WO2018143477A1 - Method of modifying genome of dicotyledonous plant - Google Patents

Abstract

A genome modification method is provided which enables inducing a DNA conversion inheritable by progeny in a target site of the dicotyledonous plant genome. The genome of a dicotyledonous plant is modified with a method involving the introduction, into dicotyledonous plant cells, of guide RNA and a fusion protein which includes a nucleic acid sequence recognition module and a nucleobase converting enzyme. Thanks to ample genome modification efficiency, it is possible to produce a plant body from the genome-modified plant cells, to produce progeny plants from the plant body, and, through selection of progeny plants that have a variation, to breed progeny plants that have the variation.

Description

Method for modifying the genome of dicotyledonous plants

The present invention relates to a method for modifying the genome of a dicotyledonous plant.

Genome editing is a new technology that enables efficient and rapid introduction of genome modification. Genome editing techniques have been applied to many types of organisms. One widely used genome editing tool is the CRISPR / Cas9 (Short Palindromic Repeats / CRISPR-related 9) system with regularly spaced clusters. In this system, Cas9 enzyme (Non-patent Document 1) and RNA called guide RNA (gRNA) are used. The gRNA includes an RNA sequence (corresponding to crRNA) corresponding to an approximately 20 bp base sequence (target sequence) of a target site into which DNA modification is introduced, and an RNA sequence corresponding to trans-activated CRISPR RNA (tracrRNA). When gRNA and Cas9 are brought into contact with genomic DNA, gRNA is specifically detected via RNA-DNA base pairing at the target site located immediately before the protospacer adjacent motif (PAM) required for Cas9 activity on the genome. It binds and mobilizes Cas9, which cleaves genomic DNA at the target site. In this system, wild-type Cas9 produces a double-strand break (DSB) upstream of the PAM sequence. Genomic DNA breaks generated by DSB are religated by error-prone non-homologous end joining (NHEJ) or homologous recombination repair (HDR). When repaired by non-homologous end joining (NHEJ), DNA insertion or deletion (insertion deletion; Indel) mutations occur frequently at the repair site, so use non-homologous end joining (NHEJ) to target An insertion deletion mutation can be induced at the site. Compared to other genome editing technologies, the CRISPR / Cas9 system has advantages such as ease of construct design and low cost. However, in the CRISPR / Cas9 system, it is known that wild-type Cas9 can cause cleavage at the target external site by off-target action. In order to reduce the off-target effect, Cas9 nickase (nCas9) in which mutation (D10A or H840A, respectively) is introduced into one of the two nuclease domains RuvC and HNH to inactivate one of the nucleases is also used. Such Cas9 nickase produces a nick in the single strand rather than a double strand break at the target site and is repaired by homologous recombination repair (HDR), thus reducing off-target effects. When using Cas9 nickase instead of wild-type Cas9 in the CRISPR / Cas9 system for genome editing, use two gRNAs that bind to adjacent target sites instead of one gRNA. A method of introducing cutting is usually employed (Non-Patent Document 2). One of the biggest limitations of these CRISPR / Cas9 systems is that the mutation is mainly limited to insertional deletions. Insertion and deletion mutations in the genome are suitable for functional disruption of target genes, but DNA conversion (base substitution) is more important for the treatment of many human diseases and the improvement of agriculturally important crop traits. is there. However, single-base editing technology that causes DNA conversion has not been well established. For example, if a single-base editing technique that can be successfully used for genome editing of plants is established, it is useful to further accelerate the breeding of crops.

Cytidine deaminase (CDA), including activation-induced cytidine deaminase (AID), catalyzes the irreversible hydrolytic deamination of cytidine (C) to uridine (U), and ultimately from base C Conversion to T can be induced (Non-patent Document 3). Catalytically inactive Cas9 (dCas9) with two mutations (D10A and H840A) that inactivate nuclease activity in both the two nuclease domains RuvC and HNH, lamprey-derived CDA1 (PmCDA), human-derived CDA (APOBEC1 ) Or by fusing with rat-derived CDA (rAPOBEC1) (dCas9-CDA) and contacting it with DNA in yeast or mammalian cells together with gRNA. Can be converted into another base (base editing) (Non-patent Documents 4 and 5). It has also been reported that genome editing was performed in rice using a fusion protein of Cas9 nickase and cytidine deaminase (Non-patent Document 6).

However, there are still few reports that DNA transformation introduced into plants can be inherited by progenies by genome editing. In order to inherit DNA conversion to a progeny, it is necessary to introduce DNA conversion into the genome of germ cells, but introduction of DNA conversion into the genome of plant germ cells using genome editing has not always been successful.

Belhaj, K., et al., (2015) V. Curr. Opin Biotech., 32, 76-84 Sanger, J.D. and Joung, J.K., (2014) Nature Biotechnology, Apr; 32 (4): 347-355 Muramatsu, M., et. Al., (2000) T. Cell 102, 553-563 Nishida, K. et al., (2016) Science 16, 353 (6305) Komor, A.C., et al., (2016) Nature, 533 (7603): 420-424 J. Li., Et al., Molecular Plant, (2016) December 8, pii: S1674-2052 (16) 30298-2.doi: 10.1016 / j.molp.2016.12.001

The present inventors have found that the genome of a dicotyledonous plant can be successfully modified by introducing a guide RNA and a fusion protein containing a nucleic acid sequence recognition module and a nucleobase converting enzyme into a plant cell. .

The present invention relates to a target-specific genome modification method for dicotyledonous plants. The present invention particularly relates to genome editing technology (for example, CRISPR (clustered regularly interspaced short palindromic repeats) -Cas9 (CRISPR-associated 9) system). The present invention relates to a method for introducing a mutation including DNA conversion into a target site on the genome of a dicotyledon. In the method of the present invention, a genome to which gRNA specifically binds is introduced by introducing a complex (eg, fusion protein) of a nucleic acid sequence recognition module and a nucleobase converting enzyme into a dicotyledonous cell together with a guide RNA (gRNA). DNA conversion can be frequently induced in the target site. In a preferred embodiment, the methods of the invention can also provide inheritance to progeny of introduced DNA conversions. The present invention introduces a fusion protein containing a guide RNA and a mutant Cas9 protein lacking at least one nuclease activity of two nuclease domains and a nucleobase converting enzyme (eg, cytidine deaminase) into dicotyledonous plant cells. , Dicotyledonous genomes using the CRISPR / Cas9 system, including inducing mutations involving DNA conversion (eg substitution of cytosine to other bases) at target sites in the genome to which guide RNA specifically binds The modification method is provided.

For example, the following items are provided in the present invention.
[Item 1] A method for modifying the genome of a dicotyledon, comprising introducing a guide RNA and a fusion protein comprising a nucleic acid sequence recognition module and a nucleobase converting enzyme into a plant cell.
[Item 2] The nucleic acid sequence recognition module according to the item, wherein the nucleic acid sequence recognition module is selected from the group consisting of a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated, a zinc finger motif, a TAL effector, and a PPR motif. Method.
[Item 3] The method according to any one of the preceding items, wherein the nucleic acid sequence recognition module is a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated.
[Item 4] The method according to any one of the preceding items, wherein the CRISPR-Cas system comprises two nuclease domains, and any one of the two nuclease domains is inactivated.
[Item 5] The method according to any one of the preceding items, wherein the nucleobase converting enzyme is a deaminase.
[Item 6] The method according to any one of the preceding items, wherein the nucleobase converting enzyme is cytidine deaminase.
[Item 7] The method according to any one of the preceding items, wherein the introduction is performed by introducing the nucleic acid sequence recognition module and a nucleic acid construct encoding the nucleobase converting enzyme into the cell.
[Item 8] The method according to any one of the preceding items, wherein the nucleic acid construct further encodes the guide RNA.
[Item 9] The method according to any one of the preceding items, wherein the nucleic acid sequence recognition module and the sequence encoding the nucleobase converting enzyme are optimized for codon usage of dicotyledonous plants.
[Item 10] The method according to any one of the preceding items, wherein the nucleic acid sequence recognition module and the sequence encoding the nucleobase converting enzyme are optimized for codon usage of Arabidopsis thaliana.
[Item 11] The method according to any one of the preceding items, wherein the guide RNA and the nucleic acid sequence recognition module target the sequence of the SlDELLA or SlETR1 gene.
[Item 12] The method according to any one of the preceding items, wherein the dicotyledonous plant is a solanaceous plant.
[Item 13] The method according to any one of the preceding items, wherein the dicotyledonous plant is a plant of the genus Eggplant.
[Item 14] The method according to any one of the preceding items, wherein the dicotyledonous plant is a tomato plant (Solanum lycopersicum).
[Item 15] The method according to any one of the preceding items, wherein the introduction is performed at about 23 to about 27 ° C.
[Item 16] The method according to any one of the preceding items, wherein the introduction is performed at about 25 ° C.
[Item 17] The method according to any one of the preceding items, further comprising culturing the introduced plant cell at about 23 to about 27 ° C.
[Item 18] The method according to any one of the preceding items, further comprising culturing the introduced plant cell at about 25 ° C.
[Item 19] The plant genome in a dicotyledonous plant cell is modified by the method according to any one of the preceding items to induce a mutation including DNA conversion, and a plant body is produced from the plant cell having the modified genome. And a method for breeding dicotyledonous plants, comprising producing a progeny plant from the plant and selecting a progeny plant having the mutation.
[Item 20] A composition comprising a nucleic acid sequence recognition module and a nucleic acid construct encoding a nucleobase converting enzyme for modifying a dicotyledon genome, wherein the nucleic acid sequence recognition module is present in the presence of a guide RNA. A composition that recognizes a target sequence on the genome.
[Item 21] The nucleic acid sequence recognition module according to the item, wherein the nucleic acid sequence recognition module is selected from the group consisting of a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated, a zinc finger motif, a TAL effector, and a PPR motif. Composition.
[Item 22] The composition according to any one of the preceding items, wherein the nucleic acid sequence recognition module is a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated.
[Item 23] The composition according to any one of the preceding items, wherein the CRISPR-Cas system comprises two nuclease domains, and any one of the two nuclease domains is inactivated.
[Item 24] The composition according to any one of the preceding items, wherein the nucleobase converting enzyme is deaminase.
[Item 25] The composition according to any one of the preceding items, wherein the nucleobase converting enzyme is cytidine deaminase.
[Item 26] The composition according to any one of the preceding items, wherein the nucleic acid construct further encodes the guide RNA.
[Item 27] The composition according to any one of the preceding items, wherein the nucleic acid sequence recognition module and the sequence encoding the nucleobase converting enzyme are optimized for codon usage of dicotyledonous plants.
[Item 28] The composition according to any one of the preceding items, wherein the nucleic acid sequence recognition module and the sequence encoding the nucleobase converting enzyme are optimized for codon usage of Arabidopsis thaliana.
[Item 29] The composition according to any one of the preceding items, wherein the guide RNA and the nucleic acid sequence recognition module target the sequence of the SlDELLA or SlETR1 gene.
[Item 30] The composition according to any of the preceding items, wherein the dicotyledonous plant is a solanaceous plant.
[Item 31] The composition according to any one of the items described above, wherein the dicotyledonous plant is a plant of the genus Eggplant.
[Item 32] The composition according to any of the preceding items, wherein the dicotyledonous plant is a tomato plant (Solanum lycopersicum).
[Item 32A] The composition according to any one of the preceding items, comprising the characteristics of the method according to any one of the preceding items.
[Item 32B] The composition or method according to any one of the preceding items, comprising the characteristics of the method according to any one of items [A1] to [A9] below.
CLAIM | ITEM 33 The plant produced by the method in any one of said claim | items.
[Item 34] A plant part produced by the method according to any one of the items described above.
[Item 35] The plant part according to the item, wherein the part is selected from fruits, roots, leaves, flowers, seeds and stems.
CLAIM | ITEM 36 The processed product which processed the plant produced by the method in any one of the said claim | items, or its part.

