Inhibition of target gene expression by genome editing of natural miRNAs

Abstract

The present invention relates to methods and compositions for reducing or inhibiting target gene expression through genome editing of natural miRNAs.

Description

[0001] Sequence Listing

[0002] A sequence listing in ASCII text format, which is submitted pursuant to 37 CFR §1.821, is named "81815_ST25.txt", is 47 kilobytes in size, and was generated on February 26, 2019. This sequence listing is hereby incorporated by reference into this specification for its disclosure. Technical Field

[0003] The present invention relates to methods and compositions for reducing or inhibiting target gene expression through genome editing of natural miRNAs. Background Art

[0004] MicroRNA (miRNA) transcribed and processed by a longer RNA (pre-miRNA) containing an incomplete hairpin is an RNA of about 20-24 nucleotides. MiRNA can accurately target and reduce or inhibit the expression of its mRNA target gene in a post-transcriptional manner (Yu et al. 2017, New Phytol. [New Plant Scientist] Vol. 216 (4), pp. 1002-1017; Gebert and MacRae 2019, Nature Reviews Molecular Cell Biology [Natural Molecular Cell Biology Review], Vol. 20, pp. 21-37). Compared with RNAi induced by small interfering RNA, miRNA-mediated gene expression inhibition has high specificity and effectiveness. MiRNA has been used, for example, to target exogenous RNA from pathogens by transgenic methods (e.g., WO 2010 / 123904), whereby artificial miRNA is ectopically overexpressed. This method can be effective; however, relying on the genetic transformation of plants requires a large number of transformation events to identify events showing good expression levels while retaining the agronomic characteristics and advantages of the recipient plant. Furthermore, these events are considered genetically modified organisms (GMOs), which are either prohibited from commercialization or must undergo costly and lengthy regulatory processes before they can enter the market.

[0005] Therefore, there is a need for improved methods that rely on the use of miRNAs to modulate target gene expression. Summary of the invention

[0006] The present disclosure provides a novel target gene silencing method that uses genome editing to exchange a 20-24 nucleotide long natural miRNA core embedded in a natural pre-miRNA with an amiRNA core sequence derived from and intended to be complementary to the target gene sequence. Modification of the natural pre-miRNA will produce an alternative artificial miRNA for other target gene transcripts, thereby conferring a novel phenotype, such as novel resistance to harmful organisms (e.g., viruses).

[0007] The present invention provides a method for reducing the expression of a target gene, the method comprising introducing a nuclease into a plant cell that is capable of site-directed DNA cleavage at a genomic site encoding a natural pre-miRNA of the plant cell; causing at least one double-strand break at or near the genomic site; selecting cells, wherein the at least one double-strand break has been repaired with an intervening DNA to replace the genomic site; and reducing the expression of the target gene, wherein the intervening DNA encodes a modified pre-miRNA comprising an amiRNA core sequence complementary to the target gene.

[0008] Among other advantages, this approach relies on genome editing technology to precisely and specifically reprogram natural pre-miRNAs to complement different target genes, which could lead to the production of plants that can be considered GMO-free because of the restricted presence of foreign DNA in the plant genome after the method is implemented.

[0009] Another advantage of this approach relies on the ability to produce plants that have one copy of a natural miRNA and one copy of a modified / edited miRNA at the same locus. This is particularly relevant to hybrid crops, which can then express newly modified copies of miRNAs to target different target genes while retaining copies of the natural mRNA and their associated biological functions. Another benefit compared to previous methods that relied on genetic transformation is that the final edited plant cells carry one copy of each miRNA (one copy of the natural miRNA and one copy of the amiRNA), while plant cells obtained according to prior art methods carry two copies of each version of the miRNA (two copies of the natural miRNA and two copies of the amiRNA), which are more demanding on plant cell metabolism and may affect plant performance.

[0010] In another embodiment, the present invention relates to the method according to the preceding embodiment, wherein the target gene is an exogenous target gene, more preferably a harmful organism gene, more preferably a viral, fungal or microbial gene.

[0011] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the target gene is a Bunyavirales gene, preferably a tospovirus gene, and more preferably a tomato spotted wilt virus (TSWV) gene.

[0012] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the target gene is an endogenous plant gene.

[0013] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the target endogenous plant gene is a gene involved in plant development, biotic or abiotic stress.

[0014] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the plant cell is a Solanaceae plant, corn, rice, canola, soybean, or sunflower cell. In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the plant cell is a tomato cell.

[0015] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the genomic site encoding a native pre-miRNA encodes a native tomato pre-miRNA.

[0016] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the genomic locus comprises SEQ ID NO:6 or SEQ ID NO:7.

[0017] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the intermediate DNA comprises any one of SEQ ID NOs: 1 to 5.

[0018] In another embodiment, the present invention relates to a method according to any one of the preceding embodiments, wherein the nuclease is selected from the group consisting of: mega nuclease (MN), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), Cas9 nuclease, Cfp1 nuclease, dCas9-FokI, dCpf1-FokI, chimeric Cas9 / Cpf1-cytosine deaminase, chimeric Cas9 / Cpf1-adenine deaminase, chimeric FEN1-FokI, and Mega-TAL, nickase Cas9 (nCas9), chimeric dCas9 non-FokI nuclease and dCpf1 non-FokI nuclease.

[0019] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the cell has a haploid, diploid, polyploid or hexaploid genome.

[0020] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the cell is heterozygous for the modified pre-miRNA.

[0021] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein one or more guide sequences are introduced together with said nuclease.

[0022] In another embodiment, the present invention relates to a plant cell, preferably a Solanaceae plant, corn, rice, canola, soybean or sunflower cell, more preferably a tomato plant cell obtained by the method of any one of the above embodiments.

[0023] In another embodiment, the invention relates to a plant cell according to the preceding embodiments, wherein the cell comprises any one of SEQ ID NOs: 1-5.

[0024] In another embodiment, the invention relates to a plant cell according to the preceding embodiments, wherein the cell comprises any one of SEQ ID NOs: 8-17.

[0025] In another embodiment, the present invention relates to a method for producing plant seeds, preferably Solanaceae, corn, rice, canola, soybean or sunflower seeds, more preferably tomato seeds, the method comprising crossing a plant comprising a plant cell obtained by the method of any one of the preceding embodiments with itself or with another plant of the same crop. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A schematic diagram showing the modification of a natural pre-miRNA by exchanging the natural miRNA core for an amiRNA core complementary to a new target gene is shown.

[0027] Figure 2 Shown are the resistance levels of Nicotiana benthamiana plants with different overexpressed viral amiRNA core sequences to TSWV.

[0028] Figure 3 Shown are pictures of TSWV-infiltrated N. benthamiana plants with different overexpressed viral amiRNA core sequences.

[0029] Figure 4 Shown are the levels of TSWV resistance in N. benthamiana plants with different native pre-miRNA sequences modified by the viral amiRNA core of SEQ ID NO:2.

[0030] Figure 5 The binary vector 17839 (SEQ ID NO: 18) used for transient experiments in Nicotiana benthamiana plants is shown.

[0031] Figure 6Shown is the binary vector 24598 (SEQ ID NO: 19) for tomato transformation with soybean codon-optimized Cas9 driven by the constitutive prAtEF1aA1-02 promoter and two gene-specific gRNAs driven by prAtU6-01 and prS1U6 to mutate the tomato SlmiR156b gene (SEQ ID NO: 6).

[0032] Brief description of the sequences in the sequence listing

[0033] SEQ ID NO: 1 is the TSWV sequence of amiTSWV_N1w_PC (used as amiRNA core in the context of the present invention)

[0034] SEQ ID NO: 2 is the TSWV sequence of amiTSWV_N2_PC (used as amiRNA core in the context of the present invention)

[0035] SEQ ID NO: 3 is the TSWV sequence of amiTSWV_N2_PC_rev (used as amiRNA core in the context of the present invention)

[0036] SEQ ID NO: 4 is the TSWV sequence of amiR159a_3p_N_GC35 (used as amiRNA core in the context of the present invention)

[0037] SEQ ID NO: 5 is the TSWV sequence of amiR159a_3p_N_GC50 (used as amiRNA core in the context of the present invention)

[0038] SEQ ID NO:6 is the tomato sequence of miR156b, including the 1 kb promoter (used as a pre-miRNA scaffold in the context of the present invention)

[0039] SEQ ID NO:7 is the tomato sequence of miR1919b, including the 1 kb promoter (used as a pre-miRNA scaffold in the context of the present invention)

[0040] SEQ ID NO:8-12 are SEQ ID NO:1, 2, 3, 4 or 5 respectively embedded in SEQ ID NO:6

[0041] SEQ ID NO: 13-17 are SEQ ID NO: 1, 2, 3, 4 or 5 respectively embedded in SEQ ID NO: 7

[0042] SEQ ID NO: 18 is the nucleotide sequence of binary vector 17839

[0043] SEQ ID NO: 19 is the nucleotide sequence of binary vector 24598.

[0044] SEQ ID NO:20 and 21 are gRNA sequences.

[0045] SEQ ID NO: 22 is the TSWV sequence of amiTSWV_N1w_PC_rev (used as amiRNA core in the context of the present invention)

[0046] SEQ ID NO: 23 is the TSWV sequence of amiR159a_3p_N_GC35_rev (used as amiRNA core in the context of the present invention)

[0047] SEQ ID NO: 24 is the TSWV sequence of amiR159a_3p_N_GC50 (used as amiRNA core in the context of the present invention) DETAILED DESCRIPTION

[0048] This description is not intended to be an exhaustive list of all the different ways in which the present invention may be implemented, or of all the features that may be added to the present invention. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be deleted from that embodiment. In addition, in view of this disclosure, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art without departing from the present invention. Therefore, the following description is intended to set forth some specific embodiments of the present invention, and does not exhaustively describe all permutations, combinations, and variations thereof.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. The terms used in the description of the invention herein are for the purpose of describing specific embodiments only and are not intended to limit the invention. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety.

[0050] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise indicated, the terms used herein should be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of general terms in molecular biology can also be found in Rieger et al., Glossary of Genetics:Classical and Molecular Glossary of Genetics: Standard and Molecular, 5th ed., Springer-Verlag, New York, 1994.

[0051] As used in the description of embodiments of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0052] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0053] As used herein, the term "about" when referring to a measurable value such as an amount of a compound, dosage, time, temperature, etc., is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0054] The terms “comprise”, “comprises” and / or “comprising” when used in this specification specify the presence of listed features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0055] As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim will be interpreted as covering the specified materials or steps mentioned in the claim and those materials or steps that do not materially affect one or more basic and novel features of the claimed invention. Therefore, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising".

[0056] As used herein, the term "amplified" means using at least one nucleic acid molecule as a template to construct multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule. See, for example, Diagnostic Molecular Microbiology: Principles and Applications, D.H. Persing et al., eds., American Society for Microbiology, Washington, D.C. (1993). The amplified product is referred to as an amplicon.

[0057] A "coding sequence" is a nucleic acid sequence that is transcribed into RNA (e.g., mRNA, rRNA, tRNA, snRNA, sense RNA, or antisense RNA). In some embodiments, the RNA is subsequently translated in vivo to produce a protein.

[0058] The term "event" of transgenic as used herein refers to a recombinant plant produced by transforming and regenerating a single plant cell with heterologous DNA (e.g., an expression cassette comprising one or more target genes (e.g., transgenic)). The term "event" refers to the original transformant and / or the progeny of the transformant comprising the heterologous DNA. The term "event" also refers to the progeny produced by sexual outcrossing (outcross) between the transformant and another strain. Even after repeated backcrossing to a recurrent parent, the inserted DNA and flanking DNA from the transformed parent are present in the same chromosomal position of the hybrid progeny. Typically, the transformation of plant tissues produces multiple events, each of which represents a different position in the genome of a plant cell inserted by a DNA construct. Based on the expression of transgenic or other desired features, a specific event is selected. Thus, as used herein, "event MIR604," "MIR604," or "MIR604 event" refers to the original MIR604 transformant and / or progeny of a MIR604 transformant (U.S. Pat. Nos. 7,361,813; 7,897,748; 8,354,519 and 8,884,102, incorporated herein by reference).

[0059] As used herein, "expression cassette" means a nucleic acid molecule capable of directing the expression of a specific nucleotide sequence in a suitable host cell, the nucleic acid molecule comprising a promoter operably connected to a target nucleotide sequence (typically a coding region), the nucleotide sequence being operably connected to a termination signal. It also typically comprises a sequence required for the appropriate translation of the nucleotide sequence. The coding region usually encodes the target protein, but can also encode a functional RNA of interest (e.g., antisense RNA or non-translated RNA) in a sense or antisense direction. The expression cassette may also be included in a sequence not required in directing the expression of the target nucleotide sequence, but it exists because of a convenient restriction site for removing the expression cassette from the expression vector. The expression cassette comprising the target nucleotide sequence may be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components. The expression cassette may also be a naturally occurring expression cassette that has been obtained in a recombinant form useful for heterologous expression. However, the expression cassette is usually heterologous relative to the host, i.e., the specific nucleic acid sequence of the expression cassette is not naturally occurring in the host cell, and must have been introduced into the host cell or the ancestor of the host cell by a transformation method known in the art. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some specific external stimulus. In the case of multicellular organisms (e.g., plants), the promoter may also be specific to a particular tissue, or organ, or developmental stage. When transformed into a plant, an expression cassette or fragment thereof may also be referred to as an "inserted sequence" or "inserted sequence."

[0060] "Gene" is a limited area located in the genome, and in addition to the aforementioned coding nucleic acid sequence, it also includes other major regulatory nucleic acid sequences responsible for controlling the expression (i.e., transcription and translation) of the coding portion. A gene can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions). A gene typically expresses mRNA, functional RNA, or specific protein, including regulatory sequences. A gene may or may not be used to produce functional proteins. In certain embodiments, a gene refers only to a coding region. The term "natural gene" refers to a gene as found in nature. The term "chimeric gene" refers to any gene comprising the following: 1) a DNA sequence, including regulatory sequences and coding sequences that are not found together in nature, or 2) a sequence encoding a portion of an unnaturally adjacent protein, or 3) a portion of an unnaturally adjacent promoter. Therefore, a chimeric gene can include regulatory sequences and coding sequences obtained from different sources, or regulatory sequences and coding sequences obtained from the same source but arranged in a different manner from those found in nature. A gene can be "isolated," meaning a nucleic acid molecule that is substantially or essentially free of components normally found associated with the nucleic acid molecule in its natural state. Such components include other cellular materials, culture medium from recombinant production, and / or various chemicals used in the chemical synthesis of the nucleic acid molecule.

[0061] The terms "express" or "expression" with reference to a polynucleotide coding sequence mean that the sequence is transcribed, and optionally translated.

[0062] "Target gene", "target nucleotide sequence" or "target sequence" refers to any gene that, when transferred to a plant, confers a desired characteristic on the plant (e.g., antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other harmful organisms, herbicide tolerance, improved nutritional value, improved performance of an industrial process, or altered breeding capacity). "Target gene" can also be a gene transferred to a plant for the production of a commercially valuable enzyme or metabolite in the plant.

[0063] As used herein, "exogenous" refers to a nucleic acid molecule or nucleotide sequence that is not naturally associated with the host cell into which it is introduced, the sequence being derived from another species or from the same species or organism, but being modified from its original form or the form primarily expressed in the cell, including multiple copies of non-naturally occurring nucleic acid sequences. Therefore, a nucleotide sequence derived from an organism or species different from the organism or species to which the cell into which it is introduced belongs is heterologous relative to that cell or the progeny of the cell. In addition, a heterologous nucleotide sequence includes a nucleotide sequence that is derived from and inserted into the same natural original cell type, but exists in a non-natural state, for example, exists in different copy numbers, and / or is under the control of regulatory sequences different from those found in the natural state of the nucleic acid molecule. Nucleic acid sequences can also be heterologous to other nucleic acid sequences associated therewith, for example in a nucleic acid construct, such as an expression vector. As a non-limiting example, a promoter can be present in a nucleic acid construct in combination with one or more regulatory elements and / or coding sequences that are not naturally present in relation to that particular promoter, i.e., they are heterologous to the promoter.

[0064] A "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with the host cell into which it is introduced. Homologous nucleic acid sequences can also be nucleic acid sequences naturally associated with other nucleic acid sequences, which can, for example, be present in a nucleic acid construct. As a non-limiting example, a promoter can be present in a nucleic acid construct in combination with one or more regulatory elements and / or coding sequences, which naturally exist in relation to that particular promoter, i.e., they are homologous to the promoter.

[0065] "Operably linked" refers to the association of nucleic acid sequences on a single nucleic acid sequence so that the function of one affects the function of another. For example, when a promoter can affect the expression of a coding sequence or a functional RNA (i.e., the coding sequence or the functional RNA is under the transcriptional control of the promoter), the promoter is operably linked to the coding sequence or the functional RNA. A coding sequence in the sense direction or the antisense direction can be operably linked to a regulatory sequence. Therefore, a regulatory or control sequence (e.g., a promoter) operably associated with a nucleotide sequence can affect the expression of the nucleotide sequence. For example, a promoter operably linked to a nucleotide sequence encoding GFP will be able to affect the expression of the GFP nucleotide sequence.

[0066] Control sequences need not be adjacent to the nucleotide sequence of interest, as long as they function to direct its expression. Thus, for example, intervening untranslated, transcribed sequences may be present between a promoter and a coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence.

[0067] As used herein, "primers" are isolated nucleic acids that are annealed to complementary target DNA strands by nucleic acid hybridization to form hybridization between the primer and the target DNA strand, and then extended along the target DNA strand by a polymerase (such as DNA polymerase). Primer pairs or primer sets can be used for amplification of nucleic acid molecules, such as by polymerase chain reaction (PCR) or other nucleic acid amplification methods.

[0068] "Probe" is a nucleic acid molecule that is complementary to the separation of a part of a target nucleic acid molecule, and is typically used to detect and / or quantify a target nucleic acid molecule. Therefore, in some embodiments, the probe can be a nucleic acid molecule that can detect a part or a reporter gene to which the separation is attached, such as a radioisotope, a ligand, a chemiluminescent agent, a fluorescent agent or an enzyme. Probe according to the present invention can not only include deoxyribonucleic acid or ribonucleic acid, but also includes polyamides and other probe materials that are specifically combined with the target nucleic acid sequence and can be used to detect the presence of the target nucleic acid sequence or the amount of the quantitative target nucleic acid sequence.

