WO2021079759A1 - Procédé de production d'une cellule végétale éditée par un adn, et kit à utiliser dans celui-ci - Google Patents

Procédé de production d'une cellule végétale éditée par un adn, et kit à utiliser dans celui-ci Download PDF

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WO2021079759A1
WO2021079759A1 PCT/JP2020/038323 JP2020038323W WO2021079759A1 WO 2021079759 A1 WO2021079759 A1 WO 2021079759A1 JP 2020038323 W JP2020038323 W JP 2020038323W WO 2021079759 A1 WO2021079759 A1 WO 2021079759A1
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base sequence
sequence
guide rna
protein
base
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Japanese (ja)
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啓明 雑賀
精一 土岐
秀隆 賀屋
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国立研究開発法人農業・食品産業技術総合研究機構
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Priority to JP2021554273A priority Critical patent/JP7452884B2/ja
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a method for producing a plant cell in which DNA has been edited, and a kit for using the method.
  • Target recombination gene targeting
  • Target recombination is a technique capable of various modifications such as deletion, insertion, and substitution from the base level to the gene level with respect to the target DNA.
  • it is possible to modify the enzyme activity by introducing an amino acid substitution into a region that controls the enzyme activity.
  • the positive / negative selection method using two types of selection markers is general-purpose, and theoretically, the target base sequence is not limited.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2015-). No. 177788, Patent Document 2: Japanese Unexamined Patent Publication No. 2015-171358).
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2015-.
  • Patent Document 2 Japanese Unexamined Patent Publication No. 2015-171358.
  • the efficiency of target recombination generally tends to be low.
  • Target mutation techniques include modified MNs in which the recognition sequence of meganucleases (MNs) that specifically recognize sequences around 20 bases is improved by techniques such as protein engineering, and a combination of existing DNA binding motifs and DNA cleavage motifs.
  • MNs meganucleases
  • the enzymes ZFNs and TALENs, and the CRISPR-Cas9 system applying the prokaryotic acquisition immune system have been developed (Non-Patent Document 1: Kim and Kim (2014) Nature Rev. Genet.).
  • the CRISPR-Cas9 system functions in a variety of species, including plants, and researchers around the world. It is a general-purpose gene modification tool used by.
  • mutations that can be generated by this method are mainly deletions or insertions of a few bases, and many of them result in gene disruption due to frameshifting. Therefore, it is not suitable for high-precision DNA editing that requires base substitution, for example, functional modification of enzyme protein by amino acid substitution or addition of new function.
  • the target base of the target DNA can be replaced by combining the Cas9 protein and the base deamination enzyme, and so far, C to T (G to A) can be used by using cytidine deaminase. ), It has been reported that the substitution from A to G (T to C) can be induced by using adenosine deaminase (Non-Patent Document 2: Kim (2016) Nat. Plants).
  • base substitution introduction techniques other than C to T (G to A) or A to G (T to C) have not yet been established, and the positions of bases that can be substituted are also limited.
  • the base on which the deamination enzyme acts cannot be determined exactly, the Cas9 protein cannot always recognize an arbitrary sequence, etc., so that the base substitution location and the type of substitution that can be introduced, etc. Is limited.
  • Non-Patent Document 3 Halperin et al. (2016) Catalyst.
  • Non-Patent Document 4 Nishizawa-Yokoi et al. (Non-Patent Document 4: Nishizawa-Yokoi et al.). 2016) Plant Physiol.).
  • one of the intracellular genomic loci or Methods of modifying (eg, introducing mutations) multiple allelic genes include methods involving the generation of chimeric splicing RNA molecules, including transcribed exons spliced into a nuclease-interacting RNA segment.
  • chimeric splicing RNA guides a DNA modifying enzyme (eg, a nuclease) to a genomic locus in a cell, resulting in modification of that locus, as said to be CRISPR-related (eg, nuclease).
  • Cas nuclease is mentioned.
  • Patent Document 4 describes the composition and method for modifying the genome at the target site in the genome of filamentous fungal cells in order to modify or modify the target site.
  • a guide polynucleotide / Cas endonuclease system for the purpose is described
  • Patent Document 5 describes a gene modification in a fungal host cell (for example, a filamentous fungal host cell).
  • a fungal host cell for example, a filamentous fungal host cell.
  • Patent Document 6 Japanese Patent Application Laid-Open No. 2017-537647 (Patent Document 6) and Japanese Patent Application Laid-Open No. 2018-504895 (Patent Document 7) are used for genome modification at a target site in the genome of a fungal cell or filamentous fungal cell.
  • Patent Document 7 a guide polynucleotide / Cas endonuclease system for facilitating the insertion of donor DNA at a target site in a fungal host cell or filamentous fungal cell genome has been described.
  • the present invention has been made in view of the above-mentioned problems of the prior art, and is a method for producing a DNA-edited plant cell capable of inserting one base with high frequency and capable of high-precision DNA editing. , And the kits used for it.
  • Cas9 Cert protein of CRISPR-Cas9
  • Cas9 CAstered regularly interspaced short palindromic repeats / CRISPR assisted protein 9
  • NmCas9 protein 1-base insertion can occur more frequently than the conventionally used target mutation using the SpCas9 protein, and T or A tends to be inserted more easily. I found that. No such report has ever been made with other Cas9 proteins.
  • the present inventors insert a plurality of desired bases (for example, 3 bases) into the target DNA region by utilizing the target mutation by the NmCas9 protein that enables high-frequency insertion of 1 base.
  • desired bases for example, 3 bases
  • a method for producing a DNA-edited plant cell which comprises the step of introducing a CRISPR-Cas9 system having a Cas9 protein and its guide RNA as a component into the plant cell, and the Cas9 protein is derived from Neisseria meningitidis. There is a way.
