Infectious plant rhabdovirus vectors and methods for non-transgenic genome site-specific editing in plants

Abstract

To provide methods for site-specifically modifying a plant chromosomal DNA using a plant rhabdovirus vector, and to provide viral vector compositions.SOLUTION: A step of systemically infecting a plant using a plant rhabdovirus vector, expressing a sequence-specific nuclease, targeting an infected plant genomic target site with a sequence-specific nuclease, and cleaving the target site; and a step of completing a site-specific editing on the target sites by a DNA repair mechanism of the plant are comprised. This method for site-specifically editing a plant genome target DNA does not require the introduction of selective marker genes. By delivering a sequence-specific nuclease using a plant rhabdovirus expression vector, not only can the editing efficiency for the target site be improved, but also a non-transgenic, and stably inheritable edited plant can be obtained.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of plant genetic engineering, and more particularly to a method for performing site-directed editing in plants using recombinant rhabdovirus vectors. Specifically, the present invention relates to a method for performing site-directed editing in plants by non-transgenic delivery of CRISPR nuclease components using recombinant rhabdovirus vectors. [Background technology]

[0002] In recent years, the emergence of targeted genome editing technology has opened up new avenues for precise crop breeding and has very broad application prospects in functional genomics research and genetic improvement of traits in various crops such as wheat, rice, and maize. Currently, targeted genome editing technology is largely based on site-specific nucleases (SSNs). A site-specific nuclease is a domain that combines a site-specific DNA-binding domain and a non-specific DNA-cleavage domain, allowing it to target and modify specific regions of the genome. After sequence-specific nucleases are introduced into cells, they can identify specific sequences in the genome, cleave those sequences to form double-stranded breaks (DSBs) in DNA, thereby activating and repairing non-homologous end joining (NHEJ) or homologous recombination (HR) within the cell, leading to deletion or replacement of gene function. Currently, commonly used sequence-specific nucleases mainly include three types: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR / Cas). Of these, CRISPR / Cas nucleases have the advantages of being easy to use, rapid, and highly efficient, and are currently the most widely applied genome editing tool.

[0003] One of the key technologies in current plant genome editing is plant delivery technology for sequence-specific nucleases, i.e., methods and means for introducing nucleic acid molecules and / or protein molecules containing sequence-specific nucleases into target cells, tissues, organs, or plant specimens. An ideal delivery technology must meet the following conditions: (1) It can introduce a complete sequence-specific nuclease nucleic acid or protein molecule into the plant material; (2) The recipient plant material to be delivered is easily obtainable, and the introduction process is simple and easy to handle; (3) The sequence-specific nuclease introduced into the plant material can exhibit high editing efficiency; (4) The mutated plant specimens after editing are easily obtainable; and (5) The obtained edited plant specimens do not contain the integration of foreign nucleic acids.

[0004] Conventional techniques for delivering CRIPSR / Cas nucleases to plants primarily rely on genetic recombination methods, specifically the introduction of recombinant genes containing CRIPSR / Cas into receptor plant cells for integration into the plant genome, and the resulting expressed nuclease producing predictable directional genetic changes to target genomic sequences. Currently, the most commonly used techniques in plants include Agrobacterium-mediated recombination and gene gun recombination, where any insertion of foreign DNA into the genome is considered genetic recombination. While these methods can deliver nucleases to plants, the following problems still exist in their breeding applications: (1) Regarding the integration of foreign genes into the plant genome, and during the transformation process, it is necessary to screen for markers using antibiotics or herbicides; (2) Unexpected phenotypic variations may exist in the resulting edited plants. (3) The process of isolating recombinant genes by means such as self-pollination or backcrossing is long, especially in plants with long breeding cycles, polyploids, and asexual reproduction; (4) In some countries or regions, the offspring of edited plantlets from which recombinant genes have been removed, and their products, may be subject to the supervision and control of genetically modified products.

[0005] Given the problems with genetically modified delivery methods, there is a need to develop a delivery method for non-transgenic CRISPR / Cas nucleases. Plant viral vectors are another promising method for CRISPR / Cas nuclease delivery. Viral vectors with systemic infectivity can spread from inoculated cells or tissues to other uninoculated tissues and cells, and can achieve systemic delivery of foreign genes at the plant organism level. Viral vectors without systemic infectivity cannot spread from the primary infected cell to other cells and therefore cannot achieve systemic delivery. Viral vectors with systemic infectivity, especially infectious RNA viral vectors, have special value in non-transgenic nuclease delivery applications because their replication process does not involve the DNA stage, and therefore there is no possibility of foreign nucleic acids being integrated into the host genome. However, the molecular weight of CRISPR / Cas nuclease genes is relatively large, reaching 4.2 thousand base pairs (Kb), and with the addition of the promoter element, it can reach 5-7 kb, posing a significant challenge to the current packaging capabilities of viral vectors. If the length of the foreign gene exceeds the loading capacity of the infectious viral vector, it can lead to loss or deletion of the foreign gene fragment, or loss of the virus's ability to infect the entire body (Avesani et al., Transgenic Res, 2007, 16:587-597).

[0006] Due to packaging limitations, some infectious plant virus vectors are used only to deliver relatively small nuclease components, such as CRISPR guide RNA (gRNA) of approximately 100-200 base pairs (bp) in length. Such vectors include, for example, cabbage leaf curl virus in plant DNA viruses (Yin et al., Sci Rep, 2015, 5:14926), tobacco stem necrosis virus in positive sense RNA viruses (Ali et al., Mol Plant, 2015, 8:1288-1291; WO 2015189693), tobacco mosaic virus (Cody et al., Plant Physiol, 2017, 175:23-35), pea browning virus (Ali et al., Virus Res, 2018, 244:333-337), and sugar beet root rot (Jiang et al., Plant Biotechnol). Examples include J, 2019, 17:1302-1315). These viral vectors are required to achieve genome site-directed editing via Cas9 recombinant inoculations, and therefore the genome of the edited inoculations obtained in this way still contains the insertion of the Cas9 nucleic acid sequence.

[0007] The paper (Mei et al., Plant Direct, 2019, 3:1-16) discloses a positive-sense RNA virus vector of *Zostera margaritacea* mosaic virus, which, when it has a green fluorescent protein gene of approximately 700 bp, completely deletes the foreign insertion fragment in infected upper leaves, making it available only for expressing a 100-200 bp CRISPR gRNA, and enabling gene editing dependent on the recombination expression of the Cas9 protein. Similar to the paper (Zhang et al., Funct & Integr Genomics, 2020, 20:471-477) and Chinese patent CN 111979262 A, a method for performing plant genome editing using this viral vector is disclosed. Due to limitations in packaging capacity, the Cas9 and gRNA were inserted into two separate viral vectors, enabling low-frequency gene editing when co-infecting plants.

[0008] The tolerance of the potato X virus, another positive-sense RNA virus, to foreign fragments was less than 2 kb (Larsen et al., BMC Biotech, 2012, 12:21; Avesani et al., Transgenic Res, 2007, 16:587-597). In a paper (Ariga et al., Plant Cell Physiol, 2020, 61:1946-1953), a method for delivering the CRISPR / Cas nuclease gene using this vector is disclosed. This viral vector can express the complete CRISPR / Cas nuclease in inoculated leaves and enable gene editing, but it cannot achieve systemic delivery of CRISPR / Cas and gene editing in uninoculated tissue.

[0009] A viral replicon system is a vector that retains the elements necessary for viral replication but removes elements related to the viral package and / or movement. This type of vector can efficiently replicate but cannot cause systemic infection, and therefore can accommodate fairly large (>10 kb) foreign insertion fragments. A paper (Baltes et al., Plant Cell, 2014, 151-163) discloses the delivery of CRISPR / Cas nucleases using a single-stranded DNA soybean dwarf virus replicon belonging to the Geminiviridae family, while a paper (Wang et al., Mol Plant, 2017, 10:1007-1010) and Chinese patent CN 109880846 A disclose the delivery of CRISPR / Cas nucleases and DNA repair templates using a Geminiviridae wheat dwarf virus replicon vector system. However, since viral repliconvectors are essentially deconstructed viruses, and it is necessary to introduce DNA constructs containing viral vectors into cells using methods such as Agrobacterium-mediated injection or gene guns, the possibility of foreign DNA integration in receptor cells is unavoidable.

[0010] Given the shortcomings of currently developed plant virus vectors, there is a need to develop a plant virus vector system that is infectious and can deliver a complete CRISPR / Cas nuclease throughout the body, particularly an infectious RNA virus vector system that does not involve the DNA stage in the replication process.

[0011] Viruses of the Rhabdoviridae family belong to the category of non-segmented single-stranded negative-sense RNA viruses. A paper (Wang et al., PLoS Pathog, 2015, 11:e 1005223) and patent CN 105039388 B disclose a method for genetically manipulating sowthistle yellow reticulum virus (SYNV), which belongs to the plant rhabdovirus family. Patent CN 104962580 B discloses a SYNV expression vector capable of carrying a green fluorescent protein (approximately 700 bp) and a β-galactosidase (1.8 Kb) binding gene, and a method for constructing such a vector, but it failed to enable systemic delivery of larger exogenous gene fragments (e.g., Cas nucleases) and a method for non-transgenic genome site-specific editing using the viral vector.

[0012] The paper (Gao et al., New Phytol, 2019, 223:2120-2133) and patent CN 110511955 A disclose an infectious clone of the plant rhabdovirus barley stripe dwarf virus (BYSMV), an expression vector, a method for constructing the same, and its use. When a fluorescent protein gene and a β-galactosidase gene are inserted, the vector can achieve systemic delivery and expression of the foreign gene in a cereal host plant. However, when carrying a larger CRISPR / Cas nuclease gene (approximately 5 Kb), the vector can only be introduced into tobacco benthamiana via Agrobacterium T-DNA transformation. While it expresses CRISPR / Cas nuclease in single cells and produces targeted gene editing, the recombinant virus cannot motility within tobacco benthamiana and cannot systemically infect planthoppers, which are hosts and vectors of cereal plants (Gao et al., New Phytol, 2019, 223: 2120-2133; original text page 12: "BYSMV-based genome edited systems cannot currently be recovered in cereals and planthoppers, as a result of their extremely large genomes, but this limitation of BYSMV and other rhabdovirus-based expression systems will be addressed by insertions of newly identified small CAS9 proteins in future studies"). As explained above, conventional technology has not been able to achieve systemic delivery of non-transgenic CRISPR / Cas nucleases. [Overview of the project] [Problems that the invention aims to solve]

[0013] To overcome the current technical challenges of performing non-transgenic genome site-specific modifications in plants using plant virus vectors, the present invention delivers a sequence-specific nuclease to a plant using a plant rhabdovirus. The sequence-specific nuclease targets a specific nucleic acid sequence in the plant genome, cleaves the target site, and completes site-specific modification of the target site via the plant's endogenous DNA repair mechanism. Furthermore, the plant rhabdovirus has systemic infectivity, and upon systemic infection with the virus, systemic delivery to the sequence-specific nuclease is achieved, allowing for the acquisition of a site-specific edited plant portion that does not involve the integration of foreign nucleic acid sequences. Moreover, after the regeneration of the site-specific edited plant portion (e.g., plant cells) that does not involve the integration of foreign sequences, a non-transgenic, stably heritable site-specific edited plant can be obtained. [Means for solving the problem]

[0014] A first aspect of the present invention provides a method for modifying plant cell genetic material that does not require the introduction of foreign gene sequences into the plant cell genome to be modified. The method comprises the steps of: a) providing at least one plant cell to be genetically modified; and b) infecting the plant cell with a recombinant plant rhabdovirus vector having systemic infectivity, wherein the recombinant rhabdovirus having systemic infectivity carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, and the sequence-specific nuclease specifically targets a plant genome nucleic acid sequence and cleaves the target site. The aforementioned cytogenetic material modification process is induced by cleavage using sequence-specific nucleases and completed by plant endogenous DNA repair mechanisms. Preferably, the method further includes step c) selecting a modified plant cell target containing genetic material, without the need to utilize a selection marker after step b).

[0015] A second aspect of the present invention provides a method for producing a plant in which a genetic material is modified without the need to introduce a foreign gene sequence into the plant cell genome to be modified. The method includes: step a) providing at least one plant cell in which a genetic material is to be modified; step b) infecting the plant cell with a recombinant plant rhabdovirus vector having systemic infectivity, wherein the recombinant rhabdovirus having systemic infectivity carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, and the sequence-specific nuclease specifically targets a plant genomic nucleic acid sequence and cleaves a target site; step c) obtaining a plant from the cell in which the genetic material is modified, without the need to use a selection marker, and step d) selecting a modified plant target containing the genetic material.

[0016] Preferably, the method of infecting the cell to be modified includes natural infection of a cell not inoculated with a recombinant virus, Ma rub-inoculation, grafting, insect-mediated transmission, or any other method of infection by a virus.

[0017] Preferably, the rhabdovirus vector having systemic infectivity is sonchus yellow net virus (SYNV) or eggplant mottled dwarf virus (EMDV). <W

[0018] Preferably, the genomic nucleic acid sequence of the sonchus yellow net virus is shown in SEQ ID NO:38, and the genomic nucleic acid sequence of the eggplant mottled dwarf virus is shown in SEQ ID NO:39. [[ID=W0000087]]

[0019] Preferably, the rhabdovirus vector having systemic infectivity includes an induced virus vector after one or more types of mutations and / or recombination modifications. Preferably, the induced virus vector is an induced virus vector in which the amino-terminal domain of the glycoprotein (G) is mutated and / or after recombination modification. [[ID=W0000090]] Preferably, the glycoprotein is mutated and / or recombinantly modified such that the glycoprotein N-terminal domain is deleted.

[0020] Preferably, the glycoprotein is mutated and / or recombinantly modified such that the nucleotide sequence of the glycoprotein N-terminal domain is replaced with a heterologous nucleic acid sequence.

[0021] Preferably, the cell genetic material modification includes at least one base deletion, or one base insertion, or one base substitution, or a combination of these modification patterns at the target site.

[0022] Preferably, the sequence-specific nuclease is a CRISPR / Cas nuclease, a TALEN nuclease, a zinc finger nuclease, or a nuclease capable of realizing all genome editing, preferably, the sequence-specific endonuclease is a CRISPR / Cas nuclease, preferably, the CRISPR / Cas nuclease is SpCas9 derived from Streptococcus pyogenes or LbCpf1 nuclease derived from Lachnospira.

[0023] Preferably, the ribonucleic acid sequence encoding the sequence-specific endonuclease includes the nucleic acid sequence of a single or multiple guide RNAs and the nucleic acid sequence of a Cas nuclease. Preferably, the guide RNA includes a sequence paired with the target gene and a sequence that binds to the Cas nuclease to form a complex. Preferably, the nucleic acid sequence encoding the sequence-specific nuclease exists in the form of an independent transcription unit in the genome of the rhabdovirus. Preferably, the transcription of the sequence-specific nuclease sequence is controlled by regulatory elements related to rhabdovirus messenger RNA transcription. Preferably, the guide RNA and the Cas nuclease sequence are controlled by the same transcription unit. Preferably, the guide RNA and Cas nuclease sequence are controlled by different transcription units. Preferably, the guide RNA transcript contains a terminal sequence derived from the virus. Preferably, the guide RNA transcript is processed to remove the virus-derived terminal sequence. Preferably, the guide RNA transcript has its virus-derived terminal sequence removed by an intracellular tRNA processing device. The aforementioned guide RNA transcript has its virus-derived terminal sequence removed by processing with Cpf1 nuclease.

[0024] Preferably, the plant is a natural or experimental host of plant rhabdovirus. Preferably, the plant is a host of sow thistle yellow reticulum virus or eggplant spot dwarf virus. Preferably, the plant is Nicotiana benthamiana. Preferably, the plant is potato (Solanum tuberosum). Preferably, the plant is a tomato (Lycopersicon esculentum). Preferably, the plant is tobacco (Nicotiana tabacum).

[0025] Preferably, the plants include eggplant (Solanum melongena), cucumber (Cucumis sativus), hydrangea (Hydrangea macrophylla), melon (Cucumis melo), Nicotiana clevelandii, Nicotiana glutinosa, Nicotiana rustica, Nicotiana edwardsonii, chili pepper (Capsicum annuum), Chenopodium amaranticolor, quinoa (Chenopodium quinoa), Datura metel, Hibiscus rosa-sinensis, honeysuckle (Lonicera), black nightshade (Solanum nigrum), sonchus oleraceus, and lettuce (Lactuca The selection is made from plants such as sativa, a hybrid variety of Nicotiana glutinosa and Nicotiana leebrand, zinnia elegans, Bidens pilosa, and quinoa (Chenopodium quinoa).

[0026] A third aspect of the present invention provides an infectious recombinant plant rhabdovirus vector comprising a polynucleotide sequence encoding at least one sequence-specific endonuclease, wherein the sequence-specific nuclease is transiently expressed in cells infected with the viral vector and specifically targets a plant genome nucleic acid sequence, and the target sequence is modified by the nuclease.

[0027] The recombinant rhabdovirus has systemic infectivity, and preferably the rhabdovirus is sonchus yellow net virus (SYNV) or eggplant mottled dwarf virus (EMDV).

[0028] Preferably, the recombinant plant rhabdovirus vector is a recombinant nucleic acid construct, and the sequence containing the viral vector is operably linked to a promoter.

[0029] Preferably, the genome nucleic acid sequence of the sow thistle yellow reticulum virus is shown in SEQ ID NO:38, and the genome nucleic acid sequence of the eggplant spot dwarf virus is shown in SEQ ID NO:39.

[0030] Preferably, the rhabdoviral vector having systemic infectivity comprises one or more types of mutated and / or recombinantly modified induced viral vectors, preferably the induced viral vector being a glycoprotein (G) amino-terminal domain mutated and / or recombinantly modified induced viral vector. Preferably, the glycoprotein is mutated and / or recombinantly modified such that the glycoprotein N-terminal domain is deleted.

[0031] Preferably, the glycoprotein is mutated and / or recombinantly modified such that the nucleotide sequence of the glycoprotein's N-terminal domain is replaced with a heterologous nucleic acid sequence.

[0032] Preferably, the cytogenetic material modification includes at least one base deletion, one base insertion, or one base substitution at the target site, or a combination of these modification patterns.

[0033] Preferably, the sequence-specific nuclease is a CRISPR / Cas nuclease, a TALEN nuclease, a zinc finger nuclease, or any nuclease capable of genome editing. Preferably, the sequence-specific endonuclease is a CRISPR / Cas nuclease. Preferably, the CRISPR / Cas nuclease is an SpCas9 nuclease derived from Streptococcus pyogenes or an LbCpf1 nuclease derived from Lachnospira. Preferably, the nucleic acid sequence of the SpCas9 nuclease is shown in SEQ ID NO:18, and the nucleic acid sequence of the LbCpf1 nuclease is shown in SEQ ID NO:19.

[0034] Preferably, the ribonucleic acid sequence encoding the sequence-specific endonuclease includes the nucleic acid sequences of one or more guide RNAs and the nucleic acid sequence of the Cas nuclease. Preferably, the guide RNA includes a sequence that pairs with the target gene and a sequence that binds to the Cas nuclease to form a complex. Preferably, the nucleic acid sequence encoding the sequence-specific nuclease exists in the genome of the rhabdovirus in the form of an independent transcription unit. Preferably, the transcription of the sequence-specific nuclease sequence is controlled by regulatory elements related to rhabdoviral messenger RNA transcription. Preferably, the guide RNA and Cas nuclease sequence are controlled by different transcription units. Preferably, the guide RNA and Cas nuclease sequence are controlled by the same transcription unit. Preferably, the guide RNA transcript contains a terminal sequence derived from the virus. Preferably, the guide RNA transcript is processed to remove the virus-derived terminal sequence. Preferably, the guide RNA transcript has its virus-derived terminal sequence removed by an intracellular tRNA processing device. The aforementioned guide RNA transcript has its virus-derived terminal sequence removed by Cas nuclease processing.

[0035] Preferably, the plant is a natural or experimental host of plant rhabdovirus. Preferably, the plant is a host of sow thistle yellow reticulum virus or eggplant spot dwarf virus. Preferably, the plant is Nicotiana benthamiana. Preferably, the plant is tobacco (Nicotiana tabacum). Preferably, the plant is potato (Solanum tuberosum). Preferably, the plant is a tomato (Lycopersicon esculentum).

[0036] Preferably, the plants include eggplant (Solanum melongena), cucumber (Cucumis sativus), hydrangea (Hydrangea macrophylla), melon (Cucumis melo), Nicotiana clevelandii, Nicotiana glutinosa, Nicotiana rustica, Nicotiana edwardsonii, chili pepper (Capsicum annuum), Chenopodium amaranticolor, quinoa (Chenopodium quinoa), Datura metel, Hibiscus rosa-sinensis, honeysuckle (Lonicera), black nightshade (Solanum nigrum), sonchus oleraceus, and lettuce (Lactuca The selection is made from plants such as sativa, a hybrid variety of Nicotiana glutinosa and Nicotiana leebrand, zinnia elegans, Bidens pilosa, and quinoa (Chenopodium quinoa).

[0037] A fifth aspect of the present invention provides a plant and offspring generation in which genetic material produced by the method described herein or by an infectious recombinant viral vector is modified. A sixth aspect of the present invention provides an infectious recombinant viral vector as described herein or in an infectious recombinant viral vector. A seventh aspect of the present invention provides a strain or recombinant virus in the method described herein or in an infectious recombinant viral vector.

[0038] An eighth aspect of the present invention provides the use of the method described herein or recombinant rhabdovirus vectors, or recombinant rhabdovirus vectors, or strains or recombinant viruses described herein in plant genome editing. Detailed description of the invention

[0039] To better define the present invention and to suggest to those skilled in the art how to carry it out, the following definitions and methods are provided, and unless expressly specified or limited, terms should be understood in accordance with the prior use of those skilled in the art in the relevant field.

[0040] In the present invention, the term "plant" includes the entire plant and any offspring generations, and plant parts that constitute the plant, including, but not limited to, plant cells, plant cell cultures, plant tissues, plant tissue cultures, plant cuttings, plant organs (e.g., pollen, embryos, flowers, fruits, buds, leaves, roots, stems, and associated explants), and plant seeds (e.g., mature seeds, immature seeds, immature embryos without seed coats). Plant cells may be in the form of isolated single cells or cell aggregates (e.g., callus and cultured cells), or they may be protoplasts, gamete-producing cells, or cells or aggregates of cells that can be regenerated into a complete plant. Plant cell cultures or tissue cultures can regenerate plants having the physiological or morphological characteristics of the plant from which the cells or tissues originated, and can regenerate plants having a genotype substantially identical to that of the plant. Regenerative cells in plant cell cultures or tissue cultures may be embryos, protoplasts, conidia, callus, pollen, leaves, stamen anthers, roots, root apices, filaments, flowers, kernels, spikes, rachis, husks, or stems.