[0069] The TaqMan probe is designed so that it anneals in the DNA region amplified by a specific primer set. Since the Taq polymerase extends the primer and synthesizes the nascent chain from the 3' to 5' single-stranded template of the complementary chain, the 5' to 3' exonuclease of the polymerase extends the nascent chain through the probe and thus degrades the probe that has been annealed to the template. The degradation of the probe releases the fluorophore therefrom and breaks the close contact with the quencher, thereby alleviating the quenching effect and allowing the fluorescence of the fluorophore. Therefore, the fluorescence detected in the quantitative PCR thermal cycler is proportional to the amount of the released fluorophore and the DNA template present in the PCR.

[0070] The length of primers and probes is generally between 5 and 100 nucleotides or more nucleotides. In certain embodiments, the length of primers and probes can be at least 20 nucleotides or more, or at least 25 nucleotides or more, or at least 30 nucleotides or more in length. These primers and probes are hybridized with the target sequence specificity under optimal hybridization conditions known in the art. Primers and probes according to the present invention can have a complete sequence complementary to the target sequence, although probes different from the target sequence and retaining the ability to hybridize with the target sequence can be designed according to conventional methods of the present invention.

[0071] Methods for making and using probes and primers are described in e.g. Molecular Cloning:A Laboratory ManualIn Molecular Cloning: A Laboratory Manual, 2nd ed., Vols. 1-3, Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. PCR primer pairs can be derived from known sequences, for example by using a computer program designed for this purpose.

[0072] Polymerase chain reaction (PCR) is a technique for "amplifying" a specific DNA fragment. In order to perform PCR, at least a portion of the nucleotide sequence of the DNA molecule to be replicated must be known. Typically, primers or short oligonucleotides complementary to the nucleotide sequence (known sequence) at the 3' end of each strand of the DNA to be amplified (e.g., substantially complementary or completely complementary) are used. The DNA sample is heated to separate its chains and mixed with these primers. These primers hybridize with complementary sequences in their DNA samples. Synthesis (5' to 3' direction) is started using the original DNA chain as a template. The reaction mixture must contain all four deoxynucleotide triphosphates (dATP, dCTP, dGTP and dTTP) and DNA polymerase. Polymerization continues until each newly synthesized chain has proceeded far enough to contain a sequence recognized by another primer. Once this occurs, two DNA molecules identical to the original molecule are produced. The two molecules are heated to separate their chains, and the process is repeated. Each cycle doubles the number of DNA molecules. Using automated equipment, replication of each cycle can be completed in less than 5 minutes. After 30 cycles, the amplification starting from a single DNA molecule has exceeded 1 billion copies (2 30 =1.02x 10 9 ).

[0073] The oligonucleotides of the oligonucleotide primer pair are complementary to the DNA sequences on the relative DNA strand and flanking the region to be amplified. The annealed primers hybridize with the newly synthesized DNA strands. The first amplification cycle will result in two new DNA strands, whose 5' ends are fixed by the positions of the oligonucleotide primers, but whose 3' ends are variable ('irregular' 3' ends). The two new strands can successively serve as templates for the synthesis of complementary strands of desired length (the 5' end is defined by the primer and the 3' end is fixed because the synthesis cannot exceed the end of the opposite primer). After several cycles, the desired fixed-length product begins to dominate.

[0074] Quantitative polymerase chain reaction (qPCR) (also referred to as real-time polymerase chain reaction) monitors the accumulation of DNA products from PCR reactions in real time. qPCR is a molecular biology laboratory technique based on polymerase chain reaction (PCR) that is used to amplify and quantify target DNA molecules simultaneously. Even one copy of a specific sequence can be amplified and detected in PCR. The PCR reaction generates copies of DNA templates in an exponential manner. This results in a quantitative relationship between the amount of the starting target sequence and the amount of PCR products accumulated under any particular cycle. Due to inhibitors of polymerase reaction found together with template, reagent limitation or accumulation of pyrophosphate molecules, the PCR reaction eventually stops generating templates (i.e., plateau phase) at an exponential rate, making the endpoint quantification of PCR products unreliable. Therefore, repeated reactions can generate variable amounts of PCR products. Only during the exponential phase of the PCR reaction is it possible to push back in order to determine the starting amount of the template sequence. Measurement during the accumulation of PCR products (i.e., real-time quantitative PCR) allows for quantification during the exponential phase of the reaction, and therefore eliminates the variability associated with conventional PCR. In real-time PCR assays, positive reactions are detected by accumulation of fluorescent signals. For one or more specific sequences in a DNA sample, quantitative PCR can perform both detection and quantification. The quantity can be the absolute number of copies or the relative amount when normalized to DNA input or an additional normalizing gene. Since the first record of real-time PCR, it has been used for an increasing number of applications, including mRNA expression studies, DNA copy number measurements in genomes or viral DNA, allele discrimination assays, expression analysis of specific splice variants of genes, and gene expression in paraffin-embedded tissues and laser-captured microdissected cells.

[0075] As used herein, the phrase "Ct value" refers to a "cycle threshold value", which is defined as "the fractional cycle number at which the amount of amplified target reaches a fixed threshold value". In some embodiments, it represents the intersection between the amplification curve and the threshold line. The amplification curve is typically in an "S" shape, which represents the change in relative fluorescence of each reaction (Y axis) at a given cycle (X axis), which is recorded during PCR by a real-time PCR instrument in some embodiments. In some embodiments, the threshold line is the detection level at which the reaction reaches a fluorescence intensity higher than the background. See Livak and Schmittgen (2001) 25 Methods ["Methods"] 402-408. It is a relative measure of the target concentration in PCR. Typically, in some embodiments, for a given reference gene, a good Ct value for quantitative determination such as qPCR is in the range of 10-40. The Ct level is inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct level, the higher the amount of detectable target nucleic acid in the sample). In addition, a good Ct value for quantitative determination such as qPCR shows a linear response range in the case of proportional dilution of the target gDNA.

[0076] In some embodiments, qPCR is performed under conditions where Ct values ​​can be collected in real time for quantitative analysis. For example, in a typical qPCR experiment, DNA amplification is monitored at each cycle of PCR during the extension phase. When DNA is in the log-linear phase of amplification, the amount of fluorescence typically increases above background. In some embodiments, Ct values ​​are collected at this time point.

[0077] As used herein, the term "cell" refers to any living cell. The cell can be a prokaryotic cell or a eukaryotic cell. The cell can be isolated. The cell may or may not be able to regenerate into an organism. The cell can be in the context of a tissue, callus, culture, organ, or part. In certain embodiments, the cell can be a plant cell. The plant cell of the present invention can be in an isolated unicellular form, or can be a cultured cell, or can be a part of a higher organizational unit (e.g., plant tissue or plant organ). The plant cell can be derived from angiosperms or gymnosperms or a part thereof. In other embodiments, the plant cell can be a monocotyledonous plant cell, a dicotyledonous plant cell. The monocotyledonous plant cell can be, for example, maize, rice, sorghum, sugarcane, barley, wheat, oats, turf grass, or an ornamental grass cell. The dicotyledonous plant cell can be, for example, a tobacco, pepper, eggplant, sunflower, cruciferous plant, flax, potato, cotton, soybean, beet, or rapeseed cell.

[0078] The term "plant part" as used herein includes, but is not limited to, embryos, pollen, ovules, seeds, leaves, stems, buds, flowers, branches, fruits, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, plant cells (including intact plant cells in plants and / or parts of plants), plant protoplasts, plant tissues, plant cell tissue cultures, plant callus, plant masses, etc. As used herein, "buds" refer to the above-ground parts including leaves and stems. In addition, as used herein, "plant cells" refer to the structural and physiological units of plants, including cell walls and may also refer to protoplasts.

[0079] In the context of cells, prokaryotic cells, bacterial cells, eukaryotic cells, vegetable cells, plants and / or plant parts, the term "introducing" (introducing or introducing) means contacting a nucleic acid molecule with the cell, eukaryotic cell, plant, plant part and / or plant cell in such a way that the nucleic acid molecule is able to enter the interior of the cell, eukaryotic cell, vegetable cell and / or plant and / or plant part. In the case of introducing more than one nucleic acid molecule, these nucleic acid molecules can be assembled into a part of a single polynucleotide or nucleic acid construct, or assembled into separate polynucleotides or nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Therefore, these polynucleotides can be introduced into plant cells in a single transformation event, in separate transformation events, or, for example, as part of a breeding scheme by conventional hybridization.

[0080] An "inversion" is a chromosomal rearrangement in which segments of a chromosome are joined end to end. An inversion occurs when a single chromosome breaks and rearranges within itself. A chromosomal "translocation" is a rearrangement of sections between non-homologous chromosomes.

[0081] As used herein, the terms "transformed" and "transgenic" refer to any cell, prokaryotic cell, eukaryotic cell, plant, plant cell, callus, plant tissue, or plant part that contains all or part of at least one recombinant (e.g., heterologous) polynucleotide. In some embodiments, all or part of the recombinant polynucleotide is stably integrated into a chromosome or a stable extrachromosomal element so that it is passed on to successive generations. For the purposes of the present invention, the term "recombinant polynucleotide" refers to a polynucleotide that has been altered, rearranged, or modified by genetic engineering. Examples include any cloned polynucleotide, or a polynucleotide linked or joined to a heterologous sequence. The term "recombinant" does not refer to polynucleotide changes that occur due to naturally occurring events (such as spontaneous mutations) or due to non-spontaneous mutagenesis followed by selective breeding.

[0082] As used herein, the term "conversion" refers to the introduction of heterologous nucleic acid into a cell. The transformation of a cell can be stable or transient. Therefore, transgenic cells, plant cells, plants and / or plant parts of the present invention can be stably transformed or transiently transformed. The term "conversion" can refer to the transfer of nucleic acid molecules into the genome of a host cell, resulting in genetically stable inheritance. In certain embodiments, the introduction of plants, plant parts and / or plant cells is via bacterial-mediated transformation, particle bombardment transformation, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, liposome-mediated transformation, nanoparticle-mediated transformation, polymer-mediated transformation, virus-mediated nucleic acid delivery, whisker-mediated nucleic acid delivery, microinjection, ultrasonic treatment, infiltration, polyethylene glycol-mediated transformation, protoplast transformation or any other electrical, chemical, physical and / or biological mechanism causing the introduction of nucleic acid into plants, plant parts and / or their cells, or any combination thereof.

[0083] Procedures for transforming plants are well known and routine in the art and are generally described in the literature. Non-limiting examples of methods for plant transformation include transformation via bacterial-mediated nucleic acid delivery (e.g., via bacteria from the genus Agrobacterium), viral-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid absorption, and any other electrical, chemical, physical (mechanical) and / or biological mechanism that allows nucleic acid to be introduced into plant cells, including any combination thereof. A general guide to various plant transformation methods known in the art includes Miki et al. ("Procedures for Introducing Foreign DNA into Plants" in Plant Molecular Biology and Biotechnology In Methods in Plant Molecular Biology and Biotechnology, Glick, BR and Thompson, JE, eds. (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7: 849-858 (2002)).

[0084] Agrobacterium-mediated transformation is a common method for transforming plants because of its high transformation efficiency and because of its wide applicability with many different species. Agrobacterium-mediated transformation typically involves transferring a binary vector carrying the exogenous DNA of interest to an appropriate Agrobacterium strain, which may depend on the complement of the vir genes carried by the host Agrobacterium strain on a co-existing Ti plasmid or chromosomally (Uknes et al., 1993, Plant Cell 5:159-169). Transferring the recombinant binary vector to Agrobacterium can be accomplished by a three-parent mating procedure using Escherichia coli carrying the recombinant binary vector, a helper Escherichia coli strain carrying a plasmid capable of moving the recombinant binary vector into the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by nucleic acid transformation ( and Willmitzer, 1988, Nucleic Acids Res. 16:9877).

[0085] Plant transformation by recombinant Agrobacterium typically involves co-cultivation of the Agrobacterium with explants from the plant and follows methods well known in the art. Transformed tissue is typically regenerated on a selection medium carrying an antibiotic or herbicide resistance marker located between the binary plasmid T-DNA borders. An exemplary method for transforming tomato plants is disclosed in Garcia D., Narváez-Vásquez J., Orozco-Cárdenas ML (2015) Tomato (Solanum lycopersicum). In: Wang K. (ed.) Agrobacterium Protocols. Methods in Molecular Biology, Vol. 1223. Springer, New York, NY. [Springer: New York, New York].

[0086] Another method for transforming plants, plant parts and plant cells involves advancing inert or biologically active particles on plant tissues and cells. See, for example, U.S. Patent Nos. 4,945,050; 5,036,006 and 5,100,792. Typically, this method involves advancing inert or biologically active particles at plant cells under conditions effective to penetrate the outer surface of the cell and provide for incorporation into its interior. When inert particles are used, the vector can be introduced into the cell by coating these particles with a vector containing a target nucleic acid. Alternatively, one or more cells can be surrounded by the vector so that the vector is brought into the cell by the excitation of the particle. Biologically active particles (e.g., dried yeast cells, dried bacteria or bacteriophages, each containing one or more nucleic acids intended to be introduced) can also be advanced into plant tissues.

[0087] "Transient transformation" in the context of a polynucleotide means that the polynucleotide is introduced into a cell and is not integrated into the genome of the cell.

[0088] As used herein, in the context of a polynucleotide introduced into a cell, "stably introducing", "stably introduced", "stable transformation" or "stably transformed" means that the introduced polynucleotide is stably integrated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide. Thus, the integrated polynucleotide can be inherited by its progeny, more specifically, by multiple successive generations of progeny. "Genome" as used herein includes nuclear and / or plastid genomes, and therefore includes integration of a polynucleotide into, for example, a chloroplast genome. Stable transformation as used herein can also refer to a polynucleotide that is maintained outside the chromosome, for example, as a minichromosome.

[0089] Transient transformation can be detected by, for example, enzyme-linked immunosorbent assay (ELISA) or protein blotting, both of which can detect the presence of peptides or polypeptides encoded by one or more nucleic acid molecules introduced into an organism. Stable transformation of cells can be detected by, for example, Southern blot hybridization assays of cellular genomic DNA and nucleic acid sequences (these sequences specifically hybridize with the nucleotide sequences of nucleic acid molecules introduced into an organism (e.g., a plant). Stable transformation of cells can be detected by, for example, Northern blot hybridization assays of cellular RNA and nucleic acid sequences (these sequences specifically hybridize with the nucleotide sequences of nucleic acid molecules introduced into a plant or other organism). Stable transformation of cells can also be detected by, for example, polymerase chain reaction (PCR) or other amplification reactions well known in the art, which employ specific primer sequences that hybridize with one or more target sequences of nucleic acid molecules, resulting in amplification of the one or more target sequences, which can be detected according to standard methods. Conversion can also be detected by direct sequencing and / or hybridization schemes well known in the art.

[0090] Thus, in specific embodiments of the invention, plant cells may be transformed by any method known in the art and as described herein and whole plants may be regenerated from these transformed cells using any of a variety of known techniques. Plant regeneration from plant cells, plant tissue cultures, and / or cultured protoplasts is described in, for example, Evans et al. ( Handbook of Plant Cell Cultures [Plant Cell Culture Manual], Volume 1, MacMilan Publishing Co. [MacMilan Publishing Co.], New York (1983)); and Vasil I.R. (ed.) (Cell Culture and Somatic Cell Genetics of Plants [plant cell culture and somatic cell genetics], Academic Press, Orlando, Volume I (1984) and Volume II (1986)). Methods for selecting transformed transgenic plants, plant cells and / or plant tissue cultures are routine in the art and can be used in the methods of the invention provided herein.

[0091] "Transformation and regeneration process" refers to the process of stably introducing a transgene into a plant cell and regenerating a plant from the transgenic plant cell. As used herein, transformation and regeneration include a selection process by which the transgene includes a selectable marker and the transformed cell has incorporated and expresses the transgene so that the transformed cell will survive and thrive in the presence of the selection agent. "Regeneration" refers to the growth of a whole plant from a plant cell, a group of plant cells, or a plant piece (such as from a protoplast, callus, or tissue portion).

[0092] The terms "nucleotide sequence", "nucleic acid", "nucleic acid sequence", "nucleic acid molecule", "oligonucleotide" and "polynucleotide" are used interchangeably herein to refer to heteropolymers of nucleotides and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and chimeras of RNA and DNA. The term nucleic acid molecule refers to a nucleotide chain, regardless of the length of the chain. These nucleotides contain a sugar, a phosphate and a base, which is a purine or a pyrimidine. Nucleic acid molecules can be double-stranded or single-stranded. When single-stranded, nucleic acid molecules can be positive strands or antisense strands. Nucleic acid molecules can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or thiophosphate nucleotides). Such oligonucleotides can, for example, be used to prepare nucleic acid molecules with altered base pairing ability or enhanced resistance to nucleases. The nucleic acid sequences provided herein are represented from left to right in the 5' to 3' direction and are represented using standard codes representing nucleotide characters, as described in U.S. Sequence Rules, 37 CFR §§1.821-1.825 and World Intellectual Property Organization (WIPO) Standard ST.25.

[0093] A "nucleic acid fragment" is a portion of a given nucleic acid molecule. An "RNA fragment" is a portion of a given RNA molecule. A "DNA fragment" is a portion of a given DNA molecule. A "nucleic acid segment" is a portion of a given nucleic acid molecule and is not isolated from the molecule. An "RNA segment" is a portion of a given RNA molecule and is not isolated from the molecule. A "DNA segment" is a portion of a given DNA molecule and is not isolated from the molecule. A segment of a polynucleotide can be any length, such as at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, or 500 or more nucleotides in length. A segment or portion of a guide sequence can be about 50%, 40%, 30%, 20%, 10% of the guide sequence, such as one-third of the guide sequence or less, such as 7, 6, 5, 4, 3, or 2 nucleotides in length.

[0094] In the context of a molecule, the term "derived from" refers to a molecule that is isolated or made using a parent molecule or information from that parent molecule. For example, the Cas9 single mutant nickase and the Cas9 double mutant null nuclease are derived from the wild-type Cas9 protein.

[0095] In higher plants, deoxyribonucleic acid (DNA) is the genetic material, while ribonucleic acid (RNA) involves the transfer of information contained in DNA to proteins. "Genome" is the entirety of the genetic material contained in each cell of an organism. Unless otherwise indicated, the specific nucleic acid sequence of the present invention also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as sequences explicitly specified. Specifically, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. [Nucleic Acids Research] 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. [Journal of Biological Chemistry] 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes [Molecular and Cellular Probes] 8: 91-98 (1994)). The term nucleic acid molecule is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0096] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or peptide sequences are invariant over the entire comparison window of components (e.g., nucleotides or amino acids). "Identity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology [Sequence Analysis in Molecular Biology]) (von Heinje, G. ed.) Academic Press (1987); and Sequence Analysis Primer [Primers for sequence analysis] (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0097] As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, "percent identity" may refer to the percentage of identical amino acids in an amino acid sequence.

