[go: up one dir, main page]

WO2018143477A1 - Procédé de modification du génome d'une plante dicotylédone - Google Patents

Procédé de modification du génome d'une plante dicotylédone Download PDF

Info

Publication number
WO2018143477A1
WO2018143477A1 PCT/JP2018/004062 JP2018004062W WO2018143477A1 WO 2018143477 A1 WO2018143477 A1 WO 2018143477A1 JP 2018004062 W JP2018004062 W JP 2018004062W WO 2018143477 A1 WO2018143477 A1 WO 2018143477A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
nucleic acid
acid sequence
recognition module
genome
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2018/004062
Other languages
English (en)
Japanese (ja)
Inventor
浩 江面
亨 有泉
謙治 三浦
幸子 嘉祥寺谷
真理子 高山
剛史 山本
敬二 西田
近藤　昭彦
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kobe University NUC
University of Tsukuba NUC
Original Assignee
Kobe University NUC
University of Tsukuba NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kobe University NUC, University of Tsukuba NUC filed Critical Kobe University NUC
Priority to JP2018566171A priority Critical patent/JPWO2018143477A1/ja
Publication of WO2018143477A1 publication Critical patent/WO2018143477A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • 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.
  • Cas9 enzyme Non-patent Document 1
  • gRNA guide RNA
  • 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).
  • gRNA and Cas9 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.
  • PAM protospacer adjacent motif
  • 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).
  • NHEJ error-prone non-homologous end joining
  • HDR homologous recombination repair
  • the CRISPR / Cas9 system has advantages such as ease of construct design and low cost.
  • wild-type Cas9 can cause cleavage at the target external site by off-target action.
  • Cas9 nickase 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.
  • HDR homologous recombination repair
  • Non-Patent Document 2 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.
  • 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 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).
  • Non-patent Documents 4 and 5 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).
  • 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).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas9 CRISPR-associated 9
  • the present invention relates to a method for introducing a mutation including DNA conversion into a target site on the genome of a dicotyledon.
  • 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).
  • gRNA guide RNA
  • DNA conversion can be frequently induced in the target site.
  • 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.
  • nucleic acid sequence recognition module 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.
  • 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.
  • nucleic acid sequence recognition module is a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated.
  • CRISPR-Cas system comprises two nuclease domains, and any one of the two nuclease domains is inactivated.
  • nucleobase converting enzyme is a deaminase.
  • nucleobase converting enzyme is cytidine deaminase.
  • nucleic acid sequence recognition module and the sequence encoding the nucleobase converting enzyme are optimized for codon usage of dicotyledonous plants.
  • nucleic acid sequence recognition module and the sequence encoding the nucleobase converting enzyme are optimized for codon usage of Arabidopsis thaliana.
  • guide RNA and the nucleic acid sequence recognition module target the sequence of the SlDELLA or SlETR1 gene.
  • dicotyledonous plant is a solanaceous plant.
  • dicotyledonous plant is a plant of the genus Eggplant.
  • [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.
  • 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.
  • 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 is provided.
  • 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.
  • 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.
  • nucleobase converting enzyme is deaminase.
  • nucleobase converting enzyme is cytidine deaminase.
  • nucleic acid construct further encodes the guide RNA.
  • nucleic acid sequence recognition module and the sequence encoding the nucleobase converting enzyme are optimized for codon usage of Arabidopsis thaliana.
  • guide RNA and the nucleic acid sequence recognition module target the sequence of the SlDELLA or SlETR1 gene.
  • composition according to any of the preceding items, wherein the dicotyledonous plant is a solanaceous plant.
  • the composition according to any one of the items described above, wherein the dicotyledonous plant is a plant of the genus Eggplant.
  • the composition according to any of the preceding items, wherein the dicotyledonous plant is a tomato plant (Solanum lycopersicum).
  • ITEM 33 The plant produced by the method in any one of said claim
  • 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.
  • ITEM 36 The processed product which processed the plant produced by the method in any one of the said claim
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • FIG. 6 shows the codon usage frequency of tomato (Sl) and Arabidopsis (At).
  • 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.
  • 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.
  • introduction refers to the presence of a desired molecule in a cell.
  • a nucleic acid encoding the protein molecule may be transferred into the cell and the protein may be expressed in the cell.
  • “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.
  • the double-stranded DNA to be modified is not particularly limited, but is preferably genomic DNA.
  • 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.
  • dicotyledonous plants can be used as the object of the method or composition for introducing a mutation into the genome.
  • mutations could be introduced into the genome with high efficiency in dicotyledonous plants.
  • Dicotyledonous plants are angiosperms with two (or more) embryonic cotyledons.
  • angiosperms with one cotyledon are called monocotyledons.
  • 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.
  • 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.
  • 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.
