JP6614622B2 - Plant genome editing method - Google Patents

Description

  The present invention relates to a plant genome editing method and the like.

  In modern times, rapid global environmental changes and food shortages due to population growth have become problems. For these measures, techniques for investigating and modifying plant tolerance and traits for sustainable crop production are needed.

  In recent years, genome editing technology using target-specific nuclease has attracted attention as a new plant breeding technology. As one of such technologies, the CRISPR / Cas system is known. Typically, target genes can be mutated in various animals and plants by introducing Cas9 and guide RNA.

  An Agrobacterium method is known as a method for introducing a target-specific nuclease gene into a plant. However, this method can be applied only to limited plants and is not versatile. In addition, since the DNA and the like introduced by this method are integrated into the genome, the obtained plant is treated as a transformant and its use is restricted. In many cases, in order to obtain a genome-edited plant, work and time-consuming operations such as callusization and redifferentiation are required.

  On the other hand, the particle bombardment method (Non-patent Document 1) can be applied to a wide range of plants, and can easily introduce genes. However, since the application target is limited to immature embryos and protoplasts so far, it takes a lot of time and labor to prepare these targets, and in order to obtain a genome-edited plant body. Requires work and time-consuming work such as callusization and regeneration.

Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, Qiu JL, Gao C (2016) Efficient and transgene-free genome editing in wheat through transient expression of CRISPR / Cas9 DNA or RNA.Nature Communications 7: 12617

  An object of the present invention is to provide a simple and efficient method for editing a plant genome, and further a method for easily and efficiently producing a genome-edited plant.

  As a result of diligent research, the present inventor has found that genome editing can be performed easily and efficiently by applying the particle bombardment method to a part of growing plants such as leaves and pollen. . Moreover, based on this knowledge, it also discovered that genome editing efficiency could be evaluated simply and efficiently using the particle bombardment method. As a result of further research based on these findings, the present inventor has completed the present invention.

  That is, the present invention includes the following aspects.

  Item 1. (1) A plant comprising a step of introducing an introduction containing a target-specific nuclease expression cassette to at least one target selected from the group consisting of a plant and a part thereof by a particle bombardment method Genome editing method.

  Item 2. Item 2. The plant genome editing method according to Item 1, wherein the object is at least one selected from the group consisting of leaves and pollen.

  Item 3. Item 3. The plant genome editing method according to Item 1 or 2, wherein the object is pollen.

  Item 4. Item 4. The target according to any one of Items 1 to 3, wherein the target-specific nuclease expression cassette is a Cas protein expression cassette, and the introduction further comprises at least one selected from the group consisting of a guide RNA and a guide RNA expression cassette. Plant genome editing method.

  Item 5. Item 5. The plant genome editing method according to any one of Items 1 to 4, wherein the introduced product further comprises a reporter expression cassette.

  Item 6. Item 6. The plant genome editing method according to Item 5, wherein the reporter expression cassette is a fluorescent protein expression cassette.

Item 7. (A) A step of introducing an introduction containing a target-specific nuclease expression cassette into the pollen by a particle bombardment method, and (B) a step of obtaining a next generation plant using the pollen obtained in step A,
A method for producing a genome-edited plant comprising:

Item 8. (A) An introduction containing the reporter-specific nuclease expression cassette is introduced by particle bombardment method into at least one target selected from the group consisting of a plant containing a reporter expression cassette and a part thereof. And mutating a reporter gene in the reporter expression cassette, and (b) evaluating the efficiency of the mutation using the reporter signal as an indicator,
A method for evaluating plant genome editing efficiency.

Item 9. Step b is
(B1) after step a, including the step of introducing the reporter gene into a cell in an expressible state; and (b2) evaluating the genome editing efficiency using the reporter signal in the cell population obtained in step b1 as an indicator. Item 9. The evaluation method according to Item 8.

  Item 10. Item 10. The evaluation method according to Item 8 or 9, wherein the object is a leaf.

  ADVANTAGE OF THE INVENTION According to this invention, the simple and efficient plant genome editing method and also the method of manufacturing the genome edited plant simply and efficiently can be provided. In addition, according to the present invention, a simple and efficient method for evaluating genome editing efficiency can also be provided.

An overview of Example 1 is shown. The result of the sequence analysis of Example 1 is shown. An overview of Example 2 is shown. The result of the sequence analysis of Example 2 is shown. An overview of Example 3 is shown. The plate observation image (Example 3) under natural light or excitation light conditions is shown.

  In this specification, the expressions “containing” and “including” include the concepts of “containing”, “including”, “consisting essentially of”, and “consisting only of”.

1． Plant Genome Editing Method The present invention provides, as one aspect thereof, (1) an introduction containing a target-specific nuclease expression cassette for at least one target selected from the group consisting of a plant body and a part thereof. The present invention also relates to a plant genome editing method (also referred to as “genome editing method of the present invention” in the present specification), which includes a step of introducing by a particle bombardment method. This will be described below.