[0098] As used herein, in the context of two nucleic acid molecules, nucleotide sequences or protein sequences, the phrase "substantially identical" refers to two or more sequences or subsequences with at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments of the present invention, there is substantial identity in a sequence region of at least about 50 residues to about 150 residues in length. Therefore, in some embodiments of the present invention, there is substantial consistency in a sequence region of at least about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, or more residues in length. In some specific embodiments, these sequences are substantially identical in at least about 150 residues. In other embodiments, the sequences are substantially identical over the entire length of the coding region. In addition, in representative embodiments, substantially identical nucleotide sequences or protein sequences perform substantially the same function (e.g., directing endonuclease cleavage to a specific genomic target table, a specific genomic target site).

[0099] For sequence comparison, typically, one sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer (if necessary, subsequence coordinates are specified), and the parameters of the sequence algorithm program are specified. The sequence comparison algorithm then calculates the sequence identity percentages of one or more test sequences relative to the reference sequence based on the specified program parameters.

[0100] Optimal alignment of sequences for comparison windows is well known to those skilled in the art and can be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and optionally by computerized implementations of these algorithms, such as Wisconsin (Accelrys Inc., San Diego, California). The "identity score" of the aligned segments of a test sequence and a reference sequence is the number of identical components common to the two aligned sequences divided by the total number of components in the reference sequence segment (i.e., a complete reference sequence or a smaller defined portion of a reference sequence). The percent sequence identity is expressed as the identity score multiplied by 100. The comparison of one or more polynucleotide sequences can be relative to a full-length polynucleotide sequence or a portion thereof, or relative to a longer polynucleotide sequence. For purposes of the present invention, "percent identity" can also be determined using BLASTX, version 2.0, for translated nucleotide sequences and BLASTN, version 2.0, for polynucleotide sequences.

[0101] Software for performing BLAST analysis can be publicly obtained through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words with length W in the query sequence, which match or meet some positive threshold scores T when compared with words with the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits are used as seeds to initiate searches to find longer HSPs containing them. Then, these word hits are extended along each sequence in both directions until the cumulative comparison score can be increased. For nucleotide sequences, the parameters M (reward score for a pair of matching residues; always>0) and N (penalty for mismatched residues; always<0) are used to calculate the cumulative score. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction ceases when the cumulative alignment score decreases by the amount X from its maximum achieved value; when the cumulative score approaches zero or below due to the accumulation of one or more negative-scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0102] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of a test nucleotide sequence to a reference nucleotide sequence is less than about 0.1 to less than about 0.001. Thus, in some embodiments of the invention, the smallest sum probability in a comparison of a test nucleotide sequence to a reference nucleotide sequence is less than about 0.001.

[0103] When two nucleotide sequences hybridize to each other under stringent conditions, the two nucleotide sequences may also be considered to be substantially identical. In some representative embodiments, two nucleotide sequences considered to be substantially identical hybridize to each other under high stringency conditions.

[0104] In the context of nucleic acid hybridization experiments (such as DNA hybridization and RNA hybridization), "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. An extensive guide to nucleic acid hybridization is found in: Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acids, Chapter 2, Part I, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York (1993). Typically, highly stringent hybridization and wash conditions are selected to be above the thermal melting point (T) for a specific sequence at a defined ionic strength and pH. m ) is about 5℃ lower.

[0105] T m It is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T for a specific probe. m. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences (which have more than 100 complementary residues on the filter in a Southern or Northern blot) is 50% formamide with 1 mg of heparin at 42°C, where hybridization is performed overnight. An example of high stringency washing conditions is 0.15M NaCl at 72°C for about 15 minutes. An example of stringent washing conditions is washing at 65°C with 0.2x SSC for 15 minutes (see Sambrook, below, for a description of SSC buffer). Typically, a low stringency wash is performed before a high stringency wash to remove background probe signals. An example of a medium stringency wash for a duplex of, for example, more than 100 nucleotides is at 45°C with 1x SSC for 15 minutes. An example of a low stringency wash for a duplex of, for example, more than 100 nucleotides is at 40°C with 4-6xSSC for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. In general, a signal-to-noise ratio of 2x (or higher) observed in a particular hybridization assay compared to an unrelated probe indicates that specific hybridization has been detected. If the proteins encoded by nucleotide sequences that do not hybridize to each other under stringent conditions are substantially identical, then the nucleotide sequences are still substantially identical. For example, this may occur when a copy of a nucleotide sequence is generated using the maximum codon degeneracy allowed by the genetic code.

[0106] The following are examples of hybridization / washing conditions that can be used to clone homologous nucleotide sequences that are substantially identical to the reference nucleotide sequences of the present invention. In one embodiment, the reference nucleotide sequence is washed at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 In another embodiment, the reference nucleotide sequence is hybridized to the "test" nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA, and washed in 2X SSC, 0.1% SDS at 50°C. 4 , 1 mM EDTA with a wash in 1X SSC, 0.1% SDS at 50°C; or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4, 1 mM EDTA, with washes in 0.5X SSC, 0.1% SDS at 50°C. In still other embodiments, the reference nucleotide sequence is hybridized at 50°C in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA with a wash in 0.1X SSC, 0.1% SDS at 50°C; or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 4 , 1 mM EDTA, and washed at 65°C in 0.1X SSC, 0.1% SDS.

[0107] "Isolated" nucleic acid molecules or nucleotide sequences or "isolated" polypeptides are nucleic acid molecules, nucleotide sequences or polypeptides that exist out of their natural environment by human hands and / or have different, modified, regulated and / or altered functions when compared to their functions in their natural environment and are therefore not natural products. Isolated nucleic acid molecules or isolated polypeptides can exist in purified form or can exist in a non-natural environment (such as, for example, a recombinant host cell). Thus, for example, with respect to a polynucleotide, the term isolated means that the polynucleotide is separated from the chromosome and / or cell in which it naturally exists. If a polynucleotide is separated from the chromosome and / or cell in which it naturally exists and then inserted into a genetic background, chromosome, chromosomal position, and / or cell in which it does not naturally exist, the polynucleotide is also isolated. Recombinant nucleic acid molecules and nucleotide sequences of the present invention can be considered as "isolated" as defined above.

[0108] Thus, an "isolated nucleic acid molecule" or "isolated nucleotide sequence" is a nucleic acid molecule or nucleotide sequence that is not adjacent to adjacent nucleotide sequences (sequences at the 5' end or sequences at the 3' end) in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately adjacent to the coding sequence. Thus, the term includes, for example, recombinant nucleic acids that are incorporated into a vector, into a self-replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or that exist as a separate molecule (e.g., cDNA or a genomic DNA fragment obtained by PCR or restriction endonuclease treatment) independent of other sequences. It also includes recombinant nucleic acids that are part of a hybrid nucleic acid molecule encoding an additional polypeptide or peptide sequence. An "isolated nucleic acid molecule" or "isolated nucleotide sequence" may also include a nucleotide sequence that is derived from and inserted into the same natural original cell type, but is present in a non-natural state, for example, in a different copy number, and / or under the control of regulatory sequences that are different from those found in the natural state of the nucleic acid molecule.

[0109] The term "isolated" may further refer to a nucleic acid molecule, nucleotide sequence, polypeptide, peptide or fragment that is substantially free of cellular material, viral material, and / or culture medium (e.g., when produced by recombinant DNA techniques), or chemical precursors or other chemicals (e.g., when chemically synthesized). Additionally, an "isolated fragment" is a fragment of a nucleic acid molecule, nucleotide sequence or polypeptide that does not naturally exist as a fragment and would not so exist in nature. "Isolated" does not necessarily mean that the preparation is industrially pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form that can be used for the intended purpose.

[0110] In representative embodiments of the invention, "isolated" nucleic acid molecules, nucleotide sequences and / or polypeptides have a purity (w / w) of at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or more. In other embodiments, "isolated" nucleic acids, nucleotide sequences and / or polypeptides represent at least about 5-fold, 10-fold, 25-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold or greater enrichment (w / w) of the nucleic acid compared to the starting material.

[0111] A "wild-type" nucleotide sequence or amino acid sequence refers to a naturally occurring ("native") or endogenous nucleotide sequence or amino acid sequence. Thus, for example, a "wild-type mRNA" is an mRNA naturally occurring in or endogenous to an organism. A "homologous" nucleotide sequence is a nucleotide sequence naturally associated with the host cell into which it is introduced.

[0112] The terms "open reading frame" and "ORF" refer to the amino acid sequence encoded between the translation start and stop codons of a coding sequence. The terms "start codon" and "stop codon" refer to a unit of three adjacent nucleotides ("codon") in a coding sequence that specifies the start and chain termination of protein synthesis (mRNA translation), respectively.

[0113] "Promoter" refers to a nucleotide sequence, usually upstream (5') of its coding sequence, which controls the expression of the coding sequence by providing recognition of the RNA polymerase and other factors required for appropriate transcription. "Promoter regulatory sequence" consists of proximal and more distal upstream elements. The promoter regulatory sequence affects the transcription, RNA processing or stability, or translation of the relevant coding sequence. Regulatory sequences include enhancers, promoters, non-translated leader sequences, introns, and polyadenylation signal sequences. They include natural sequences and synthetic sequences, together with sequences that may be combinations of synthetic sequences and natural sequences. "Enhancer" is a DNA sequence that can stimulate the activity of a promoter and can be an intrinsic element of the promoter or an inserted heterologous element to enhance the level or tissue specificity of a promoter. It can operate in two directions (normal or flipped), and can even play a role when moved upstream or downstream of the promoter. The meaning of the term "promoter" includes "promoter regulatory sequence".

[0114] "Primary transformants" and "E0 generation" refer to transgenic plants of the same genetic generation as the tissue that was initially transformed (i.e., has not undergone meiosis and fertilization since transformation). "Secondary transformants" and "E1, E2, E3, etc. generations" refer to transgenic plants that are derived from primary transformants through one or more meiotic and fertilization cycles. They can be derived by self-fertilization of primary or secondary transformants or hybridization of primary or secondary transformants with other transformed or untransformed plants.

[0115] "Transgenic" refers to a nucleic acid molecule that has been introduced into the genome by transformation and is stably maintained. A transgenic may include at least one expression cassette, typically at least two expression cassettes, and may include ten or more expression cassettes. A transgenic may include, for example, genes that are heterologous or homologous to the genes of a particular plant to be transformed. In addition, a transgenic may include a natural gene that is inserted into a non-natural organism, or a chimeric gene. The term "endogenous gene" refers to a natural gene in its natural location in the genome of an organism. An "exogenous" gene refers to a gene that is not normally found in the host organism but is introduced into the organism by gene transfer.

[0116] "Intron" refers to an intervening segment of DNA that occurs almost exclusively in eukaryotic genes, but which is not translated into an amino acid sequence in the gene product. These introns are removed from the immature mRNA by a process called splicing, which leaves the exons untouched, thereby forming the mRNA. For the purposes of the present invention, the definition of the term "intron" includes modifications to the nucleotide sequence derived from an intron of a target gene, provided that the modified intron does not significantly reduce the activity of its associated 5' regulatory sequence.

[0117] "Exon" refers to a segment of DNA that carries a coding sequence for a protein or a portion thereof. Exons are separated by intervening, non-coding sequences (introns). For the purposes of the present invention, the definition of the term "exon" includes modifications to the nucleotide sequence of an exon derived from a target gene, provided that the modified exon does not significantly reduce the activity of its associated 5' regulatory sequence.

[0118] The term "cleavage" or "cleaving" refers to the break of the covalent phosphodiester linkage in the ribosylphosphodiester backbone of a polynucleotide. The term "cleavage" or "cleaving" covers both single-strand breaks and double-strand breaks. As a result of two different single-strand cleavage events, double-strand cleavage can occur. Cutting can result in the production of flat ends or staggered ends. "Nuclease cleavage site" or "genomic nuclease cleavage site" is a nucleotide region including a nuclease cleavage sequence, which is recognized by a specific nuclease, and the nuclease is used to cut the nucleotide sequence of genomic DNA in one or two chains. This cutting by the nuclease triggers the DNA repair mechanism in the cell, which establishes an environment for the occurrence of homologous recombination.

[0119] A "donor molecule" or "donor sequence" is a nucleotide polymer or oligomer intended for insertion at a target polynucleotide (typically a target genomic site). The donor sequence may be one or more transgenes of interest, expression cassettes, or nucleotide sequences. The donor molecule may be a donor DNA molecule, single-stranded, partially double-stranded, or double-stranded. The donor polynucleotide may be a natural or modified polynucleotide, an RNA-DNA chimera, or a DNA fragment, a single-stranded, or at least partially double-stranded, or fully double-stranded DNA molecule, or a PGR-amplified ssDNA, or at least a portion of a dsDNA fragment. In some embodiments, the donor DNA molecule is part of a circularized DNA molecule. A fully double-stranded donor DNA is advantageous because it may provide increased stability because dsDNA fragments are generally more resistant to nuclease degradation than ssDNA. In some embodiments, the donor polynucleotide molecule may comprise at least about 100, 150, 200, 250, 300, 250, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 7500, 10000, 15,000 or 20,000 nucleotides, including any value within this range that is not explicitly described herein. In some embodiments, the donor DNA molecule comprises a heterologous nucleic acid sequence. In some embodiments, the donor DNA molecule comprises at least one expression cassette. In some embodiments, the donor DNA molecule may comprise a transgenic comprising at least one expression cassette. In some embodiments, the donor DNA molecule comprises an allele modification of a gene originating from a target genome. The allele modification may comprise at least one nucleotide insertion, at least one nucleotide deletion, and / or at least one nucleotide substitution. In some embodiments, the allele modification may comprise an insertion-deletion (INDEL). In some embodiments, the donor DNA molecule comprises arms homologous to the target genomic site. In some embodiments, the donor DNA molecule comprises at least 100 consecutive nucleotides with at least 90% identity to the genomic nucleic acid sequence, and optionally may further comprise a heterologous nucleic acid sequence, such as a transgene. In some embodiments, the "donor DNA molecule" is an "intermediate DNA".

[0120] As used herein, the terms "vicinity" or "proximal" or "vicinity of or proximal to" with respect to one or more nucleotide sequences of the invention means immediately adjacent to or separated by from about 1 base to about 2000 bases (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, or 2000 bases), including any value contained within this range but not expressly recited herein.

[0121] "MicroRNAs" (abbreviated as miRNAs) are small non-coding RNA molecules (containing between about 20 and about 24 nucleotides, usually about 22 nucleotides) found in plants, animals, and some viruses, which function as RNA silencing and post-transcriptional regulation of gene expression. miRNA genes are usually transcribed by RNA polymerase II (Pol II). The polymerase often binds to a promoter found near a DNA sequence encoding a hairpin loop that will become the pre-miRNA. The resulting transcript is capped at the 5' end with specially modified nucleotides, polyadenylated with multiple adenosines (poly-A tail), and spliced.

[0122] "Pre-miRNA" is a miRNA precursor with a stem-loop structure, with the 5' cap and 3' ploy-A removed. It is a natural structure that helps produce miRNA. Sometimes this term is used to distinguish it from mature miRNA (between about 20 to about 24 nucleotides, usually about 22 nucleotide sequences). In this way, it means the structure, rather than the final functional short sequence. The term "miRNA scaffold" or "miRNA backbone" is also used to refer to the pre-miRNA structure in the context of the present invention.

[0123] As used herein, the term "amiRNA" (artificial miRNA) generally refers to a natural miRNA scaffold whose core sequence (mature miRNA sequence and corresponding miRNA* sequence) is replaced by an "amiRNA core" sequence to redirect targeting (silencing) to a new gene. The term "amiRNA core" refers to the artificial (designed) part of this method, a short sequence of about 20 to 24 nucleotides that is complementary to a new target gene. In this context, the term complementary refers to the ability of amiRNA to bind to a target RNA molecule. In some embodiments, the amiRNA core is 90% complementary to the new target gene molecule and retains its ability to bind to the target RNA molecule.

[0124] As used herein, the term "guide RNA" or "gRNA" generally refers to an RNA molecule (or a group of total RNA molecules) that can bind to a CRISPR system effector (such as Cas or Cpf1 protein) and help target the Cas or Cpf1 protein to a specific position within a target polynucleotide (such as DNA). The guide RNA of the present invention can be an engineered single RNA molecule (sgRNA), wherein, for example, the sgRNA comprises a crRNA segment and an optional tracrRNA segment. The guide RNA of the present invention can also be a dual-guide system, in which the crRNA and tracrRNA molecules are physically different molecules, which then interact to form a duplex for recruiting CRISPR system effectors (such as Cas9) and for targeting the protein to a target polynucleotide.

[0125] As used herein, the term "crRNA" or "crRNA segment" refers to an RNA molecule or a portion of an RNA molecule, which includes a polynucleotide targeting guide sequence, a stem sequence involved in protein binding, and an optional 3'-overhang sequence. The polynucleotide targeting guide sequence is a nucleic acid sequence complementary to a sequence in a target DNA. This polynucleotide targeting guide sequence is also referred to as a "pre-spacer sequence". In other words, the polynucleotides of the guide sequence of the targeting crRNA molecule interact with the target DNA via hybridization (i.e., base pairing) in a sequence-specific manner. In this way, the nucleotide sequence of the polynucleotide targeting guide sequence of the crRNA molecule can vary, and determines the position in the target DNA where the guide RNA and the target DNA will interact.

[0126] The polynucleotide targeting guide sequence of crRNA molecule can be modified (for example, by genetic engineering) so as to hybridize with any desired sequence in target DNA. The polynucleotide targeting guide sequence of crRNA molecule of the present invention can have a length from about 12 nucleotides to about 100 nucleotides. For example, the polynucleotide targeting guide sequence of crRNA can have the following length: from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 40 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt or from about 12 nt to about 19 nt. For example, the polynucleotide targeting guide sequence of crRNA can have a length from about 17 nt to about 27 nt. For example, the polynucleotide targeting guide sequence of the crRNA can have a length of from about 19 nt to about 20 nt, from about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 19 nt to about 70 nt, from about 19 nt to about 80 nt, from about 19 nt to about 90 nt, In some embodiments, the polynucleotide targeting guide sequence of crRNA is 20 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, from about 20 nt to about 60 nt, from about 20 nt to about 70 nt, from about 20 nt to about 80 nt, from about 20 nt to about 90 nt, or from about 20 nt to about 100 nt. The nucleotide sequence of the polynucleotide targeting guide sequence of crRNA can have a length of at least about 12 nt. In some embodiments, the length of the polynucleotide targeting guide sequence of crRNA is 20 nucleotides. In some embodiments, the length of the polynucleotide targeting guide sequence of crRNA is 19 nucleotides.