For example, in an aspect of the present invention, the following items are provided.
[A1] A guide RNA and a fusion protein containing a mutant Cas9 protein lacking at least one nuclease activity of two nuclease domains RuvC and HNH and cytidine deaminase are introduced into tomato plant cells, whereby the guide RNA is specific. A method for modifying a tomato plant genome using the CRISPR / Cas9 system, comprising inducing a mutation including DNA conversion at a target site in the genome that binds to the.
[A2] The method according to [A1] above, which comprises introducing a nucleic acid construct containing an expression unit containing a base sequence encoding the fusion protein into tomato plant cells and expressing the fusion protein.
[A3] The base sequence encoding the fusion protein, wherein the base sequence encoding the mutant Cas9 protein and the base sequence encoding cytidine deaminase are optimized for codon usage of Arabidopsis thaliana Method.
[A4] The method according to [A2] or [A3] above, wherein the nucleic acid construct further comprises an expression unit comprising a base sequence encoding a guide RNA.
[A5] The method according to [A4] above, wherein the base sequence encoding the guide RNA is arranged under the control of an Arabidopsis thaliana-derived U6 promoter.
[A6] The method according to [A2] above, wherein the expression unit consists of the base sequence represented by SEQ ID NO: 21, 25, 27, or 40.
[A7] The method according to any one of [A2] to [A6] above, wherein the nucleic acid construct is T-DNA.
[A8] The method according to any one of [A1] to [A7] above, wherein the DNA conversion comprises cytosine substitution.
[A9] Using the method described in any of [A1] to [A8] above, a tomato plant having a genome in which a mutation including DNA conversion has been induced is produced, and progeny is generated by crossing using the plant A method for breeding a tomato plant, comprising producing a plant and selecting a progeny plant having the mutation.

According to the present invention, mutation can be induced with high efficiency at a target site in the genome of a dicotyledonous plant. High-efficiency mutagenesis allows mutations to be inherited to progeny. In addition, the present invention can induce DNA conversion (base substitution) particularly frequently as a mutation.

FIG. 1 is a diagram schematically showing a target site for mutagenesis by the CRISPR / Cas9 system. A shows the target site on Solyc11g011260 (SlDELLA gene), and B shows the target site on Solyc12g011330 (SlETR1 gene). TGG is a PAM sequence, and a 20-base long sequence located immediately before in the genome is a target sequence. The target sequence of SlETR1 ^site3 and the adjacent PAM sequence are represented by complementary sequences in the figure. FIG. 2 is a diagram schematically showing the structure of T-DNA containing gRNA and a Cas9 or nCas9-CDA expression unit in a vector. Cas9, nCas9-PmCDA, nCas9-PmCDA ^opt and nCas9-stop-PmCDA Cas9 or nCas9 coding sequences are under the control of the PcUbi promoter, and pea3A terminator from pea (Pisum sativum; P. sativum) is used for transcription termination. Using. Although the gRNA expression unit in the figure is shown in the forward direction for convenience, it may be inserted in the reverse direction during actual vector production. Note that the region containing PmCDA downstream of the stop codon (stop) of nCas9-stop-PmCDA (“non-functional sequence” in the figure) does not function in protein expression due to insertion of the stop codon in this vector. FIG. 3 is a photograph showing the leaf phenotypes of T ₀ and T ₁ plants into which nCas9-PmCDA ^opt targeting SlDELLA was introduced and DNA modification occurred. Arrows indicate wild type (WT) leaf saw blades. Figure 4 is a photograph showing the results of PCR analysis to determine the presence or absence of the kanamycin resistance marker gene in T ₁ plants and F ₁ plants carrying a DNA modification (NPTII). PC: positive control, NC: negative control, WT: wild-type tomato plant. # Represents the parent plant and the following number represents the individual progeny plant. Line # 3BC1_6 is an F ₁ plant obtained by crossing between line # 3 (T ₀ plant) and a wild type plant. All progeny plants except # 3BC1_6 are T ₁ plants. FIG. 5 shows changes in amino acid sequences in SlDELLA or SlETR1-targeted T ₁ plants. It shows that the DNA conversion occurring at the target sequence causes an amino acid substitution. Amino acid substitutions observed in these T ₁ plants, homozygous, heterozygous, or a biallelic. Plants that have been shown to be free of the selectable marker gene are noted. The “-” in the right column indicates a DNA deletion, and the number following it indicates the length of the deletion (bp). “C” indicates DNA conversion, and the number immediately following indicates the number of converted bases. “WT” indicates wild type (no modification). Bases that have undergone DNA conversion in the sequence are shown in lower case. FIG. 6 shows the codon usage frequency of tomato (Sl) and Arabidopsis (At).

Hereinafter, the present invention will be described while showing the best mode. Throughout this specification, it should be understood that expression in the singular also includes the concept of the plural unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

The term “about” is understood by those skilled in the art and is understood to vary to some extent depending on the content in which the term is used. There is a use of this term that is unclear to those skilled in the art, and given the context in which this term is used, the term “about” is given unless the meaning of the term is specifically defined in the art. Means up to ± 10% of a given number or rounded to the nearest significant figure.

(1. Explanation of definition and basic technology)
Hereinafter, definitions of terms particularly used in the present specification and / or basic technical contents will be described as appropriate.

In the present invention, the “nucleic acid sequence recognition module” means a molecule or molecular complex having the ability to specifically recognize and bind to a specific nucleotide sequence (ie, a target nucleotide sequence) on a DNA strand. Binding of the nucleic acid sequence recognition module to the target nucleotide sequence allows the nucleobase converting enzyme linked to the module to specifically act on the targeted site of double stranded DNA.

In the present invention, “nucleobase converting enzyme” refers to a target nucleotide without cleaving a DNA strand by catalyzing a reaction for converting a substituent on a purine or pyrimidine ring of a DNA base into another group or atom. Is an enzyme capable of converting to other nucleotides.

In the present invention, “introduction” refers to the presence of a desired molecule in a cell. When introducing a protein molecule into a cell, in addition to transferring the protein molecule into the cell, a nucleic acid encoding the protein molecule may be transferred into the cell and the protein may be expressed in the cell.

In the present invention, “modification” of double-stranded DNA means that a certain nucleotide (eg, dC) on the DNA strand is converted to another nucleotide (eg, dT, dA or dG) or deleted. Or a nucleotide or nucleotide sequence inserted between certain nucleotides on a DNA strand. Here, the double-stranded DNA to be modified is not particularly limited, but is preferably genomic DNA. In addition, the “targeted site” of double-stranded DNA refers to all or part of the “target nucleotide sequence” to which the nucleic acid sequence recognition module specifically recognizes and binds, or in the vicinity of the target nucleotide sequence ( One or both of 5 ′ upstream and 3 ′ downstream), and the range thereof can be appropriately adjusted between 1 base and several hundred bases depending on the purpose.

Hereinafter, preferred embodiments of the present disclosure will be described. It is understood that the embodiments provided below are provided for a better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present disclosure with reference to the description in the present specification. Also, it is understood that the following embodiments of the present disclosure may be used alone or in combination.

In addition, each of the embodiments described below shows a comprehensive or specific example. Numerical values, shapes, materials, components, arrangement positions and connection forms of components, steps, order of steps, and the like shown in the following embodiments are merely examples, and are not intended to limit the scope of the claims. In addition, among the constituent elements in the following embodiments, constituent elements that are not described in the independent claims indicating the highest concept are described as optional constituent elements.

(2. Plant)
In the present invention, dicotyledonous plants can be used as the object of the method or composition for introducing a mutation into the genome. In the examples herein, it has been demonstrated that mutations could be introduced into the genome with high efficiency in dicotyledonous plants.

Dicotyledonous plants are angiosperms with two (or more) embryonic cotyledons. In contrast, angiosperms with one cotyledon are called monocotyledons. Although the separation between dicotyledonous and monocotyledonous plants has been proposed based on the number of cotyledons, these two groups are considered to be genetically distinct lines. In addition to the number of cotyledons, there are differences in traits such as the way the veins are divided (stratification), the arrangement of stem vascular bundles, and the basic number of organs that make up the flower. In dicotyledonous plants, the veins branch like feathers (or palms), and the branching branches have so-called reticulated veins that connect to the mesh, whereas in monocotyledons, they run almost in parallel while branching at the base of the leaves. It becomes a parallel pulse and the communication between the branches is also simple. The arrangement of vascular bundles is generally called the true central column in dicotyledonous plants, with the xylem at the center of the stem and the phloem at the periphery. On the other hand, monocotyledonous plants are scattered central pillars, and vascular bundles are scattered irregularly inside the stems. Dicotyledonous plants are further merged into joints such as rhododendrons, perennials, asteraceae, solanaceae, etc., where the petals coalesce in a tubular shape (genital flowering pods) and roses, legumes, celery, etc. It is sometimes divided roughly into non-leaflet flowers (Paleoflora).

Dicotyledonous plants include Amborelae, Waterlily, Austrobailea, Senleo, Canela, Pepper, Camphora, Magnolia, Drosophila, Omodaka, Yam, Taconidae, Lily, Pheasant , Dashipogonidae, palm eyes, rice eyes, commune eyes, ginger eyes, pine eyes, buttercup eyes, abalone departments, antaceae eyes, box eyes, corn borer eyes, gunnera eyes, terrestris eyes, asterisidae, kintrano eyes, caterpillar eyes, legumes Eyes, rose eyes, cucumber eyes, beech eyes, burdock eyes, black peach eyes, crosso eyes, mukuroji eyes, fuertea eyes, cruciferous eyes, mallow eyes, grape eyes, cynosididae, bivalamidae, berberidopsis eyes, sandalwood eyes, urchinaceae, Dogwood, Azalea, Gallia, Gentian, Perilla, Eggplant, Ilex Apiales include calycerales and plants belonging to dipsacales but not limited thereto.

Examples of solanaceous plants include Montinaceae, Nagano Nourushiaceae, Ceylon Jacobeaceae, Convolvulaceae, and Solanaceae.

Examples of solanaceous plants include plants belonging to the genus Solanum (for example, Solanum aethiopicum, Solanum americanum, Solanum carolinense, Solanum betaceum, tomato (Solanum lycopersicum (Lycopersicon esculentum)), Solanum lyratum, hornet (Solanum mammola) melongena), Solanum muricatum, Solanum nigrum, Solanum pseudocapsicum, potatoes (Solanum tuberosum, etc.), plants belonging to the genus Capsicum (for example, capsicum annuum), Capsicum baccatum, ensecapsicums , Capsicum pubescens, etc.), plants belonging to the genus Tobacco (eg, Nicotiana alata), tobacco (Nicotiana spp.), Etc., plants belonging to the genus Datura (eg, Datura metel), American Datura (Datura i noxia), Datura stramonium, etc.), plants belonging to the genus Pleurotus genus, plants belonging to the genus physalis, plants belonging to the genus physalis, plants belonging to the genus physalis, plants belonging to the petunia genus, genus corydalis Plants belonging to the genus Hyos, a plant belonging to the genus Belladonna, a plant belonging to the genus Mandragora, a plant belonging to the genus Cucurbita, and a plant belonging to the genus Calibracore, but are not limited thereto.