  • a method of inserting n consecutive bases into the DNA of a plant cell which comprises the step of introducing a CRISPR-Cas9 system having a Cas9 protein and its guide RNA as components into the plant cell.
  • the Cas9 protein is derived from Neisseria meningitidis and
  • the guide RNA is a combination of n guide RNAs.
  • n is a natural number of 2 or more
  • the first guide RNA contains a targeting base sequence 1 homologous to the target base sequence 1 into which the base is inserted, and the nth guide RNA has n-1 in the target base sequence 1. It contains a targeting base sequence n that is homologous to the target base sequence n into which the base is inserted.
  • Method. It is a method of substituting a base in the DNA of a plant cell, and in the plant cell, a Cas9 protein derived from Neisseria meningitidis and a CRISPR-Cas9 system having a guide RNA thereof as a constituent, and a Cas9 protein other than the Cas9 protein derived from Neisseria meningitidis are added.
  • the guide RNA of the Cas9 protein derived from Neisseria meningitidis contains a targeting base sequence 1 homologous to the target base sequence 1 into which the base is inserted, and the guide RNA of the other Cas9 protein is the target base. It contains a targeting base sequence 2 that is homologous to the target base sequence 2 in which one base is inserted into the sequence 1.
  • the guide RNA is a combination of n guide RNAs. n is a natural number of 2 or more,
  • the first guide RNA contains a targeting base sequence 1 homologous to the target base sequence 1 into which the base is inserted, and the nth guide RNA has n-1 in the target base sequence 1.
  • the kit contains a targeting base sequence n that is homologous to the target base sequence n into which the base is inserted.
  • the kit according to [5]. [7] The following (C) and (D): (C) Cas9 protein other than Cas9 protein derived from Neisseria meningitidis, a polynucleotide encoding the protein, or a vector expressing the polynucleotide, (D) A guide RNA of the other Cas9 protein, a polynucleotide encoding the guide RNA, or a vector expressing the polynucleotide.
  • the guide RNA of the Cas9 protein derived from Neisseria meningitidis contains a targeting base sequence 1 homologous to the target base sequence 1 into which the base is inserted, and the guide RNA of the other Cas9 protein is the target base. It contains a targeting base sequence 2 that is homologous to the target base sequence 2 in which one base is inserted into the sequence 1.
  • a CRISPR-Cas9 system which is particularly prone to single base deletion, is used in combination to delete one base existing in the target base sequence after inserting one base into the target base sequence. , It is also possible to replace the base efficiently.
  • the present invention comprises the step of introducing a CRISPR-Cas9 system having a Cas9 protein and its guide RNA as a component into a plant cell, and a DNA-edited plant cell in which the Cas9 protein is derived from Neisseria meningitidis.
  • a method for producing hereinafter, in some cases, referred to as "DNA-edited plant cell manufacturing method").
  • Plant cells examples of “plant cells” that edit DNA in the present invention, that is, introduce the CRISPR-Cas9 system, include cells of cereals, oil crops, forage crops, fruits, and vegetables.
  • the "plant cell” includes, for example, cells constituting an individual plant, cells constituting an organ or tissue separated from a plant, and cultured cells derived from a plant tissue.
  • plant organs and tissues include leaves, stems, shoot apex (growth point), roots, tubers, tubers, seeds and callus.
  • Examples of plants include rice, barley, wheat, rye, barnyard millet, sorghum, corn, banana, peanut, sunflower, tomato, abrana, tobacco, potato, soybean, cotton and carnation.
  • grasses such as rice, barley, wheat, rye, barnyard grass, sorghum, and corn are preferable, and rice is particularly preferable.
  • the DNA (target DNA) edited by the present invention is not particularly limited as long as it contains the PAM sequence of the following NmCas9 protein. As will be described later, it is also possible to modify the recognition specificity of PAM according to the base sequence of the target DNA by modifying the Cas9 protein (for example, introducing a mutation).
  • the CRISPR-Cas9 system contains at least Cas9 protein and its guide RNA as components.
  • the guide RNA is added to a complementary sequence (a sequence on the antisense strand of the sense strand) of the target base sequence (for example, a sequence on the gene to be deleted and on the strand (sense strand) containing the PAM sequence). It consists of crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) having a complementary base sequence (that is, a sequence homologous to the target base sequence).
  • Cas9 protein and its guide RNA are introduced into cells so that the guide RNA binds to the complementary sequence of the target base sequence of genomic DNA and forms a complex with the guide RNA. Induces cleavage of the target DNA by the protein. In the cleaved target DNA, deletion / insertion of a base in the base sequence is caused in the repair process, and as a result, the gene can be edited.
  • NmCas9 protein In the present invention, as the CRISPR-Cas9 system, at least a Cas9 protein derived from Neisseria meningitidis is used.
  • the Cas9 protein derived from Neisseria meningitidis is referred to as "NmCas9 protein”.
  • the typical amino acid sequence of the "NmCas9 protein" used in the present invention is shown in SEQ ID NO: 1, and the base sequence of the DNA encoding the protein is shown in SEQ ID NO: 2.
  • Table 1 shows the size (number of amino acids) of the wild-type NmCas9 protein (NmCas9), the PAM (proto-spacer adaptive motif) sequence, and the DNA cleavage end (DNA end).
  • the PAM sequence of a typical NmCas9 protein is "5'-NNNNGATT".
  • Cas9 protein (SpCas9) derived from Streptococcus pyogenes, Cpf1 protein (FnCpf1) derived from Francisella novicida, Cas9 protein derived from Staphylococcus aureus (Staphylococcus aureus)
  • CjCas9 protein For the derived Cas9 protein (CjCas9), typical size (number of amino acids), PAM sequence, DNA cleavage end (DNA end), and reference reference name in some cases are also shown.
