WO2021079759A1

WO2021079759A1 - Method for producing dna-edited plant cell, and kit to be used therein

Info

Publication number: WO2021079759A1
Application number: PCT/JP2020/038323
Authority: WO
Inventors: 啓明雑賀; 精一土岐; 秀隆賀屋
Original assignee: 国立研究開発法人農業・食品産業技術総合研究機構
Priority date: 2019-10-23
Filing date: 2020-10-09
Publication date: 2021-04-29
Also published as: JP7452884B2; JPWO2021079759A1

Abstract

A method for producing a DNA-edited plant cell, the method comprising a step for introducing a CRISPR-Cas9 system, which contains Cas9 protein and a guide RNA thereof as constituting elements, into a plant cell, wherein the Cas9 protein is derived from Neisseria meningitidis.

Description

Methods for producing DNA-edited plant cells and kits for use in them

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.

As a DNA editing technology, the development of mutation breeding technology that introduces mutations into genes using radiation and DNA damaging agents has been underway for nearly 100 years. However, it is difficult to introduce a mutation specifically into a target gene by this method with the current technique. Therefore, for example, in the case of crop breeding, it is necessary to select a strain showing the desired trait by preparing a large number of mutated samples and investigating them individually.

In addition, as a DNA editing technique, there is target recombination (gene targeting) in which the sequence of the target DNA is rewritten using a foreign DNA as a template. 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. For example, it is possible to modify the enzyme activity by introducing an amino acid substitution into a region that controls the enzyme activity. In particular, the positive / negative selection method using two types of selection markers is general-purpose, and theoretically, the target base sequence is not limited. Further, by combining the positive / negative selection method and the marker removal method that does not leave a footprint, it is possible to introduce any kind of base substitution into the sequence of the target DNA (Patent Document 1: Japanese Patent Application Laid-Open No. 2015-). No. 177788, Patent Document 2: Japanese Unexamined Patent Publication No. 2015-171358). However, in plants, the efficiency of target recombination generally tends to be low.

Further, as a DNA editing technique, there is a target mutation technique in which a target DNA is cleaved by a restriction enzyme and a repair mechanism is used when the target DNA is repaired. 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. 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.).

In particular, since it was reported in 2012 that the CRISPR-Cas9 system is available as a restriction enzyme, it has become clear that 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. However, in current technology, 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.

Further, it is known that 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 (2018) Nat. Plants). However, 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. Furthermore, with the current technology, 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.

In addition, as a target mutation technology, a technology for introducing various base substitutions at random positions has been developed by using a debase enzyme that catalyzes a base debase reaction and DNA polymerase having a reduced proofreading function. (Non-Patent Document 3: Halperin et al. (2018) Catalyst). However, it is difficult to control the position of the base into which the substitution is introduced and the type of base substitution that can be introduced by this method with the current technique.

In addition, it has been reported that the mutation introduction efficiency can be improved and the type of mutation introduced can be modified (deletion length can be lengthened, etc.) by modifying a part of the DNA repair mechanism using a restriction enzyme. .. For example, it has been reported that the efficiency of target mutation by TALENs can be enhanced by suppressing non-homologous end binding (NHEJ), which is one of the DNA repair pathways (Non-Patent Document 4: Nishizawa-Yokoi et al. (Non-Patent Document 4: Nishizawa-Yokoi et al.). 2016) Plant Physiol.). However, when the SpCas9 protein is used, deletions and insertions of about 1 to several bases are introduced at the cleavage site, but what kind of repair mechanism works at the cleavage site and what kind of mutation as a result. Since it depends on the sequence of the target DNA and the state of the cell, a technique capable of highly accurate DNA editing, such as inserting one base with high frequency, has not yet been developed.

In addition, the CRISPR-Cas9 system has been studied for various purposes so far. For example, in Japanese Patent Application Laid-Open No. 2017-502683 (Patent Document 3), 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. , The same document describes that 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.

Further, for example, Japanese Patent Application Laid-Open No. 2017-538425 (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, and Japanese Patent Application Laid-Open No. 2017-538424 (Patent Document 5) describes a gene modification in a fungal host cell (for example, a filamentous fungal host cell). In compositions and methods using the helper strain system, it is described that the Cas / guide RNA complex is introduced into a heterokaryon.