For example, examples of the plant to be subjected to the method or composition for genome modification include, but are not limited to, tomato, eggplant, potato, pepper, pepper and tobacco. The dicotyledonous flowering plants are: Aoi eyes: Aoiaceae (for example, cabbage, okra, sea bream, sardine blue, roselle, etc.), Lindenaceae (such as Morohea); (Such as lotus), coralidae (Junsai, water lily, etc.); Violet eyes: cucurbitaceae (winter melon, cantaloupe, cucumber, black rice pumpkin, fresh pumpkin, shiroi, watermelon, zucchini, cactus pumpkin, one Melon, Tongan, Tokachi Hechi, Nigeri, Nihon Pumpkin, Net Melon, Hayatouri, Gourd, Hechima, Snake, Pepo Pumpkin, Makiwauri, Melon, Yugao, etc .; Mushrooms, celery family (Ashitaba, Italian parsley, coriander, soup celery, soup mint, Ri, Celery, Chervil, Dill, Carrot, Parsnip, Parsley, Hamabufu, Fennel, Mitsuba, etc .; Tadeidae: Tadeidae (Yanagi, Aidae, Rhubarb, etc.); Table beats, everyday, broom, spinach, matsuna, mountain spinach, etc., scorpion family (such as Tatsusurihiyu), tsuruna (such as vine), tsurumurasaki (such as tsurumurasaki), ayu (family amaranth, hinoki) Roses: Rose family (Strawberry, Salad Barnet, Wild Strawberry, etc.); Fusaulaceae: Brassicaceae (Brassic, Fishy, Kairan, Turnip, Mustard, Cauliflower, Cabbage, Watercress, Kale, Kohlrabi , Kousaitai, Pepper Seed, Komatsuna, Immediately, Ta Lee, Daikon, Daisai, Daisaisai, Takana, Takutaka, Chingensai, Nozomi, Hakusai, Hakura, Hatsudaiiko, Hamana, Petit Veil, Broccoli, Mizuna, Mibuna, Meji cabbage , Rutabaga, arugula, wasabi, wasabi daisou; fusoso: Nozenhalenidae (such as kinenka); myrtaceae: cynosumidae (such as pelvis); legumes: legumes (red bean, American potato, green bean, enamel) How, pigtails, shikama-dame, jujuku-dango, broad-bellied, broad-bellied, tuna-tama-me, bun-ma-me, tama-ma-me, nante-hagi, hashi-sho-mame, chick-bama, hira-ma-me, fuji-ma-me, bean-baba, (Lima bean, Rakase, Ryokuto, etc.); Mucurodia: Citrus family (Sansho, Henruda, etc.) It is not limited to these. Dicotyledonous joint-flower plants include the following: chrysanthemum: asteraceae (artichoke, endive, Dutch senchi, oysters, chamomile, cardon, curry plant, kiku, kikuimo, burdock, salsify, sanchu, (Shungyoku, Shokuyo Dandelion, Stewart, Stevia, Tachisha, Chicory, Chisha, Tsububuki, Trevis, Wipe, Yamagobo, Leaf Lettuce, Lettuce, etc.); Scorpionidae: Sesameaceae (sesame, etc.); Lamiaceae (apple mint, winter savory, sesame, oregano, perilla, spearmint, sage, thyme, chorogi, bran, pineapple mint, bran, basil, peppermint, marjoram, lavender, lemon thyme, lemon balm, rosemary, etc.) Purple family (Comfrey etc.) Eggplant: There are solanaceae (currant tomatoes, shiso pepper, potatoes, shrimp cheeks, white peppers, red peppers, tomatoes, eggplants, peppers, hiranasu, pepino, etc.), convolvulaceae (sweet potatoes, sweet potatoes, etc.) However, it is not limited to these.

The genome modification method or composition of the present invention may target tomato plants. Examples of tomato plants to be subjected to the genome modification method or composition of the present invention include Solanum lycopersicum, Lycopersicon cerasiforme, Lycopersicon pimpinellifolium, Lycopersicon y impericon cheesmanii), Lycopersicon perviflorum, Lycopersicon chmielewskiy (Lycopersicon hirsutum), Lycopersicon perumenperi , Solanum lycopersicoides and Solanum habrochaites but not limited to tomato lines and varieties belonging to chaites), etc. or their derivatives. Wild-type tomato varieties Microtom (Solanum lycopersicum cv. Micro-Tom) (Scott JW, Harbaugh BK (1989) Micro-Tom A miniature dwarf tomato. Florida Agr. Expt. Sta. Circ. 370, p. 1-6) is commercially available and can also be obtained from Tomato Genetics Resource Center (TGRC) (USA) under accession number LA3911. Wild-type tomato cultivar Microtom is fertile (about 10 to 20 cm), has small leaves and fruits, and can be crossed with conventional tomato varieties. The whole genome sequence of the wild-type tomato variety Microtom has been determined (Kobayashi M, et al., (2014) Plant Cell Physiol. 2014 Feb; 55 (2): 445-454). In the present invention, a derivative strain refers to a progeny plant obtained through one or more crosses between the original plant and another plant line / variety, or through mutagenesis or mutagenesis.

(3. Nucleic acid sequence recognition module)
Examples of the nucleic acid sequence recognition module in the present invention include a CRISPR-Cas system (preferably, a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated (CRISPR-mutated Cas)), zinc finger motif, TAL effector In addition to the PPR motif and the like, a restriction enzyme, a transcription factor, a DNA binding domain of a protein that can specifically bind to DNA such as RNA polymerase, etc., and a fragment that does not have the ability to cleave DNA double strands can be used. It is not limited to these. Preferably, CRISPR-mutated Cas, zinc finger motif, TAL effector, PPR motif and the like can be mentioned. A nucleic acid sequence recognition module that does not have the ability to cleave DNA double strands is suitable for introducing mutations other than deletion insertion, for example, base substitution, by combining with a nucleobase converting enzyme or the like.

The zinc finger motif is a linkage of 3 to 6 different zinc finger units of the Cys2His2 type (one finger recognizes about 3 bases), and can recognize a target nucleotide sequence of 9 to 18 bases. Zinc finger motifs include Modular assembly method (Nat Biotechnol (2002) 20:-135-141), OPEN method (Mol Cell (2008) 31: 294-301), CoDA method (Nat Methods (2011) 8: 67-69) In addition, it can be prepared by a known method such as E. coli one-hybrid method (Nat Biotechnol (2008) 26: 695-701). For details of the production of the zinc finger motif, reference can be made to Japanese Patent No. 4968498.

The TAL effector has a repeating structure of about 34 amino acid units, and the binding stability and base specificity are determined by the 12th and 13th amino acid residues (called RVD) of one module. The Since each module is highly independent, it is possible to create a TAL effector specific to the target nucleotide sequence simply by connecting the modules. TAL effectors can be created using open resources (REAL method (Curr, Protoc, Mol, Biol, (2012), Chapter, 12: 15, Unit, 12.15), FLASH method (Nat, Biotechnol, (2012), 30: 460-465), Golden Gate method (Nucleic, Acids Res 2011 (2011) 39: e82) etc. have been established, and a TAL effector for a target nucleotide sequence can be designed relatively easily. For details of the production of the TAL effector, reference can be made to JP 2013-513389 A.

The PPR motif consists of 35 amino acids, and is constructed to recognize a specific nucleotide sequence by a series of PPR motifs that recognize one nucleobase. The 1st, 4th, and ii (-2) th amino acids of each motif Only recognize the target base. Since there is no dependence on the motif structure and there is no interference from the motifs on both sides, it is possible to produce a PPR protein specific to the target nucleotide sequence by linking the PPR motifs just like the TAL effector. JP, 2013-128413, A, etc. can be referred for the details of preparation of a PPR motif.

In addition, when using fragments such as restriction enzymes, transcription factors, RNA polymerase, etc., the DNA-binding domain of these proteins is well known, so it is easy to design a fragment that contains this domain and does not have the ability to cleave DNA double strands. And can be built.

In the present invention, the CRISPR / Cas9 system refers to a guide RNA (gRNA) containing a target recognition sequence corresponding to a target sequence located immediately before the protospacer adjacent motif (PAM) of any strand of genomic DNA and Cas9 nuclease or its Genome editing technology that promotes the introduction of mutations (insertion, deletion, base substitution, etc.) into target sites by binding complexes containing mutants to target sites in genomic DNA in cells Say.

In the present invention, a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated can be used as a nucleic acid sequence recognition module. Inactivation of at least one DNA cleavage ability of Cas is realized, for example, by using a mutant of Cas9 nuclease (mutant Cas9 protein). As an example, the mutant Cas9 protein used in the present invention is a mutant Cas9 protein lacking at least one nuclease activity of two nuclease domains RuvC and HNH. Cas9 nuclease has RuvC domain and HNH domain which are nuclease domains. The mutant Cas9 protein that can be used in the present invention may have a mutation that inactivates nuclease activity in one or both of the RuvC domain and the HNH domain. Mutant Cas9 protein with mutation that inactivates nuclease activity of one of RuvC domain and HNH domain nicks single strand without breaking DNA double strand (cuts only single strand) It has nickase activity and is called Cas9 nickase (or nCas9). Examples of mutations that inactivate the nuclease activity of the RuvC domain include D10A mutations in Cas9, including type II Cas9 in Streptococcus pyogenes (S. pyogenes) (aspartic acid to alanine at position 10 of Cas9) Substitution). Examples of mutations that inactivate the nuclease activity of the HNH domain include H840A mutations in Cas9, including type II Cas9 of Streptococcus pyogenes (S. pyogenes) (histidine at position 840 of Cas9 to alanine). Substitution). On the other hand, mutant Cas9 protein (dCas9) having a mutation that inactivates nuclease activity of both RuvC domain and HNH domain does not have nuclease activity and does not cleave DNA. The mutant Cas9 protein used in the present invention usually retains gRNA binding ability regardless of the presence or absence of DNA cleavage activity. The mutant Cas9 protein used in the present invention may be a mutant of Cas9 derived from any biological species, and typically may be a mutant of Cas9 derived from bacteria. Preferable examples of the bacterium to be derived include Streptococcus pyogenes (also referred to as group A hemolytic streptococci) and Streptococcus thermophilus.