[0127] The present invention also provides a guide RNA comprising an engineered crRNA, wherein the crRNA comprises a bait RNA segment capable of hybridizing with a genomic target sequence. This engineered crRNA can be a physically different molecule, just as in a dual-guide system.

[0128] As used herein, the term "tracrRNA" or "tracrRNA segment" refers to an RNA molecule or portion thereof that includes a protein binding segment (e.g., a protein binding segment that can interact with a CRISPR-associated protein (such as Cas9)). The present invention also provides a guide RNA comprising an engineered tracrRNA, wherein the tracrRNA further comprises a bait RNA segment that can bind to a donor DNA molecule. The engineered tracrRNA can be a physically different molecule (such as in a dual-guide system), or can be a segment of an sgRNA molecule.

[0129] In some embodiments, the guide RNA as sgRNA or as two or more RNA molecules does not contain tracrRNA, because some CRISPR-related nucleases (such as Cpf1 (also known as Cas12a)) are known in the art for their RNA-mediated endonuclease activity without tracrRNA (Qi et al., 2013, Cell [Cell], 152: 1173-1183; Zetsche et al., 2015, Cell [Cell] 163: 759-771). Such guide RNAs of the present invention may include crRNA, wherein the bait RNA is operably connected to the 5' or 3' end of the crRNA. Cpf1 also has RNase activity to its homologous pre-crRNA (Fonfara et al., 2016, Nature [Nature], doi.org / 10.1038 / nature17945). The guide RNA of the present invention may include multiple crRNAs in which Cpf1 is processed into mature crRNA. In some embodiments, each of these crRNAs is operably connected to the bait RNA. In other embodiments, at least one of these crRNAs is operably linked to a bait RNA. The bait RNA can be specific to a sequence of interest (SOI) or a target genomic site, as described in the examples herein.

[0130] The present invention also provides nucleic acid molecules comprising nucleic acid sequences encoding guide RNAs of the present invention. The nucleic acid molecules may be DNA or RNA molecules. In some embodiments, the nucleic acid molecules are cyclized. In other embodiments, the nucleic acid molecules are linear. In some embodiments, the nucleic acid molecules are single-stranded, partially double-stranded, or double-stranded. In some embodiments, nucleic acid molecules are compounded with at least one polypeptide. The polypeptide may have a nucleic acid recognition domain or a nucleic acid binding domain. In some embodiments, the polypeptide is a shuttle for mediating delivery of, for example, chimeric RNAs, nucleases, and optional donor molecules of the present invention. In some embodiments, the polypeptide is a Feldan shuttle (U.S. Patent Publication No. 20160298078, incorporated herein by reference). Nucleic acid molecules may include an expression cassette capable of driving expression of chimeric RNA. Nucleic acid molecules may also include additional expression cassettes capable of expressing, for example, nucleases (such as CRISPR-associated nucleases). The present invention also provides expression cassettes comprising nucleic acid sequences encoding chimeric RNAs of the present invention.

[0131] A "site-directed modifying polypeptide" modifies a target DNA (e.g., cleavage or methylation of a target DNA) and / or a polypeptide associated with a target DNA (e.g., methylation or acetylation of a histone tail). A site-directed modifying polypeptide is also referred to herein as a "site-directed polypeptide" or "RNA-binding site-directed modifying polypeptide." Due to the association of a site-directed modifying polypeptide with a guide RNA, the site-directed modifying polypeptide interacts with the guide RNA (the guide RNA is a single RNA molecule or an RNA duplex of at least two RNA molecules) and is guided to a DNA sequence (e.g., a chromosomal sequence or an extrachromosomal sequence, such as an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.).

[0132] In some cases, the site-directed modifying polypeptide is a naturally occurring modified polypeptide. In other cases, the site-directed modifying polypeptide is not a naturally occurring modified polypeptide (e.g., a chimeric polypeptide or a modified (e.g., mutated, deleted, inserted) naturally occurring polypeptide). Exemplary naturally occurring site-directed modifying polypeptides are known in the art (see, e.g., Makarova et al., 2017, Cell 168:328-328.e1, and Shmakov et al., 2017, Nat Rev Microbiol 15(3):169-182, both of which are incorporated herein by reference). These naturally occurring polypeptides bind to the DNA targeting RNA and are thereby directed to a specific sequence within the target DNA and cut the target DNA, thereby producing a double-strand break.

[0133] The site-directed modifying polypeptide comprises two parts, namely an RNA binding part and an active part. In some embodiments, the site-directed modifying polypeptide comprises: (i) an RNA binding part that interacts with a DNA-targeting RNA, wherein the DNA-targeting RNA comprises a nucleotide sequence complementary to a sequence in the target DNA; and (ii) an active part that exhibits site-directed enzymatic activity (e.g., DNA methylation activity, DNA cleavage activity, histone acetylation activity, histone methylation activity, etc.), wherein the site of enzymatic activity is determined by the DNA-targeting RNA. In other embodiments, the site-directed modifying polypeptide comprises: (i) an RNA binding part that interacts with a DNA-targeting RNA, wherein the DNA-targeting RNA comprises a nucleotide sequence complementary to a sequence in the target DNA; and (ii) an active part that regulates transcription (e.g., increases or decreases transcription) in the target DNA, wherein the site of regulated transcription in the target DNA is determined by the DNA-targeting RNA.

[0134] In some cases, the site-directed modifying polypeptide has an enzyme activity that modifies the target DNA (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity). In other cases, the site-directed modifying polypeptide has an enzyme activity that modifies a polypeptide (e.g., histone) associated with the target DNA (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity).

[0135] In some cases, different site-directed modification polypeptides, such as different Cas9 proteins (i.e., Cas9 proteins from a variety of species) can be advantageously used in a variety of methods provided by the present invention to utilize a variety of enzyme features of different Cas9 proteins (e.g., for different pre-spacer adjacent motif (PAM) sequence preferences; for increased or decreased enzyme activity; for increased or decreased cytotoxicity levels; for changing the balance between NHEJ, homology-directed repair, single-strand breaks, double-strand breaks, etc.). Cas9 proteins from a variety of species (e.g., those disclosed in Shmakov et al., 2017, or polypeptides derived therefrom) may require different PAM sequences in the target DNA. Therefore, for a particular Cas9 enzyme selected, the PAM sequence requirement may be different from the 5'-NGG-3' sequence (where N is A, T, C, or G) known to be required for Cas9 activity. Many Cas9 orthologs from a variety of species have been identified herein, and the protein shares only a few identical amino acids. All identified Cas9 orthologs have the same domain architecture with a central HNH endonuclease domain and separate RuvC / RNase H domains. Cas9 proteins share 4 key motifs with conserved architecture; motifs 1, 2, and 4 are RuvC-like motifs, while motif 3 is a HNH motif.

[0136] The site-directed modification polypeptide can also be a chimeric and modified Cas9 nuclease. For example, it can be a modified Cas9 "base editor". Base editing enables a target DNA base to be directly and irreversibly converted to another base in a programmable manner without the need for DNA cutting or donor DNA molecules. For example, Komor et al. (2016, Nature [Nature], 533: 420-424) teach Cas9-cytidine deaminase fusion, in which Cas9 has also been engineered to be inactive and does not induce double-stranded DNA breaks. In addition, Gaudelli et al. (2017, Nature [Nature], doi: 10.1038 / nature24644) teach Cas9 fused to the catalytic activity weakened tRNA adenosine deaminase, which can mediate the conversion of A / T to G / C in the target DNA sequence. Another class of engineered Cas9 nucleases that can serve as site-directed modifying polypeptides in the methods and compositions of the present invention are variants that recognize a broad range of PAM sequences, including NG, GAA, and GAT (Hu et al., 2018, Nature, doi: 10.1038 / nature26155).

[0137] Any Cas9 protein (including those that occur naturally and / or are mutated or modified from naturally occurring Cas9 proteins) can be used as a site-directed modified polypeptide in the methods and compositions of the present invention. The catalytically active Cas9 nuclease cuts the target DNA, thereby generating double-strand breaks. These breaks are then repaired by the cell in one of two ways: non-homologous end joining, and homology-directed repair.

[0138] In non-homologous end joining (NHEJ), double-strand breaks are repaired by directly connecting to each other through the broken ends. In this way, no new nucleic acid material is inserted into the site, although some nucleic acid materials can be lost, resulting in deletion. In homology-directed repair, a donor DNA molecule or an intermediate DNA homologous to the target DNA sequence of cutting is used as a template, for the repair of the target DNA sequence of cutting, resulting in genetic information being transferred to the target DNA from the donor polynucleotide. In this way, new nucleic acid material can be inserted / copied into the site. In some cases, the target DNA is contacted with a donor molecule (such as a donor DNA molecule or an intermediate DNA molecule). In some cases, a donor DNA molecule or an intermediate DNA molecule is introduced into a cell. In some cases, at least one section of a donor DNA molecule or an intermediate DNA molecule is integrated into the genome of the cell.

[0139] Modification of the target DNA due to NHEJ and / or homology-directed repair results in, for example, gene correction, gene replacement, gene tagging, transgenic insertion, nucleotide deletion, gene disruption, gene mutation, etc. Thus, in the absence of an exogenously provided donor polynucleotide, by cleaving the target DNA sequence and allowing the cell to repair the sequence, cleavage of DNA by a site-directed modifying polypeptide can be used to delete nucleic acid material from a target DNA sequence (e.g., to destroy genes that make cells susceptible to infection (e.g., CCR5 or CXCR4 genes, which make T cells susceptible to HIV infection), to remove pathogenic trinucleotide repeat sequences in neurons, to generate gene knockouts and mutations as disease models for study, etc.). Thus, the subject methods can be used to knock out a gene (resulting in a complete lack of transcription or altered transcription), or to knock genetic material into a selected locus in the target DNA. Alternatively, if a DNA-targeting RNA duplex and a site-directed modifying polypeptide are co-administered to a cell with a donor molecule that includes at least a segment homologous to a target DNA sequence, the subject methods can be used to add, i.e., insert or replace nucleic acid material into a target DNA sequence (e.g., to "knock in" a nucleic acid encoding a protein, siRNA, miRNA, etc.), to add a tag (e.g., 6xHis, a fluorescent protein (e.g., green fluorescent protein; yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a gene (e.g., a promoter, a polyadenylation signal, an internal ribosome entry sequence (IRES), a 2A peptide, a start codon, a stop codon, a splicing signal, a localization signal, etc.), to modify a nucleic acid sequence (e.g., to introduce a mutation), etc. Thus, the complex comprising a DNA-targeting RNA duplex and a site-directed modifying polypeptide can be used in any in vitro or in vivo application in which it is desired to modify DNA in a site-specific, i.e., "targeted," manner, such as gene knockout, gene knockin, gene editing, gene tagging, etc., such as for use in, for example, gene therapy (e.g., for treating a disease), or as an antiviral, antipathogenic, or anticancer therapeutic, in the production of genetically modified organisms in agriculture, in the large-scale production of proteins by cells for therapeutic, diagnostic, or research purposes, inducing iPS cells, in biological research, in targeting genes of pathogens for deletion or replacement, etc.

[0140] The term "CRISPR-associated protein", "Cas protein", "CRISPR-associated nuclease" or "Cas nuclease" refers to a wild-type Cas protein, a fragment thereof, or a mutant or variant thereof. The term "Cas mutant" or "Cas variant" refers to a protein or polypeptide derivative of a wild-type Cas protein, such as a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or a combination thereof. In certain embodiments, the Cas mutant or Cas variant substantially retains the nuclease activity of the Cas protein, such as a Cas9 variant described herein that is operably linked to a plant-derived nuclear localization signal (NLS). In certain embodiments, the Cas nuclease is mutated so that one or two nuclease domains are inactive, such as a Cas9 that is not catalytically active, called dCas9, which is still able to target a specific genomic location, but does not have endonuclease activity (Qi et al., 2013, Cell [Cell], 152: 1173-1183, hereby incorporated herein). In some embodiments, the Cas nuclease is mutated so that it lacks some or all of the nuclease activity of its wild-type counterpart. The Cas protein can be Cas9, Cpf1 (Zetsche et al., 2015, Cell, 163:759-771, hereby incorporated herein), or any other CRISPR-associated nuclease.

[0141] Argonaute proteins from bacteria such as Thermus thermophilus can also be used for genome editing in a manner similar to CRISPR / Cas9. Similar to Cas9, Argonaute proteins are thought to use oligonucleotides as guides to degrade invading genomes. The complex of these guides and thermophilic Argonaute proteins cuts complementary DNA strands at high temperatures (75 degrees Celsius). WO 2014 / 189628 describes a method in which this system can be used for genome editing. Other examples include WO 2014 / 189628, WO 2016 / 161375, and WO 2016 / 166268.

[0142] The present invention provides a method for reducing the expression of a target gene, the method comprising introducing a nuclease into a plant cell that is capable of site-directed DNA cleavage at a genomic site encoding a natural pre-miRNA of the plant cell; causing at least one double-strand break at or near the genomic site; selecting cells, wherein the at least one double-strand break has been repaired with an intervening DNA to replace the genomic site; and reducing the expression of the target gene, wherein the intervening DNA encodes a modified pre-miRNA comprising an amiRNA core sequence complementary to the target gene.

[0143] The genomic locus encodes the native pre-miRNA of the plant cell being modified by the method of the present invention. The intermediate DNA is a DNA identical to the genomic locus encoding the native pre-miRNA of the plant cell, but with the amiRNA core sequence complementary to the new target gene replacing the native miRNA core sequence. The intermediate DNA is introduced into the plant cell together with the nuclease.

[0144] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the nuclease capable of site-directed DNA cleavage at the genomic site encoding the natural pre-miRNA makes a double-stranded break at the genomic site sequence.

[0145] In another embodiment, the present invention relates to the method according to the preceding embodiments, wherein the nuclease capable of site-directed DNA cleavage at a genomic site encoding a natural pre-miRNA causes a double-stranded break near the genomic site, preferably within 2kb upstream or downstream of the genomic site.

[0146] In another embodiment, the present invention relates to the method according to the preceding embodiments, wherein the nuclease capable of site-directed DNA cleavage at a genomic site encoding a natural pre-miRNA causes a double-stranded break near the genomic site, preferably within 500 nucleotides upstream or downstream of the genomic site.

[0147] In another embodiment, the invention relates to the method according to the preceding embodiments, wherein the nuclease capable of site-directed DNA cleavage at the genomic site encoding the natural pre-miRNA makes a double-stranded break within 100 nucleotides upstream or downstream of the genomic site.

[0148] In another embodiment, the invention relates to the method according to the preceding embodiments, wherein the nuclease capable of site-directed DNA cleavage at the genomic site encoding the native pre-miRNA of the plant cell causes at least two double-stranded breaks at or near the genomic site.

[0149] In another embodiment, the present invention relates to the method according to the preceding embodiment, wherein the target gene is an exogenous target gene, more preferably a harmful organism gene, more preferably a viral, fungal or microbial gene.

[0150] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the target gene is a pest gene or a nematode pest gene.

[0151] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the target gene is a Bunyavirales gene, preferably a tospovirus gene, and more preferably a tomato spotted wilt virus (TSWV) gene.

[0152] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the target gene is an endogenous plant gene.

[0153] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the target endogenous plant gene is a gene involved in plant development, biotic or abiotic stress.

[0154] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the plant cell is a Solanaceae plant, corn, rice, canola, soybean, or sunflower cell. In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the plant cell is a tomato cell.

[0155] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the genomic site encoding a native pre-miRNA encodes a native tomato pre-miRNA.

[0156] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the genomic locus comprises SEQ ID NO:6 or SEQ ID NO:7.

[0157] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the genomic locus consists of SEQ ID NO:6 or SEQ ID NO:7.

[0158] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the genomic locus encodes the SlmiR156b or SlmiR1919b gene.

[0159] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the intermediate DNA comprises any one of SEQ ID NOs: 1 to 5.

[0160] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the intermediate DNA comprises any one of SEQ ID NOs: 22 to 24.

[0161] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the intermediate DNA comprises any one of SEQ ID NOs: 8 to 17.

[0162] In another embodiment, the present invention relates to a method according to any one of the preceding embodiments, wherein the nuclease is selected from the group consisting of: mega nuclease (MN), zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), Cas9 nuclease, Cfp1 nuclease, dCas9-FokI, dCpf1-FokI, chimeric Cas9 / Cpf1-cytosine deaminase, chimeric Cas9 / Cpf1-adenine deaminase, chimeric FEN1-FokI, and Mega-TAL, nickase Cas9 (nCas9), chimeric dCas9 non-FokI nuclease and dCpf1 non-FokI nuclease.

[0163] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the cell has a haploid, diploid, polyploid or hexaploid genome.

[0164] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the cell is heterozygous for the modified pre-miRNA.

[0165] In another embodiment, the invention relates to the method according to any one of the preceding embodiments, wherein the cell has one copy of the modified pre-miRNA and one copy of the native pre-miRNA.

[0166] In the context of the present invention, haploid plant cells comprising one copy of the modified pre-miRNA have utility in, for example, breeding processes and seed production methods.

[0167] In another embodiment, the present invention relates to a method for producing plant seeds, preferably Solanaceae, corn, rice, canola, soybean or sunflower seeds, more preferably tomato seeds, the method comprising crossing a plant comprising a plant cell obtained by the method of any one of the preceding embodiments with itself or with another plant of the same crop.

[0168] In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein the method further comprises the use of one or more guide sequences. In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein one or more guide sequences are introduced into the cell together with the nuclease. In another embodiment, the present invention relates to the method according to any one of the preceding embodiments, wherein one or more guide sequences are derived from a target genomic site.

[0169] In another embodiment, the method of any of the preceding embodiments confers resistance to a plant pest.

[0170] In another embodiment, the present invention relates to a plant cell, preferably a Solanaceae plant, corn, rice, canola, soybean or sunflower cell, more preferably a tomato plant cell obtained by the method of any one of the above embodiments.

[0171] In another embodiment, the invention relates to a plant cell according to the preceding embodiments, wherein the cell comprises any one of SEQ ID NOs: 1-5.

[0172] In another embodiment, the invention relates to a plant cell according to the preceding embodiments, wherein the cell comprises any one of SEQ ID NOs: 22-24.

[0173] In another embodiment, the invention relates to a plant cell according to the preceding embodiments, wherein the cell comprises any one of SEQ ID NOs: 8-17.

[0174] In another embodiment, the present invention relates to a plant cell comprising any one of SEQ ID NOs: 1-5.

[0175] In another embodiment, the present invention relates to a plant cell comprising any one of SEQ ID NOs: 22-24.

[0176] In another embodiment, the present invention relates to a plant cell comprising any one of SEQ ID NOs: 8-17.

[0177] In another embodiment, the present invention relates to a diploid plant cell comprising one copy of SEQ ID NO: 6 and one copy of any one of SEQ ID NOs: 8-12.