[0041] The term "rhabdovirus" refers to a member of the Rhabdoviridae family of negative-sense RNA viruses. Negative-sense RNA viruses, also known as negative-strand RNA viruses, are viruses that infect humans, animals, plants, and fungi. They include the order Mononegavirale, which has an unsegmented genome, and encompasses families such as Paramyxoviridae, Filoviridae, Nyamiviridae, Bornaviridae, and Rhabdoviridae. ), Mymonaviridae, Nyamiviridae and Sunviridae, and other families of the Bunyavirales order whose genomes are segmented, such as Bunyaviridae, Ferraviridae, Fimoviridae, Hantaviridae, Jonviridae, Naiviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae and Tospoviridae. Also, families with segmented genomes that are not classified for the purpose of this study, such as Arenaviridae, Aspiviridae and Orthomyxoviridae. Here, rhabdoviruses have typical rod-shaped or bullet-shaped viral particles and include rhabdoviridae families that infect animals and rhabdoviridae families that infect plants. Rhabdoviridae families that infect animals include, but are not limited to, the genera Lyssavirus, Vesiculovirus, Ephemerovirus, and Novirhabdivirus.Rhabdoviruses that infect plants mainly belong to the following four genera: Nucleorhabdovirus, Cytorhabdovirus, Dichorhavirus, and Varicosavirus. Members of the genus Rhabdovirus include, but are not limited to, sonchus yellow net virus (SYNV), maize fine streak virus (MFSV), maiziranian mosaic virus (MIMV), maize mosaic virus (MMV), eggplant mottled dwarf virus (EMDV), potato yellow dwarf virus (PYDV), wheat yellow striate virus (WYSV), rice yellow stunt viruses (RYSV), taro vein chlorosis virus (TVCV), black currant-associated rhabdovirus (BCARV), and datura yellow vein virus (DYVV). Members of the cytoplasmic rhabdovirus genus include, but are not limited to, rice stripe mosaic virus (RSMV), northern cereal mosaic virus (NCMV), maize yellow striate virus (MYSV), and barley yellow striate mosaic virus (BYSMV).Members of the genus Valicosavirus include, but are not limited to, alopecurus myosuroides varicosavirus (AMVV), lettuce big vein associated virus (LBVaV), and red clover varicosavirus (RCVV). The genus Bipartite Rhabdovirus includes, but are not limited to, orchid fleck virus (OFV), cirus leprosis virus (CiLV), citrus chlorotic spot virus (CCSV), coffee ringspot virus (CRV), and tiger chlorotic spot virus (clerodendrum chlorotic spot virus (CLCSV).

[0042] The structure of the Rhabdoviridae virus genome is conserved, encoding five conserved structural proteins in the order of 3'-NPMGL-5' from the 3' end to the 5' end of the genome: nucleocapsid protein N, phosphoprotein P, matrix protein M, glycoprotein G, and RNA-dependent RNA polymerase large subunit L. Here, the minimal infectious unit of rhabdoviruses, the ribonucleoprotein core complex (RNP), is composed of genomic RNA surrounded by the viral core proteins, which are usually nucleocapsid proteins N and L. The core proteins of some viral members may also be other types of proteins; for example, the core protein of sonchus yellow net virus (SYNV) contains phosphoprotein P. In addition to encoding the five conserved structural proteins mentioned above, some rhabdovirus members encode additional non-structural proteins; for example, SYNV encodes one additional non-structural protein, sc4. The rhabdovirus genome contains 3'-leader and 5'-trailer sequences at both ends, respectively. Both the leader and trailer sequences are non-protein-coding sequences, but they play important regulatory roles in genome replication and mRNA transcription. Each gene in the rhabdovirus contains conserved gene spacer sequences; for example, the spacer between the N and P genes is the NPJ (N / P junction) sequence. The function of the gene spacer sequence is to terminate the transcription of the upstream gene and initiate the transcription of the downstream gene. It consists of three parts: an element that serves as the upstream mRNA transcription termination signal and polyadenyle template, an element consisting of sequences that are not transcribed during mRNA synthesis (usually including a continuous uracil nucleotide U), and a downstream mRNA transcription initiation element.

[0043] Rhabdovirus transcription exhibits both sequential and polar transcription characteristics. Sequential transcription means that each viral gene is transcribed sequentially according to its relative position on the genome, while polar transcription means that the amount of sequentially transcribed mRNA produced gradually decreases according to its relative position on the genome. The rhabdovirus transcription process is carried out under the action of viral polymerase, which first transcribes the leader mRNA from the 3' end of the viral genome, then sequentially transcribes the mRNAs encoding the viral proteins. The transcription process is controlled by gene spacer sequences. When viral polymerase moves to the gene spacer between each gene, it terminates the transcription of the gene located upstream of the gene spacer. The polymerase complex then adds a poly-A sequence to the end of the mRNA of the upstream gene and releases mRNA with a 5' cap structure and a 3' poly-A structure. At this point, the polymerase complex continues to move downstream until it encounters the transcription initiation signal for the next gene, initiating the transcription of the next gene, and finally transcribing to the trailer mRNA. As a result, when performing vector modification using this type of virus, it is necessary to follow the transcriptional characteristics of this type of virus. In addition to adding an exotic nucleic acid sequence, it is also necessary to add a transcriptional regulatory element, i.e., the gene spacer mentioned above, so that the exotic nucleic acid sequence can be made into an independent transcriptional unit and transcribed and expressed under the action of viral polymerase.

[0044] The term "vector" in genetic engineering refers to a self-replicating DNA molecule that transfers a foreign nucleic acid fragment to a receptor cell, and generally contains a foreign coding sequence and the appropriate nucleic acid sequence required to express the coding sequence in a specific host; plasmids are exemplary vectors. "Recombinant viral vector," "recombinant viral expression vector," and "recombinant viral clone" are interchangeable as used herein and are constructs formed by delivering a specific nucleic acid sequence mediated by a viral vector to a target cell, tissue, organ, or plant. "Infectious viral vector" and "virally infectious clone" refer to constructs formed by delivering a specific nucleic acid sequence mediated by a viral vector to a target cell, tissue, organ, or plant, and by rescuing an active recombinant virus, thereby achieving systemic infection.

[0045] The terms “exotic” and “other” refer to sequences from exotic species, or, if from the same species, sequences whose composition and / or loci have been significantly altered from their natural form due to intentional human intervention. The term “genetic material” refers to the substance that transmits genetic information between parental and offspring generations. “Genetic material to be modified” includes, but is not limited to, single or multiple deoxyribonucleotide substitutions, deletions, additions, or combinations thereof. The terms “nucleic acid,” “nucleic acid molecule,” and “nucleic acid sequence” are interchangeable and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutated, or synthetic DNA or RNA molecules, and DNA or RNA analogs produced using nucleotide analogs, which may be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences, antisense sequences, and non-coding regulatory sequences that do not code for mRNA or protein products of structural genes. The terms “gene” and “gene sequence” refer to DNA nucleic acids relating to biological function. Therefore, a gene may include introns and exons in a genome sequence, or it may include only the coding sequence in a cDNA, and / or cDNA combined with regulatory sequences.

[0046] The term "sequence-specific nuclease" (SSss) refers to an endonuclease that can cleave a specific DNA sequence and create a double-strand break (DSB). Currently, commonly used sequence-specific nucleases include, but are not limited to, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats, and CRISPR-associated systems (CRISPR / Cas). A "zinc finger nuclease" refers to an artificial restriction enzyme produced by binding a zinc finger DNA-binding domain to a DNA-cleaving domain. ZFNs contain synthetic proteins of engineered and modified zinc finger DNA-binding domains bound to the cleaving element of a FokI restriction endonuclease, and can be used to achieve locus-specific targeting of genome sequences by inducing double-strand breaks at specific DNA sequences. A "transcription activator-like effector nuclease" refers to an artificial restriction enzyme produced by binding a DNA-recognizing element that induces Xanthomonas to the catalytic element of a nuclease. Specifically, the TALEN system mainly consists of a DNA-binding domain containing the TALE protein and a FokI endonuclease domain. The TALE protein contains multiple duplicate peptide fragments consisting of 33-35 amino acids, and each peptide fragment can recognize one base. For TALENs to function, they need to recognize DNA strands in the 5′-to-3′ and 3′-to-5′ directions in dimer form, and then use the bound FokI endonuclease to cleave the target sequence.The terms "clustered, regular, short-sequence palindromic sequence repeats and CRISPR-related systems," "CRISPR nuclease," "CRISPR / Cas nuclease," and "Cas nuclease" are interchangeable and generally refer to nucleases present in the CRISPR system, their mutants (e.g., NIC enzyme mutants, inactivating mutants, etc.), and derivatives that bind to other functional proteins. The term encompasses any nuclease capable of achieving genome editing in cells within the CRISPR system.

[0047] In this invention, the terms "guide RNA," "gRNA," and "sgRNA" are interchangeable and generally refer to RNA molecules that can bind to a CRISPR nuclease to form a complex and guide that complex to a target site. In Cas9-based genome editing systems, gRNA consists of crRNA and tracrRNA molecules, of which crRNA contains a sequence complementary to the target sequence and can guide the CRISPR complex to specifically bind to the target sequence. Furthermore, as is well known in the art, a single guide RNA can be designed by binding crRNA and tracrRNA. On the other hand, in CpF1-based genome editing systems, gRNA generally refers only to crRNA.

[0048] In the present invention, the terms “target,” “target site,” “target sequence,” and “target site sequence” are used interchangeably and generally refer to any desired nucleic acid sequence on which to act, including, but not limited to, coding or non-coding sequences, genes, exons, introns, regulatory sequences, gene spacer sequences, synthetic sequences, and intracellular parasitic sequences.

[0049] In the present invention, the term "delivery" refers to means of delivering nucleic acid sequences and / or amino acid sequences to target cells, tissues, organs or plants, and the delivery methods include, but are not limited to, gene gun methods, protoplast transformation, Agrobacterium inoculation, mechanical friction, vacuum penetration, high-pressure spray gun injection, and chemical delivery.

[0050] In this invention, the term "tRNA" refers to a small RNA molecule that has the function of carrying and transporting amino acids, which can fold into a clover-shaped secondary structure, and in eukaryotes, tRNA precursors produced by tRNA gene transcription are recognized by endogenous RNase P and RNase Z and processed into mature tRNA. In some embodiments of this invention, the tRNA sequence used is the tRNA sequence described in "Methods and Compositions for Multi-Induced Genome Editing and Other RNA Technologies" (Patent Publication No. WO 2016 / 061481) Gly Selected from a code array.

[0051] In this invention, the terms "tissue culture" and "organ culture" are interchangeable and refer to techniques for isolating cells, tissues, organs, or protoplasts from a plant body that meet specific requirements, culturing them under artificially controlled conditions using sterile techniques to obtain complete strains for regeneration or to produce other economically valuable products.

[0052] In this invention, the terms "gene editing," "genome editing," "genome site-specific editing," "genome-directed editing," "genome modification," "genome site-specific modification," and "genome-directed modification" are interchangeable and refer to techniques for modifying specific target genes in the genome of an organism. Generally, this involves first producing a locus-specific double-strand break at a specific location in the genome, inducing the organism to repair the double-strand break via non-homologous end joining or homologous recombination, and then producing the targeted modification. Furthermore, as used herein, the singular terms “one / one kind” and “the aforementioned” include multiple referents unless otherwise indicated by the specific context.

[0053] The present invention provides a method for modifying plant cell genetic material that does not require the introduction of foreign gene sequences into the plant cell genome to be modified. The method comprises the steps of: a) providing at least one plant cell to be genetically modified; and b) infecting the plant cell with a recombinant plant rhabdovirus vector having systemic infectivity, wherein the recombinant rhabdovirus having systemic infectivity carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, and the sequence-specific nuclease specifically targets a plant genome nucleic acid sequence and cleaves the target site. The aforementioned cytogenetic material modification process is induced by cleavage using sequence-specific nucleases and completed by plant endogenous DNA repair mechanisms. Preferably, the method further includes step c) selecting a modified plant cell target containing genetic material, without the need to utilize a selection marker after step b).

[0054] The present invention further provides a method for producing genetically modified plants that do not require the introduction of foreign gene sequences into the plant cell genome to be modified. The method comprises the steps of: a) providing at least one plant cell to be genetically modified; b) infecting the plant cell with a recombinant plant rhabdovirus vector having systemic infectivity, wherein the recombinant rhabdovirus having systemic infectivity carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, the sequence-specific nuclease specifically targets a plant genome nucleic acid sequence and cleaves the target site; and c) selecting a target plant cell containing the genetic material without requiring the use of a selection marker.

[0055] The present invention further provides a method for producing genetically modified plant callus that does not require the introduction of foreign gene sequences into the genome of plant cells to be modified. The method comprises the steps of: a) providing at least one plant cell to be genetically modified; b) infecting the plant cell with a recombinant plant rhabdovirus vector having systemic infectivity, wherein the recombinant rhabdovirus having systemic infectivity carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, the sequence-specific nuclease specifically targets a plant genome nucleic acid sequence and cleaves the target site; and c) selecting a modified callus target containing the genetic material without requiring the use of a selection marker.

[0056] The method for infecting the cells to be modified is natural infection of recombinant virus-uninoculated cells. Ma This includes inoculation by rubbing, grafting, insect-borne transmission, or any other method of infection that does not involve viral DNA constructs.

[0057] The cytogenetic material modification includes at least one base deletion, one base insertion, or one base substitution at the target site, or a combination of these modification patterns.

[0058] The present invention further provides an infectious recombinant plant rhabdovirus vector characterized by a) being able to systemically infect a certain type of plant host, and b) carrying a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, wherein the sequence-specific nuclease specifically targets a plant genome nucleic acid sequence and cleaves the target site.

[0059] The recombinant rhabdovirus has systemic infectivity, and preferably the rhabdovirus is sow thistle reticulata virus. The sow thistle reticulata virus genome nucleic acid sequence used in the embodiments of the present invention is shown as SEQ ID NO:38, and the above sequence is the nucleic acid sequence of an isolate of the virus on sow thistle. In addition to sow thistle, the virus can infect several types of plants such as lettuce, chicory, quinoa, and tobacco, and nucleic acid sequences of virus isolates derived from other hosts are also applicable to the method of the present invention. The total length of the viral genome is 13.7 kb, and the encoding order of each gene is 3'-NP-sc4-MGL-5'. According to the rhabdovirus transcription scheme, heterologous nucleic acid sequences can be inserted between any two adjacent genes in the viral genome (e.g., between N and P, between P and sc4, between sc4 and M, between M and G, or between G and L), or after the leader region (between the 3'-leader and N protein), or before the trailer region (between the L protein and 5'-trailer). In further embodiments of the present invention, the insertion site of the heterologous nucleic acid sequence is between the 3'-leader and the N protein. In some embodiments of the present invention, the insertion site of the heterologous nucleic acid sequence is between N and P, and the insertion of the heterologous nucleic acid sequence must conform to the virus's own coding policy, and additional spacers should be added simultaneously with the addition of the heterologous nucleic acid sequence to form an independent transcription unit. The length of each gene spacer in SYNV is between 100 and 200 nt and contains approximately 16 base-conserved sequences (5'-TATAAGAAAAACCAAC-3'), and when spacers are added additionally, these conserved sequences should generally be included, but the remaining sequences can be mutated, truncated, or lengthened. In a preferred embodiment of the present invention, a complete gene spacer should be added simultaneously with the addition of the heterologous nucleic acid sequence.

[0060] Preferably, the recombinant rhabdovirus is eggplant spot dwarf virus, and the eggplant spot dwarf virus genome nucleic acid sequence used in embodiments of the present invention is shown as SEQ ID NO:39. The above nucleic acid sequence is the nucleic acid sequence of an isolate of the virus on eggplant, and in addition to eggplant, the virus can infect many plants such as cucumber, hydrangea, and tomato, and nucleic acid sequences of virus isolates derived from other hosts are also applicable to the method of the present invention. The total length of the viral genome is approximately 13.2 kb, and the encoding order of each gene is 3'-NXPYMGL-5', and similar to sow thistle yellow reticulata virus SYNV, heterologous nucleic acid sequences can be inserted between any two adjacent genes in the viral genome (e.g., between N and X, between X and P, between P and Y, between Y and M, between M and G, or between G and L), or after the leader region (between the 3'-leader and N protein), or before the trailer region (between the L protein and 5'-trailer). The insertion of heterologous nucleic acid sequences must similarly adhere to the virus's coding policy, and normal transcription and expression can be performed after the addition of additional spacers. Each gene spacer in EMDV is between 47 and 217 nt in length and contains approximately 17 base-conserved sequences (5'-TTTAATAAAAACCCAAC-3'). When additional transcription units are added, these conserved sequences generally need to be retained, while the remaining sequences can be mutated, truncated, or lengthened. In some embodiments of the present invention, the insertion site of the heterologous nucleic acid sequence is between Leader and N, and in further embodiments of the present invention, the insertion site of the heterologous nucleic acid sequence is between N and X.

[0061] The rhabdoviral vector having systemic infectivity further comprises one or more induced viral vectors after various mutations and / or recombinant modifications. Preferably, the modified viral vector is a viral vector in which the glycoprotein (G) has been mutated and / or recombinantly modified. The G protein structure encoded by negative sense RNA viruses is conserved and consists of a signal peptide, an N-terminal domain, a transmembrane domain, and a C-terminal domain, and is mosaicized on the surface of the viral particle in the form of trimers. Existing research has shown that the G protein plays an important role in the recognition and transmission process of the virus and its vector, and for example, one non-synonymous mutation (C1375A) in the G protein of tomato yellow spot necrosis virus did not affect systemic infection of the virus. However, since it cannot be transmitted by the vector thrips, partially deleting, mutating, or substituting the G protein of the recombinant rhabdoviral vector affects insect-borne transmission of the virus and provides greater biosafety. In some embodiments of the present invention, recombinant viral vectors with systemic infectivity can be obtained by deleting or substituting the N-terminal domain of the G protein of sow thistle (Sonchus oleraceus) virus without affecting plant site-specific editing.

[0062] The sequence-specific endonuclease is a CRISPR / Cas nuclease, a TALEN nuclease, a zinc finger nuclease, or a nuclease capable of all genome editing, preferably a CRISPR / Cas nuclease. Specifically, the nuclease is a nuclease capable of all genome editing, such as Cas9 (e.g., SpCas9, SaCas9), Cpf1 (also called Cas12a, such as AsCpf1, LbCpf1, FnCpf1), CasX, or CasY, and a mutation, variant, or derivative of some amino acids based on the CRISPR nuclease, such as the hi-fi Cas nuclease espCast9, the Cas9 nic enzyme nCas9, the Cas9 inactivating enzyme dCas9, and the base editors ABE, CBE, PBE, etc.

[0063] Preferably, the nuclease is a nucleotide sequence containing the nucleic acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 19, or SpCas9 derived from Streptococcus pyogenes or LbCpF1 derived from Lachnospira having 20% ​​or more sequence identity (e.g., 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity). "Identity" refers to the degree of invariance of a polynucleotide or protein segment in matching a sequence (e.g., a nucleotide sequence or an amino acid sequence). Sequence matching is generated by the following steps: manually comparing two sequences, for example, a reference sequence and another sequence as provided herein, to generate a maximum number of matching elements, such as individual nucleotides or amino acids, while simultaneously allowing the introduction of nicks into any sequence. The "identity score" of the sequence matched with the reference sequence is the number of matching elements divided by the total length of the reference sequence, and does not include the nicks introduced into the reference sequence by the matching process. As used herein, "percentage identity" is the identity score multiplied by 100.

[0064] Those skilled in the art will see that examples of codon optimization for specific host species are feasible and known, and that to obtain more efficient Cas nuclease expression, the sequence encoding the Cas nuclease can be codon-optimized according to the specific species. Codon optimization is a method of modifying the nucleic acid sequence to enhance expression in the host cell of interest by substituting at least one codon in the native sequence, for example, about 1, 2, 3, 4, 5, 10, 15, 20, 25, or 50 or more codons, via codons that are more frequently or most frequently used in the host cell's gene, while maintaining the native amino acids. Some embodiments of the present invention provide examples of Cas9 nuclease sequences for codon optimization in humans, and according to further embodiments of the present invention, examples of Cas9 nuclease sequences for codon optimization in tobacco benthamiana are provided.

[0065] The sequence-specific nuclease may further comprise one or more subcellular localization domains, which may include, but are not limited to, nuclear localization signals, mitochondrial targeting signals, or chloroplast targeting signals. In some embodiments of the present invention, the subcellular structural localization domain is a nuclear localization sequence (NLS). Generally, an NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the surface of a protein, although other types of NLSs are also known. The NLS sequence may be present at the N-terminus and / or C-terminus of the nuclease, and about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs may be bound to the N-terminus, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs may be bound to the C-terminus, or a combination of the binding methods described above may be employed. In some preferred embodiments of the present invention, the NLS is an SV40 nuclear localization signal simultaneously bound to the N-terminus and C-terminus of the nuclease.

[0066] The rhabdoviral vector carries a nucleic acid sequence encoding at least one sequence-specific nuclease, and if the sequence-specific nuclease is a Cas nuclease, the nucleic acid sequence carried by the rhabdoviral vector is a nucleic acid sequence encoding gRNA (or both crRNA and tracrRNA) and a Cas nuclease. The gRNA includes a nucleotide sequence Nx that can specifically hybridize with a complementary sequence of the target sequence, and a sequence that binds to the Cas nuclease to form a complex, where Nx represents a nucleotide sequence consisting of X consecutive nucleotides, N is independently selected from A, G, T, and C, and in the formula, X is an integer between 18 and 35, preferably X = 20.

[0067] In currently used CRISPR / Cas9 genome editing systems, Cas9 and gRNA are generally constructed as independent transcription and expression units. The Cas9 protein expression unit consists of a Pol II promoter, Cas9 ORF, and terminator, while the gRNA transcription unit consists of a Pol III promoter (often using small RNA transcription promoters such as U6 or U3), a gRNA unit, and a PolyT terminator. The Cas9 protein expression unit and gRNA transcription unit can be constructed on two independent vectors and co-transformed, or they can be constructed as two independent units on a single vector and transformed, or the Cas9 protein expression unit and gRNA transcription unit can be placed on the same unit via several binding sequences and constructed on a single vector for transformation. Here, Cas9 protein expression can be controlled by selecting an appropriate Pol II type promoter depending on the characteristics of the transformation target and experimental requirements, such as the CMV, Hsp70, and SV40 promoters commonly used in animal cells and CaMV35S, ZmUb1, and AtUb10 promoters commonly used in plants. Generally, the gRNA transcription unit uses specific small RNA transcription promoters such as U6 and U3 in the target genome, depending on the transformation target. However, in the case of rhabdovirus vectors, due to the transcriptional characteristics of rhabdoviruses, it is only necessary to add gene spacers to the additional expression unit, making the operation extremely simple and convenient. Furthermore, the guide RNA and Cas nuclease sequence can be controlled by the same transcription unit or by different transcription units. In a further embodiment of the present invention, the nucleic acid sequence encoding the Cas nuclease and the nucleic acid sequence encoding the guide RNA can be controlled by the same transcription unit, and they are bound together by a known binding sequence. Specifically, the binding sequence is 5'-AAGCCATGGGATATC-3' (using the sequence reported by Mikami et al. in 2017), and it is known that, in addition to this binding sequence, the nucleic acid sequence encoding the Cas nuclease and the nucleic acid sequence encoding the guide RNA can be placed in the same transcription unit in various ways.For example, RNA-modified elements such as ribozymes (hammerhead ribozymes, hairpin ribozymes, hepatitis D ribozymes, VS ribozymes, I-class introns, RNase P, etc.), tRNA sequences, and Csyh4 sequences are included, but are not limited to these. In further embodiments of the present invention, the nucleic acid sequence encoding the guide RNA and the nucleic acid sequence encoding the Cas nuclease are each ligated with additional gene spacers so that they each become independent transcription units.