The mutant Cas9 protein that can be used in the present invention includes, for example, one mutation (amino acid substitution, addition, deletion, or insertion) that inactivates nuclease activity in one or both of the RuvC domain and the HNH domain of the Cas9 protein. Alternatively, it may have two or more and retain the binding ability to gRNA. In a preferred embodiment, the mutant Cas9 protein used in the present invention is i) a protein consisting of the amino acid sequence represented by SEQ ID NO: 6, ii) one or more (typically) amino acid sequences represented by SEQ ID NO: 6 (typically Consisting of an amino acid sequence having 1-50, preferably 1-30, more preferably 1-10, eg 1-5, mutations (amino acid substitutions, additions, deletions, insertions, etc.) A protein in which the nuclease activity of at least one of the domain and the HNH domain is inactivated, or iii) 70% or more (for example, 80% or more, 90% or more, 95% or more) with respect to the amino acid sequence represented by SEQ ID NO: 6, Or a protein having an inactivated nuclease activity of at least one of the RuvC domain and the HNH domain. The protein in which the nuclease activity of the RuvC domain is inactivated has, for example, alanine as an amino acid corresponding to the 10th amino acid of the amino acid sequence represented by SEQ ID NO: 6. The mutant Cas9 protein used in the present invention preferably has both nickase activity and gRNA binding ability, or gRNA binding ability alone. The 1373 to 1379th amino acid sequence shown in SEQ ID NO: 6 is a nuclear localization signal (NLS). The mutant Cas9 protein used in the present invention preferably has a nuclear localization signal having nuclear localization ability at the N-terminus or C-terminus. The nuclear localization signal may have a mutation in the amino acid sequence from 1373 to 1379 of SEQ ID NO: 6 as long as it retains the nuclear localization ability. The sequence identity defined in the context of the present invention means the percent identity over the full length of both amino acid sequences being compared. The gRNA binding ability is the gRNA that contacts the genomic DNA containing the target site of gRNA and gRNA that is known to bind to wild-type Cas9 to form a complex and the mutant Cas9 protein. And the mutant Cas9 protein can be examined by confirming whether they formed a complex.

Any of the above nucleic acid sequence recognition modules can be provided as a fusion protein with a nucleobase converting enzyme, or a protein binding domain such as SH3 domain, PDZ domain, GK domain, GB domain and their binding partners. May be fused to a nucleic acid sequence recognition module and a nucleobase converting enzyme, respectively, and provided as a protein complex through the interaction between the domain and its binding partner. Alternatively, intein can be fused to the nucleic acid sequence recognition module and the nucleobase converting enzyme, and both can be linked by ligation after protein synthesis.

In the present invention, guide RNA (gRNA) is RNA used for binding (inducing) Cas9 nuclease or a mutant thereof to a target site in the CRISPR / Cas9 system by binding to the target site on genomic DNA. gRNA has an RNA sequence (crRNA) containing a target recognition sequence that binds to a target site in genomic DNA on the 5 ′ end side and an RNA sequence having a scaffold function (tracrRNA; trans-activating crRNA). The “side sequence” and the 5 ′ side sequence of tracrRNA have complementary sequences to each other and form base pairs. The gRNA may be a single-stranded gRNA (single guide RNA; sgRNA) in which crRNA and tracrRNA are linked, or a complex of crRNA and tracrRNA, which are separate single-stranded RNAs. The target site to which gRNA specifically binds is located immediately before the PAM sequence of either strand of genomic DNA, and its target sequence is designed to be approximately 20 bases long (usually 17 to 24 bases long). Can do. gRNA contains a target recognition sequence (RNA sequence) corresponding to such a target sequence. gRNA binds to the complementary strand sequence of the target sequence in genomic DNA by RNA-DNA base pairing.

The protospacer adjacent motif (PAM) sequence varies depending on the species and type from which the Cas9 nuclease or variant thereof is derived, such as 5'-NGG-3 for Cas9 of Streptococcus pyogenes (S. pyogenes). '(N = A, T, G or C). In addition, for example, Streptococcus thermophilus Cas9 recognizes 5'-NGGNG-3 'or 5'-NNAGAA-3' as a PAM sequence. The design method and production method of gRNA are well known. For example, gRNA can be prepared by incorporating a target sequence into a commercially available gRNA vector and expressing it. The target sequence can also be designed easily using, for example, commercially available software for gRNA design published on the web. gRNA design software can be used from websites such as CHOPCHOP (https://chopchop.rc.fas.harvard.edu/index.php) and CRISPRdirect (http://crispr.dbcls.jp/). .

(4. Nucleobase converting enzyme)
The nucleobase converting enzyme used in the present invention is not particularly limited as long as it can catalyze the reaction of converting a substituent on the purine or pyrimidine ring of a DNA base into another group or atom. For example, an amino group And a deaminase belonging to the nucleic acid / nucleotide deaminase superfamily that catalyzes a deamination reaction for converting a carbonyl group to a carbonyl group. Preferred examples include cytidine deaminase that can convert cytosine or 5-methylcytosine to uracil or thymine, adenosine deaminase that can convert adenine to hypoxanthine, and guanosine deaminase that can convert guanine to xanthine. More preferred examples of cytidine deaminase include activation-induced cytidine deaminase (hereinafter also referred to as AID), which is an enzyme that introduces a mutation into an immunoglobulin gene in acquired immunity of vertebrates.

The origin of the nucleobase converting enzyme is not particularly limited. For example, PmCDA1 derived from lamprey (Petromyzon marinus cytosine deaminase 1), AID (Activation-induced cytidine derived from mammals (eg, human, pig, cow, horse, monkey, etc.) deaminase; AICDA) can be used. Nucleobase conversion enzymes are considered to have different optimum temperatures depending on their origin, and base conversion efficiency can be optimized by using them at an appropriate reaction temperature. Lamprey-derived PmCDA1 can be used at about 23-27 ° C, for example at about 25 ° C.

In the present invention, cytidine deaminase can be used together with the nucleic acid sequence recognition module. As one example, in the present invention, a fusion protein containing a mutant Cas9 protein and cytidine deaminase can be introduced into a cell. Cytidine deaminase (CDA) can convert cytidine to uridine by deaminase activity, and ultimately lead to the conversion of base cytosine (C) in DNA to thymine (T). In a preferred embodiment, the cytidine deaminase is activation-induced cytidine deaminase (AID). The cytidine deaminase used in the present invention may be derived from any biological species, for example, derived from animals such as fish, mammals and birds. In one embodiment of the present invention, the cytidine deaminase may be derived from fish such as lamprey, primates such as humans, cloven hoofs such as pigs, cows and camels, rodents such as rats, and the like.

The cytidine deaminase used in the present invention may be wild type cytidine deaminase, or one or more mutations (amino acid substitution, addition, deletion, insertion, etc.) in the amino acid sequence of wild type cytidine deaminase. It may be a protein that has and retains cytidine deaminase activity. In a preferred embodiment, the cytidine deaminase used in the present invention is i) a protein consisting of the amino acid sequence represented by SEQ ID NO: 9, ii) one or a plurality (typically 1 to 2) of the amino acid sequence represented by SEQ ID NO: 9. Cytidine deaminase comprising an amino acid sequence having 50 mutations (preferably 1 to 30 mutations, amino acid substitutions, additions, deletions, insertions, etc.), preferably 1 to 30, more preferably 1 to 10, for example 1 to 5 A protein having activity, or iii) an amino acid sequence having sequence identity of 70% or more (eg, 80% or more, 90% or more, 95% or more, or 99% or more) with respect to the amino acid sequence represented by SEQ ID NO: 9 And a protein having cytidine deaminase activity.

The nucleobase converting enzyme used in the present invention may be provided with a nuclear localization signal on the N-terminal side and / or C-terminal side. The nuclear localization signal may be directly linked to the nucleobase converting enzyme, or may be linked to the nucleobase converting enzyme via another polypeptide such as a linker peptide. For example, in the protein consisting of the amino acid sequence represented by SEQ ID NO: 30, a nuclear localization signal is added to the C-terminus of cytidine deaminase. In the present invention, the protein consisting of the amino acid sequence represented by SEQ ID NO: 30 or the amino acid sequence thereof includes one or more (typically 1 to 50, preferably 1 to 30, more preferably 1 to 10, for example, A protein having an amino acid sequence having 1 to 5) mutations (amino acid substitution, addition, deletion, insertion, etc.) and having cytidine deaminase activity and nuclear localization ability; or iii) shown in SEQ ID NO: 9 70% or more (for example, 80%, 90%, 95%, or 99% or more) of amino acid sequences, and cytidine deaminase activity and nuclear localization ability A protein having can be preferably used.

The nucleic acid sequence recognition module and the nucleobase converting enzyme can be linked by any method to form a fusion protein. In a preferred embodiment, a fusion protein in which a nucleic acid sequence recognition module (for example, a mutant Cas9 protein) is arranged on the N-terminal side and a nucleobase converting enzyme (for example, cytidine deaminase) is arranged on the C-terminal side can be used. A nucleic acid sequence recognition module (for example, mutant Cas9 protein) and a nucleobase converting enzyme (for example, cytidine deaminase) can be linked via a linker peptide. A nuclear localization signal and / or SH3 domain may be included between the nucleic acid sequence recognition module of the fusion protein and the nucleobase converting enzyme (eg, cytidine deaminase). A glycine-serine linker and / or a tag (such as a 3xFlag tag) may also be included between the nucleic acid sequence recognition module of the fusion protein and the nucleobase converting enzyme. A fusion protein in which another protein such as a linker peptide or marker protein is further linked to the nucleic acid sequence recognition module and the nucleobase converting enzyme can also be used.

The target site to which gRNA specifically binds may be in the untranslated region or in the translated region, but more preferably in the translated region. The target site may be in any target gene and may be in an intron or exon, but is preferably in an exon.

In the method of the present invention, one type of gRNA may be used, or two or more types of gRNA may be used. Two or more types of gRNAs are gRNAs that specifically bind to two or more target sites, respectively.

(5. Nucleic acid construct)
In the method of the present invention, the fusion protein can be introduced into a genomic plant cell by introducing a nucleic acid construct encoding the fusion protein into the plant cell. Also provided in the present invention is a composition comprising a nucleic acid construct encoding a fusion protein for use in such a method.

“Expression unit” refers to a nucleic acid fragment capable of inducing the expression of a target gene product (here, gRNA or mRNA encoding a fusion protein). Typically, an expression unit includes a promoter, a coding sequence placed under the control of the promoter, and a terminator in this order. The promoter may be a constitutive promoter, a transient promoter, a tissue or time specific promoter, and the like. Examples of promoters include, but are not limited to, Arabidopsis derived U6 promoter, PcUbi promoter, CaMV 35S promoter and the like. The terminator is not particularly limited as long as it functions in plant cells, and examples thereof include pea3A terminator and Oshsp17.3 terminator.

The “nucleic acid construct” may be, for example, a DNA vector such as a plasmid having autonomous replication ability, or a nucleic acid not having autonomous replication ability such as T-DNA that can be incorporated into a plant genome by the Agrobacterium method. It may be. T-DNA is a DNA fragment sandwiched between an RB sequence at the 5 ′ end and an LB sequence at the 3 ′ end. A “nucleic acid construct” can include one or more expression units. The nucleic acid construct is typically a DNA construct.

Expression units and nucleic acid constructs include additional genes (selective marker genes such as drug resistance genes and reporter genes), expression units containing them, 2A peptide linker coding sequences, restriction enzyme cleavage sites and multiple cloning sites, nuclear localization signals Additional DNA sequences such as (NLS), poly A addition signal may be included. Examples of selectable marker genes include kanamycin resistance marker gene (NPTII), gentamicin resistance gene, neomycin resistance gene, hygromycin resistance gene, puromycin resistance gene, zeocin resistance gene, blasticidin resistance gene, and ampicillin resistance gene. It is done. In the nucleic acid construct, the nuclear localization signal can be incorporated before and after each protein, and can be arranged as NLS-nucleic acid sequence recognition module-NLS-nucleobase converting enzyme-NLS, including the leading NLS The arrangement of nucleic acid sequence recognition module-NLS-nucleobase converting enzyme-NLS is more preferable. While not wishing to be bound by theory, protein expression may be improved by reducing the size of the expression unit.

