttcttaagat atgaagataa tcttcaaaag gcccctggga atctgaaaga 300

agagaagcag gcccatttat atgggaaaga acaatagtat ttcttatata ggcccattta 360

agttgaaaac aatcttcaaa agtcccacat cgcttagata agaaaacgaa gctgagttta 420

tatacagcta gagtcgaagt agtgccgtta atttgagagt ccagttttag agctagaaat 480

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 540

tttttt 546

Claims (36)

1. A plant expression system for expressing a complete genome editing GE system for editing a target sequence in a plant, characterized in that the plant expression system comprises at least one nucleotide sequence comprising:

(i) a plant-active promoter operably linked to a FoMV cDNA;

(ii) (ii) a FoMV cDNA encoding one or more recombinant foxtail mosaic virus FoMV viral vectors comprising at least one subgenomic promoter SGP operably linked to a heterologous nucleic acid encoding the GE system, and a terminator sequence;

(iii) a terminator sequence.

2. The plant expression system of claim 1, wherein the plant promoter is a cauliflower mosaic virus 35S gene promoter or repetitive 35S promoter and/or the terminator sequence is a NOS terminator.

3. An in vitro expression system for expressing one or more RNA transcripts collectively encoding a complete genome editing GE system for editing a target sequence in a plant, characterized in that said expression system comprises at least one nucleotide sequence comprising:

(i) a promoter operably linked to a FoMV cDNA;

(ii) FoMV cDNA encoding one or more recombinant Fomv viral vectors of foxtail mosaic virus comprising at least one subgenomic promoter SGP operably linked to a heterologous nucleic acid encoding the GE system.

4. The expression system of any one of claims 1 to 3, characterized in that the heterologous nucleic acid further comprises an RNAi repressor.

5. The expression system of claim 4, wherein the RNAi inhibitor is p19 or a derivative thereof.

6. Expression system according to claim 5, characterized in that said p19 derivative is shown in SEQ ID No:2 in (c).

7. The expression system according to any one of claims 1 to 6, characterized in that the expression system comprises first and second expression vectors encoding first and second FoMV viral vectors, respectively, which together encode the GE system.

8. Expression system according to any one of claims 1 to 7, characterized in that the GE system comprises at least:

(a)Cas9；

(b) a small guide RNA, sgRNA, for targeting the GE system to the target sequence.

9. The expression system of claim 8, in which the Cas9 comprises FLAG and/or nuclear localization signal.

10. The expression system according to claim 8 or 9, characterized in that the sgRNA is operably linked to a DNA-dependent RNA polymerase III, Pol III, promoter sequence, which is an optional U6 promoter nucleotide sequence.

11. The expression system according to any one of claims 8 to 10, characterized in that the sgRNA includes a target sequence corresponding to a target sequence in a target gene and includes a protospacer adjacent motif PAM.

12. The expression system according to any one of claims 7 to 11, characterized in that the expression system comprises first and second FoMV viral vectors which together encode the GExit, and wherein the first vector expresses Cas9 and the second vector expresses the sgRNA and optionally the repressor.

13. The expression system according to any one of claims 1 to 12, characterized in that the FoMV cDNA comprises a first and a second FoMV SGP, wherein:

the first SGP is operably linked to the heterologous nucleic acid encoding the GE system, and

the second SGP subgenomic promoter is operably linked to its native FoMV ORF.

14. The expression system of claim 13, wherein the first and second CP subgenomic promoters comprise the same sequence, optionally the same sequence being the native FoMV coat protein subgenomic promoter.

15. The expression system of any one of claims 13 to 14, wherein the heterologous nucleic acid is located 5' to the second subgenomic promoter.

16. The expression system according to any one of claims 1 to 15, characterized in that the system comprises a FoMV vector as shown in SEQ ID No:1 into which a CRISPR sequence as shown in SEQ ID No:3 is inserted.

17. The expression system according to any one of claims 1 to 16, characterized in that the system comprises a FoMV vector as shown in SEQ ID No:1 into which an At U6 scaffold as shown in SEQ ID No:4 and a further sgRNA sequence have been inserted.

18. A process for producing the expression system according to any one of claims 1 to 17, characterized in that the process comprises:

(1) providing:

(i) a promoter operably linked to a FoMV cDNA;

(ii) a FoMV cDNA comprising at least one FoMV subgenomic promoter SGP operably linked to a multiple cloning site MCS;

(2) cloning a heterologous nucleic acid encoding the GE system into the MCS.

19. One or more isolated RNA transcripts, characterized in that said one or more isolated RNA transcripts, together encoding a complete genome editing GE system for editing target sequences in plants, optionally encoded by an expression system according to any one of claims 1 to 17, encode one or more recombinant foxtail mosaic virus FoMV viral vectors comprising at least one subgenomic promoter SGP operably linked to a heterologous nucleic acid encoding said GE system.

20. A method comprising introducing the plant expression system of any one of claims 1 to 17 or the isolated RNA transcript of claim 19 into plant tissue.

21. A method of editing a target sequence in a plant tissue, comprising the steps of: introducing the expression system according to any one of claims 1 to 17 or the isolated RNA transcript according to claim 19 into said plant tissue or an elite thereof.

22. The method of claim 21, wherein said editing alters the sequence or expression of a gene product of said target sequence, wherein said target sequence is a target gene.

23. The method of claim 22, wherein the gene product is characterized by one or more of the following: herbicide tolerance, drought resistance, male sterility, insect resistance, abiotic stress tolerance, modified fatty acid metabolism, modified carbohydrate metabolism, improved seed yield, improved oil percentage, improved protein percentage, and resistance to bacterial, fungal, or viral diseases.

24. Method according to any one of claims 20 to 23, characterized in that the system comprises a first and a second FoMV viral vector which together encode the GE system and optionally an RNAi repressor, and the two viral vectors are applied simultaneously.

25. The method according to any one of claims 20 to 24, wherein the plant tissue is a leaf or a seed, wherein the seed coat has been disrupted.

26. Method according to any one of claims 20 to 24, characterized in that said plant tissue is part of a plant at the 2 or 3 leaf development stage.

27. The method according to any one of claims 20 to 26, characterized in that the expression system is introduced by agrobacterium-mediated T-DNA transfer.

28. Method according to any one of claims 20 to 27, characterized in that said plant is a monocotyledonous plant.

29. Method according to claim 28, characterized in that said plant is selected from the list consisting of: tobacco, lentil, alfalfa, barley, wheat, grass, corn, rice, soybean, cotton, rape, canola, tomato, potato.

30. A method comprising providing progeny or seed of a elite plant, characterized in that a target sequence has been edited in a tissue of the elite plant according to the method according to any one of claims 20 to 29, wherein the progeny or seed comprise the edited target sequence.

31. A kit comprising the expression system according to any one of claims 1 to 17.

32. An isolated RNA transcript from the expression system of any one of claims 1 to 17.

33. A virus or viral particle encapsulating the RNA transcript according to claim 19 or 32.

34. A plant host cell comprising the expression system of any one of claims 1 to 17 or the transcript or virus or viral particle of claim 19, 32 or 33.

35. Plant tissue comprising or transiently transformed with the expression system of any one of claims 1 to 17 or the transcript or virus or viral particle of claim 19, 32 or 33.

36. A plant comprising the plant cell of claim 34 or the plant tissue of claim 35.

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