Further, for example, 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. In compositions and methods, 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.

Japanese Unexamined Patent Publication No. 2015-177788 Japanese Unexamined Patent Publication No. 2015-171358 Special Table 2017-502683 Special Table 2017-538425 Special Table 2017-538424 Special Table 2017-537647 Special Table 2018-504895

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.

As a result of intensive studies to achieve the above object, the present inventors have conducted Cas9 (Casserid protein of CRISPR-Cas9) system as Cas9 (crustered regularly interspaced short palindromic repeats / CRISPR assisted protein 9) system in the target mutation of plant cells. By using the 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. Furthermore, 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. We have also found that it is possible to substitute a base as intended, and have completed the present invention.

The aspects of the present invention obtained from such findings are as follows.
[1]
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.
[2]
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.
[3]
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. Including the step of introducing the CRISPR-Cas9 system having the guide RNA as a component and the guide RNA thereof.
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.
Method.
[4]
The method according to any one of [1] to [3], wherein the plant cell is a rice cell.
[5]
A kit for use in the production of DNA-edited plant cells.
The following (A) and (B):
(A) Cas9 protein derived from Neisseria meningitidis, a polynucleotide encoding the protein, or a vector expressing the polynucleotide,
(B) A guide RNA of the Cas9 protein, a polynucleotide encoding the guide RNA, or a vector expressing the polynucleotide.
Including, kit.
[6]
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.
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.
Including
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 kit according to [5].
[8]
The kit according to any one of [5] to [7], wherein the plant cell is a rice cell.

According to the present invention, it is possible to provide 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. Become.

Further, according to the present invention, 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.

It is the schematic which shows an example of the aspect of the continuous base insertion method of this invention. It is the schematic which shows an example of the aspect of the base substitution method of this invention. It is a figure which shows the frequency which the insertion / deletion of the sequence and the base obtained by the sequence analysis in Example 1 (NmCas9) and Comparative Example 1 (SpCas9) occurred. It is a figure which shows the frequency which the insertion / deletion of the sequence and the base obtained by the sequence analysis in Example 2 (NmCas9) and Comparative Example 2 (SpCas9) occurred.

Hereinafter, the present invention will be described in detail according to its preferred embodiment.

<Method of producing DNA-edited plant cells>
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. Provided is 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. Examples of 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. Among these, 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).

(CRISPR-Cas9 system)
The CRISPR-Cas9 system according to the present invention 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). Generally, in the CRISPR-Cas9 system, 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. Hereinafter, 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 (amino acid sequence of the wild-type NmCas9 protein) is shown in SEQ ID NO: 1, and the base sequence of the DNA encoding the protein is shown in SEQ ID NO: 2. In addition, Table 1 below 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". However, as will be described later, it is also possible to modify the recognition specificity of PAM by modifying the Cas9 protein (for example, introducing a mutation).

In Table 1, for reference, Cas9 protein (SpCas9) derived from Streptococcus pyogenes, Cpf1 protein (FnCpf1) derived from Francisella novicida, Cas9 protein derived from Staphylococcus aureus (Staphylococcus aureus) 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).

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. Here, "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). Examples of variants include the NmCas9 protein, which has modified the recognition specificity of PAM by introducing a mutation into a specific amino acid residue. Techniques for modifying the recognition specificity of PAM in Cas9 proteins are known (eg, Benjamin, P. et al., Nature 523, 481-485 (2015), Hirano, S. et al., Molecular Cell 61, 886-894 (2016). )etc). By utilizing the NmCas9 protein modified in the recognition specificity of PAM, it becomes possible to set more target DNA regions on the genomic DNA of plants.

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.

[Guide RNA]
In the CRISPR-Cas9 system according to the present invention, 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.

In the present invention, 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. When the first base sequence and the second base sequence are both DNA or RNA, the base sequences are in the same relationship with each other, and either the first base sequence or the second base sequence When 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.

Here, 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. On the other hand, since 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.

In the target DNA region, the site that causes the target gene modification (that is, the site where the target DNA is cleaved) does not have to be contained in the target base sequence, but may be contained in the target base sequence. preferable. Typically, 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.