[0178] In another embodiment, the present invention relates to a diploid plant cell comprising one copy of SEQ ID NO:7 and one copy of any one of SEQ ID NOs:13-17.

[0179] In another embodiment, the present invention relates to a method for producing plant seeds, preferably Solanaceae, corn, rice, canola, soybean or sunflower seeds, more preferably tomato seeds, the method comprising crossing a plant comprising the plant cell according to any one of the preceding embodiments with itself or with another plant of the same crop.

[0180] In another embodiment, the present invention relates to a plant comprising the plant cell according to any one of the preceding embodiments. In another embodiment, the present invention relates to a tomato plant comprising the plant cell according to any one of the preceding embodiments.

[0181] In another embodiment, the present invention relates to a plant part comprising the plant cell according to any one of the preceding embodiments. In another embodiment, the present invention relates to a tomato plant part comprising the plant cell according to any one of the preceding embodiments. In another embodiment, the plant part is a plant seed, preferably a tomato plant seed.

[0182] In another embodiment, the plant or plant part according to any of the preceding embodiments provides pest resistance. In another embodiment, the plant or plant part according to any of the preceding embodiments provides pest resistance to tospovirus. In another embodiment, the plant or plant part according to any of the preceding embodiments provides resistance to TSWV.

[0183] In another embodiment, the present invention relates to a method for producing plant seeds, preferably Solanaceae, corn, rice, canola, soybean or sunflower seeds, more preferably tomato seeds, the method comprising crossing the plant according to any one of the preceding embodiments with itself or with another plant of the same crop.

[0184] In another embodiment, the present invention relates to a method for producing a plant, preferably a Solanaceae plant, corn, rice, canola, soybean or sunflower plant, more preferably a tomato plant, the method comprising crossing the plant according to any one of the preceding embodiments with itself or with another plant of the same crop to produce progeny plants comprising the amiRNA of the present invention and exhibiting a novel phenotype.

[0185] The methods of the present invention have been implemented and exemplified in the model crop tomato and the model virus Tomato Spotted Wilt Virus (TSWV). A skilled artisan with the information disclosed herein can easily transfer the knowledge and implement the methods of the present invention in different plants and different target types.

[0186] Examples

[0187] Example 1: Identification of TSWV sequences suitable for use as amiRNA cores

[0188] The publicly available TSWV genomes (Table 1) were collected and aligned.

[0189] Table 1 lists the data from NCBI (available at the World Wide Web www.ncbi.nlm.nih.gov / nuccore / TSWV genomes collected from

[0190]

[0191]

[0192]

[0193] Conserved TSWV regions with high similarity were selected. The 21-nt sequence in the tomato plant genome was analyzed for GC content, secondary structure, specific position and off-target (TSWV 21-nt sequence compared to the tomato genome). TSWV sequences with 30% to 60% GC content and no less than 3 mismatched hits on the tomato genome were preferred.

[0194] To test whether a given amiRNA core viral sequence can effectively control the virus, potential targets were identified in the TSWV viral genome as described above and tested in transient experiments. The Arabidopsis native pre-miRNA AtmiR159a was used as a scaffold. Modified miRNAs were directly synthesized by replacing the native AtmiR159a core sequence with a designed 21-nt sequence complementary to the TSWV target gene. The modified miRNAs were compared with the native miRNAs in terms of structure and stability (MFE), and the miRNAs with the least changes were selected for experimental evaluation and validation in transient viral assays. For these transient assays, the binary vector 17839 ( Figure 5 ) to express the designed amiRNA. Both the binary vector 17839 and the synthesized AtmiR159a-amiRNA fragment were cut by BamHI / NcoI and gel purified. The two fragments were ligated together and transformed into DH5α cells. Positive clones were verified by BamHI / NcoI digestion, and all connections were sequenced.

[0195] Table 2 lists all TSWV sequences tested as amiRNA cores within the AtmiR159a scaffold. Five of them (SEQ ID NOs: 1-5) have been identified as suitable for providing high resistance to TSWV in transient assays ( Figure 2 and 3 ).

[0196] example amiRNA Core Resistance effect SEQ ID NO: ET-16 amiRNA_RdRp_GC52 Moderate resistance ET-17 amiRNA_RdRp_GC42 Susceptibility ET-18 amiRNA_NSs_GC52 Susceptibility ET-19 amiRNA_N_GC42 Moderate resistance ET-20 amiRNA_GnGc_GC52 Susceptibility ET-21 amiRNA_GnGc_GC40 Moderate resistance ET-22 amiRNA_NSm_GC30 Moderate resistance ET-23 amiTSWV_N1w_PC High resistance 1 ET-24 amiTSWV_N2_PC High resistance 2 ET-26 amiTSWV_N2_PC_rev High resistance 3 ET-27 amiRNA_NSs_GC52_rev Susceptibility ET-36 amiR159a_3p_N_GC42 Susceptibility ET-37 amiR159a_3p_N_GC25 Susceptibility ET-38 amiR159a_3p_N_GC35 High resistance 4 ET-39 amiR159a_3p_N_GC50 High resistance 5 ET-40 amiR159a_3p_N_GC43 Susceptibility ET-41 amiR159a_3p_NSs_GC35 Susceptibility ET-42 amiR159a_3p_RdRP_GC25 Susceptibility ET-43 amiR159a_3p_GnGc_GC30 Moderate resistance ET-44 amiR159a_3p_NSm_GC40 Susceptibility

[0197] Examples ET-23, ET-24, ET-26, ET-38 and ET-39 provide high levels of resistance to TSWV. Thus, this method described in Example 1 allows the identification of suitable amiRNA core sequences homologous to new target genes and can be effectively used to obtain novel phenotypes. Notably, ET-26 (the reverse complement of ET-24) also provided high levels of resistance, indicating that once an effective amiRNA core sequence is identified, its reverse complement can also be successfully used using the methods of the present invention.

[0198] Example 2: Identification of suitable native tomato pre-miRNA sequences

[0199] To test whether a given native tomato pre-miRNA sequence could be effectively used as a container for the TSWV amiRNA core sequence for virus control, potential pre-miRNA scaffolds were identified in the tomato genome and tested using ET-24 (SEQ ID NO: 2) as the TSWV amiRNA core sequence (see Example 1).

[0200] Public tomato sRNA-seq data (Table 3) were collected to examine native miRNA expression.

[0201] Table 3 lists the data from the NCBI SRA database ( Available on the World Wide Web at www.ncbi.nlm.nih.gov / sra / ) Collected tomato sRNA-seq dataset.

[0202] run experiment length Total number of spots SRR039920 SRX019222 36 5299195 SRR039921 SRX019223 36 4574008 SRR2039800 SRX1038192 37 6202076 SRR2989577 SRX1478064 36 11026240 SRR2989578 SRX1478065 36 18528550 SRR4013313 SRX2008739 50 23760631 SRR4346447 SRX2213272 51 46872476 SRR5031857 SRX2356906 51 2655264 SRR5031858 SRX2356907 51 4954975 SRR5031859 SRX2356908 51 4375546 SRR786979 SRX252396 36 15573561 SRR786980 SRX252397 36 13077046 SRR1463412 SRX627473 49 18158256 SRR1777738 SRX833690 50 10309183 SRR1795959 SRX871216 51 73080323

[0203] The abundance of mature miRNAs in these datasets was analyzed and compared with miRBase ( Available on the World Wide Web www.mirbase.org / The following criteria were used to select tomato native miRNAs for modification, including miRNAs with multiple family members, production of identical mature miRNAs and high expression levels, especially in green tissues.

[0204] Some excellent candidates listed in Table 3 were selected for further experiments. The amiRNA core sequence ET24 (SEQ ID NO: 2) was first used to verify these candidates, and then the new 21-nt sequence was also used. The binary vector 17839 was first cut by Kpn1 / Nco1, and the 5762bp fragment was gel purified. The 1kb promoter region and the modified pre-miRNA (the miRNA core sequence was replaced by the identified amiRNA core sequence ET-24) were directly synthesized and cut with Kpn1 / Nco1. The two fragments were connected together and transformed into DH5α cells. Positive clones were verified by Kpn1 / Nco1 digestion, and all connections were sequenced.

[0205] Table 4 lists all the sequences tested as pre-miRNA scaffolds. Two of them (SEQ ID NOs: 9 and 14) have been identified as suitable for providing high resistance to TSWV in transient assays ( Figure 4 ).

[0206] example pre-miRNA Resistance effect SEQ ID NO: ET-28 miR156a_N2_PC Susceptibility ET-29 miR156b_N2_PC Resistance 9 ET-30 miR168a_N2_PC NA ET-31 miR168b_N2_PC Susceptibility ET-32 miR172a_N2_PC Susceptibility ET-33 miR395b1_N2_PC Susceptibility ET-34 miR395b2_N2_PC Susceptibility ET-35 miR1919b_N2_PC Resistance 14

[0207] Tomato pre-miRNA scaffolds ET-29 and ET-35 holding the amiRNA core TSWV sequence ET-24 (SEQ ID No 9 and 14, respectively) showed good resistance levels against TSWV, indicating that they are suitable for use in the methods of the present invention.

[0208] Example 3: Design of genome editing constructs to modify native tomato pre-miRNA by replacing the amiRNA core sequence To target tomato viral pathogen gene targets.

[0209] To test whether editing the tomato native miRNA to target viral genes could confer resistance to the virus on tomatoes, the following constructs were designed to edit the native tomato miRNA SlmiR156b. The target viral genes tested were the RNA-dependent RNA polymerase (RdRp), glycoprotein precursor (Gn / Gc), nonstructural movement protein (NSm), nonstructural silencing suppressor protein (NSs), and nucleocapsid protein (N) from TSWV. Cas9 was used with two gRNAs to generate double-strand breaks around the native SlmiR156b locus in tomato and provide a modified amiRNA donor for replacement.

