Infectious plant rhabdovirus vectors and methods for non-transgenic site-specific genome 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, in particular to a method for site-specific editing in plants using a recombinant rhabdovirus vector. Specifically, the present invention relates to a method for site-specific editing in plants by non-transgenic delivery of CRISPR nuclease components using a recombinant rhabdovirus vector. [Background technology]

[0002] In recent years, the emergence of targeted genome editing (TGE) has opened up new avenues for precision crop breeding and has broad application prospects in functional genomics research and trait genetic improvement of a variety of crops, including wheat, rice, and maize. Currently, targeted genome editing is primarily accomplished by using sequence-specific nucleases (SSNs). SSNs combine a site-specific DNA binding domain with a nonspecific DNA cleavage domain, enabling them to target and modify specific regions of the genome. After being introduced into cells, sequence-specific nucleases can recognize specific sequences in the genome and cleave those sequences to form double-stranded breaks (DSBs), which then activate non-homologous end joining (NHEJ) or homologous recombination (HR) in the cell to repair and cause deletion or replacement of gene function. Currently, there are three types of sequence-specific nucleases commonly used: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (CRISPR / Cas). Among these, CRISPR / Cas nucleases have the advantages of being easy to operate, rapid, and highly efficient, and are currently the most widely used genome editing tool.

[0003] One of the key technologies currently in plant genome editing is plant delivery of 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 plantlets. An ideal delivery technology must meet the following requirements: (1) the complete sequence-specific nuclease nucleic acid or protein molecule can be introduced into plant material; (2) the recipient plant material is easy to obtain, and the introduction process is simple and easy to operate; (3) the sequence-specific nuclease introduced into the plant material can achieve high editing efficiency; (4) it is easy to obtain mutant regenerated plantlets after editing; and (5) the edited plantlets obtained do not contain the integration of foreign nucleic acids.

[0004] In conventional technology, delivery of CRIPSR / Cas nucleases into plants mainly relies on genetic engineering, i.e., introducing a recombinant gene containing CRIPSR / Cas into recipient plant cells to integrate into the plant genome, where the expressed nuclease produces predictable, targeted genetic changes to the genomic target sequence. Currently, the most commonly used techniques for plants include Agrobacterium-mediated transfection and gene gun transfection, and any insertion of foreign DNA into the genome is considered genetic engineering. While these methods can deliver nucleases into plants, the following problems remain in their breeding applications: (1) the integration of foreign genes into the plant genome and the need for marker screening using antibiotics or herbicides during the transformation process; and (2) the possibility of unexpected phenotypic variations in the resulting edited plants. (3) The process of isolating the recombinant gene by means of self-breeding or backcrossing is long, especially in the case of polyploid and asexually reproduced plants with long breeding cycles; (4) In some countries or regions, the offspring of the edited plant from which the recombinant gene has been removed and its products may be subject to the supervision and control of genetically modified products.

[0005] Given the current limitations of recombinant gene delivery methods, there is a need to develop non-transgenic CRISPR / Cas nuclease delivery methods. Plant viral vectors offer another promising method for CRISPR / Cas nuclease delivery. Viral vectors with systemic infection capabilities can spread from inoculated cells or tissues to other uninoculated tissues and cells, achieving systemic delivery of foreign genes at the plant level. Viral vectors without systemic infection capabilities cannot spread from the initially infected cell to other cells and therefore cannot achieve systemic delivery. Viral vectors with systemic infection capabilities, especially infectious RNA viral vectors, have special value in non-transgenic nuclease delivery applications because their replication process does not involve the DNA stage, eliminating the possibility of foreign nucleic acids integrating into the host genome. However, the molecular weight of the CRISPR / Cas nuclease gene is relatively large, reaching 4.2 thousand base pairs (kb), which could potentially reach 5-7 kb with the addition of promoter elements, posing a significant challenge to the packaging capacity of current viral vectors. If the length of the foreign gene exceeds the limiting loading capacity of an infectious viral vector, the foreign gene fragment may be lost, deleted, or the virus may lose its ability to infect the whole body (Avesani et al., Transgenic Res, 2007, 16:587-597).

[0006] Due to limited packaging capacity, some infectious plant virus vectors are only used to deliver relatively small nuclease components, such as CRISPR guide RNAs (gRNAs) of approximately 100–200 base pairs (bp) in length. Such vectors have been used to deliver a wide range of plant DNA viruses, including cabbage leaf curl virus (Yin et al., Sci Rep, 2015, 5:14926), tobacco rattle virus (Ali et al., Mol Plant, 2015, 8:1288–1291; WO 2015189693), tobacco mosaic virus (Cody et al., Plant Physiol, 2017, 175:23–35), pea early browning virus (Ali et al., Virus Res, 2018, 244:333–337), and sugar beet root disease (Jiang et al., Plant Biotechnol, 2018, 244:333–337). J, 2019, 17:1302-1315). These viral vectors are required to achieve site-specific genome editing via Cas9 transgenic plants, so the genome of the edited plants obtained in this way still contains the Cas9 nucleic acid sequence inserted.

[0007] A paper (Mei et al., Plant Direct, 2019, 3:1-16) discloses a Muskgrass mosaic virus (M. mosaic virus), a positive-sense RNA virus, that, when carrying a 700-bp green fluorescent protein gene, completely deletes the foreign inserted fragment in infected upper leaves, leaving only a 100-200-bp CRISPR gRNA available for expression. Gene editing can be achieved by transfecting the Cas9 protein. Similarly, a paper (Zhang et al., Funct & Integr Genomics, 2020, 20:471-477) and Chinese Patent CN 111979262 A disclose a method for plant genome editing using this viral vector. Due to packaging capacity limitations, Cas9 and gRNA were inserted into two viral vectors, respectively, and low-frequency gene editing was achieved when plants were co-infected.

[0008] Potato virus X, another positive-sense RNA virus, has a tolerance for foreign fragments of less than 2 kb (Larsen et al., BMC Biotech, 2012, 12:21; Avesani et al., Transgenic Res, 2007, 16:587-597). A paper (Ariga et al., Plant Cell Physiol, 2020, 61:1946-1953) discloses a method for delivering CRISPR / Cas nuclease genes using this vector. This viral vector expresses intact CRISPR / Cas nuclease and can achieve gene editing in inoculated leaves, but cannot achieve systemic CRISPR / Cas delivery and gene editing in uninoculated tissues.

[0009] Viral replicon systems are vectors that retain elements required for viral replication but remove elements related to viral packaging and / or motility. These vectors can replicate efficiently but are unable to infect the system, allowing for the accommodation of fairly large (>10 kb) foreign insert fragments. A paper (Baltes et al., Plant Cell, 2014, pp. 151-163) discloses the use of the soybean dwarf virus replicon, a single-stranded DNA Geminiviridae, to deliver CRISPR / Cas nucleases. A paper (Wang et al., Mol Plant, 2017, 10:1007-1010) and Chinese Patent CN 109880846 A disclose the use of the wheat dwarf virus replicon vector system, a Geminiviridae, to deliver CRISPR / Cas nucleases and DNA repair templates. However, since viral replicon vectors are essentially deconstructed viruses, and DNA constructs containing viral vectors must be introduced into cells using techniques such as Agrobacterium-mediated or gene gun delivery, the possibility of foreign DNA integrating into recipient cells is unavoidable.

[0010] Given the deficiencies of currently available plant viral vectors, it is necessary to develop a plant viral vector system that is infectious and capable of delivering intact CRISPR / Cas nucleases throughout the body, particularly an infectious RNA viral vector system that does not involve the DNA stage in the replication process.

[0011] Rhabdoviridae viruses belong to the non-segmented, single-stranded, negative-sense RNA virus family. A paper (Wang et al., PLoS Pathog, 2015, 11:e 1005223) and patent CN 105039388 B disclose a genetic manipulation method for the plant rhabdovirus, Sonchus oleracea yellow net virus (SYNV). Patent CN 104962580 B discloses a SYNV expression vector capable of carrying green fluorescent protein (approximately 700 bp) and β-galactosidase (1.8 Kb)-binding genes and a method for constructing the vector. However, systemic delivery of larger exogenous gene fragments (e.g., Cas nuclease) and non-transgenic site-specific genome editing using the viral vector have not been achieved.

[0012] A paper (Gao et al., New Phytol. 2019, 223:2120-2133) and patent CN 110511955 A disclose an infectious clone and expression vector of the plant rhabdovirus Barley yellow dwarf virus (BYSMV), as well as methods for constructing and using the vector. When a fluorescent protein gene and a β-galactosidase gene are inserted, the vector can achieve systemic delivery and expression of foreign genes in barley plant hosts. However, when carrying a larger CRISPR / Cas nuclease gene (approximately 5 kb), the vector can only be introduced into N. benthamiana via Agrobacterium T-DNA transformation. Although the CRISPR / Cas nuclease is expressed in single cells and produces targeted gene editing, the recombinant virus cannot move within N. benthamiana or systemically infect planthoppers, which serve as hosts and vectors for wheat plants (Gao et al., New Phytol, 2019, 223: 2120-2133; see page 12 of the original text, "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 described above, conventional techniques have not been able to achieve non-transgenic systemic delivery of CRISPR / Cas nucleases. Summary of the Invention [Problem to be solved by the invention]

[0013] In order to overcome the technical difficulties of using current plant virus vectors to carry out non-transgenic genome site-specific modification in plants, the present invention uses plant rhabdovirus to deliver sequence-specific nuclease to plants.This sequence-specific nuclease can target specific nucleic acid sequence in plant genome, cut target site, and complete the site-specific modification of this target site through plant endogenous DNA repair mechanism.In addition, this plant rhabdovirus has systemic infection ability, and with the systemic infection of virus, it can realize the systemic delivery of sequence-specific nuclease, and can obtain site-specific editing plant parts that do not contain foreign nucleic acid sequence integration, and after regenerating site-specific editing plant parts (such as plant cells) that do not contain foreign sequence integration, can obtain non-transgenic, stable and inheritable site-specific editing plants. [Means for solving the problem]

[0014] The first aspect of the present invention provides a method for modifying plant cell genetic material, which does not need to introduce foreign gene sequence into the plant cell genome to be modified.The method comprises the following steps: a) providing at least one plant cell to be modified genetic material; and b) using a recombinant plant rhabdovirus vector with systemic infection ability to infect the plant cell, wherein the recombinant rhabdovirus with systemic infection ability carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, and the sequence-specific nuclease specifically targets the plant genome nucleic acid sequence and cuts the target site. The process of modifying the cellular genetic material is induced by cleavage by sequence-specific nucleases and completed by the plant's endogenous DNA repair machinery. Preferably, the method further comprises, after step b), a step c) of selecting modified plant cell subjects containing the genetic material without the need for the use of a selectable marker.

[0015] The second aspect of the present invention provides a method for producing a plant whose genetic material is modified, and does not need to introduce a foreign gene sequence into the plant cell genome to be modified.The method comprises the following steps: a) providing at least one plant cell to be modified with genetic material; b) infecting the plant cell with a recombinant plant rhabdovirus vector capable of systemic infection, wherein the recombinant rhabdovirus capable of systemic infection carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, and the sequence-specific nuclease specifically targets the plant genome nucleic acid sequence and cleaves the target site; c) obtaining a plant from the cell whose genetic material is modified; and d) selecting the modified plant subject comprising the genetic material without the need to use a selection marker.

[0016] Preferably, the method of infecting the cells to be modified includes natural infection of uninoculated cells with the recombinant virus, trituration inoculation, grafting, insect-mediated transmission, or any other method of infection with a virus.

[0017] Preferably, the rhabdovirus vector capable of systemic infection is sonchus yellow net virus (SYNV) or eggplant mottled dwarf virus (EMDV).

[0018] Preferably, the Sonchus sonchii yellow net virus genome nucleic acid sequence is shown in SEQ ID NO:38, and the Eggplant spotted dwarf virus genome nucleic acid sequence is shown in SEQ ID NO:39.

[0019] Preferably, the rhabdovirus vector capable of systemic infection comprises a derived viral vector after one or more types of mutation and / or recombination modification, and preferably, the derived viral vector is a derived viral vector after mutation and / or recombination modification of the glycoprotein (G) amino-terminal domain. 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 cellular genetic material modification comprises at least one base deletion, one base insertion, 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 any nuclease capable of 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 comprises a nucleic acid sequence for one or more guide RNAs and a nucleic acid sequence for a Cas nuclease. Preferably, the guide RNA comprises a sequence that pairs with a target gene and a sequence that binds to a Cas nuclease to form a complex. Preferably, the nucleic acid sequence encoding the sequence-specific nuclease is present in the genome of the rhabdovirus in the form of an independent transcription unit. Preferably, transcription of said sequence-specific nuclease sequence is controlled by regulatory elements for 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 the Cas nuclease sequence are controlled by different transcription units. Preferably, the guide RNA transcript comprises terminal sequences derived from a virus. Preferably, the guide RNA transcript is processed to remove viral-derived terminal sequences. Preferably, the guide RNA transcript has viral-derived terminal sequences removed by the cell's endogenous tRNA processing machinery. The guide RNA transcript is processed by Cpf1 nuclease to remove viral-derived terminal sequences.

[0024] Preferably, the plant is a natural or experimental host for a plant rhabdovirus, preferably a host for Sonchus yellow net virus or Eggplant spotted 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 plant is selected from the group consisting of eggplant (Solanum melongena), cucumber (Cucumis sativus), hydrangea (Hydrangea macrophylla), melon (Cucumis melo), Nicotiana clevelandii, Nicotiana glutinosa, Nicotiana rustica, Nicotiana edwardsonii, capsicum annuum, red seaweed (Chenopodium amaranticolor), quinoa (Chenopodium quinoa), Datura metel, Hibiscus rosa-sinensis, honeysuckle (Lonicera), night jasmine (Solanum nigrum), spring sowweed (Sonchus oleraceus), lettuce (Lactuca sativa), a hybrid variety of Nicotiana glutinosa and Nicotiana liebrand, 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 endonuclease is transiently expressed in cells infected with the viral vector and specifically targets a plant genomic nucleic acid sequence, and the target sequence is modified by the nuclease.

[0027] The recombinant rhabdovirus has the ability to infect the whole body, 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, wherein the sequence comprising the viral vector is operably linked to a promoter.

[0029] Preferably, the Sonchus sonchii yellow net virus genome nucleic acid sequence is shown in SEQ ID NO:38, and the Eggplant spotted dwarf virus genome nucleic acid sequence is shown in SEQ ID NO:39.

[0030] Preferably, the rhabdovirus vector capable of systemic infection comprises a derived viral vector after one or more types of mutation and / or recombination modification, and preferably, the derived viral vector is a derived viral vector after mutation and / or recombination modification of the glycoprotein (G) amino-terminal domain. 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 N-terminal domain is replaced with a heterologous nucleic acid sequence.

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

[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 SpCas9 derived from Streptococcus pyogenes or LbCpf1 nuclease derived from Lachnospira; preferably, the nucleic acid sequence of the SpCas9 nuclease is set forth in SEQ ID NO: 18, and the nucleic acid sequence of the LbCpf1 nuclease is set forth in SEQ ID NO: 19.

[0034] Preferably, the ribonucleic acid sequence encoding the sequence-specific endonuclease comprises a nucleic acid sequence for one or more guide RNAs and a nucleic acid sequence for a Cas nuclease. Preferably, the guide RNA comprises a sequence that pairs with a target gene and a sequence that binds to a Cas nuclease to form a complex. Preferably, the nucleic acid sequence encoding the sequence-specific nuclease is present in the genome of the rhabdovirus in the form of an independent transcription unit. Preferably, transcription of said sequence-specific nuclease sequence is controlled by regulatory elements for rhabdovirus messenger RNA transcription. Preferably, the guide RNA and the Cas nuclease sequence are controlled by different transcription units. Preferably, the guide RNA and the Cas nuclease sequence are controlled by the same transcription unit. Preferably, the guide RNA transcript comprises terminal sequences derived from a virus. Preferably, the guide RNA transcript is processed to remove viral-derived terminal sequences. Preferably, the guide RNA transcript has viral-derived terminal sequences removed by the cell's endogenous tRNA processing machinery. The guide RNA transcript is processed by Cas nuclease to remove viral-derived terminal sequences.

[0035] Preferably, the plant is a natural or experimental host for a plant rhabdovirus, preferably a host for Sonchus yellow net virus or Eggplant spotted 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 plant is selected from the group consisting of eggplant (Solanum melongena), cucumber (Cucumis sativus), hydrangea (Hydrangea macrophylla), melon (Cucumis melo), Nicotiana clevelandii, Nicotiana glutinosa, Nicotiana rustica, Nicotiana edwardsonii, capsicum annuum, red seaweed (Chenopodium amaranticolor), quinoa (Chenopodium quinoa), Datura metel, Hibiscus rosa-sinensis, honeysuckle (Lonicera), night jasmine (Solanum nigrum), spring sowweed (Sonchus oleraceus), lettuce (Lactuca sativa), a hybrid variety of Nicotiana glutinosa and Nicotiana liebrand, Zinnia elegans, Bidens pilosa, and quinoa (Chenopodium quinoa).

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

[0038] An eighth aspect of the present invention provides the use of a method or a recombinant rhabdovirus vector described herein, or a recombinant rhabdovirus vector, or a strain or recombinant virus described herein in plant genome editing. Invention Description

[0039] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in practicing the same, and unless expressly specified or limited, terms should be understood in accordance with conventional usage by those of ordinary skill in the relevant art.

[0040] As used herein, the term "plant" includes the entire plant and any offspring, as well as 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 related explants), plant seeds (e.g., mature seeds, immature seeds, and immature embryos without seed coats), and the like. Plant cells may be in the form of isolated single cells or cell aggregates (e.g., callus and cultured cells), protoplasts, gamete-producing cells, or cells or collections of cells that can regenerate into whole plants. Plant cell cultures or tissue cultures can regenerate plants having physiological or morphological characteristics of the plant from which the cells or tissues are derived, and can regenerate plants having substantially the same genotype as the plant. The regenerable cells in the plant cell culture or tissue culture may be embryos, protoplasts, blastocysts, callus, pollen, leaves, stamen anthers, roots, root tips, threads, flowers, kernels, panicles, cobs, husks, or stems.

[0041] The term "rhabdovirus" refers to a member of the Rhabdoviridae family of negative-sense RNA viruses. "Negative-sense RNA viruses" are also called "negative-strand RNA viruses." These viruses infect humans, animals, plants, and fungi, and have a negative-sense viral genome (opposite to the polarity of the mRNA) packaged in the viral particle. These viruses include the Mononegavirales, which have non-segmented genomes, such as Paramyxoviridae, Filoviridae, Nyamiviridae, Bornaviridae, and Rhabdoviridae. The families Bunyavirales, which have segmented genomes, include the families Bunyaviridae, Feraviridae, Fimoviridae, Hantaviridae, Jonviridae, Naiviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae, and Tomato spotted wilt virus (Tospoviridae). The families Arenaviridae, Aspiviridae, and Orthomyxoviridae, which have segmented genomes and are not classified for this purpose, include the families Arenaviridae, Aspiviridae, and Orthomyxoviridae. Here, rhabdovirus has typical rod-shaped or bullet-shaped virus particles and includes Rhabdoviridae that infect animals and Rhabdoviridae that infect plants, and Rhabdoviridae that infect animals include, but are not limited to, Lyssavirus, Vesiculovirus, Ephemerovirus, Novirhabdivirus, etc.There are four main genera of plant-infecting rhabdoviruses: 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 virus (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), barley yellow striate mosaic virus (BYSMV), and the like.Members of the genus Varicosavirus include, but are not limited to, Alopecurus myosuroides varicosavirus (AMVV), Lettuce big vein associated virus (LBVaV), Red clover varicosavirus (RCVV), etc. Members of the genus Bisecting Rhabdovirus include, but are not limited to, Orchid fleck virus (OFV), Cirus leprosis virus (CiLV), Citrus chlorotic spot virus (CCSV), Coffee ringspot virus (CRV), Clerodendrum chlorotic spot virus (CLCSV), etc.