  • the "NmCas9 protein” according to the present invention is a homologue, a mutant, or a variant of the above-mentioned typical NmCas9 protein as long as it has an activity (nuclease activity) of forming a complex with a guide RNA and cleaving the target DNA. It may be a partial peptide.
  • the homologs include, for example, the typical amino acid sequence of the NmCas9 protein (eg, the amino acid sequence of SEQ ID NO: 1) and 85% or more, preferably 90% or more, more preferably 95% or more (eg, 96% or more).
  • a protein consisting of an amino acid sequence having 97% or more, 98% or more, 99% or more) identity is included. Sequence identity can be evaluated numerically as calculated using BLAST or the like (eg, default or default parameters).
  • NmCas9 protein As a variant, from an amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted into a typical amino acid sequence of NmCas9 protein (for example, the amino acid sequence of SEQ ID NO: 1). It contains proteins that have the activity of forming a complex with the guide RNA and cleaving the target DNA.
  • “plurality” means, for example, 2 to 150 pieces, preferably 2 to 100 pieces, more preferably 2 to 50 pieces (for example, 2 to 30 pieces, 2 to 10 pieces, 2 to 5 pieces, 2). ⁇ 3 pieces, 2 pieces).
  • variants include the NmCas9 protein, which has modified the recognition specificity of PAM by introducing a mutation into a specific amino acid residue.
  • the NmCas9 protein according to the present invention is preferably one to which a nuclear localization signal is added. This promotes localization to the nucleus in the cell, resulting in efficient DNA editing.
  • the "guide RNA” is a combination of crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA).
  • the guide RNA of the NmCas9 protein according to the present invention has a base sequence complementary to the base sequence of the target DNA region (complementary sequence of the target base sequence) (hereinafter, sometimes referred to as “targeted base sequence”) in crRNA. Including. That is, the guide RNA of the NmCas9 protein according to the present invention contains a targeting base sequence that is homologous to the target base sequence.
  • the term "homologous" with respect to the first base sequence means that the second base sequence is complementary to the complementary sequence of the first base sequence.
  • the base sequences are in the same relationship with each other, and either the first base sequence or the second base sequence
  • one is DNA and the other is RNA it means that their base sequences are in the same relationship with each other except that the base which is timine (T) in DNA is uracil (U) in RNA.
  • the "target DNA region” means a region on the target DNA of a plant cell that includes a target base sequence and a site that causes a target gene modification (insertion / substitution of a base).
  • the target DNA region is adjacent to the PAM sequence on its 3'side.
  • the targeting base sequence in crRNA is usually a base sequence consisting of 12 to 50 bases, preferably 17 to 30 bases, and more preferably 17 to 25 bases.
  • the crRNA further contains a base sequence capable of interacting (hybridizing) with tracrRNA on the 3'side.
  • tracrRNA contains a base sequence capable of interacting (hybridizing) with a part of the base sequence of crRNA on the 5'side
  • the guide RNA interacts with the NmCas9 protein by the interaction of these base sequences. Form double-stranded RNA. Therefore, the guide RNA of the NmCas9 protein according to the present invention binds to the complementary sequence of the target base sequence of the target DNA region and forms a complex with the NmCas9 protein to induce the NmCas9 protein into the target DNA region.
  • the induced NmCas9 protein cleaves the target DNA by its endonuclease activity.
  • the site that causes the target gene modification does not have to be contained in the target base sequence, but may be contained in the target base sequence. preferable.
  • cleavage of the target DNA is due to both the complementarity of base pairing between the guide RNA and the complementary sequence of the target base sequence and the PAM sequence present on the 3'side of the target base sequence. Occurs at the determined position.
  • the NmCas9 protein generally cleaves between the 3rd and 4th bases upstream of the PAM sequence in the target DNA region.
  • the guide RNA of the CRISPR-Cas9 system of the present invention may be a single-molecule guide RNA (sgRNA) containing crRNA and tracrRNA, or a two-molecule guideRNA composed of a crRNA fragment and a tracrRNA fragment.
  • sgRNA single-molecule guide RNA
  • the Cas9 protein is edited by DNA editing of plant cells.
  • NmCas9 protein as a syrup, one base insertion occurs at a surprisingly higher frequency than before.
  • the frequency of mutations other than single nucleotide insertion is about 1.1 to 25%, which is the same as the SpCas9 protein conventionally used as the Cas9 protein of the CRISPR-Cas9 system.
  • the frequency of single base insertion could be significantly increased.
  • the frequency of mutations other than single-base insertion of T or A decreased to 1 / 3.4 to 1 / 2.3, and the frequency of single-base insertion of T or A could be particularly high. (Examples 1 and 2, Comparative Examples 1 and 2).
  • the Cas9 protein (NmCas9 protein and, if necessary, other Cas9 proteins) encodes the protein even in the form of the protein. It may be in the form of RNA or DNA (polynucleotide) or in the form of a vector (expression vector) expressing the polynucleotide. Independently of this, whether the guide RNA is in the form of RNA or the form of DNA (polynucleotide) encoding the RNA, the form of the vector (expression vector) expressing the polynucleotide is used. It may be.
  • a vector expressing Cas9 protein and a vector expressing its guide RNA may be introduced into cells, respectively, or both Cas9 protein and its guide RNA may be introduced.
  • the vector to be expressed may be introduced into cells.
  • these guide RNAs may be loaded on the same expression vector or on different expression vectors.
  • the NmCas9 protein is combined with another Cas9 protein, the NmCas9 protein and the other Cas9 protein may be loaded on the same expression vector or on different expression vectors.
  • the polynucleotide encoding the Cas9 protein may be, for example, an appropriately codon-optimized one for plant cells.
  • the expression vector when adopting the form of an expression vector, preferably contains one or more regulatory elements that are operably linked to the polynucleotide to be expressed.