  The target of the genome editing method of the present invention is at least one selected from the group consisting of plant bodies and parts thereof.

  The plant is not particularly limited as long as it is a plant capable of genome editing with a target-specific nuclease. Examples of plants include moss plants, fern plants, gymnosperms, angiosperm magnolias, monocots, true dicotyledons (roses I, roses II, chrysanthemum I, chrysanthemum II and their outer groups). A wide range of plants including can be mentioned. Of these, gymnosperms and angiosperms are preferable. More specific examples of plants include eggplants such as tomatoes, peppers, peppers, eggplants and tobacco; cucumbers, pumpkins, watermelons and other cucumbers; cabbages, broccoli, Chinese cabbage, Arabidopsis and other vegetables; celery, parsley, Fresh vegetables such as lettuce and spicy vegetables; Green onions such as leeks, onions and garlic; Beans such as soybeans, peanuts, green beans, peas, azuki bean, mungbean, cowpea and broad beans; Other fruit vegetables such as strawberries and melons; Roots such as carrot and burdock; potatoes such as taro, cassava, potato, sweet potato and yam; Soft vegetables such as eustoma, stock, Carnations, chrysanthemums, etc .; turf such as bentgrass, corn borer; oil crops such as rapeseed, peanut, oilseed rape, oilseed rape; fiber crops such as cotton, rush; feed such as clover, dent corn, talum Crops; Deciduous fruit trees such as apples, pears, grapes, peaches, kiwifruits; Citrus fruits such as Satsuma mandarin, oranges, lemons, grapefruits; Woods such as Satsuki, Azalea, Sugi, Poplar, Para rubber, Ginkgo, Pine Etc.

  A plant body shall mean the whole plant individual. An individual plant is an individual found in the normal life cycle of the plant, and typically includes roots, stems, leaves, and sometimes flowers. The plant body may be a plant body growing in a growth environment (for example, soil) and a part thereof, or may be a plant body separated from the growth environment.

  The part of the plant is not particularly limited as long as it is a part of the plant. Specific examples include pollen, leaves, stems, roots, buds, flowers, fruits, seeds and the like. Among these, pollen and leaves are preferable, and pollen is more preferable. A part of the plant body may be in a state not separated from the plant body, or may be in a state separated from the plant body.

  One type of object may be used alone, or a combination of two or more types may be used.

  The introduction into the object in the genome editing method of the present invention includes a target-specific nuclease expression cassette.

  The target-specific nuclease is not particularly limited as long as it is a nuclease that can specifically cleave a specific site on genomic DNA to induce mutation. Examples of target-specific nucleases include Cas protein, TALEN protein, and ZFN protein.

  The CRISPR / Cas system using Cas protein uses a Cas protein that is a nuclease (RGN; RNA-guided nuclease) and a guide RNA. By introducing the system into the cell, the guide RNA binds to the target site, and the DNA can be cleaved by the Cas protein called into the binding site.

  The TALEN system using the TALEN protein uses an artificial nuclease (TALEN) that contains the DNA-binding domain of a transcriptional activator-like (TAL) effector in addition to a DNA cleavage domain (eg, FokI domain). By introducing the system into the cell, TALEN binds to the target site via the DNA binding domain, where it cleaves the DNA. The DNA binding domain that binds to the target site can be designed according to known schemes (eg, Zhang F et al. (2011) Nature Biotechnology 29 (2); this paper is incorporated herein by reference). .

  The ZFN system using the ZFN protein uses an artificial nuclease (ZFN) comprising a nucleic acid cleavage domain conjugated to a DNA binding domain comprising a zinc finger array. By introducing the system into the cell, ZFN binds to the target site via the DNA binding domain, where it cleaves the DNA. The DNA binding domain that binds to the target site can be designed according to known schemes.

  Among target-specific nucleases, Cas protein is preferable from the viewpoint that the cleavage site can be determined more freely. The Cas protein is not particularly limited as long as it is used in the CRISPR / Cas system. For example, the Cas protein can bind to a target site of genomic DNA in a complex with a guide RNA and can cleave the target site into double strands. Can be used in various ways.

  In the present specification, the “target site” means a PAM (Proto-spacer Adjacent Motif) sequence and a 17-30 base length (preferably 18-25 base length, more preferably 19-22 bases) adjacent to the 5 ′ side thereof. It is a site on genomic DNA consisting of a DNA strand (target strand) consisting of a sequence of a length (particularly preferably 20 bases) and its complementary DNA strand (non-target strand).