[0042] The genome structure of Rhabdoviridae viruses is conserved, encoding five conserved structural proteins, 3'-NPMGL-5', from the 3' end to the 5' end: nucleocapsid protein N, phosphoprotein P, matrix protein M, glycoprotein G, and RNA-dependent RNA polymerase large subunit L. The ribonucleoprotein core complex (RNP), the minimal infectious unit of rhabdoviruses, is composed of genomic RNA wrapped in viral core proteins. These core proteins are typically nucleocapsid proteins N and L, although some viral core proteins also contain other types of proteins, such as the core protein of sonchus yellow net virus (SYNV), which contains phosphoprotein P. In addition to encoding the five conserved structural proteins mentioned above, some rhabdoviruses encode additional nonstructural proteins; for example, SYNV encodes an additional nonstructural protein, sc4. Rhabdovirus genomes contain 3'-leader and 5'-trailer sequences at both ends. Although both leader and trailer sequences are non-protein-coding, they play important regulatory roles in genome replication and mRNA transcription. Each gene in rhabdoviruses contains a conserved gene spacer sequence; for example, the spacer between the N and P genes is the N / P junction (NPJ) sequence. The gene spacer sequence functions to terminate transcription of the upstream gene and initiate transcription of the downstream gene. It consists of three parts: an upstream mRNA transcription termination signal and polyadenylation template element; a non-transcribed sequence (usually containing consecutive uracil nucleotides, U) during mRNA synthesis; and a downstream mRNA transcription initiation element.

[0043] The transcription of rhabdovirus has the characteristics of sequential transcription and polar transcription. Sequential transcription means that in the process of viral transcription, each viral gene is transcribed sequentially according to its relative position on the genome; polar transcription means that the amount of sequentially transcribed mRNA produced gradually decreases according to its relative position on the genome. The transcription process of rhabdovirus is carried out under the action of viral polymerase, which first transcribes leader mRNA from the 3' end of the viral genome, and then sequentially transcribes each mRNA encoding viral protein. The transcription process is controlled by gene spacer sequence. When viral polymerase moves to the gene spacer between each gene, it ends the transcription of the gene located upstream of the gene spacer, and the polymerase complex 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 time, the polymerase complex continues to move downstream until it encounters the transcription start signal of the next gene, and starts the transcription of the next gene, and finally transcribes into trailer mRNA. Therefore, when using such a virus to modify a vector, it is necessary to follow the transcription characteristics of that virus. At the same time as adding the foreign nucleic acid sequence, it is necessary to add a transcription control element, i.e., the above-mentioned gene spacer, so that the foreign nucleic acid sequence becomes an independent transcription unit and can be transcribed and expressed under the action of the viral polymerase.

[0044] The term "vector" refers to a self-replicating DNA molecule used in genetic engineering to transfer foreign nucleic acid fragments into recipient cells. It generally contains a foreign coding sequence and appropriate nucleic acid sequences required for expressing the coding sequence in a specific host. A plasmid is an exemplary vector. The terms "recombinant viral vector," "recombinant viral expression vector," and "recombinant viral clone" are used interchangeably herein and refer to constructs formed by delivering specific nucleic acid sequences mediated by a viral vector to target cells, tissues, organs, or plants. The terms "infectious viral vector" and "viral infectious clone" refer to constructs formed by delivering specific nucleic acid sequences mediated by a viral vector to target cells, tissues, organs, or plants, and capable of rescuing active recombinant viruses and achieving systemic infection.

[0045] The terms "foreign" and "heterologous" refer to sequences from a foreign species, or, if from the same species, sequences that have undergone significant changes in composition and / or gene loci from their native form due to deliberate human intervention. The term "genetic material" refers to material that transmits genetic information between parental and progeny generations. "Modified genetic material" includes, but is not limited to, single or multiple deoxyribonucleotide substitutions, deletions, additions, or combinations of the above. The terms "nucleic acid," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably 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 analogs of DNA or RNA 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 of structural genes, antisense sequences, and non-coding regulatory sequences that do not encode mRNA or protein products. The terms "gene" and "gene sequence" refer to DNA nucleic acids associated with biological functions. Thus, a gene can include introns and exons in a genomic sequence, or can include only the coding sequence in a cDNA, and / or can include cDNA in combination with regulatory sequences.

[0046] The term "sequence-specific nucleases" (SSss) refers to endonucleases that can cleave specific DNA sequences and create DNA double-strand breaks (DSBs). 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 (CRISPR) and CRISPR-associated proteins (CRISPR / Cas). "Zinc finger nuclease" refers to an artificial restriction enzyme created by combining a zinc finger DNA-binding domain with a DNA cleavage domain. ZFNs contain synthetic proteins with engineered zinc finger DNA-binding domains combined with the cleavage element of the FokI restriction endonuclease. These can be used to achieve locus-specific targeted manipulation of genomic sequences by inducing double-strand breaks at specific DNA sequences. "Transcription activator-like effector nucleases" refer to artificial restriction enzymes created by combining a Xanthomonas-inducible DNA recognition element with the catalytic element of a nuclease. Specifically, the TALEN system primarily consists of a DNA-binding domain and a FokI endonuclease domain containing a TALE protein. The TALE protein contains multiple overlapping peptide fragments of 33–35 amino acids, each capable of recognizing a single base. To function, TALENs must form dimers that recognize the 5' to 3' and 3' to 5' DNA strands, respectively, and cleave the target sequence using the bound FokI endonuclease.The terms "clustered regularly interspaced short palindromic repeats and CRISPR-associated system," "CRISPR nuclease," "CRISPR / Cas nuclease," and "Cas nuclease" are used interchangeably and generally refer to nucleases present in CRISPR systems, as well as mutants thereof (e.g., nicking mutants, inactivation mutants, etc.) and derivatives thereof that bind to other functional proteins. The terms encompass any nuclease capable of achieving genome editing in a cell in a CRISPR system.

[0047] In the present invention, the terms "guide RNA," "gRNA," and "sgRNA" are used interchangeably and generally refer to an RNA molecule that can bind to a CRISPR nuclease to form a complex and guide the complex to a target site. In a Cas9-based genome editing system, the gRNA consists of a crRNA and a tracrRNA molecule, of which the crRNA contains a sequence complementary to the target sequence and can guide the CRISPR complex to specifically bind to the target sequence. Alternatively, as is well known in the art, a single guide RNA can be designed by combining the crRNA and tracrRNA. In contrast, in a CpF1-based genome editing system, the gRNA generally refers to only the 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 to be acted upon, including, but not limited to, coding or non-coding sequences, genes, exons, introns, regulatory sequences, gene spacer sequences, synthetic sequences, and intracellular parasite sequences.

[0049] In the present invention, the term "delivery" refers to a means of delivering a nucleic acid sequence and / or an amino acid sequence to a target cell, tissue, organ, or plant, and delivery methods include, but are not limited to, gene gun techniques, protoplast transformation, Agrobacterium inoculation, mechanical friction, vacuum infiltration, high-pressure spray gun injection, chemical delivery, etc.

[0050] In the present invention, the term "tRNA" refers to a small RNA molecule that has the function of carrying and transporting amino acids and can fold into a cloverleaf-shaped secondary structure. In eukaryotic organisms, tRNA precursors produced by tRNA gene transcription are recognized by endogenous RNase P and RNase Z and processed into mature tRNAs. In some embodiments of the present invention, the tRNA sequence used is the tRNA sequence described in "Methods and Compositions for Multiple-Directed Genome Editing and Other RNA Technologies" (Patent Publication No. WO 2016 / 061481). Gly The coding sequence is selected from:

[0051] In the present invention, the terms "tissue culture" and "tissue culture" are used interchangeably and refer to a technique for isolating desired cells, tissues, organs, protoplasts, etc. from a plant body and culturing them under artificially controlled conditions using sterile procedures to obtain intact plants or to produce other products of economic value.

[0052] In the present 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 used interchangeably and refer to a technique for modifying a specific target gene in the genome of an organism. Generally, a locus-specific double-strand break is first generated at a specific location in the genome, and the organism is then induced to repair the double-strand break via non-homologous end joining or homologous recombination, thereby generating the target modification. Also, as used herein, the singular forms "a," "an," and "said" include plural referents unless the specific context dictates otherwise.

[0053] The present invention provides a method for modifying plant cell genetic material, which does not need to introduce foreign gene sequence into the plant cell genome to be modified.The method comprises the following steps: a) providing at least one plant cell to be modified genetic material; and b) infecting the plant cell with a recombinant plant rhabdovirus vector capable of systemic infection, wherein the recombinant rhabdovirus capable of systemic infection carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, and the sequence-specific nuclease specifically targets the plant genome nucleic acid sequence and cleaves the target site. The process of modifying the cellular genetic material is induced by cleavage by sequence-specific nucleases and completed by the plant's endogenous DNA repair machinery. Preferably, the method further comprises, after step b), a step c) of selecting modified plant cell subjects containing the genetic material without the need for the use of a selectable marker.

[0054] The present invention also provides a method for producing a plant whose genetic material is modified, and does not require the introduction of a foreign gene sequence into the plant cell genome to be modified.The method comprises the following steps: a) providing at least one plant cell to be modified with genetic material; b) infecting the plant cell with a recombinant plant rhabdovirus vector capable of systemic infection, wherein the recombinant rhabdovirus capable of systemic infection carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, and the sequence-specific endonuclease specifically targets the plant genome nucleic acid sequence and cleaves the target site; and c) selecting the modified plant cell target containing the genetic material without the need for the use of a selection marker.

[0055] The present invention also provides a method for producing plant callus whose genetic material is modified, which does not require the introduction of a foreign gene sequence into the plant cell genome to be modified. The method includes the following steps: a) providing at least one plant cell to be modified with genetic material; b) infecting the plant cell with a recombinant plant rhabdovirus vector capable of systemic infection, wherein the recombinant rhabdovirus capable of systemic infection carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, and the sequence-specific nuclease specifically targets the plant genome nucleic acid sequence and cleaves the target site; and c) selecting the modified callus target containing the genetic material without the need for a selection marker.

[0056] Methods for infecting the cells to be modified include natural infection of uninoculated cells with the recombinant virus, trituration inoculation, grafting, insect-mediated propagation, or any other infection method not mediated by a viral DNA construct.

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

[0058] The present invention further provides an infectious recombinant plant rhabdovirus vector that is characterized in that: a) is capable of systemically infecting a certain type of plant host; and b) carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, wherein the sequence-specific nuclease specifically targets a plant genomic nucleic acid sequence and cleaves the target site.

[0059] The recombinant rhabdovirus has systemic infection capability, and preferably, the rhabdovirus is a sowbuckthorn yellow net virus. The nucleic acid sequence of the sowbuckthorn yellow net virus genome used in the present invention is set forth in SEQ ID NO: 38. The above sequence is the nucleic acid sequence of the virus isolated from sowbuckthorn. In addition to sowbuckthorn, the virus can infect multiple types of plants, such as lettuce, lettuce, quinoa, and tobacco. The nucleic acid sequences of virus isolates from other hosts are also applicable to the method described in the present invention. The total length of the viral genome is 13.7 kb, and the coding order of each gene is 3'-NP-sc4-MGL-5'. According to the rhabdovirus transcription method, the heterologous nucleic acid sequence 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), after the leader region (between the 3'-leader and N protein), or before the trailer region (between the L protein and the 5'-trailer). In a further embodiment of the present invention, the heterologous nucleic acid sequence is inserted between the 3'-leader and the N protein. In some embodiments of the present invention, the heterologous nucleic acid sequence is inserted between the N and P regions. Insertion of the heterologous nucleic acid sequence must comply with the virus's own coding sequence. An additional spacer should be added simultaneously with the addition of the heterologous nucleic acid sequence to form an independent transcription unit. Each SYNV gene spacer is between 100 and 200 nt in length and contains approximately 16 conserved bases (5'-TATAAGAAAAACCAAC-3'). When additional spacers are added, these conserved sequences generally must be included, but the remaining sequences can be mutated, truncated, or elongated. In a preferred embodiment of the present invention, the complete gene spacer should be added simultaneously with the addition of the heterologous nucleic acid sequence.

[0060] Preferably, the recombinant rhabdovirus is eggplant spotted dwarf virus, and the eggplant spotted dwarf virus genome nucleic acid sequence used in the embodiments of the present invention is set forth in SEQ ID NO: 39. The nucleic acid sequence is that of an eggplant isolate of the virus. In addition to eggplant, the virus can infect many plants, including cucumber, hydrangea, and tomato. Nucleic acid sequences of virus isolates from other hosts are also applicable to the methods described herein. The total length of the viral genome is approximately 13.2 kb, and the coding order of each gene is 3'-NXPYMGL-5'. Similar to the common sowberry yellow net virus SYNV, a heterologous nucleic acid sequence 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), after the leader region (between the 3'-leader and N protein), or before the trailer region (between the L protein and the 5'-trailer). The insertion of a heterologous nucleic acid sequence must also comply with the viral coding policy, and an additional spacer must be added before normal transcription and expression can occur. Each EMDV gene spacer is between 47 and 217 nt in length and contains a conserved sequence of approximately 17 bases (5'-TTTAATAAAAACCCAAC-3'). When adding an additional transcription unit, this conserved sequence generally needs to be retained, but the remaining sequence can be mutated, truncated, or elongated. In some embodiments of the present invention, the insertion position of the heterologous nucleic acid sequence is between Leader and N. In further embodiments of the present invention, the insertion position of the heterologous nucleic acid sequence is between N and X.

[0061] The rhabdovirus vector capable of systemic infection further includes a derivative viral vector after one or more 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 encoded by negative-sense RNA viruses has a conserved structure, consisting of a signal peptide, an N-terminal domain, a transmembrane domain, and a C-terminal domain, and is mosaic-likely formed on the surface of the virus particle in a trimer form. Existing research has shown that the G protein plays an important role in the process of virus-vector discrimination and transmission. For example, a nonsynonymous mutation (C1375A) in the G protein of Tomato spotted wilt virus does not affect systemic viral infection. However, since it cannot be transmitted by the vector, the oriental flower thrips, partial deletion, mutation, or substitution of the G protein of the recombinant rhabdovirus vector can affect the insect-vector transmission of the virus and improve biosafety. In some embodiments of the present invention, by deleting or substituting the N-terminal domain of the G protein of Sonchus oleracea virus, a recombinant viral vector capable of systemic infection can be obtained without affecting site-specific editing in plants.

[0062] The sequence-specific endonuclease is CRISPR / Cas nuclease, TALEN nuclease, zinc finger nuclease, or any nuclease that can achieve genome editing, and preferably, the sequence-specific endonuclease is CRISPR / Cas nuclease.Specifically, the nuclease is any nuclease that can achieve genome editing, such as Cas9 (for example, SpCas9, SaCas9), Cpf1 (also known as Cas12a, such as AsCpf1, LbCpf1, FnCpf1), CasX or CasY, and some amino acid mutations, variants or derivatives based on the CRISPR nuclease, such as high-fidelity Cas nuclease espCast9, Cas9 nick enzyme nCas9, Cas9 inactivation enzyme dCas9, base editor ABE, CBE, PBE, etc.

[0063] Preferably, the nuclease is SpCas9 from Streptococcus pyogenes or LbCpF1 from Lachnospira that comprises the nucleic acid sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 19, or has 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) to the nucleotide sequence. "Identity" refers to the degree of consistency of polynucleotide or protein segments in matching sequences (e.g., nucleotide sequences or amino acid sequences). Sequence matching is generated by manually comparing two sequences, for example, a reference sequence as provided herein and another sequence, to generate the maximum number of matching elements, such as individual nucleotides or amino acids, while allowing for the introduction of nicks into any sequence; the "identity score" of a sequence matched to a reference sequence is the number of matching elements divided by the total length of the reference sequence, not including nicks introduced into the reference sequence by the matching process. As used herein, "percent identity" is the identity score multiplied by 100.

[0064] Those skilled in the art will recognize that examples of codon optimization for specific host species are feasible and known, and that sequences encoding Cas nucleases can be codon-optimized for specific species to achieve more efficient Cas nuclease expression. Codon optimization refers to the process of replacing at least one codon, e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, of a native sequence with a codon that is more or most frequently used in the host cell's genes, while maintaining the native amino acid sequence and modifying the nucleic acid sequence to enhance expression in the host cell of interest. In some embodiments of the present invention, an example of a Cas9 nuclease sequence that is codon-optimized for humans is provided, and in accordance with further embodiments of the present invention, an example of a Cas9 nuclease sequence that is codon-optimized for Nicotiana benthamiana is provided.

[0065] The sequence-specific nuclease can further comprise one or more subcellular localization domains, including, but not limited to, a nuclear localization signal, a mitochondrial targeting signal, or a chloroplast targeting signal. In some embodiments of the present invention, the subcellular localization domain is a nuclear localization sequence (NLS). Generally, an NLS consists of one or more short sequences of positively charged lysines or arginines exposed on the surface of a protein, although other types of NLSs are also known. The NLS sequences can 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 can be attached to the N-terminus, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs can be attached to the C-terminus, or a combination of the above-mentioned attachment methods can be used. In some preferred embodiments of the invention, the NLS is an SV40 nuclear localization signal simultaneously attached to the N-terminus and C-terminus of the nuclease.

[0066] Said rhabdovirus vector carries the nucleic acid sequence that encodes at least one sequence-specific nuclease, and when said sequence-specific nuclease is Cas nuclease, the nucleic acid sequence that said rhabdovirus vector carries is the nucleic acid sequence that encodes gRNA (or both crRNA and tracrRNA) and Cas nuclease.Described gRNA comprises the nucleotide sequence Nx that can specifically hybridize with the complementary sequence of target sequence and the sequence that binds with Cas nuclease to form complex, wherein Nx represents the nucleotide sequence that consists of X consecutive nucleotides, and N each independently selects A, G, T, C, wherein X is an integer that 18≦X≦35, and 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 is composed of a Pol II promoter, Cas9 ORF, and terminator, in that order, while the gRNA transcription unit is composed of a Pol III promoter (often using small RNA transcription promoters such as U6 or U3), gRNA unit, and PolyT terminator, in that order. The Cas9 protein expression unit and gRNA transcription unit can be constructed on two independent vectors and then cotransformed, or they can be constructed on a single vector as two independent units and then transformed, or they can be constructed on a single vector and then transformed, with some linkage sequences between them. Here, the expression of Cas9 protein can be selected according to the characteristics of the transformation target and experimental requirements, such as CMV, Hsp70, SV40 promoters commonly used in animal cells, and CaMV35S, ZmUb1, AtUb10 promoters commonly used in plants. Generally, the gRNA transcription unit uses a specific small RNA transcription promoter such as U6 or U3 in the target genome according to the transformation target. However, in the case of rhabdovirus vectors, due to the transcription characteristics of rhabdovirus, it is only necessary to add a gene spacer to the additional expression unit, which is very simple and convenient to operate, and the guide RNA and the Cas nuclease sequence can be controlled by the same transcription unit or by different transcription units. In another embodiment of the present invention, the nucleic acid sequence encoding Cas nuclease and the nucleic acid sequence encoding guide RNA can be controlled by the same transcription unit, and they are connected by a known linker 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 the binding sequence, a nucleic acid sequence encoding a Cas nuclease and a nucleic acid sequence encoding a guide RNA can be arranged in the same transcription unit in various ways.Examples include, but are not limited to, RNA processing elements such as ribozymes (hammerhead ribozymes, hairpin ribozymes, hepatitis D ribozymes, VS ribozymes, I-class introns, RNase P, etc.), tRNA sequences, and Csyh4 sequences. In a further embodiment of the present invention, the nucleic acid sequence encoding the guide RNA and the nucleic acid sequence encoding the Cas nuclease are each linked to an additional gene spacer so as to form independent transcription units.