  • operably bound means that the polynucleotide is expressively bound to the regulatory element.
  • regulatory elements include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, transcription termination signals (polyadenylation signals, polyU sequences, etc.)).
  • the expression vector is one that can stably express the encoding protein without being integrated into the host genome.
  • the CRISPR-Cas9 system is introduced into plant cells by appropriately selecting a known method such as an Agrobacterium method, a particle gun method, an electroporation method, a method using a cell membrane penetrating peptide, or a plasma method. be able to.
  • the method for producing DNA-edited plant cells of the present invention by introducing the CRISPR-Cas9 system containing the NmCas9 protein as a component into plant cells, insertion of one base occurs with high frequency, and therefore, one base is frequently inserted into the above-mentioned target DNA region. Edited plant cells into which one base is inserted can be obtained. Further, in the method for producing a DNA-edited plant cell of the present invention, a plant in which DNA has been edited can be produced by regenerating a plant from a plant cell into which the CRISPR-Cas9 system has been introduced.
  • a method for obtaining an individual by redifferentiating a plant tissue by tissue culture a method established in the present technical field can be used (for example, transformation protocol [plant edition] Yutaka Tabei, ed. Kagaku-Dojin pp. 340-347 (2012)). Once a plant is obtained in this way, it is possible to obtain offspring from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain breeding materials (for example, seeds, fruits, cut ears, strains, curls, protoplasts) from the plants and their progeny or clones, and mass-produce the plants based on them.
  • breeding materials for example, seeds, fruits, cut ears, strains, curls, protoplasts
  • one base can be inserted into the target base sequence of a plant cell more frequently than before by the CRISPR-Cas9 system using NmCas9 protein. Therefore, in the CRISPR-Cas9 system according to the present invention, it is possible to insert a plurality of consecutive bases into the target base sequence of a plant cell by using a combination of a plurality of guide RNAs.
  • the present invention includes a step of introducing a CRISPR-Cas9 system having a Cas9 protein and its guide RNA as a constituent element into a plant cell as a method utilizing a DNA-edited plant cell production method.
  • the Cas9 protein is derived from Neisseria meningitidis and
  • the guide RNA is a combination of n guide RNAs. n is a natural number of 2 or more,
  • the first guide RNA contains a targeting base sequence 1 homologous to the target base sequence 1 into which the base is inserted, and the nth guide RNA has n-1 in the target base sequence 1. It contains a targeting base sequence n that is homologous to the target base sequence n into which the base is inserted.
  • a method of inserting n consecutive bases into the DNA of a plant cell hereinafter, in some cases, referred to as a "continuous base insertion method").
  • the introduction of the CRISPR-Cas9 system into plant cells, the NmCas9 protein, its guide RNA, and the CRISPR-Cas9 system, and the CRISPR-Cas9 system into the plant cells each independently includes preferred embodiments thereof. As described in the method for producing DNA-edited plant cells.
  • n guide RNAs is used as a guide RNA for the NmCas9 protein.
  • n is a natural number of 2 or more, and is a multiple of 3 when base insertion is performed in codon units corresponding to amino acids.
  • FIG. 1 shows a schematic view of an example of the aspect of the continuous base insertion method of the present invention.
  • guide RNAs sgRNA1, sgRNA2, sgRNA3 of three NmCas9 proteins are introduced into plant cells by the same expression vector ((a) in FIG. 1) as the NmCas9 protein (NmCas9). To do.
  • the first guide RNA is homologous to the target base sequence 1 (target sequence) into which the base is inserted (that is, complementary to the complementary sequence of the target base sequence 1).
  • targets base sequence 1 that is, in the continuous base insertion method of the present invention, the targeted base sequence 1 of the first guide RNA is a base sequence (target base sequence 1) containing a target site for inserting a base (that is, a cleavage site of the target DNA). It is a base sequence homologous to.
  • the cleavage site of the target base sequence 1 by the NmCas9 protein Since one base is inserted at a high frequency, the target base sequence 2 in which one base is inserted into the target base sequence 1 can be obtained.
  • the gap between the 3rd and 4th bases upstream of the PAM sequence on the target DNA (SEQ ID NO: 7, 1st stage of FIG. 1 (b)) is cleaved, and the cleavage site is cleaved.
  • T is inserted between the 3rd to 4th bases of the target base sequence 1 (SEQ ID NO:: SEQ ID NO:: 8) can be obtained (second stage of (b) in FIG. 1).
  • the second guide RNA contains a target base sequence 2 homologous to the target base sequence 2 in which one base is inserted into the target base sequence 1. Therefore, when the above target base sequence 2 is obtained by the first guide RNA, the second guide RNA (sgRNA2) binds to the complementary sequence of the target base sequence 2 and forms a complex with the NmCas9 protein. By doing so, the NmCas9 protein is induced to the target base sequence 2, and the induced NmCas9 protein cleaves the target base sequence 2 by its endonuclease activity, and one base is frequently inserted into the cleavage site.
  • sgRNA2 binds to the complementary sequence of the target base sequence 2 and forms a complex with the NmCas9 protein.
  • the target base sequence 3 in which one base is inserted in the target base sequence 2 (that is, the target base sequence 3 in which two bases are inserted in the target base sequence 1) can be obtained.
  • the gap between the 3rd and 4th bases upstream of the PAM sequence is cleaved, and 1 base, particularly T or A, is frequently inserted into the cleaved portion.
  • the target base sequence 3 (SEQ ID NO: 9) in which A is inserted between the 3rd and 4th bases from the end can be obtained (the third stage of (b) in FIG. 1).