[0068] When a heterologous nucleic acid sequence encoding guide RNA is expressed as an independent transcription unit, the transcript contains extra sequences derived from the viral vector, depending on the transcriptional characteristics of the rhabdovirus itself. In some embodiments of the present invention, the editing efficiency at the target site of a recombinant rhabdovirus vector (SYNV-gGFP2-Cas9) carrying the guide RNA nucleic acid sequence and the Cas9 nuclease nucleic acid sequence was approximately 20%. In a further embodiment of the present invention, to obtain a relatively accurate guide RNA transcript, tRNA is added on both sides of the heterologous nucleic acid sequence encoding the guide RNA. Gly A precursor sequence is added, and the precursor sequence is identified by a plant endogenous tRNA processing device, allowing for more precise cleavage and processing of the guide RNA transcript. The editing efficiency at the target site of a recombinant rhabdovirus vector (SYNV-tgtGFP2-Cas9) carrying the nucleic acid sequence of the guide RNA and the nucleic acid sequence of the Cas9 nuclease was approximately 90%. Therefore, recombinant viral vectors having RNA processing elements are preferred vectors of the present invention, and common RNA processing elements include, but are not limited to, ribozymes (hammerhead ribozymes, hairpin ribozymes, hepatitis D ribozymes, VS ribozymes, I-class introns, RNase P, etc.), tRNA sequences, Csyh4 sequences, etc.

[0069] The guide RNA can target a single gene or multiple genes, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. In a further embodiment of the present invention, the guide RNA targets a single gene. Specifically, the targeted gene is the endogenous PDS gene of Citrus benthamiana, the selected target is PDS target 1, the sequence-specific nuclease used is CRISPR / Cas9 nuclease, the recombinant rhabdovirus used is SYNV, the resulting recombinant viral expression vector is SYNV-tgtPDS1-Cas9, and the editing efficiency of the vector was approximately 79%. In a further embodiment of the present invention, the guide RNA targets two genes simultaneously. Specifically, the policy employs a serial linking of multiple tRNA sequences and gRNA sequences (tRNA-gRNA1-tRNA-gRNA2-tRNA). When the tRNA sequence is identified and cleaved by a plant endogenous processing mechanism, two guide RNA transcripts can be released simultaneously. Specifically, the genes are the endogenous genes RDR6 and SGS3 of Tobacco benthamiana, and the two target sites are target 1 of RDR6 and target 1 of SGS3, respectively. The above targets can target two copies of RDR6 and two copies of SGS3 in heterotetraploid Tobacco benthamiana, respectively. More specifically, the target site sequences are 5'-AGTTGGGTAAGAGTCAGGAG-3' (SEQ ID NO:22) and 5'-ACAAGAGTGGAAGCAGTGCT-3' (SEQ ID NO:23), respectively, and the heterogeneous nucleic acid sequence containing the tRNA-RDR6 target1-gRNA scaffold-tRNA-SGS3 target1-gRNA scaffold-tRNA is shown in SEQ ID NO:1010. The resulting recombinant rhabdovirus vector is SYNV-tgtRDR6-1 / SGS3-1-Cas9, and the targeting efficiencies of this recombinant rhabdovirus vector for RDR6 and SGS3 were approximately 96% and 93%, respectively. In a further specific embodiment of the present invention, partial deletion of genomic chromosome fragments was achieved using a SYNV multi-target editing vector.Specifically, two target sites of the endogenous PDS gene targeting Nicotiana benthamiana are linked in series using tRNA sequences, releasing two guide RNA transcripts that target PDS target 1 and target 3, respectively (both targets can simultaneously target two copies of the PDS targeting Nicotiana benthamiana). Specifically, the target sequences are 5'-GCCGTTAATTTGAGAGTCCA-3' (SEQ ID NO:21) and 5'-TTAACTGTATTGTCTAGCTCTGG-3' (SEQ ID NO:24), and the heterologous nucleic acid sequence containing the tRNA-PDS target1-gRNA scaffold-tRNA-PDS target3-gRNA scaffold-tRNA is shown in SEQ ID NO:12. The resulting recombinant rhabdovirus vector is SYNV-tgtPDS1 / 3-Cas9, and the efficiency of partial deletion of chromosomal fragments produced by this vector was approximately 48%.

[0070] Those skilled in the art will see that the embodiments described above are merely one preferred form of multi-target editing, and that according to the rhabdovirus transcription method, multiple guide RNA transcription units can be expressed in other different ways, such as by having heterogeneous nucleic acid sequences of multiple guide RNA transcription units each bind a gene spacer, making each an independent transcription unit, or by having transcription units encoding multiple guide RNAs bind and express according to the binding sequences described above, and that these methods are feasible and known. For example, in a further embodiment of the present invention, multi-target editing of a plant genome can be achieved by serially linking multiple heterogeneous crRNA nucleic acid sequences. Specifically, the crRNA serialization method is direct repeat (DR)-guide sequence 1 (Guide 1)-direct repeat (DR)-guide sequence 2 (Guide 2)-direct repeat (DR), i.e., DR-guide1-DR-guide2-DR. When the DR sequence is identified and cleaved by the crRNA processing mechanism of Cpf1, two crRNA transcripts can be released, specifically the two target genes being the 16c endogenous genes GFP and PDS, and the two target sites being target 1 of GFP and target 1 of PDS, respectively, the above targets can target one copy of GFP and two copies of PDS in the heterotetraploid 16c strain, more specifically the target site sequences being 5'-AAGATACCCAGATCATATGAAGC-3' (SEQ ID NO: 25) and 5'-TGCCGTTAATTTGAGAGTCCAAG-3' (SEQ ID NO: 27), and the heterologous nucleic acid sequence containing DR-GFP1-DR-PDS1-DR is SEQ ID As shown in NO:17, the obtained recombinant rhabdovirus vector was pSYNV-crGFP-1 / PDS-1-Cpf1, and the targeting efficiencies of this vector for GFP and PDS were 81% and 82%, respectively.

[0071] The aforementioned plants are plants that can be host plants of any rhabdovirus, and include most monocots and dicots, as well as plants of the families Graminea, Solanaceae, Fabaceae, Cucurbitaceae, Compositae, Amaranthaceae, Chenopodiaceae, and Caprifoliaceae, such as tobacco (Nicotiana tabacum), benthamiana tobacco (Nicotiana benthamiana), potato (Solanum tuberosum), wheat (Triticum aestivum), soybean (Glycine max), maize (Zea mays), rice (Oryza sativa), coffee (Coffea spp.), tomato (Lycopersicon esculentum), lettuce (Lactuca sativa), and sow thistle (Sonchus). oleraceus), zinnia elegans, Bidens pilosa, quinoa (Chenopodium quinoa), eggplant (Solanum melongena), cucumber (Cucumis sativus), hydrangea (Hydrangea macrophylla), melon (Cucumis melo), Nicotiana clevelandii, Nicotiana glutinosa, Nicotiana rustica, Nicotiana edwardsonii, chili pepper (Capsicum annuum), Chenopodium amaranticolor, quinoa (Chenopodium quinoa), Datura metel, Hibiscus This includes, but is not limited to, plants such as rosa-sinensis, honeysuckle (Lonicera), or black nightshade (Solanum nigrum). In some embodiments of the present invention, the plant is the tobacco benthamiana, tobacco, potato, tomato, eggplant, and the like.

[0072] The methods for infecting plants via the rhabdovirus include, but are not limited to, any method known in the art, such as gene gun inoculation, protoplast transformation, Agrobacterium inoculation, mechanical friction, grafting, vacuum penetration, and high-pressure spray gun injection. In a preferred embodiment of the present invention, systemic infection with recombinant rhabdovirus is achieved using the Agrobacterium infiltration inoculation method. In a further embodiment of the present invention, the friction inoculation method is used to initiate infection with recombinant rhabdovirus. In another embodiment of the present invention, systemic infection with rhabdovirus is achieved using the grafting method.

[0073] The method for obtaining the non-transgenic, stably heritable, directionally modified plant includes tissue culture of a portion of the plant to be edited to obtain a site-specifically edited and stably heritable strain. For example, one embodiment of the present invention provides a method for obtaining a non-transgenic edited plant by encoding a Cas9 nuclease using a rhabdovirus vector. The recombinant rhabdovirus is sowthistle yellow reticulum virus, the sequence-specific nuclease is CRISPR / Cas9 nuclease, the gene spacer is a spacer between N and P, the guide RNA heteronucleotide sequence containing the tRNA flanking sequence and the heteronucleotide sequence for expressing Cas9 are each joined by an NPJ sequence to form independent transcription units which are inserted between N and P, the target gene is the endogenous PDS gene of tobacco benthamiana, the target site is 5'-GCCGTTAATTTGAGAGTCCA-3' (SEQ ID NO:21), and the obtained recombinant rhabdovirus vector is pSYNV-tgtPDS1-Cas9. The vector can be used to obtain virus-free, non-transgenic edited inoculum in the M0 generation after tissue culture of systemically infected leaves following inoculation into tobacco benthamiana, and can stably inherit mutations to offspring, thereby obtaining virus-free, non-transgenic edited inoculum in both the M1 and M2 generations. In a further embodiment of the present invention, a method is provided for obtaining non-transgenic edited inoculum by encoding the LbCpf1 nuclease using a rhabdovirus vector. The gene spacers are N 5'UTR and NPJ, and the heterologous nucleic acid sequence for transcribing the crRNA and the heterologous nucleic acid sequence for expressing LbCpf1 are joined to the gene spacers to form two independent transcription units, both of which are inserted before the N gene transcription unit, the target gene is the endogenous PDS gene of Citrus benthamiana, the target site is 5'-TGCCGTTAATTTGAGAGTCCAAG-3' (SEQ ID NO:27), and the heterologous nucleic acid sequence for expressing the crRNA is as shown in SEQ ID NO:15.The resulting recombinant rhabdovirus vector is SYNV-crPDS1-Cpf1. This vector can be used to obtain virus-free, non-transgenic edited plants in the M0 generation after tissue culture of systemically infected leaves following inoculation into Citrus benthamiana, and it can stably pass mutations to offspring. The present invention further provides plants and offspring generations in which genetic material produced by the methods described herein or by infectious recombinant viral vectors has been modified. The present invention further provides infectious recombinant viral vectors in the manner described herein or in infectious recombinant viral vectors. The present invention further provides bacterial strains or recombinant viruses in the methods described herein or in infectious recombinant viral vectors.

[0074] The present invention further provides the use of recombinant rhabdovirus vectors, recombinant rhabdovirus vectors, strains, or recombinant viruses described herein in plant genome editing. [Effects of the Invention]

[0075] This expression has the following beneficial effects compared to conventional technologies. (1)

[0076] (2) The recombinant rhabdoviral vector has a stronger exogenous fragment-accommodating capacity that allows it to simultaneously accommodate a heterogeneous nucleic acid sequence for transcribing guide RNA and a heterogeneous nucleic acid sequence for expressing Cas nuclease. (3)

[0077] (4) After inoculating plants with the recombinant rhabdovirus vector, a large amount of sequence-specific nuclease is produced as the virus replicates, editing the target gene and achieving higher editing efficiency. (5)

[0078] (6) Since the production of the guide RNA transcript depends on the virus's own transcription policy, the first base of the target sequence is not limited to G or A as required by the conventional U6 or U3 promoter. (7) The recombinant rhabdovirus vector can express multiple nucleases, so it can identify multiple PAM sequences and expand the range of targets. (8) The recombinant rhabdovirus vector can be subjected to multi-target editing by simultaneously adding multiple transcription units or by using a tRNA processing method. (9) The recombinant rhabdovirus vector has good stability, and the obtained recombinant rhabdovirus can be used to obtain edited inoculum by friction inoculation. (10) According to this application, non-transgenic edited plants can be directly obtained in the M0 generation, and the editing can be stably inherited to the offspring generation. [Brief explanation of the drawing]