[0068] When the heterologous nucleic acid sequence encoding guide RNA is expressed as an independent transcription unit, the transcription product contains extra sequences derived from the viral vector according to the transcription characteristics of the rhabdovirus itself. In some embodiments of the present invention, the recombinant rhabdovirus vector (SYNV-gGFP2-Cas9) carrying the nucleic acid sequence of the above-mentioned guide RNA and the nucleic acid sequence of Cas9 nuclease has an editing efficiency of about 20% for the target site. In another embodiment of the present invention, in order to obtain a relatively accurate guide RNA transcription product, the heterologous nucleic acid sequence encoding guide RNA is flanked by tRNAs. Gly Add precursor sequence, this precursor sequence can be recognized by plant endogenous tRNA processing machinery, and can cut and process more precise guide RNA transcript.The recombinant rhabdovirus vector (SYNV-tgtGFP2-Cas9) carrying the nucleic acid sequence of above-mentioned guide RNA and the nucleic acid sequence of Cas9 nuclease has an editing efficiency of about 90% for target site.Therefore, recombinant virus vector with RNA processing element is the preferred vector of the present invention, and common RNA processing element includes but is not limited to ribozyme (hammerhead ribozyme, hairpin ribozyme, hepatitis D ribozyme, VS ribozyme, I class intron, RNase P etc.), tRNA sequence, Csyh4 sequence etc.

[0069] Said guide RNA can target a single gene or multiple genes, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In a further embodiment of the present invention, said guide RNA targets a single gene. Specifically, the targeted gene is the endogenous PDS gene of Nicotiana benthamiana, the selected target is PDS target 1, the sequence-specific nuclease used is CRISPR / Cas9 nuclease, the recombinant rhabdovirus used is SYNV, and the resulting recombinant virus expression vector is SYNV-tgtPDS1-Cas9, and the editing efficiency of this vector is around 79%. In a further embodiment of the present invention, said guide RNA simultaneously targets two genes. Specifically, a strategy of tandemly connecting multiple tRNA sequences and gRNA sequences (tRNA-gRNA1-tRNA-gRNA2-tRNA) is adopted, and when the tRNA sequences are recognized and cleaved by the plant's endogenous processing machinery, two guide RNA transcripts can be simultaneously released. Specifically, the genes are the endogenous genes RDR6 and SGS3 of N. 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 the allotetraploid N. benthamiana. 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 heterologous nucleic acid sequence comprising tRNA-RDR6 target1-gRNA scaffold-tRNA-SGS3 target1-gRNA scaffold-tRNA is as shown in SEQ ID NO: 1010. The resulting recombinant rhabdovirus vector is SYNV-tgtRDR6-1 / SGS3-1-Cas9, and the targeting efficiencies of the recombinant rhabdovirus vector for RDR6 and SGS3 are approximately 96% and 93%, respectively. In another specific embodiment of the present invention, the SYNV multi-target editing vector is used to achieve partial deletion of genomic chromosome fragments.Specifically, a tRNA sequence was used to tandemly connect two target sites of the endogenous PDS gene targeted to N. benthamiana, releasing two guide RNA transcripts that target PDS target 1 and target 3, respectively (each of which can simultaneously target two copies of the PDS targeted to N. benthamiana). Specifically, the target sequences were 5'-GCCGTTAATTTGAGAGTCCA-3' (SEQ ID NO: 21) and 5'-TTAACTGTATTGTCTAGCTCTGG-3' (SEQ ID NO: 24), respectively. The heterologous nucleic acid sequence comprising the tRNA-PDS target 1-gRNA scaffold-tRNA-PDS target 3-gRNA scaffold-tRNA was as shown in SEQ ID NO: 12. The resulting recombinant rhabdovirus vector was SYNV-tgtPDS1 / 3-Cas9, and the vector produced a partial deletion of a chromosomal fragment with an efficiency of approximately 48%.

[0070] Those skilled in the art will understand that the above embodiment is merely one preferred form of the multi-target editing method, and that according to the transcription method of rhabdovirus, multiple guide RNA transcription units can be expressed in other different ways, such as a method in which the heterologous nucleic acid sequences of multiple guide RNA transcription units are each linked to a gene spacer to form independent transcription units, or a method in which transcription units encoding multiple guide RNAs are linked and expressed according to the above-mentioned linking sequence, and these methods are feasible and known. For example, in a further embodiment of the present invention, multiple crRNA heterologous nucleic acid sequences can be tandemly connected to achieve multi-target editing of plant genomes. Specifically, the tandem arrangement of the crRNAs 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. The DR sequence can be recognized and cleaved by the Cpf1 crRNA processing machinery to release two crRNA transcripts. Specifically, the two target genes are the 16c endogenous genes GFP and PDS, and the two target sites are 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 allotetraploid 16c strain, respectively. More specifically, the target site sequences are 5'-AAGATACCCAGATCATATGAAGC-3' (SEQ ID NO:25) and 5'-TGCCGTTAATTTGAGAGTCCAAG-3' (SEQ ID NO:27), respectively. The heterologous nucleic acid sequence comprising DR-GFP1-DR-PDS1-DR is represented by SEQ ID NO:28. As shown in NO:17, the resulting recombinant rhabdovirus vector was pSYNV-crGFP-1 / PDS-1-Cpf1, and the targeting efficiencies of the vector for GFP and PDS were 81% and 82%, respectively.

[0071] The plant may be any plant that can serve as a host for rhabdoviruses, including most monocotyledonous and dicotyledonous plants, plants of the Graminea, Solanaceae, Fabaceae, Cucurbitaceae, Compositae, Amaranthaceae, Chenopodiaceae, and Caprifoliaceae families, such as tobacco (Nicotiana tabacum), Nicotiana benthamiana, potato (Solanum tuberosum), wheat (Triticum aestivum), soybean (Glycine max), corn (Zea mays), rice (Oryza sativa), coffee (Coffea spp.), tomato (Lycopersicon esculentum), lettuce (Lactuca sativa), and sowweed (Sonchus oleracea). 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, Capsicum annuum, Red seaweed (Chenopodium amaranticolor), Quinoa (Chenopodium quinoa), Datura metel, Hibiscus Examples of suitable plants include, but are not limited to, plants such as rosa-sinensis, honeysuckle (Lonicera), or night jasmine (Solanum nigrum). In some embodiments of the invention, the plant is Nicotiana benthamiana, tobacco, potato, tomato, eggplant, or the like.

[0072] The method of infecting plants with the rhabdovirus is by any method known in the art, including but not limited to gene gun method, protoplast transformation, Agrobacterium inoculation, mechanical friction, plant grafting, vacuum infiltration, high-pressure spray gun spraying, etc. In the preferred embodiment of the present invention, Agrobacterium infiltration inoculation method is used to achieve systemic infection of recombinant rhabdovirus.In another embodiment of the present invention, friction inoculation method is used to start infection of recombinant rhabdovirus.In another embodiment of the present invention, grafting method is used to achieve systemic infection of rhabdovirus.

[0073] The method for obtaining a non-transgenic, stably inheritable, directional-modified plant includes tissue culturing a part of the plant to be edited, and obtaining a plant capable of site-specific editing of the target site and stably inheriting the target site. For example, in one embodiment of the present invention, a method for obtaining a non-transgenic, edited plant is provided, using a rhabdovirus vector to encode a Cas9 nuclease. The recombinant rhabdovirus is Sonchus oleracea virus, the sequence-specific nuclease is CRISPR / Cas9 nuclease, the gene spacer is a spacer between N and P, the guide RNA heterologous nucleic acid sequence including the tRNA flanking sequence and the heterologous nucleic acid sequence for expressing Cas9 are each linked with an NPJ sequence to form independent transcription units and inserted between N and P, the target gene is the Nicotiana benthamiana endogenous PDS gene, the target site is 5'-GCCGTTAATTTGAGAGTCCA-3' (SEQ ID NO: 21), and the resulting recombinant rhabdovirus vector is pSYNV-tgtPDS1-Cas9. This vector can be infiltrated into Nicotiana benthamiana and then subjected to tissue culture of the systemically infected leaves to obtain virus-free non-transgenic edited plantlets in the M0 generation, and can stably transmit the mutation to offspring generations, thereby obtaining virus-free non-transgenic edited plantlets in both the M1 and M2 generations. In another embodiment of the present invention, a method for obtaining non-transgenic edited plantlets is provided, using a rhabdovirus vector to encode LbCpf1 nuclease. The gene spacers are N 5'UTR and NPJ, the heterologous nucleic acid sequence for transcribing the crRNA and the heterologous nucleic acid sequence for expressing LbCpf1 are linked by the gene spacers to form two independent transcription units, and both are inserted before the N gene transcription unit, the target gene is the Nicotiana benthamiana endogenous PDS gene, 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 was SYNV-crPDS1-Cpf1, and this vector could be used to obtain virus-free, non-transgenic, edited plantlets in the M0 generation after infiltration and inoculation into Nicotiana benthamiana and subsequent tissue culture of the systemically infected leaves, and the mutation could be stably transmitted to subsequent generations. The present invention further provides plants and offspring in which the genetic material produced by the methods or infectious recombinant viral vectors described herein has been modified. The present invention further provides infectious recombinant viral vectors in the methods or infectious recombinant viral vectors described herein. The present invention further provides strains or recombinant viruses in the methods or infectious recombinant viral vectors described herein.

[0074] The present invention further provides for the use of a recombinant rhabdovirus vector, recombinant rhabdovirus vector, strain, or recombinant virus described herein in plant genome editing. [Effects of the Invention]

[0075] This invention has the following advantageous effects compared to the prior art.

[0076] The recombinant rhabdovirus vector has a stronger foreign fragment accommodation capacity, which can simultaneously accommodate a heterologous nucleic acid sequence for transcribing a guide RNA and a heterologous nucleic acid sequence for expressing a Cas nuclease.

[0077] After the recombinant rhabdovirus vector is inoculated into a plant, a large amount of sequence-specific nuclease is produced along with viral replication, which can edit the target gene and achieve higher editing efficiency.

[0078] Because the production of guide RNA transcripts depends on the virus's own transcription policy, the first base of the target sequence is not limited to G or A, which are required for conventional U6 or U3 promoters. The recombinant rhabdovirus vectors can express multiple nucleases and therefore can distinguish between multiple PAM sequences, expanding their targeting range. The recombinant rhabdovirus vector can carry out multi-target editing by simultaneously adding multiple transcription units or by using tRNA processing strategies. The recombinant rhabdovirus vector has good stability, and the resulting recombinant rhabdovirus can be used to obtain edited plantlets by trituration. According to the present application, non-transgenic edited plants can be directly obtained in the M0 generation, and the edits can be stably inherited to progeny generations. [Brief explanation of the drawings]