In the present invention, the base sequence encoding the fusion protein or the base sequence encoding one or more proteins or polypeptide regions constituting the fusion protein is optimized for codon usage of dicotyledonous plants, preferably tomato or It may be optimized for Arabidopsis codon usage. The term “optimized for codon usage” refers to a base sequence that has been modified so that a codon that is less frequently used in a certain plant is replaced with a codon that is frequently used in that plant. The codon usage frequency of tomato and Arabidopsis is shown in FIG. Codon usage in dicotyledonous plants is known in many plants. For example, tomatoes are reported in The Sol Genomics Network (SGN) database (https://solgenomics.net/misc/codon_usage/codon_usage.pl). .

The base sequence encoding the fusion protein, or the base sequence encoding one or more proteins or polypeptide regions constituting the fusion protein, includes the use of codons for dicotyledonous plants, codon usage for solanaceous plants, It can be used by optimizing for codon usage or codon usage of cruciferous plants. The base sequence encoding the fusion protein, or the base sequence encoding one or more proteins or polypeptide regions constituting the fusion protein is, for example, Arabidopsis thaliana, Brassica napus, soybean (Glycine). max), tomato (Lycopersicon esculentum), Bensamiana tobacco (Nicotiana benthamiana), tobacco (Nicotiana tabacum) and other codon usage can be optimized. In addition, the base sequence encoding the fusion protein or the base sequence encoding one or more proteins or polypeptide regions constituting the fusion protein may be used for any plant codon usage described herein. Can be optimized and used.

In a preferred embodiment, the base sequence encoding the nucleic acid sequence recognition module used in the present invention and / or the base sequence encoding the nucleobase converting enzyme is optimized for codon usage of dicotyledonous plants, preferably tomato or Arabidopsis thaliana. . An example of a base sequence encoding a mutant Cas9 protein optimized for codon usage in Arabidopsis thaliana is shown in SEQ ID NO: 5. An example of a base sequence encoding cytidine deaminase optimized for codon usage in Arabidopsis thaliana is shown in SEQ ID NO: 8. Examples of base sequences encoding the fusion protein optimized for Arabidopsis codon usage are shown in SEQ ID NOs: 25, 27, and 40. An example of a base sequence encoding a polypeptide containing cytidine deaminase and a C-terminal nuclear localization signal optimized for codon usage in Arabidopsis thaliana is shown in SEQ ID NO: 29. In addition to the above codon optimization, an arbitrary base sequence that can be included between the base sequence encoding the nucleic acid sequence recognition module and the base sequence encoding the nucleobase converting enzyme, for example, a linker peptide coding sequence and its components It may be optimized for codon usage of dicotyledonous plants, preferably tomato or Arabidopsis. For example, an SH3 domain optimized for Arabidopsis codon usage (for example, 5084 of the base sequence shown in SEQ ID NO: 25) between a base sequence encoding a nucleic acid sequence recognition module and a base sequence encoding a nucleobase converting enzyme. To 5260th sequence). The nucleic acid construct to be introduced into the dicotyledonous plant cell may contain these base sequences.

In the present invention, a base sequence encoding a fusion protein comprising a nucleic acid sequence recognition module and a nucleobase converting enzyme has a base sequence encoding a nucleic acid sequence recognition module at the 5 ′ end and a base sequence encoding a nucleobase converting enzyme as 3 It is preferable to include at the end. The base sequence encoding the nucleic acid sequence recognition module may be a sequence encoding the above-mentioned mutant Cas9 protein, for example, the base sequence shown by SEQ ID NO: 5 or the base sequence shown by SEQ ID NO: 5 70% or more (for example, 80% or more, 90% or more, 95% or more, or 99% or more) of the nucleotide sequence, and at least one of the RuvC domain and HNH domain nuclease activity is inactivated It may be a base sequence encoding the protein being processed. The protein preferably has both nickase activity and gRNA binding ability, or gRNA binding ability alone. The protein also preferably has a nuclear localization signal having nuclear localization ability (for example, 1373 to 1379th in the amino acid sequence shown in SEQ ID NO: 6) at the N-terminus or C-terminus. The base sequence represented by SEQ ID NO: 5 is optimized for use of Arabidopsis codons. The base sequence encoding cytidine deaminase may be a sequence encoding the above cytidine deaminase, for example, the base sequence represented by SEQ ID NO: 7 or 8, or the base sequence represented by SEQ ID NO: 7 or 8. It is a base sequence encoding a protein having a cytidine deaminase activity, comprising a base sequence having sequence identity of 70% or more (for example, 80% or more, 90% or more, 95% or more, or 99% or more). obtain. The base sequence represented by SEQ ID NO: 8 is optimized for use of Arabidopsis codons.

The gRNA can be introduced into dicotyledonous cells by introducing a nucleic acid construct containing an expression unit containing a base sequence encoding gRNA to express gRNA, or by directly introducing gRNA. More preferably, the gRNA is introduced into a dicotyledonous cell by introducing a nucleic acid construct containing an expression unit containing a base sequence encoding gRNA into the dicotyledonous plant cell to express the gRNA. In a preferred embodiment, a single nucleic acid construct comprising both an expression unit comprising a base sequence encoding a fusion protein comprising a nucleic acid sequence recognition module and a nucleobase converting enzyme and an expression unit comprising a base sequence encoding gRNA. Can be introduced into tomato plant cells.

In an expression unit containing a base sequence encoding gRNA, the base sequence encoding gRNA is preferably arranged under the control of an Arabidopsis promoter. In an expression unit including a base sequence encoding RNA, the base sequence encoding gRNA is also preferably arranged under the control of the U6 promoter. A preferred example of such a promoter is the Arabidopsis thaliana U6 promoter. An example of the base sequence of the U6 promoter derived from Arabidopsis thaliana is shown in SEQ ID NO: 39. The U6 promoter has one or more (preferably 1 to 30, more preferably 1 to 10, for example, 1 to 5) base mutations (base substitutions, additions, deletions) in the base sequence represented by SEQ ID NO: 39. 70% or more (for example, 80% or more, 90% or more, 95% or more) with respect to a nucleic acid comprising a nucleotide sequence having a promoter activity or a nucleotide sequence represented by SEQ ID NO: 39 Or a nucleic acid having a promoter activity and having a promoter activity.

In one embodiment, the base sequence encoding a fusion protein comprising a nucleic acid sequence recognition module and a nucleobase converting enzyme is preferably arranged under the control of a ubiquitin promoter, preferably a PcUbi promoter.

The nucleic acid construct of the present invention may include an expression unit that can express Cas9 and the like, including the base sequence represented by SEQ ID NO: 21, 25, or 27. The nucleic acid construct of the present invention may include a base sequence encoding a fusion protein including Cas9, cytidine deaminase, and the like, including the amino acid sequence represented by SEQ ID NO: 22, 26, or 28.

(6. Method)
In the present invention, there is provided a method for modifying the genome of a dicotyledon, which comprises introducing a guide RNA and a fusion protein comprising a nucleic acid sequence recognition module and a nucleobase converting enzyme into a plant cell. . The introduction can be performed by introducing a nucleic acid sequence recognition module and a nucleic acid construct encoding a nucleobase converting enzyme into a cell.

Introduction of a nucleic acid such as the above nucleic acid construct, gRNA, or mRNA encoding the above fusion protein into a dicotyledonous plant cell can be performed by a conventional method. For example, nucleic acid can be introduced by Agrobacterium-mediated transformation method, whisker method, particle gun method, electroporation method, polyethylene glycol (PEG) method, microinjection method, protoplast fusion method and the like. Details of the plant transformation method include descriptions of general textbooks such as Yutaka Tabei, Transformation Protocol [Plant Edition], (2012) Chemistry Doujin, etc., and Sun, HJ, et al., (2006) A highly efficient. You can refer to documents such as transformation protocol for Micro-Tom, a model cultivar of tomato functional genomics. Plant Cell Physiol. 47, 426-431.

Any Agrobacterium-mediated transformation method can be used. For example, a vector prepared by incorporating the above-described expression unit into T-DNA in a vector suitable for the Agrobacterium-mediated transformation method can be used as a suitable Agrobacterium. Infected by inoculating cells, calli, or cotyledon sections of dicotyledonous plants with the obtained recombinant Agrobacterium, for example, by introduction into an Agrobacterium tumefaciens by electroporation. That's fine. Suitable examples of Agrobacterium include, but are not limited to, strains such as GV2260, GV3101, C58, C58C1Rif (R), EHA101, EHA105, AGL1, and LBA4404. By the infection with the recombinant Agrobacterium, T-DNA containing the above expression unit can be integrated into the genomic DNA of dicotyledonous cells.

In the particle gun method and the electroporation method, a nucleic acid construct such as a vector containing the above expression unit can be directly introduced into a dicotyledonous plant cell. For introduction of the nucleic acid construct, tissue sections derived from cells, calli, leaves and cotyledons of dicotyledonous plants, or protoplasts may be used (Christou P, et al., Bio / technology (1991) 9: 957- 962). For example, in the particle gun method, a nucleic acid delivery device (for example, PDS-1000 (BIO-RAD), etc.) is used according to the manufacturer's instructions, and metal particles coated with a nucleic acid construct are implanted into such a sample. By introducing into a plant cell, a transformed plant cell can be obtained. The operating conditions are, for example, a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.

The gRNA introduced into dicotyledonous cells binds to the target site of genomic DNA. The mRNA encoding the above fusion protein is translated into a fusion protein in dicotyledonous cells and mobilized to the target site of genomic DNA. The nucleic acid construct encoding gRNA or the above fusion protein induces the expression of gRNA or the above fusion protein, the expressed gRNA binds to the target site of genomic DNA, and the expressed fusion protein binds to the target site of genomic DNA. Mobilized.

When the fusion protein mobilized with the gRNA bound to the target site of genomic DNA forms a complex, the fusion protein induces a mutation at the target site upstream of the PAM sequence. When Cas9 nickase (nCas9) is used, both insertion deletion and DNA conversion (base substitution) mutations can be induced at the target site, and mutations, particularly DNA conversion, can be induced at a high frequency. When dCas9 in which nuclease activity is completely inactivated is used, DNA conversion (base substitution) can be mainly induced at the target site. The methods of the invention can typically cause substitution of the base cytosine with another base (eg, primarily thymine or guanine) at the target site. The insertion deletion may be an insertion or deletion of one base, or may be an insertion or deletion of two or more consecutive bases. The mutation induced in the target site may be either insertion deletion or DNA conversion, or both. At the target site, more than one insertion deletion and / or more than one DNA conversion may be induced.

Next, dicotyledonous cells, tissue sections and the like into which the nucleic acid construct has been introduced are cultured, and cultured in a selective medium according to, for example, a conventionally known plant tissue culture method. (Including auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.) can be used to regenerate a plant that has been introduced with a nucleic acid construct and transformed. In this way, a transgenic plant (T ₀ plant) having a mutation (for example, cytosine substitution) at a target site on the genome can be produced. When plant cells or plant tissues after introduction of the nucleic acid construct are cultured, they can be cultured at a temperature suitable for plant growth, but in addition, the culture should be performed at a temperature that optimizes the activity of the introduced protein. Is also possible. In addition, for example, when introduction is performed using callus, if the time in callus state is short, the possibility of incomplete mutagenesis increases. In some cases, even if the individuals are destabilized and regenerate, the next generation of seeds may not be obtained, and there may be sterilized individuals. By selecting the culture time, the efficiency of mutagenesis that can be inherited by progenies can be further optimized. Is possible. For example, dicotyledonous plants, particularly tomatoes, can be cultured in a callus state for about 2 to 4 days, preferably about 3 days after introduction of the nucleic acid construct.