  • 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, Egg
  • solanaceous plants examples include Montinaceae, Nagano Nourushiaceae, Ceylon Jacobeaceae, Convolvulaceae, and Solanaceae.
  • 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
  • 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 (Ashitab
  • 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.)
  • the genome modification method or composition of the present invention may target tomato plants.
  • 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.
  • nucleic acid sequence recognition module examples 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
  • 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 preferably, a CRISPR-Cas system in which at least one DNA cleavage ability of Cas is inactivated (CRISPR-mutated Cas)
  • zinc finger motif preferably, zinc finger motif, TAL effector
  • 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.
  • 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).
  • E. coli one-hybrid method Naat Biotechnol (2008) 26: 695-701.
  • 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.
  • RVD 12th and 13th amino acid residues
  • 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.
  • 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.
  • 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.
  • gRNA guide RNA
  • PAM protospacer adjacent motif
  • 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).
  • 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.
  • 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.
  • 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.)
  • 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.
  • 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.
  • 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.
  • guide RNA 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).
  • crRNA RNA sequence
  • tracrRNA RNA sequence having a scaffold function
  • 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.
  • 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.
  • 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/). .
  • 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.
  • 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.
  • 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.
  • cytidine deaminase can be used together with the nucleic acid sequence recognition module.
  • 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).
  • 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.
  • 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.
  • 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.
  • a nuclear localization signal is added to the C-terminus of cytidine deaminase.
  • 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.
  • 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
  • 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).
  • 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.
  • 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.
  • the fusion protein in the method of the present invention, 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).
  • a target gene product here, gRNA or mRNA encoding a fusion protein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleic acid construct to be introduced into the dicotyledonous plant cell may contain these base sequences.
  • 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.
  • 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.
  • 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.
  • 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.
  • the base sequence encoding gRNA is preferably arranged under the control of an Arabidopsis promoter.
  • 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.
  • 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.
  • 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.
  • nucleic acid such as the above nucleic acid construct, gRNA, or mRNA encoding the above fusion protein into a dicotyledonous plant cell
  • 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.
  • PEG polyethylene glycol
  • Any Agrobacterium-mediated transformation method can be used.
  • 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.
  • T-DNA containing the above expression unit can be integrated into the genomic DNA of dicotyledonous cells.
  • a nucleic acid construct such as a vector containing the above expression unit can be directly introduced into a dicotyledonous plant cell.
  • 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).
  • a nucleic acid delivery device for example, PDS-1000 (BIO-RAD), etc.
  • metal particles coated with a nucleic acid construct are implanted into such a sample.
  • 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.
  • the fusion protein 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.
  • Cas9 nickase nCas9
  • base substitution 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.
  • DNA conversion base substitution
  • 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.
  • 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.
  • a conventionally known plant tissue culture method including auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.
  • 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.
  • a transgenic plant (T 0 plant) having a mutation (for example, cytosine substitution) at a target site on the genome can be produced.
  • plant cells or plant tissues after introduction of the nucleic acid construct 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.
  • 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.
  • the efficiency of mutagenesis that can be inherited by progenies can be further optimized. Is possible.
  • 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.
  • the sequence of the gene product of the target gene can be changed with high efficiency by DNA conversion in the dicotyledonous genome.
  • 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.
  • mutations such as insertion deletion and DNA conversion can be induced in the genome of dicotyledonous plants to modify the genome.
  • a dicotyledonous plant in which the mutation is introduced into a target site on the genome can be produced with high efficiency.
  • a 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