[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. Bsu36I and NheI are single enzyme cleavage sites located on the N protein and P protein, respectively, and NPJ is the NP gene spacer (NP gene junction). (B) shows the fluorescence expression status after inoculation of N. benthamiana with the recombinant rhabdovirus vectors SYNV-GFP and SYNV-GFP-RFP. (C) shows the viral symptoms produced after inoculation of N. benthamiana with the recombinant rhabdovirus vectors SYNV-GFP and SYNV-GFP-RFP. (D) shows the expression status of SYNV structural proteins, GFP protein, and RFP protein detected by Western blot. [Figure 2]Figure 2 shows the construction of SYNV-based CRISPR / Cas9 gene editing vectors. (A) Schematic diagram of SYNV-Cas9, SYNV-gRNA-Cas9, and SYNV-tgtRNA-Cas9 clones. Bsu36I, NheI, and AhdI are single enzyme cleavage sites located on the N protein, P protein, and Cas9 protein, respectively. NPJ is the 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 between the NPJ and the NheI locus on the P gene. gRNA is the 20-nt target sequence and gRNA backbone sequence. NPJ-AhdI is the locus from the NPJ region to the AhdI locus on the Cas9 gene. tgtRNA is the pre-tRNA sequence attached to both ends of the gRNA. The above fragments are used in the clone construction process. (B) Schematic diagram of gRNA release. Due to the characteristics of SYNV replication and transcription, the gRNA transcript contains virus-derived non-coding sequences (5'UTR, 3'UTR sequences, and polyA tail) at both ends. (C) A schematic diagram of the tRNA processing policy used to obtain accurate gRNA transcripts shows that the tRNA sequences attached to both ends of the gRNA are processed by plant endogenous RNase P and RNase Z, which removes the virus-derived non-coding sequences and releases accurate gRNA transcripts. [Figure 3]Figure 3 shows the infectivity of the SYNV CRISPR / Cas9 editing vector targeting GFP transgenic plantlets. (A) Schematic diagram of the symptoms observed after inoculation of 16c transgenic tobacco plants with the recombinant viral vectors SYNV-Cas9, SYNV-tgtGFP1-Cas9, and SYNV-tgtGFP2-Cas9. GFP expression in systemic leaves of plantlets inoculated with the recombinant viruses was observed using a UV lamp and laser confocal microscope. Mock represents a healthy control plant, while V-Cas9, V-tgtGFP1-Cas9, and V-tgtGFP2-Cas9 represent the recombinant viral vectors, respectively. The symptom images were taken 15 days after systemic infection of the inoculated plantlets. (B) Western blot analysis of the expression of SYNV viral structural proteins, Cas9 protein, and GFP protein. (C) Electron microscopy of the morphology of recombinant viral particles. The length of the viral particles was measured and averaged over 20 viral particles. [Figure 4] Figure 4 shows the mutation frequency analysis of gene recombination targeting GFP with the SYNV CRISPR / Cas9 editing vector. (A) GFP site-specific editing efficiency was detected using the PCR / RE method. In the figure, + and - indicate whether or not the corresponding restriction endonuclease was added to the enzyme digestion reaction. Indel (%) represents the percentage of PCR products that cannot be digested with enzymes. (B) GFP site-specific editing status was detected using Sanger sequencing. The underlined sequences are the recognition sequences of the corresponding restriction endonuclease. WT indicates that the sequencing sequence matches the wild-type sequence. d indicates a base deletion, d# indicates a #-th base deletion, and X# indicates the #th occurrence of the sequence in the sequencing results. (C) shows a Sanger sequencing peak diagram. [Figure 5]Figure 5 shows the effect of gRNA processing expressed by the SYNV CRISPR / Ca9 vector on editing efficiency. (A) Schematic diagram of the principle of loop reverse transcription PCR (cRT-PCR). (B) cRT-PCR was used to detect the size of gRNA transcripts produced by the recombinant viral vectors SYNV-gGFP2-Cas9 and SYNV-tgtGFP2-Cas9. (C) PCR / RE analysis was used to detect the efficiency of targeted gene editing. (D) Sanger sequencing was used to analyze the editing status of GFP produced by imprecise gRNA transcripts. [Figure 6]Figure 6 shows the mutation frequency analysis of the endogenous PDS gene targeted by the SYNV CRISPR / Cas9 editing vector in N. benthamiana. (A) is a diagram of the NbPDS gene. In the figure, PDSa and PDSb represent the two copies of the endogenous PDS gene in allotetraploid N. benthamiana. The vertical lines indicate the locations of the target sequences. gPDS1, gPDS2, and gPDS3 represent the three PDS target sequences. All of the above target sequences can simultaneously target PDSa and PDSb. The underlines indicate the corresponding restriction endonuclease recognition sequences in the target sequences. (B) shows the symptoms observed after inoculation of N. benthamiana with the recombinant viral vector SYNV-tgtRNA-Cas9. gPDS1, gPDS2, and gPDS3 represent the symptoms observed around 50 days after inoculation of N. benthamiana with the recombinant viral vector containing the above target sites. (C) The site-specific editing efficiency of PDS was detected using PCR / RE. In the figure, M stands for marker, WT / U stands for WT / undigested, i.e., the wild-type control PCR product was not subjected to the corresponding restriction endonuclease digestion, and WT / D stands for WT / digested, i.e., the wild-type control PCR product was subjected to the corresponding restriction endonuclease digestion. gPDS1, gPDS2, and gPDS3 represent three randomly selected plantlets after inoculation of N. benthamiana with an editing vector containing the above target site. Indel (%) represents the percentage of PCR products that cannot be digested with the enzyme. (D) Sanger sequencing was used to verify the PDS target mutation. gPDS1 / a and gPDS1 / b represent the editing status of PDSa and PDSb after inoculation of N. benthamiana with a recombinant viral vector targeting PDS1, respectively. [Figure 7]Figure 7 shows the targeting of the endogenous RDR6 gene in N. benthamiana by the SYNV CRISPR / Cas9 editing vector. (A) is a schematic diagram of the NbRDR6 gene. In the figure, RDR6a and RDR6b represent the two copies of the endogenous RDR6 gene in allotetraploid N. benthamiana, and gR6-1 and gR6-4 represent the two target sequences of RDR6. Both of these target sequences can simultaneously target RDR6a and RDR6b. (B) shows the symptoms observed after inoculation of N. benthamiana with the recombinant viral vector SYNV-tgtRNA-Cas9. gR6-1 and gR6-4 represent the symptoms observed after inoculation of N. benthamiana with the recombinant viral vectors containing the above target sites, respectively. (C) shows the efficiency of site-specific editing of RDR6 detected using PCR / RE analysis. (D) shows the verification of the RDR6 target mutation using Sanger sequencing. [Figure 8] Figure 8 shows the targeting of the N. benthamiana endogenous gene SGS3 by the SYNV CRISPR / Cas9 editing vector. (A) is a schematic diagram of the NbSGS3 gene. In the figure, SGS3a and SGS3b represent the two copies of the allotetraploid N. benthamiana endogenous gene SGS3, and gS3-1 and gS3-2 represent the two target sequences of SGS3. Both of these target sequences can simultaneously target SGS3a and SGS3b. (B) shows the symptoms observed after inoculation of N. benthamiana with the recombinant viral vector SYNV-tgtRNA-Cas9. gS3-1 and gS3-2 represent the symptoms observed after inoculation of N. benthamiana with the recombinant viral vectors containing the above target sites, respectively. (C) shows the site-specific editing efficiency of SGS3 detected using PCR / RE analysis. (D) shows the verification of the SGS3 target mutation using Sanger sequencing. [Figure 9]Figure 9 shows a schematic diagram of the construction of the SYNV CRISPR / Cas9 dual-target editing vector. t stands for tRNA. Using a tandem tRNA-gRNA (tRNA-gRNA-tRNA-tRNA-tRNA) approach, the plant's endogenous tRNA processing machinery is used to identify and process the precursor tRNA sequence, releasing two gRNA transcripts. [Figure 10] Figure 10 shows the SYNV CRISPR / Cas9 dual-target editing vector simultaneously targeting the endogenous genes RDR6 and SGS3 in N. benthamiana. (A) shows the symptoms observed after inoculation of N. benthamiana with the recombinant rhabdovirus vector SYNV-tgtgtRNA-Cas9. In the figure, gR6-1&gS3-1 and gR6-4&gS3-2 represent the symptoms observed after inoculation of N. benthamiana with the recombinant rhabdovirus vectors SYNV-tgtgtRDR6-1 / SGS3-1-Cas9 and SYNV-tgtgtRDR6-4 / SGS3-2-Cas9, respectively. (B) shows the site-specific editing efficiency detected by PCR / RE analysis when the dual-target SYNV editing vector simultaneously targets RDR6 and SGS3. 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 the editing status of RDR6 target site 4 and SGS3 target site 2, respectively, after inoculation of N. benthamiana with the dual-target editing vectors SYNV-tgtgtRDR6-1 / SGS3-1-Cas9 and SYNV-tgtgtRDR6-4 / SGS3-2-Cas9. (C) Sanger sequencing analysis of the editing status when gR6-4 and gS3-2 are simultaneously targeted. In the figure, gR6-4 / gS3-2 / a represents the editing status of RDR6 copy a when RDR6 and SSG3 are simultaneously targeted, and gR6-4 / gS3-2 / b represents the editing status of RDR6 copy b when RDR6 and SSG3 are simultaneously targeted. 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 the SYNV CRISPR / Cas9 dual-target editing vector, which simultaneously targets two PDSs to produce chromosomal fragment deletions. (A) The symptoms observed after inoculation of N. benthamiana with the recombinant viral vector SYNV-tgtgtPDS1 / 3-Cas9. (B) Chromosomal deletions were detected using PCR / RE analysis after inoculation of N. benthamiana with the recombinant viral vector SYNV-tgtgtPDS1 / 3-Cas9. In the figure, the arrows indicate the fragments resulting from chromosomal deletions by simultaneously targeting gPDS-1 and gPDS-3. Indels (%) represent the percentage of PCR products resulting from chromosomal deletions relative to the total PCR product. (C) Sanger sequencing was used to verify the chromosomal deletion status when gPDS-1 and gPDS-3 were simultaneously targeted. [Figure 12]Figure 12 shows the leaf regeneration and M0 generation genotype analysis of plantlets infected with the SYNV CRISPR / Cas9 vector. (A) shows regenerated plants obtained by tissue culture of systemic leaves after infection of Nicotiana benthamiana with the recombinant viral vector SYNV-tgtPDS1-Cas9. The left image shows regenerated plants obtained on differentiation medium, the middle image shows albino plants obtained on rooting medium, and the right image shows green plants obtained on rooting medium. (B) PCR / RE analysis was used to detect the editing status of regenerated plants. In the figure, Mock / U stands for Mock / undigested, i.e., the PCR product of the wild-type control was not digested with HinfI enzyme. Mock / D stands for Mock / digested, i.e., the PCR product of the wild-type control was digested with HinfI enzyme. Lane Albino represents the PCR product of six albino seedlings obtained by tissue culture that was digested with HinfI enzyme. Lane Normal represents the PCR product of 24 green seedlings obtained by tissue culture that was digested with HinfI enzyme. M represents the marker. (C) RT-PCR analysis was used to detect the virus content of tissue cultured plantlets. In the figure, virus represents the viral genome detection primer, and actin represents the N. benthamiana reference primer. SYNV represents wild-type SYNV systemically infected inoculum as a positive control, Mock represents healthy inoculum as a negative control, and M is the marker. [Figure 13] Figure 13 shows the phenotypic inheritance analysis of the M1 generation of Cas9-edited regenerants. The left panel shows the phenotypic segregation of representative M1 generation plantlets, with M0-8 and M0-22 showing segregation ratios of 3:1 and 15:1, respectively. The right panel shows the M1 generation phenotypic segregation statistics, with the observed values ​​representing the actual segregation ratios and the expected segregation ratios of 3:1 and 15:1, respectively. P values ​​were calculated using a chi-square test; a P value of >0.5 indicates that the observed segregation ratios matched the expected segregation ratios. [Figure 14]Figure 14 shows the genotypic analysis of the progeny of the Cas9-edited M0-8 plantlets. (A) shows the phenotype and genotype of the M0-8 plantlets. (B) shows the PCR / RE analysis and genotyping results of the M1 generation plantlets of the M0-8 plantlets. In the figure, lanes M8-1 to M8-10 represent 10 M1 generation plantlets of M0-8, M8-1 to M8-5 represent five randomly selected albino plantlets, and M8-6 to M8-10 represent five randomly selected green seedlings. The right panel shows the genotypic sequencing results of randomly selected plantlets, where M8-1 represents an albino plantlet and M8-9 represents a green seedling. (C) shows the PCR / RE analysis and genotyping results of the M2 generation plantlets of the M0-8-9 plantlets. In the figure, lanes M8-9-1 to M8-9-10 are M2 generation plantlets of 10 plants of M0-8-9, M8-9-1 to M8-9-5 are five randomly selected albino plants, and M8-9-6 to M8-9-10 are five randomly selected green seedlings. The figure on the right shows the genotype sequencing results of the randomly selected plantlets, where M8-9-1 is an albino seedling and M8-9-10 is a green seedling. [Figure 15] Figure 15 shows the results of RT-PCR to verify the removal of the viral vector from Cas9-edited M1 generation plantlets. The detected plantlets were four to five M1 generation plantlets randomly selected from strains M0-8, -18, -22, -29, -42, -6, -7, and -34. The CK+ lane shows the SYNV-inoculated plantlets as a positive control. SYNV indicates the primers used to detect the viral genome, and Actin indicates the primers used to detect the Nicotiana benthamiana reference primer. [Figure 16] Figure 16 shows the off-target analysis of the SYNV CRISPR / Cas9 genome editing system. (A) Potential off-target loci of the target site gPDS1. The underlined sequence in the figure is the recognition sequence for HinfI endonuclease. (B) PCR / RE analysis results of potential off-target loci of the target site gPDS1. In the figure, 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 viral editing ability and stability in progeny after friction inoculation with SYNV CRISPR / Cas9. (A) Symptoms of friction-inoculated explants. V-Cas9 and V-tgtGFP1-Cas9 represent systemically infected leaves after infiltration inoculation of 16c tobacco plants with SYNV-Cas9 or SYNV-tgtGFP1-Cas9, respectively. (B) Western blot analysis of friction-inoculated explants. (C) RT-PCR analysis of friction-inoculated explants. In the figure, the Mock lane represents a healthy 16c explant, while V-WT, V-Cas9, and V-tgtGFP1-Cas9 represent friction-inoculated 16c explants. The inoculum was systemically infected leaves after infiltration inoculation of 16c tobacco plants with SYNV, SYNV-Cas9, and SYNV-tgtGFP1-Cas9, respectively. gRNA, Cas9, SYNV, and Actin represent the detection primers used, respectively. (D) PCR / RE analysis of the friction-inoculated strains. In the figure, lane V-tgtGFP1-Cas9 represents the 16c strain friction-inoculated with three randomly selected SYNV-tgtGFP1-Cas9 strains. [Figure 18]Figure 18 shows the construction of a SYNV-based CRISPR / Cpf1 gene editing vector. (A) Schematic diagram of SYNV-Cpf1 and SYNV-crRNA-Cpf1 clones. PvuI and Bsu36I are single enzyme cleavage sites located on the N protein before the leader sequence, respectively. NPJ is the 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 from the NPJ sequence to the Bsu36I enzyme cleavage site on the N gene. NPJ-Cpf1-Bsu36I is the sequence from the NPJ sequence to the Bsu36I enzyme cleavage site on the N gene, including the Cpf1 sequence. The above fragments were used in the clone construction process. (B) Schematic diagram of crRNA release. Due to the characteristics of SYNV replication and transcription, the crRNA transcript contains viral-derived non-coding sequences (5'UTR, 3'UTR sequences, and polyA tail) at both ends. The guide sequences are bound to both ends of the guide, allowing the crRNA transcript to be processed by Cpf1, which removes the excess non-coding sequences and releases the correct crRNA. [Figure 19] Figure 19 shows an infectivity assay using the SYNV CRISPR / Cpf1 editing vector targeting GFP transgenic plantlets. (A) shows the symptoms observed after inoculation of 16c transgenic tobacco plants with the recombinant viral vectors SYNV-Cpf1, SYNV-crGFP1-Cpf1, and SYNV-crGFP2-Cpf1. GFP expression in systemic leaves of inoculated plantlets was observed using a UV lamp and laser confocal microscope. Mock represents a healthy control plant. The above symptom images were taken 15 days after systemic infection of the inoculated plantlets. (B) Western blot analysis shows the expression of SYNV viral proteins, Cpf1 protein, and GFP protein. [Figure 20]Figure 20 shows the mutation frequency analysis of gene recombination targeting GFP with the SYNV CRISPR / Cpf1 editing vector. (A) GFP site-specific editing efficiency was detected using the PCR / RE method. In the figure, + and - indicate whether or not the corresponding restriction endonuclease was added to the enzyme digestion reaction. Indel (%) indicates the percentage of PCR products that cannot be digested with enzymes. (B) GFP site-specific editing status was detected using Sanger sequencing. The underlined sequence is the GFP target sequence, wt indicates the sequence matches the wild-type sequence, d indicates deletion, d# indicates a deletion of # bases, and X# indicates the occurrence of the sequence # times in the sequencing results. (C) shows a Sanger sequencing peak diagram. [Figure 21]Figure 21 shows the mutation frequency analysis of the endogenous PDS gene targeted by the SYNV CRISPR / Cpf1 editing vector in N. benthamiana. (A) is a schematic diagram of the NbPDS gene. In the figure, PDSa and PDSb represent the two copies of the endogenous PDS gene in allotetraploid N. benthamiana. The vertical lines indicate the locations of the target sequences, and crPDS1 and crPDS2 represent the two target sequences of the PDS. Both of these target sequences can simultaneously target PDSa and PDSb. The underlines indicate the corresponding restriction endonuclease recognition sequences in the target sequences. (B) shows the symptoms observed after inoculation of N. benthamiana with the recombinant viral vectors SYNV-Cpf1, SYNV-crPDS1-Cpf1, and SYNV-crPDS2-Cpf1. (C) shows the site-specific editing efficiency of the PDS gene detected using PCR / RE analysis. In the figure, M stands for marker, Mock / U stands for Mock / undigested, i.e., the wild-type control PCR product was not cleaved by the corresponding endonuclease, and Mock / D stands for Mock / digested, i.e., the wild-type control PCR product was cleaved by the corresponding endonuclease. V-crPDS1-Cpf1 and V-crPDS2-Cpf1 represent three randomly selected inoculation lines after inoculation of N. benthamiana with an editing vector containing the above target site. Indel (%) represents the percentage of PCR products that cannot be cleaved by the enzyme. (D) Sanger sequencing was used to analyze the site-specific editing of PDS. PDSa and PDSb represent the editing status of PDSa and PDSb, respectively, after inoculation of N. benthamiana with a recombinant viral vector targeting the PDS1 site. [Figure 22] Figure 22 shows the construction of the SYNV CRISPR / Cpf1 dual-target editing vector. By sequentially connecting the DR and guide sequences (DR-guide1-DR-guide2-DR), the Cpf1 crRNA processing mechanism can be used to remove excess non-coding sequences and release precise crRNA. [Figure 23]Figure 23 shows the simultaneous targeting of the GFP and PDS genes by the SYNV CRISPR / Cpf1 dual-target editing vector in 16c transgenic N. benthamiana. (A) shows the symptoms observed after inoculation of N. benthamiana with the recombinant rhabdovirus vector SYNV-crGFP-1 / PDS-1-Cpf1. (B) shows the site-specific editing efficiency detected by PCR / RE analysis when the dual-target SYNV editing vector simultaneously targets GFP and PDS. 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 16c transgenic N. benthamiana with the dual-target editing vector SYNV-crGFP-1 / PDS-1-Cpf1. [Figure 24] Figure 24 shows the regeneration of systemic leaves from plantlets infected with the SYNV CRISPR / Cpf1 vector and the analysis of the genotype of the M0 generation. (A) shows the regenerated plants obtained by tissue culture of systemic leaves after infection of Nicotiana benthamiana with the recombinant viral vector SYNV-crPDS1-Cpf1. The top image shows the regenerated plants obtained on differentiation medium, and the bottom image shows the albino plants obtained on rooting medium. (B) PCR / RE analysis of the editing status of regenerated plants. In the figure, Mock / U stands for Mock / undigested, i.e., the wild-type control PCR product was not digested with HinfI. Mock / D stands for Mock / digested, i.e., the wild-type control PCR product was digested with HinfI. Lane Albino represents the PCR product of four albino seedlings obtained by tissue culture that was digested with HinfI. Lane Normal represents the PCR product of 27 green seedlings obtained by tissue culture that was digested with HinfI. M represents the marker. (C) RT-PCR analysis of the virus content of tissue cultured plants. N. benthamiana actin is used as an internal control. [Figure 25]Figure 25 shows the phenotypic inheritance analysis of the M1 generation of Cpf1-edited regenerates. (A) shows the phenotypic segregation of representative M1 generation plantlets. M0-15 in the figure shows no albino seedling segregation, while M0-11 and M0-9 show segregation ratios of 3:1 and 15:1, respectively. (B) shows the M1 generation phenotypic segregation statistics. The observed values ​​in the figure represent the actual segregation ratios, while the expected segregation ratios are 0, 3:1, or 15:1. [Figure 26] Figure 26 shows the infectivity analysis of SYNV editing vectors with G protein N-terminal deletions or substitutions. (A) Schematic diagram of the clones of the recombinant rhabdoviruses 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) Symptoms and mCherry expression in systemic leaves after inoculation of Nicotiana benthamiana with the recombinant rhabdoviruses SYNV-tgtPDS1-Cas9, SYNV-tgtPDS1-Cas9-dGN, or SYNV-tgtPDS1-Cas9-mCherry:GN. (C) Western blot analysis of the recombinant rhabdovirus SYNV-tgtPDS1-Cas9, SYNV-tgtPDS1-Cas9-dGN, or SYNV-tgtPDS1-Cas9-mCherry:GN inoculated strains using a G protein monoclonal antibody. (D) PCR / RE detection. In the figure, M stands for marker, Mock / U stands for Mock / undigested, i.e., the wild-type control PCR product was not subjected to the corresponding restriction endonuclease reaction, and Mock / D stands for Mock / digested, i.e., the wild-type control PCR product was subjected to the corresponding restriction endonuclease reaction. Lanes SYNV-tgtPDS1-Cas9, SYNV-tgtPDS1-Cas9-dGN, and SYNV-tgtPDS1-Cas9-mCherry:GN represent systemically infected inoculum after inoculation of the recombinant viruses into Nicotiana benthamiana, respectively. [Figure 27]Figure 27 shows the infectivity and editing efficiency of the SYNV editing vectors codon-optimized for N. benthamiana Cas9. (A) Schematic diagram of the cloning of the recombinant rhabdoviruses SYNV-tgtPDS2-Cas9 and SYNV-tgtPDS2-oCas9. oCas9 is the Cas9 nuclease sequence codon-optimized for N. benthamiana. (B) Symptoms appearing in systemic leaves after inoculation of N. benthamiana with the recombinant rhabdoviruses SYNV-tgtPDS2-Cas9 and SYNV-tgtPDS2-oCas9. (C) Site-specific editing of the PDS was detected using PCR / RE analysis. In the figure, lanes SYNV-tgtPDS2-Cas9 and SYNV-tgtPDS2-oCas9 represent 10 randomly selected systemically infected inoculations after inoculation of N. benthamiana with the recombinant viral vectors. [Figure 28] Figure 28 shows the infectivity and editing efficiency of the SYNV single transcriptional unit editing vector. (A) Schematic diagram of the recombinant rhabdovirus SYNV-oCas9gPDS2 clone, in which the oCas9 nuclease sequence and gRNA sequence are linked using the indicated 15-bp spacer sequence (5'-AAGCCATGGGATATC-3'). (B) Represents symptoms observed in systemic leaves after inoculation of N. benthamiana with the recombinant rhabdoviruses SYNV-oCas9gPDS2 and SYNV-tgtPDS2-oCas9. (C) Detects site-specific editing of the NbPDS using PCR / RE analysis. In this figure, lanes SYNV-oCas9gPDS2 and SYNV-tgtPDS2-oCas9 represent three randomly selected systemically infected inoculations after inoculation of N. benthamiana with the recombinant viral vectors. [Figure 29]Figure 29 shows an analysis of the infection status of N. benthamiana with EMDV fluorescent expression vectors. (A) is a diagram illustrating the construction process of the EMDV-GFP green fluorescent and EMDV-RFP red fluorescent expression vectors. The N3'+N5' vectors consist of the N3'UTR+ggg+N5'UTR between the GFP gene and the N gene, while the RFP gene has two repeat gene spacers on either side, forming an NX junction. (B) shows the symptoms of systemic infection of N. benthamiana with EMDV-GFP recombinant virus. The left panel shows the expression of viral structural proteins and green fluorescent protein in leaves of infected N. benthamiana lines using a Western blot detection system, and the right panel shows the symptoms of systemic infection of N. benthamiana with EMDV-GFP. (C) shows the onset of symptoms after systemic infection of N. benthamiana with EMDV-RFP recombinant virus. The left panel shows the expression of viral structural proteins and red fluorescent protein in the leaves of affected N. benthamiana lines using a Western blot detection system, and the right panel shows the symptoms of systemic infection of N. benthamiana with EMDV-RFP. [Figure 30]Figure 30 shows the infection status of tobacco, potato, eggplant, and tomato with EMDV fluorescent expression vectors. (A) Shows the symptoms of systemic infection of tobacco with EMDV-RFP recombinant virus. The left panel shows the expression of viral structural proteins and red fluorescent protein in leaves of affected tobacco lines using a Western blot detection system. The right panel shows the symptoms of systemic infection of tobacco with EMDV-RFP recombinant virus. Columns 1 and 2 show the status of plantlets 45 days after friction inoculation of systemically affected tobacco bearing EMDV-RFP and healthy control tobacco, as well as the corresponding fluorescence status of systemic leaves. (B) EMDV-RFP or EMDV-GFP recombinant virus can be infected in eggplant, tomato, and potato by mechanical friction. (C) Shows the systemic infection of EMDV-RFP recombinant virus after grafting potato and N. benthamiana. The left figure is a schematic diagram of a grafted plant, with N. benthamiana as the rootstock and potato as the scion. The right figure shows the state of the grafted plant at the time of onset of symptoms in the N. benthamiana rootstock and potato scion, approximately 14 days after the onset of symptoms, when the grafted plant was mechanically inoculated with EMDV-RFP by friction onto the N. benthamiana rootstock, and the fluorescence expression status of the corresponding leaves. [Figure 31] Figure 31 shows infection and expression analysis of pepper plants with an EMDV fluorescent expression vector. (A) Western blot analysis measured the expression of viral structural proteins (upper panel) and red fluorescent protein (middle panel) in systemically infected pepper leaf tissue. Rub L: Rubisco large subunit was used as a loading control. (B) Symptoms (upper panels) and fluorescent imaging (lower panel) developed 35 days after systemic infection of pepper plants with EMDV-RFP. Mock: Control plant inoculated with healthy juice. [Figure 32]Figure 32 shows the analysis of mutation frequency in transgenic N. benthamiana plants in which the EMDV CRISPR / Cas9 editing vector targets GFP. (A) Schematic diagram of the construction of the EMDV-tgtGFP2-Cas9 editing vector. (B) Left panel shows Western blot analysis of viral and Cas9 protein expression after systemic infection of N. benthamiana plants with the EMDV-tgtGFP2-Cas9 recombinant virus. The right panel shows the systemic infection of N. benthamiana plants with the EMDV-tgtGFP2-Cas9 recombinant virus. Under a portable UV lamp, it was clearly observed that after highly efficient editing of the EMDV editing vector, the green fluorescence production of the plantlets was significantly reduced. (C) PCR / RE analysis of site-specific editing efficiency of the GFP2 target. In the figure, + and - indicate whether or not the corresponding restriction endonuclease was added in the enzymatic digestion reaction. In the Sanger sequencing results, 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, i indicates a base insertion, d# indicates # base deletions, i# indicates # base insertions, and X# indicates the # occurrences of the sequence in the sequencing results. (D) Peak diagram of the sequencing results in Figure C. [Figure 33]Figure 33 shows an analysis of the mutation frequency of the PDS gene targeted by the EMDV CRISPR / Cas9 editing vector in N. benthamiana. (A) is a schematic diagram of the construction of the EMDV-tgtPDS4-Cas9 editing vector. (B) shows the onset of disease after systemic infection of N. benthamiana with EMDV-tgtPDS4-Cas9. The left panel shows Western blot analysis of viral structural protein expression after systemic infection of N. benthamiana with the EMDV-tgtPDS4-Cas9 recombinant virus. The right panel shows a photograph taken 40 days after systemic infection of N. benthamiana with the EMDV-tgtPDS4-Cas9 recombinant virus. (C) The site-specific editing efficiency of the PDS4 target was detected using the PCR / RE method. The left panel shows that the target is completely conserved in the PDS genes of N. benthamiana, potato, tomato, pepper, and tobacco. The right panel shows the editing situation of the endogenous PDS genes-a and PDS-b in N. benthamiana systemically infected with the EMDV-tgtPDS4-Cas9 recombinant virus. + and - indicate whether or not the corresponding restriction endonuclease was added in the enzymatic digestion reaction. [Figure 34]Figure 34 shows the M0 plantlets obtained by regenerating plant leaves infected with the EMDV CRISPR / Cas9 vector and their genotypic analysis. (A) Regenerated plantlets obtained by systemic leaf tissue culture after infection of N. benthamiana with EMDV-tgtPDS 4-Cas9. The left image shows callus and shoots differentiated on MS medium, and the right image shows albino plantlets obtained on MS rooting medium. (B) Gene editing in the M0 regenerated plantlets was detected by PCR / RE. Mock / U and Mock / D represent PCR products amplified by the wild-type control plant that were not cleaved with T7EI enzyme and those cleaved with T7EI enzyme, respectively. Albino: albino plantlets; Normal: normal green plantlets. (C) RT-PCR was used to detect the presence of EMDV in the M0 plantlets. The "virus" primers used were viral genome detection primers, and the "GAPDH" primers were N. benthamiana internal control primers. EMDV represents wild-type EMDV systemically infected inoculum and served as a positive control, and Mock represents healthy inoculum and served as a negative control. [Figure 35] Figure 35 shows an analysis of mutation frequencies of PDS genes targeted by EMDV CRISPR / Cas9 editing vectors in tobacco, tomato, and potato. (A) shows a sequence peak map of editing results for EMDV-tgtPDS4-Cas9 in tobacco, with a summary of monoclonal sequencing results on the left and corresponding Sanger sequencing peak map on the right. (B) shows a sequence peak map of editing results for EMDV-tgtPDS4-Cas9 in tomato, with a summary of monoclonal sequencing results on the left and corresponding Sanger sequencing peak map on the right. (C) shows a sequence peak map of editing results for EMDV-tgtPDS4-Cas9 in potato, with a summary of monoclonal sequencing results on the left and corresponding Sanger sequencing peak map on the right. DETAILED DESCRIPTION OF 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 will fall within the protection scope of the present invention.