  Cas proteins derived from various organisms are known. Examples of Cas proteins include Cas9 protein derived from S. pyogenes (type II), Cas protein derived from S. solfataricus (types I-A1 and I-A2), H. Cas protein from walsbyi (type IB), Cas protein from E. coli (IE type, IF type), Cas protein from P. aeruginosa (IF type), Cas9 protein from S. thermophilus (type II), S Examples include Cas9 protein derived from agalactiae (type II-A), Cas9 protein derived from S. aureus, Cas9 protein derived from N. meningitidis, Cas9 protein derived from T. denticola, and the like. Among these, Cas9 protein is preferable, and Cas9 protein inherently contained in bacteria belonging to the genus Streptococcus is more preferable. Information on the amino acid sequences of various Cas proteins and their coding sequences can be easily obtained on various databases such as NCBI.

  The PAM sequence varies depending on the type of Cas protein used. For example, the PAM sequence corresponding to the Cas9 protein derived from S. pyogenes (type II) is 5'-NGG, and the PAM sequence corresponding to the Cas protein derived from S. solfataricus (type I-A1) is 5'-CCN. Yes, the PAM sequence corresponding to the Cas protein derived from S. solfataricus (type I-A2) is 5'-TCN, and the PAM sequence corresponding to the Cas protein derived from H. walsbyi (type IB) is 5'-TTC Yes, the PAM sequence corresponding to the E. coli-derived Cas protein (IE type) is 5′-AWG, and the PAM sequence corresponding to the E. coli-derived or P. aeruginosa-derived Cas protein (IF type) is 5 ′. -CC, the PAM sequence corresponding to Cas9 protein (type II) from S. thermophilus is 5'-NNAGAAW, and the PAM sequence corresponding to Cas9 protein (type II-A) from S. agalactiae is 5 ' The PAM sequence corresponding to the Cas9 protein from S. aureus, which is -NGG, is 5'-NNGRRT or 5'-NNGRR (N) and corresponds to the Cas9 protein from N. meningitidis PAM sequence, 5 'are -NNNNGATT, PAM sequence corresponding to Cas9 protein from T. Denticola is 5' -NAAAAC.

  The target-specific nuclease may have a mutation as long as its activity is not impaired. From this viewpoint, the target-specific nuclease is, for example, 85% or more, preferably 90% or more, more preferably 95% or more, and still more preferably 98% or more with the amino acid sequence of the original wild-type target-specific nuclease. It may be a protein having an amino acid sequence having identity and having the activity. Alternatively, from the same viewpoint, the target-specific nuclease may be one or more (for example, 2 to 100, preferably 2 to 50, more preferably, the amino acid sequence of the original wild-type target-specific nuclease. 2-20, more preferably 2-10, even more preferably 2-5, particularly preferably 2) amino acid sequences comprising amino acid substitutions, deletions, additions or insertions, and their activity It may be a protein having an activity of binding to a target site of genomic DNA in a complex with a guide RNA and cleaving the target strand or non-target strand of the target site. The “activity” can be evaluated in vitro or in vivo according to or according to a known method. The mutation is preferably a conservative substitution.

  As long as the target-specific nuclease has the above “activity”, a protein having a known protein tag, signal sequence, enzyme protein or the like may be added thereto. Examples of protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, and PA tag. Examples of the signal sequence include a nuclear translocation signal.

  The target-specific nuclease expression cassette is not particularly limited as long as it is DNA capable of expressing the target-specific nuclease in the target cell of the genome editing method of the present invention. A typical example of a target-specific nuclease expression cassette is a DNA comprising a promoter and a target-specific nuclease coding sequence placed under the control of the promoter. As a specific arrangement mode, for example, a mode in which a target-specific nuclease coding sequence is arranged immediately below the 3 ′ side of the promoter (for example, from the base at the 3 ′ end of the promoter to the 5 ′ end of the target-specific nuclease coding sequence) An embodiment in which the number of bases up to the base is 100 or less, preferably 50 or less).

  The promoter included in the target-specific nuclease expression cassette is not particularly limited, and for example, various types of Pol II promoters can be used. The Pol II promoter is not particularly limited. For example, RPS5A (ribosomal protein) promoter, UBQ (ubiquitin) promoter, ACT (actin) promoter, tubulin promoter, histone promoter, CaMV35S promoter, NOS promoter, WOX promoter, Examples include LAT52 promoter and DUO promoter. Among these, RPS5A promoter, UBQ promoter and the like are preferable.

  The target-specific nuclease expression cassette may contain other elements (eg, multiple cloning site (MCS), transcription termination signal, etc.) as necessary. For example, in the target-specific nuclease expression cassette, if the promoter and the target-specific nuclease coding sequence are arranged in this order from the 5 ′ side, preferably between the promoter and the target-specific nuclease coding sequence (preferably Examples include an embodiment in which MCS is arranged adjacent to one or both), or 3 ′ side (preferably adjacent to) the coding sequence of the target-specific nuclease. MCS is not particularly limited as long as it contains a plurality (for example, 2 to 50, preferably 2 to 20, more preferably 2 to 10) restriction enzyme sites.