Generally, in the CRISPR-Cas9 system, when the cleavage of the target DNA by the Cas9 protein is repaired intracellularly, the insertion or deletion of the base mainly occurs, but in the present invention, the Cas9 protein is edited by DNA editing of plant cells. By using the NmCas9 protein as a syrup, one base insertion occurs at a surprisingly higher frequency than before. For example, according to the DNA-edited plant cell production method of the present invention, when a sequence of an endogenous gene of rice is used as a target DNA, as an example, the frequency of mutations other than single nucleotide insertion (total mutation (insertion / absence of base) The ratio of the number of samples with mutations other than single nucleotide insertion to the number of samples with loss) 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. Compared with the case of using it for the target DNA, it was reduced to 1 / 8.7 to 1 / 2.3, and the frequency of single base insertion could be significantly increased. In particular, 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).

(Introduction of CRISPR-Cas9 system into plant cells)
In the present invention, as the CRISPR-Cas9 system introduced into plant cells, 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.

When the form of the expression vector is adopted, for example, 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. When a plurality of guide RNAs are used, these guide RNAs may be loaded on the same expression vector or on different expression vectors. Furthermore, when 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.

Further, when the form of the expression vector is adopted, the polynucleotide encoding the Cas9 protein may be, for example, an appropriately codon-optimized one for plant cells.

Furthermore, when adopting the form of an expression vector, the expression vector preferably contains one or more regulatory elements that are operably linked to the polynucleotide to be expressed. Here, "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.)). Moreover, it is preferable that 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.

In 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. As 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.

<Method of inserting n consecutive bases into the DNA of plant cells>
According to the DNA-edited plant cell production method of the present invention, 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.

Therefore, 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.
Also provided is 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").

In the continuous base insertion method of the present invention, 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.

In the continuous base insertion method of the present invention, 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, 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. For example, in the embodiment shown in FIG. 1, 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.

In the continuous base insertion method of the present invention, the first guide RNA (sgRNA1) 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). Includes targeting 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. By introducing the CRISPR-Cas9 system, which comprises the first guide RNA and the NmCas9 protein, into the plant cells, as described in the method for producing DNA-edited plant cells, 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. For example, in FIG. 1 (b), 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. Since 1 base, particularly T or A, is frequently inserted into the target base sequence 2, 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).

Further, in the continuous base insertion method of the present invention, 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. As a result, 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. For example, in FIG. 1, 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).

Further, in the continuous base insertion method of the present invention, 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. As a result, 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. For example, in FIG. 1, 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).

As described above, in the continuous base insertion method of the present invention, a combination of 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. By including 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. In particular, when the CRISPR-Cas9 system containing 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, so that TAA, which is a stop codon, can be easily inserted from beginning to end. It becomes.

In FIG. 1, n is 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. However, according to the present invention, 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. ..

<Method of replacing bases in DNA of plant cells>
The DNA-edited plant cell production method of the present invention may be combined with other CRISPR-Cas9 systems. For example, 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.

Therefore, 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. Contains a targeting base sequence 2 that is homologous to the target base sequence 2 into which the base is inserted.
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.

As 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.

In the base substitution method of the present invention, 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.

Further, in the base substitution method of the present invention, 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. -In the Cas9 system, 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. It is the same as the CRISPR-Cas9 system having NmCas9 protein and guide RNA as components except that it is replaced with SpCas9) and a guide RNA corresponding thereto is used, and it can be appropriately carried out by a known technique.

FIG. 2 shows a schematic view of an example of the aspect of the base substitution method of the present invention. For example, in the embodiment shown in FIG. 2, NmCas9 protein (NmCas9) and its guide RNA (sgRNA for NmCas9), SpCas9 protein (SpCas9) and its guide RNA (sgRNA for SpCas9) are expressed in each expression vector (FIG. 2). Each is introduced into a plant cell according to (a)).

In the base substitution method of the present invention, 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). Contains the modified base sequence 1. That is, in the base substitution method of the present invention, the target base sequence 1 of the guide RNA of the NmCas9 protein is arranged in the base sequence (target base sequence 1) containing the target site for inserting the base (that is, the cleavage site of the target DNA). On the other hand, it is a homologous base sequence. By introducing the CRISPR-Cas9 system containing the guide RNA and the NmCas9 protein into plant cells, as described in the method for producing DNA-edited plant cells, 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. For example, in FIG. 2B, 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. Since 1 base, especially T or A, is frequently inserted into the cleavage site, the target base sequence in which A is inserted between the 3'end to the 4th base of the target base sequence 1 is inserted. 2 (SEQ ID NO: 12) can be obtained (second stage of (b) in FIG. 2).