[0079] [Figure 1] Figure 1 shows the construction of a SYNV vector to simultaneously express two reporter genes. (A) is a schematic diagram of the construction of the dual reporter gene expression vector SYNV-GFP-RFP, where Bsu36I and NheI are single enzyme cleavage sites located on the N protein and P protein, respectively, and NPJ is an NP gene spacer (NP gene junction). (B) shows the fluorescence expression status of recombinant rhabdovirus vectors SYNV-GFP and SYNV-GFP-RFP after inoculation with tobacco benthamiana, respectively. (C) shows the viral symptoms produced after inoculation with recombinant rhabdovirus vectors SYNV-GFP and SYNV-GFP-RFP with tobacco benthamiana, respectively. (D) shows the expression status of SYNV structural protein, GFP protein, and RFP protein detected by Western blot. [Figure 2]Figure 2 shows the construction of a CRISPR / Cas9 gene editing vector based on SYNV. (A) is a schematic clone diagram of SYNV-Cas9, SYNV-gRNA-Cas9, and SYNV-tgtRNA-Cas9. Bsu36I, NheI, and AhdI are single enzyme cleavage sites located on the N protein, P protein, and Cas9 protein, respectively. NPJ is an NP gene spacer (NP gene junction). Bsu36I-NPJ is the sequence from the Bsu36I locus on the N gene to the downstream NPJ. Cas9 is the Cas9 ORF sequence. NPJ-NheI is the sequence from NPJ to the NheI locus on the P gene. gRNA is a 20nt target sequence and a gRNA backbone sequence. NPJ-AhdI is the locus from the NPJ region to AhdI on the Cas9 gene. tgtRNA is the bilateral tRNA precursor sequence of gRNA. The aforementioned fragments are used in the clonal construction process. (B) is a schematic gRNA release diagram. Due to the characteristics of SYNV replication and transcription, the gRNA transcript contains virus-derived non-coding region sequences (5'UTR, 3'UTR sequences, and polyA tail) at both ends. (C) is a schematic diagram of obtaining an accurate gRNA transcript using a tRNA processing policy, in which the tRNA sequences bound to both ends of the gRNA are processed by plant endogenous RNase P and RNase Z, after which the virus-derived non-coding region sequences are removed, and an accurate gRNA transcript is released. [Figure 3]Figure 3 shows an analysis of the infectivity of the SYNV CRISPR / Cas9 edited vector targeting GFP genetically modified plantlets. (A) shows the symptoms that occur after inoculation of 16c genetically modified tobacco with the recombinant viral vectors SYNV-Cas9, SYNV-tgtGFP1-Cas9, and SYNV-tgtGFP2-Cas9. When GFP expression was observed in the whole leaves of plantlets inoculated with the recombinant viruses using a UV ultraviolet lamp and laser confocal microscope, Mock represents a healthy control strain, and V-Cas9, V-tgtGFP1-Cas9, and V-tgtGFP2-Cas9 represent the above-mentioned recombinant viral vectors, respectively. The symptom diagram was taken 15 days after systemic infection of the inoculated plantlets. (B) shows the expression status of SYNV viral structural protein, Cas9 protein, and GFP protein detected by Western blot. (C) shows the morphological structure of recombinant viral particles observed with an electron microscope. The length of the viral particles was calculated as the average value after measuring 20 viral particles. [Figure 4] Figure 4 shows an analysis of the mutation frequency of genetic recombinations targeting GFP by the SYNV CRISPR / Cas9 editing vector. (A) shows the detection of site-directed editing efficiency of GFP using the PCR / RE method. In the figure, + and - indicate whether or not the corresponding restriction endonuclease was added in the enzyme cleavage reaction, respectively. Indel(%) represents the percentage of PCR products that could not be cleaved by the enzyme out of the total PCR product. (B) shows the detection of site-directed editing status of GFP using Sanger sequencing. The underlined sequences are the recognition sequences of the corresponding restriction endonucleases, WT indicates that the sequencing sequence matches the wild-type sequence, d indicates a base deletion, d# indicates # base deletions, and X# indicates # occurrences of the sequence in the sequencing result. (C) is a Sanger sequencing peak diagram. [Figure 5]Figure 5 shows an analysis of the effect of gRNA processing expressed by the SYNV CRISPR / Ca9 vector on editing efficiency. (A) is a diagram illustrating the principle of loop reverse transcription PCR (cRT-PCR). (B) shows the size of gRNA transcripts produced by recombinant viral vectors SYNV-gGFP2-Cas9 and SYNV-tgtGFP2-Cas9 detected using the cRT-PCR method. (C) shows the target gene editing efficiency detected using the PCR / RE method. (D) shows the editing status of GFP generated by imprecise gRNA transcripts analyzed using Sanger sequencing. [Figure 6]Figure 6 shows the mutation frequency analysis of the endogenous PDS gene targeting Nicotiana benthamiana with the SYNV CRISPR / Cas9 edited vector. (A) is a diagram of the NbPDS gene. In the figure, PDSa and PDSb represent two copies of the heterotetraploid Nicotiana benthamiana endogenous PDS gene, the vertical lines represent the location of the target sequence, and gPDS1, gPDS2, and gPDS3 represent the three target sequences of PDS, all of which can simultaneously target PDSa and PDSb. The underlines represent the corresponding restriction endonuclease recognition sequences in the target sequences. (B) shows the symptoms that occur after inoculation of Nicotiana benthamiana with the recombinant viral vector SYNV-tgtRNA-Cas9. gPDS1, gPDS2, and gPDS3 represent the symptoms that occur approximately 50 days after inoculation of Nicotiana benthamiana with the recombinant viral vector containing the above target sites. (C) shows the detection of site-specific editing efficiency of PDS using the PCR / RE method. In the figure, M represents the marker, WT / U represents WT / undigested, i.e., the result when the PCR product of the wild-type control did not undergo the corresponding restriction endonuclease reaction, WT / D represents WT / digested, i.e., the result when the PCR product of the wild-type control underwent the corresponding restriction endonuclease reaction, and gPDS1, gPDS2, and gPDS3 represent three randomly selected inoculated strains of tobacco benthamiana after inoculation with editing vectors containing the aforementioned target sites. Indel(%) represents the percentage of the total PCR product that could not be cleaved by enzymes. (D) shows the verification of PDS target mutations using Sanger sequencing. gPDS1 / a and gPDS1 / b represent the editing status of PDSa and PDSb after inoculation with recombinant viral vectors targeting PDS1, respectively, in tobacco benthamiana. [Figure 7]Figure 7 shows the SYNV CRISPR / Cas9 editing vector targeting the endogenous gene RDR6 in Tobacco benthamiana. (A) is a diagram of the NbRDR6 gene. In the figure, RDR6a and RDR6b represent two copies of the heterotetraploid endogenous gene RDR6 in Tobacco benthamiana, and gR6-1 and gR6-4 represent two target sequences of RDR6, both of which can simultaneously target RDR6a and RDR6b. (B) shows the symptoms that occur after inoculation of Tobacco benthamiana with the recombinant viral vector SYNV-tgtRNA-Cas9, and gR6-1 and gR6-4 represent the symptoms that occur after inoculation of Tobacco benthamiana with recombinant viral vectors containing the above target sites, respectively. (C) shows the detection of site-directed editing efficiency of RDR6 using the PCR / RE method. (D) shows the verification of RDR6 target mutations using Sanger sequencing. [Figure 8] Figure 8 shows the SYNV CRISPR / Cas9 editing vector targeting the endogenous SGS3 gene in Tobacco benthamiana. (A) is a diagram of the NbSGS3 gene. In the figure, SGS3a and SGS3b represent two copies of the heterotetraploid endogenous SGS3 gene in Tobacco benthamiana, and gS3-1 and gS3-2 represent two target sequences of SGS3, both of which can simultaneously target SGS3a and SGS3b. (B) shows the symptoms that occur after inoculation of Tobacco benthamiana with the recombinant viral vector SYNV-tgtRNA-Cas9, and gS3-1 and gS3-2 represent the symptoms that occur after inoculation of Tobacco benthamiana with recombinant viral vectors containing the above target sites, respectively. (C) shows the detection of site-directed editing efficiency of SGS3 using the PCR / RE method. (D) shows the verification of SGS3 target mutations using Sanger sequencing. [Figure 9]Figure 9 is a schematic diagram of the construction of a SYNV CRISPR / Cas9 dual-target editing vector. 't' represents tRNA, and a method of sequentially linking tRNA and gRNA in series (tRNA-gRNA-tRNA-gRNA-tRNA) is used to identify and process the tRNA precursor sequence using a plant endogenous tRNA processing device, and then release two gRNA transcripts. [Figure 10] Figure 10 shows a SYNV CRISPR / Cas9 dual-target editing vector that simultaneously targets the endogenous genes RDR6 and SGS3 in tobacco plants. (A) shows the symptoms that occur after inoculation of tobacco plants with the recombinant rhabdovirus vector SYNV-tgtgtRNA-Cas9. In the figure, gR6-1&gS3-1 and gR6-4&gS3-2 represent the symptoms that occur after inoculation of tobacco plants with the recombinant rhabdovirus vectors SYNV-tgtgtRDR6-1 / SGS3-1-Cas9 and SYNV-tgtgtRDR6-4 / SGS3-2-Cas9, respectively. (B) shows the detection of site-directed editing efficiency when the dual-target SYNV editing vector simultaneously targets RDR6 and SGS3 using the PCR / RE method. In the figure, gR6-1&gS3-1 and gR6-4&gS3-2 represent the editing status of RDR6 target site 1 and SGS3 target site 1, and RDR6 target site 4 and SGS3 target site 2, respectively, after inoculation of tobacco benthamiana with the dual-target editing vectors SYNV-tgtgtRDR6-1 / SGS3-1-Cas9 and SYNV-tgtgtRDR6-4 / SGS3-2-Cas9. (C) shows the editing status when gR6-4 and gS3-2 are targeted simultaneously using Sanger sequencing. In the figure, gR6-4 / gS3-2 / a represents the editing status of RDR6 copy a when RDR6 and SSG3 are targeted simultaneously, and gR6-4 / gS3-2 / b represents the editing status of RDR6 copy b when RDR6 and SSG3 are targeted simultaneously. gR6-4 / gS3-2 / a represents the editing status of SSG3 copy a when RDR6 and SSG3 are targeted simultaneously, and gR6-4 / gS3-2 / b represents the editing status of SSG3 copy b when RDR6 and SSG3 are targeted simultaneously. [Figure 11]Figure 11 shows that the SYNV CRISPR / Cas9 dual-target editing vector produces chromosomal fragment deletions by simultaneously targeting two targets of PDS. (A) shows the symptoms that occur after inoculation of tobacco benthamiana with the recombinant viral vector SYNV-tgtgtPDS1 / 3-Cas9. (B) shows the detection of chromosomal deletions after inoculation of tobacco benthamiana with the recombinant viral vector SYNV-tgtgtPDS1 / 3-Cas9 using the PCR / RE method. In the figure, the arrows represent the fragments after chromosomal deletion by simultaneously targeting gPDS-1 and gPDS-3. Indel(%) represents the percentage of the PCR product after chromosomal deletion in the total PCR product. (C) shows the chromosomal deletion status when gPDS-1 and gPDS-3 are simultaneously targeted using Sanger sequencing. [Figure 12]Figure 12 shows the regeneration of leaves and analysis of M0 generation genotypes of plantlets infected with the SYNV CRISPR / Cas9 vector. (A) are regenerated plants obtained by tissue culture of whole leaves after infection of tobacco benthamiana with the recombinant viral vector SYNV-tgtPDS1-Cas9. The left panel shows regenerated plants obtained in differentiation medium, the middle panel shows albinos obtained in rooting medium, and the right panel shows green plants obtained in rooting medium. (B) shows the results of detecting the editing status of regenerated strains using the PCR / RE method. In the figure, Mock / U represents Mock / undigested, meaning the PCR product of the wild-type control was not cleaved by HinfI enzyme, and Mock / D represents Mock / digested, meaning the PCR product of the wild-type control was cleaved by HinfI enzyme. The Albino lane shows the results of HinfI enzyme cleavage in the PCR products of 6 albino seedlings obtained from tissue culture, and the Normal lane shows the results of HinfI enzyme cleavage in the PCR products of 24 green seedlings obtained from tissue culture. M is the marker. (C) shows the results of detecting the virus content of tissue cultured plants using the RT-PCR method. In the figure, virus indicates that the primer used was a viral genome detection primer, and actin indicates that the primer used was a Benthamiana tobacco reference primer. SYNV represents a plant with systemic infection of wild-type SYNV and serves as a positive control, while Mock represents a healthy plant and serves as a negative control. M is the marker. [Figure 13] Figure 13 shows the phenotypic inheritance analysis of the M1 generation of regenerated plants edited by Cas9. The left figure shows the phenotypic segregation of representative M1 generation plantations, with M0-8 and M0-22 indicating segregation ratios of 3:1 and 15:1, respectively. The right figure shows the M1 generation phenotypic segregation statistics, where the observed values ​​represent the actual segregation ratio, and the expected segregation ratio is 3:1 or 15:1. The p-value is calculated using a chi-squared test, and a p-value > 0.5 indicates that the observed segregation ratio is consistent with the expected segregation ratio. [Figure 14]Figure 14 shows the analysis of the offspring generation genotypes of M0-8 plantlets edited by Cas9. (A) shows the phenotype and genotype of the M0-8 plantlets. (B) shows the results of PCR / RE analysis and genotyping of M1 generation plantlets from the M0-8 strain system. In the figure, lanes M8-1 to M8-10 are M1 generation plantlets of 10 M0-8 plants, M8-1 to M8-5 are 5 randomly selected albino plants, and M8-6 to M8-10 are 5 randomly selected green seedlings. The right figure shows the genotyping results of the randomly selected plantlets, with M8-1 being an albino seedling and M8-9 being a green seedling. (C) shows the results of PCR / RE analysis and genotyping of M2 generation plantlets from the M0-8-9 plantlets. In the figure, lanes M8-9-1 to M8-9-10 are M2 generation plantings of 10 M0-8-9 plants, M8-9-1 to M8-9-5 are 5 randomly selected albino plants, and M8-9-6 to M8-9-10 are 5 randomly selected green seedlings. The figure on the right shows the genotype sequencing results of the randomly selected plantings, with M8-9-1 being an albino seedling and M8-9-10 being a green seedling. [Figure 15] Figure 15 shows the results of verifying the removal of the viral vector from Cas9-edited M1 generation inoculations using RT-PCR. The detected strains were 4-5 M1 generation inoculations randomly selected from strain lines such as M0-8, -18, -22, -29, -42, -6, -7, and -34. The CK+ lane represents SYNV-infiltrated inoculations as the positive control, SYNV indicates that the primer used was a viral genome detection primer, and Actin indicates that the primer used was a Benthamiana tobacco reference primer. [Figure 16] Figure 16 shows the off-target analysis of the SYNV CRISPR / Cas9 genome editing system. (A) shows the potential off-target loci of the target site gPDS1. In the figure, the underlined sequences are the recognition sequences of the HinfI endonuclease. (B) shows the PCR / RE analysis results of the potential off-target loci of the target site gPDS1, where T7E1 and HinfI represent the endonucleases used, respectively, and OT1-13 represent 13 potential off-target loci. [Figure 17] Figure 17 shows the analysis of offspring virus editing ability and stability after friction inoculation with SYNV CRISPR / Cas9. (A) shows the symptoms of friction-inoculated plants. V-Cas9 and V-tgtGFP1-Cas9 represent leaves of systemic infection after infiltration inoculation of 16c tobacco with SYNV-Cas9 or SYNV-tgtGFP1-Cas9, respectively. (B) is the Western blot analysis of friction-inoculated plants. (C) is the RT-PCR analysis of friction-inoculated plants. In the figure, Lane Mock represents healthy 16c plants, and V-WT, V-Cas9, and V-tgtGFP1-Cas9 represent 16c plants after friction inoculation, with their inoculation sources being leaves of systemic infection after infiltration inoculation of SYNV, SYNV-Cas9, and SYNV-tgtGFP1-Cas9, respectively. gRNA, Cas9, SYNV, and Actin represent the detection primers used. (D) shows the PCR / RE analysis of friction-inoculated plantlets. In the figure, lane V-tgtGFP1-Cas9 represents the 16c strain, which was friction-inoculated with SYNV-tgtGFP1-Cas9 from three randomly selected strains. [Figure 18]Figure 18 shows the construction of a CRISPR / Cpf1 gene editing vector based on SYNV. (A) is a schematic diagram of clones of SYNV-Cpf1 and SYNV-crRNA-Cpf1. PvuI and Bsu36I are single enzyme cleavage sites located on the N protein before the leader sequence, respectively. NPJ is an NP gene spacer (NP gene junction). The PvuI-N5'UTR fragment is the sequence from the PvuI enzyme cleavage site to the 5' non-coding region of the N gene. NPJ-Bsu36I is the sequence before the NPJ sequence to the Bsu36I enzyme cleavage site on the N gene. NPJ-Cpf1-Bsu36I is the sequence before the NPJ sequence to the Bsu36I enzyme cleavage site on the N gene, including the Cpf1 sequence. The aforementioned fragments are used in the clonal construction process. (B) is a schematic diagram of crRNA release. Due to the characteristics of SYNV replication and transcription, the crRNA transcript contains virus-derived non-coding region sequences (5'UTR, 3'UTR sequences, and polyA tail) at both ends. By ligating DR sequences to both ends of the guide, the crRNA transcript can be processed with Cpf1, and the excess non-coding region sequences can be removed, releasing the accurate crRNA. [Figure 19] Figure 19 shows the infectivity analysis of SYNV CRISPR / Cpf1 edited vectors targeting GFP genetically modified plantlets. (A) shows the symptoms that occur after inoculation of 16c genetically modified tobacco plants with recombinant virus vectors SYNV-Cpf1, SYNV-crGFP1-Cpf1, and SYNV-crGFP2-Cpf1. GFP expression in the entire leaf system of the inoculated plantlets was observed using a UV ultraviolet lamp and laser confocal microscope. Mock represents a healthy control strain, and the symptom diagrams shown above were taken 15 days after systemic infection of the inoculated plantlets. (B) shows the expression status of SYNV virus protein, Cpf1 protein, and GFP protein detected by Western blot. [Figure 20]Figure 20 shows an analysis of the mutation frequency of genetic recombinations targeting GFP by the SYNV CRISPR / Cpf1 editing vector. (A) shows the detection of site-directed editing efficiency of GFP using the PCR / RE method. In the figure, + and - indicate whether or not the corresponding restriction endonuclease was added in the enzyme cleavage reaction, respectively. Indel(%) represents the percentage of PCR products that could not be cleaved by the enzyme out of the total PCR product. (B) shows the detection of site-directed editing status of GFP using Sanger sequencing. The underlined sequence is the GFP target sequence, wt indicates that the sequencing sequence matches the wild-type sequence, d indicates a delete, d# indicates # base deletions, and X# indicates # occurrences of the sequence in the sequencing result. (C) is a Sanger sequencing peak diagram. [Figure 21]Figure 21 shows the mutation frequency analysis of the endogenous PDS gene targeting Nicotiana benthamiana with the SYNV CRISPR / Cpf1 editing vector. (A) is a diagram of the NbPDS gene. In the figure, PDSa and PDSb represent two copies of the heterotetraploid Nicotiana benthamiana endogenous PDS gene, the vertical lines represent the location of the target sequence, and crPDS1 and crPDS2 represent the two target sequences of PDS, all of which can simultaneously target PDSa and PDSb. The underlined lines represent the corresponding restriction endonuclease recognition sequences in the target sequences. (B) shows the symptoms that occur after inoculation of Nicotiana benthamiana with the recombinant viral vectors SYNV-Cpf1, SYNV-crPDS1-Cpf1, and SYNV-crPDS2-Cpf1. (C) shows the detection of site-specific editing efficiency of PDS using the PCR / RE method. In the figure, M represents a marker, Mock / U represents Mock / undigested, i.e., the result of the wild-type control PCR product not undergoing the corresponding endonuclease enzyme cleavage, Mock / D represents Mock / digested, i.e., the result of the wild-type control PCR product undergoing the corresponding endonuclease enzyme cleavage, and V-crPDS1-Cpf1 and V-crPDS2-Cpf1 represent three randomly selected inoculated strains of tobacco benthamiana after inoculation with the edit vectors containing the aforementioned target sites. Indel(%) represents the percentage of the total PCR product that could not be cleaved by the enzyme. (D) shows the analysis of the site-specific editing status of PDS using Sanger sequencing. PDSa and PDSb represent the editing status of PDSa and PDSb after inoculation with recombinant viral vectors targeting the PDS1 site in tobacco benthamiana, respectively. [Figure 22] Figure 22 is a schematic diagram of the construction of a SYNV CRISPR / Cpf1 dual-target editing vector. By using a method of sequentially connecting DR and guide sequences in series (DR-guide1-DR-guide2-DR), it is possible to remove extra non-coding regions after Cpf1 processing using the Cpf1 crRNA processing mechanism, thereby releasing accurate crRNA. [Figure 23]Figure 23 shows the SYNV CRISPR / Cpf1 dual-target editing vector simultaneously targeting the GFP and PDS genes in recombinant 16c tobacco plants. (A) shows the symptoms that occur after inoculation of tobacco plants with the recombinant rhabdovirus vector SYNV-crGFP-1 / PDS-1-Cpf1. (B) shows the site-specific editing efficiency detected when the dual-target SYNV editing vector simultaneously targets GFP and PDS using the PCR / RE method. In the figure, crGFP-1 / PDS-1 and crGFP-1 / PDS-1 represent the editing status of GFP target site 1 and PDS target site 1, respectively, after inoculation of recombinant 16c tobacco plants with the dual-target editing vector SYNV-crGFP-1 / PDS-1-Cpf1. [Figure 24] Figure 24 shows the regeneration of whole leaves of plantlets infected with the SYNV CRISPR / Cpf1 vector and the analysis of M0 generation genotypes. (A) is a regenerated strain obtained from tissue culture of whole leaves after infection of Tobacco benthamiana with the recombinant viral vector SYNV-crPDS1-Cpf1. The upper figure shows a regenerated strain obtained in differentiation medium, and the lower figure shows albino strains obtained in rooting medium. (B) shows the results of detecting the editing status of regenerated strains using the PCR / RE method. In the figure, Mock / U represents Mock / undigested, meaning the PCR product of the wild-type control was not cleaved by HinfI enzyme, and Mock / D represents Mock / digested, meaning the PCR product of the wild-type control was cleaved by HinfI enzyme. The Albino lane shows the results of HinfI enzyme cleavage in the PCR products of 4 albino seedlings obtained from tissue culture, and the Normal lane shows the results of HinfI enzyme cleavage in the PCR products of 27 green seedlings obtained from tissue culture. M is the marker. (C) shows the results of detecting the virus content of tissue cultured plants using the RT-PCR method. In the figure, tobacco benthamiana actin is used as the internal control. [Figure 25]Figure 25 shows the phenotypic genetic analysis of the M1 generation of regenerated plants edited by Cpf1. (A) shows the phenotypic segregation of representative M1 generation plants, with M0-15 showing no segregation of albinos, and M0-11 and M0-9 showing segregation ratios of 3:1 and 15:1, respectively. (B) shows the M1 generation phenotypic segregation statistics, with the observed values ​​in the figure representing the actual segregation ratio, and the expected segregation ratios being 0, 3:1, or 15:1. [Figure 26] Figure 26 shows the infectivity analysis of SYNV-edited vectors with G protein N-terminal deletion or substitution. (A) is a schematic clone of recombinant rhabdovirus SYNV-tgtPDS1-Cas9, SYNV-tgtPDS1-Cas9-dGN, and SYNV-tgtPDS1-Cas9-mCherry:GN. In the figure, SP, NT, TM, and CT represent the signal peptide, N-terminal domain, transmembrane domain, and C-terminal domain, respectively. (B) shows the symptoms and mCherry expression status obtained from whole leaves after inoculation of tobacco benthamiana with recombinant rhabdovirus SYNV-tgtPDS1-Cas9, SYNV-tgtPDS1-Cas9-dGN, or SYNV-tgtPDS1-Cas9-mCherry:GN. (C) shows the results of Western blot analysis performed on inoculated strains of recombinant rhabdovirus SYNV-tgtPDS1-Cas9, SYNV-tgtPDS1-Cas9-dGN, or SYNV-tgtPDS1-Cas9-mCherry:GN using G protein monoclonal antibodies. (D) represents PCR / RE detection, where M represents a marker, Mock / U represents Mock / undigested, i.e., the PCR product of the wild-type control did not undergo the corresponding restriction endonuclease reaction, Mock / D represents Mock / digested, i.e., the PCR product of the wild-type control underwent the corresponding restriction endonuclease reaction, and the lanes SYNV-tgtPDS1-Cas9, SYNV-tgtPDS1-Cas9-dGN, and SYNV-tgtPDS1-Cas9-mCherry:GN represent systemically infected inoculations of tobacco benthamiana after inoculation with the recombinant viruses, respectively. [Figure 27]Figure 27 shows the analysis of the infectivity and editing efficiency of SYNV edited vectors with codon optimization for Cas9 in tobacco benthamiana. (A) is a schematic clone of recombinant rhabdovirus SYNV-tgtPDS2-Cas9 and SYNV-tgtPDS2-oCas9. In the figure, oCas9 is the Cas9 nuclease sequence with codon optimization for tobacco benthamiana. (B) shows the symptoms that appear from the whole leaves after inoculation of tobacco benthamiana with recombinant rhabdovirus SYNV-tgtPDS2-Cas9 and SYNV-tgtPDS2-oCas9. (C) shows the detection of site-specific editing of PDS using the PCR / RE method. In the figure, lanes SYNV-tgtPDS2-Cas9 and SYNV-tgtPDS2-oCas9 represent 10 randomly selected systemically infected plantlets, respectively, after inoculation of tobacco benthamiana with the recombinant viral vectors described above. [Figure 28] Figure 28 shows the analysis of the infectivity and editing efficiency of the SYNV single transcription unit editing vector. (A) is a schematic clone of recombinant rhabdovirus SYNV-oCas9gPDS2, where the oCas9 nuclease sequence and gRNA sequence are linked using the 15 bp spacer sequence (5'-AAGCCATGGGATATC-3') shown. (B) shows the symptoms that occur from whole leaves after inoculation of tobacco benthamiana with recombinant rhabdovirus SYNV-oCas9gPDS2 and SYNV-tgtPDS2-oCas9. (C) shows the detection of site-directed editing of NbPDS using the PCR / RE method. In the figure, lanes SYNV-oCas9gPDS2 and SYNV-tgtPDS2-oCas9 represent three randomly selected strains of systemically infected tobacco plants after inoculation of tobacco benthamiana with the recombinant viral vectors described above. [Figure 29]Figure 29 shows an analysis of the infection status of EMDV fluorescent expression vectors in Tobacco benthamiana. (A) is a diagram illustrating the construction process of EMDV-GFP green fluorescent and EMDV-RFP red fluorescent expression vectors. N3'+N5' means that the space between the GFP gene and the N gene consists of N3'UTR+ggg+N5'UTR, while the bilateral gene spacers of the RFP gene are two repeating NX junctions. (B) shows the disease state after systemic infection of Tobacco benthamiana with recombinant EMDV-GFP virus. The left figure shows the expression status of viral structural proteins and green fluorescent proteins in the leaves of diseased Tobacco benthamiana plants using a Western blot detection system, and the right figure shows the symptoms after systemic infection of Tobacco benthamiana with EMDV-GFP. (C) shows the disease state when recombinant EMDV-RFP virus systemically infects Tobacco benthamiana. The left figure shows the expression of viral structural proteins and red fluorescent proteins in diseased Tobacco benthamiana leaves using a Western blot detection system. The right figure shows the symptoms of systemic infection of Tobacco benthamiana with EMDV-RFP. [Figure 30]Figure 30 shows an analysis of the infection status of tobacco, potato, eggplant, and tomato with EMDV fluorescence expression vectors. (A) shows the symptoms of systemic infection of tobacco with recombinant EMDV-RFP virus. The left figure shows the expression status of viral structural proteins and red fluorescent proteins in the leaves of affected tobacco plants using a Western blot detection system. The right figure shows the symptoms of systemic infection of tobacco with recombinant EMDV-RFP virus. The first and second columns show the plant condition 45 days after friction inoculation of systemically affected tobacco with EMDV-RFP and a control of healthy tobacco, and the corresponding fluorescence status of whole leaves. (B) shows that EMDV-RFP or EMDV-GFP recombinant viruses can infect eggplant, tomato, and potato by mechanical friction. (C) shows that potato can be systemically infected with recombinant EMDV-RFP virus after grafting with tobacco benthamiana. The left figure is a schematic diagram of a grafted plant, with tobacco benthamiana as the rootstock and potato as the scion. The right figure shows the condition of the grafted plant and the corresponding leaf fluorescence expression at approximately 14 days after the onset of disease, when the tobacco benthamiana rootstock was mechanically inoculated with EMDV-RFP. [Figure 31] Figure 31 shows the infection and expression analysis of peppers using an EMDV fluorescence expression vector. (A) Western blot was used to measure the expression of viral structural proteins (upper panel) and red fluorescent proteins (middle panel) in the systemically infected leaf tissue of peppers. Rub L: The large Rubisco subunit was used as the loading control. (B) Symptoms (upper panels) and fluorescence imaging (lower panel) that occurred 35 days after systemic infection of peppers with EMDV-RFP. Mock: Control strain inoculated with healthy juice. [Figure 32]Figure 32 shows the analysis of mutation frequency in recombinant tobacco benthamiana targeting GFP with the EMDV CRISPR / Cas9 editing vector. (A) is a schematic diagram of the EMDV-tgtGFP2-Cas9 editing vector. The left figure of (B) shows the expression status of viral proteins and Cas9 proteins after systemic infection of tobacco benthamiana with the EMDV-tgtGFP2-Cas9 recombinant virus detected by Western blot, and the right figure shows the situation after systemic infection of tobacco benthamiana with the EMDV-tgtGFP2-Cas9 recombinant virus. Under a portable ultraviolet lamp, it was clearly observed that the green fluorescence production of the plant was significantly reduced after highly efficient editing by the EMDV editing vector. (C) shows the detection of site-specific editing efficiency of the GFP2 target using the PCR / RE method. In the figure, + and - indicate whether or not the corresponding restriction endonuclease was added in the enzyme cleavage reaction, respectively. In the Sanger sequencing results, the underlined sequences are the recognition sequences of the corresponding restriction endonucleases, where wt indicates that the sequencing sequence matches the wild-type sequence, d indicates a base deletion, i indicates a base insertion, d# indicates # base deletions, i# indicates # base insertions, and X# indicates # occurrences of the sequence in the sequencing results. (D) This is a peak plot of the sequencing results in Figure C. [Figure 33]Figure 33 shows the analysis of the mutation frequency of the PDS gene targeting Nicotiana benthamiana by the EMDV CRISPR / Cas9 editing vector. (A) is a schematic diagram of the EMDV-tgtPDS4-Cas9 editing vector. (B) shows the disease state after systemic infection of Nicotiana benthamiana with EMDV-tgtPDS4-Cas9. The left figure shows the expression status of viral structural proteins after systemic infection of Nicotiana benthamiana with the EMDV-tgtPDS4-Cas9 recombinant virus detected by Western blot, and the right figure is a photograph taken 40 days after systemic infection of Nicotiana benthamiana with the EMDV-tgtPDS4-Cas9 recombinant virus. (C) shows the site-directed editing efficiency of the PDS4 target detected using the PCR / RE method. The left figure shows that the target is completely conserved in the PDS genes of tobacco benthamiana, potato, tomato, chili pepper, and tobacco. The right figure shows the editing conditions for endogenous PDS genes-a and PDS-b in tobacco benthamiana systemically infected with the EMDV-tgtPDS4-Cas9 recombinant virus, where + and - indicate whether or not the corresponding restriction endonuclease is added in the enzymatic cleavage reaction. [Figure 34]Figure 34 shows the analysis of M0 plantlets and their genotypes obtained by regenerating plant leaves infected with the EMDV CRISPR / Cas 9 vector. (A) Regenerated plantlets obtained by whole-body leaf tissue culture after infection of Tobacco benthamiana with EMDV-tgtPDS4-Cas 9. The left figure shows differentiated callus and shoots in MS medium, and the right figure shows albino plantlets obtained in MS rooting medium. (B) Results of detecting gene editing in M0 regenerated plants by PCR / RE method. Mock / U and Mock / D are those in which the PCR product amplified by the wild-type control plantlet was either not cleaved by the T7 EI enzyme or was cleaved by the T7 EI enzyme, respectively. Albino: albino seedling; Normal: normal green seedling. (C) Results of detecting the presence or absence of EMDV in M0 strains by RT-PCR. For virus, the primer used was a viral genome detection primer, and for GAPDH, the primer used was the Tobacco benthamiana internal standard primer. EMDV represented plantlets infected with wild-type EMDV systemically and was used as a positive control, while Mock represented healthy plantlets and was used as a negative control. [Figure 35] Figure 35 shows the analysis of mutation frequencies in the PDS gene targeted by the EMDV CRISPR / Cas9 editing vector in tobacco, tomato, and potato. (A) is the editing result sequencing peak diagram of EMDV-tgtPDS4-Cas9 in tobacco, with the left side being a summary of the monoclonal sequencing results and the right side being the corresponding Sanger sequencing peak diagram. (B) is the editing result sequencing peak diagram of EMDV-tgtPDS4-Cas9 in tomato, with the left side being a summary of the monoclonal sequencing results and the right side being the corresponding Sanger sequencing peak diagram. (C) is the editing result sequencing peak diagram of EMDV-tgtPDS4-Cas9 in potato, with the left side being a summary of the monoclonal sequencing results and the right side being the corresponding Sanger sequencing peak diagram. [Modes for carrying out the invention]

[0080] The present invention will be further described below based on examples, but it should be noted that such examples are merely illustrative and should not be considered limiting. Any other modifications or improvements obtained based on the described examples of the present invention are all within the scope of protection of the present invention.