[0210] Binary vector 24598 ( Figure 6 ) contains a soybean codon-optimized Cas9 driven by the constitutive prAtEF1aA1-02 promoter and two gene-specific gRNAs driven by prAtU6-01 and prSlU6 to edit the tomato SlmiR156b gene. The construct is designed to replace the natural SlmiR156b core sequence with an artificial core sequence targeting the TSWV viral genome. A 1.5kb donor sequence is also included, which contains a 1kb promoter, a pre-SlmiR156b with an artificial core, and a 0.5kb terminator. cSpec-03 driven by prGmEF-01 is used as a selectable marker. The donor DNA fragment and two gRNA boxes of prAtU6-01-rsgRNASlmiR156b-A (SEQ ID NO: 20) and prSlU6-rsgRNASlmiR156b-B (SEQ ID NO: 21) were synthesized by Generalbiol. All four boxes in this binary vector are part of a single transgene. Sequence Listing <110> Syngenta Crop Protection AG Syngenta Biotechnology China Co. Ltd. LIU, Juntao XU, Jianping CHEN, Yanhui LIU, Zhiqiang CHEN, Xi <120> Inhibition of target gene expression by genome editing of natural miRNAs <130> 81815-CN-REG-ORG-P-1 <160> twenty four <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Tomato spotted wilt virus <400> 1 cagtgttgtc tgtgctatat a 21 <210> 2 <211> twenty one <212> DNA <213> Tomato spotted wilt virus <400> 2 atgaaatgtt cggggttaaa a 21 <210> 3 <211> twenty one <212> DNA <213> Tomato spotted wilt virus <400> 3 ttttaacccc gaacatttca t 21 <210> 4 <211> twenty one <212> DNA <213> Tomato spotted wilt virus <400> 4 ttcaaatgct ttgcttttca g 21 <210> 5 <211> twenty one <212> DNA <213> Tomato spotted wilt virus <400> 5 tagcagcata ctctttcccc t 21 <210> 6 <211> 1084 <212> DNA <213> Tomato (Solanum lycopersicum) <400> 6 attcggttac ctctctttcc tatgtaacta aatgtctgct aatgtattca caagtccaag 60 tgatgtattc gaaattataa aatttaagga attcttataa tttgaaaaag aagtagaaaa 120 taatgtaatt agctcttaac gctatgaaat ttatgtaaat tatataatta ttatgtactc 180 cttccgattc atatgacata tcttactttt aacctttaca ttttgttcaa aataagtaat 240 tttattgtaa ctaagaatgt attactatta tttagttttt caaatttacg ccttcttttg 300 ataagtgggt tttaactttt aacgtaacca agaaatgata ttaaatatgt actatataat 360 taagaataat tagtaaaaac aatttttaat attttaggac ctaaactttt tatttttttg 420 tgcgacatgt tacctaaaag atagtaaaaa aataattgcc aataataaat ggaataattt 480 tactagaaaa taaacatagg aaaagaaata tacgtaacac attaaattat atcaacggat 540 cattaaaatt cttttgtatt gtctatataa tactatataa aagtaaagaa ttctataaaa 600 ttaatttgag ttgacataga aaaactgttt tgggttaaat tttttactag ttgtgcacta 660 tttatcttcg atctataaat agatcgacat gttggaaaac actcaaacca tcctatgcta 720 taagataata tatagctaca tttcttagat aactagaaac ctccattagc ttcctattct 780 cataagcaaa tctccaatca taatttacaa actgagactc gatgtatgat cagtgataga 840 tttaaaattt agatatcaca agtgatatgt ttagatcata agggtctaga aatgcatatc 900 taactcgatg tattctatgt tgcactttgt cccgcatcac ctcacaactg taagtataaa 960 ttatttcaaa gagagcagga aagtattggg tgagatattg ttgacagaag atagagagca 1020 cgaataatga ggtgctaatt ggaagctgca ccttaattct ttgtgctctc tattcttctg 1080 tcat 1084 <210> 7 <211> 1207 <212> DNA <213> Solanum lycopersicum <400> 7 agcgaattat acagaacata attatgcaaa ttttgctata acatacaaat atgaatttta 60 tgtttgatat atgtgaaagt tgcccattat ggaattagct atgaaattta tggtaattttt 120 aagggacaat tacgcggtga agcaaactta tactacttaa atattcatca tagctatagt 180 ttgctataat taacactcgc gactaatatt atacattaat tatgtggcct gacttcgagt 240 ttgtataatt agtcagaata aaaataca tgtataata tacaattatc taaccgatat 300 acataaaaa tttaccctct tcccactctt tgccctctct cgctcgtctc tctcccaatc 360 tcgttcttct cttcctccct tcccagtat tgccgccact ctcccaatct ctctctcctc 420 tctcctccct ctcccaatct cctttgccat atatacaaat acatatgtat aatacaat 480 tatataacca atatacatat acaatgcacc tctccccctc tctttgccct ctctcctctc 540 tctcccagtc tcgcttgcct gtctcttctc tataacatgt agttacagat tgtaattatc 600 aaactgtaac tatgaagagt attaaacta tttttgagtg actatacgtg aaagttcctc 660 taattttaat caattcatca caaatccata tctaaatgaa atgaacaaag aaaaattatt 720 attgtttagt tatgaatttt atcaatcact aattcacgtg aatattaggg aataaaaaat 780 gactactttg gcataatcta aacttgctag tagaaatttg aagttgcaaa aagaaaaaga 840 gaagcaaaag aagtgaaaga aaaagaggcg ttattgtttt ttactttatt cagtataaag 900 tgcgttttac tcttctattt cttgtagctc acaaatcgtc tttactgacc ctacaaattc 960 tcttccggca agttttcagg ttcctccgaa tcgctccgac gcctttgatg ttcacatctt 1020 ccggtagtcc tgtcgcagat gactttcgcc catttatgga accacacttt ctttaatttg 1080 aattctatgt ggtaggacga gagtcatctg tgacaggata atggaagatc gagttatcaa 1140 aggcttattg ggcgtttcct ttttcatctt gagttcgtac cagattaatg caaaaccgaa 1200 gaagtag 1207 <210> 8 <211> 1083 <212> DNA <213> Artificial Sequence <220> <223> Tomato / Tomato spotted wilt virus <400> 8 attcggttac ctctctttcc tatgtaacta aatgtctgct aatgtattca caagtccaag 60 tgatgtattc gaaattataa aatttaagga attcttataa tttgaaaaag aagtagaaaa 120 taatgtaatt agctcttaac gctatgaaat ttatgtaaat tatataatta ttatgtactc 180 cttccgattc atatgacata tcttactttt aacctttaca tttgttca aaagtaat 240 tttattgtaa ctaagaatgt attactatta tttagtttt caatttacg ccttctttg 300 ataagtgggt tttaactttt aacgtaacca agaaatgata ttaaatatgt actatataat 360 taggaataat tgtaaaac aatttttaat atttaggac ctaaactttt tattttttg 420 tgcgacatgt tacctaaag atagtaaaa aaattgcc aaataat ggaataattt 480 tactgaaaa taaacatagg aaagaata tacgtacac atttaattat atcaacggat 540 cattaaaatt cttttgtatt gtctatata tactataata aagtaaaa ttcttaaaa 600 ttaatttgag ttgacataga aaaactgttt tggttaaat tttttactag ttgtgcacta 660 tttatcttcg atcttaaat agatcgacat gttggaaac actcaaacca tcctatgcta 720 taagatata tatagctaca tttcttagat aactagaaac ctccattagc ttcctattct 780 catagcaa tctccaatca taatttaca actgagactc catgtatgat cagtgataga 840 tttaaaattt agatatcaca agtgatatgt ttagatcata agggtctaga aatgcatatc 900 taactcgatg tattctatgt tgcactttgt cccgcatcac ctcacaactg taagtataaa 960 ttatttcaaa gagagcagga aagtattggg tgagatattg cagtgttgtc tgtgctatat 1020 agaataatga ggtgctaatt ggaagctgca ccttaattct tttatatagc acagacaaca 1080 ctg 1083 <210> 9 <211> 1083 <212> DNA <213> Artificial Sequence <220> <223> Tomato / Tomato spotted wilt virus <400> 9 attcggttac ctctctttcc tatgtaacta aatgtctgct aatgtattca caagtccaag 60 tgatgtattc gaaattataa aatttaagga attcttataa tttgaaaaag aagtagaaaa 120 taatgtaatt agctcttaac gctatgaaat ttatgtaaat tatataatta ttatgtactc 180 cttccgattc atatgacata tcttactttt aacctttaca ttttgttcaa aataagtaat 240 tttattgtaa ctaagaatgt attactatta tttagttttt caaatttacg ccttcttttg 300 ataagtgggt tttaactttt aacgtaacca agaaatgata ttaaatatgt actatataat 360 taagaataat tagtaaaaac aatttttaat attttaggac ctaaactttt tatttttttg 420 tgcgacatgt tacctaaaag atagtaaaaa aataattgcc aataataaat ggaataattt 480 tactagaaaa taaacatagg aaaagaaata tacgtaacac attaaattat atcaacggat 540 cattaaaatt cttttgtatt gtctatataa tactatataa aagtaaagaa ttctataaaa 600 ttaatttgag ttgacataga aaaactgttt tgggttaaat tttttactag ttgtgcacta 660 tttatcttcg atctataaat agatcgacat gttggaaaac actcaaacca tcctatgcta 720 taagataata tatagctaca tttcttagat aactagaaac ctccattagc ttcctattct 780 cataagcaaa tctccaatca taatttacaa actgagactc gatgtatgat cagtgataga 840 tttaaaattt agatatcaca agtgatatgt ttagatcata agggtctaga aatgcatatc 900 taactcgatg tattctatgt tgcactttgt cccgcatcac ctcacaactg taagtataaa 960 ttatttcaaa gagagcagga aagtattggg tgagatattg atgaaatgtt cggggttaaa 1020 agaataatga ggtgctaatt ggaagctgca ccttaattct ttttttaacc ccgaacattt 1080 cat 1083 <210> 10 <211> 1083 <212> DNA <213> Artificial Sequence <220> <223> Tomato / Tomato spotted wilt virus <400> 10 attcggttac ctctctttcc tatgtaacta aatgtctgct aatgtattca caagtccaag 60 tgatgtattc gaaattataa aatttaagga attcttataa tttgaaaaag aagtagaaaa 120 taatgtaatt agctcttaac gctatgaaat ttatgtaaat tatataatta ttatgtactc 180 cttccgattc atatgacata tcttactttt aacctttaca ttttgttcaa aataagtaat 240 tttattgtaa ctaagaatgt attactatta tttagttttt caaatttacg ccttcttttg 300 ataagtgggt tttaactttt aacgtaacca agaaatgata ttaaatatgt actatataat 360 taagaataat tagtaaaaac aatttttaat attttaggac ctaaactttt tatttttttg 420 tgcgacatgt tacctaaaag atagtaaaaa aataattgcc aataataaat ggaataattt 480 tactagaaaa taaacatagg aaaagaaata tacgtaacac attaaattat atcaacggat 540 cattaaaatt cttttgtatt gtctatataa tactatataa aagtaaagaa ttctataaaa 600 ttaatttgag ttgacataga aaaactgttt tgggttaaat tttttactag ttgtgcacta 660 tttatcttcg atctataaat agatcgacat gttggaaaac actcaaacca tcctatgcta 720 taagataata tatagctaca tttcttagat aactagaaac ctccattagc ttcctattct 780 cataagcaaa tctccaatca taatttacaa actgagactc gatgtatgat cagtgataga 840 tttaaaattt agatatcaca agtgatatgt ttagatcata agggtctaga aatgcatatc 900 taactcgatg tattctatgt tgcactttgt cccgcatcac ctcacaactg taagtataaa 960 ttatttcaaa gagagcagga aagtattggg tgagatattg ttttaacccc gaacatttca 1020 tgaataatga ggtgctaatt ggaagctgca ccttaattct ttatgaaatg ttcggggtta 1080 aaa 1083 <210> 11 <211> 1083 <212> DNA <213> Artificial Sequence <220> <223> Tomato / Tomato spotted wilt virus <400> 11 attcggttac ctctctttcc tatgtaacta aatgtctgct aatgtattca caagtccaag 60 tgatgtattc gaaattataa aatttaagga attcttataa tttgaaaaag aagtagaaaa 120 taatgtaatt agctcttaac gctatgaaat ttatgtaaat tatataatta ttatgtactc 180 cttccgattc atatgacata tcttactttt aacctttaca tttgttca aaagtaat 240 tttattgtaa ctaagaatgt attactatta tttagtttt caatttacg ccttctttg 300 ataagtgggt tttaactttt aacgtaacca agaaatgata ttaaatatgt actatataat 360 taggaataat tgtaaaac aatttttaat atttaggac ctaaactttt tattttttg 420 tgcgacatgt tacctaaag atagtaaaa aaattgcc aaataat ggaataattt 480 tactgaaaa taaacatagg aaagaata tacgtacac atttaattat atcaacggat 540 cattaaaatt cttttgtatt gtctatata tactataata aagtaaaa ttcttaaaa 600 ttaatttgag ttgacataga aaaactgttt tggttaaat tttttactag ttgtgcacta 660 tttatcttcg atcttaaat agatcgacat gttggaaac actcaaacca tcctatgcta 720 taagatata tatagctaca tttcttagat aactagaaac ctccattagc ttcctattct 780 catagcaa tctccaatca taatttaca actgagactc catgtatgat cagtgataga 840 tttaaaattt agatatcaca agtgatatgt ttagatcata agggtctaga aatgcatatc 900 taactcgatg tattctatgt tgcactttgt cccgcatcac ctcacaactg taagtataaa 960 ttatttcaaa gagagcagga aagtattggg tgagatattg ttcaaatgct ttgcttttca 1020 ggaataatga ggtgctaatt ggaagctgca ccttaattct ttctgaaaag caaagcattt 1080 gaa 1083 <210> 12 <211> 1083 <212> DNA <213> Artificial Sequence <220> <223> Tomato / Tomato spotted wilt virus <400> 12 attcggttac ctctctttcc tatgtaacta aatgtctgct aatgtattca caagtccaag 60 tgatgtattc gaaattataa aatttaagga attcttataa tttgaaaaag aagtagaaaa 120 taatgtaatt agctcttaac gctatgaaat ttatgtaaat tatataatta ttatgtactc 180 cttccgattc atatgacata tcttactttt aacctttaca ttttgttcaa aataagtaat 240 tttattgtaa ctaagaatgt attactatta tttagttttt caaatttacg ccttcttttg 300 ataagtgggt tttaactttt aacgtaacca agaaatgata ttaaatatgt actatataat 360 taagaataat tagtaaaaac aatttttaat attttaggac ctaaactttt tatttttttg 420 tgcgacatgt tacctaaaag atagtaaaaa aataattgcc aataataaat ggaataattt 480 tactagaaaa taaacatagg aaaagaaata tacgtaacac attaaattat atcaacggat 540 cattaaaatt cttttgtatt gtctatataa tactatataa aagtaaagaa ttctataaaa 600 ttaatttgag ttgacataga aaaactgttt tgggttaaat tttttactag ttgtgcacta 660 tttatcttcg atctataaat agatcgacat gttggaaaac actcaaacca tcctatgcta 720 taagataata tatagctaca tttcttagat aactagaaac ctccattagc ttcctattct 780 cataagcaaa tctccaatca taatttacaa actgagactc gatgtatgat cagtgataga 840 tttaaaattt agatatcaca agtgatatgt ttagatcata agggtctaga aatgcatatc 900 taactcgatg tattctatgt tgcactttgt cccgcatcac ctcacaactg taagtataaa 960 ttatttcaaa gagagcagga aagtattggg tgagatattg tagcagcata ctctttcccc 1020 tgaataatga ggtgctaatt ggaagctgca ccttaattct ttaggggaaa gagtatgctg 1080 cta 1083 <210> 13 <211> 1144 <212> DNA <213> Artificial Sequence <220> <223> Tomato / Tomato spotted wilt virus <400> 13 agcgaattat acagaacata attatgcaaa ttttgctata acatacaaat atgaatttta 60 tgtttgatat atgtgaaagt tgcccattat ggaattagct atgaaattta tggtaatttt 120 aagggacaat tacgcggtga agcaaactta tactacttaa atattcatca tagctatagt 180 ttgctataat taacactcgc gactaatatt atacattaat tatgtggcct gacttcgagt 240 ttgtataatt agtcagaata aacaaataca tgttataata tacaattatc taaccgatat 300 acataaacaa tttacctctc tcccactctt tgccctctct cgctcgtctc tctcccaatc 360 tcgttcttct cttcctccct ttcccagtat tgccgccact ctcccaatct ctctctcctc 420 tctcctccct ctcccaatct ctcttgccat atatacaaat acatatgtat aatatacaat 480 tatataacca atatacatat acaatgcacc tctccccctc tctttgccct ctctcctctc 540 tctcccagtc tcgcttgcct gtctcttctc tataacatgt agttacagat tgtaattatc 600 aaactgtaac tatgaagagt aattaaacta tttttgagtg actatacgtg aaagttcctc 660 taattttaat caattcatca caaatccata tctaaatgaa atgaacaaag aaaaattatt 720 attgtttagt tatgaatttt atcaatcact aattcacgtg aatattaggg aataaaaaat 780 gactactttg gcataatcta aacttgctag tagaaatttg aagttgcaaa aagaaaaaga 840 gaagcaaaag aagtgaaaga aaaagaggcg ttattgtttt ttactttatt cagtataaag 900 tgcgttttac tcttctattt cttgtagctc acaaatcgtc tttactgacc ctacaaattc 960 tcttccggca agttttcagg ttcctccgaa tcgctccgac gcctttgatg ttcacatctt 1020 ccggtagtcc cagtgttgtc tgtgctatat aatttatgga accacacttt ctttaatttg 1080 aattctatgt ggtatatata gcacagacaa cactgggata atggaagatc gagttatcaa 1140 aggc 1144 <210> 14 <211> 1144 <212> DNA <213> Artificial Sequence <220> <223> Tomato / Tomato spotted wilt virus <400> 14 agcgaattat acagaacata attatgcaaa ttttgctata acatacaaat atgaatttta 60 tgtttgatat atgtgaaagt tgcccattat ggaattagct atgaaattta tggtaatttt 120 aagggacaat tacgcggtga agcaaactta tactacttaa atattcatca tagctatagt 180 ttgctataat taacactcgc gactaatatt atacattaat tatgtggcct gacttcgagt 240 ttgtataatt agtcagaata aaaataca tgtataata tacaattatc taaccgatat 300 acataaaaa tttaccctct tcccactctt tgccctctct cgctcgtctc tctcccaatc 360 tcgttcttct cttcctccct tcccagtat tgccgccact ctcccaatct ctctctcctc 420 tctcctccct ctcccaatct cctttgccat atatacaaat acatatgtat aatacaat 480 tatataacca atatacatat acaatgcacc tctccccctc tctttgccct ctctcctctc 540 tctcccagtc tcgcttgcct gtctcttctc tataacatgt agttacagat tgtaattatc 600 aaactgtaac tatgaagagt attaaacta tttttgagtg actatacgtg aaagttcctc 660 taattttaat caattcatca caaatccata tctaaatgaa atgaacaaag aaaaattatt 720 attgtttagt tatgaatttt atcaatcact aattcacgtg aatattaggg aataaaaaat 780 gactactttg gcataatcta aacttgctag tagaaatttg aagttgcaaa aagaaaaaga 840 gaagcaaaag aagtgaaaga aaaagaggcg ttattgtttt ttactttatt cagtataaag 900 tgcgttttac tcttctattt cttgtagctc acaaatcgtc tttactgacc ctacaaattc 960 tcttccggca agttttcagg ttcctccgaa tcgctccgac gcctttgatg ttcacatctt 1020 ccggtagtcc atgaaatgtt cggggttaaa aatttatgga accacacttt ctttaatttg 1080 aattctatgt ggtattttaa ccccgaacat ttcatggata atggaagatc gagttatcaa 1140 aggc 1144 <210> 15 <211> 1144 <212> DNA <213> Artificial Sequence <220> <223> Tomato / Tomato spotted wilt virus <400> 15 agcgaattat acagaacata attatgcaaa ttttgctata acatacaaat atgaatttta 60 tgtttgatat atgtgaaagt tgcccattat ggaattagct atgaaattta tggtaatttt 120 aagggacaat tacgcggtga agcaaactta tactacttaa atattcatca tagctatagt 180 ttgctataat taacactcgc gactaatatt atacattaat tatgtggcct gacttcgagt 240 ttgtataatt agtcagaata aacaaataca tgttataata tacaattatc taaccgatat 300 acataaaaa tttaccctct tcccactctt tgccctctct cgctcgtctc tctcccaatc 360 tcgttcttct cttcctccct tcccagtat tgccgccact ctcccaatct ctctctcctc 420 tctcctccct ctcccaatct cctttgccat atatacaaat acatatgtat aatacaat 480 tatataacca atatacatat acaatgcacc tctccccctc tctttgccct ctctcctctc 540 tctcccagtc tcgcttgcct gtctcttctc tataacatgt agttacagat tgtaattatc 600 aaactgtaac tatgaagagt attaaacta tttttgagtg actatacgtg aaagttcctc 660 taattttaat caattcatca caaatccata tctaaatgaa atgaacaaag aaaaattatt 720 attgtttagt tatgaatttt atcaatcact aattcacgtg aatattaggg aataaaaaat 780 gactactttg gcataatcta aacttgctag tagaaatttg aagttgcaaa aagaaaaaga 840 gaagcaaaag aagtgaaaga aaaagaggcg ttattgtttt ttactttatt cagtataaag 900 tgcgttttac tcttctattt cttgtagctc acaaatcgtc tttactgacc ctacaaattc 960 tcttccggca agttttcagg ttcctccgaa tcgctccgac gcctttgatg ttcacatctt 1020 ccggtagtcc ttttaacccc gaacatttca tatttatgga accacacttt ctttaatttg 1080 aattctatgt ggtaatgaaa tgttcggggt taaaaggata atggaagatc gagttatcaa 1140 aggc 1144 <210> 16 <211> 1144 <212> DNA <213> Artificial sequence <220> <223> Tomato / tomato spotted wilt virus <400> 16 agcgaattat acagaacata attatgcaaa ttttgctata acatacaaat atgaatttta 60 tgtttgatat atgtgaaagt tgcccattat ggaattagct atgaaattta tggtaatttt 120 aagggacaat tacgcggtga agcaaactta tactacttaa atattcatca tagctatagt 180 ttgctataat taacactcgc gactaatatt atacattaat tatgtggcct gacttcgagt 240 ttgtataatt agtcagaata aacaaataca tgttataata tacaattatc taaccgatat 300 acataaacaa tttacctctc tcccactctt tgccctctct cgctcgtctc tctcccaatc 360 tcgttcttct cttcctccct ttcccagtat tgccgccact ctcccaatct ctctctcctc 420 tctcctccct ctcccaatct ctcttgccat atatacaaat acatatgtat aatatacaat 480 tatataacca atatacatat acaatgcacc tctccccctc tctttgccct ctctcctctc 540 tctcccagtc tcgcttgcct gtctcttctc tataacatgt agttacagat tgtaattatc 600 aaactgtaac tatgaagagt attaaacta tttttgagtg actatacgtg aaagttcctc 660 taattttaat caattcatca caaatccata tctaaatgaa atgaacaaag aaaaattatt 720 attgtttagt tatgaatttt atcaatcact aattcacgtg aatattaggg aataaaaaat 780 gactactttg gcataatcta aacttgctag tagaaatttg aagttgcaaa aagaaaaaga 840 gaagcaaaag aagtgaaaga aaaagaggcg ttattgtttt ttactttatt cagtataaag 900 tgcgttttac tcttctattt cttgtagctc acaaatcgtc tttactgacc ctacaaattc 960 tcttccggca agttttcagg ttcctccgaa tcgctccgac gcctttgatg ttcacatctt 1020 ccggtagtcc ttcaaatgct ttgcttttca gatttatgga accacacttt cttaatttg 1080 aattctatgt ggtactgaaa agcaaagcat ttgaaggata atggaagatc gagttatcaa 1140 aggc 1144 <210> 17 <211> 1144 <212> DNA <213> Artificial Sequence <220> <223> Tomato / Tomato spotted wilt virus <400> 17 agcgaattat acagaacata attatgcaaa ttttgctata acatacaaat atgaatttta 60 tgtttgatat atgtgaaagt tgcccattat ggaattagct atgaaattta tggtaatttt 120 aagggacaat tacgcggtga agcaaactta tactacttaa atattcatca tagctatagt 180 ttgctataat taacactcgc gactaatatt atacattaat tatgtggcct gacttcgagt 240 ttgtataatt agtcagaata aacaaataca tgttataata tacaattatc taaccgatat 300 acataaacaa tttacctctc tcccactctt tgccctctct cgctcgtctc tctcccaatc 360 tcgttcttct cttcctccct ttcccagtat tgccgccact ctcccaatct ctctctcctc 420 tctcctccct ctcccaatct ctcttgccat atatacaaat acatatgtat aatatacaat 480 tatataacca atatacatat acaatgcacc tctccccctc tctttgccct ctctcctctc 540 tctcccagtc tcgcttgcct gtctcttctc tataacatgt agttacagat tgtaattatc 600 aaactgtaac tatgaagagt aattaaacta tttttgagtg actatacgtg aaagttcctc 660 taattttaat caattcatca caaatccata tctaaatgaa atgaacaaag aaaaattatt 720 attgtttagt tatgaatttt atcaatcact aattcacgtg aatattaggg aataaaaaat 780 gactactttg gcataatcta aacttgctag tagaaatttg aagttgcaaa aagaaaaaga 840 gaagcaaaag aagtgaaaga aaaagaggcg ttattgtttt ttactttatt cagtataaag 900 tgcgttttac tcttctattt cttgtagctc acaaatcgtc tttactgacc ctacaaattc 960 tcttccggca agttttcagg ttcctccgaa tcgctccgac gcctttgatg ttcacatctt 1020 ccggtagtcc tagcagcata ctctttcccc tatttatgga accacacttt ctttaatttg 1080 aattctatgt ggtaagggga aagagtatgc tgctaggata atggaagatc gagttatcaa 1140 aggc 1144 <210> 18 <211> 6727 <212> DNA <213> Artificial Sequence <220> <223> Binary Vector 17839 <400> 18 attcctgtgg ttggcatgca catacaaatg gacgaacgga taaacctttt cacgcccttt 60 taaatatccg attattctaa taaacgctct tttctcttag gtttacccgc caatatatcc 120 tgtcaacac tgatagttta aacgggaccc ggcgcccat ttaaatgta ccggtccgct 180 ggcagacaaa gtggcagaca tactgtccca caatgaga tggaatctgt aaagaaaac 240 gcgtgaaata atgcgtctga caaggttag gtcggctgcc tttaatcaat accaagtgg 300 tccctaccac gatggaaaaa ctgtgcagtc ggttttggctt tttctgacga acaataaga 360 ttcgtggccg acaggtgggg gtccaccatg tgaggcatc ttcagactcc ataatggag 420 caatgacgta agggcttacg aaatagtaa gggtagttg ggaatgtcc actcacccgt 480 cagtcttaa atacttagcc cctccctcat tgttaaggga gcaaatctc agagagatag 540 tcctagagag agaagagag caagtagcct agaagtagga tccatgtctc cagagagaag 600 gccagttgag attagacctg ctactgcggc cgatatggca gctgtttgtg atattgttaa 660 ccattatatt gagacttcta ctgttaactt cagaactgag ccacaactc ctcaagagtg 720 gattgatgat cttgagagac ttcaagatag atacccttgg cttgttgctg aggttgaggg 780 agttgttgct ggaattgctt atgctggacc tggaggct agaacgctt atgattggac 840 tgttgagtct actgtttatg tttctcatag acatcaaaga cttggacttg gatctactct ttatactcat cttcttaagt ctatggaggc tcaaggattc aagtctgttg ttgctgttat tggacttcca aacgatccat ctgttagact tcatgaggct cttggatata ctgctagagg aactcttaga gctgctggat attagcatgg aggatggcat gatgttggat tctggcaaag agatttcgag cttccagctc caccaagacc agttagacca gttactcaaa tttgaccatg ggtcgacctg cagatcgttc aaacatttgg caataagtt tcttaagatt gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta 1380. 1380. 1380. 1380. 1380. 1380. 1380. 1380. 1380. 