  • a progeny plant having the mutation can be produced.
  • 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.
  • 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).
  • 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.
  • 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.
  • the introduction treatment condition for example, the culture can be performed at about 23 to 27 ° C. (eg, about 25 ° C.).
  • 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.
  • 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.
  • 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 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.
  • gRNA and its fusion protein into tomato plant cells can be performed by any method.
  • 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.
  • 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.
  • 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.
  • generation of progeny plants by crossing and selection of progeny plants having mutations may be further repeated.
  • 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.
  • 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.
  • 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).
  • pZK_FFnCas9 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).
  • 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.
  • 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.
  • pZK_FFnCas9 D10A
  • D10A pZK_FFnCas9
  • the target sequence of guide RNA was inserted between the AtU6-26 promoter and the chimeric gRNA scaffold in the gRNA expression unit vector pUC19_AtU6oligo by the PCR method.
  • the two endogenous tomato genes SlDELLA gene Solyc11g011260
  • SlETR1 gene Solyc12g011330
  • 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.
  • 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.
  • nCas9 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.
  • 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).
  • 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.
  • T-DNA in each vector prepared as described above is shown in FIG.
  • the AtU6 promoter Arabidopsis U6 promoter; SEQ ID NO: 39
  • the ubiquitin promoter PcUbi promoter
  • the CaMV 35S promoter was used for the expression induction of the NPTII expression unit.
  • SlDELLA 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
  • 560th to 655th are gRNA coding sequences. It is.
  • 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)
  • 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).
  • 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 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
  • 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
  • the 9444th to 10541th are the 3'UTR and terminator of Oshsp17.3.
  • 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).
  • 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
  • the 5138th position 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
  • 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.
  • 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
  • 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
  • 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.
  • SEQ ID NO: 26 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.
  • 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
  • 6070 to 6867 are The NPTII gene, 6874th to 7971st, is the 3 ′ UTR and terminator of Oshsp17.3.
  • 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.
  • 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 ⁇ M acetosyringone and zeatin 1.5 mg / L.
  • 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.
  • regenerated shoots from callus were cut out and rooted on MS solid medium containing kanamycin 50 mg / L to regenerate the plant body.
  • 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.
  • 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 ⁇ molm ⁇ 2 s ⁇ 1 light conditions (light conditions 16 hours / dark conditions 8 hours) .
  • Example 3 Mutation analysis in transgenic plants
  • 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 ⁇ g / 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.
  • 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).
  • nCas9-PmCDA 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.
  • 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).
  • 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.
  • 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).
  • a selection marker gene was contained in the genome of a transgenic plant having a stable DNA modification.
  • kanamycin PCR using a resistance marker gene (NPTII) amplification primer was performed.
  • NPTII resistance marker gene
  • NPTII amplification primer NPTII-F 5'-ATGATTGAACAAGATGGATTGCAC-3 '(SEQ ID NO: 35)
  • NPTII-R 5'-TCAGAAGAACTCGTCAAGAAGGCG-3 '(SEQ ID NO: 36)
  • Actin-F 5'-GATGGATCCTCCAATCCAGACACTGTA'-3 '(SEQ ID NO: 37)
  • Actin-R 5'-GTATTGTGTTGGACTCTGGTGATGGTGT'-3 '(SEQ ID NO: 38)
  • 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.
  • 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.
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'invention concerne un procédé de modification de génome qui permet d'induire une conversion d'ADN pouvant être héritée par la descendance dans un site cible du génome de plante dicotylédone. Le génome d'une plante dicotylédone est modifié avec un procédé impliquant l'introduction, dans des cellules de plante dicotylédone, d'ARN guide et d'une protéine de fusion qui comprend un module de reconnaissance de séquence d'acide nucléique et une enzyme de conversion de nucléobase. Grâce à une grande efficacité de modification de génome, il est possible de produire un corps de plante à partir des cellules de plante à génome modifié, de produire des plantes de descendance à partir du corps de plante, et, à travers la sélection de plantes de descendance qui présentent une variation, de cultiver des plantes de descendance qui présentent la variation.
PCT/JP2018/004062 2017-02-06 2018-02-06 Procédé de modification du génome d'une plante dicotylédone Ceased WO2018143477A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2018566171A JPWO2018143477A1 (ja) 2017-02-06 2018-02-06 双子葉植物のゲノムの改変方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2017-019921 2017-02-06
JP2017019921 2017-02-06