  • the third guide RNA contains a target base sequence 3 homologous to the target base sequence 3 in which one base is inserted into the target base sequence 2. Therefore, when the above target base sequence 3 is obtained by the second guide RNA, the third guide RNA (sgRNA3) binds to the complementary sequence of the target base sequence 3 and forms a complex with the NmCas9 protein. By doing so, the NmCas9 protein is induced to the target base sequence 3, and the induced NmCas9 protein cleaves the target base sequence 3 by its endonuclease activity, and one base is frequently inserted into the cleavage site.
  • sgRNA3 binds to the complementary sequence of the target base sequence 3 and forms a complex with the NmCas9 protein.
  • a base sequence in which one base is inserted in the target base sequence 3 (that is, a base sequence in which three bases are inserted in the target base sequence 1) can be obtained.
  • the gap between the 3rd and 4th bases upstream of the PAM sequence is cleaved, and 1 base, particularly T or A, is frequently inserted into the cleaved portion, so that 3 of the target base sequence 3 'It is possible to obtain a base sequence (SEQ ID NO: 10) in which A is inserted between the 3rd and 4th bases from the end, and as a result, TAA, which is a stop codon, is efficiently inserted into the target DNA. Can be done (fourth row in (b) of FIG. 1).
  • n guide RNAs is used as the guide RNA of the NmCas9 protein, and the nth guide RNA has n-1 bases in the target base sequence 1.
  • the targeting base sequence n that is homologous to the inserted target base sequence n (that is, complementary to the complementary sequence of the target base sequence n)
  • the above-mentioned one-base insertion can be continued n times. , It becomes possible to insert n consecutive bases into the target DNA region of a plant cell.
  • the frequency of inserting one base of T or A is high, so that TAA, which is a stop codon, can be easily inserted from beginning to end. It becomes.
  • n 3 and all three guide RNAs are introduced into plant cells as sgRNA by the same expression vector as NmCas9 protein ((a) in FIG. 1).
  • the mode of the continuous base insertion method is not limited to this, and for example, n guide RNAs may be sequentially introduced into plant cells separately, and cloning is performed between the introductions at this time. It may include steps and the like.
  • the target base sequence n in which one base is frequently inserted into the target base sequence 1 and n-1 bases (particularly T or A) are inserted frequently is used. Therefore, by simultaneously introducing n guide RNAs designed in accordance with this into plant cells, it is possible to easily insert n consecutive bases with a single introduction operation. ..
  • the DNA-edited plant cell production method of the present invention may be combined with other CRISPR-Cas9 systems.
  • the CRISPR-Cas9 system using the NmCas9 protein according to the present invention may be used in combination with the CRISPR-Cas9 system using other Cas9 proteins (for example, Cas9 proteins derived from other bacteria). Good.
  • the present invention replaces the base of the DNA of a plant cell by using another CRISPR-Cas9 system in combination with the CRISPR-Cas9 system using the NmCas9 protein as a method utilizing the DNA-edited plant cell production method.
  • Method for example Including a step of introducing a CRISPR-Cas9 system having NmCas9 protein and its guide RNA as a component and a CRISPR-Cas9 system having another Cas9 protein other than NmCas9 protein and its guide RNA as a component into plant cells.
  • the guide RNA of the NmCas9 protein contains a targeting base sequence 1 homologous to the target base sequence 1 into which the base is inserted, and one guide RNA of the other Cas9 protein is included in the target base sequence 1.
  • a method for substituting a base in DNA of a plant cell (hereinafter, in some cases, referred to as a “base replacement method”) is also provided.
  • the CRISPR-Cas9 system whose constituents are Cas9 proteins other than the NmCas9 protein and its guide RNA, it is preferable that the CRISPR-Cas9 system is prone to single-base deletion, and the configuration of such a CRISPR-Cas9 system is preferable.
  • Examples of other Cas9 proteins as elements include, for example, SpCas9 protein and SaCas9 protein shown in Table 1 above, and SpCas9 protein is preferable, but the present invention is not limited thereto.
  • the CRISPR-Cas9 system having plant cells, NmCas9 protein and its guide RNA as components, and its introduction into plant cells are independently, including their preferred embodiments. As described in the method for producing DNA-edited plant cells.
  • the CRISPR-Cas9 system containing the other Cas9 protein and its guide RNA as components, and the CRISPR using the NmCas9 protein according to the present invention as its introduction into plant cells.
  • the above-mentioned typical NmCas9 protein is replaced with the above-mentioned other Cas9 protein, for example, a Cas9 protein derived from Streptococcus pyogenes whose typical amino acid sequence is shown by UniProtKB / Swiss-Prot accession number: Q99ZW2.1.
  • FIG. 2 shows a schematic view of an example of the aspect of the base substitution method of the present invention.
  • NmCas9 protein NmCas9 protein and its guide RNA
  • SpCas9 protein SpCas9 protein
  • sgRNA for SpCas9 are expressed in each expression vector (FIG. 2).
  • Each is introduced into a plant cell according to (a)).
  • the guide RNA of the NmCas9 protein is a target homologous to the target base sequence 1 (target sequence) into which the base is inserted (that is, complementary to the complementary sequence of the target base sequence 1).
  • the frequency of cleavage by the NmCas9 protein in the target base sequence 1 is high. Since one base is inserted in, the target base sequence 2 in which one base is inserted into the target base sequence 1 can be obtained.
  • the gap between the 3rd and 4th bases upstream of the PAM sequence of the NmCas9 protein on the target DNA (SEQ ID NO: 11, the first stage of FIG. 2B) is cleaved.
  • the guide RNA of another Cas9 protein (SpCas9 protein in FIG. 2) is homologous to the target base sequence 2 in which one base is inserted into the target base sequence 1 (that is, that is). It contains a targeted base sequence 2 that is complementary to the complementary sequence of the target base sequence 2. Therefore, when the above-mentioned target base sequence 2 is obtained by the NmCas9 protein and its guide RNA, the guide RNA of the SpCas9 protein binds to the complementary sequence of the target base sequence 2 and forms a complex with the SpCas9 protein.