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

[0082] All of the Nicotiana benthamiana and virus materials used were stored in our laboratory, and all of the expression vectors used were constructed and stored in our laboratory.

[0083] The recombinant rhabdovirus vectors SYNV, SYNV-GFP, the core protein expression vector pGD-NPL, and the RNA silencing suppressor 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 and SYNV-RFP-dG N , SYNV-RFP-mCherry:G NThis is disclosed in the document "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 viral infection. Molecular Plant, 2018, 11:269-287."

[0086] The plasmid pYQ230 is disclosed in the document "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 SYNV viral vectors Step 1: Construction of SYNV dual reporter gene expression vector

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

[0088] In step (1), the Bsu36I-NPJ fragment, GFP fragment, and RFP-NheI fragment were amplified using SYNV, SYNV-GFP, and SYNV-RFP as templates, respectively, with N-Bsu36I / F and NPJ-GFP / R, GFP / F and NPJ-RFP / R, and RFP / F and P-NheI / R in Table 6. Here, the Bsu36I-NPJ fragment contains the NPJ region from the Bsu36I enzyme cleavage site on the N gene to downstream, and the RFP-NheI fragment contains the entire 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 the adjacent fragments, allowing the three fragments to be connected in series by homologous recombination. Fragment 1N-GFP and fragment 3RFP-P each contain a sequence homologous to the linearized SYNV vector (linearized after double digestion with Bsu36I and NheI enzymes) and are used for recombination with the SYNV vector.

[0089] In step (2), the SYNV vector was digested with restriction endonucleases Bsu36I and NheI, and the vector fragment was recovered after electrophoresis detection to obtain the linearized vector, which was purified and used.

[0090] In step (3), the purified Bsu36I-NPJ fragment, GFP fragment, and RFP-NheI fragment were simultaneously cloned into the linearized SYNV vector using the in-fusion recombination method. Positive clones of SYNV-GFP-RFP were screened by colony PCR to identify the enzyme cleavage sites. The clones without any enzyme cleavage site identification errors were sequenced to verify that the three fragments were correctly assembled into the SYNV vector in sequence. Step 2: Agrobacterium transformation, culture, and infiltration inoculation into Nicotiana benthamiana SYNV-GFP-RFP was introduced into Agrobacterium strain EHA105 using the electric shock method.

[0091] At the same time, the core protein expression vector pGD-NPL, which is required for SYNV systemic infection, and the viral RNA silencing suppressor subprotein expression vectors (pGD-Hc-Pro, pGD-p19, and pGD-γb) were blitz-transformed into Agrobacterium strain EHA105.

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

[0093] After incubation, Agrobacterium containing the viral expression vector, core protein expression vector, and RNA silencing suppressor subprotein expression vector was mixed in equal volumes at a 1:1:1 ratio and infiltrated into Nicotiana benthamiana plants. It is recommended to select plants at the 4- to 6-leaf stage. Infiltration was performed using a 1ml disposable syringe without a needle, infiltrating the dorsum of the leaf, so that 3-4 leaves per plant were infiltrated. After inoculation, the plantlets were cultured in an isolated greenhouse at 25°C and used as controls. A similar method was used to infiltrate SYNV-GFP, a recombinant viral vector that expresses only the GFP protein. Step 3: Detect whether the recombinant SYNV virus vector can simultaneously express two reporter genes

[0094] Around 6 days after inoculation of Nicotiana benthamiana plants with the recombinant SYNV virus vector SYNV-GFP-RFP, prominent GFP and RFP fluorescence was observed in the inoculated leaves. Around 12 days after inoculation, the fluorescent reporter gene diffused between cells. Around 30 days after inoculation, strong GFP and RFP fluorescence was observed in the veins and mesophyll cells of upper systemic leaves (Fig. 1B). However, only GFP fluorescence was observed in the control plants inoculated with SYNV-GFP. After systemic infection, all inoculated plants showed typical symptoms of virus infection, such as dwarfism, leaf curl, yellowing of leaf veins, and curling of calyx (Fig. 1C). Furthermore, the petals of the SYNV-GFP-RFP-inoculated plants gradually became pink due to the accumulation of red fluorescent protein RFP. To confirm that the above-mentioned symptoms were due to SYNV virus infection, we harvested systemic leaves from plantlets inoculated with the recombinant SYNV-GFP-RFP vector. Leaf total protein was extracted and subjected to Western blot analysis using a SYNV polyclonal antibody, a GFP monoclonal antibody, and an RFP monoclonal antibody. The results showed that SYNV virus protein bands, GFP protein bands, and RFP protein bands were detected in the total leaf protein. In control leaves inoculated with SYNV-GFP, only SYNV virus protein bands and GFP bands were detected, with no RFP band (Figure 1D). These results demonstrate that the recombinant SYNV virus vector can simultaneously express two exogenous reporter genes, GFP and RFP. Example 2: Construction of SYNV CRISPR / Cas9 editing vector

[0095] This process involves simultaneously inserting gRNA and Cas9 into the SYNV genome in the form of independent expression frames, with the insertion position including, but not limited to, between the N and P genes. Below, we take the simultaneous insertion of a gRNA transcription unit and a Cas9 expression frame between the N and P genes as an example (Figure 2A), to describe the construction process of a SYNV-based CRISPR / Cas9 genome editing vector in detail. The constructed clones are as follows: 1) SYNV-Cas9: This is a SYNV vector that expresses only the Cas9 protein and served as a control. 2) SYNV-gRNA-Cas9: A SYNV vector that transcribes sgRNA and expresses the Cas9 protein. 3) SYNV-tgtRNA-Cas9: A SYNV vector that accurately transcribes sgRNA and expresses the Cas9 protein. The specific steps to construct a clone are as follows: 1) SYNV-Cas9

[0096] The Bsu36I-NPJ fragment, Cas9 ORF fragment, and NPJ-NheI fragment (Figure 2A) were amplified using SYNV, pBGK01-gRNA-Cas9, and SYNV as templates with primers N-Bsu36I-F and Flag-NPJ / R, Cas9 / F and Cas9 / R, and Cas9-NPJ / F and P-NheI / R, respectively, as shown in Table 6. The Bsu36I-NPJ fragment encompasses the NPJ region from the Bsu36I enzyme cleavage site on the N gene downstream, and the NPJ-NheI fragment encompasses 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 fragments. Fragment 1, Bsu36I-NPJ, and fragment 3, NPJ-NheI, contain sequences homologous to the linearized SYNV vector after double digestion with Bsu36I and NheI, respectively. Therefore, the SYNV-Cas9 vector can be obtained by sequentially joining the above three fragments by in-fusion ligation. 2) SYNV-gRNA-Cas9

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

[0098] To achieve precise transcription of the gRNA, tRNA precursor sequences (i.e., tRNA-gRNA-tRNA) are attached to both ends of the gRNA, where the tRNA sequence can be recognized and cleaved by the plant's endogenous processing machinery to release a relatively precise gRNA transcript (Figure 2B). We synthesized sequences containing different target sites according to the sequences provided by SEQ IDs 1-9, adding a 15-nt sequence (5'-TTATTTGTCTAGGCC-3') derived from the 3' end of NPJ (5'-TTATTTGTCTAGGCC-3') and a 15-nt sequence (5'-TAAACTACAGCCACA-3') to the 5' and 3' ends, respectively, to facilitate amplification and clone construction in the next step. The target sites selected were GFP, NbPDS, NbRDR6, and NbSGS3, in the order of two, three, two, and two targets. Each of these target sites contains a restriction endonuclease, facilitating detection of editing efficiency by PCR / RE. Furthermore, these target sites are perfectly matched in the two homologous copies (a and b) of NbPDS, NbRDR6, and NbSGS3, allowing the selected target sites to be simultaneously targeted in the above genes.The synthesized sequences were used as templates for amplification using the primers NPJ-tRNA / F and NPJ-tRNA / R in Table 6 to obtain tRNA-gRNA-tRNA sequences containing different target sites. At the same time, SYNV-Cas9 was used as a template to amplify the Bsu36I-NPJ fragment and the NPJ-AhdI fragment for N-Bsu36I / F and NPJ / R, and NPJ / F and Cas9-AhdI / R, respectively. Here, the Bsu36I-NPJ fragment encompassed the NPJ region from the Bsu36I enzyme cleavage site on the N gene downstream, and the NPJ-AhdI fragment encompassed the NPJ region. By in-fusion cloning the above three fragments, including the AhdI enzyme cleavage site from the J sequence to the Cas9 gene, we obtained 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, respectively.

[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 IDs 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 the GFP gene in 16c transgenic tobacco Step 1: Inoculation of Agrobacterium-infiltrated 16c transgenic tobacco with the SYNV CRISPR / Cas 9 vector

[0100] A 20-nt target sequence (5'-GATACCCAGAT) capable of targeting GFP in the 16c plantlets was designed by CRISPR-P (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR) online design software. 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 described in Example 1, Agrobacterium was electrophoretically transformed with the recombinant viral expression vectors SYNV-Cas9, SYNV-tgtGFP1-Cas9, and SYNV-tgtGFP2-Cas9 and infiltrated into 16c transgenic tobacco plants. Approximately 30 days after inoculation, the infiltrated plants exhibited viral symptoms similar to those observed with wild-type virus, including leaf curl and yellowing of leaf veins. Under portable UV light and laser confocal microscopy, the inoculated plants exhibited significantly reduced levels of green GFP fluorescence, with almost no GFP expression in mesophyll cells, and only green guard cells. In the control SYNV-Cas9-inoculated systemic leaves, the GFP fluorescence intensity was consistent with that of wild-type 16c (Figure 3A).

[0102] To verify that the above-mentioned symptoms were due to systemic SYNV infection, systemic leaves from affected plantlets were harvested, total leaf protein extracted, and Western blot analysis was performed using SYNV polyclonal antibody, Flag tag antibody, and GFP monoclonal antibody, respectively. Results showed that SYNV viral protein bands and Cas9 protein bands were detected in each plantlet inoculated with the SYNV editing vector, and GFP protein expression levels were significantly reduced. The GFP protein expression levels in tobacco leaves inoculated with the control vector SYNV-Cas9 were essentially consistent with those of wild-type 16c plantlets (Figure 3B). These results demonstrate that the SYNV viral vector can simultaneously accommodate both gRNA and Cas9 expression frames, and that the recombinant viral vector containing the above-mentioned exogenous expression frames still possesses systemic infectivity.

[0103] To determine whether the exogenous expression vector could be integrated into the viral genome, viral particles were extracted from the inoculated cultures and examined under an electron microscope for their morphology. The recombinant viral particles exhibited a typical ball- or rod-shaped morphology, and the length of the viral particles containing the edited vector was significantly longer, reaching approximately 333 nm, compared with wild-type SYNV viral particles (approximately 247 nm) (Figure 3C). The length of the viral particles was proportional to the size of the recombinant viral genome, indicating that the 4.8 kb exogenous fragment had integrated into the SYNV genome and was packaged into mature viral particles. Step 2: GFP-targeted mutation efficiency analysis

[0104] To verify the editing efficiency of the GFP target, PCR / RE (Polymerase Chain Reaction / Restriction Digestion) was used to measure the systemic leaf quality of the inoculated plants. PCR was performed using primers GFP / F and GFP / R (Table 7), followed by enzyme digestion analysis using the corresponding restriction endonucleases. Uncleavable PCR products indicate mutations at the target site, and the extent to which the uncleaved bands reflect high editing efficiency. After enzyme digestion of the PCR products, GFP target 1 and target 2 were found to have varying degrees of mutation, with a mutation efficiency of approximately 91% (Figure 4A). To characterize the GFP editing types, Sanger sequencing was performed on the PCR products. The PCR products were ligated into a T vector, and 10 monoclonals were identified at each of the two GFP target sites. Seven and eight monoclonals were found to have mutated GFP target 1 and target 2, respectively (Figure 4B), which was essentially consistent with the PCR / RE results. The majority of the edits generated were single- or multiple-base deletions (Figure 4C). Example 4: Improving editing efficiency by precision engineering of SYNV CRISPR / Cas9-generated gRNA transcripts

[0105] To assess the accuracy of the gRNA transcripts generated by the SYNV editing vector SYNV-tgtRNA-Cas9, we measured the size of the gRNA transcripts using cRT-PCR, and simultaneously used the gRNA transcripts generated by the recombinant viral vector SYNV-gRNA-Cas9 as a control. Based on the viral transcription pattern, the gRNA transcripts generated 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, and the transcripts were approximately 208–300 nt in size (208 nt transcript and a polyA sequence of unknown length). The gRNA transcripts produced by the recombinant viral vector SYNV-tgtGFP2-Cas9 were engineered to contain tRNA recognition sequences at both ends. After the endogenous plant RNase enzyme recognition and processing, gRNA transcripts of approximately 100 nt were produced. Without this recognition and processing, the size of the gRNA transcripts produced was approximately 362–400 nt (a 362-nt transcript and a polyA sequence of unknown length) (Figure 5A).

[0106] The 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 plants, respectively. Total RNA was extracted from the systemic leaves of the vector-inoculated plants and subjected to cRT-PCR. The results showed that the gRNA transcripts generated by the recombinant viral vector SYNV-tgtGFP2-Cas9 were all approximately 100 nt in size (Figure 5B), indicating that the tRNA sequence was fully recognized and processed by the plant's endogenous RNase enzyme. To further validate the accurate release of gRNA transcripts and the residual sequences at both ends of the gRNA transcripts, the PCR products were ligated into a T vector and then Sanger sequenced. The results showed that the generated gRNA-scaffold transcripts had no residual bases at the 5' end, but 1-3 nt at the 3' end, with the residual bases derived from the tRNA sequence attached to the 3' end (Figure 5D). The recombinant viral vector SYNV-gGFP2-Cas9 produced transcripts of the desired size, ranging from 208 nt to 300 nt, as well as a transcript of approximately 100 nt (Figure 5B). Sequencing analysis of this small fragment revealed that it was a gRNA transcript after removal of viral sequences, and that it lacked one base from the viral 5'UTR region or 4-5 bases from the scaffold sequence at its 3' end (Figure 5D).

[0107] To compare the editing efficiency of the two recombinant viral vectors mentioned above, we randomly selected two diseased strains around 30 days after inoculation, collected systemically infected leaves, and measured the editing efficiency of the GFP target using PCR / RE and Sanger sequencing. The editing efficiency of the GFP target was found to be around 11–20% (Figure 5C).

[0108] These results demonstrate that when a SYNV editing vector does not employ a tRNA processing mechanism, it can utilize a single processing mechanism to produce a small amount of precise gRNA transcripts, resulting in a certain degree of editing of the target gene (approximately 11-20%). However, when the plant's endogenous tRNA processing mechanism is employed, the initial gRNA transcript can be fully recognized and cleaved into precise gRNA transcripts by the plant's endogenous RNase enzyme, significantly increasing the editing efficiency of the vector (approximately 90%). Therefore, the SYNV editing vector SYNV-tgtRNA-Cas9, which incorporates tRNA recognition sequences at both ends of the gRNA transcript, is the preferred vector of the present invention. Example 5: SYNV CRISPR / Cas9 vector targets the endogenous PDS gene in Nicotiana benthamiana The N. benthamiana endogenous PDS gene was used to further validate the viral vector editing system of the present invention.

[0109] A 20-nt target sequence (5'-GCCGTTAATTTGA) capable of targeting PDS was designed by CRISPR-P (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR) online design software. GAGTC CA-3', 5'-TTGGTAGTAGCGACT CCATGG -3', 5'-TTAACTGTATTGT CTAG CTC-3' (the underlined parts are the corresponding enzyme cleavage sites, HinfI, NcoI, and BfaI) was selected, and all of the above targets can simultaneously target PDS copies a and b in allotetraploid Nicotiana 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, the recombinant virus expression vectors SYNV-tgtPDS1-Cas9, SYNV-tgtPDS2-Cas9, and SYNV-tgtPDS3-Cas9 were electrophoretically transformed into Agrobacterium, which was then infiltrated into N. benthamiana plants. Thirty days after inoculation of the recombinant virus expression vectors into N. benthamiana plants, the plants showed symptoms such as leaf curling and yellowing of leaf veins, similar to those observed with the wild-type virus (Figure 6B).

[0111] We measured the target editing efficiency of the PDS using PCR / RE. All primers used simultaneously amplified PDS copies a and b. The results showed that the three target sites of the PDS all generated varying degrees of mutation, with the maximum editing efficiency reaching approximately 79% (Figure 6C). For example, in target 1, the editing efficiencies of three randomly selected plant lines were 79%, 70%, and 56%, respectively. The difference in editing efficiency between different lines may be related to the expression levels of gRNA transcripts and Cas9 protein.