Publications (1)

Publication Number Publication Date
WO2018143477A1 true WO2018143477A1 (fr) 2018-08-09

Family

ID=63040840

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2018/004062 Ceased WO2018143477A1 (fr) 2017-02-06 2018-02-06 Procédé de modification du génome d'une plante dicotylédone

Country Status (2)

Country Link
JP (1) JPWO2018143477A1 (fr)
WO (1) WO2018143477A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110607320A (zh) * 2018-11-23 2019-12-24 电子科技大学 一种植物基因组定向碱基编辑骨架载体及其应用
CN112251464A (zh) * 2020-10-19 2021-01-22 复旦大学附属中山医院 一种基因点突变的诱导方法
WO2022181796A1 (fr) * 2021-02-26 2022-09-01 国立大学法人神戸大学 Procédé semi-rationnel d'ingénierie de l'évolution du génome des plantes
JP2023526035A (ja) * 2020-05-13 2023-06-20 ヌンヘムス ビー．ブイ． 標的突然変異生成によって変異体植物を得るための方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015133554A1 (fr) * 2014-03-05 2015-09-11 国立大学法人神戸大学 Procédé de modification de séquence génomique permettant la conversion de façon spécifique de bases d'acide nucléique de séquences d'adn ciblées et complexe moléculaire destiné à être utilisée dans ce dernier
WO2017090761A1 (fr) * 2015-11-27 2017-06-01 国立大学法人神戸大学 Procédé de conversion de séquence de génome de monocotylédone convertissant spécifiquement une base d'acide nucléique d'une séquence adn ciblée, et complexe moléculaire mettant en œuvre celui-ci

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015133554A1 (fr) * 2014-03-05 2015-09-11 国立大学法人神戸大学 Procédé de modification de séquence génomique permettant la conversion de façon spécifique de bases d'acide nucléique de séquences d'adn ciblées et complexe moléculaire destiné à être utilisée dans ce dernier
WO2017090761A1 (fr) * 2015-11-27 2017-06-01 国立大学法人神戸大学 Procédé de conversion de séquence de génome de monocotylédone convertissant spécifiquement une base d'acide nucléique d'une séquence adn ciblée, et complexe moléculaire mettant en œuvre celui-ci