  The termination signal is particularly limited as long as it is a nucleotide sequence capable of terminating transcription from the target-specific nuclease coding sequence (preferably, adding a polyA sequence to mRNA transcribed from the target-specific nuclease coding sequence). Not. Examples of the termination signal include heat shock protein termination signal (HspT: Heat shock protein Terminator), NosT (Noparin synthase Terminator), 35sT (CaMV35S Terminator) and the like. Of these, the heat shock protein termination signal is preferred.

  The target-specific nuclease expression cassette alone may constitute a vector together with other sequences (for example, drug resistance gene, replication origin, etc.). The type of vector is not particularly limited.

  Examples of drug resistance genes include chloramphenicol resistance gene, tetracycline resistance gene, neomycin resistance gene, erythromycin resistance gene, spectinomycin resistance gene, kanamycin resistance gene, hygromycin resistance gene, puromycin resistance gene and the like.

  When the target-specific nuclease is Cas protein, the introduction into the object in the genome editing method of the present invention includes a guide RNA expression cassette. In this case, the target-specific nuclease expression cassette may be contained on the same DNA (one molecule of DNA) as the guide RNA expression cassette described below, or on a separate DNA (different DNA molecule). May be. In the latter case, the guide RNA expression cassette may constitute a vector alone or together with other elements, other sequences, and the like. The guide RNA expression cassette is not particularly limited as long as it is a polynucleotide used for the purpose of expressing guide RNA in the target of the genome editing method of the present invention. A typical example of the expression cassette includes a promoter and a polynucleotide containing a coding sequence of the entire guide RNA or a part of the guide RNA placed under the control of the promoter.

  The promoter of the guide RNA expression cassette is not particularly limited, and a Pol II promoter can be used, but a Pol III promoter is preferable from the viewpoint of allowing a relatively short RNA to be transcribed more accurately. Examples of the Pol II promoter include RPS5A promoter, UBQ promoter, CaMV35S promoter, NOS promoter and the like. Examples of the Pol III promoter include U6-snRNA (eg, U6.1-snRNA, U6.26-snRNA) promoter, U3-snRNA promoter, and the like.

  The guide RNA is not particularly limited as long as it is used in the CRISPR / Cas system. For example, the guide RNA can be induced to the target site of the genomic DNA by binding to the target site of the genomic DNA and binding to the Cas protein. Various things can be used.

  The guide RNA has a sequence (sometimes referred to as a crRNA (CRISPR RNA) sequence) that is involved in binding to the target site of genomic DNA. This crRNA sequence excludes the PAM sequence complementary sequence of the non-target strand. The guide RNA can bind to the target site of the genomic DNA by binding complementarily (preferably complementary and specific) to the sequence.

  Note that “complementary” binding is not only based on perfect complementarity (A and T, and G and C), but also complementary so that it can hybridize under stringent conditions. The case of combining based on the above is also included. Stringent conditions are taught in known schemes (Berger and Kimmel (1987) Guide to molecular cloning techniques, Methods in Enzymology, 152: 1-812; this reference is incorporated herein by reference). Thus, it can be determined based on the melting temperature (Tm) of the nucleic acid binding the complex or probe. For example, as washing conditions after hybridization, the conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” can be mentioned. It is preferable that the hybridized state be maintained even when washed under such conditions. Although there is no particular limitation, the more stringent hybridization conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.”, and the more severe hybridization conditions are “0.1 × SSC, 0.1% SDS, 65 ° C.”. Can do.

  Specifically, among the crRNA sequences, the sequence that binds to the target sequence is, for example, 90% or more, preferably 95% or more, more preferably 98% or more, still more preferably 99% or more, and particularly preferably 100% or more. % Identity. In addition, it is said that 12 bases on the 3 'side of the sequence that binds to the target sequence are important for binding of the guide RNA to the target site. For this reason, when the sequence that binds to the target sequence in the crRNA sequence is not completely identical to the target strand, the base that is different from the target strand is 12 bases on the 3 ′ side of the sequence that binds to the target sequence in the crRNA sequence. It is preferable to exist other than.

  The guide RNA has a sequence (sometimes referred to as a tracrRNA (trans-activating crRNA) sequence) that is involved in the binding to the Cas protein, and this tracrRNA sequence binds to the Cas protein, thereby It can be directed to the target site of genomic DNA.

  The tracrRNA sequence is not particularly limited. The tracrRNA sequence is typically an RNA consisting of a sequence of about 50 to 100 bases in length that can form multiple (usually three) stem loops, and the sequence varies depending on the type of Cas protein used . As the tracrRNA sequence, various known sequences can be adopted depending on the type of Cas protein to be used.