[0112] Sanger sequencing was used to verify the editing status of PDS target site 1. PDS copies a and b were amplified using the PDS-specific primers listed in Table 7, the PCR products were ligated into a T vector, and 10 clones per copy were analyzed. The results showed that the number of mutant clones in PDS copies a and b was 8 and 7, respectively, which was essentially consistent with the editing efficiency measured by PCR / RE. The editing types were mainly single-base and multi-base deletions and single-base insertions (Figure 6D). Example 6: SYNV CRISPR / Cas9 vector targets the Nicotiana benthamiana endogenous gene RDR6 The N. benthamiana endogenous gene RDR6 was used to further validate the viral vector editing system of the present invention.

[0113] A 20-nt target sequence (5'-AGTTGGGTAAGAG) capable of targeting RDR6 was designed by CRISPR-P (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR) online design software. TCAGGA G-3', 5'- ATTCTCAGCTAAC CAGCTG A-3', where the underlined parts are the corresponding enzyme cleavage sites Hpy188III and PvuII), was selected, and both of the above targets can simultaneously target two copies of RDR6, a and b, in allotetraploid Nicotiana benthamiana (Figure 7A).

[0114] The 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 electrophoretically transformed into Agrobacterium using the method described in Example 1, and then inoculated into N. benthamiana. Thirty days after inoculation of the recombinant virus expression vectors into N. benthamiana, they exhibited symptoms such as leaf curling and yellowing of the leaf veins, similar to those observed with the wild-type virus (Figure 7B).

[0115] The PCR / RE method was used to measure the target editing efficiency of RDR6. All primers used were capable of simultaneously amplifying RDR6 copies a and b. The results showed that mutations occurred at the two target sites of RDR6 to different degrees, with the maximum editing efficiency reaching around 91% (Figure 7C).

[0116] Sanger sequencing was used to verify the editing status of RDR6 target site 1. RDR6 copies a and b were amplified using the RDR6-specific primers listed in Table 7, the PCR products were ligated into a T vector, and 10 clones per copy were analyzed. The results showed that the number of mutant clones in RDR6 copies a and b was 6 and 7, respectively, which was essentially consistent with the editing efficiency measured by PCR / RE. The editing types were mainly single- or multiple-base deletions, single-base substitutions, or insertions (Figure 7D). Example 7: SYNV CRISPR / Cas9 vector targets the Nicotiana benthamiana endogenous gene SGS3 The N. benthamiana endogenous gene SGS3 was used to further validate the viral vector editing system of the present invention.

[0117] A 20-nt target sequence (5'-ACAAGAGTGGAAG) capable of targeting SGS3 was designed using CRISPR-P (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR) online design software. CAGTG CT-3', 5'- TGCCTCAACTGATC CCAAGG The target sequences were selected from the two SGS3 gene clones a and b (the underlined parts are the corresponding enzyme cleavage sites, TscAI and Eco130I, respectively). Both of the above targets can simultaneously target the two copies of SGS3, a and b, in allotetraploid Nicotiana benthamiana (Figure 8A).

[0118] The 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 electrophoretically transformed into Agrobacterium using the method described in Example 1, and then inoculated into N. benthamiana. Both of the recombinant virus expression vectors exhibited symptoms such as leaf curling and yellowing of the vein, similar to those observed with the wild-type virus, before and after 30 days of inoculation into N. benthamiana (Figure 8B).

[0119] The targeted editing efficiency of SGS3 was measured using PCR / RE. All primers used were capable of simultaneously amplifying SGS3 copies a and b. The results showed that mutations occurred at the two targeted sites of SGS3 to varying degrees, with the maximum editing efficiency reaching approximately 91% (Figure 8C).

[0120] Sanger sequencing was used to verify the editing status of SGS3 target site 2. SGS3-specific primers listed in Table 7 were used to amplify SGS3 copies a and b, respectively. The PCR products were then ligated into a T vector, and 10 clones per copy were analyzed. The results showed that the number of mutant clones in SGS3 copies a and b was 8 and 6, respectively, which was essentially consistent with the editing efficiency measured by PCR / RE. The editing types were mainly single-base and multi-base deletions and single-base insertions (Figure 8D). Example 8: SYNV CRISPR / Cas9 multi-targeting vector simultaneously targets the endogenous genes RDR6 and SGS3 of Nicotiana benthamiana

[0121] To verify whether the SYNV vector-based genome editing system can perform multi-target editing of plant genomes, we constructed a SYNV dual-target editing vector using a tRNA-gRNA tandem connection method (tRNA-gRNA1-tRNA-gRNA2-tRNA). The primary transcript produced can be recognized and cleaved by endogenous plant RNase enzymes, after which two precise gRNA transcripts can be simultaneously released (Figure 9).

[0122] The 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 described in Example 1, the recombinant virus expression vectors SYNV-tgtgtRDR6-1 / SGS3-1-Cas9 and SYNV-tgtgtRDR6-4 / SGS3-2-Cas9 were electrophoretically transformed into Agrobacterium, which was then inoculated into N. benthamiana plants. Both vectors showed symptoms similar to those of the wild-type virus, such as leaf curling and yellowing of the vein, 30 days after inoculation into N. benthamiana plants (Figure 10A).

[0123] The targeted editing efficiencies of RDR6 and SGS3 were measured using PCR / RE analysis. The 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). These results demonstrate that the SYNV vector can be used for simultaneous editing of multiple targets in the plant genome without affecting the efficiency of single-target editing (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 the specific primers listed in Table 7, and the PCR products were ligated into a T vector. Ten clones per copy were analyzed. The results showed that the number of mutant clones for RDR6 copies a and b was 5 and 5, respectively, and for SGS3 copies a and b was 7 and 8, respectively. These results were essentially consistent with the results measured using PCR / RE. The editing types were mainly single- and multiple-base deletions, single- and multiple-base substitutions, and single-base insertions (Figure 10C). Example 9: Chromosomal deletions caused by targeting the endogenous PDS gene in Nicotiana benthamiana with the SYNV CRISPR / Cas9 multitargeting vector

[0125] To verify whether the SYNV multi-target editing vector can cause deletion of chromosomal fragments in plant genomes, the recombinant virus clone SYNV-tgtgtPDS1 / 3-Cas9 was constructed according to the method described in Example 2. This vector can simultaneously target PDS targets 1 and 3 (Example 5). Using the method described in Example 1, the recombinant virus expression vector SYNV-tgtgtPDS1 / 3-Cas9 was electrophoresed into Agrobacterium, which was then inoculated into N. benthamiana plants. Thirty days after inoculation, the recombinant virus expression vector exhibited symptoms similar to those of the wild-type virus, such as leaf curling and yellowing of leaf veins (Figure 11A).

[0126] To determine whether the above-mentioned inoculated plantlets could produce chromosomal fragment deletions, total DNA was extracted from the inoculated plantlets, along with total DNA extracted from a wild-type plantlet as a control. PCR amplification was performed using primers PDS-1 / 3 / F and PDS-1 / 3 / R (Table 7). The primers simultaneously amplified PDS copies a and b. The amplified product of PDS copy a was 492 bp, and the amplified product of PDS copy b was 486 bp. Three randomly selected SYNV-tgtgtPDS1 / 3-Cas9 systemically infected plantlets showed a small band of approximately 200 bp in addition to the wild-type band. This size corresponded to the size of the chromosomal fragment deletion resulting from simultaneous editing of PDS target sites 1 and 3 (Figure 11B).

[0127] To further verify that the bands were generated by simultaneous editing of the PDS double targets, we recovered the fragments and performed Sanger sequencing. The results indicated that the bands were indeed due to a chromosomal fragment deletion between PDS target sites 1 and 3. Further analysis of these results revealed that both PDS copies a and b produced fragment deletions (Figure 11C), and the resulting sequencing peaks are shown in Figure 11 (Figure 11D). Example 10: Regeneration of SYNV CRISPR / Cas9-infected leaf tissue and M0 generation genotype analysis

[0128] To obtain regenerated plantlets, whole-body leaves inoculated with the recombinant virus vector SYNV-tgtPDS1-Cas9 were collected and washed with sterile water. The surface was then disinfected with 70% ethanol for 30 seconds, then disinfected with 0.1% mercury for 1-2 minutes, and washed with sterile water at least three times. After disinfection, the disinfected leaves were removed and the prominent mid-vein and leaf edges were cut off. The leaves were then cut into 1 x 1 cm pieces. 2The tissue cultures were cut into small pieces and cultured on antibiotic-free MS differentiation medium (containing 1 mg / L 6-BA). After 2-3 weeks, the differentiated tissue culture lines showed two phenotypes: albino seedlings and green seedlings (Figure 12A). The differentiated shoots were transferred to a medium containing live roots and cultured. After about 3 weeks, sterile ddH2O was added to the culture bottles and the bottles were covered with plastic wrap. After 4-5 days, the tissue culture seedlings were transplanted into soil.

[0129] To detect the editing status of the PDS target site in the tissue culture plants, DNA was extracted from the M0 generation albino and green seedlings and subjected to PCR / RE analysis (Figure 12B) and Sanger sequencing. The results showed that the PCR products from the albino seedlings were not completely digested, while the PCR products from the tissue culture green seedlings showed three types of digestion: complete digestion, partial digestion, and complete indigestion. The statistical results for each type of plantlet are shown in Table 1. 1) PDS copies a and b were completely edited in albino plants (M0-1, -2, -4, -5, -9, and -11); 2) PDS copies a and b were not edited in tissue culture green plantlets (M0-3 and -6) in which the PCR product was completely digested with HinfI; 3) PDS copies a and b were partially edited in tissue culture green plantlets (M0-7, -10, -12, -13, -15, -23, -28, -34, -35, 39, and 41) in which the PCR product was partially digested with HinfI; and 4) PDS copies a and b were both edited in tissue culture green plantlets (M0-8, -18, -21, -22, -27, -29, -31, -32, -33, -36, and -42) in which the PCR product was not completely digested with HinfI. The phenotype of such plants is green, and the PDS function is not completely lost due to mutations, but rather to non-frameshift editing types, e.g., d3, d6, d9, etc., where d stands for deletion.

[0130] To detect the virus content of tissue culture explants, total RNA was extracted from the M0 explants and subjected to RT-PCR using SYNV primers. The results showed that a small number of regenerated seedlings had already eliminated the virus, allowing the generation of non-transgenic explants that had been virus-free at the M0 generation, such as M0-7 and M0-42 (Figure 12C). Example 11: Verification that Cas9-generated mutations can be stably transmitted to offspring plants

[0131] To determine whether mutations can be transmitted from the M0 generation to the M1 generation, seeds from green seedlings (M1 generation) were collected from the M0 generation. Because albino plants cannot flower and set seeds, we selected the fourth type of plant in Example 10, i.e., M0 plantlets in which PDS copies a and b were fully edited and contained mutations but not frameshift editing types, for progeny analysis. The progeny of 11 M0 lineages, including M0-8, were sown on MS medium. The results showed that the M1 generation plantlets exhibited two types of segregation: 1) when the M0 generation genotype contained a single mutation in either PDS a or b but not a frameshift editing type, the progeny segregation ratio was 3:1; and 2) when the M0 generation genotype contained a single mutation in PDS a and b but not a frameshift editing type, the progeny segregation ratio was 15:1 (Figure 13).

[0132] We will use M0-8 as an example to explain the inheritance of editing types. The genotype of this plant was d1i1d5d3 (Figure 14A), where d represents deletion, i represents insertion, and d3 represents a mutation, but not a frameshift, in the editing type. After screening 114 M1 generation plantlets of M0-8, 84 green seedlings and 30 albino seedlings emerged, essentially conforming to the 3:1 Mendelian rule. Four albino seedlings and six green seedlings were randomly selected for PCR / RE analysis and sequencing (Figure 14B). The results showed that the PDS editing type in the M1 generation plants was completely derived from the M0 generation, with no new editing types emerging. The PDS target site in albino seedlings was fully edited and frameshifted (e.g., M8-1, genotype d1d1d5d5). Green seedlings contained a mutant, but non-frameshift, editing type d3, which did not completely lose PDS function (e.g., M8-9, genotype d1i1d5d3). Furthermore, we further examined the M1 generation genotypes and phenotypes (Table 2) using selected lines, including M0-18, M0-22, M0-29, and M0-42. The results showed that all editing types in the M0 generation plants were stably inherited in the M1 generation, and that the transmission of editing types conformed to Mendelian law.

[0133] To detect the transmission rule of mutations from the M1 generation to the M2 generation, seeds from the M1 generation (M2 generation) were collected and the editing status of the M2 generation plantlets was detected. Taking M8-9 as an example, the genotype was d1i1d5d3. 151 M2 generation plants were detected, and 116 green seedlings and 35 albino seedlings emerged, basically conforming to the 3:1 Mendelian law. Five albino seedlings and five green seedlings were randomly selected for PCR / RE analysis and sequencing analysis. The test results showed that the editing type of the PDS gene in the M2 generation plantlets was completely derived from the M1 generation, and the genotypes of the albino seedlings were all mutation and frameshift editing types, for example, M8-9-1, genotype is d1d1d5d5, and the genotypes of the green seedlings included the editing type d3, which was mutation but not frameshift, for example, M8-9-6, genotype is i1i1d5d3 (Figure 14C).

[0134] To further verify the transmission of edited genes from the M1 generation to the M2 generation, homozygous plantlets were selected in the M1 generation. For example, M8-3 had the genotype i1i1d3d3. Ten M2 generation plantlets from this line were randomly selected and sequenced for verification. The results showed that the genotypes of all M2 generation plantlets were i1i1d3d3. Furthermore, M1 generation homozygous plantlets M18-3, M22-4, M29-5, and M42-4 were selected for genotyping of the offspring. The results showed that all M2 generation plantlets from the above lineages were homozygous, indicating that edited genes were stably inherited from the M1 generation to the M2 generation and that no new edited genes were produced (Table 3).

[0135] The above results demonstrated that the editing types of tissue culture regenerated plants obtained using the SYNV editing system can be stably inherited to offspring (see Tables 4 and 5 for editing types), and that the inheritance laws follow Mendelian laws. Example 12: Verifying removal of viral vectors from Cas9-edited M1 generation plants

[0136] Currently, there are no reports that the plant lavender virus can be transmitted via seeds. In this example, we further examined whether SYNV virus infection was present in the M1 generation edited plantlets. Eight plant lines, namely, M0-8, -18, -22, -19, -42, -6, -7, and -42, were selected. Four to five M1 generation plantlets from each line were randomly selected, total RNA was extracted, and RT-PCR was performed using SYNV-specific primers. The test results showed that all M1 generation plantlets were SYNV-free, and the resulting T1 generation plantlets were all virus-free, non-transgenic edited plantlets (Figure 15). Example 13: Off-target analysis of SYNV CRISPR / Cas9 editing vectors

[0137] To detect off-target effects in the SYNV editing system, PDS target site 1 was selected for off-target analysis. Potential off-target loci were predicted for PDS target site 1 using the Cas-OFFinder online tool (http: / / www.rgenome.net / cas-offinder / ). The results revealed one potential off-target locus with two mismatched bases, two potential off-target loci with three mismatched bases, and 93 potential off-target loci with four mismatched bases. 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. A total of 13 potential off-target loci were selected for analysis (Figure 16A).

[0138] The T7E1 method was used to detect off-target activity at PDS target site 1. Because the virus is constantly present during the growth of tissue culture-derived plantlets, potentially resulting in a higher off-target probability, the tissue culture albino plantlet M0-11 was selected for off-target detection. PCR amplification of the above-mentioned potential off-target loci was performed using the primers listed in Table 8. The PCR products for all potential sites in M0-11 were cleaved by T7E1 enzyme, and all were identical in brightness to the bands in the wild-type control plantlet. No bands recognized or cleaved by the T7E1 endonuclease were observed. This result demonstrated that none of the above-mentioned potential off-target loci were off-target. To further verify this result, PCR / RE analysis was performed on the above-mentioned potential off-target loci using HinfI endonuclease. Except for 2 and 3 of the 13 potential off-target loci, all of the remaining sites contained HinfI enzyme cleavage sites. Therefore, after HinFI enzyme cleavage of the above-mentioned 11 potential off-target loci, all sites were completely cleaved with the enzyme, and the enzyme cleavage bands were essentially identical to those of the wild-type control, with no off-target bands being observed (Figure 16B). The above results demonstrated that the SYNV vector-based CRISPR / Cas9 genome editing system has relatively high specificity in plants. Example 14: Recombinant SYNV CRISPR / Cas9 Vector Friction Inoculation Passage and Progeny Virus Stability Analysis

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

[0140] Western blot analysis of total protein extracted from systemic leaves of the above-mentioned inoculated plantlets revealed that the infected plantlets contained large amounts of the viral structural proteins N, P, M, and G. Both SYNV-Cas9 and SYNV-tgtGFP1-Cas9-infected plantlets expressed Cas9 protein. Compared to control plantlets, SYNV-tgtGFP1-Cas9-infected plantlets expressed significantly less GFP protein (Figure 17B). RT-PCR analysis of total RNA extracted from systemic leaves of these inoculated plantlets revealed that the gRNA transcription unit and Cas9 nuclease sequence were stably present in the progeny viral genome in SYNV-tgtGFP1-Cas9-infected plantlets (Figure 17C), indicating that the virus remained stable even after friction passage.

[0141] We measured the editing efficiency of the GFP target using PCR / RE method. We found that in three randomly selected plantlets inoculated with SYNV-tgtGFP1-Cas9, mutations occurred at the GFP target site to different degrees, with the maximum editing efficiency reaching approximately 83% (Figure 17D). Example 15: Construction of SYNV CRISPR / Cpf1 editing vector

[0142] The process involves simultaneously inserting crRNA and Cpf1 into the SYNV genome in the form of independent expression frames, with the insertion location including, but not limited to, between the leader and N genes. The construction process of the SYNV-based CRISPR / Cpf1 genome editing vector is detailed below, taking the simultaneous insertion of the crRNA transcription unit and Cpf1 expression frame between the leader and N genes as an example (Figure 18A). The constructed clones are as follows: 1) SYNV-Cpf1: SYNV vector expressing only the Cpf1 protein, which served as a control. 2) SYNV-crRNA-Cpf1: A SYNV vector that accurately transcribes crRNA and accurately expresses Cpf1 protein. The specific steps to construct a clone are as follows: 1) SYNV-Cpf1

[0143] The primers PvuI / F and N5'UTR / R, Cpf1 / F and Cpf1 / R, and NPJ / F and N-Bsu36I / R in Table 6 were used to detect SYNV-GFP. le / N , YQ230, SYNV-GFP le / N Using this as a template, we amplified the PvuI-N5'UTR fragment, the Cpf1 ORF fragment, and the NPJ-Bsu36I fragment (Figure 18A). 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 sequence from the NPJ sequence to the Bsu36I enzyme cleavage site of the N gene. These fragments each contain 15-20 bp of homology with the adjacent fragments. Fragment 1, PvuI-N5'UTR, and Fragment 3, NPJ-Bsu36I, each contain sequences homologous to the linearized SYNV vector after double digestion with PvuI and Bsu36I. Therefore, the SYNV-Cpf1 vector can be obtained by sequentially ligating these three fragments by in-fusion ligation. 2) SYNV-crRNA-Cpf1

[0144] The primers in Table 6, N-PvuI / F and N5'UTR / R, N5'UTR-GFP1 / F and NPJ-GFP1 / R, and NPJ / F and N-Bsu36I / R, were used to identify SYNV-GFP. le / N Using the primers themselves and SYNV-Cpf1 as a template, we amplified the PvuI-N5'UTR fragment, crRNA fragment, and NPJ-Cpf1-Bsu36I fragment (Figure 18A). The PvuI-N5'UTR fragment encompasses 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. The NPJ-Cpf1-Bsu36I fragment encompasses the sequence between the NPJ region and the Bsu36I site of the N gene, including the Cpf1 sequence. These fragments undergo in-fusion recombination to obtain the vector SYNV-crGFP1-Cpf1. Due to the viral transcriptional characteristics, a crRNA transcript containing viral-derived sequences at both ends is released, which can then be cleaved with Cpf1 to produce the correct crRNA (Figure 18B).