[0081] Unless otherwise explicitly specified or limited, the experimental methods used in the following examples are all commonly used procedures, and all reagents and materials used in the experiments may be purchased from standard biochemical reagent companies.

[0082] All of the tobacco benthamiana and viral materials used were stored in this laboratory, and all of the expression vectors used were constructed and stored in this laboratory.

[0083] Recombinant rhabdovirus vectors SYNV and SYNV-GFP, core protein expression vector pGD-NPL, and RNA silencing repression subprotein expression vectors (pGD-Hc-Pro, pGD-p19, pGD-γb) are disclosed in the paper "Wang Q, Ma X, Qian S, et al. Rescue of a plant negative-strand RNA virus from cloned cDNA: Insights into enveloped plant virus movement and morphogenesis. PLoS pathogens, 2015, 11: e1005223".

[0084] Recombinant rhabdovirus vectors SYNV-RFP, SYNV-RFP-dG N SYNV-RFP-mCherry:G NHowever, this is disclosed in the paper "Sun K, Zhou X, Lin W, et al. Matrix-glycoprotein interactions required for budding of a plant nucleorhabdovirus and induction of inner nuclear membrane invagination. Molecular Plant Pathology, 2018, 19:2288-2301".

[0085] The plant dual expression vector pBGK01-gRNA-Cas9 is disclosed in the paper "Fu S, Xu Y, Li C, et al. Rice stripe virus interferes with S-acylation of remorin and induces its autophagic degradation to facilitate virus infection. Molecular Plant, 2018, 11:269-287".

[0086] Plasmid pYQ230 is disclosed in the paper "Tang, X., Lowder, LG, Zhang, T., et al. A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nature Plants, 2017, 3:17018". Example 1: Construction of a dual reporter gene expression system using a SYNV viral vector Step 1: Construction of a SYNV dual reporter gene expression vector.

[0087] The process involves simultaneously inserting the GFP and RFP reporter genes into the SYNV genome in the form of independent expression frames, with insertion sites including, but not limited to, the space between the N and P genes. Below, the construction process of SYNV-GFP-RFP will be described in detail, using the simultaneous insertion of the GFP and RFP expression frames between the N and P genes as an example (Figure 1A). The above construction process mainly includes the following steps.

[0088] In step (1), Bsu36I-NPJ fragments, GFP fragments, and RFP-NheI fragments were amplified using SYNV, SYNV-GFP, and SYNV-RFP as templates for N-Bsu36I / F and NPJ-GFP / R, GFP / F and NPJ-RFP / R, and RFP / F and P-NheI / R from Table 6, respectively. Here, the Bsu36I-NPJ fragment includes the NPJ region from the Bsu36I enzyme cleavage site on the N gene downstream, and the RFP-NheI fragment includes the complete RFP ORF and the NheI enzyme cleavage site from the downstream NPJ sequence to the P gene. Each of these fragments contains a 15-20 bp sequence homologous to an adjacent fragment, and the three fragments can be sequentially linked in series by homologous recombination. Fragment 1, N-GFP, and Fragment 3, RFP-P, each contain sequences homologous to a linearized SYNV vector (linearized after double enzyme cleavage of Bsu36I and NheI) and are used for recombination with the SYNV vector.

[0089] In step (2), the SYNV vector was digested using restriction endonucleases Bsu36I and NheI, and after electrophoretic detection, the vector fragment was recovered to obtain a linearized vector, which was then purified and set aside.

[0090] In step (3), purified Bsu36I-NPJ fragment, GFP fragment, and RFP-NheI fragment were simultaneously cloned into a linearized SYNV vector using an in-fusion recombination method. Positive clones SYNV-GFP-RFP were screened for enzymatic cleavage identification from colony PCR, and sequencing was performed on the clones without errors in enzymatic cleavage identification to verify that the three fragments were correctly and sequentially assembled into the SYNV vector. Step 2: Agrobacterium transformation, culture, and inoculation into Citrus benthamiana. SYNV-GFP-RFP was introduced into Agrobacterium strain EHA105 using electroshock therapy.

[0091] Simultaneously, Agrobacterium strain EHA105 was electrocuted with the core protein expression vector pGD-NPL and the viral RNA silencing suppression subprotein expression vectors (pGD-Hc-Pro, pGD-p19, pGD-γb) required for systemic SYNV infection.

[0092] Agrobacterium containing the recombinant virus expression vector SYNV-GFP-RFP, the core protein expression vector pGD-NPL, and the RNA silencing suppression subprotein expression vectors (pGD-Hc-Pro, pGD-p19, pGD-γb) were inoculated into 4 mL of YEP liquid medium (containing 50 μg / mL kanamycin + 25 μg / mL rifampicin), respectively, and OD was performed. 600 Incubate overnight at 28°C with shaking at 220 rpm until the pH reaches 0.8-1.2, then centrifuge at 5500 rpm for 10 minutes, discard the supernatant, and resuspend the cells in infiltration buffer (containing 10 mM MgCl2, 10 mM MES, and 200 mM acetosyringone), then OD 600 The value was adjusted to around 1.0, and the sample was left to stand for 2-3 hours.

[0093] Agrobacterium, consisting of a virus expression vector, a core protein expression vector, and an RNA silencing suppression subprotein expression vector, was mixed in an equivolume ratio of 1:1:1 after standing, and then inoculated into Citrus benthamiana. It was preferable to select strains at the 4-6 leaf stage. Inoculation was performed by inoculating the back of the leaves with a 1 ml disposable syringe without a needle, inoculating 3-4 leaves per strain. After inoculation, the inoculated plants were cultured in a 25°C isolated greenhouse and used as a control. Using a similar method, recombinant virus vector SYNV-GFP, which expresses only the GFP protein, was inoculated into the plants. Step 3: Detect whether the recombinant SYNV viral vector can simultaneously express two reporter genes.

[0094] Six days after inoculation of tobacco plants with the recombinant SYNV virus vector SYNV-GFP-RFP, significant GFP and RFP fluorescence was observed in the inoculated leaves. Around 12 days after inoculation, the fluorescent reporter gene diffused between cells, and around 30 days after inoculation, strong GFP and RFP fluorescence was observed in the veins and mesophyll cells of the upper whole leaves (Figure 1B). However, only GFP fluorescence was observed in the control SYNV-GFP inoculated plants. After systemic viral infection, all inoculated plants showed typical viral infection symptoms such as dwarfism, leaf curling, leaf vein yellowing, and calyx curling (Figure 1C). In addition, as red fluorescent protein RFP accumulated, the petals of the plants inoculated with SYNV-GFP-RFP gradually showed pink symptoms. To confirm that the symptoms described above were caused by SYNV virus infection, whole leaves were collected from plants inoculated with the recombinant viral vector SYNV-GFP-RFP, and total protein was extracted from the leaves. Western blot analysis was performed using SYNV polyclonal antibody, GFP monoclonal antibody, and RFP monoclonal antibody, respectively. The test results showed that viral protein bands corresponding to SYNV, GFP protein bands, and RFP protein bands were detected in the total protein of the leaves. In contrast, only SYNV viral protein bands and GFP bands were detected in the control leaves inoculated with SYNV-GFP, and no RFP band was detected (Figure 1D). These results demonstrate that the recombinant SYNV viral vector can simultaneously express two exogenous reporter genes, GFP and RFP. Example 2: Construction of a SYNV CRISPR / Cas9 editing vector

[0095] The SYNV process involves simultaneously inserting gRNA and Cas9 into the genome in the form of independent expression frames, with insertion sites including, but not limited to, the space between N and P. Below, using the simultaneous insertion of a gRNA transcription unit and a Cas9 expression frame between the N and P genes as an example (Figure 2A), the construction process of a SYNV-based CRISPR / Cas9 genome editing vector is described in detail, and the constructed clone is as follows: 1) SYNV-Cas9: This SYNV vector expresses only the Cas9 protein and was used as a control. 2) SYNV-gRNA-Cas9: A SYNV vector that transcribes sgRNA and expresses the Cas9 protein. 3) SYNV-tgRNA-Cas9: A SYNV vector that accurately transcribes sgRNA and expresses the Cas9 protein. The specific method for constructing a clone is as follows: 1) SYNV-Cas9

[0096] Using the primers N-Bsu36I-F and Flag-NPJ / R, Cas9 / F and Cas9 / R, and Cas9-NPJ / F and P-NheI / R from Table 6, SYNV, pBGK01-gRNA-Cas9, and SYNV were used as templates to amplify the Bsu36I-NPJ fragment, Cas9 ORF fragment, and NPJ-NheI fragment (Figure 2A). Here, the Bsu36I-NPJ fragment contains the NPJ region from the Bsu36I enzyme cleavage site on the N gene downstream, and the NPJ-NheI fragment contains the NheI enzyme cleavage site from the NPJ sequence to the P gene. Each of these fragments contains a 15-20 bp sequence homologous to the adjacent fragment. Fragment 1, Bsu36I-NPJ, and Fragment 3, NPJ-NheI, each contain sequences homologous to the linearized SYNV vector after double enzymatic cleavage via Bsu36I and NheI, respectively. Therefore, by sequentially binding these three fragments via in-fusion, the SYNV-Cas9 vector can be obtained. 2) SYNV-gRNA-Cas9

[0097] Using the primers N-Bsu36I / F and GFP2-NPJ / R, GFP2-gRNA / F and gRNA / R, and gRNA-NPJ / F and Cas9-AhdI / R from Table 6, SYNV, pBGK01-gRNA-Cas9, and SYNV-Cas9 were used as templates to amplify the Bsu36I-NPJ fragment, gRNA fragment, and NPJ-AhdI fragment (Figure 2A). The Bsu36I-NPJ fragment contains the NPJ region from the Bsu36I enzyme cleavage site on the N gene downstream, the gRNA fragment contains the GFP target2 sequence and the gRNA scaffold sequence, and the NPJ-AhdI fragment contains the enzyme cleavage site from the NPJ sequence to the Cas9 gene. After in-fusion recombination of these fragments, the vector SYNV-gGFP2-Cas9 can be obtained. 3) SYNV-tgRNA-Cas9

[0098] To ensure accurate gRNA transcription, tRNA precursor sequences, i.e., tRNA-gRNA-tRNA, are attached to both ends of the gRNA. Here, the tRNA sequences are identified and cleaved by a plant endogenous processing device, allowing for the release of a relatively accurate gRNA transcript (Figure 2B). Sequences containing different target sites are synthesized according to the sequences provided by SEQ IDs 1-9. A 15nt sequence derived from the NPJ3' end (5'-TTATTTGTCTAGGCC-3') and a 15nt sequence at the 5' end (5'-TAAACTACAGCCACA-3') are added to the 5' and 3' ends of the sequence, respectively, to facilitate amplification and clone construction in the next step. The selected target sites were GFP, NbPDS, NbRDR6, and NbSGS3, in the order of 2, 3, 2, and 2 targets, respectively. All of these target sites contain restriction endonucleases, facilitating the detection of editing efficiency by PCR / RE. Furthermore, these target sites are completely identical in two homologous copies (a and b) of NbPDS, NbRDR6, and NbSGS3, allowing the selected target sites to be simultaneously targeted by the aforementioned genes.Using the synthesized sequence described above as a template, amplification was performed using the primers NPJ-tRNA / F and NPJ-tRNA / R in Table 6 to obtain tRNA-gRNA-tRNA sequences containing different target sites. Simultaneously, using SYNV-Cas9 as a template, the Bsu36I-NPJ fragment and the NPJ-AhdI fragment were amplified using primers for N-Bsu36I / F and NPJ / R, and for NPJ / F and Cas9-AhdI / R, respectively. Here, the Bsu36I-NPJ fragment contains the NPJ region from the Bsu36I enzyme cleavage site downstream on the N gene, and the NPJ-AhdI fragment contains the NP The three fragments, each containing an AhdI enzyme cleavage site from the J sequence to the Cas9 gene, were in-fusion cloned to obtain SYNV-tgtRNA-Cas9 vectors containing different target sites: SYNV-tgtGFP1-Cas9, SYNV-tgtGFP2-Cas9, SYNV-tgtPDS1-Cas9, SYNV-tgtPDS2-Cas9, SYNV-tgtPDS3-Cas9, SYNV-tgtRDR6-1-Cas9, SYNV-tgtRDR6-4-Cas9, SYNV-tgtSGS3-1-Cas9, and SYNV-tgtSGS3-2-Cas9.

[0099] To construct SYNV editing vectors containing two target sites simultaneously, tRNA-gRNA-tRNA-gRNA-tRNA fragments were synthesized according to the sequences provided in SEQ ID 10-12, and SYNV-tgtgtRDR6-1 / SGS3-1-Cas9, SYNV-tgtRDR6-4 / SGS3-2-Cas9, and SYNV-tgtPDS1 / 3-Cas9 were constructed using the same method. These vectors can simultaneously target NbRDR6 target 1 and NbSGS3 target 1, NbRDR6 target 4 and NbSGS3 target 2, and NbPDS target 1 and target 3, respectively. Example 3: SYNV CRISPR / Cas9 vector targets GFP gene in 16c genetically modified tobacco. Step 1: Inoculation of Agrobacterium-infected 16c genetically modified tobacco with a SYNV CRISPR / Cas 9 vector.

[0100] The CRISPR-P (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR) online design software allows for the targeting of GFP in 16c plantations using a 20nt target sequence (5'-GATACCCAGAT CATATG AAG-3' and 5'-ACAAGTGTTGG CCATGG AAC-3' (where the underlined parts are the corresponding enzyme cleavage sites NdeI and NcoI) was selected, and clones SYNV-tgtGFP1-Cas9 and SYNV-tgtGFP2-Cas9 were constructed according to the method described in Example 2.

[0101] Using the method of Example 1, recombinant virus expression vectors SYNV-Cas9, SYNV-tgtGFP1-Cas9, and SYNV-tgtGFP2-Cas9 were electrocuted into Agrobacterium and inoculated into 16c genetically modified tobacco plants. Approximately 30 days after inoculation, the inoculated plants showed viral symptoms similar to wild-type virus, such as leaf curling and leaf vein yellowing. Under portable UV wrap and laser confocal microscopy, in plants inoculated with SYNV-edited vectors SYNV-tgtGFP1-Cas9 and SYNV-tgtGFP2-Cas9, the expression level of GFP green fluorescence was clearly reduced, and GFP fluorescence expression was hardly observed in mesophyll cells, with only green guard cells being observed. In whole leaves inoculated with SYNV-Cas9, the GFP fluorescence intensity was consistent with wild-type 16c (Figure 3A).

[0102] To verify that the above-mentioned symptoms were caused by systemic SYNV infection, whole leaves were collected from affected plants, and total protein was extracted from the leaves. Western blot analysis was performed using SYNV polyclonal antibody, Flag-tagged antibody, and GFP monoclonal antibody, respectively. The test results showed that viral protein bands corresponding to SYNV and Cas9 protein bands could be detected in each plant inoculated with the SYNV edit vector, and that the expression level of GFP protein was clearly reduced. The expression level of GFP protein in tobacco leaves inoculated with the control vector SYNV-Cas9 was basically the same as that of wild-type 16c plants (Figure 3B). These results demonstrate that the SYNV viral vector can simultaneously accommodate gRNA expression frames and Cas9 expression frames, and that recombinant viral vectors with the aforementioned exogenous expression frames inserted still possess systemic infectivity.

[0103] To explore whether the exogenous expression frame could be integrated into the viral genome, virus particles were extracted from inoculated plants, and the morphological characteristics of the viral particles were observed under an electron microscope. The recombinant viral particles exhibited a typical elliptical or rod-shaped morphology, and it was found that the length of the viral particles of the edited vector was significantly longer than that of wild-type SYNV viral particles (approximately 247 nm), reaching approximately 333 nm (Figure 3C). The length of the viral particles was in positive proportion to the size of the recombinant viral genome, indicating that the 4.8 kb exogenous fragment was integrated into the SYNV genome and packaged into mature viral particles. Step 2: GFP Target Mutation Efficiency Analysis

[0104] To verify the editing efficiency of GFP targets, whole leaves of inoculated plants were measured using the PCR / RE method (Polymerase Chain Reaction / Restriction digestion). PCR was performed using primers GFP / F and GFP / R from Primer Table 7, and then enzymatic cleavage analysis was performed by selecting the corresponding restriction endonuclease at the target sequence. It was shown that if the PCR amplification product could not be cleaved, a mutation occurred at the target site, and the degree to which the band could not be cleaved reflected the high editing efficiency. After enzymatic cleavage of the above PCR product, mutations of different degrees occurred at both GFP target 1 and target 2, and the mutation efficiency reached approximately 91% (Figure 4A). To measure the editing type of GFP, Sanger sequencing analysis was performed on the above PCR amplification product. The PCR product was bound to a T vector, and 10 monoclonals were measured at each of the two GFP target sites. The number of monoclonals produced by mutations at GFP target 1 and target 2 was 7 and 8, respectively (Figure 4B), which was basically consistent with the detection results of PCR / RE, and most of the generated editing types were single base deletions or multiple base deletions (Figure 4C). Example 4: Improvement of editing efficiency through precise processing of gRNA transcripts produced by SYNV CRISPR / Cas9

[0105] To measure the accuracy of the gRNA transcript produced by the SYNV-tgtRNA-Cas9 SYNV editing vector, the size of the gRNA transcript was measured using the cRT-PCR method, while simultaneously using the gRNA transcript produced by the recombinant viral vector SYNV-gRNA-Cas9 as a control. According to the transcription pattern of the virus itself, the gRNA transcript produced by the recombinant viral vector SYNV-gRNA-Cas9 contained the 5'UTR and 3'UTR sequences of the viral gene spacer NPJ at both ends, respectively, and the size of the transcript was approximately 208-300 nt (208 nt transcript and polyA sequence of unknown length). The recombinant viral vector SYNV-tgtGFP2-Cas9 should produce gRNA transcripts with tRNA recognition sequences attached to both ends, followed by recognition processing using a plant endogenous RNase enzyme to produce gRNA transcripts of approximately 100 nt. Without recognition processing, the size of the produced gRNA transcripts was approximately 362-400 nt (362 nt transcript and polyA sequence of unknown length) (Figure 5A).

[0106] Recombinant viral vector SYNV-gGFP2-Cas9 was constructed according to the method described in Example 2. SYNV-gGFP2-Cas9 and SYNV-tgtGFP2-Cas9 were inoculated into 16c strains, and total RNA was extracted from the whole leaves of the vector-inoculated strains and performed cRT-PCR. The test results showed that the size of the gRNA transcripts produced by the recombinant viral vector SYNV-tgtGFP2-Cas9 was all around 100 nt (Figure 5B), indicating that the tRNA sequence was fully recognized and processed by the plant endogenous RNase enzyme. To release more precise gRNA transcripts and further examine the residual sequences at both ends of the gRNA transcripts, Sanger sequencing was performed after the PCR products were bound to a T vector. The test results showed that there were no residual bases at the 5' end of the generated gRNA-scaffold transcript, and 1-3 nt remained at the 3' end, with the residual bases originating from the tRNA sequence bound to the 3' end (Figure 5D). The recombinant viral vector SYNV-gGFP2-Cas9 produces transcripts ranging from the desired size of 208nt to 300nt, as well as transcripts of approximately 100nt in size (Figure 5B). Sequencing analysis of these smaller fragments revealed that they are gRNA transcripts after the removal of viral-derived sequences, and that they have a deletion of one base in the viral 5'UTR region or 4-5 bases in the scaffold sequence at their 3' end (Figure 5D).

[0107] To compare the editing efficiency of the two recombinant viral vectors described above, two disease-causing strains were randomly selected approximately 30 days after inoculation, and whole-body infected leaves were collected. The editing efficiency of the GFP target was measured using PCR / RE and Sanger sequencing, and it was found that the editing efficiency of the GFP target was approximately 11-20% (Figure 5C).

[0108] The results described above demonstrate that when a SYNV editing vector does not employ a tRNA processing mechanism, it can produce a small amount of accurate gRNA transcript using a single processing mechanism, resulting in some degree of editing (around 11-20%) of the target gene. However, when a plant endogenous tRNA processing mechanism is employed, the initial gRNA transcript is completely recognized by a plant endogenous RNase enzyme and can be cleaved into an accurate gRNA transcript, thereby significantly increasing the editing efficiency of the vector (around 90%). Therefore, the SYNV-tgtRNA-Cas9 SYNV editing vector, which ligates tRNA recognition sequences to both ends of a gRNA transcript, is the preferred vector of the present invention. Example 5: SYNV CRISPR / Cas9 vector targets endogenous PDS gene in Citrus benthamiana. The viral vector editing system of the present invention was further validated by selectively using the endogenous PDS gene of Citrus benthamiana.

[0109] The CRISPR-P (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR) online design software allows for the design of a 20nt target sequence (5'-GCCGTTAATTTGA) that can target PDS. GAGTC CA-3', 5'-TTGGTAGTAGCGACT CCATGG -3', 5'-TTAACTGTATTGT CTAG By selecting CTC-3' (where the underlined parts correspond to the enzyme cleavage sites HinfI, NcoI, and BfaI), the above targets can simultaneously target copies a and b of PDS in heterotetraploid tobacco benthamiana (Figure 6A). Recombinant virus clones SYNV-tgtPDS1-Cas9, SYNV-tgtPDS2-Cas9, and SYNV-tgtPDS3-Cas9 were constructed according to the method described in Example 2.

[0110] Using the method of Example 1, recombinant virus expression vectors SYNV-tgtPDS1-Cas9, SYNV-tgtPDS2-Cas9, and SYNV-tgtPDS3-Cas9 were electrocuted into Agrobacterium and inoculated into Citrus benthamiana. Approximately 30 days after inoculation, the recombinant virus expression vectors exhibited symptoms similar to those of wild-type virus, such as leaf curling and yellowing of leaf veins (Figure 6B).

[0111] Using the PCR / RE method, the target editing efficiency of PDS was measured. All primers used were able to simultaneously amplify copies a and b of PDS. As can be seen from the results, mutations of varying degrees occurred at all three target sites of PDS, and the editing efficiency reached a maximum of approximately 79% (Figure 6C). Using target 1 as an example, the editing efficiencies in three randomly selected plant strains were 79%, 70%, and 56%, respectively. The differences in editing efficiency between different strains may be related to the expression levels of gRNA transcripts and Cas9 protein.

[0112] Using Sanger sequencing, the editing status of PDS target site 1 was verified. PDS copies a and b were amplified using PDS-specific primers from Primer Table 7, and the PCR products were bound to a T vector. Ten clones were measured for each copy. The number of mutant clones in PDS copies a and b was 8 and 7, respectively, which was basically consistent with the results of the editing efficiency measured by the PCR / RE method. Furthermore, the editing types mainly consisted of single-base and multi-base deletions and single-base insertions (Figure 6D). Example 6: SYNV CRISPR / Cas9 vector targets the endogenous gene RDR6 in Citrus benthamiana. The viral vector editing system of the present invention was further validated by selectively using the endogenous gene RDR6 from Citrus benthamiana.