In the method of the present invention, the sequence of the gene product of the target gene can be changed with high efficiency by DNA conversion in the dicotyledonous genome. When the target gene is a protein coding sequence, substitution of bases, for example, substitution of the base cytosine with thymine, frequently causes amino acid substitutions in the protein. In this way, mutations such as insertion deletion and DNA conversion can be induced in the genome of dicotyledonous plants to modify the genome. According to the method of the present invention, a dicotyledonous plant in which the mutation is introduced into a target site on the genome can be produced with high efficiency.

Using a plant (T ₀ plant) in which a mutation is induced in a target site on the genome, crossing (self-crossing or cross-breeding), collecting seeds, raising it, and progeny plants having the mutation By selecting, a progeny plant having the mutation can be produced. In the method of the present invention, a mutation can be induced in a target site on the genome of a plant, and it can be genetically passaged to progeny plants (T ₁ or F ₁ generations and beyond; also referred to as progeny). This is made possible by the fact that the efficiency of the mutation introduction into the target site according to the present invention is high enough to be inherited by progeny.

In the present invention, by using a nucleic acid construct and / or a fusion protein suitable for efficient mutation introduction into the plant germ cell genome, DNA conversion and other mutations are introduced into the plant germ cell genome, and the progeny is efficiently generated. Can be inherited. The generation of a progeny having a mutation involves modifying the plant genome in the plant cell by the method described herein, inducing a mutation including DNA conversion, and removing the plant from the plant cell having the modified genome. Producing a progeny plant from the plant body, and selecting a progeny plant having the mutation.

The present disclosure provides plants, portions thereof, and combinations thereof that have been genomically modified or produced by the methods described herein. Such plants include not only modern plants but also their progeny plants. Plant parts provided by the present disclosure include fruits, roots (including, for example, root variants such as tuberous roots), leaves, flowers, seeds, grains and stems (eg, stem variants such as rhizomes, tubers). Etc.) or variations thereof. The present disclosure also includes a plant that has been genetically modified or produced by the methods described herein, processed parts thereof (eg, materials such as bioethanol, processed foods, and enzymes, Including processed materials such as starch and sugar). In the present specification, “processing” is interpreted in its broadest sense, and it is understood that it includes various processing processes, cooking with or without heating, salting, fermentation and the like.

(7. Preferred embodiment)
1 illustrates a preferred embodiment of the present invention. The present invention provides a method for modifying a plant genome by introducing a guide RNA and a fusion protein containing a nucleic acid sequence recognition module and a nucleobase converting enzyme into a plant cell. The plant is preferably a dicotyledonous plant, more preferably a solanaceous plant, preferably a solanaceous plant, more preferably a solanaceous plant, and still more preferably a tomato plant. The sequence recognition module is preferably selected from the group consisting of a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated, a zinc finger motif, a TAL effector and a PPR motif. The CRISPR-Cas system in which at least one DNA-cleaving ability of Cas is inactivated may include two nuclease domains, and any one of the two nuclease domains may be inactivated. The nucleobase converting enzyme is preferably deaminase, more preferably cytidine deaminase. The introduction is preferably performed by introducing a nucleic acid construct encoding the nucleic acid sequence recognition module and the nucleobase converting enzyme into the cell, and more preferably, the nucleic acid construct further encodes a guide RNA, These are introduced simultaneously. The nucleic acid construct or the coding sequence therein can be optimized for codon usage in dicotyledonous plants, eg, Arabidopsis. The target of the guide RNA and the nucleic acid sequence recognition module is not particularly limited, and examples thereof include the sequence of SlDELLA or SlETR1 gene. The introduced plant cell or tissue (for example, callus) containing the plant cell can be cultured. As the introduction treatment condition, for example, the culture can be performed at about 23 to 27 ° C. (eg, about 25 ° C.). As the culture conditions, for example, the culture can be performed at about 23 to 27 ° C. (for example, about 25 ° C.), and the culture period can be about 2 to 4 days (for example, about 3 days). A plant genome in a plant cell is modified by a method having any of the above characteristics, a mutation including DNA conversion is induced, a plant body is produced from the plant cell having the modified genome, and a progeny from the plant body By producing a plant and selecting a progeny plant having the mutation, it is possible to produce a progeny plant having the mutation.

In the present invention, preferred embodiments in the case of targeting tomato plants are exemplified below. In the method of the present invention, tomato plant cells are introduced by introducing a fusion protein containing gRNA and a mutant Cas9 protein lacking at least one nuclease activity of two nuclease domains RuvC and HNH and cytidine deaminase into the tomato plant cells. Causes contact of the genomic DNA with gRNA and its fusion protein. Introduction of gRNA and its fusion protein into tomato plant cells can be performed by any method. For example, a nucleic acid construct containing an expression unit containing a base sequence encoding a fusion protein containing a mutant Cas9 protein and cytidine deaminase is introduced into a tomato plant cell, or a fusion protein containing a mutant Cas9 protein and a cytidine deaminase is encoded. By directly introducing mRNA to be expressed and expressing the fusion protein, the fusion protein can be introduced into tomato plant cells. A tomato plant cell is introduced with a nucleic acid construct containing an expression unit containing a base sequence encoding a fusion protein containing a mutant Cas9 protein and cytidine deaminase, and the fusion protein is expressed in the tomato plant cell. It is more preferable to introduce. *

The present invention induces mutations, particularly DNA conversion, in the target site of the genome of a tomato plant by the above-described method, and produces a plant body (T ₀ plant) having the genome in which such mutation is induced. Also provided is a method for breeding a tomato plant, which comprises producing a progeny plant by crossing using and selecting a progeny plant having the mutation. The mating may be self-mating, cross-breeding, or backcrossing. In this method, generation of progeny plants by crossing and selection of progeny plants having mutations may be further repeated. In the selection of progeny plants, it is particularly preferable to select progeny plants having homozygous mutations. Selection of a progeny plant having a mutation can be performed by determining a base sequence of a target site of genomic DNA. Alternatively, selection of a progeny plant having a mutation may be performed based on a phenotypic change caused by introducing a mutation into the target gene. By using the breeding method of the present invention, it is possible to efficiently produce a plant in which a mutation introduced into the target site of the genome of a tomato plant is genetically stably passed to a progeny plant. Therefore, the breeding method of the present invention can also be used for production of a new tomato variety.

(General technology)
Molecular biological methods, biochemical methods, microbiological methods, general methods of genome editing, bioinformatics, etc. used in the present specification are known in the art, and any known or commonly used Can be used. References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described. The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration and are not provided for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the claims.

Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[Example 1] Preparation of T-DNA vector A T-DNA vector used in the CRISPR-Cas9 system was prepared as follows.
The CRISPER / Cas9 vectors pZK_FFCas9 and pUC19_AtU6oligo with Cas9 coding sequences optimized for Arabidopsis codon usage are pCAS9-TPC and pChimera (Fauser et al., (2014) Plant J., 79, p.348, respectively. -59) and was provided by Dr. Masaki Endo.

The Cas9 nuclease-inactivated D10A mutation was introduced into the vector pZK_FFCas9 using PCR and Gibson assembly method to create pZK_FFnCas9 (D10A). The following primers were used for the preparation of pZK_FFnCas9 (D10A).
kn1091_HolCas9D10A_F (5'-CTCTATCGGACTCGcTATCGGAACTAACTCTG-3 '; SEQ ID NO: 1)
kn1092_HolCas9D10A_R (5'-GAGTTAGTTCCGATAgCGAGTCCGATAGAGTAC-3 '; SEQ ID NO: 2)
kn1095_HolCas9D10A_F (5'-TGTATGTGCAGCGAATTCGGCGCGCaATGGATAAGAAGTACTCTATCGGACTCGcTATC-3 '; SEQ ID NO: 3)
kn1083_ApaI-HolCas9_R (5'-CTGGGAGGCCTGGAtCaGGGCCCtCCTCcAACCTTCCTCTTCTTCTTAGG-3 '; SEQ ID NO: 4)

pZK_FFnCas9 (D10A) is a coding sequence optimized for codon usage of Arabidopsis (SEQ ID NO: 5) encoding a Cas9 protein having a D10A mutation (hereinafter referred to as Cas9 (D10A) or nCas9; SEQ ID NO: 6). including. Cas9 protein is derived from Streptococcus pyogenes. Cas9 having a D10A mutation is deficient in nuclease activity and can nick only one strand without causing double-strand breaks. It functions as a nickase that can be used.

Next, a DNA sequence (SEQ ID NO: 7) optimized for human codon usage encoding Arabidopsis, an activation-inducing cytidine deaminase (PmCDA) (SEQ ID NO: 9) derived from lamprey, a base converting enzyme, and Arabidopsis A vector prepared as described above so that cytidine deaminase (CDA) is linked to the C-terminal side of nCas9 via a linker peptide, each of which is a DNA sequence (PmCDA ^opt ) (SEQ ID NO: 8) optimized for codon usage. and inserted into pZK_FFnCas9 (D10A), respectively to prepare a vector pZK_FFnCas9 (D10A) -PmCDA and pZK_FFnCas9 (D10A) -PmCDA ^opt.

On the other hand, the target sequence of guide RNA (gRNA) was inserted between the AtU6-26 promoter and the chimeric gRNA scaffold in the gRNA expression unit vector pUC19_AtU6oligo by the PCR method. In this example, the two endogenous tomato genes SlDELLA gene (Solyc11g011260) and SlETR1 gene (Solyc12g011330) that control the development of tomato (Solanum lycopersicum) fruit are targeted genes, and within the SlDELLA gene protein coding sequence and the SlETR1 gene three loci protein coding sequence ^{(SlETR1 site1, SlETR1 site2, SlETR1} site3) , design any one of the nucleotide sequences of 20 bases in length located TGG sequence immediately preceding the DNA strand as a target sequence for guide RNA (gRNA) did. The primer sequences used for inserting each target sequence into the vector are shown in Table 1 below.

From pUC19_AtU6oligo insertion of the target sequence, the gRNA expression unit excised with I-SceI treated and inserted into pZK_FFnCas9 (D10A) -PmCDA and pZK_FFnCas9 (D10A) -PmCDA ^opt by ligation. was inserted GRNA expression unit pZK_FFnCas9 (D10A) -PmCDA and pZK_FFnCas9 (D10A) -PmCDA ^opt includes a GRNA, capable of expressing the nCas9-PmCDA fusion protein. The generated nCas9-PmCDA fusion protein forms a complex of nCas9 and PmCDA and is induced (targeted) to a target site in the genome by gRNA.

For comparison, create a vector with the same structure except that it does not contain a sequence encoding PmCDA and uses a DNA sequence optimized for codon usage of Arabidopsis instead of nCas9. did. In addition, to express nCas9 without PmCDA, a vector that prevents the translation of PmCDA by inserting a stop codon immediately upstream of the PmCDA coding sequence in pZK_FFnCas9 (D10A) -PmCDA with a gRNA expression unit inserted Was also prepared (nCas9-stop-PmCDA). These were used as comparative objects in the examples described later to examine the effects of nCas9 and the fusion of PmCDA to nCas9 on DNA modification based on the CRISPR system.

In some vectors, a 2A peptide linker, ie, a sequence encoding the foot-and-mouth disease virus 2A peptide (Ryan et al., (1991) J Gen Virol., 72 (Pt 11): 2727-2732) is fused in the vector. The gene was inserted between nCas9-PmCDA ^opt and NPT II coding sequence. Note that the targeting of SlETR ^site3, using nCas9-PmCDA ^opt vector backbone was inserted 2A peptide linker coding sequence.