[0145] To construct a SYNV editing vector containing two target sites simultaneously, we synthesized the DR-guide1-DR-guide2-DR fragment according to the sequence provided in SEQ ID NO: 17 and used the same method to construct SYNV-crGFP-1 / PDS-1-Cpf1. 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 transgenic tobacco Step 1: Inoculation of Agrobacterium-infiltrated 16c transgenic tobacco with the SYNV CRIPSR / Cpf1 vector

[0146] A 23-nt target sequence (5'-AAGATACCCAGAT) capable of targeting GFP in the 16c plant line was designed by CRISPR RGEN Tools (http: / / www.rgenome.nt) online design software. CATATGAAGC-3' and 5'-AGGGAGACACCCTC GTCAAC AGG-3', the underlined parts are the corresponding enzyme cleavage sites NdeI and HincII) was selected, and clones SYNV-crGFP1-Cpf1 and SYNV-crGFP2-Cpf1 were constructed using the method of Example 15.

[0147] Using the method described in Example 1, Agrobacterium was electrophoretically transformed with the recombinant viral expression vectors SYNV-Cpf1, SYNV-crGFP1-Cpf1, and SYNV-crGFP2-Cpf1 and infiltrated into 16c transgenic tobacco plants. Approximately 30 days after inoculation, each infiltrated plant exhibited viral symptoms similar to those observed with wild-type virus, including leaf curling and yellowing of leaf veins. Furthermore, in plantlets inoculated with the SYNV editing vectors SYNV-crGFP1-Cpf1 and SYNV-crGFP2-Cpf19 under portable UV protection, the expression of green GFP fluorescence was significantly reduced, with almost no GFP expression observed in mesophyll cells, and only green guard cells. In the control plant, SYNV-Cpf1-inoculated systemic leaves, the GFP fluorescence intensity was consistent with that of wild-type 16c (Figure 19A).

[0148] To verify that the above-mentioned symptoms were due to systemic SYNV infection, systemic leaves from affected plantlets were collected, total leaf protein extracted, and Western blot analysis was performed using SYNV polyclonal antibody, Flag tag antibody, and GFP monoclonal antibody, respectively. The results showed that SYNV viral protein bands and Cpf1 protein bands were detected in all plantlets inoculated with the SYNV editing vector, and GFP protein expression levels were significantly reduced. The GFP protein expression levels in tobacco leaves inoculated with the control vector SYNV-Cpf1 were essentially consistent with those of wild-type 16c plantlets (Figure 19B). These results demonstrate that the SYNV viral vector can simultaneously accommodate both the crRNA and Cpf1 expression frames, and that the recombinant viral vector containing the above-mentioned exogenous expression frames still possesses systemic infectivity. Step 2: GFP target site mutation efficiency analysis

[0149] To verify the editing efficiency of the GFP target, PCR / RE was used to measure the GFP gene editing efficiency in systemic leaves of the inoculated plants. PCR was performed on target site DNA using primers GFP / F and GFP / R (Table 7). Enzyme digestion analysis was performed using the corresponding restriction endonucleases NdeI and HincII in the target sequence. Mutations occurred to varying degrees in GFP target 1 and target 2, with mutation efficiencies reaching approximately 90% (Figure 20A). To determine the editing types of the GFP target, the PCR amplification products were cloned and ligated into a T vector. Ten positive monoclonals were selected from each target site and subjected to Sanger sequencing. Seven and eight monoclonals were generated by mutations in GFP target 1 and target 2, respectively (Figure 20B), which was essentially consistent with the PCR / RE results. The majority of the edits generated were single- or multiple-base deletions (Figure 20C). Example 17: SYNV CRISPR / Cpf1 vector targets the endogenous PDS gene in N. benthamiana The N. benthamiana endogenous PDS gene was used to further validate the SYNV CRISPR / Cpf1 viral vector editing system of the present invention.

[0150] A 23-nt target sequence (PDS1: 5'-TGCCGTTAATTTGA) capable of targeting the PDS gene was designed using the CRISPR RGEN Tools (http: / / www.rgenome.nt) online design software. GAGTC CAAG-3', PDS-2:5'- GTAGTAGCGACT CCATGG The target genes were selected as GGCAT-3' (the underlined parts are the corresponding enzyme cleavage sites, HinfI and NcoI), and both of these targets could simultaneously target PDS copies a and b in heterotetraploid Nicotiana benthamiana (Figure 21A). Recombinant virus clones SYNV-crPDS1-Cpf1 and SYNV-crPDS2-Cpf1, capable of targeting the above targets, were constructed according to the method described in Example 2.

[0151] Using the method of Example 1, the recombinant virus expression vectors SYNV-crPDS1-Cpf1 and SYNV-crPDS2-Cpf1 were electrophoretically transformed into Agrobacterium, which was then inoculated into N. benthamiana by infiltration. Thirty days after inoculation of the recombinant virus expression vectors into N. benthamiana, symptoms such as leaf curling and yellowing of leaf veins were observed, similar to those observed with the wild-type virus (Figure 21B).

[0152] The PCR / RE method was used to measure the target editing efficiency of PDS. All primers used were capable of simultaneously amplifying PDS copies a and b. The test results showed that the two target sites of PDS generated different degrees of mutation, with the maximum editing efficiency reaching approximately 79% (Figure 21C). Taking target 1 as an example, the editing efficiencies of three randomly selected plant lines were 79%, 70%, and 73%, respectively.

[0153] Sanger sequencing was used to verify the editing status of PDS target site 1. PDS copies a and b were amplified using the PDS-specific primers listed in Primer Table 7, the PCR products were ligated into a T vector, and 10 clones per copy were analyzed. The results showed that the number of mutant clones in PDS copies a and b was 8 and 7, respectively, which was essentially consistent with the editing efficiency measured by PCR / RE. The editing types were mainly single-base and multiple-base deletions (Figure 21D). Example 18: SYNV CRISPR / Cpf1 multi-targeting vector simultaneously targets GFP and PDS genes

[0154] To verify whether the SYNV vector-based CRISPR / Cpf1 system can perform multitarget editing of plant genomes, we constructed a SYNV double-target editing vector using a tandem DR-guide1-DR-guide2-DR strategy. The primary transcripts then underwent Cpf1 processing, which removed excess non-coding sequences and released precise crRNAs (Figure 22).

[0155] The 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 described in Example 1, the recombinant rhabdovirus expression vector SYNV-crGFP-1 / PDS-1-Cpf1 was electrophoresed into Agrobacterium, which was then inoculated into N. benthamiana by infiltration. 30 days after inoculation of the recombinant rhabdovirus expression vector into N. benthamiana, symptoms such as leaf curling and yellowing of the vein, similar to those of the wild-type virus, were observed (Figure 23A).

[0156] The PCR / RE method was used to measure the target editing efficiencies of GFP and PDS, respectively. The 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). These results demonstrated that the SYNV vector can be used for simultaneous editing of multiple targets in the plant genome without affecting the editing efficiency of a single target (Figure 23B). Example 19: Regeneration of SYNV CRISPR / Cpf1-infected leaf tissue and M0 generation genotype analysis

[0157] To obtain regenerated plantlets, systemic leaves infected with the recombinant viral vector SYNV-crPDS1-Cpf1 were collected and regenerated by tissue culture according to the method described in Example 10.

[0158] To detect the editing status of the PDS target site in tissue culture plantlets, DNA was extracted from M0 generation albino and green plantlets and subjected to PCR / RE analysis (Figure 24B) and Sanger sequencing. The results showed that the PCR products of albino plantlets were not completely digested, while the PCR products of tissue culture green plantlets showed three types of digestion: complete digestion, partial digestion, and complete indigestion. The results of Sanger sequencing of each type of plantlet are shown in Tables 9 and 10. 1) PDS copies a and b were completely edited in albino plantlets (M0-1, -2, -3, -4); 2) PDS copies a and b were not edited in tissue culture green plantlets (M0-5, -7, -10, -12, -20, -25, -27, -28, -30, -31) whose PCR products were completely digested with HinfI. 3) In tissue culture green plantlets where the PCR product was partially cleavable with HinfI (M0-6, -8, -13, -18, and -19), PDS copies a and b were partially edited; and 4) in tissue culture green plantlets where the PCR product was not completely cleavable with HinfI (M0-9, -11, -14, -15, -16, -17, -21, -22, -23, -24, 26, and -29), PDS copies a and b were both edited. These plantlets were phenotypically green, and the lack of complete loss of PDS function was primarily due to mutations, but not frameshifts, resulting in edit types such as d3, d6, and d9 (where d stands for deletion).

[0159] To detect the virus content of tissue culture explants, total RNA was extracted from the M0 generation explants and subjected to RT-PCR using SYNV primers. The results showed that some regenerated seedlings had already eliminated the virus, and non-transgenic edited explants that had been virus-free at the M0 generation, such as M0-6, M0-17, M0-18, M0-22, M0-23, M0-24, M0-27, and M0-28, were obtained (Figure 24C). Example 20: Verification that mutations generated by Cpf1 can be stably transmitted to offspring plants

[0160] Seeds from green seedlings (M1 generation) edited with SYNV-crPDS1-Cpf1 in the M0 generation were collected, and the fourth type of plantlets described in Example 19, i.e., M0 generation plantlets in which PDS copies a and b were fully edited and contained a mutation but a non-frameshift editing type, were selected for progeny analysis. The progeny of this type of M0 plantlet was sown on MS medium. After sprouting into the M1 generation, three types of segregation patterns emerged: 1) when the M0 generation PDSa or b genotype contained a homozygous non-frameshift editing type, there were no albino seedlings, as in the M0-15 progeny; 2) when the M0 generation genotype contained a single mutation but a non-frameshift editing type, the segregation ratio in the progeny was 3:1; and 3) when the M0 generation genotype contained a single mutation but a non-frameshift editing type, the segregation ratio in the progeny was 15:1 (Figure 25).

[0161] The above results demonstrated that the edited type of tissue culture regenerated plantlets obtained using the SYNV editing system can be stably inherited to subsequent generations, and that the inheritance laws follow Mendelian laws. Example 21: SYNV editing vectors for G protein N-terminal deletion or substitution enable site-specific editing of the Nicotiana 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 mosaicked on the surface of virus particles in a trimer form. Existing research results have revealed that the G protein plays an important role in the virus-vector recognition and transmission process, and that deletion or mutation of the SYNV G protein may affect the virus's insect vector recognition or transmission, resulting in higher biological safety.

[0163] The SYNV G protein N-terminal domain was deleted (SYNV-RFP-dGN) or the N-terminal domain was transfected with the reporter gene mCherry (SYNV-RFP-mCherry:G N), and then SYNV-tgtPDS1-Cas9 was cleaved with a single enzyme cleavage site, Bsu36I, on N and a single enzyme cleavage site, NheI, on P, to recover the Bsu36I-NheI fragment, and the linearized vector SYNV-RFP-dG was generated via Bsu36I and NheI. N and SYNV-RFP-mCherry:G N The resulting clone was SYNV-tgtPDS1-Cas9-dG N and SYNV-tgtPDS1-Cas9-mCherry:G N (Figure 26A).

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

[0165] To verify the targeted editing efficiency of the above vectors against PDS, PCR / RE was used to detect the systemic leaves of the inoculated plants. All primers used were capable of simultaneously amplifying PDS copy a and copy b. The test results showed that all of the above recombinant vectors were able to produce effective editing against PDS target site 1, and the editing efficiency was comparable to that of existing SYNV editing vectors (Figure 26D). Deletion or substitution of the N-terminal domain of SYNV G protein did not affect the editing efficiency of the recombinant virus. The above results demonstrated that site-specific editing of the Nicotiana benthamiana genome was achieved using SYNV editing vectors with G protein N-terminal deletion or substitution. Example 22: SYNV CRISPR / oCas9 vector targets the N. benthamiana endogenous PDS gene.

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

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

[0168] First, a SYNV vector expressing only oCas9 was constructed. The N. benthamiana codon-optimized oCas9 sequence was synthesized according to the sequence provided in SEQ ID: 36. The Bsu36I-NPJ fragment, oCas9 ORF fragment, and NPJ-NheI fragment were amplified using the primers N-Bsu36I / F and NPJ-oCas9 / R, oCas9 / F and Cas9 / R, and Cas9-NPJ / F and P-NheI / R, respectively, using SYNV, oCas9, and SYNV as templates. The Bsu36I-NPJ fragment encompasses the NPJ region from the Bsu36I enzyme cleavage site on the N gene downstream, and the NPJ-NheI fragment encompasses the NheI enzyme cleavage site from the NPJ sequence to the P gene. Each of these fragments contains 15-20 bp of homology to the adjacent fragments. Fragment 1, Bsu36I-NPJ, and fragment 3, NPJ-NheI, contain sequences homologous to the SYNV vector linearized by double digestion with Bsu36I and NheI, respectively. The three fragments were sequentially ligated by in-fusion ligation to obtain the SYNV-oCas9 vector. Next, the N / Bsu36I-PDS2 fragment and the PDS2-oCas9 / Bsu36I fragment were amplified using primers N-Bsu36I / F and P2 20nt / R, and P2 20nt / F and oCas9 / Bsu36I / R (Table 6) as templates for SYNV-tgtPDS2-Cas9 and SYNV-oCas9, respectively. These fragments were then in-fusion ligated with the SYNV-oCas9 vector linearized with Bsu36I, resulting in the vector SYNV-tgtPDS2-oCas9.

[0169] Following the method described in Example 1, Agrobacterium was electrophoretically transformed with the recombinant viral editing vector SYNV-tgtPDS2-oCas9 and infiltrated into Nicotiana benthamiana plants. Simultaneously, SYNV-tgtPDS2-Cas9 was inoculated as a control. Thirty days after inoculation, each inoculated plant exhibited viral symptoms, such as leaf curling and yellowing of leaf veins, similar to those observed with wild-type virus (Figure 27B). To verify the targeted editing efficiency of the above-mentioned vectors against the PDS, 10 systemically infected plantlets were randomly selected and subjected to PCR / RE analysis. The primers used were capable of simultaneously amplifying PDS copy a and copy b. The results showed that all of the above-mentioned recombinant vectors were able to effectively edit PDS target site 1, with editing efficiencies comparable to those of existing SYNV editing vectors (Figure 27C). These results demonstrate the feasibility of species-specific codon optimization for Cas nucleases. Example 23: SYNV CRISPR / Cas9 editing system based on a SYNV single transcription unit editing vector

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

[0171] According to the method described in Example 1, the single transcriptional unit editing vector SYNV-oCas9gPDS2 was electrophoretically transfected into Agrobacterium, which was then inoculated into Nicotiana benthamiana plants. At the same time, an existing SYNV-tgtPDS2-oCas9 clone was inoculated as a control. Around 30 days after inoculation, each of the inoculated plants showed symptoms such as leaf curling and yellowing of the leaf veins, similar to those seen with the wild-type virus (Figure 28B).

[0172] The PCR / RE method was used to detect the mutation efficiency of the PDS target, and the primers used were capable of simultaneously amplifying PDS copy a and copy b. The results showed that the single-transcription unit SYNV editing vector was able to effectively edit PDS target site 2, with editing efficiency comparable to that of existing double-transcription unit SYNV editing vectors (Figure 28C). These results demonstrate the feasibility of using the same transcription unit to express sgRNA and Cas9 nuclease in a SYNV editing vector. Example 24: Measurement of the infectivity of EMDV fluorescent expression vectors in Nicotiana benthamiana Step 1: Construction of EMDV fluorescent expression vector

[0173] The process involves inserting the GFP or RFP fluorescent reporter gene into the EMDV genome in the form of an independent expression frame, and the insertion position includes, but is not limited to, between N and X. The following describes the construction process of the EMDV fluorescent expression vector in detail, taking 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 vectors are as follows: EMDV-GFP: An EMDV fluorescent expression vector carrying the green fluorescent protein gene GFP between the EMDV leader and N genes. EMDV-RFP: an EMDV fluorescent expression vector carrying the red fluorescent protein gene RFP between the EMDV N and X genes Specifically, the clone construction method is as follows:

[0174] For EMDV-GFP, primers EMDV-AatII / F and EMDV-N3' / R, and EMDV-N5' / F and EMDV-AatII / R in Table 6 were used to amplify EMDV MR and EMDV-WT, respectively, to generate the EMDV-GFP fluorescent expression vector by multi-fragment in-fusion ligation with the EMDV-WT vector backbone, which had been digested with AatII and recovered from the vector backbone (Figure 29A).

[0175] EMDV-RFP was amplified using the primers EMDV-SpeI / F and EMDV-A / R, EMDV-RFP / F and EMDV-RFP / R, and EMDV-C / F and EMDV-SpeI / R in Table 6, respectively, to obtain 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 region to near the SpeI enzyme cleavage site on the M gene. These fragments, together with the EMDV-WT vector backbone digested and recovered by single SpeI enzyme cleavage, were ligated by multi-fragment in-fusion to obtain an EMDV-RFP fluorescent-tagged vector (Figure 29A). Step 2: Infiltrate Nicotiana benthamiana with Agrobacterium and inoculate it with the EMDV fluorescent expression vector

[0176] According to the method described in Example 1, Agrobacterium was electrophoretically infected with the recombinant rhabdovirus expression vector EMDV-GFP or EMDV-RFP, and then inoculated into N. benthamiana plants. For N. benthamiana plants inoculated with EMDV-GFP, 23 days after inoculation, diseased plantlets showed phenotypes such as whole-body leaf curl, yellowing of leaf veins, and significant plant dwarfism. Under a fluorescence microscope, the whole systemic leaf infected with the virus emitted relatively strong green fluorescence (Figure 29B). For N. benthamiana plants inoculated with EMDV-RFP, approximately 30 days after inoculation, diseased plantlets showed viral symptoms similar to those of the wild-type virus, such as whole-body leaf curl and plant dwarfism. Furthermore, due to the overexpression of red fluorescent protein, the stems, upper systemic leaves, and petals of the plantlets also exhibited a pale red color under visible light, and these plant tissues emitted strong red fluorescence under a fluorescence microscope (Figure 29C).