[0113] The CRISPR-P (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR) online design software allows for the targeting of RDR6 with a 20nt target sequence (5'-AGTTGGGTAAGAG). TCAGGA G-3', 5'- ATTCTCAGCTAAC CAGCTG By selecting A-3' (where the underlined parts correspond to the enzyme cleavage sites HyP188III and PvuII), the aforementioned targets can simultaneously target two copies a and b of RDR6 in heterotetraploid tobacco benthamiana (Figure 7A).

[0114] Recombinant virus clones SYNV-tgtRDR6-1-Cas9 and SYNV-tgtRDR6-4-Cas9 were constructed according to the method described in Example 2. The recombinant virus expression vectors SYNV-tgtRDR6-1-Cas9 and SYNV-tgtRDR6-4-Cas9 were electrocuted into Agrobacterium using the method described in Example 1 and inoculated into Citrus benthamiana. Approximately 30 days after inoculation of Citrus benthamiana, the recombinant virus expression vectors exhibited symptoms similar to those of wild-type virus, such as leaf curling and yellowing of leaf veins (Figure 7B).

[0115] The target editing efficiency of RDR6 was measured using the PCR / RE method. All primers used were able to simultaneously amplify copies a and b of RDR6, and as can be seen from the results, mutations of different degrees occurred at both target sites of RDR6, with editing efficiencies reaching up to approximately 91% (Figure 7C).

[0116] Using Sanger sequencing, we verified the editing status of RDR6 target site 1. RDR6 copies a and b were amplified using RDR6-specific primers from Primer Table 7, and the PCR products were bound to a T vector. Ten clones were measured for each copy. The number of mutant clones in RDR6 copies a and b was 6 and 7, respectively, which was basically consistent with the results of the editing efficiency measured by the PCR / RE method. Furthermore, the editing types were mainly single or multi-base deletions, single base substitutions, or insertions (Figure 7D). Example 7: SYNV CRISPR / Cas9 vector targets the endogenous gene SGS3 in Citrus benthamiana. The viral vector editing system of the present invention was further validated by selectively using the endogenous gene SGS3 from Citrus benthamiana.

[0117] The CRISPR-P (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR) online design software allows for the design of a 20nt target sequence (5'-ACAAGAGTGGAAG) that can target SGS3. CAGTG CT-3', 5'- TGCCTCAACTGATC CCAAGG By selecting -3' (where the underlined portion corresponds to the enzyme cleavage site TscAI, Eco130I), the aforementioned targets can simultaneously target two copies a and b of SGS3 in heterotetraploid tobacco benthamiana (Figure 8A).

[0118] Recombinant virus clones SYNV-tgtSGS3-1-Cas9 and SYNV-tgtSGS3-2-Cas9 were constructed according to the method described in Example 2. The recombinant virus expression vectors SYNV-tgtSGS3-1-Cas9 and SYNV-tgtSGS3-2-Cas9 were electrocuted into Agrobacterium using the method described in Example 1 and inoculated into Citrus benthamiana. Around 30 days after inoculation, the recombinant virus expression vectors exhibited symptoms similar to those of wild-type virus, such as leaf curling and yellowing of leaf veins (Figure 8B).

[0119] The target editing efficiency of SGS3 was measured using the PCR / RE method. All primers used were able to simultaneously amplify copies a and b of SGS3, and as can be seen from the results, mutations of different degrees occurred at both target sites of SGS3, with the editing efficiency reaching a maximum of approximately 91% (Figure 8C).

[0120] Using Sanger sequencing, we verified the editing status of SGS3 target site 2. Using SGS3-specific primers from Primer Table 7, we amplified SGS3 copies a and b, respectively. The PCR products were bound to a T vector, and 10 clones were measured for each copy. The number of mutant clones in SGS3 copies a and b was 8 and 6, respectively, which was basically consistent with the results of the editing efficiency measured by the PCR / RE method. Furthermore, the editing types mainly consisted of single-base and multi-base deletions and single-base insertions (Figure 8D). Example 8: SYNV CRISPR / Cas9 multi-target vector simultaneously targets endogenous genes RDR6 and SGS3 in Citrus benthamiana.

[0121] To verify whether a genome editing system based on a SYNV vector can perform multi-target editing of plant genomes, a SYNV dual-target editing vector was constructed using a method in which tRNA is linked to gRNA transcripts in series (tRNA-gRNA1-tRNA-gRNA2-tRNA). The resulting primary transcript can be identified and cleaved by a plant endogenous RNase enzyme, after which two precise gRNA transcripts can be released simultaneously (Figure 9).

[0122] Recombinant virus clones SYNV-tgtgtRDR6-1 / SGS3-1-Cas9 and SYNV-tgtgtRDR6-4 / SGS3-2-Cas9 were constructed according to the method described in Example 2, where the recombinant virus clone SYNV-tgtgtRDR6-1 / SGS3-1-Cas9 can simultaneously target RDR6 target 1 (Example 6) and SSG3 target 1 (Example 7), and the recombinant virus clone SYNV-tgtgtRDR6-4 / SGS3-2-Cas9 can simultaneously target RDR6 target 4 (Example 6) and SSG3 target 2 (Example 7). Using the method of Example 1, recombinant virus expression vectors SYNV-tgtgtRDR6-1 / SGS3-1-Cas9 and SYNV-tgtgtRDR6-4 / SGS3-2-Cas9 were electrocuted into Agrobacterium and inoculated into Citrus benthamiana. Around 30 days after inoculation, both recombinant virus expression vectors showed symptoms similar to those of wild-type virus, such as leaf curling and yellowing of leaf veins (Figure 10A).

[0123] Using the PCR / RE method, the target editing efficiencies of RDR6 and SGS3 were measured. The test results showed that when the SYNV dual-target editing vector simultaneously targeted RDR6 and SGS3, the editing efficiency of RDR6 or SGS3 was essentially the same as that of the SYNV single-target editing vector (Example 6 or Example 7). This result demonstrates that the SYNV vector can be used for simultaneous editing of multiple targets in the plant genome without affecting the editing efficiency of single targets (Figure 10B).

[0124] The targeted editing efficiency of RDR6 and SGS3 was further verified using Sanger sequencing. Single copies of RDR6 and SGS3 were amplified using specific primers from Primer Table 7, and the PCR products were bound to a T vector. Ten clones were measured for each copy. The number of mutant clones in RDR6 copies a and b was 5 and 5, respectively, and the number of mutant clones in SGS3 copies a and b was 7 and 8, respectively. These results were basically consistent with the editing efficiency measured by the PCR / RE method, and the editing types were mainly single or multiple base deletions, single or multiple base substitutions, and single base insertions (Figure 10C). Example 9: Chromosomal deletion caused by targeting the endogenous PDS gene in tobacco benthamiana with a SYNV CRISPR / Cas9 multi-target vector.

[0125] To verify whether the SYNV multi-target editing vector can induce deletion of chromosomal fragments in the plant genome, recombinant virus clone SYNV-tgtgtPDS1 / 3-Cas9 was constructed according to the method described in Example 2. This vector was able to simultaneously target PDS target 1 and target 3 (Example 5). The recombinant virus expression vector SYNV-tgtgtPDS1 / 3-Cas9 was electrocuted into Agrobacterium using the method of Example 1 and inoculated into Tobacco benthamiana. Approximately 30 days after inoculation of Tobacco benthamiana, the recombinant virus expression vector showed symptoms similar to those of wild-type virus, such as leaf curling and leaf vein yellowing (Figure 11A).

[0126] To measure whether the inoculated plants described above could produce chromosomal fragment deletions, the total DNA of the inoculated plants was extracted, and the total DNA of the wild-type plants was extracted as a control. PCR amplification was performed using the primers PDS-1 / 3 / F and PDS-1 / 3 / R from Primer Table 7. The primers described above were able to simultaneously amplify PDS copies a and b, with the size of the amplified product of PDS copy a being 492 bp and the size of the amplified product of PDS copy b being 486 bp. Three SYNV-tgtgtPDS1 / 3-Cas9 systemically infected plants were randomly selected, and the test results showed that the PCR amplification products of the above plants showed a small band of approximately 200 bp in addition to the wild-type band. This size was found to be consistent with the size after chromosomal fragment deletion by simultaneous editing of PDS target sites 1 and 3 (Figure 11B).

[0127] To further verify that the aforementioned band was generated by simultaneous editing of the PDS dual target, the fragment was recovered and Sanger sequencing was performed. The test results confirmed that the band was indeed due to a chromosomal fragment deletion between PDS target sites 1 and 3. Further analysis of these results showed that both PDS copies a and b produced fragment deletions (Figure 11C), and their sequencing peak diagrams are shown in Figure 11 (Figure 11D). Example 10: Regeneration of leaf tissue infected with SYNV CRISPR / Cas9 and analysis of M0 generation genotypes

[0128] To obtain regenerated and edited plantlets, whole leaves were collected after inoculation with the recombinant virus vector SYNV-tgtPDS1-Cas9, washed with sterile water, disinfected with 70% ethanol for 30 seconds, disinfected with 0.1% liter of mercury for 1-2 minutes, washed with sterile water at least three times, removed the disinfected leaves, cut off the obvious midrib and leaf edges, and then cut the leaves to 1 x 1 cm. 2The tissue was cut to size and cultured on MS differentiation medium (containing 1 mg / L6-BA) without antibiotics. After about 2-3 weeks, differentiated tissue cultures showed two phenotypes: albino and green (Figure 12A). The differentiated buds were then transferred to a medium containing live roots and cultured for about 3 weeks. Sterile ddH2O was added to the culture bottles and covered with plastic wrap. After 4-5 days, the tissue culture seedlings were transplanted into soil.

[0129] To detect the editing status of PDS target sites in tissue-cultured plants, DNA was extracted from M0 generation albino and green seedlings and subjected to PCR / RE analysis (Figure 12B) and Sanger sequencing analysis. The test results showed that the PCR product from albino seedlings could not be completely cleaved, but the PCR product from tissue-cultured green seedlings showed three different states after enzymatic cleavage: complete cleavage, partial cleavage, and complete failure to cleave. As shown in Table 1, the statistical results for each type of plant described above are as follows: 1) In albino plants (M0-1, -2, -4, -5, -9, -11), PDS copies a and b were completely edited; 2) In tissue culture green plants (M0-3, -6) where the PCR product was completely cleaved with HinfI, PDS copies a and b were not edited; 3) In tissue culture green plants (M0-7, -10, -12, -13, -15, -23, -28, -34, -35, 39, 41) where the PCR product was partially cleaved with HinfI, PDS copies a and b were partially edited; 4) In tissue culture green plants (M0-8, -18, -21, -22, -27, -29, -31, -32, -33, -36, -42) where the PCR product could not be completely cleaved with HinfI, both PDS copies a and b were edited. The phenotype of these plantlets is green, and the fact that the PDS function is not completely lost is mainly due to mutation, but because it was produced by an editing type that is not a frameshift, such as d3, d6, d9, etc., where d stands for delete.

[0130] To detect the virus content of tissue culture inoculations, total RNA was extracted from M0 generation inoculations and RT-PCR was performed using SYNV primers. The test results showed that a small number of regenerated seedlings had already eliminated the virus, and non-transgenic edited inoculations with virus elimination in the M0 generation, such as M0-7 and M0-42, could be obtained (Figure 12C). Example 11: Verification that Cas9-generated mutations can be stably transmitted to offspring plantings.

[0131] To detect whether mutations can be transmitted from the M0 generation to the M1 generation, seeds (M1 generation) were collected from green seedlings of the M0 generation. Since albino plants cannot flower and produce seeds, the fourth type of plant in Example 10, namely M0 generation plant in which PDS copies a and b were all completely edited and contained mutations but not frameshift edit types, was selected and used for analysis of the offspring generation. The offspring generation of 11 M0 strains, including M0-8, was sown in MS medium, and as can be seen from the results, two types of segregation were observed in the M1 generation plant in which 1) when the M0 generation genotype contained one mutation of PDSa or b but not a frameshift edit type, the segregation ratio of the offspring generation was 3:1; and 2) when the M0 generation genotype contained one mutation of PDSa and b each but not a frameshift edit type, the segregation ratio of the offspring generation was 15:1 (Figure 13).

[0132] The genetic status of editing types will be explained using M0-8 as an example. The genotype of the plant in question is d1i1d5d3 (Figure 14A), where d represents delete, i represents insert, and d3 represents a mutation but an editing type that is not a frameshift. When M1 generation plantations of 114 M0-8 plants were detected, 84 green seedlings and 30 albino seedlings appeared, basically conforming to Mendel's law of 3:1. Four albino seedlings and six green seedlings were randomly selected and subjected to PCR / RE analysis and sequencing analysis (Figure 14B). As the results show, the editing type of PDS in M1 generation plantings is entirely derived from the M0 generation, and no new editing types appear. In this case, the PDS target site in the albino seedlings is completely edited and frameshifted, for example M8-1, with the genotype d1d1d5d5. In the green seedlings, although there is a mutation, the editing type d3 is not frameshifted, so the PDS function is not completely lost, for example M8-9, with the genotype d1i1d5d3. Furthermore, by selecting strains such as M0-18, M0-22, M0-29, and M0-42 and further examining the M1 generation genotypes and phenotypes (Table 2), it was revealed that all editing types in M0 generation plantings can be stably inherited to the M1 generation, and that the transmission of editing types conforms to Mendel's laws.

[0133] To detect the rules governing the transmission of mutations from the M1 generation to the M2 generation, M1 generation seeds (M2 generation) were collected, and the editing status of the M2 generation plants was detected. Using M8-9 as an example, the genotype was d1i1d5d3, and 151 M2 generation plants were detected. As a result, 116 green seedlings and 35 albino seedlings appeared, basically conforming to Mendel's law of 3:1. Five albino seedlings and five green seedlings were randomly selected and subjected to PCR / RE analysis and sequencing analysis. The test results showed that the editing type of the PDS gene in M2 generation planted plants was entirely derived from the M1 generation, and all albino seedling genotypes were mutations and frameshift editing types, for example, M8-9-1 with genotype d1d1d5d5. The green seedling genotypes included a mutation but not a frameshift editing type d3, for example, M8-9-6 with genotype i1i1d5d3 (Figure 14C).

[0134] To further investigate the transmission rules of editing types from the M1 generation to the M2 generation, homozygous plantlets in the M1 generation were selected, for example, M8-3, with the genotype i1i1d3d3. Ten M2 generation plantlets from this strain were randomly selected and sequenced for verification. The test results showed that all M2 generation plantlets had the genotype i1i1d3d3. In addition, homozygous M1 generation plants M18-3, M22-4, M29-5, and M42-4 were selected and their offspring genotypes were analyzed. The test results showed that all M2 generation plantlets from the above strains were homozygous, indicating that editing types can be stably inherited from the M1 generation to the M2 generation, and that no new editing types are produced (Table 3).

[0135] The results above demonstrate that the editing type of tissue culture regenerated plantlets obtained using the SYNV editing system can be stably inherited by the offspring generation (see Tables 4 and 5 for editing types), and that the inheritance laws follow Mendel's laws. Example 12: Verifying the removal of the viral vector from Cas9-edited M1 generation plantlets.

[0136] Currently, there are no reports on the transmission of plant labruvirus through seeds. This example further investigated whether SYNV virus infection was present in M1 generation edited inoculations. Eight strains were selected, namely M0-8, -18, -22, -19, -42, -6, -7, and -42. Four to five M1 generation inoculations were randomly selected from each strain, and total RNA was extracted. RT-PCR was performed using primers specific to SYNV. The test results showed that all M1 generation inoculations were free of SYNV virus, and all obtained T1 generation inoculations were non-transgenic edited inoculations with virus removal (Figure 15). Example 13: Off-target analysis of SYNV CRISPR / Cas9 edited vectors

[0137] To detect whether off-target effects exist in the SYNV editing system, off-target analysis was performed on PDS target site 1, and potential off-target loci were predicted for PDS target site 1 using the Cas-OFFinder online tool (http: / / www.rgenome.net / cas-offinder / ). From the test results, potential off-target loci were found with 2 base pairs missing from one target sequence, 3 base pairs missing from two target sequences, and 4 base pairs missing from 93 target sequences. The CRISPR-P online design tool (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR) was used to analyze the off-target probability of each potential off-target locus, and a total of 13 potential off-target loci were selected for analysis (Figure 16A).

[0138] Using the T7E1 method, we detected the off-target status of PDS target site 1. Since the virus is always present during the growth process of tissue culture plantations, and there is a possibility of a higher off-target probability, we selected tissue culture albino plantations M0-11 for off-target measurement and performed PCR amplification on the aforementioned potential off-target gene loci using the primers in Table 8. As a result, after enzymatic cleavage by T7E1, the PCR products of all potential sites in M0-11 all matched the band brightness of the control wild-type plantations, and no bands were identified or cleaved by the T7E1 endonuclease. This result clarifies that none of the aforementioned potential off-target gene loci are off-target. To further verify these results, PCR / RE analysis was performed on the aforementioned potential off-target gene loci using HinfI endonuclease. With the exception of 2 and 3 of the 13 potential off-target gene loci, all remaining sites contained HinfI enzyme cleavage sites. After performing HinFI enzyme cleavage on the aforementioned 11 potential off-target gene loci, all sites were completely cleaved, and the enzyme cleavage bands were essentially identical to those of the wild-type control, with no off-target bands observed (Figure 16B). The results described above demonstrate that the SYNV vector-based CRISPR / Cas9 genome editing system exhibits relatively high specificity in plants. Example 14: Viral stability analysis of the recombinant SYNV CRISPR / Cas9 vector after friction inoculation and in subsequent generations.

[0139] In the above examples, SYNV CRISPR / Cas9 vector virus infection was initiated using Agrobacterium infiltration inoculation. To verify the feasibility of other inoculation methods, whole leaves of SYNV-tgtGFP1-Cas9 infected plants were collected as a virus source and inoculated into healthy 16c genetically modified tobacco plants using a mechanical friction method. At the same time, wild-type SYNV and recombinant vector SYNV-Cas9 were inoculated as controls. Approximately 30 days after inoculation, all of these plants showed typical symptoms of SYNV systemic infection. When observed under a portable ultraviolet lamp, the expression level of green fluorescent protein was clearly reduced in the plants inoculated with the SYNV-tgtGFP1-Cas9 edited vector compared to the control plants, and the plants also exhibited spontaneous red fluorescence of chloroplasts (Figure 17A).

[0140] Western blot analysis was performed on the total protein of the whole leaves of the inoculated plants described above. Large amounts of viral structural proteins N, P, M, and G were found in each of the infected plants described above, and the plants infected with SYNV-Cas9 and SYNV-tgtGFP1-Cas9 expressed the Cas9 protein. Compared with the control plants, the expression level of GFP protein was clearly reduced in the SYNV-tgtGFP1-Cas9 infected plants (Figure 17B). RT-PCR analysis was performed on the total RNA of the whole leaves of these inoculated plants. In the plants infected with SYNV-tgtGFP1-Cas9, the gRNA transcription unit and Cas9 nuclease sequence were stably present in the offspring viral genome (Figure 17C), indicating that the virus remained stable even after frictional passaging.

[0141] Using the PCR / RE method, we measured the editing efficiency of the GFP target and found that in three randomly selected strains inoculated with SYNV-tgtGFP1-Cas9, mutations of varying degrees occurred at the GFP target site, with editing efficiencies reaching up to approximately 83% (Figure 17D). Example 15: Construction of a SYNV CRISPR / Cpf1 editing vector

[0142] The step of simultaneously inserting crRNA and Cpf1 into the SYNV genome in the form of independent expression frames, where the insertion position includes but is not limited to the region between the leader and N. Hereinafter, taking the simultaneous insertion of the crRNA transcription unit and the Cpf1 expression frame between the leader and the N gene as an example (Figure 18A), the construction process of the CRISPR / Cpf1 genome editing vector based on SYNV will be described in detail, and the constructed clones are as follows: 1) SYNV-Cpf1: A SYNV vector that expresses only the Cpf1 protein, which was used as a control. 2) SYNV-crRNA-Cpf1: A SYNV vector that accurately transcribes crRNA and accurately expresses the Cpf1 protein. The specific construction method of the clones is as follows: 1) SYNV-Cpf1

[0143] Using the primers PvuI / F and N5’UTR / R, Cpf1 / F and Cpf1 / R, NPJ / F and N-Bsu36I / R in Table 6, SYNV-GFP le / N , YQ230, SYNV-GFP le / N as templates, the PvuI-N5’UTR fragment, the Cpf1 ORF fragment, and the NPJ-Bsu36I fragment (Figure 18A) were amplified. Here, the PvuI-N5’UTR fragment contains the sequence from the PvuI enzyme cleavage site to the 5’ non-coding region of the N gene, and the NPJ-Bsu36I fragment contains the Bsu36I enzyme cleavage site of the N gene from the NPJ sequence. All of these fragments contain 15 - 20 bp sequences homologous to adjacent fragments. Since the fragment one PvuI-N5’UTR and the fragment three NPJ-Bsu36I contain sequences homologous to the linearized SYNV vector after double enzyme cleavage via PvuI and Bsu36I respectively, the vector SYNV-Cpf1 can be obtained by sequentially ligating the above three fragments by in-fusion ligation. 2) SYNV-crRNA-Cpf1

[0144] In Table 6, primers N-PvuI / F and N5'UTR / R, N5'UTR-GFP1 / F and NPJ-GFP1 / R, and NPJ / F and N-Bsu36I / R each produce SYNV-GFP. le / N Using the primer itself, SYNV-Cpf1, as a template, the PvuI-N5'UTR fragment, crRNA fragment, and NPJ-Cpf1-Bsu36I fragment (Figure 18A) were amplified. The PvuI-N5'UTR fragment contains the sequence from the PvuI enzyme cleavage site to the 5' non-coding region of the N gene, the Cpf1 fragment contains the GFP target1 sequence, and NPJ-Cpf1-Bsu36I is the sequence between the NPJ region and the Bsu36I site of the N gene, where the Cpf1 sequence is included. After in-fusion recombination, the aforementioned fragments can acquire the vector SYNV-crGFP1-Cpf1. Due to the transcriptional characteristics of the virus itself, it releases crRNA transcripts containing virus-derived sequences at both ends, and after Cpf1 cleavage, it can produce accurate crRNA (Figure 18B).

[0145] To construct a SYNV-edited vector containing two target sites simultaneously, the DR-guide1-DR-guide2-DR fragment was synthesized according to the sequence provided in SEQ ID NO:17, and SYNV-crGFP-1 / PDS-1-Cpf1 was constructed using the same method. These vectors can simultaneously target GFP target 1 and PDS target 1, respectively. Example 16: SYNV CRISPR / Cpf1 vector targets the GFP gene in 16c genetically modified tobacco. Step 1: Inoculation of Agrobacterium-infested 16c genetically modified tobacco with the SYNV CRIPSR / Cpf1 vector.

[0146] The CRISPR RGEN Tools (http: / / www.rgenome.nt) online design software allows for the targeting of GFP in 16c plantations using a 23nt target sequence (5'-AAGATACCCAGAT). CATATGAAGC-3' and 5'-AGGGAGACACCCTC GTCAAC We selected AGG-3' (where the underlined parts are the corresponding enzyme cleavage sites NdeI and HincII) and constructed clones SYNV-crGFP1-Cpf1 and SYNV-crGFP2-Cpf1 using the method of Example 15.