The structure of T-DNA in each vector prepared as described above is shown in FIG. In any T-DNA, the AtU6 promoter (Arabidopsis U6 promoter; SEQ ID NO: 39) was used in the gRNA expression unit, and the ubiquitin promoter (PcUbi promoter) was used in the Cas9 expression unit. The CaMV 35S promoter was used for the expression induction of the NPTII expression unit.

Contained in the above ^{T-DNA, SlDELLA, SlETR1 site1} , SlETR1 site2, or SlETR1 shows ^site3 the nucleotide sequence of the gRNA expression unit that targets in SEQ ID NO: 15-18. 1st to 18th of the nucleotide sequence shown in SEQ ID NOs: 15 to 18 are I-SecI recognition sites, 172nd to 559th are AtU6 promoters, 560th to 579th are target sequences, and 560th to 655th are gRNA coding sequences. It is.

The above T-DNA, namely Cas9 [PcUbi (P) _Cas9_Pea3A (T)], nCas9-PmCDA [PcUbi (P) _nCas9-PmCDA_Pea3A (T)], nCas9-stop-PmCDA [PcUbi (P) _nCas9A_P3_A (T)], Cas9 / NPTII expression unit contained in nCas9-PmCDA ^opt [PcUbi (P) _nCas9-PmCDAopt_Pea3A (T)], nCas9-PmCDA ^opt _2A [PcUbi (P) _nCas9-PmCDA ^opt _2A] Are shown in SEQ ID NOs: 19, 21, 23, 25, and 27, respectively.

In the nucleotide sequence represented by SEQ ID NO: 19, the 1st to 917th positions are the PcUbi promoter, the 932th to 5047th positions are the Arabidopsis codon optimized Cas9 gene, the 5048th to 5068th positions are nuclear localization signals (NLS), and the 5069th to 5071 positions. Is the stop codon, 5086 to 5554 is the Pea3A terminator, 6865 to 7699 is the CaMV 35s promoter, 7712 to 8509 is the NPTII gene, 8516 to 9613 is the 3'UTR and terminator of Oshsp17.3 . The amino acid sequence of Cas9-NLS encoded in the base sequence represented by SEQ ID NO: 19 is shown in SEQ ID NO: 20 (the first to 1372th positions of SEQ ID NO: 20 are Cas9, and the 1373th to 1379th positions are NLS).

In the nucleotide sequence shown in SEQ ID NO: 21, the 1st to 917th positions are the PcUbi promoter, the 932th to 5047th positions are the nCas9 gene, the 5048th to 5068th positions are nuclear localization signals, and the 5078th to 5098th positions are nuclear localization signals. (NLS40), 5099 to 5128 are glycine-serine linker, 5129 to 5305 are SH3 domains, 5306 to 5371 are 3xFlag tags, 5078 to 6001 are PmCDA genes, and 6002 to 6004 are stop codons The 6014th to 6482th are the Pea3A terminator, the 7793th to 8627th are the CaMV 35s promoter, the 8640th to 9437th are the NPTII genes, and the 9444th to 10541th are the 3'UTR and terminator of Oshsp17.3. The amino acid sequence from nCas9 to PmCDA encoded in the base sequence shown in SEQ ID NO: 21 is shown in SEQ ID NO: 22 (the first to 1372th positions of SEQ ID NO: 22 are nCas9, and the 1483th to 1695th positions are PmCDA).

In the nucleotide sequence shown in SEQ ID NO: 23, the 1st to 917th positions are PcUbi promoter, the 932th to 5047th positions are nCas9 genes, the 5048th to 5068th positions are nuclear localization signals, the 5078th to 5238th positions are SH3 domains, and the 5138th position. The 5140th is a stop codon. Downstream from this, the function has been lost due to insertion of a stop codon, but the 5239th to 5304th are 3xFlag tags, the 5311th to 5943th are PmCDA genes, the 5947th to 6415th are Pea3A terminators, and the 7726th to 8560th are CaMV. The 35s promoter corresponds to the NPTII gene from 8573th to 9370th and the 3′UTR and terminator of Oshsp17.3 from 9377th to 10474th. The amino acid sequence containing nCas9 encoded in the base sequence shown in SEQ ID NO: 23 is shown in SEQ ID NO: 24.

In the nucleotide sequence shown in SEQ ID NO: 25, the 1st to 917th are the PcUbi promoter, the 932th to 5047th are the nCas9 gene, the 5048th to 5068th are the nuclear localization signals, and the 5084th to 5260th are the SH3 domain (Arabidopsis codon) Optimization), 5261 to 5326 are 3xFlag tags, 5333 to 5959 are PmCDA ^OPT genes, 5960 to 5980 are nuclear localization signals, 5992 to 6460 are Pea3A terminators, and 7771 to 8605 are The CaMV 35s promoter, 8618th to 9415th are the NPTII gene, 9422th to 10519th are the 3 'UTR and terminator of Oshsp17.3. The amino acid sequence from nCas9 encoded in the nucleotide sequence shown in SEQ ID NO: 25 to PmCDA ^opt and nuclear localization signal is shown in SEQ ID NO: 26 (the first to 1372th positions of SEQ ID NO: 26 are nCas9, and the 1468th to 1685 positions). The second is PmCDA ^opt ). Further, the base sequence of the first to 6460th base sequence (from PcUbi promoter to Pea3A terminator) of the base sequence shown in SEQ ID NO: 25 is shown in SEQ ID NO: 40.

In the nucleotide sequence represented by SEQ ID NO: 27, the 1st to 917th positions are the PcUbi promoter, the 932th to 5047th positions are the nCas9 gene, the 5048th to 5068th positions are nuclear localization signals, the 5084th to 5260th positions are SH3 domains (Arabidopsis codons) Optimization), 5261 to 5326 are 3xFlag tags, 5333 to 5959 are PmCDA ^OPT genes, 5960 to 5980 are nuclear localization signals, 6001 to 6060 are 2A peptides, and 6070 to 6867 are The NPTII gene, 6874th to 7971st, is the 3 ′ UTR and terminator of Oshsp17.3. First to 1372 th nCas9,1468 th to 1676 th PmCDA ^opt of SEQ ID NO: The amino acid sequence from nCas9 encoded in the nucleotide sequence shown up NPTII 27 shown in SEQ ID NO: 28 (SEQ ID NO: 28, 1710 th The 1974th is NPTII).

Further, the DNA sequence of PmCDA ^opt and the subsequent nuclear localization signal coding sequence contained in SEQ ID NOs: 25 and 27 is represented by SEQ ID NO: 29, and the fusion polypeptide (CDA having a nuclear localization signal) encoded thereby The amino acid sequence is shown in SEQ ID NO: 30.

[Example 2] Production of transgenic plant T-DNA in each vector produced in Example 1 was transformed into Agrobacterium-mediated transformation method using Agrobacterium GV2260 (Sun et al. (2006) Plant Cell Physiol. 47, 426-431) introduced into a tomato (Solanum lycopersicum) plant, and selected a transgenic tomato plant (primary transgenic plant) in which T-DNA was inserted into the genome based on kanamycin resistance. To play. The variety Micro-Tom was used as the tomato plant.

Briefly, first, microtom seeds were aseptically sown on MS solid medium and germinated in a culture room at 25 ° C. for 16 hours. The germinated cotyledons were cut with scissors, immersed in an Agrobacterium solution holding the vector prepared in Experimental Example 1, and then cultured for 3 days on an MS solid medium containing 10 μM acetosyringone and zeatin 1.5 mg / L. Subsequently, callus was induced on an MS solid medium containing zeatin 1.5 mg / L and kanamycin 100 mg / L, and shoots were further induced on an MS solid medium containing zeatin 1 mg / L and kanamycin 100 mg / L. Finally, regenerated shoots from callus were cut out and rooted on MS solid medium containing kanamycin 50 mg / L to regenerate the plant body.

In addition, the regenerated plant body (primary plant T ₀ ) was cultivated and self-mated to obtain self-propagating seeds. A plant body was grown from the self-propagated seed (T ₁ ), and further self-crossed to obtain a self-propagated seed (T ₂ ), and the plant body was grown. The crossed a wild-type Micro-Tom plant with primary plant T _0, to obtain a F ₁ plants. All primary plants T ₀ and progeny plants (T ₁ , T ₂ and F ₁ ) were cultivated under a constant temperature of 25 ° C. under 200 μmolm ⁻² s ⁻¹ light conditions (light conditions 16 hours / dark conditions 8 hours) .

[Example 3] Mutation analysis in transgenic plants In order to analyze mutations occurring in transgenic plants, first, genomic DNA was extracted from the leaves of the wild-type microtom plant and the transgenic plant obtained in Example 2. PCR amplification was performed using the obtained genomic DNA as a template and a target gene-specific primer. Table 2 shows the target gene-specific primers used.

The amplified fragment was subcloned into plasmid pGEM-T Easy (Promega), introduced into E. coli, transformed, and cultured on an LB agar plate containing 100 μg / ml ampicillin. About the colony formed on the LB agar plate, the base sequence of the inserted amplified fragment was determined by the Sanger method using the target gene specific primer.

In the target gene in the genome of the obtained transgenic plant, both insertion deletion (Indel) and DNA conversion (base substitution) mutations were observed in each line. Therefore, the number of transgenic plants having insertion deletions and transgenic plants having DNA conversion was counted. The results are shown in Table 3. We also identified the mutation pattern in the target sequence. The patterns of mutations observed in T ₀ plants are shown in Tables 4 and 5. Also shows the mutation pattern observed by T ₁ plants in Tables 6 and 7.

In primary transgenic plants targeted to SlDELLA (T ₀ ), wild-type Cas9 mainly induced insertional deletions and only slight DNA conversion was detected (Table 3). Also, 4 of the 5 transgenic lines (# 2, # 21, # 26, and # 31) progeny plants (T ₁ ) also had frequent insertion deletions (Tables 3, 4 and 6). This result indicates that wild-type Cas9 almost exclusively induces heritable insertion deletions due to error-prone non-homologous end joining (NHEJ).

In contrast, nCas9-PmCDA-introduced T ₀ transgenic plants targeted with SlDELLA induced both insertional deletion and DNA conversion, particularly DNA conversion more frequently (Tables 3 and 4). DNA conversion was clearly shown as a C to T or C to G transition within the target sequence (Table 4). Further also mutated in both the insert deletions and DNA transformation observed by T ₁ plants, each mutant was shown to be inherited successfully in T ₁ plants (Table 3,6). This demonstrates that the base editing technology using gRNA and nCas9-PmCDA functions in tomato plant cells.

On the other hand, in the nCas9-stop-PmCDA-introduced T ₀ plant targeting SlDELLA, although a slight DNA modification was observed, the inheritance of the DNA modification to the T ₁ plant was not observed (Tables 4 and 6). This result indicates that nickase alone cannot induce NHEJ-mediated targeted mutagenesis or DNA conversion, and that CDA functions are required for base editing.

The target the SlDELLA, retained both also transgenic T _O and T ₁ plants were introduced optimized nCas9-PmCDA ^opt to codon usage of the inserted deletions and DNA conversion plant (Arabidopsis thaliana) (Table 3, 4 and 6).