  The guide RNA usually includes the above-described crRNA sequence and tracr RNA sequence. The mode of the guide RNA may be a single-stranded RNA (sgRNA) containing a crRNA sequence and a tracr RNA sequence, or an RNA complex formed by complementary binding of an RNA containing a crRNA sequence and an RNA containing a tracrRNA sequence. It may be a body.

  As an aspect of “the entire guide RNA or a part of the coding sequence arranged under the control of the promoter”, for example, an aspect in which the whole guide RNA or a part of the coding sequence is arranged immediately below the 3 ′ side of the promoter (for example, An embodiment in which the number of bases between the base at the 3 ′ end of the promoter and the base at the 5 ′ end of the guide RNA coding sequence is 100 or less, preferably 50 or less).

  Specific examples of guide RNA expression cassettes include, for example, when a guide RNA is an sgRNA containing a crRNA sequence and a tracr RNA sequence, a promoter, a site for inserting a crRNA coding sequence placed under the control of the promoter, and the site And a polynucleotide containing a tracrRNA coding sequence located downstream of the promoter, a polynucleotide containing a promoter and an sgRNA coding sequence placed under the control of the promoter, and the like. As another example, when the guide RNA is an RNA complex formed by complementary binding of RNA containing a crRNA sequence and RNA containing a tracrRNA sequence, typical examples of the guide RNA expression cassette include a promoter, and An expression cassette (crRNA expression cassette) containing a “RNA containing crRNA sequence” coding sequence (or a site for inserting a crRNA coding sequence) placed under the control of the promoter, a promoter, and placed under the control of the promoter Further, a combination with an expression cassette (tracrRNA expression cassette) containing a “RNA containing a tracrRNA sequence” coding sequence can be mentioned.

  The site for inserting a crRNA coding sequence is not particularly limited as long as it has a sequence suitable for insertion of a polynucleotide containing an arbitrary crRNA coding sequence. Examples of the site include a sequence containing one or a plurality of restriction enzyme sites.

  The introduction into the target in the genome editing method of the present invention preferably further includes a reporter expression cassette. In this case, by using the reporter signal as an index, it can be easily confirmed that the introduced product has been introduced into the target. The reporter expression cassette is not particularly limited as long as it is a polynucleotide capable of expressing the reporter in the subject of the genome editing method of the present invention. A typical example of the expression cassette is a polynucleotide comprising a promoter and a reporter coding sequence placed under the control of the promoter.

  The promoter of the reporter expression cassette is not particularly limited, and examples thereof include RPS5A promoter, UBQ promoter, CaMV35S promoter, NOS promoter, LAT52 promoter, ACT promoter, tubulin promoter, histone promoter, and DUO promoter.

  The reporter is not particularly limited, and examples thereof include a luminescent (colored) protein that emits light (colored) by reacting with a specific substrate, or a fluorescent protein that emits fluorescence by excitation light. Examples of the luminescent (coloring) protein include luciferase, β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase and the like, and examples of the fluorescent protein include GFP, Azami-Green, ZsGreen, GFP2, Clover, HyPer, Sirius, BFP, CFP, Turquoise, Cyan, TFP, YFP, Venus, ZsYellow, Banana, Orange, Apple, KusabiraOrange, Tomato, RFP, DsRed, AsRed, Strawberry, JRed, KillerRed, Cherry, HcRed, Plum and so on. In addition, a light-emitting (color-developing) protein or a fluorescent protein and a fusion protein of another protein, a light-emitting (color-developing) protein or a fluorescent protein with a known protein tag, a known signal sequence, etc. added to the reporter Are also included.

  Examples of the “reporter coding sequence arranged under the control of the promoter” include, for example, an embodiment in which the reporter coding sequence is arranged immediately below the 3 ′ side of the promoter (for example, 5 to 5 of the reporter coding sequence from the base at the 3 ′ end of the promoter). 'The embodiment in which the number of bases up to the terminal base is 100 or less, preferably 50 or less).

  In the genome editing method of the present invention, the introduced substance is introduced into the object by a particle bombardment method.

  The aspect of the particle bombardment method is not particularly limited. The method typically includes a step of injecting a fine particle-coated article (coating particle) onto an object at a high speed.

  The material of the fine particles is not particularly limited, and examples thereof include gold, tungsten, magnetic particles, that is, metals such as triiron tetroxide. Among these, gold is preferable.

  The particle diameter of the fine particles is not particularly limited. The particle diameter is, for example, 50 nm to 5 μm, preferably 100 nm to 3 μm, more preferably 200 nm to 1.5 μm, and still more preferably 400 nm to 800 nm.