[0147] Using the method of Example 1, recombinant virus expression vectors SYNV-Cpf1, SYNV-crGFP1-Cpf1, and SYNV-crGFP2-Cpf1 were electrocuted into Agrobacterium and inoculated into 16c genetically modified tobacco plants. Approximately 30 days after inoculation, each inoculated plant showed viral symptoms similar to wild-type virus, such as leaf curling and leaf vein yellowing. Furthermore, in plants inoculated with SYNV-edited vectors SYNV-crGFP1-Cpf1 and SYNV-crGFP2-Cpf19 under portable UV wrapping, the expression level of GFP green fluorescence was clearly reduced, and GFP fluorescence expression was hardly observed in mesophyll cells, with only green guard cells being observed. In whole leaves inoculated with SYNV-Cpf1, the GFP fluorescence intensity was consistent with wild-type 16c (Figure 19A).

[0148] To verify that the above-mentioned symptoms were caused by systemic SYNV infection, whole leaves were collected from affected plants, and total protein was extracted from the leaves. Western blot analysis was performed using SYNV polyclonal antibody, Flag-tagged antibody, and GFP monoclonal antibody, respectively. The test results showed that in plants inoculated with the SYNV edit vector, viral protein bands corresponding to SYNV and Cpf1 protein bands could be detected, and the expression level of GFP protein was clearly reduced. The expression level of GFP protein in tobacco leaves inoculated with the control vector SYNV-Cpf1 was basically the same as that of wild-type 16c plants (Figure 19B). These results demonstrate that the SYNV viral vector can simultaneously accommodate both the crRNA expression frame and the Cpf1 expression frame, and that recombinant viral vectors with the aforementioned exogenous expression frame inserted still possess systemic infectivity. Step 2: Analysis of GFP target site mutation efficiency

[0149] To verify the editing efficiency of GFP targets, the GFP gene editing efficiency in the whole leaves of inoculated plants was measured using the PCR / RE method. PCR was performed on target site DNA using primers GFP / F and GFP / R from Primer Table 7, and the corresponding restriction endonucleases NdeI and HincII in the target sequence were selected for enzymatic cleavage analysis. Different degrees of mutation occurred in both GFP target 1 and target 2, and the mutation efficiency reached approximately 90% (Figure 20A). To measure the editing type of GFP targets, the PCR amplification products described above were cloned, bound to a T vector, and 10 positive monoclonals were selected from each target site for Sanger sequencing. The number of monoclonals generated by mutations in GFP target 1 and target 2 was 7 and 8, respectively (Figure 20B), which was basically consistent with the detection results of PCR / RE, and most of the generated editing types were single base deletions or multiple base deletions (Figure 20C). Example 17: SYNV CRISPR / Cpf1 vector targets endogenous PDS gene in Citrus benthamiana. The SYNV CRISPR / Cpf1 viral vector editing system of the present invention was further validated by selectively using the endogenous PDS gene of Citrus benthamiana.

[0150] The CRISPR RGEN Tools (http: / / www.rgenome.nt) online design software allows for the targeting of the PDS gene using a 23nt target sequence (PDS1:5'-TGCCGTTAATTTGA GAGTC CAAG-3', PDS-2:5'- GTAGTAGCGACT CCATGG By selecting GGCAT-3' (where the underlined parts correspond to the enzyme cleavage sites HinfI and NcoI), the above targets can simultaneously target copies a and b of PDS in heterotetraploid tobacco benthamiana (Figure 21A). Recombinant virus clones SYNV-crPDS1-Cpf1 and SYNV-crPDS2-Cpf1, respectively, capable of targeting the above targets were constructed according to the method described in Example 2.

[0151] Using the method of Example 1, recombinant virus expression vectors SYNV-crPDS1-Cpf1 and SYNV-crPDS2-Cpf1 were electrocuted into Agrobacterium and inoculated into Citrus benthamiana. Approximately 30 days after inoculation, the recombinant virus expression vectors exhibited symptoms similar to those of wild-type virus, such as leaf curling and yellowing of leaf veins (Figure 21B).

[0152] Using the PCR / RE method, the target editing efficiency of PDS was measured. All primers used were able to simultaneously amplify copies a and b of PDS. The test results showed that mutations of different degrees occurred at both target sites of PDS, with editing efficiencies reaching a maximum of approximately 79% (Figure 21C). Using target 1 as an example, the editing efficiencies in three randomly selected plant strains were found to be 79%, 70%, and 73%, respectively.

[0153] Using Sanger sequencing, the editing status of PDS target site 1 was verified. PDS copies a and b were amplified using PDS-specific primers from Primer Table 7, and the PCR products were bound to a T vector. Ten clones were measured for each copy. The number of mutant clones in PDS copies a and b was 8 and 7, respectively, which was basically consistent with the results of the editing efficiency measured by the PCR / RE method. Furthermore, the editing types were mainly single base deletions and multi-base deletions (Figure 21D). Example 18: SYNV CRISPR / Cpf1 multi-target vector simultaneously targets GFP and PDS genes.

[0154] To verify whether a CRISPR / Cpf1 system based on a SYNV vector can perform multi-target editing of plant genomes, a SYNV dual-target editing vector was constructed using a method in which DR guide transcripts are sequentially linked (DR-guide1-DR-guide2-DR). The resulting primary transcript, after Cpf1 processing, was able to remove excess non-coding regions and release precise crRNA (Figure 22).

[0155] Recombinant rhabdovirus clone SYNV-crGFP-1 / PDS-1-Cpf1 was constructed according to the method described in Example 15, and this vector can simultaneously target GFP target 1 (Example 16) and PDS target 1 (Example 17). Using the method of Example 1, the recombinant rhabdovirus expression vector SYNV-crGFP-1 / PDS-1-Cpf1 was electrocuted into Agrobacterium and inoculated into Citrus benthamiana. Around 30 days after inoculation of Citrus benthamiana, the recombinant rhabdovirus expression vector exhibited symptoms similar to wild-type virus, such as leaf curling and yellowing of leaf veins (Figure 23A).

[0156] Using the PCR / RE method, the target editing efficiencies of GFP and PDS were measured separately. The test results showed that when the SYNV dual-target editing vector simultaneously targeted GFP and PDS, the editing efficiency of GFP or PDS was essentially the same as that of the SYNV single-target editing vector (Example 16 or Example 17). This result demonstrates that the SYNV vector can be used for simultaneous editing of multiple targets in the plant genome without affecting the editing efficiency of single targets (Figure 23B). Example 19: Regeneration of leaf tissue infected with SYNV CRISPR / Cpf1 and M0 generation genotyping analysis

[0157] To obtain regenerated and edited plantlets, whole leaves were collected after infection with the recombinant viral vector SYNV-crPDS1-Cpf1, and tissue culture regeneration was performed according to the method described in Example 10.

[0158] To detect the editing status of PDS target sites in tissue cultured plants, DNA was extracted from M0 generation albino and green seedlings and subjected to PCR / RE analysis (Figure 24B) and Sanger sequencing analysis. The test results showed that the PCR product from albino seedlings could not be completely cleaved, but the PCR product from tissue cultured green seedlings exhibited three states after enzymatic cleavage: complete cleavage, partial cleavage, and complete failure to cleave. The results of Sanger sequencing performed on each type of plant are shown in Tables 9 and 10: 1) In albino plants (M0-1, -2, -3, -4), PDS copies a and b were completely edited; 2) In tissue cultured green plants (M0-5, -7, -10, -12, -20, -25, -27, -28, -30, -31) where the PCR product was completely cleaved with HinfI, PDS copies a and b were edited. 3) In tissue-cultured green plants (M0-6, -8, -13, -18, -19) where the PCR product was partially cleavable with HinfI, PDS copies a and b were partially edited; 4) In tissue-cultured green plantations (M0-9, -11, -14, -15, -16, -17, -21, -22, -23, -24, 26, -29) where the PCR product was not completely cleavable with HinfI, both PDS copies a and b were edited. The phenotype of such plantations is green, and the fact that PDS function is not completely lost is mainly due to mutations, but because they were produced by editing types that are not frameshift, such as d3, d6, d9, etc., where d represents delete.

[0159] To detect the virus content of tissue culture inoculations, total RNA was extracted from M0 generation inoculations and RT-PCR was performed using SYNV primers. The test results showed that some regenerated seedlings had already eliminated the virus, and it was possible to obtain non-transgenic edited inoculations with virus elimination in the M0 generation, such as M0-6, M0-17, M0-18, M0-22, M0-23, M0-24, M0-27, and M0-28 (Figure 24C). Example 20: Verification that mutations generated by Cpf1 can be stably transmitted to offspring plantings.

[0160] Seeds (M1 generation) of green seedlings of the M0 generation edited with SYNV-crPDS1-Cpf1 were collected, and the fourth type of planting in Example 19, i.e., M0 generation plantings in which PDS copies a and b were all completely edited and contained a mutation but not a frameshift editing type were selected and the offspring generation was analyzed. The offspring generation of this type of M0 plant was sown in MS medium, and after the M1 generation germinated, three types of segregation situations appeared: 1) when the M0 generation PDSa or b genotype contained a homozygous non-frameshift editing type, there were no albino seedlings like in the M0-15 offspring generation; 2) when PDSa or b in the M0 generation genotype contained one mutation but not a frameshift editing type, the segregation ratio of the offspring generation was 3:1; and 3) when PDSa or b in the M0 generation genotype each contained one mutation but not a frameshift editing type, the segregation ratio of the offspring generation was 15:1 (Figure 25).

[0161] The results above demonstrate that the editing type of tissue culture regenerated plantlets obtained using the SYNV editing system can be stably inherited by the offspring generation, and that the inheritance laws follow Mendel's laws. Example 21: SYNV editing vectors with N-terminal deletions or substitutions of G proteins enable site-directed editing of the Citrus benthamiana genome.

[0162] The G protein encoded by rhabdoviruses consists of a signal peptide, an N-terminal domain, a transmembrane domain, and a C-terminal domain, and is mosaic-like on the surface of the virus particle in the form of trimmers. Existing research has shown that the G protein plays a crucial role in the recognition and transmission process of the virus and its vector, and that deletion or mutation in the SYNV G protein may affect the insect-borne recognition or transmission of the virus, thus providing greater biosafety.

[0163] The G protein of SYNV has a deletion of (SYNV-RFP-dGN) at its N-terminus or the N-terminal domain is modified in the reporter gene mCherry (SYNV-RFP-mCherry:G NAfter substitution, the Bsu36I-NheI fragment was recovered using SYNV-tgtPDS1-Cas9, which performs monoenzyme cleavage at the Bsu36I site on N and the NheI site on P, and the vector SYNV-RFP-dG was linearized via Bsu36I and NheI. N and SYNV-RFP-mCherry:G N It binds to and the resulting clone is SYNV-tgtPDS1-Cas9-dG N and SYNV-tgtPDS1-Cas9-mCherry:G N This is the case (Figure 26A).

[0164] Recombinant virus editing vector SYNV-tgtPDS1-Cas9-dG according to the method described in Example 1 N and SYNV-tgtPDS1-Cas9-mCherry:G N Agrobacterium was electrocuted and infiltrated into Tobacco benthamiana, and as a control, the SYNV-tgtPDS1-Cas9 edited vector was inoculated. Approximately 30 days after inoculation, each inoculated strain showed viral symptoms similar to wild-type virus, such as leaf curling and leaf vein yellowing, and SYNV-tgtPDS1-Cas9-mCherry:G N Clear mCherry red fluorescence was observed in the entire leaf of the inoculated strain under a fluorescence microscope (Figure 26B). Western blot analysis was performed on total protein extracted from the entire leaf of the diseased strain using a G protein monoclonal antibody. The test results showed that in the strain inoculated with the SYNV editing vector, a G protein band of the same size as wild-type SYNV was detected. However, when the N-terminal domain of the G protein was replaced with the reporter gene mCherry, a 36.7 kD mChery:GN binding protein was detected, which was consistent with the expected size (Figure 26C). These results demonstrate that deletion or substitution of the N-terminal domain of the SYNV G protein does not affect the systemic infectivity of the recombinant virus.

[0165] To verify the targeted editing efficiency of the above vectors against PDS, whole leaves of inoculated plants were detected using the PCR / RE method. All primers used were able to simultaneously amplify copies a and b of PDS. The test results showed that all of the above recombinant vectors could produce effective editing against PDS target site 1, and their editing efficiency was equivalent to that of existing SYNV editing vectors (Figure 26D). Furthermore, deletion or substitution of the N-terminal domain of the G protein of SYNV did not affect the editing efficiency of the recombinant virus. The results above demonstrate that site-directed editing of the *Cigarette benthamiana* genome was achieved using a SYNV editing vector with N-terminal deletion or substitution of a G protein. Example 22: The SYNV CRISPR / oCas9 vector targets the endogenous PDS gene in Citrus benthamiana.

[0166] To verify the feasibility of implementing Cas nuclease codon optimization for specific species, we performed codon optimization for Cas9 nuclease against tobacco benthamiana and verified the target editing efficiency of the SYNV CRISPR / oCas9 edited vector against endogenous PDS in tobacco benthamiana.

[0167] Based on the existing editing vector SYNV-tgtPDS2-Cas9, the Cas9 nucleic acid sequence was replaced with the oCas9 sequence, and this was modified into SYNV-tgtPDS2-oCas9 (Figure 27A). The specific construction process is as follows:

[0168] First, a SYNV vector expressing only oCas9 was constructed. The construction process involved synthesizing a benthamiana tobacco codon-optimized oCas9 sequence according to the sequence provided by SEQ ID: 36. Using the primers N-Bsu36I / F and NPJ-oCas9 / R, oCas9 / F and Cas9 / R, Cas9-NPJ / F and P-NheI / R from Table 6, SYNV, oCas9, and SYNV were used as templates to amplify the Bsu36I-NPJ fragment, the oCas9 ORF fragment, and the NPJ-NheI fragment, respectively. Here, the Bsu36I-NPJ fragment contains the NPJ region from the Bsu36I enzyme cleavage site on the N gene downstream, and the NPJ-NheI fragment contains the NheI enzyme cleavage site from the NPJ sequence to the P gene. Each of these fragments contains a 15-20 bp sequence homologous to the adjacent fragment. Fragment 1, Bsu36I-NPJ, and Fragment 3, NPJ-NheI, each contain sequences homologous to the linearized SYNV vector after double enzymatic cleavage of Bsu36I and NheI, respectively. By sequentially binding these three fragments via in-fusion, the vector SYNV-oCas9 can be obtained. Next, using the primers N-Bsu36I / F and P2 20nt / R, and P2 20nt / F and oCas9 / Bsu36I / R from Table 6, SYNV-tgtPDS2-Cas9 and SYNV-oCas9 were used as templates to amplify the N / Bsu36I-PDS2 fragment and the PDS2-oCas9 / Bsu36I fragment, respectively. These were then in-fusion-bound with the Bsu36I-linearized SYNV-oCas9 vector, and the resulting vector was SYNV-tgtPDS2-oCas9.

[0169] According to the method described in Example 1, recombinant viral editing vector SYNV-tgtPDS2-oCas9 was electrocuted into Agrobacterium and inoculated into Tobacco benthamiana. Simultaneously, SYNV-tgtPDS2-oCas9 was inoculated as a control. Approximately 30 days after inoculation, each inoculated plant showed viral symptoms similar to wild-type virus, such as leaf curling and leaf vein yellowing (Figure 27B). To verify the targeted editing efficiency of the above vectors against PDS, 10 systemically infected plantlets were randomly selected, and whole leaves were detected using the PCR / RE method. All primers used were capable of simultaneously amplifying PDS copy a and copy b. From the test results, it was found that all of the above recombinant vectors could produce effective editing against PDS target site 1, and that the editing efficiency was equivalent to that of existing SYNV editing vectors (Figure 27C). The above results demonstrate that specific species codon optimization for Cas nucleases is feasible. Example 23: SYNV CRISPR / Cas 9 editing system based on SYNV single-transfer unit editing vector

[0170] To verify that sgRNA and Cas9 can be controlled by the same transcription unit within a SYNV editing vector, a single transcription unit SYNV-oCas9gPDS2 (Figure 28A) was constructed by ligating a benthamiana tobacco codon-optimized Cas nuclease sequence with an sgRNA sequence using a 15 bp spacer sequence (5'-AAGCCATGGGATATC-3'). The clonal construction process was as follows: Using the primers oCas9 / BstEII / F and Cas9 / R, oCas9 / linker P2 / F and gRNA / R, gRNA / NPJ / F and P / NheI / R from Table 6, oCas9 / BstEII-linker, linker-Scaffold, and NPJ-NheI fragments were amplified using SYNV-oCas9, SYNV-tgtPDS2-Cas9, and SYNV as templates. These fragments were then in-fusion coupled with the SYNV-oCas9 vector linearized with BstEII and NheI, resulting in the vector SYNV-oCas9gPDS2.

[0171] According to the method described in Example 1, the single transcription unit edited vector SYNV-oCas9gPDS2 was electrocuted into Agrobacterium and inoculated into Tobacco benthamiana. Simultaneously, an existing SYNV-tgtPDS2-oCas9 clone was inoculated as a control. Approximately 30 days after inoculation, each of the inoculated plants showed symptoms similar to those of the wild-type virus, such as leaf curling and leaf vein yellowing (Figure 28B).

[0172] The mutation efficiency of the PDS target was detected using the PCR / RE method, and all primers used were capable of simultaneously amplifying PDS copy a and copy b. The test results showed that the single transcription unit SYNV editing vector could produce effective editing at PDS target site 2, and that the editing efficiency was comparable to that of existing double transcription unit SYNV editing vectors (Figure 28C). The above results demonstrate the feasibility of expressing sgRNA and Cas9 nuclease using the same transcription unit in a SYNV editing vector. Example 24: Measurement of the infectivity of EMDV fluorescence-expressing vector to Citrus benthamiana. Step 1: Construction of the EMDV fluorescence expression vector

[0173] The process involves inserting a GFP or RFP fluorescent reporter gene into the EMDV genome in the form of an independent expression frame, where the insertion site includes, but is not limited to, the space between N and X. Below, the construction process of the EMDV fluorescent expression vector is detailed, using the insertion of the GFP gene between Leader and N, and the insertion of the RFP gene between N and X genes as examples (Figure 29A). The constructed vector is as follows: EMDV-GFP: An EMDV fluorescence expression vector that carries the green fluorescent protein gene GFP between the EMDV leader and the N gene. EMDV-RFP: An EMDV fluorescence expression vector that carries the red fluorescent protein gene RFP between the EMDV N gene and X gene. Specifically, the method for constructing the clone is as follows:

[0174] For EMDV-GFP, using the primers EMDV-AatII / F and EMDV-N3' / R, EMDV-N5' / F and EMDV-AatII / R from Table 6, and EMDV-WT as templates, an A fragment containing the GFP gene with EMDV N gene UTRs at both ends, and a B fragment containing the second AatII enzyme cleavage site region from the N gene 5'UTR were amplified, respectively. These fragments were then digested and recovered by AatII monoenzyme cleavage to obtain an EMDV-GFP fluorescence expression vector by multi-fragment in-fusion with the EMDV-WT vector backbone (Figure 29A).

[0175] For EMDV-RFP, amplification was performed using the primers EMDV-SpeI / F and EMDV-A / R, EMDV-RFP / F and EMDV-RFP / R, EMDV-C / F and EMDV-SpeI / R shown in Table 6. This yielded a bound A fragment containing the region from near the SpeI enzyme cleavage site on the N gene to the N-X junction, a B fragment containing the RFP gene sequence, and a C fragment containing the region from the start of the NX junction to near the SpeI enzyme cleavage site on the M gene. These fragments were then combined with the EMDV-WT vector skeleton, which was recovered by digestion via SpeI monoenzyme cleavage, to obtain the EMDV-RFP fluorescent tag vector through multi-fragment in-fusion (Figure 29A). Step 2: Infuse Citrus benthamiana with Agrobacterium and inoculate with an EMDV fluorescence expression vector.

[0176] According to the method described in Example 1, recombinant rhabdovirus expression vectors EMDV-GFP or EMDV-RFP were electrocuted into Agrobacterium and inoculated into Tobacco benthamiana. In Tobacco benthamiana inoculated with EMDV-GFP, 23 days after inoculation, infected plants showed phenotypes such as whole-body leaf curling, yellowing of leaf veins, and significant plant dwarfism, and relatively strong green fluorescence could be emitted from the entire body of infected leaves under a fluorescence microscope (Figure 29B). In Tobacco benthamiana inoculated with EMDV-RFP, around 30 days after inoculation, infected plants showed viral symptoms similar to wild-type virus, such as whole-body leaf curling and plant dwarfism. Furthermore, due to the overexpression of red fluorescent protein, the stems, upper leaves, and petals of the plants also appeared pale red under visible light, and these plant tissues could emit strong red fluorescence under a fluorescence microscope (Figure 29C).

[0177] To verify that the above-mentioned symptoms were caused by systemic EMDV infection, whole leaves were collected from affected plants, and total leaf protein was extracted. Western blot analysis was performed using EMDV virus particle antibody, GFP, or RFP monoclonal antibody, respectively. The test results showed that plants inoculated with EMDV-GFP allowed detection of the EMDV-corresponding viral structural protein band and GFP protein band (Figure 29B), while plants inoculated with EMDV-RFP allowed detection of the EMDV viral structural protein band and RFP protein band (Figure 29C). These results indicate that EMDV, by carrying GFP or RFP fluorescent proteins, can successfully complete systemic infection of tobacco benthamiana, express large amounts of the fluorescent proteins in various tissues of tobacco benthamiana, and that this does not affect the infection characteristics of EMDV itself, greatly facilitating subsequent EMDV inoculation and measurement on various crops. Example 25: Measurement of the infectivity of EMDV fluorescence expression vectors against tobacco, potatoes, eggplants, and tomatoes.

[0178] Existing research has revealed that some scientists have obtained EMDV virus through isolation in multiple types of agricultural and commercial crops, including potatoes, tobacco, tomatoes, eggplants, and cucumbers, under natural conditions. Using recombinant EMDV-RFP or EMDV-GFP viruses rescued from tobacco benthamiana, infection was measured in potatoes, tobacco, tomatoes, and eggplants via methods such as mechanical friction, stem injection, spray gun application, and grafting. The specific results are as follows.

[0179] In tobacco plants, systemic infection with the EMDV-RFP recombinant virus can be achieved through mechanical friction. Systemic symptoms appear approximately 30 days after inoculation, with yellowing of the leaf veins occurring throughout the entire leaf system of infected plants. Around 45 days later, the yellowing of the leaf veins worsens, and twisted malformations appear throughout the entire leaf system. Under a fluorescence microscope, strong red fluorescence can be emitted from both the entire leaf system and the tobacco stems of infected plants. Western blot allows detection of EMDV virus structural proteins and RFP red fluorescent protein bands from the entire leaf system of infected tobacco plants (Figure 30A).