SlDELLA throughout transgenic plants targeting, system # 1 for lines # 1 and # 9, nCas9-PmCDA ^{opt-introduced} plant for five (nCas9-PmCDA transgenic plants of the transgenic lines 11, # 3 and # 27 ) Produced T ₁ progeny plants with DNA conversion. This further shows that nCas9-PmCDA can induce heritable DNA conversion.

Further, even in nCas9-PmCDA or nCas9-PmCDA ^opt Transgenic plants targeting different target gene SlETR1 the SlDELLA, both inserts deletions and DNA transformation was observed (Table 3, 5 and 7). Specifically, T ₁ transgenic line # 3 was introduced nCas9-PmCDA that target the ^{SlETR1 site1, # 4, # 8} , # 9, # 11, # 13, and # 14, SlETR1 ^site2 were targeted nCas9-PmCDA ^opt the introduced T ₁ transgenic line # 6-1, # 6-2, # 8-1 and # 8-2, and T ₁ transgenic a SlETR1 ^site3 was introduced Cas9-PmCDA ^opt was targeted Insertion deletion and / or DNA conversion were confirmed in lines # 3, # 8, # 11, # 25a, # 25b, # 30, # 57 and # 72 (Table 7). Some of those mutations observed in T ₀ plants were inherited in T ₁ plants. SlETR1 ^site1 T ₁ was introduced nCas9-PmCDA that target the transgenic lines # 13, SlETR1 ^site2 T ₁ transgenic line # 6-1 was introduced nCas9-PmCDA ^opt that target the, # 6-2, # 8 -1 and # 8-2, as well SlETR1 ^site3 was targeted nCas9-PmCDA ^opt to introduce T ₁ transgenic line # 3, # 8, # 11 , # 25a, # 25b, # 30, # 57 and # 72 Inheritance of insertion deletion and / or DNA conversion from T ₀ plant to T ₁ plant was observed (Table 5 and Table 7). The above results indicate that this base editing system can be applied to genes other than SlDELLA.

In Tables 4 to 7, “+” and “−” described in the pattern column indicate DNA insertion and deletion, respectively, and the subsequent numbers indicate the length (bp) of the insertion deletion. “C” indicates DNA conversion, and the number following it indicates the number of converted bases. Bases that have undergone DNA conversion in the sequence are shown in lower case. “WT” indicates wild type (no modification).

In addition, nCas9-PmCDA ^opt targeting SlDELLA was introduced, and T ₁ plants (# 27_9) and T ₂ plants (with a 12 base deletion and a 2 base substitution (replacement from CAC to tAt)) in the target sequence ( # 3_2_4) showed a phenotype similar to the tomato mutant procera with the SlDELLA-deficient allele (eg, leaflets with reduced saw blades) (FIG. 3).

Subsequently, it was tested whether a selection marker gene was contained in the genome of a transgenic plant having a stable DNA modification. Using the obtained transgenic T ₁ plant and the genomic DNA extracted from the F ₁ plant obtained by crossing between the nCas9-PmCDA-introduced T ₀ plant targeting SlDELLA and the wild-type microtom plant as a template, kanamycin PCR using a resistance marker gene (NPTII) amplification primer was performed. As a positive control for genomic DNA extraction, PCR using primers for amplifying endogenous actin gene was also performed. The primers used are as follows.

NPTII amplification primer NPTII-F: 5'-ATGATTGAACAAGATGGATTGCAC-3 '(SEQ ID NO: 35)
NPTII-R: 5'-TCAGAAGAACTCGTCAAGAAGGCG-3 '(SEQ ID NO: 36)
Primer for amplifying endogenous actin gene Actin-F: 5'-GATGGATCCTCCAATCCAGACACTGTA'-3 '(SEQ ID NO: 37)
Actin-R: 5'-GTATTGTGTTGGACTCTGGTGATGGTGT'-3 '(SEQ ID NO: 38)

Cas9, nCas9-PmCDA and nCas9-PmCDA ^opt- introduced transgenic T ₁ plants targeting SlDELLA, and F ₁ plants (# 3BC1_6) retained heritable insertion deletion and DNA conversion, but PCR analysis Did not detect a kanamycin resistance gene in the plant genome (FIG. 4). Similarly, SlETR1 ^site2 and SlETR1 ^site3 a nCas9-PmCDA ^opt Transgenic T ₁ plants targeted is retained the DNA transformation of hereditary, kanamycin resistance gene into the plant genome by PCR analysis not detected (FIG. 4).

Further, SlDELLA have DNA transformation of the target sequence in homozygous or heterozygous or biallelic, SlETR1 ^site1, SlETR1 ^site2 or SlETR1 ^site3 transgenic T ₁ that target the plants and T ₂ plants, DNA transformation It was shown to have an amino acid substitution caused by. For example, transgenic line # 1 (T ₁ ) targeting SlDELLA and introducing nCas9-PmCDA, and transgenic line # 1 (T ₂ ) targeting SlDELLA and introducing nCas9-PmCDA ^opt , Amino acid substitution, ie PL (proline-leucine) → LV (leucine-valine) was shown (FIG. 5). This indicates that the base editing technique used in the present invention can induce amino acid sequence substitution. Moreover, it was shown that the mutation can be retained while the marker gene is excluded in the progeny of some of the plants into which the mutation has been introduced.

(Note)
As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, it is understood that the scope of this invention should be construed only by the claims. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood. This application is a patent application claiming priority to Japanese Patent Application No. 2017-019921 filed on February 6, 2017 with the Japan Patent Office. It is understood that the contents of which are to be incorporated by reference in their entirety as if specifically set forth herein.

The method of the present invention can induce DNA conversion at a target site, and can promote induction of site-specific genetic mutations in tomato plants. Since many agronomically important traits are dominated by single nucleotide polymorphisms, the method of the present invention can be advantageously used in crop breeding.

SEQ ID NOs: 1-4: primers SEQ ID NO: 5: Arabidopsis codon optimized nCas9-NLS coding sequence
SEQ ID NO: 6: nCas9-NLS protein SEQ ID NO: 7: human codon optimized PmCDA coding sequence SEQ ID NO: 8: Arabidopsis codon optimized PmCDA coding sequence SEQ ID NO: 9: PmCDA protein SEQ ID NO: 10-14: primer SEQ ID NO: 15: gRNA expression unit ( SlDELLA)
SEQ ID NO: 16: gRNA expression unit (SlETR1 ^site1 )
SEQ ID NO 17: gRNA expression unit (SlETR1 ^site2)
SEQ ID NO: 18: gRNA expression unit (SlETR1 ^site3 )
SEQ ID NO: 19, 21, 23, 25, 27: Cas9 / NPTII expression unit SEQ ID NO: 20, 22, 24, 26, 28: Synthetic construct SEQ ID NO: 29: PmCDA ^opt- NLS coding sequence SEQ ID NO: 30: PmCDA-NLS protein sequence No. 31-38: Primer SEQ ID NO: 39: Arabidopsis U6 primer SEQ ID NO: 40: DNA sequence from PcUbi (P) to Pea3A (T) of SEQ ID NO: 25

Claims (35)

A method for modifying the genome of a dicotyledonous plant, comprising:
A method comprising introducing a guide RNA and a fusion protein comprising a nucleic acid sequence recognition module and a nucleobase converting enzyme into a plant cell. The method according to claim 1, wherein the nucleic acid sequence recognition module is selected from the group consisting of a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated, a zinc finger motif, a TAL effector, and a PPR motif. The method according to claim 2, wherein the nucleic acid sequence recognition module is a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated. The method according to claim 3, wherein the CRISPR-Cas system includes two nuclease domains, and any one of the two nuclease domains is inactivated. The method according to any one of claims 1 to 4, wherein the nucleobase converting enzyme is deaminase. The method according to claim 5, wherein the nucleobase converting enzyme is cytidine deaminase. The method according to any one of claims 1 to 6, wherein the introduction is performed by introducing the nucleic acid sequence recognition module and a nucleic acid construct encoding the nucleobase converting enzyme into the cell. The method of claim 6, wherein the nucleic acid construct further encodes the guide RNA. The method according to claim 6 or 7, wherein the nucleic acid sequence recognition module and the sequence encoding the nucleobase converting enzyme are optimized for codon usage of dicotyledonous plants. The method according to claim 9, wherein the nucleic acid sequence recognition module and the sequence encoding the nucleobase converting enzyme are optimized for use in Arabidopsis codons. The method according to any one of claims 1 to 10, wherein the guide RNA and the nucleic acid sequence recognition module target the sequence of the SlDELLA or SlETR1 gene. The method according to any one of claims 1 to 11, wherein the dicotyledonous plant is a solanaceous plant. The method according to any one of claims 1 to 12, wherein the dicotyledonous plant is a plant of the genus Eggplant. The method according to any one of claims 1 to 13, wherein the dicotyledonous plant is a tomato plant (Solanum lycopersicum). The method according to any one of claims 1 to 14, wherein the introduction is performed at about 23 to about 27 ° C. The method according to any one of claims 1 to 15, wherein the introduction is performed at about 25 ° C. The method according to any one of claims 1 to 16, further comprising culturing the introduced plant cell at about 23 to about 27 ° C. The method according to any one of claims 1 to 17, further comprising culturing the introduced plant cell at about 25 ° C. Modifying the plant genome in dicotyledonous cells by the method of any one of claims 1 to 18 to induce mutations including DNA conversion;
Producing a plant from the plant cell whose genome has been modified;
A method for breeding a dicotyledon, comprising producing a progeny plant from the plant and selecting a progeny plant having the mutation. A composition comprising a nucleic acid sequence recognition module and a nucleic acid construct encoding a nucleobase converting enzyme for modifying a dicotyledon genome, wherein the nucleic acid sequence recognition module is a target on the genome in the presence of a guide RNA. A composition that recognizes a sequence. 21. The composition according to claim 20, wherein the nucleic acid sequence recognition module is selected from the group consisting of a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated, a zinc finger motif, a TAL effector, and a PPR motif. The composition according to claim 21, wherein the nucleic acid sequence recognition module is a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated. The composition according to claim 22, wherein the CRISPR-Cas system includes two nuclease domains, and any one of the two nuclease domains is inactivated. The composition according to any one of claims 20 to 23, wherein the nucleobase converting enzyme is deaminase. The composition according to claim 24, wherein the nucleobase converting enzyme is cytidine deaminase. The composition according to any one of claims 20 to 25, wherein the nucleic acid construct further encodes the guide RNA. The composition according to any one of claims 20 to 26, wherein the nucleic acid sequence recognition module and the sequence encoding the nucleobase converting enzyme are optimized for codon usage of dicotyledonous plants. 28. The composition of claim 27, wherein the nucleic acid sequence recognition module and the sequence encoding the nucleobase converting enzyme are optimized for Arabidopsis codon usage. The composition according to any one of claims 20 to 28, wherein the guide RNA and the nucleic acid sequence recognition module target a sequence of SlDELLA or SlETR1 gene. The composition according to any one of claims 20 to 29, wherein the dicotyledonous plant is a solanaceous plant. The composition according to any one of claims 20 to 30, wherein the dicotyledonous plant is a plant of the genus Eggplant. The composition according to any one of claims 20 to 31, wherein the dicotyledonous plant is a tomato plant (Solanum lycopersicum). A plant produced by the method according to any one of claims 1 to 19. A plant part produced by the method according to any one of claims 1 to 19. 35. A plant part according to claim 34, wherein said part is selected from fruits, roots, leaves, flowers, seeds and stems.

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