[0177] To verify that the above-mentioned symptoms were due to systemic EMDV infection, systemic leaves from affected plantlets were harvested, total leaf protein extracted, and Western blot analysis was performed using EMDV virion antibodies and GFP or RFP monoclonal antibodies, respectively. The results showed that EMDV-GFP-inoculated plantlets were able to detect EMDV viral structural protein bands and GFP protein bands (Figure 29B), while EMDV-RFP-inoculated plantlets were able to detect EMDV viral structural protein bands and RFP protein bands (Figure 29C). These results demonstrate that EMDV carrying GFP or RFP fluorescent proteins can successfully achieve systemic infection in N. benthamiana and express high amounts of the fluorescent proteins in various tissues without affecting the infection characteristics of EMDV itself, providing great convenience for subsequent EMDV inoculation assays on various crops. Example 25: Measurement of the infectivity of EMDV fluorescent expression vectors to tobacco, potato, eggplant, and tomato

[0178] Previous research has revealed that some researchers have isolated EMDV virus under natural conditions in several agricultural and economic crops, including potato, tobacco, tomato, eggplant, and cucumber. Using EMDV-RFP or EMDV-GFP recombinant viruses rescued from Nicotiana benthamiana, infection tests were conducted on potato, tobacco, tomato, and eggplant using methods such as mechanical friction, stem injection, spray gun injection, and grafting. The specific results are as follows:

[0179] In tobacco, EMDV-RFP recombinant virus can be systemically infected by mechanical friction. Approximately 30 days after inoculation, systemic disease develops, with yellowing of the veins in the systemic leaves of affected plantlets. Approximately 45 days later, the yellowing of the veins becomes more severe, and twisted and deformed leaves appear in the systemic leaves. Under a fluorescence microscope, both the systemic leaves and tobacco stems with relatively severe symptoms emit strong red fluorescence. Western blot analysis detects EMDV viral structural proteins and RFP red fluorescent protein bands in affected tobacco systemic leaves (Figure 30A).

[0180] In the case of potato, virus transmission and systemic infection of potato scions can be achieved through heterologous grafting using virus-containing N. benthamiana rootstocks. First, healthy N. benthamiana and potato plants of the appropriate seedling age are selected and used. The upper tissue of the N. benthamiana is removed, and the N. benthamiana with 3-4 leaves is used as the rootstock. The stem tip of a young potato in good condition is used as the scion. The cross section of the circular stem of the rootstock is cut along the diameter, and excess tissue is removed from both sides of the stem of the scion, finally forming it into a wedge shape. The wedge-shaped scion was then gently inserted into a pre-cut opening along the rootstock stem and secured in place. The grafted plantlets were carefully and completely covered with a pre-wetted thin plastic bag to slow dehydration. After incubation in a thermostatic culture chamber at 27°C / 24 h / day with high light for 7 days, the moisture-retaining plastic bag was removed. The N. benthamiana rootstock portion of relatively well-grown plantlets was inoculated with the EMDV recombinant virus by Agrobacterium infiltration and mechanical friction. After incubation at 24°C / 24 h / day with low light for approximately 1 month, the plantlets were transferred from the culture chamber and tested for virus infection. Using this method, we successfully achieved systemic infection of potato scions with EMDV-RFP, and strong red fluorescence was observed from the rootstock and scion leaves (Figure 30C). It is also possible to successfully infect eggplant, tomato, and potato with EMDV by mechanical friction (Figure 30B). Example 26: Measurement of pepper infectivity of EMDV fluorescent expression vector.

[0181] For pepper, we used a mechanical friction inoculation method. We homogenized leaves from Nicotiana benthamiana plants systemically infected with EMDV-RFP as the toxin source, obtained a virus juice, and inoculated it into pepper plants approximately 2 weeks old. The plants were cultured at 23°C under 16 h of light and 8 h of darkness. EMDV-RFP infection points were observed on the inoculated leaves 7 days after inoculation, and systemic infection symptoms appeared 25 days after inoculation. Vein yellowing due to virus infection was observed in the upper systemic leaves, and strong red fluorescence was observed under a fluorescence microscope (Fig. 31B). Western blots also detected EMDV viral structural proteins and RFP red fluorescent protein in pepper systemic leaves (Fig. 31A). Example 27: EMDV CRISPR / Cas9 vector targets the GFP gene in 16c transgenic tobacco Step 1: Construction of the EMDV-tgtGFP2-Cas9 editing vector

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

[0183] EMDV-tgtGFP2-Cas9: Contains an EMDV vector expressing sgRNA and Cas9 protein targeting GFP2 in Nicotiana benthamiana 16c transgenic lines The specific method for constructing a 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, the underlined part is the corresponding NcoI enzyme cleavage site) targeting GFP in the 16c plant strain, as in Example 3. First, an intermediate vector was constructed that was cleavable with AarI and allowed for flexible target exchange by primer annealing. The intermediate vector contained the sgRNA and full-length Cas9 sequence, including two EMDV NX junctions and two AarI enzyme cleavage sites, and was single-cleavable with AarI. Primers PDS4 / F and PDS4 / R (Table 6) were annealed to the intermediate vector using T4 cleavage. This vector was then used as a template to generate 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, and EMDV-III / F and EMDV-SpeI / R in Table 6 to obtain fragment I, which contains the 3'UTR from near the SpeI enzyme cleavage site on the N gene to the N gene; fragment II, which contains two EMDV NX junctions, the tgt-GFP2 sequence, and the Cas9 ORF; and fragment III, which contains one EMDV NX junction and the X 5'UTR from near the SpeI enzyme cleavage site on the M gene. These fragments were then fused together with the EMDV-WT vector backbone, which had been digested and recovered by single SpeI enzyme cleavage, to obtain the EMDV-tgtGFP2-Cas9 editing vector. Step 2: Infiltrate N. benthamiana with Agrobacterium and inoculate with the EMDV-tgtGFP2-Cas9 editing vector

[0185] According to the method described in Example 1, the recombinant rhabdovirus expression vector EMDV-tgtGFP2-Cas9 was electrophoretically transfected into Agrobacterium, which was then infiltrated into 16c transgenic baco. 27 days after inoculation, the infiltrated plantlets showed viral symptoms similar to those of the wild-type virus, such as systemic leaf curling and plantlet dwarfism. Furthermore, under a portable ultraviolet lamp, the green fluorescence in the veins of the systemic leaves of the plantlets was significantly weakened. 35 days after inoculation, the green fluorescence in the veins and mesophyll of the top leaves of healthy 16c plantlets was significantly weakened, while the green fluorescence in the veins and mesophyll of the top leaves remained unchanged (Figure 32B).

[0186] To verify that the above symptoms were due to systemic EMDV infection, systemic leaves from affected plantlets were harvested, total leaf protein extracted, and Western blot analysis was performed using EMDV virion antibodies and Flag tag antibodies, respectively. The results showed that EMDV viral protein bands and Cas9 protein bands (Figure 32B) were detected in plantlets inoculated with the EMDV editing vector. These results demonstrate that the EMDV viral vector is similar to the SYNV editing vector, and both can simultaneously accommodate the sgRNA expression frame and the 4.2 kb Cas9 expression frame. The recombinant rhabdovirus vector containing the above exogenous expression frame still possesses systemic infectivity. Step 3: GFP-targeted mutation efficiency analysis

[0187] To verify the editing efficiency of the GFP target, PCR / RE (Polymerase Chain Reaction / Restriction Digestion) was used to measure the systemic leaf quality of the inoculated plants. PCR was performed using primers GFP2 / F and GFP2 / R (Table 7), followed by enzyme digestion analysis using the corresponding restriction endonuclease NcoI in the target sequence. The results showed that the selected GFP2 target generated relatively high gene mutation efficiency, with the editing efficiency calculated from the NcoI digestion results being approximately 90%. To detect the GFP editing type, the PCR amplified product was ligated into a T vector, and monoclonal clones were isolated and subjected to Sanger sequencing. Results showed that eight of the nine monoclonal colonies detected showed different types of base mutations, which was generally consistent with the PCR / RE detection results. The editing types generated consisted mainly of single- and multiple-base deletions, with some base insertions (Figure 32C). Example 28: EMDV CRISPR / Cas9 vector targets the PDS gene in Nicotiana benthamiana Step 1: Construction of the EMDV-tgtPDS4-Cas9 editing vector

[0188] To test whether the EMDV CRISPR / Cas9 editing vector, like the EMDV fluorescent expression vector, can successfully infect multiple crops such as potato and tomato and produce the corresponding types of editing in the crops, a conserved 20-nt target sequence (5'-CAATACAGTTAACTATTTGG-3', SEQ ID NO:37) in the PDS genes of the above crops was selected and inserted between the EMDV N and X genes in the same manner as a single transcription unit. The resulting vector is shown below.

[0189] EMDV-tgtPDS4-Cas9: Contains an EMDV vector expressing sgRNA and Cas9 protein targeting multiple plant PDS genes, PDS4.

[0190] The construction process of the EMDV-tgtPDS4-Cas9 editing vector was essentially the same as that of EMDV-tgtGFP2-Cas9, except that the II fragment was replaced with the II-PDS4 fragment (Figure 33A). Step 2: Editing the PDS gene in N. benthamiana with the EMDV-tgtPDS4-Cas9 editing vector

[0191] Agrobacterium was electrophoretically transformed with the EMDV-tgtPDS4-Cas9 vector and inoculated into wild-type N. benthamiana plants. At 40 days postinoculation, the infected plants also showed viral symptoms, including systemic leaf vein yellowing, leaf curl, and plantlet dwarfism. Western blot analysis of infected leaves revealed the corresponding viral structural protein bands (Figure 33B). Two pairs of primers, PDS-4a / F and PDS-4a / R, and PDS-4b / F and PDS-4b / R, listed in Table 7, were used to amplify the two PD alleles, PDS-a and PDS-b, respectively, in N. benthamiana plants. Following T7EI digestion, both alleles demonstrated some degree of gene editing (Figure 33C). Example 29 EMDV CRISPR / Cas 9 infected leaf tissue regeneration and M 0 generation genotyping.

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

[0193] To measure the editing status of the PDS target site in regenerated plantlets, total DNA was extracted from M0 generation seedlings and subjected to PCR / RE analysis (Figure 34B). 100 ng of the wild-type PDS fragment amplification product was mixed with 100 ng of M0 generation seedling PDS fragment amplification primers, annealed, and subjected to T7EI enzyme digestion analysis. Target site editing was detected in all albino seedlings, and target site editing was detected in M0-19, 20, 21, and 27 plantlets among the green seedlings.

[0194] To detect whether the regenerated plants were persistently infected with the EMDV-edited vector, total RNA from the M0 generation plants was extracted and subjected to RT-PCR using EMDV primers. All albino seedlings carried the EMDV vector, while some regenerated green seedlings, such as M0-19 and -27, contained the edited vector but had already been cleared of the virus (Figure 34C). This means that virus-cleared, non-transgenic, edited plantlets could be obtained in the M0 generation. Example 30: EMDV-tgtPDS4-Cas9 editing vector edits tobacco, potato, and tomato PDS genes

[0195] The EMDV-tgtPDS4-Cas9 recombinant virus rescued from N. benthamiana was inoculated into tobacco, potato, and tomato plants, respectively, as described in Example 24. After systemic infection, fragments corresponding to the PDS gene of tobacco, potato, and tomato were amplified using primers PDS-Nt / F and PDS-Nt / R, PDS-ST / F and PDS-ST / R, and PDS-SL / F and PDS-SL / R, respectively, and ligated into T vectors. Six to seven monoclonal antibodies were isolated from each plant and sent for testing. Mutations at the PDS4 target site in these plants were detected, primarily single- and multiple-base deletions and base substitutions (Figure 35). The above results demonstrated that the EMDV editing vector successfully achieved site-specific genome editing in tobacco, potato, and tomato. Table 1: Genotypic and phenotypic analysis of SYNV CRISPR / Cas9 vector editing plant line M0 generation Table 2: Genotypic and phenotypic analysis of SYNV CRISPR / Cas9 vector editing in the M1 generation of plantlets. JPEG0007824611000002.jpg145170Table 3: SYNV CRISPR / Cas9 edited plant M2 generation plant genotype analysis JPEG0007824611000003.jpg39170Table 4: List of editing types that SYNV CRISPR / Cas9 uses to edit PDS-1 targets in the M0 generation of plantlets. JPEG0007824611000004.jpg195170Table 5. List of editing types of SYNV CRISPR / Cas9-mediated PDS editing in M1 generation plantlets JPEG0007824611000005.jpg221170Table 6. Clone construction primers and RT-PCR and cRT-PCR detection primers JPEG0007824611000006.jpg216170JPEG0007824611000007.jpg175170Note: The sequences in lowercase are 15-20 nt fragments for homologous recombination. Table 7. Primers for detecting target gene editing efficiency JPEG0007824611000008.jpg142170Note: / represents different PCR product sizes for copy a / copy b. Table 8. PDS1 Potential Off-Target Locus Detection Primers JPEG0007824611000009.jpg128170Table 9Genotypic and phenotypic analysis of the SYNV CRISPR / Cpf1 vector editing the plantlets of the M0 generation JPEG0007824611000010.jpg229170Table 10: List of editing types of PDS-1 targets edited by SYNV CRISPR / Cpf1 in M0 generation plants JPEG0007824611000011.jpg207170

[0196] Although the 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 easily modify the present invention using the contents of the specification and drawings of the present invention, and it should be understood that these modifications are included within the scope of the claims of the present invention.

[0197] It should be noted that the specific technical features described in the above specific embodiments can be combined in any suitable manner if not contradictory, and various possible combination manners will not be described here in order to avoid unnecessary duplication.

[0198] Introduction of the nucleotide sequences 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 sequences SEQ ID NOs 34-35: sequences derived from NPJ SEQ ID NO 36: oCas9 sequence codon-optimized for bean syrup

[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. 1. A method for modifying the genetic material of a plant cell that does not require the introduction of a foreign gene sequence into the genome of the cell to be modified, comprising: a) providing at least one plant cell to be modified with genetic material; b) infecting the plant cells with a recombinant plant rhabdovirus vector capable of systemic infection, wherein the recombinant rhabdovirus capable of systemic infection 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 viral vector is sonchus yellow net virus (SYNV) or eggplant mottled dwarf virus (EMDV); when the viral vector is sonchus yellow net virus (SYNV), the plant is tobacco; when the viral vector is eggplant mottled dwarf virus (EMDV), the plant is tobacco, potato, or tomato; the sequence-specific endonuclease is a CRISPR / Cas nuclease; A method for modifying the genetic material of a plant cell, wherein the process of modifying the genetic material of the plant cell is cleavage-induced by a sequence-specific endonuclease and completed by the plant's endogenous DNA repair machinery.

2. 1. A method for producing a plant having modified genetic material that does not require the introduction of a foreign gene sequence into the genome of the plant cell to be modified, comprising: a) providing at least one plant cell to be modified with genetic material; Infecting the plant cells with a recombinant plant rhabdovirus vector capable of systemic infection, wherein the recombinant rhabdovirus capable of systemic infection carries a ribonucleic acid sequence encoding at least one sequence-specific endonuclease, wherein the sequence-specific endonuclease specifically targets a plant genome nucleic acid sequence and cleaves the target site; c) obtaining a plant from the cells in which the genetic material has been modified; and step d) selecting modified plant subjects containing said genetic material without the need for a selection marker; the viral vector is sonchus yellow net virus (SYNV) or eggplant mottled dwarf virus (EMDV); when the viral vector is sonchus yellow net virus (SYNV), the plant is tobacco; when the viral vector is eggplant mottled dwarf virus (EMDV), the plant is tobacco, potato, or tomato; A method for producing a plant having modified genetic material, wherein the sequence-specific endonuclease is a CRISPR / Cas nuclease.

3. The method of claim 1, wherein the method of infecting the cells to be modified includes natural infection of uninoculated cells with the recombinant virus, friction inoculation, grafting, insect-mediated transmission, or any other viral infection method.

4. The method of claim 1, wherein the rhabdovirus vector capable of systemic infection comprises a derived virus vector after one or more types of mutation and / or recombination modification.

5. 5. The method of claim 4, wherein the inducible viral vector is an inducible viral vector in which the glycoprotein (G) amino-terminal domain has been mutated and / or recombinantly modified.

6. 2. The method of claim 1, wherein the modification of the plant cell genetic material is selected from at least one base deletion, one base insertion, one base substitution, or a combination of these modification patterns at the target site.

7. 2. The method of claim 1, wherein the ribonucleic acid sequence encoding the sequence-specific endonuclease comprises a nucleic acid sequence for one or more guide RNAs and a nucleic acid sequence for a Cas nuclease.

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

9. 8. The method of claim 7, wherein the transcription of the sequence-specific endonuclease sequence is controlled by a regulatory element for rhabdovirus messenger RNA transcription.

10. 8. The method of claim 7, wherein the guide RNA and the Cas nuclease sequence are controlled by the same transcription unit.

11. 8. The method of claim 7, wherein the guide RNA and the Cas nuclease sequence are controlled by different transcription units.

12. 8. The method of claim 7, wherein the guide RNA transcript has virus-derived terminal sequences removed by the cell's endogenous tRNA processing machinery.

13. 8. The method of claim 7, wherein the guide RNA transcript has viral-derived terminal sequences removed by Cas nuclease processing.

14. 2. The method of claim 1, wherein the plant is selected from Nicotiana benthamiana, potato (Solanum tuberosum), tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), and eggplant (Solanum melongena).

15. 1. An infectious recombinant plant rhabdovirus vector, comprising: the vector comprises a polynucleotide sequence encoding at least one sequence-specific endonuclease, the sequence-specific endonuclease being transiently expressed in a cell infected with the viral vector and specifically targeting a plant genomic nucleic acid sequence, the target sequence being modified by the nuclease; the viral vector is sonchus yellow net virus (SYNV) or eggplant mottled dwarf virus (EMDV); when the viral vector is sonchus yellow net virus (SYNV), the plant is tobacco; when the viral vector is eggplant mottled dwarf virus (EMDV), the plant is tobacco, potato, or tomato; An infectious recombinant plant rhabdovirus vector, wherein the sequence-specific endonuclease is a CRISPR / Cas nuclease.

16. The infectious recombinant plant rhabdovirus vector of claim 15, characterized in that the infectious recombinant plant rhabdovirus vector comprises one or more types of mutation and / or recombinantly modified derivative virus vectors.

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

18. The infectious recombinant plant rhabdovirus vector of claim 15, characterized in that the sequence carried by the rhabdovirus vector transcribes and produces a single or multiple guide RNA nucleic acid sequences and a Cas nuclease nucleic acid sequence.

19. 16. The infectious recombinant plant rhabdovirus vector of claim 15, wherein the sequence-specific endonuclease sequence is present in the rhabdovirus genome in the form of an independent transcription unit.

20. The infectious recombinant plant rhabdovirus vector of claim 18, characterized in that the nucleic acid sequence of the guide RNA and the nucleic acid sequence of the Cas nuclease are controlled by different transcription units.

21. An infectious recombinant plant rhabdovirus vector as described in claim 18, characterized in that the nucleic acid sequence of the guide RNA and the nucleic acid sequence of the Cas nuclease are controlled by the same transcription unit.

22. 16. The infectious recombinant plant rhabdovirus vector of claim 15, wherein transcription of the sequence-specific endonuclease sequence is controlled by a regulatory element for rhabdovirus messenger RNA transcription.

23. The infectious recombinant plant rhabdovirus vector of claim 18, characterized in that the transcription product of the nucleic acid sequence of the guide RNA has virus-derived terminal sequences removed by the cell's endogenous tRNA processing machinery.

24. The infectious recombinant plant rhabdovirus vector described in Claim 18, characterized in that the transcription product of the nucleic acid sequence of the guide RNA has virus-derived terminal sequences removed by Cas nuclease processing.

25. 16. The infectious recombinant plant rhabdovirus vector of claim 15, wherein the viral vector is a recombinant nucleic acid construct, and the sequence comprising the viral vector is operably linked to a promoter.

26. 16. The infectious recombinant plant rhabdovirus vector of claim 15, which transiently expresses the sequence-specific endonuclease in infected natural or experimental host cells.

27. 16. The infectious recombinant plant rhabdovirus vector of claim 15, wherein the plant host is selected from Nicotiana benthamiana, potato (Solanum tuberosum), tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), and eggplant (Solanum melongena).

28. Use of the infectious recombinant plant rhabdovirus vector of any one of claims 15 to 27 in plant genome editing.

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