  • the SpCas9 protein is induced into the target base sequence 2, and the induced SpCas9 protein cleaves the target base sequence 2 by its endonuclease activity, and one base is deleted at the cleavage site with a relatively high frequency.
  • a base sequence in which one base is deleted from the target base sequence 2 (that is, a base sequence in which one base of the target base sequence 1 is substituted) can be obtained.
  • the upstream 3rd and 4th bases of the PAM sequence of the SpCas9 protein are cleaved, and 1 base is frequently deleted at the cleavage site, so that 3'of the target base sequence 2'.
  • a base sequence (SEQ ID NO: 13) in which C at the third base from the end is deleted can be obtained, and as a result, the base at the third base from the 3'end of the target base sequence 1 is replaced with C from A. (Third stage of (b) in FIG. 2).
  • the base substitution method of the present invention by combining the CRISPR-Cas9 system using the NmCas9 protein with the CRISPR-Cas9 system, which is particularly prone to single nucleotide deletion, after insertion of one base into the target base sequence. , One base existing in the same target base sequence can be deleted, and the base can be replaced efficiently.
  • the CRISPR-Cas9 system using the NmCas9 protein according to the present invention is introduced into a plant cell, the frequency of inserting one base of T or A is high, and therefore, after inserting one base of T or A into the target base sequence.
  • By deleting one base existing in the same target base sequence it is possible to efficiently replace the base from A / C / G to T or from C / G / T to A.
  • each guide RNA is introduced into a plant cell as an sgRNA by the same expression vector as each Cas9 protein ((a) in FIG. 2), but the embodiment of the base substitution method of the present invention. Is not limited to this, for example, even if they are all introduced into a plant cell by the same expression vector, the NmCas9 protein and its guide RNA are introduced into the plant cell, and then the SpCas9 protein and its guide RNA are introduced. It may be introduced into a plant cell, and a cloning step or the like may be included between the introduction at this time.
  • the target base sequence 2 in which one base (particularly T or A) is inserted with high frequency with respect to the target base sequence 1 can be obtained, these are simultaneously introduced into plant cells. As a result, it is possible to easily replace the base with a single introduction operation.
  • the present invention also provides a kit for use in the method of the present invention.
  • the kit of the present invention comprises (A) NmCas9 protein, a polynucleotide encoding the protein, or a vector expressing the polynucleotide, and (B) a guide RNA of the NmCas9 protein, a polynucleotide encoding the guide RNA, or the said.
  • the vectors (A) and (B) may be different vectors from each other or may be the same vector.
  • n guide RNAs For the purpose of inserting n consecutive bases into the DNA of plant cells, a combination of n guide RNAs is used as a guide RNA for the NmCas9 protein.
  • n is a natural number of 2 or more
  • the first guide RNA contains a targeting base sequence 1 homologous to the target base sequence 1 into which the base is inserted, and is the nth guide RNA.
  • kits of the present invention express Cas9 proteins other than (C) NmCas9 proteins (eg, SpCas9 proteins), polynucleotides encoding the proteins, or the polynucleotides. It is preferable to further include (D) a guide RNA of the other Cas9 protein (for example, SpCas9 protein), a polynucleotide encoding the guide RNA, or a vector expressing the polynucleotide.
  • C NmCas9 proteins
  • D a guide RNA of the other Cas9 protein
  • a polynucleotide encoding the guide RNA for example, SpCas9 protein
  • a vector expressing the polynucleotide for example, a vector expressing the polynucleotide.
  • the guide RNA of the NmCas9 protein contains a targeting base sequence 1 that is homologous to the target base sequence 1 into which the base is inserted, and the guide RNA of the other Cas9 protein is in the target base sequence 1. It contains a targeting base sequence 2 that is homologous to the target base sequence 2 in which one base is inserted.
  • the vectors (C) and (D) may be different vectors from each other or may be the same vector. Further, the vectors (C) and / or (D) may be the same vectors as the vectors (A) and / or (B).
  • the kit of the present invention may further include one or more additional reagents.
  • additional reagents include, but are limited to, for example, dilution buffers, reconstruction solutions, wash buffers, nucleic acid transfer reagents, protein transfer reagents, control reagents (eg, control guide RNAs). It's not a thing.
  • the kit may further include an instruction manual for carrying out the method of the present invention.
  • Example 1 [Plasid construction] First, the DNA (SEQ ID NO:) of the NmCas9 protein gene optimized for the codon of Shiroinu clawa by GeneArt Gene Synthesis (manufactured by Thermo Fisher Scientific) and added with a FLAG tag and a nuclear localization signal derived from Simian Virus40 (SV40) on the N-terminal side. : The base sequence shown in SEQ ID NO: 4, which encodes the amino acid sequence shown in 3, was synthesized. Next, an expression vector for the NmCas9 protein (NmCas9) was constructed using the binary vector pRI-SpCas9 (Kaya et al. (2016) Sci. Rep. 6: 26871) as the backbone.
  • the canamycin resistance gene expression cassette (promoter of NOS gene of agrobacterium and neomycin phosphotransferase gene) in pRI-SpCas9 is used as a hyglomycin resistance gene expression cassette (35S promoter of cauliflower mosaic virus and hyglomycin phosphotransferase gene).
  • the pRI-PcUBI-pro :: SpCas9 vector was cleaved with SmaI and SacI, and the DNA of the SpCas9 protein gene in the vector was replaced with the DNA of the NmCas9 protein gene synthesized above to construct an NmCas9 expression vector.