1380 gcggtgtcat ctatgttact agatctgcta gccctgcagg aaatttaccg gtgcccgggc 1440 ggccagcatg gccgtatccg caatgtgtta ttagttgtc tagcgtcaa tttgtttaca ccacaatata tcctgccacc agccagcca cagctccccg accggcagct cggcacaaaa tcaccactcg atacaggcag cccatcagaa ttaattctca tgtttgacag cttatcatcg 1620 actgcacggt gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg 1680 tgcaggtcgt aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa 1740 tgttttttgc gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa 1800 ttaatcatcc ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa 1860 acagaccatg agggaagcgt tgatcgccga agtatcgact caactatcag aggtagttgg 1920 cgtcatcgag cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt 1980 ggatggcggc ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct 2040 tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg 2100 agagagcgag attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc 2160 gtggcgttat ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct 2220 tgcaggtatc ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc 2280 aagagaacat agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat ctatttgagg cgctaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc 2520 ccagtatcag cccgtcatac ttgaagctag gcaggcttat cttggacaag aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt tgttcactac gtgaaaggcg agatcaccaa agtagtcggc aaataaagct ctagtggatc tccgtaccca gggatctggc tcgcggcgga 2700. cgcacgacgc cggggcgaga ccataggcga tctcctaaat caatagtagc tgtaacctcg aagcgtttca cttgtaacaa cgattgagaa tttttgtcat aaaattgaaa tacttggttc gcatttttgt catccgcggt cagccgcaat tctgacgaac tgcccattta gctggagatg 2880. attgtacatc cttcacgtga aaatttctca agcgctgtga acaagggttc agttttaga ttgaaaggtg agccgttgaa acacgttctt cttgtcgatg acgacgtcgc tatgcggcat cttattattg aataccttac gatccacgcc ttcaaagtga ccgcggtagc cgacagcacc 3060 cagttcacaa gagtactctc ttccgcgacg gtcgatgtcg tggttgttga tctagattta 3120 ggtcgtgaag atgggctcga gatcgttcgt aatctggcgg caaagtctga tattccaatc 3180 ataattatca gtggcgaccg ccttgaggag acggataaag ttgttgcact cgagctagga 3240 gcaagtgatt ttatcgctaa gccgttcagt atcagagagt ttctagcacg cattcgggtt 3300 gccttgcgcg tgcgccccaa cgttgtccgc tccaaagacc gacggtcttt ttgttttact 3360 gactggacac ttaatctcag gcaacgtcgc ttgatgtccg aagctggcgg tgaggtgaaa 3420 cttacggcag gtgagttcaa tcttctcctc gcgtttttag agaaaccccg cgacgttcta 3480 tcgcgcgagc aacttctcat tgccagtcga gtacgcgacg aggaggttta tgacaggagt 3540 atagatgttc tcattttgag gctgcgccgc aaacttgagg cagatccgtc aagccctcaa 3600 ctgataaaaa cagcaagagg tgccggttat ttctttgacg cggacgtgca ggtttcgcac 3660 ggggggacga tggcagcctg agccaattcc cagatccccg aggaatcggc gtgagcggtc 3720 gcaaaccatc cggcccggta caaatcggcg cggcgctggg tgatgacctg gtggagaagt 3780 tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc agaagcacgc cccggtgaat 3840 cgtggcaagc ggccgctgat cgaatccgca aagaatcccg gcaaccgccg gcagccggtg 3900 cgccgtcgat taggaagccg cccaagggcg acgagcaacc agattttttc gttccgatgc 3960 tctatgacgt gggcacccgc gatagtcgca gcatcatgga cgtggccgtt ttccgtctgt 4020 cgaagcgtga ccgacgagct ggcgaggtga tccgctacga gcttccagac gggcacgtag 4080 aggtttccgc agggccggcc ggcatggcca gtgtgtggga ttacgacctg gtactgatgg 4140 cggtttccca tctaaccgaa tccatgaacc gataccggga agggaaggga gacaagcccg 4200 gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt ctgccggcga gccgatggcg 4260 gaaggaa agaacgacctg gtagaaacct gcattcggtt aaacaccacg cacgttgcca 4320 tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac ggtatccgag ggtgaagcct 4380 tgattagccg ctacaagatc gtaaagagcg aaaccgggcg gccggagtac atcgagatcg 4440 agctggctga ttggatgtac cgcgagatca cagaaggcaa gaacccggac gtgctgacgg 4500 ttcaccccga ttactttttg atcgatcccg gcatcggccg ttttctctac cgcctggcac 4560 gccgcgccgc aggcaaggca gaagccagat ggttgttcaa gacgatctac gaacgcagtg 4620 gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg caagctgatc gggtcaaatg 4680 acctgccgga gtacgatttg aaggaggagg cggggcaggc tggcccgatc ctagtcatgc 4740 gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc ctaatgtacg gagcagatgc 4800 tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg tctctttcct gtggatagca 4860 cgtacattgg gaacccaaag ccgtacattg ggaaccggaa cccgtacatt gggaacccaa 4920 agccgtacat tgggaaccgg tcacacatgt aagtgactga tataaagag aaaaaaggcg 4980 atttttccgc ctaaaactct ttaaaactta ttaaaactct taaaacccgc ctggcctgtg 5040 cataactgtc tggccagcgc acagccgaag agctgcaaaa agcgcctacc cttcggtcgc 5100 tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc ggccgctggc cgctcaaaaa 5160 tggctggcct acggccaggc aatctaccag ggcgcggaca agccgcgccg tcgccactcg 5220 accgccggcg ctgaggtctg cctcgtgaag aaggtgttgc tgactcatac caggcctgaa 5280 tcgccccatc atccagccag aaagtgaggg agccacggtt gatgagagct ttgttgtagg 5340 tggaccagtt ggtgattttg aacttttgct ttgccacgga acggtctgcg ttgtcgggaa 5400 gatgcgtgat ctgatccttc aactcagcaa aagttcgatt tattcaacaa agccgccgtc 5460 ccgtcaagtc agcgtaatgc tctgccagtg ttacaaccaa ttaaccaatt ctgattagaa 5520 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata 5580 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat 5640 ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa 5700 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc 5760 cggtgagaat ggcaaaagct ctgcattaat gaatcggcca acgcgcgggg agaggcggtt 5820 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 5880 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 5940 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 6000 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 6060 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 6120 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 6180 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 6240 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 6300 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 6360 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 6420 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc 6480 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 6540 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 6600 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 6660 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttgatcc 6720 ggaatta 6727 <210> 19 <211> 17512 <212> DNA <213> Artificial sequence <220> <223> Binary vector 24598 <400> 19 attcctgtgg ttggcatgca catacaaatg gacgaacgga taaacctttt cacgcccttt 60 taaatatccg attattctaa taaacgctct tttctcttag gtttacccgc caatatatcc 120 tgtcaaacac tgatagttta aacgggaccg ggcgccaagc ttgatatcgg aagtttctct 180 cttgagggag gttgctcgtg gaatgggaca catatggttg ttataataaa ccatttccat 240 tgtcatgaga ttttgaggtt aatatatact ttacttgttc attattttat ttggtgtttg 300 aataaatgat ataaatggct cttgataatc tgcattcatt gagatatcaa atatttactc 360 tagagaagag tgtcatatag attgatggtc cacaatcaat gaaatttttg ggagacgaac 420 atgtataacc atttgcttga ataaccttaa ttaaaaggtg tgattaaatg atgtttgtaa 480 catgtagtac taaacattca taaaacacaa ccaacccaag aggtattgag tattcacggc 540 taaacagggg cataatggta atttaaagaa tgatattatt ttatgttaaa ccctaacatt 600 ggtttcggat tcaacgctat aaataaaacc actctcgttg ctgattccat ttatcgttct 660 tattgaccct agccgctaca cacttttctg cgatatctct gaggtaagcg ttaacgtacc 720 cttagatcgt tctttttctt tttcgtctgc tgatcgttgc tcatattatt tcgatgattg 780 ttggattcga tgctctttgt tgattgatcg ttctgaaaat tctgatctgt tgtttagatt 840 ttatcgattg ttaatatcaa cgtttcactg cttctaaacg ataatttatt catgaaacta 900 ttttcccatt ctgatcgatc ttgttttgag attttaattt gttcgattga ttgttggttg 960 gtggatctat atacgagtga acttgttgat ttgcgtattt aagatgtatg tcgatttgaa 1020 ttgtgattgg gtaattctgg agtagcataa caaatccagt gttccctttt tctaagggta 1080 attctcggat tgtttgcttt atatctcttg aaattgccga tttgattgaa tttagctcgc 1140 ttagctcaga tgatagagca ccacaatttt tgtggtagaa atcggtttga ctccgatagc 1200 ggctttttac tatgattgtt ttgtgttaaa gatgattttc ataatggtta tatatgtcta ctgtttttat tgattcaata tttgattgtt cttttttttg cagatttgtt gaccagacta gtgctaaaat ggataagaag tattctattg gacttgatat tggaaccaac tctgtgggat gggctgttat tactgacgag tataaggttc catctaaga gttcaaggtt cttggaaaca ctgatagaca ctctattaag aagaacctta ttggtgctct tcttttcgat tctggagaga ctgctgaggc tactagactt aagagaactg ctagaagaag atatactaga agaaagaaca gaatttgcta tcttcaagag attttctcta acgagatggc taaggttgac gattctttct tccacagact tgaggagtct ttccttgttg aggaggataa gaagcacgag agacaccaa ttttcggaa cattgttgac gaggttgctt atcacgaga gtatccaact atttatcacc ttagaaaga gctcgttgat tctactgata aggctgatct tagacttatt tatcttgctc ttgctcacat gattaagttc agaggacact tccttattga gggagatctt aacccagata actctgacgt tgataagctc ttcattcaac ttgttcaaac ttataaccaa cttttcgagg agaacccaat taacgcttct ggagttgacg ctaaggctat tctttctgct agactttcta 1980 agtctagaag gcttgagaac cttattgctc aacttccagg agagaagaag aacggacttt 2040 tcggaaacct tattgctctt tctcttggac ttactccaaa cttcaagtct aacttcgatc 2100 ttgctgagga cgctaagctc caactttcta aggatactta cgacgatgat cttgataacc 2160 ttcttgctca aattggagat caatacgctg atctttcct tgctgctaag aacctttctg 2220 acgctattct tctttctgat attcttagag ttaacactga gattactaag gctccacttt 2280 ctgcttctat gattaagaga tacgacgagc accaccaaga tcttactctt cttaaggctc 2340 ttgttagaca acaacttcca gagaagtata aggagatttt cttcgatcaa tctaagaacg 2400 gatacgctgg atatattgac ggaggagctt ctcaagagga gttctataag ttcattaagc 2460 caattcttga gaagatggac ggaactgagg agcttcttgt taagctcaac agagaggatc 2520 ttcttagaaa gcaaagaact ttcgataacg gatctattcc acaccaaatt caccttggag 2580 agcttcacgc tattcttaga aggcaagagg atttctatcc attccttaag gataacagag 2640 agaagattga gaagattctt actttccgta ttccatatta cgttggacca cttgctagag 2700 gaaactctag attcgcttgg atgactagaa agtctgagga gactattact ccttggaact 2760 tcgaggaggt tgttgataag ggagcttctg ctcaatcttt cattgagaga atgactaact 2820 tcgataagaa ccttccaaac gagaaggttc ttccaaagca ctctcttctt tacgagtatt 2880 tcactgttta taacgagctt actaaggtta agtacgttac tgagggaatg agaaagccag 2940 ctttcctttc tggagagcaa aagaaggcta ttgttgatct tcttttcaag actaacagaa 3000 aggttactgt taagcaactt aaggaggatt atttcaagaa gattgagtgc ttcgattctg 3060 ttgagatttc tggagttgag gatagattca acgcttctct tggaacttat cacgatcttc 3120 ttaagattat taaggataag gatttccttg ataacgagga gaacgaggat attcttgagg 3180 atattgttct tactcttact cttttcgagg atagagat gattgaggag agacttaaga 3240 cttacgctca ccttttcgac gataaggtta tgaagcaact taagagaaga agatatactg 3300 gatggggtag actttctaga aagctcatta acggaattag agataagcaa tctggaaaga 3360 ctattcttga tttccttaag tctgacggat tcgctaacag aaacttcatg caacttattc 3420 acgacgattc tcttactttc aggaggata ttcaaaaggc tcaagtttct ggacaaggag 3480 attctcttca cgagcacatt gctaaccttg ctggatctcc agctattaag aagggaattc 3540 ttcaaactgt taaggttgtt gacgagcttg ttaaggttat gggtagacac aagccagaga 3600 acattgttat tgagatggct agagagaacc aaactactca aaagggacaa aagaactcta 3660 gagagagaat gaagagaatt gaggagggaa ttaaggagct tggatctcaa attcttaagg 3720 agcacccagt tgagaacact caacttcaaa acgagaagct ctatctttat tatcttcaaa 3780 acggaagaga tatgtacgtt gatcaagagc ttgatattaa cagactttct gattacgacg 3840 ttgatcacat tgttccacaa tctttcctta aggacgattc tattgataac aaggttctta 3900 ctagatctga taagaacaga ggaaagtctg ataacgttcc atctgaggag gttgttaaga 3960 agatgaagaa ctattggaga caacttctta acgctaagct cattactcaa agaaagttcg 4020 ataaccttac taaggctgag agaggagac tttctgagct tgataaggct ggattcatta 4080 agagacaact tgttgagact agacaaatta ctaagcacgt tgctcaaatt cttgattcta 4140 gaatgaacac taagtacgac gagaacgata agctcattag agaggttaag gttattactc 4200 ttaagtctaa gctcgtttct gatttcagaa agatttcca attctataag gttagagaga 4260 ttaacaacta tcaccacgct cacgacgctt atcttaacgc tgttgttgga actgctctta 4320 ttaagaagta tccaaaactt gagtctgagt tcgtttacgg agattataag gtttacgacg 4380 ttagaaagat gattgctaag tctgagcaag agattggaaa ggctactgct aagtatttct 4440 tctattctaa cattatgaac ttcttcaaga ctgagattac tcttgctaac ggagagatta 4500 gaaagaggcc acttattgag actaacggag agactggaga gattgtttgg gataagggaa 4560 gagattcgc tactgttaga aaggttcttt ctatgccaca agttaacatt gttaagaaaa 4620 ctgaggttca aactggagga ttctctaagg agtctattct tccaaagaga aactctgata 4680 agctcattgc tagaaagaag gattgggacc caaagaagta cggaggattc gattctccaa 4740 ctgttgctta ttctgttctt gttgttgcta aggttgagaa gggaaagtct aagaagctca 4800 agtctgttaa ggagcttgtt ggaattacta tttggagag atcttctttc gagaagaacc 4860 cagttgattt ccttgaggct aagggatata aggaggttaa gaaggatctt attattaagc 4920 tcccaaagta ttctcttttc gagcttgaga acggaagaaa gagaatgctt gcttctgctg 4980 gagagcttca aaagggaaac gagcttgctc ttccatctaa gtacgttaac ttcctttatc 5040 ttgcttctca ctacgagaag ctcaagggat ctccagagga taacgagcaa aagcaacttt 5100 tcgttgagca acacaagcac tatcttgacg agattattga gcaaatttct gagttctcta 5160 agagagttat tcttgctgac gctaaccttg ataaggttct ttctgcttat aacaagcaca 5220 gagataagcc aattagagag caagctgaga acattattca ccttttcact cttactaacc 5280 ttggtgctcc agctgctttc aagtatttcg atactactat tgatagaaag agatatactt 5340 ctactaagga ggttcttgac gctactctta ttcaccaatc tattactgga ctttacgaga 5400 ctagaattga tctttctcaa cttggaggag attcttcc accaaagaag aagagaaagg 5460 tttcttggaa ggacgcttct ggatggtcta gaatgtgacg tcgcgtgatc gttcaaacat 5520 ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata 5580 atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat 5640 gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa 5700 aatatagcgc gcaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg 5760 gcgcgccaag cttcgttgaa caacggaaac tcgacttgcc ttccgcacaa tacatcattt 5820 cttcttagct ttttttttc ttcttcgttc atacagtttt tttttgttta tcagcttaca 5880 ttttcttgaa ccgtagcttt cgttttcttt tttttaactt tccattcgga gttttgtat 5940 cttgtttcat agtttgtccc aggattagaa tgattaggca tcgaaccttc aagaatttga 6000 ttgaataaaa catcttcatt cttaagatat gaagataatc ttcaaaaggc ccctgggaat 6060 ctgaaagaag agaagcaggc ccatttatat gggaaagaac aatagtattt cttatatagg 6120 cccatttaag ttgaaaacaa tcttcaaaag tcccacatcg cttagataag aaaacgaagc 6180 tgagtttata tacagctaga gtcgaagtag tgattgagag gtaaccgaat agagagtttt 6240 aggctagaa atagcaagtt aaaataggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg cttttttttt actgatgcat tgtattataa gtacgttaga atgtgcaata fathers ctatcattag aacttgaatt fathers tttttgtaat atgaattaaa agtgtattaa acatgtatta acggtgatca attggttaa aaaaaagttta ttttaaaat gataaatctt tttaatttt agtatatt tgtaagtttt cacgttgagt aaatagcgaa gaagttgggc ccaaccaagt aaaatagaa ggccgggcca ttacaattaa gtcgtcacac aactgggctt cattgaaaaa agcgcaaaac cgattccagg cccgtgttag catgaagact caactcaacc agagatttct ccctcatcgc ttacagaaaa aagctatatg ctgtttatat tgcgaaatct aacagtgtag tttgaattca gggactccaa tgagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttttct gcagccgaga cacttgtgtg attgagaga acactaatct tgtgaggact gaagtttggt gattattct tgtgatctgt cgacaaaaat atcaaatggg gtttcttta caattattt acctaatga atctgttttg aaatattta ctccatgggt 7020 ctatttttt attacaaagc gtctccctga agggcgcgtt ccccgtgaaa gtgacacgtg 7080 gcaggacttg ggacgtgccc tgcgtacagg cgcgatagtt agtgttgtta cagcaggcgc 7140 atcgggtcgt gttggggacc aggtacgac aggtcgcgct ggggacccag acacgaccca 7200 attgggtcgc acttttatta tattttgtat attgtttttta tttaatatat 7260 ttttattatta ttttatta ttttttatta ttttattaa tagttttctat attataaa 7320 ttcttagcat tatgtatgat tttaagtca taaataattt ttatattgt ttttattac 7380 tattatttt atattttatt taatatttat atattaaata aatccttcat attagaaaaa 7440 aaaaaaaaaaaaaaaaaaaaagt aaaaaaaatt aaaaaaat 7500 attaaaaaaaaaaaaaaaaaaaaaaaaataaaaaaaaaaa 7560 aaaaatatta ttttaaata attattatta gactttaaac tctaaagttg aattttaaaa 7620 aaataattt tttttacgat tttagtaaa aaaaaataca agccgcacaa tacaagtcgc 7680 cttctcaaac ccttcctcac gacattctcg gaccttatga caccgtcacc aaaacaatga 7740 tccacgcgat attaggcgcg tgcaaatcac tctaatccga aactagtaga catgggaagc 7800 acgagctata cgcgagcgtt tcaattgccg ccacgaaagc agagaaggcc agaaacggaa 7860 ccacggtaaa atggtaaggg tattttcgta aacagaagaa aagagttgta gctataaata 7920 aaccctctaa cccacggcgc actatttctc ttcactcctt cgttcactct tcttctttg 7980 cggctagggt tttagcgcag cttcttctag gttcgttctc ttccgccgct ctatggattt 8040 taaaccttcg aatcatgttt attccattga attatgttgc ttgcagttta tattttctga 8100 atctgtagtt gttgtcttca attatccta tgctttatag atcaatcttt tgtgtgtgta 8160 gtacgtaatt tttgttcttt ttgctttcg ttcaagttgt tgggaataat cggggtatca 8220 tgttttgata ttgtttgttt tctttttga ctgcttaata atttttaagt tggttttggt 8280 tttggggttt tatgtgcttg ttatattcaa atctttggat ccagatctta caaaagtttt 8340 gggtttaagg atgtttttgg ctgatgatga atagatctat aaactgttcc ttttaatcga 8400 ttcaagctta ggattttact aggcttttgc gaataaatac gtgacagtaa gctaattatg 8460 tccttttttt gtctcaatca tatctgtctg ggtgtgccat aatttgtgat atgtctatct 8520 ggtagaatct tgtgttttat gctttacgat ttggtatacc tgtttttgaa cttgttgtat 8580 gatgggtatt tagatcaccc tatctttttt atgcttctgg aagttttatg taaatgtcga 8640 atatcttaat gttgttgaac ttataatgtt gtgttgatgt atgtatgatg gttttgacaa 8700 cttttttcac tggttctgaa agttttatgt aaattgcaaa tatgttaatg ttgttgaact 8760 tatttttttt ccttcgatgt tgttttgatg tatgtatgat ggttttcacc gtagtttcta 8820 tggctaatat cttaatgttg ttgagcttat ttttttcctt atatgttgtg ttgatgtatg 8880 tatgatggtt ttgacaactt ttttagtttc tttgcagatt taaggaagat cgatggcgca 8940 agttagcaga atctgcaatg gtgtgcagaa cccatctctt atctccaatc tctcgaaatc 9000 cagtcaacgc aaatctccct tatcggtttc tctgaagacg cagcagcatc cacgagctta 9060 tccgatttcg tcgtcgtggg gattgaagaa gagtgggatg acgttaattg gctctgagct 9120 tcgtcctctt aaggtcatgt cttctgtttc cacggcgtgc atgagggaag cgttgatcgc 9180 cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac 9240 gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga 9300 tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat 9360 caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga 9420 agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact 9480 gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat 9540 cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg 9600 tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa 9660 tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt 9720 gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt 9780 cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc 9840 taggcaggct tatcttggac aagaagatcg cttggcctcg cgcgcagatc agttggaaga 9900 atttgttcac tacgtgaaag gcgagatcac caaagtagtc ggcaaataat gagctcatct 9960 agctagagct ttcgttcgta tcatcggttt cgacaacgtt cgtcaagttc aatgcatcag 10020 tttcattgcg cacacaccag aatcctactg agtttgagta ttatggcatt gggaaaactg 10080 ttttcttgt accatttgtt gtgcttgtaa tttactgtgt ttttattcg gttttcgcta 10140 tcgaactgtg aaatggaaat ggatggagaa gagttaatga atgatatggt ccttttgttc 10200 attctcaaat tatattatt tgttttttct cttatttgtt gtgtgttgaa tttgaaatta 10260 taagagatat gcaaacattt tgttttgagt aaaaatgtgt caaatcgtgg cctctaatga 10320 ccgaagttaa tatgaggagt aaaacacttg tagttgtacc attatgctta ttcactaggc 10380 aaaatata ttttcagacc tagaaaagct gcaaatgtta ctgaatacaa gtatgtcctc 10440 ttgtgtttta gacatttatg aactttcctt tatgtaattt tccagaatcc ttgtcagatt 10500 ctaatcattg ctttataatt atagtatac tcatggattt gtagttgagt atgaaaatat 10560 tttttaatgc attttatgac ttgccaattg attgacaaca tgcatcaatc ccgggcggcc 10620 agcatggccg tatccggatg tcatattccc tatctgatcg tgagaggtaa ccgaatagag 10680 agggtttcct atgtaactaa atgtctgcta atgtattcac aagtccaagt gatgtattcg 10740 aaattataaa atttaaggaa ttcttataat ttgaaaaaga agtagaaaat aatgtaatta 10800 gctcttaacg ctatgaaatt tatgtaaatt atataattat tatgtactcc ttccgattca 10860 tatgacatat cttactttta acctttacat tttgttcaaa ataagtaatt ttattgtaac 10920 taagaatgta ttactattat ttagtttttc aaatttacgc cttcttttga taagtgggtt 10980 ttaactttta acgtaaccaa gaaatgatat taaatatgta ctatataatt aagaataatt 11040 agtaaaaaaca atttttaata ttttaggacc taaacttttt attttttgt gcgacatgtt 11100 acctaaaaga tagtaaaaaa ataattgcca gaataaaatg gaataatttt actagaaaat 11160 aaacatagga aaagaaatat acgtaacaca ttaaattata tcaacggatc attaaaattc 11220 ttttgtattg tctatataat actatataaa agtaaagaat tctataaaat taatttgagt 11280 tgacatagaa aaactgtttt gggttaaatt ttttactagt tgtgcactat ttatcttcga 11340 tctataaata gatcgacatg ttggaaaaca ctcaaaccat cctatgctat aagataatat 11400 atagctacat ttcttagata actagaaacc tccattagct tcctattctc ataagcaaat 11460 ctccaatcat aatttacaaa ctgagactcg atgtatgatc agtgatagat ttaaaattta 11520 gatatcacaa gtgatatgtt tagatcataa gggtctagaa atgcatatct aactcgatgt 11580 attctatgtt gcactttgtc ccgcatcacc tcacaactgt aagtataaat tatttcaaag 11640 agagcaggaa agtattgggt gagatattgt tttaaccccg aacatttcat gaataatgag 11700 gtgctaattg gaagctgcac cttaattctt tatgaaatgt tcggggttaa aacatcttca 11760 gtccctcccc gaccctctct accttaattt atttctacgt ttatgtatt taaatttccc 11820 tatatgtcct cctttatctt caaaatcgaa aaatgaagtt atattaattt gtttagtgta 11880 acttaactct tgaccatgct gcttccgatc aagaaagggt tttattgatg atagttaatt 11940 12000. 12000. 12000. 12000. 12000. 12000. 12000. 12000 aattcacatc gatgtaatga aagtgaaaaa ttcataata ttatagaaaa tcatgaata tcgattcgtt tgacaactat aatatagtct cacaaaatct tttatctttg ccttaaatta catctttgcc ttaattaca tcaaaaaatg atttgtaac tttattatga tcacgaattc agggactcca atgaaggcat cattaagaag tgtatccata gtttcttgta ctaatttcgt atccgcaatg tgttattaag ttgtctaagc gtcaatttgt ttacaccaca atatatcctg ccccgcca gccaacagct ccccgaccgg cagctcggca caaatcacc actcgataca ggcagcccat cagattaat tctcatgttt cagagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa attachment atgagctgtt catcataat catccggctc gtataatgtg tggaattgtg agcggataac aatttcacac aggaaacaga ccatgaggga 12660 agcgttgatc gccgaagtat cgactcaact atcagaggta gttggcgtca tcgagcgcca 12720 tctcgaaccg acgttgctgg ccgtacattt gtacggctcc gcagtggatg gcggcctgaa 12780 gccacacagt gatattgatt tgctggttac ggtgaccgta aggcttgatg aaacaacgcg 12840 gcgagctttg atcaacgacc ttttggaaac ttcggcttcc cctggagaga gcgagattct 12900 ccgcgctgta gaagtcacca ttgttgtgca cgacgacatc attccgtggc gttatccagc 12960 taagcgcgaa ctgcaatttg gagaatggca gcgcaatgac attcttgcag gtatcttcga 13020 gccagccacg atcgacattg atctggctat cttgctgaca aaagcaagag aacatagcgt 13080 tgccttggta ggtccagcgg cggaggaact ctttgatccg gttcctgaac aggatctatt 13140 tgaggcgcta aatgaaacct taacgctatg gaactcgccg cccgactggg ctggcgatga 13200 gcgaaatgta gtgcttacgt tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc 13260 gccgaaggat gtcgctgccg actgggcaat ggagcgcctg ccggcccagt atcagcccgt 13320 catacttgaa gctaggcagg cttatcttgg acaagaagat cgcttggcct cgcgcgcaga 13380 tcagttggaa gaatttgttc actacgtgaa aggcgagatc accaaagtag tcggcaaata 13440 aagctctagt ggatctccgt acccagggat ctggctcgcg gcggacgcac gacgccgggg 13500 cgagaccata ggcgatctcc taaatcaata gtagctgtaa cctcgaagcg tttcacttgt 13560 aacaacgatt gagaattttt gtcataaaat tgaaatactt ggttcgcatt tttgtcatcc 13620 gcggtcagcc gcaattctga cgaactgccc atttagctgg agatgattgt acatccttca 13680 cgtgaaaatt tctcaagcgc tgtgaacaag ggttcagatt ttagattgaa aggtgagccg 13740 ttgaaacacg ttcttcttgt cgatgacgac gtcgctatgc ggcatcttat tattgaatac 13800 cttacgatcc acgccttcaa agtgaccgcg gtagccgaca gcacccagtt cacaagagta 13860 ctctcttccg cgacggtcga tgtcgtggtt gttgatctag atttaggtcg tgaagatggg 13920 ctcgagatcg ttcgtaatct ggcggcaaag tctgatattc caatcataat tatcagtggc 13980 gaccgccttg aggagacgga taaagttgtt gcactcgagc taggagcaag tgattttatc gctaagccgt tcagtatcag agagtttcta gcacgcattc gggttgcctt gcgcgtgcgc 14100. cccaacgttg tccgctccaa agaccgacgg tctttttgtt ttactgactg gacacttaat ctcaggcaac gtcgcttgat gtccgaagct ggcggtgagg tgaaacttac ggcaggtgag ttcaatcttc tcctcgcgtt tttagagaaa ccccgcgacg ttctatcgcg cgagcaactt ctcattgcca gtcgagtacg cgacgaggag gtttatgaca ggagtataga tgttctcatt ttgaggctgc gccgcaaact tgaggcagat ccgtcaagcc ctcaactgat aaaaacagca agaggtgccg gttatttctt tgacgcggac gtgcaggttt cgcacggggg gacgatggca 14460 gcctgagcca attcccagat ccccgagga tcggcgtgag cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga gaagttgag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagag cacgccccgg tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc gatgctctat gacgtgggca 14760 cccgcgatag tcgcagcatc atggacgtgg ccgttttccg tctgtcgaag cgtgaccgac 14820 gagctggcga ggtgatccgc tacgagcttc cagacgggca cgtagaggtt tccgcagggc 14880 cggccggcat ggccagtgtg tgggattacg acctggtact gatggcggtt tcccatctaa 14940 ccgaatccat gaaccgatac cgggaaggga agggagacaa gcccggccgc gtgttccgtc 15000 cacacgttgc ggacgtactc aagttctgcc ggcgagccga tggcggaaag cagaaagacg 15060 acctggtaga aacctgcatt cggttaaaca ccacgcacgt tgccatgcag cgtacgaaga 15120 aggccaagaa cggccgcctg gtgacggtat ccgagggtga agccttgatt agccgctaca 15180 agatcgtaaa gagcgaaacc gggcggccgg agtacatcga gatcgagctg gctgattgga 15240 tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct gacggttcac cccgattact 15300 ttttgatcga tcccggcatc ggccgttttc tctaccgcct ggcacgccgc gccgcaggca 15360 15420 tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc aaatgacctg ccggagtacg 15480 atttgaagga ggaggcgggg caggctggcc cgatcctagt catgcgctac cgcaacctga 15540 tcgagggcga agcatccgcc ggttcctaat gtacggagca gatgctaggg caaattgccc 15600 tagcagggga aaaaggtcga aaaggtctct ttcctgtgga tagcacgtac attgggaacc 15660 caaagccgta cattgggaac cggaacccgt acattgggaa cccaaagccg tacattggga 15720 accggtcaca catgtaagtg actgatataa aagagaaaaa aggcgatttt tccgcctaaa 15780 actctttaaa acttattaaa actctttaaaa cccgcctggc ctgtgcataa ctgtctggcc 15840 agcgcacagc cgaagagctg caaaaagcgc ctacccttcg gtcgctgcgc tccctacgcc 15900 ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc aaaaatggct ggcctacggc 15960 caggcaatct accagggcgc ggacaagccg cgccgtcgcc actcgaccgc cggcgctgag 16020 gtctgcctcg tgaagaaggt gttgctgact cataccaggc ctgaatcgcc ccatcatcca 16080 gccagaaagt gagggagcca cggttgatga gagctttgtt gtaggtggac cagttggtga 16140 ttttgaactt ttgctttgcc acggaacggt ctgcgttgtc gggaagatgc gtgatctgat 16200 ccttcaactc agcaaaagtt cgatttattc aacaaagccg ccgtcccgtc aagtcagcgt 16260 aatgctctgc cagtgttaca accaattaac caattctgat tagaaaaact catcgagcat 16320 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg 16380 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta 16440 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa 16500 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa 16560 aagctctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 16620 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 16680 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 16740 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 16800 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 16860 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 16920 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 16980 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 17040 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 17100 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 17160 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 17220 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 17280 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 17340 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 17400 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 17460 tcatgagatt atcaaaaagg atcttcacct agatcctttt gatccggaat ta 17512 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> gRNA sequences <400> 20 gagaggtaac cgaatagaga 20 <210> twenty one <211> 20 <212> DNA <213> Artificial sequence <220> <223> gRNA sequences <400> twenty one gaattcaggg actccaatga 20 <210> twenty two <211> twenty one <212> DNA <213> Tomato spotted wilt virus <400> twenty two 21 <210> twenty three <211> twenty one <212> DNA <213> Tomato spotted wilt virus <400> twenty three ctgaaaagca aagcatttga a 21 <210> twenty four <211> twenty one <212> DNA <213> Tomato spotted wilt virus <400> twenty four aggggaaaga gtatgctgct a 21