Further, in the base substitution method of the present invention, 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. As a result, 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. As a result, 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. For example, in FIG. 2, 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).

As described above, in 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. In particular, when 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.

In FIG. 2, 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. However, according to the present invention, since 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.

<Kit>
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. Contains a vector that expresses a polynucleotide. The vectors (A) and (B) may be different vectors from each other or may be the same vector.

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. Here, n is a natural number of 2 or more, and 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. Contains a targeting base sequence n that is homologous to the target base sequence n in which n-1 bases are inserted into the target base sequence 1.

For the purpose of substituting bases in DNA of plant cells, the 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. Here, 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).

Also, the kit of the present invention may further include one or more additional reagents. Such 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. In addition, the kit may further include an instruction manual for carrying out the method of the present invention.

Hereinafter, the present invention will be described in more detail based on Examples and Comparative Examples, but the present invention is not limited to the following Examples.

(Example 1)
[Plasid construction]
First, the DNA (SEQ ID NO:) of the NmCas9 protein gene optimized for the codon of Shiroinu nazuna 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. That is, the 35S promoter of cauliflower mosaic virus in pRI-SpCas9 and the 5'-UTR portion of the alcohol dehydrogenase gene of white inunazuna, the promoter of the ubiquitin 4-2 gene of parsley (PcUBI) and the alcohol dehydrogenase gene of rice. It was replaced with 5'-UTR of. In addition, 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). Substitution was used to construct the pRI-PcUBI-pro :: SpCas9 vector. Next, 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.

In addition, 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.

[Transformation to Inekals]
Using the above sgRNA-NmCas9 expression vector, according to the method of Toki et al. (Toki (1997) Plant Mol. Biol. Rep. 15: 16-21, Toki et al. (2006) Plant J 47: 969-976). Inecarus was transformed with Agrobacterium. First, an sgRNA-NmCas9 expression vector was introduced into Agrobacterium (strain: EHA105), and blastoderm-derived rice karus (rice: Oryza sativa L. cultivar: Nihonharu) was cultured for one month. Next, after co-culturing these Agrobacterium and rice callus for 3 days, 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.

[CAPS analysis]
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). Using KOD-FX neo DNA polymerase (manufactured by Toyobo Co., Ltd.), the target nucleotide sequence (target sequenceA correspending to sgRNA of NmCas9) 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.

[Sequence analysis]
For strains in which mutation introduction was confirmed by CAPS analysis, the PCR product of the target nucleotide sequence A (target sequencingA cloning to sgRNA of NmCas9) obtained in the same manner as in the above CAPS analysis was used as pCR-bluntII-TOPO (Thermo Fisher Scientific). Clone to Fick). This plasmid was sequenced using a BigDye Terminator v3.1 cycle sequencing kit (manufactured by Thermo Fisher Scientific). Sequence data was analyzed using SnapGene software (manufactured by GSL Biotech LLC).

(Comparative Example 1)
An sgRNA-SpCas9 expression vector was constructed according to the method of Kaya et al. (Kaya et al. (2016) Sci. Rep. 6: 26871). In the sgRNA, 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. .. In addition, 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.

(Example 2)
The target base sequence of guide RNA (sgRNA) 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) of 22 nucleotides upstream of the PAM sequence of NmCas9 protein in the promoter sequence of Os07g0583800 (rice target DNA_B)). 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).

Further, 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. In the CAPS analysis, XhoI was used instead of SacI to cleave the rice target DNA_B.

(Comparative Example 2)
The target base sequence of the guide RNA (sgRNA) 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. In addition, 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.

As a result of CAPS analysis in Examples 1 and 2 and Comparative Examples 1 and 2, no significant difference was observed in the mutagenesis efficiency between the SpCas9 protein and the NmCas9 protein in both the rice target DNA_A and the rice target DNA_B. It was.