  • a rice endogenous gene sequence (SEQ ID NO:) containing both a PAM sequence of NmCas9 protein (5'-NNNGATT) and a PAM sequence of SpCas9 protein (5'-NGG) by GeneArt Gene Synthesis (manufactured by Thermo Fisher Scientific Co., Ltd.) : PAM sequence of NmCas9 protein in the sequence containing the nucleotide sequence according to 5 and the gene ID (“RAP-DB”, https: //rapdb.dna.affrc.go.jp/): Os01g0899200 (rice target DNA_A)).
  • a guide RNA (sgRNA) containing a base sequence (targeted base sequence) homologous to the target base sequence A (target sequence A corresponding to sgRNA of NmCas9) of 22 nucleotides upstream of the above is synthesized, and pOsU6-sgRNA_SaCas9 (Kaya et al.) (2016) Sci. Rep. 6: 26871) was replaced with the guide RNA of the SaCas9 protein to construct a guide RNA expression cassette.
  • the guide RNA expression cassette was then cleaved with BbsI and cloned. Using PacI and AscI, the cloned guide RNA expression cassette was incorporated into the above NmCas9 expression vector to construct an sgRNA-NmCas9 expression vector.
  • the callus was washed with a 25 mg / L meropenem (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) solution, and a medium containing 50 mg / L hygromycin B and 25 mg / L meropenem. Incubated for 4-5 weeks.
  • a 25 mg / L meropenem manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.
  • a medium containing 50 mg / L hygromycin B and 25 mg / L meropenem Incubated for 4-5 weeks.
  • Genomic DNA was extracted from 24 samples of rice cults cultured for 4 to 5 weeks after transformation by a simple DNA extraction method (Kasazima et al. (2004) Plant Mol. Biol. Rep. 22: 49-52).
  • KOD-FX neo DNA polymerase manufactured by Toyobo Co., Ltd.
  • the target nucleotide sequence was amplified by the PCR method.
  • the obtained PCR product was cleaved overnight by restriction enzyme treatment with SacI, and using the MCE-202 MultiNA with a DNA-500 kit (manufactured by Shimadzu Corporation), the CAPS (Cleared Applied Polymorphic Sequences) method was used. It was investigated whether a mutation was introduced into the target base sequence.
  • sgRNA-SpCas9 expression vector was constructed according to the method of Kaya et al. (Kaya et al. (2016) Sci. Rep. 6: 26871).
  • the targeting base sequence was a sequence containing a base sequence homologous to the target base sequence A (target sequenceA correspending to sgRNA of SpCas9) of 20 nucleotides upstream of the PAM sequence of the SpCas9 protein in the rice target DNA_A. ..
  • transformation to rice carrus was performed in the same manner as in Example 1 except that the sgRNA-SpCas9 expression vector was used, and CAPS analysis and sequence analysis were performed on 24 samples.
  • the target base sequence of guide RNA is a rice endogenous gene sequence containing the PAM sequence of NmCas9 protein and the PAM sequence of SpCas9 protein (sequence including the base sequence shown in SEQ ID NO: 6), gene ID ("RAP-DB").
  • Https //rapdb.dna.affrc.go.jp/
  • Target base sequence B target sequenceB correspring
  • An sgRNA-NmCas9 expression vector was constructed in the same manner as in Example 1 except that the nucleotide sequence was homologous to sgRNA of NmCas9).
  • transformation to rice carrus was performed in the same manner as in Example 1 except that the sgRNA-NmCas9 expression vector was used, and CAPS analysis and sequence analysis were performed on 24 samples.
  • CAPS analysis XhoI was used instead of SacI to cleave the rice target DNA_B.
  • the target base sequence of the guide RNA was set to be a base sequence homologous to the target base sequence B (target sequenceB correspending to sgRNA of SpCas9) of 20 nucleotides upstream of the PAM sequence of the SpCas9 protein in the rice target DNA_B.
  • An sgRNA-SpCas9 expression vector was constructed in the same manner as in Comparative Example 1 except for the above.
  • transformation to rice carrus was performed in the same manner as in Example 2 except that the sgRNA-SpCas9 expression vector was used, and CAPS analysis and sequence analysis were performed on 24 samples.
  • FIGS. 3 to 4 show a part of the sequence of the rice target DNA_A (WT of FIG. 3, SEQ ID NO: 5) or a part of the rice target DNA_B (WT of FIG. 4, SEQ ID NO: 6), respectively.
  • the sequence in which the base is inserted / deleted (indel) is shown in the sequence of the WT, and the frequency of the insertion / deletion of the base (number of samples in which the mutation occurred * 100 / total number of samples). (%)) are shown respectively.
  • FIGS. 3 to 4 show a part of the sequence of the rice target DNA_A (WT of FIG. 3, SEQ ID NO: 5) or a part of the rice target DNA_B (WT of FIG. 4, SEQ ID NO: 6), respectively.
  • the sequence in which the base is inserted / deleted (indel) is shown in the sequence of the WT, and the frequency of the insertion / deletion of the base (number of samples in which the mutation occurred * 100 / total number of samples). (%))
  • the base sequences shown in bold are the respective target base sequences (target searchA corresponding to sgRNA of NmCas9, targetsequenceA corresponding to sgRNA of SpCasceptNest To sgRNA of SpCas9 (target sequence)) is shown, the base sequence shown by the underline shows the PAM sequence of each Cas9 protein (NmCas9 protein or SpCas9 protein), and the base shown in lower case indicates the inserted base. ..
  • the target base sequence of the guide RNA is the rice endogenous gene sequence containing the PAM sequence of the NmCas9 protein (sequence containing the base sequence set forth in SEQ ID NO: 7, gene ID (“RAP-DB”, https: // rapdb.dna.affrc.go.jp/): Intron (rice target DNA_C) of Os12g0224000), 22 nucleotides upstream of the PAM sequence of the NmCas9 protein target base sequence 1 (target sequence1 corresponding to sgRNA1 ofN A guide RNA1 (sgRNA1) expression cassette was constructed in the same manner as in Example 1 except that the nucleotide sequences were homologous.