Claims

1. A method for reducing the expression of a target gene, the method comprising the following: a) introducing into a plant cell a nuclease capable of site-directed DNA cleavage at a genomic site encoding a native pre-miRNA of the plant cell; b) creating at least one double-stranded DNA break at or near said genomic locus; c) selecting cells, wherein the at least one double strand break has been repaired by replacing the genomic site with an intermediate DNA; d) reducing the expression of the target gene; wherein the intermediate DNA encodes a modified pre-miRNA comprising an amiRNA core sequence complementary to the target gene, wherein the native pre-miRNA is replaced by the modified pre-miRNA, and wherein the cell is heterozygous for the modified pre-miRNA. The method according to claim 1 , wherein the target gene is an exogenous target gene. The method according to claim 1 , wherein the target gene is a pest gene.

4. The method of claim 1, wherein the target gene is a viral, fungal or microbial gene.

5. The method of claim 1, wherein the target gene is a bunyavirus gene.

6. The method of claim 1, wherein the target gene is a tomato spotted wilt virus gene.

7. The method of claim 1, wherein the target gene is a tomato spotted wilt virus gene.

8. The method of claim 1, wherein the target gene is an endogenous plant gene.

9. The method of claim 8, wherein the target endogenous plant gene is a gene involved in plant development, biotic stress or abiotic stress.

10. The method of any one of claims 1-9, wherein the plant cell is a Solanaceae, corn, rice, canola, soybean, or sunflower cell.

11. The method of claim 10, wherein the cells are tomato cells.

12. The method of claim 11, wherein the genomic site encoding a native pre-miRNA encodes a native tomato pre-miRNA.

13. The method of claim 8, wherein the genomic site comprises SEQ ID NO:6 or SEQ ID NO:

7.

14. The method of any one of claims 1-9, wherein the intermediate DNA comprises any one of SEQ ID NOs: 1 to 5.

15. The method of any one of claims 1-9, wherein the nuclease is selected from the group consisting of: a mega-nuclease, a zinc finger nuclease, a transcription activator-like effector nuclease, a Cas9 nuclease, a Cfp1 nuclease, a dCas9-FokI, a dCpf1-FokI, a chimeric Cas9 / Cpf1-cytosine deaminase, a chimeric Cas9 / Cpf1-adenine deaminase, a chimeric FEN1-FokI, a Mega-TAL, a nickase Cas9, a chimeric dCas9 non-FokI nuclease, and a dCpf1 non-FokI nuclease.

16. The method of any one of claims 1-9, wherein the cell has a haploid, diploid, polyploid, or hexaploid genome.

17. The method of any one of claims 1-9, wherein one or more guide sequences are introduced together with the nuclease.

18. A method for producing plant seeds, the method comprising crossing a plant comprising a plant cell obtained by the method of any one of claims 1 to 17 with itself or with another plant of the same crop.

19. The method of claim 18, wherein the seed is a Solanaceae, corn, rice, canola, soybean or sunflower seed.

20. The method of claim 18, wherein the seeds are tomato seeds.

Images