  The method of coating the introduced substance on the fine particles is not particularly limited, and a method according to a known method can be adopted. For example, coating can be performed by bringing the fine particles into contact with the introduced substance. At this time, a positively charged substance (for example, polyamine such as spermidine) or a substance (for example, a salt such as calcium chloride) that promotes adhesion of the introduced substance to the fine particles or precipitation of the fine particles to eliminate the three-dimensional structure of the plasmid vector. ) Can be coated more efficiently. The obtained coating particles are used for introduction into an object.

  The method for injecting the coating particles is not particularly limited, but a method of injecting with a gas set at a constant pressure is usually employed.

  As the gas, an inert gas is preferably used. For example, helium gas is employed.

  The gas pressure is preferably 30-2200 psi, more preferably 450-1200 psi.

  After the injection, the target is cultured for a certain period of time, so that a target-specific nuclease is expressed in the target and genome editing occurs. The culture time is, for example, 5 to 72 hours, preferably 8 to 48 hours, and more preferably 10 to 24 hours.

  The mode of culture is not particularly limited. For example, when the object is a plant growing in a growth environment or a part thereof, the growth may be continued as it is. In addition, when the target object is a plant body separated from the growth environment or a part thereof, or is separated from the plant body, the target object is placed in an appropriate environment (for example, in a moisturizing environment or at a constant temperature). In an environment that maintains the temperature).

  Confirmation of the presence or absence of genome editing can be easily performed using the presence or absence of a reporter signal in an object as an index when a reporter expression cassette is introduced. Alternatively, the presence or absence of genome editing can be confirmed using a nucleic acid obtained from the target according to a known method, for example, detecting a mutation in the base sequence around the site to be edited (around the target-specific nuclease cleavage site). (For example, a method using a primer designed at a predicted mutation position, sequence analysis, etc.).

  A genome-edited plant can be produced by the genome editing method of the present invention. When the target is pollen, a genome-edited plant (plant) can also be produced by obtaining next-generation plants using genome-edited pollen. The method for obtaining a next-generation plant using pollen is not particularly limited, and a next-generation plant (plant) can be obtained by pollinating pollen and cultivating the obtained seed.

2． Method for evaluating plant genome editing efficiency In one aspect of the present invention, the reporter-specificity is used for at least one target selected from the group consisting of (a) a plant containing a reporter expression cassette and a part thereof. A step of introducing an introduced product containing a target nuclease expression cassette by a particle bombardment method, and mutating a reporter gene in the reporter expression cassette; and (b) evaluating the efficiency of the mutation using the reporter signal as an indicator. The present invention relates to a method for evaluating plant genome editing efficiency (sometimes referred to as “the evaluation method of the present invention” in the present specification). This will be described below.

  For the reporter expression cassette, the plant body and a part thereof, the object, the introduced product, the particle bombardment method and the like, the definition in the above “1. Plant genome editing method” can be used. In the evaluation method of the present invention, the object is preferably a leaf.

  The reporter-specific nuclease is a kind of “target-specific nuclease” in the above “1. Plant genome editing method”, and specifically cleaves a specific site on the reporter gene in the reporter expression cassette included in the object, There is no particular limitation as long as it is a nuclease capable of inducing mutation.

  In step a, after introducing the introduced product into the target, the reporter gene in the reporter expression cassette can be mutated by culturing for a certain period of time in the same manner as described in “1. Plant genome editing method” above.

  The embodiment of step b is not particularly limited, and includes various embodiments using the reporter signal as an indicator. For example, in step b, (b1) after step a, the step of introducing the reporter gene into a cell in an expressible state, and (b2) genome editing efficiency using the reporter signal in the cell population obtained in step b1 as an index. The process of evaluating is included.

  Step b1 is performed as follows, for example.

  A full-length amplification product of the reporter gene is obtained from the nucleic acid obtained from the object by PCR. This reporter gene is a reporter gene derived from the genomes of a plurality of cells in an object, and is usually a heterogeneous population including those with a mutation introduced and those without a mutation introduced.

  When introducing the obtained reporter gene into a cell in an expressible state, an expression vector for the reporter gene is usually prepared. The method of introducing this into a cell is a method of cloning each reporter gene that is a heterogeneous population from the viewpoint that the genome editing efficiency can be more accurately evaluated. Specifically, for example, a drug resistance gene expression cassette is added to an expression vector. A method is employed in which the expression vector is introduced into a cell (eg, a bacterium used for gene cloning such as Escherichia coli) and transformed, and then cultured on a solid medium containing a drug. Thereby, the colony of the cell containing each reporter gene in the reporter gene group which is a heterogeneous group is obtained.