  • the target base sequence of the guide RNA is the target base sequence 2 (SEQ ID NO:) in which T is inserted between the 3'end to the 3rd base and the 4th base of the 2 to 22 nucleotide sequence of the target base sequence 1.
  • a guide RNA2 (sgRNA2) expression cassette was constructed in the same manner as in Example 1 except that the base sequence was homologous to the base sequence described in: 8.
  • the target base sequence of the guide RNA is the target base sequence 3 (SEQ ID NO:) in which A is inserted between the 3'end to the 3rd base and the 4th base of the 2 to 22 nucleotide sequence of the target base sequence 2.
  • a guide RNA3 (sgRNA3) expression cassette was constructed in the same manner as in Example 1 except that the base sequence was homologous to the base sequence described in: 9.
  • an sgRNA1-sgRNA2-sgRNA3-NmCas9 expression vector was constructed in the same manner as in Example 1 except that all of these were used.
  • transformation to rice carrus was performed in the same manner as in Example 1 except that the above-mentioned sgRNA1-sgRNA2-sgRNA3-NmCas9 expression vector was used, and CAPS analysis and sequence analysis were performed on 344 samples.
  • CAPS analysis HpaI was used instead of SacI to cleave the rice target DNA_C.
  • Table 2 shows the frequency of base insertion / deletion (indel) in the sequence of rice target DNA_C (WT, no mutation) (number of samples with the mutation * 100 / total number of samples (%)). Shown. As shown in Table 2, the frequency of introduction of 3 bases was 26.2% in total, and the frequency of introduction of the target 3 bases (TAA or TAT) was 88.9%. It was expensive. Therefore, by using the NmCas9 protein in combination with three types of guide RNAs in which the target base sequence is shifted by one base, three bases can be introduced with high accuracy by the system shown in FIG. 1, and a stop codon can be introduced. Was confirmed.
  • NmCas9 expression vector sgRNA-NmCas9 expression vector constructed in Example 1 [sgRNA for NmCas9 (targeted base sequence: target base sequence A in rice target DNA_A (target base sequence 1 in FIG. 2)) homologous base sequence ) + NmCas9] was used.
  • -SpCas9 expression vector First, using pZH_MMCas9 (Mikami et al. (2015) Plant Mol. Biol. 88-561) as a backbone, SpCas9 is expressed by the promoter of the corn ubiquitin gene, and canamycin is used as an antibiotic marker for plant selection. A binary vector having a resistance gene expression cassette (cauliflower mosaic virus 35S promoter and neomycin phosphotransferase gene) was constructed.
  • the target base sequence of the guide RNA is 20 nucleotides in which A is inserted between the 3'end to the 4th base of 19 nucleotides upstream of the PAM sequence of SpCas9 protein in the rice target DNA_A.
  • a guide RNA (sgRNA for SpCas9) expression cassette was constructed in the same manner as in Example 1 except that the base sequence was homologous to the target base sequence 2 (base sequence shown in SEQ ID NO: 12).
  • the guide RNA expression cassette was amplified by PCR using an F-primer (base sequence shown in SEQ ID NO: 14) and an R-primer (base sequence shown in SEQ ID NO: 15). After cleaving the binary vector constructed above with AscI, the PCR product of the guide RNA expression cassette was cloned using In-Fusion HD Cloning Kit (Clontech) to construct a SpCas9 expression vector.
  • Table 3 below shows the results of sequence analysis for strains in which base substitution was confirmed by direct sequence analysis.
  • the frequency of insertion of A or C base into the sequence of rice target DNA_A WT, no mutation
  • the total was 44.2%.
  • the target frequency of base substitution from C to A was 37.2%. From this, it was confirmed that the desired base substitution can be introduced with high accuracy by combining the single base insertion by NmCas9 and the single base deletion by SpCas9.
  • a method for producing a plant cell in which DNA has been edited, which can insert one base with high frequency and can edit DNA with high accuracy, and a kit used for the method are provided. It will be possible to provide.
  • a CRISPR-Cas9 system which is particularly prone to single base deletion, is used in combination to delete one base existing in the target base sequence after inserting one base into the target base sequence. , It is also possible to replace the base efficiently.
  • the present invention can be used in various studies from basics to applications.
  • base substitution technology that can control the introduction of three bases can modify not only gene disruption but also gene function as compared to conventional target mutations that disrupt genes, and it is considered that there is an extremely high need for it. Be done. Therefore, it can be widely used as a new method in the research and development field in which DNA editing technology is currently actively used, such as development of agriculture, forestry and fishery products with new traits, and gene therapy.

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Abstract

L'invention concerne un procédé de production d'une cellule végétale éditée par un ADN, le procédé comprenant une étape d'introduction d'un système CRISPR-Cas9, qui contient une protéine Cas9 et un ARN guide de celle-ci en tant qu'éléments constitutifs, dans une cellule végétale, la protéine Cas9 étant dérivée de Neisseria meningitidis.
PCT/JP2020/038323 2019-10-23 2020-10-09 Procédé de production d'une cellule végétale éditée par un adn, et kit à utiliser dans celui-ci WO2021079759A1 (fr)

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JP2018057407A (ja) * 2013-11-18 2018-04-12 クリスパー セラピューティクス アーゲー Ｃｒｉｓｐｒ−ｃａｓシステムの材料及び方法

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JP2018057407A (ja) * 2013-11-18 2018-04-12 クリスパー セラピューティクス アーゲー Ｃｒｉｓｐｒ−ｃａｓシステムの材料及び方法

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