[0180] In the case of potatoes, virus transmission and systemic infection to potato scions can be achieved by using virus-containing tobacco benthamiana rootstock through cross-grafting. First, healthy tobacco benthamiana and potato plants of suitable age are selected and used. The upper tissue of the tobacco benthamiana is removed, and 3-4 leaves are retained to form the tobacco benthamiana rootstock. The stem tip of a young potato with good growth is used as the scion. The circular cross-section of the rootstock stem is cut along the radial direction, and excess tissue is removed from both sides of the scion stem to finally shape it into a wedge shape. Subsequently, the wedge-shaped scion was slowly inserted into a pre-cut incision along the rootstock stem and secured. The completed grafted plant was then carefully and completely covered with a pre-moistened thin plastic bag to reduce the rate of dehydration. After incubation in a constant temperature culture box at 27°C / 24 hours under high light for 7 days, the moisturizing plastic bag was removed, and the relatively well-grown Tocopherol benthamiana rootstock portion was inoculated with recombinant EMDV virus via Agrobacterium infiltration / mechanical friction. After incubation at 24°C / 24 hours under low light conditions for approximately one month, the plant was transferred from the culture box and viral infection was measured. Using the above method, systemic infection of potato scions with EMDV-RFP was successfully achieved, and strong red fluorescence was observed from the rootstock and scion leaves (Figure 30C). Furthermore, it is possible to successfully infect eggplants, tomatoes, and potatoes with EMDV through mechanical friction (Figure 30B). Example 26: Measurement of the infectivity of capsicum plants using an EMDV fluorescence-expressing vector.

[0181] For chili peppers, a mechanical friction inoculation method was used, with the leaves of tobacco plants infected with EMDV-RFP systemically used as the toxin source. The mixture was homogenized to obtain the virus juice, which was then inoculated into chili pepper seedlings approximately two weeks old. The plants were cultured at 23°C under conditions of 16 hours of light and 8 hours of darkness. Seven days after inoculation, infection points of EMDV-RFP were observed on the inoculated leaves, and systemic infection symptoms appeared 25 days after inoculation. Yellowing of the leaf veins due to viral infection was observed in the upper leaves, and strong red fluorescence was detected by fluorescence microscopy (Figure 31 B). Western blot also detected EMDV virus structural protein and RFP red fluorescent protein from the entire leaves of chili peppers (Figure 31 A). Example 27: EMDV CRISPR / Cas9 vector targets GFP gene in 16c genetically modified tobacco. Step 1: Construction of the EMDV-tgtGFP2-Cas9 edited vector

[0182] This process involves simultaneously inserting sgRNA and Cas9 into the EMDV genome in the form of independent expression frames, with insertion sites including, but not limited to, the space between N and X. Below, using the simultaneous insertion of an sgRNA transcription unit and a Cas9 expression frame between the N and X genes as an example (Figure 32A), the construction process of an EMDV-based CRISPR / Cas9 genome editing vector is described in detail, and the constructed vector is as follows:

[0183] EMDV-tgtGFP2-Cas9: Contains an EMDV vector expressing sgRNA and Cas9 protein targeting the GFP2 target in the recombinant tobacco benthamiana 16c strain. The specific method for constructing the clone is as follows:

[0184] The EMDV-tgtGFP2-Cas9 vector was constructed using a 20-nt target sequence (5'-ACAAGTGTTGGCCATGGAAC-3', SEQ ID NO:20, underlined portion is the corresponding enzyme cleavage site NcoI) targeting GFP in 16c inoculations, similar to Example 3. First, an intermediate vector was constructed that was enzymatically cleavable with AarI and allowed for flexible target exchange via primer annealing. This intermediate vector contained a full-length Cas9 sequence and sgRNA with two EMDV NX junctions and two AarI enzymatic cleavage sites, and was single-enzyme cleavable with AarI. After annealing with primers PDS4 / F and PDS4 / R in Table 6, it was bound to the intermediate vector by T4, and this vector could be used as a template to obtain a II-PDS4 fragment containing the PDS4 target. Subsequently, amplification was performed using the primers EMDV-SpeI / F and EMDV-I / R, EMDV-II / F and EMDV-II / R, EMDV-III / F and EMDV-SpeI / R shown in Table 6. This yielded fragment I, containing the 3'UTR from near the SpeI enzyme cleavage site on the N gene to the N gene; fragment II, containing two EMDV NX junctions, a tgt-GFP2 sequence, and a Cas9 ORF; and fragment III, containing one EMDV NX junction and the area from the X 5'UTR to the SpeI enzyme cleavage site on the M gene. These fragments, along with the EMDV-WT vector skeleton recovered by mono-enzyme cleavage of SpeI, were then combined in-fusion of multiple fragments to obtain the EMDV-tgtGFP2-Cas9 edited vector. Step 2: Infuse tobacco plants with Agrobacterium and then inoculate them with an EMDV-tgtGFP2-Cas9 edited vector.

[0185] According to the method described in Example 1, recombinant rhabdovirus expression vector EMDV-tgtGFP2-Cas9 was electrocuted into Agrobacterium and inoculated into genetically modified 16c Bacopa. 27 days after inoculation, the infected plants showed viral symptoms similar to wild-type virus, including whole-body leaf curling and plant dwarfism. Furthermore, under a portable ultraviolet lamp, the green fluorescence in the veins of the leaves of the plants was clearly weakened. 35 days after inoculation, a clear decrease in green fluorescence was observed in all leaves of the upper part of the plants, but the green fluorescence in the veins or mesophyll of the leaves at the top of healthy 16c plants remained unchanged (Figure 32B).

[0186] To verify that the symptoms described above were caused by systemic EMDV infection, whole leaves were collected from affected plants, total protein was extracted from the leaves, and Western blot analysis was performed using EMDV virus particle antibodies and Flag tag antibodies, respectively. From the test results, it was possible to detect viral protein bands corresponding to EMDV and Cas9 protein bands (Figure 32B) from plants inoculated with the EMDV edit vector. These results indicate that the EMDV virus vector is similar to the SYNV edit vector, and both can simultaneously accommodate an sgRNA expression frame and a Cas9 expression frame with a length of 4.2kb. This revealed that the recombinant rhabdovirus vector with the aforementioned exogenous expression frame inserted still possesses systemic infectivity. Step 3: GFP Target Mutation Efficiency Analysis

[0187] To verify the editing efficiency of GFP targets, whole leaves of inoculated plants were measured using the PCR / RE method (Polymerase Chain Reaction / Restriction digestion). PCR was performed using primers GFP2 / F and GFP2 / R from Primer Table 7, and then the corresponding restriction endonuclease NcoI in the target sequence was selected for enzymatic cleavage analysis. The results showed that the selected GFP2 target was able to induce relatively efficient gene mutations, and the editing efficiency calculated from the NcoI enzymatic cleavage results was approximately 90%. To detect the editing type of GFP, the above PCR amplification product was bound to a T vector, and monoclonals were collected and subjected to Sanger sequencing analysis. From the test results, eight of the nine monoclonal colonies detected showed different types of base mutations, which were basically consistent with the PCR / RE detection results. The generated editing types were mostly single base or multiple base deletions, and were accompanied by base insertions (Figure 32C). Example 28: EMDV CRISPR / Cas9 vector targets PDS gene in Citrus benthamiana Step 1: Construction of EMDV-tgtPDS4-Cas9 editing vector

[0188] To test whether the EMDV CRISPR / Cas 9 editing vector can successfully infect multiple types of crops, such as potatoes and tomatoes, like the EMDV fluorescence expression vector, and whether it can produce the corresponding type of editing on the crops, we selected a conserved 20 nt target sequence (5'-CAATACAGTTAACTATTTGG-3', SEQ ID NO:37) in the PDS gene of the aforementioned crops and inserted it between the EMDV N gene and X gene in a single transcription unit manner. The constructed vector is as follows.

[0189] EMDV-tgtPDS4-Cas9: Contains an EMDV vector expressing sgRNAs targeting multiple types of plant PDS genes (PDS4 targets) and the Cas9 protein.

[0190] The construction process for the EMDV-tgtPDS4-Cas9 edited vector is essentially the same as that for EMDV-tgtGFP2-Cas9, with the only difference being the replacement of the II fragment with the II-PDS4 fragment (Figure 33A). Step 2: The EMDV-tgtPDS4-Cas9 editing vector edits the PDS gene of Citrus benthamiana.

[0191] The vector EMDV-tgtPDS4-Cas9 was electrocuted into Agrobacterium and inoculated into wild-type tobacco plants. 40 days after inoculation, the infected plants similarly exhibited viral symptoms including whole-body leaf vein yellowing, leaf curling, and plant dwarfism. Western blot analysis of whole leaves from infected plants similarly detected the corresponding viral structural protein bands (Figure 33B). Using two pairs of primers from Primer Table 7, PDS-4a / F and PDS-4a / R, and PDS-4b / F and PDS-4b / R, the two PD alleles, PDS-a and PDS-b, in tobacco plants were amplified. The resulting fragments contained target region fragments, and after T7EI enzyme cleavage, both alleles exhibited some degree of gene editing (Figure 33C). Example 29: Regeneration of leaf tissue infected with EMDV CRISPR / Cas 9 and genotyping analysis of the M0 generation.

[0192] To obtain regenerated and edited plants, young upper leaves of tobacco benthamiana infected systemically with EMDV-tgtPDS 4-Cas 9 were used as explants, and regenerated plants were obtained using the tissue culture method described in Example 10. Two phenotypes were observed in the regenerated seedlings: whitening and normal green (Figure 34 A).

[0193] To measure the editing status of PDS target sites in regenerated seedlings, total DNA was extracted from M0 generation seedlings and subjected to PCR / RE analysis (Figure 34 B). 100 ng of wild-type PDS fragment amplification product and 100 ng of M0 generation seedling PDS fragment amplification primer were uniformly mixed, annealed, and T7 EI enzyme cleavage analysis was performed. Target site editing could be detected in all bleached seedlings, and target site editing could be detected in M0-19, 20, 21, and 27 seedlings among the green seedlings.

[0194] To detect whether persistent infection with the EMDV editing vector was present in the regenerated plants, total RNA of M0 generation plants was extracted and detected by RT-PCR using EMDV primers. All whitened seedlings carried the EMDV vector, and some regenerated green seedlings, such as M0-19 and -27, contained editing, but the virus had already been removed (Figure 34 C), thus obtaining non-genetically modified edited plants with virus removal in the M0 generation. Example 30: EMDV-tgtPDS4-Cas9 editing vector edits tobacco, potato, and tomato PDS genes.

[0195] EMDV-tgtPDS4-Cas9 recombinant virus, rescued from tobacco benthamiana, was inoculated into tobacco, potato, and tomato according to the method described in Example 24. After systemic onset, fragments corresponding to the PDS genes of tobacco, potato, and tomato were amplified using primers PDS-Nt / F and PDS-Nt / R, PDS-ST / F and PDS-ST / R, PDS-SL / F and PDS-SL / R from the primer sheet, respectively. After binding to a T vector, 6-7 monoclonals were collected from each plant and sent for testing. The results showed that mutations occurred at the PDS4 target site in these plants, and the mutation types were mainly single or multiple base deletions or base substitutions (Figure 35). The results above demonstrate that the EMDV editing vector successfully achieved genome site-specific editing in tobacco, potato, and tomato. Table 1. Genotypic and phenotypic analysis of SYNV CRISPR / Cas9 vectors editing the M0 generation of planted plants. JPEG0007870925000001.jpg176170 Table 2: Genotypic and phenotypic analysis of the SYNV CRISPR / Cas9 vector editing the M1 generation of planted plants JPEG0007870925000002.jpg145170 Table 3: Plant genotype analysis of plant M2 generation edited by SYNV CRISPR / Cas9 JPEG0007870925000003.jpg39170 Table 4: List of editing types used by SYNV CRISPR / Cas9 to edit planted M0 generation PDS-1 targets JPEG0007870925000004.jpg195170 Table 5 SYNV CRISPR / Cas9 List of editing types for editing plant M1 generation plant PDS JPEG0007870925000005.jpg221170 Table 6 Clone construction primers and RT-PCR, cRT-PCR detection primers JPEG0007870925000006.jpg216170JPEG0007870925000007.jpg175170Note: The sequences written in lowercase are 15-20 nt fragments for homologous recombination. Table 7 Target gene editing efficiency detection primers JPEG0007870925000008.jpg142170 Note: / represents different PCR product sizes for copy a / copy b. Table 8. PDS1 potential off-target gene locus detection primers JPEG0007870925000009.jpg128170 Table 9 Genotypic and phenotypic analysis of the SYNV CRISPR / Cpf1 vector editing the M0 generation of planted plants JPEG0007870925000010.jpg229170 Table 10 List of editing types used by SYNV CRISPR / Cpf1 to edit plant M0 generation PDS-1 targets JPEG0007870925000011.jpg207170

[0196] Although preferred embodiments of the present invention have been described in detail above, the present invention is not limited to these examples. Furthermore, those skilled in the art can make various simple modifications to the present invention using the contents of the specification and drawings, and it should be understood that these modifications fall within the scope of the claims of the present invention.

[0197] Furthermore, each specific technical feature described in the above specific embodiments can be combined in any appropriate manner, provided they do not contradict each other. To avoid unnecessary redundancy, various possible combinations will not be described here.

[0198] Introduction of the nucleotide sequence according to the present invention SEQ ID NO 1: partial sequence of SYNV-tgtGFP1-Cas9(tRNA-GFP target1-scaffold-tRNA)

[0199] SEQ ID NO 2: partial sequence of SYNV-tgtGFP2-Cas9(tRNA-GFP target2-scaffold-tRNA)

[0200] SEQ ID NO 3: partial sequence of SYNV-tgtPDS1-Cas9(tRNA-PDS target1-scaffold-tRNA)

[0201] SEQ ID NO 4: partial sequence of SYNV-tgtPDS2-Cas9(tRNA-PDS target2-scaffold-tRNA)

[0202] SEQ ID NO 5: partial sequence of SYNV-tgtPDS3-Cas9(tRNA-PDS target3-scaffold-tRNA)

[0203] SEQ ID NO 6: partial sequence of SYNV-tgtRDR6-1-Cas9(tRNA-RDR6 target1-scaffold-tRNA)

[0204] SEQ ID NO 7: partial sequence of SYNV-tgtRDR6-4-Cas9(tRNA-RDR6 target4-scaffold-tRNA)

[0205] SEQ ID NO 8: partial sequence of SYNV-tgtSGS3-1-Cas9(tRNA-SGS3 target1-scaffold-tRNA)

[0206] SEQ ID NO 9: partial sequence of SYNV-tgtSGS3-2-Cas9(tRNA-SGS3 target2-scaffold-tRNA) SEQ ID NO 10: partial sequence of SYNV-tgtgtRDR6-1 / SGS3-1-Cas9 (tRNA-RDR6 target1-scaffold-tRNA-SGS3 target1-scaffold-tRNA) SEQ ID NO 11: partial sequence of SYNV-tgtgtRDR6-4 / SGS3-2-Cas9 (tRNA-RDR6 target4-scaffold-tRNA-SGS3 target2-ffffold-tRNA) SEQ ID NO 12: partial sequence of SYNV-tgtgtPDS1 / 3-Cas9 (tRNA-PDS target1-scaffold-tRNA-PDS target3-scaffold-tRNA) SEQ ID NO 13: partial sequence of SYNV-crGFP1-Cpf1(DR-GFP target1-DR) SEQ ID NO 14: partial sequence of SYNV-crGFP2-Cpf1(DR-GFP target2-DR) SEQ ID NO 15: partial sequence of SYNV-crPDS1-Cpf1(DR-PDS target1-DR) SEQ ID NO 16: partial sequence of SYNV-crPDS2-Cpf1(DR-PDS target2-DR) SEQ ID NO 17: partial sequence of SYNV-crGFP-1 / PDS-1-Cpf1 (DR-GFP target1-DR-PDS target1-DR) SEQ ID NO 18: Cas9 nucleic acid sequence SEQ ID NO 19: Cpf1 nucleic acid sequence SEQ ID NO 20-33: Target site sequence SEQ ID NO 34-35: Sequences derived from NPJ SEQ ID NO 36: oCas9 sequence with codon optimization for Benzamianatabako

[0207] SEQ ID NO 37: partial sequence of EMDV-tgtPDS4-Cas9(tRNA-PDS target4-scaffold-tRNA) SEQ ID NO 38: SYNV genome cDNA sequence SEQ ID NO 39: EMDV genome cDNA sequence

Claims

1. A method for modifying plant cytogenetic material that does not require the introduction of foreign gene sequences into the cell genome to be modified, Step a) providing at least one plant cell to be genetically modified, The steps include: b) infecting plant cells with a recombinant plant rhabdovirus vector having systemic infectivity, wherein the recombinant rhabdovirus having systemic infectivity carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, and the sequence-specific endonuclease specifically targets a plant genome nucleic acid sequence and cleaves the target site; The aforementioned rhabdovirus vectors with systemic infectivity are sonchus yellow net virus (SYNV) or eggplant mottled dwarf virus (EMDV), The sequence-specific endonuclease is a CRISPR / Cas nuclease expressed from the nucleic acid sequence indicated by SEQ ID NO: 18 or SEQ ID NO:

19. The aforementioned plants are Nicotiana benthamiana, potato (Solanum tuberosum), tomato (Lycopersicon esculentum), or tobacco (Nicotiana tabacum), A method for modifying plant cytogenetic material, wherein the modification process of plant cytogenetic material is induced by cleavage by a sequence-specific endonuclease and completed by a plant endogenous DNA repair mechanism.

2. A method for producing plants with modified genetic material that does not require the introduction of foreign gene sequences into the plant cell genome to be modified, Step a) providing at least one plant cell to be genetically modified, Step a) infecting plant cells with a recombinant plant rhabdovirus vector having systemic infectivity, wherein the recombinant rhabdovirus having systemic infectivity carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, and the sequence-specific endonuclease specifically targets a plant genome nucleic acid sequence and cleaves the target site. Step c) obtaining a plant from cells modified with the aforementioned genetic material, The step includes selecting a modified plant target containing the genetic material without needing to use a selection marker, The aforementioned rhabdovirus vectors with systemic infectivity are sonchus yellow net virus (SYNV) or eggplant mottled dwarf virus (EMDV), The sequence-specific endonuclease is a CRISPR / Cas nuclease expressed from the nucleic acid sequence indicated by SEQ ID NO: 18 or SEQ ID NO:

19. A method for producing genetically modified plants, wherein the plant is Nicotiana benthamiana, Solanum tuberosum, Lycopersicon esculentum, or tobacco (Nicotiana tabacum).

3. The method according to claim 1, characterized in that the method for infecting the cells to be modified includes natural infection of recombinant virus-uninoculated cells, friction inoculation, grafting, insect-borne transmission, or infection by any other virus.

4. The method according to claim 1, characterized in that it comprises one or more types of mutated and / or recombinantly modified rhabdovirus vectors having systemic infectivity.

5. The method according to claim 4, characterized in that the inducing viral vector is an inducing viral vector having a mutated and / or recombinantly modified amino-terminal domain of glycoprotein (G).

6. The method according to claim 1, characterized in that the modification of the plant cytogenetic material is selected from at least one base deletion, one base insertion, or one base substitution at a target site, or a combination of these modification patterns.

7. The method according to claim 1, characterized in that the ribonucleic acid sequence encoding the sequence-specific endonuclease comprises the nucleic acid sequences of one or more guide RNAs and the nucleic acid sequence of a Cas nuclease.

8. The method according to claim 7, characterized in that the sequence of the sequence-specific endonuclease is present in the genome of the rhabdovirus in the form of an independent transcription unit.

9. The method according to claim 7, characterized in that the transcription of the sequence-specific endonuclease sequence is controlled by a regulatory element related to rhabdoviral messenger RNA transcription.

10. The method according to claim 7, characterized in that the guide RNA and Cas nuclease sequence are controlled by the same transcription unit.

11. The method according to claim 7, characterized in that the guide RNA and Cas nuclease sequences are controlled by different transcription units.

12. The method according to 7, characterized in that the guide RNA transcript has its virus-derived terminal sequence removed by an intracellular tRNA processing device.

13. The method according to 7, characterized in that the guide RNA transcript is subjected to processing with a Cas nuclease to remove the terminal sequence derived from the virus.

14. Infectious recombinant plant rhabdovirus vector, The invention comprises a polynucleotide sequence encoding at least one sequence-specific endonuclease, the sequence-specific endonuclease transiently expresses in cells infected with the viral vector and specifically targets plant genome nucleic acid sequences, the target sequence being modified by the nuclease, The aforementioned viral vector is sonchus yellow net virus (SYNV) or eggplant mottled dwarf virus (EMDV), The sequence-specific endonuclease is a CRISPR / Cas nuclease expressed from the nucleic acid sequence indicated by SEQ ID NO: 18 or SEQ ID NO:

19. An infectious recombinant plant rhabdovirus vector characterized in that the plant is Nicotiana benthamiana, Solanum tuberosum, Lycopersicon esculentum, or tobacco (Nicotiana tabacum).

15. The infectious recombinant plant rhabdovirus vector according to claim 14, characterized in that the infectious recombinant plant rhabdovirus vector comprises one or more types of induced viral vectors after mutation and / or recombinant modification.

16. The infectious recombinant plant rhabdovirus vector according to claim 15, characterized in that the modified viral vector is a viral vector in which the amino-terminal domain of a glycoprotein (G) has been mutated and / or recombinantly modified.

17. The infectious recombinant plant rhabdovirus vector according to claim 14, characterized in that the sequence supported by the rhabdovirus vector transcribes and produces the nucleic acid sequences of one or more guide RNAs and the nucleic acid sequence of a Cas nuclease.

18. The infectious recombinant plant rhabdovirus vector according to claim 14, characterized in that the sequence of the sequence-specific endonuclease is present in the genome of the rhabdovirus in the form of an independent transcription unit.

19. The infectious recombinant plant rhabdovirus vector according to claim 14, characterized in that the guide RNA and Cas nuclease sequences are controlled by different transcription units.

20. The infectious recombinant plant rhabdovirus vector according to claim 14, characterized in that the guide RNA and Cas nuclease sequence are controlled by the same transcription unit.

21. The infectious recombinant plant rhabdovirus vector according to claim 14, characterized in that the transcription of the sequence-specific endonuclease sequence is controlled by a regulatory element related to rhabdovirus messenger RNA transcription.

22. The infectious recombinant plant rhabdovirus vector according to claim 14, characterized in that the guide RNA transcript has its virus-derived terminal sequence removed by an intracellular tRNA processing device.

23. The infectious recombinant plant rhabdovirus vector according to claim 14, characterized in that the guide RNA transcript has its virus-derived terminal sequence removed by Cas nuclease processing.

24. The infectious recombinant plant rhabdovirus vector according to claim 14, characterized in that the viral vector is a recombinant nucleic acid construct, and the sequence containing the viral vector is operably linked to a promoter.

25. The infectious recombinant plant rhabdovirus vector according to claim 14, characterized by transient expression of the sequence-specific endonuclease in infected natural or experimental host cells.

26. Use of an infectious recombinant plant rhabdovirus vector according to any one of claims 14 to 25 in plant genome editing.

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