On the other hand, the results of sequence analysis are shown in Figures 3-4. As shown in FIGS. 3 to 4, when the NmCas9 protein was introduced in both the rice target DNA_A and the rice target DNA_B as compared with the case where the SpCas9 protein was introduced (Comparative Examples 1 and 2) (Example 1, In 2), it was confirmed that the insertion of one base, particularly A or T, occurred frequently.

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. In addition, in FIGS. 3 to 4, 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. ..

(Example 3 Insertion of stop codon by insertion of 3 bases)
The target base sequence of the guide RNA (sgRNA) 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. In addition, 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. Further, 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. Next, an sgRNA1-sgRNA2-sgRNA3-NmCas9 expression vector was constructed in the same manner as in Example 1 except that all of these were used.

Further, 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. In the CAPS analysis, HpaI was used instead of SacI to cleave the rice target DNA_C.

The results of sequence analysis are shown in Table 2 below. Table 2 also 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.

(Example 4 Base substitution by insertion of 1 base and deletion of adjacent 1 base)
[Plasid construction]
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. In addition, 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). Using this as a template, 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.

[Transformation to Inekals]
Using the above NmCas9 expression vector and SpCas9 expression vector, the culture for callus selection was carried out in the same manner as in Example 1 except that the culture was carried out in a medium containing 35 mg / LG418 and 25 mg / L meropenem for 2 weeks. Inecallus was transformed.

[Sequence analysis]
For 24 samples of Inekals cultured for 2 weeks after transformation, the target nucleotide sequence was amplified by the PCR method in the same manner as in Example 1. The obtained PCR product was subjected to direct sequence analysis by the direct sequence method.

Table 3 below shows the results of sequence analysis for strains in which base substitution was confirmed by direct sequence analysis. As shown in Table 3, the frequency of insertion of A or C base into the sequence of rice target DNA_A (WT, no mutation) (number of samples with the mutation * 100 / total number of samples (%)) is determined. The total was 44.2%. On the other hand, 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.

As described above, according to the present invention, 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.

The present invention can be used in various studies from basics to applications. In particular, 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.

SEQ ID NO: 3
<223> Modified sequence of Neisseria meningitidis Cas9 SEQ ID NO: 4
<223> Modified sequence of Neisseria meningitidis Cas9 SEQ ID NO: 5
<223> Target base sequence A corresponding to the guide RNA of NmCas9 protein
<223> Target base sequence A corresponding to the guide RNA of SpCas9 protein
SEQ ID NO: 6
<223> Target base sequence B corresponding to the guide RNA of NmCas9 protein
<223> Target base sequence B corresponding to the guide RNA of SpCas9 protein
SEQ ID NO: 7
<223> Target base sequence 1 corresponding to the guide RNA1 of the NmCas9 protein
SEQ ID NO: 8
<223> Target base sequence 2 corresponding to the guide RNA2 of the NmCas9 protein
SEQ ID NO: 9
<223> Target base sequence 3 corresponding to the guide RNA3 of the NmCas9 protein
SEQ ID NO: 10
<223> Target DNA with TAA inserted
SEQ ID NO: 11
<223> Target base sequence 1 corresponding to the guide RNA of NmCas9 protein
SEQ ID NO: 12
<223> Target base sequence 2 corresponding to the guide RNA of SpCas9 protein
SEQ ID NO: 13
<223> Target DNA in which C is replaced with A
SEQ ID NO: 14
<223> F-primer SEQ ID NO: 15
<224> R-primer

Claims

A method for producing a plant cell in which DNA has been edited, which comprises a 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. Including the step of introducing the CRISPR-Cas9 system having the guide RNA as a component and the guide RNA thereof.
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.
Method.
The method according to any one of claims 1 to 3, wherein the plant cell is a rice cell.
A kit for use in the production of DNA-edited plant cells.
The following (A) and (B):
(A) Cas9 protein derived from Neisseria meningitidis, a polynucleotide encoding the protein, or a vector expressing the polynucleotide,
(B) A guide RNA of the Cas9 protein, a polynucleotide encoding the guide RNA, or a vector expressing the polynucleotide.
Including, kit.
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.
The kit according to claim 5.
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.
Including
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 kit according to claim 5.
The kit according to any one of claims 5 to 7, wherein the plant cell is a rice cell.