  In step b2, genome editing efficiency is evaluated using the reporter signal as an index. That is, the reporter signal intensity of the expression product of the gene changes depending on the presence or absence of a mutation in the reporter gene (usually, it is attenuated or disappears due to the mutation). The percentage of colonies with weak or no reporter signal can be measured, and the obtained value can be used as genome editing efficiency.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1: Plant genome editing test 1
For the leaves of Nicotiana benthamiana or tobacco (Nicotiana tabacum), a combination of a Cas protein expression cassette and a guide RNA expression cassette that functions specifically to the endogenous gene, and a fluorescent protein expression cassette, particle bombardment Introduced by law. An outline of this embodiment is shown in FIG. Specifically, it was performed as follows.

  pKIR1.1 (addgene ID: 85758) was modified to prepare plasmid DNA (plasmid DNA1) expressing Cas9 and guide RNA expressed in plant cells. The plasmid DNA contains sequences of Cas9 driven by the promoter of Arabidopsis RPS5A (RIBOSOMAL PROTEIN S5A; At3g11940) gene or UBQ10 (UBIQUITIN 10; At4g05320) gene, and guide RNA driven by the Arabidopsis U6 (U6.26) promoter. Has a cassette to contain. As the guide RNA, a sequence targeting the tobacco PDS3 (PHYTOENE DESATURASE 3) gene was used.

  Moreover, plasmid DNA (plasmid DNA 2) containing 35Sp :: mTFP1 sequence for labeling cytoplasm was prepared as plasmid DNA for expressing fluorescent protein in plant cells. By using this, a cell into which a gene has been introduced can be discriminated.

  0.6 μm diameter gold particles (BIO-RAD) were washed with 100% ethanol and suspended in sterilized water to prepare a 30 mg / mL gold solution. A gold solution was used at 10 μL per shot, and a solution containing plasmid DNA1 and plasmid DNA2 was mixed and added to 100 to 1000 ng each and stirred. 4 μL of 0.1 M spermidine and 10 μL of 2.5 M calcium chloride were added to the gold solution while stirring, and gold particles coated with plasmid DNA were precipitated by spin down. Thereafter, the supernatant was removed and resuspended in 10 μL of 100% ethanol.

  For each macrocarrier used for introduction by particle bombardment, 6 to 10 μL of DNA-coated gold solution was used. The gold solution was air-dried together with the macrocarrier and placed on the top of the main body of the introduction device (PDS-1000 / He, BIO-RAD). One true leaf cut from the growing plant was attached to the center of the petri dish with masking tape, placed on the target table, and placed on the second or third stage from the top of the main body. Using a rupture disk for 1100 psi, a helium gas pressure of 1100 psi and a reduced pressure of -25 to 27 inHg was shot once per leaf. Thereafter, the cells were cultured for 10 to 24 hours while being kept moist and protected from light.

  The expression of mTFP1 was observed with a fluorescence microscope, and a region having a radius of 0.75 cm including cells in which fluorescence was detected was cut out, and genomic DNA was extracted. Genomic DNA was cleaved with a restriction enzyme (MlyI) that recognizes a sequence predicted to be mutated by guide RNA, and the region containing the target sequence of the guide RNA was amplified by PCR using the cleaved genomic DNA as a template. Again, the PCR product was cleaved with a restriction enzyme (HinfI) that recognizes the sequence predicted to be mutated, and Nested PCR was performed to exclude non-specifically amplified DNA fragments. Thereafter, the nested PCR product was inserted into the pCR-Blunt II-TOPO vector (Thermo Fisher Scientific). The vector was introduced into E. coli for transformation, seeded on an LB agar plate containing kanamycin, and cultured overnight at 37 ° C. in the dark. On the next day, colony PCR was performed on each colony on the plate using a primer complementary to the sequence on the TOPO vector and a primer complementary to the mutation insertion site. The primer complementary to the mutation insertion site was designed so that the position where the mutation is expected to be inserted is the 3 'end. In this colony PCR, the wild-type sequence is amplified because it matches completely, but when the mutation is inserted, it is not amplified because the 3 'end of the primer does not match. Thereafter, plasmid vectors were extracted from colonies of E. coli that could not be amplified by colony PCR, and mutations in the target sequence of the guide RNA were confirmed by sequence analysis. A part of the result of sequence analysis is shown in FIG. As shown in FIG. 2, it was confirmed that the wild type (WT) was mutated (1-base insertion, 8-base deletion, or 9-base deletion).

  From the above, it was found that genome editing can be performed in leaves by introducing a target-specific nuclease expression cassette such as Cas into the leaves by the particle bombardment method.

Example 2: Plant genome editing test 2
A combination of a Cas protein expression cassette and a guide RNA expression cassette that functions specifically to the endogenous gene, and a fluorescent protein expression cassette were introduced into mature pollen of N. benthamiana tobacco by the particle bombardment method. An outline of the present embodiment is shown in FIG. Specifically, it was performed as follows.