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BROOKS, C. ET AL.: "Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short palindromic Repeats/CRISPR-Associated9 System", PLANT PHYSIOLOGY, vol. 166, no. 3, 15 September 2014 (2014-09-15), pages 1292 - 1297, XP055529788, Retrieved from the Internet <URL:doi:[10.1104/pp.114.247577]> *
OSAKABE, Y. ET AL.: "Genome Editing with Engineered Nucleases in Plants", PLANT CELL PHYSIOL., vol. 56, 2015, pages 389 - 400, XP055385775 *
PAN, C. ET AL.: "CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations", SCIENTIFIC REPORTS, vol. 6, 21 April 2016 (2016-04-21), pages 24765, XP055396882 *
SHIMATANI, Z. ET AL.: "Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion", NATURE BIOTECHNOLOGY, vol. 35, 27 March 2017 (2017-03-27), pages 441 - 443, XP055529795 *
ZONG, Y. ET AL.: "Precice base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion", NATURE BIOTECHNOLOGY, vol. 35, 27 February 2017 (2017-02-27), pages 438 - 440, XP055482699 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110607320A (zh) * 2018-11-23 2019-12-24 电子科技大学 一种植物基因组定向碱基编辑骨架载体及其应用
CN110607320B (zh) * 2018-11-23 2023-05-12 电子科技大学 一种植物基因组定向碱基编辑骨架载体及其应用
JP2023526035A (ja) * 2020-05-13 2023-06-20 ヌンヘムス ビー．ブイ． 標的突然変異生成によって変異体植物を得るための方法
CN112251464A (zh) * 2020-10-19 2021-01-22 复旦大学附属中山医院 一种基因点突变的诱导方法
CN112251464B (zh) * 2020-10-19 2023-09-12 复旦大学附属中山医院 一种基因点突变的诱导方法
WO2022181796A1 (fr) * 2021-02-26 2022-09-01 国立大学法人神戸大学 Procédé semi-rationnel d'ingénierie de l'évolution du génome des plantes
US20240229050A9 (en) * 2021-02-26 2024-07-11 National University Corporation Kobe University Semi-rational genome evolution engineering method for plants

Also Published As

Publication number Publication date
JPWO2018143477A1 (ja) 2019-04-25

Similar Documents

Publication Publication Date Title
Salonia et al. New plant breeding techniques in citrus for the improvement of important agronomic traits. A Review
Jaganathan et al. CRISPR for crop improvement: an update review
US11447785B2 (en) Method for base editing in plants
CN106164272B (zh) 修饰的植物
Kuluev et al. Delivery of CRISPR/Cas components into higher plant cells for genome editing
US20220348943A1 (en) Methods and compositions for genome editing via haploid induction
JP7679411B2 (ja) バナナの貯蔵寿命を延長するための組成物及び方法
JPWO2017090761A1 (ja) 標的化したｄｎａ配列の核酸塩基を特異的に変換する、単子葉植物のゲノム配列の変換方法、及びそれに用いる分子複合体
JP2022511508A (ja) ゲノム編集による遺伝子サイレンシング
CN114364805A (zh) 生产具有改变的果实发育的植物的方法及由其衍生的植物
KR20230021743A (ko) 이형접합 cenh3 외떡잎식물 및 반수체 유도 및 동시 게놈 편집을 위한 이의 사용 방법
US20180273972A1 (en) Methods of increasing virus resistance in cucumber using genome editing and plants generated thereby
JP7282382B2 (ja) ゲノム編集植物の生産方法
WO2018143477A1 (fr) Procédé de modification du génome d&#39;une plante dicotylédone
CN108026540A (zh) 抗白粉病的小麦植物
Singh et al. Improvement of crop’s stress tolerance by gene editing CRISPR/CAS9 system
CN112805385B (zh) 基于人apobec3a脱氨酶的碱基编辑器及其用途
Tang et al. Applications and roles of the CRISPR system in genome editing of plants
JPWO2020111149A1 (ja) トマトに黄化葉巻様症状を呈するベゴモウイルス属ウイルスに抵抗性のナス科植物、ナス科植物細胞、およびナス科植物の作出方法
JP2018529348A (ja) 半数体及びそれに続く倍加半数体植物の産生方法
JP2019516373A (ja) 半数体及びそれに続く倍加半数体植物の産生方法
CN115942868A (zh) 产量提高的大麻植物
Flaishman et al. Advanced molecular tools for breeding in Mediterranean fruit trees: genome editing approach of Ficus carica L.
WO2022036074A2 (fr) Génération rapide de plantes présentant des caractéristiques souhaitées
Hasley et al. Genome editing of papaya using both Cas9 and Cas12a

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18748417

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2018566171

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18748417

Country of ref document: EP

Kind code of ref document: A1