  Plasmid DNAs containing LAT52p :: mApple for labeling cytoplasm and UBQ10p :: H2B-Clover sequences for labeling nuclei were prepared as plasmid DNAs encoding fluorescent proteins expressed in pollen and pollen tubes. Particle bombardment was performed in the same manner as in Example 1 except that this plasmid DNA was used instead of plasmid DNA2.

  2-5 pods of pollen were collected from the flowers of Bensamiana tobacco, sprayed on the pollen germination medium on a petri dish, and placed on the second stage from the top of the PDS-1000 / He body. Under the same conditions as in Example 1, bombardment was performed once for each petri dish. Thereafter, the cells were cultured for 20 to 24 hours while being moisturized and protected from light.

  The fluorescence expression of pollen and pollen tube mApple and Clover fluorescence was observed. Pollen and pollen tubes expressing the fluorescent protein were collected in a tube together with the medium, and frozen with liquid nitrogen. After thawing, the cells were crushed by ultrasonic waves and friction, and genomic DNA was extracted. Restriction enzyme treatment and PCR were performed in the same procedure as in Example 1, and the Nested PCR product was inserted into the pCR-Blunt II-TOPO vector. The vector was introduced into E. coli and transformed. Thereafter, the cells were seeded on LB agar plates containing kanamycin and cultured overnight at 37 ° C. in the dark. On the next day, colony PCR and sequence analysis were performed on each colony on the plate, and mutations in the target sequence of the guide RNA were confirmed. A part of the result of the sequence analysis is shown in FIG. As shown in FIG. 4, it was confirmed that the wild type (WT) was mutated (one base substitution or one base insertion).

  From the above, it was found that genome editing can be performed in pollen by introducing a target-specific nuclease expression cassette such as Cas into the pollen by the particle bombardment method.

Example 3: Evaluation test of genome editing efficiency A combination of a Cas protein expression cassette and a guide RNA expression cassette that function specifically to the reporter was introduced by a particle bombardment method into Arabidopsis leaves containing the reporter expression cassette, Mutation efficiency was evaluated using reporter activity as an index. An outline of this embodiment is shown in FIG. Specifically, it was performed as follows.

  10 μL each of the above 30 mg / mL gold solution prepared by suspending 0.6 μm diameter gold particles in sterilized water was used. Guide RNA targeting sequences common to yellow, green and blue fluorescent proteins such as sGFP, Clover, Venus, EYFP, CFP, mCerulean, mTurquiose, and plasmid DNA expressing Cas9 should be 400 ng per shot. Stir in addition to the gold solution. As in Examples 1 and 2, Cas9 is driven by the Arabidopsis RPS5A gene or UBQ10 gene promoter, and the cassette containing the guide RNA is driven by the Arabidopsis U6 promoter.

  One true leaf of an Arabidopsis transformant expressing the LAT52p :: Venus gene was attached to the center of the petri dish with a masking tape and placed on the target table, and placed on the third stage from the top of the PDS-1000 / He body. As in Example 1, a rupture disk for 1100 psi was used, and a helium gas pressure of 1100 psi and a reduced pressure of −27 inHg were shot once per leaf. Thereafter, the cells were cultured for 24 hours while being moisturized and protected from light.

  A region with a radius of 0.5 cm where gold was most adhered was cut out and genomic DNA was extracted. The full-length Venus sequence was amplified from genomic DNA by PCR using primers with restriction enzyme recognition sequences at the ends. For cloning, a modified pGLO / R in which an arabinose-inducible protein expression pGLO vector (BIO-RAD) was modified and a sequence encoding a red fluorescent protein was introduced instead of GFP. The gene sequence of red fluorescent protein was removed by restriction enzyme cleavage, and a PCR amplified Venus sequence was inserted. The inserted vector was introduced into E. coli and transformed. Thereafter, the cells were seeded on LB agar plates containing 6 mg / mL L-(+)-arabinose and 200 μg / mL ampicillin, and cultured overnight at 37 ° C. in the dark. The next day, the colonies on the plate were irradiated with a 500 nm Blue / Green LED to confirm the presence or absence of fluorescence in each colony. Alternatively, the plate was stored at 4 ° C. for 2 to 3 days, and the fluorescent protein was stabilized to visually confirm the fluorescence under natural light without applying excitation light.

  The plate observation image under natural light or excitation light conditions is shown in FIG. Colonies into which the pGLO / R vector itself has been incorporated without being cleaved by restriction enzymes emit red fluorescence. Colonies that incorporate a vector containing a wild-type sequence that has not been genome-edited fluoresce Venus. On the other hand, colonies that have incorporated a vector with a mutation inserted into the Venus sequence by genome editing do not fluoresce. Colonies that did not emit fluorescence were selected, plasmid vectors were extracted, and sequence analysis was performed to confirm mutations in the target sequence of the guide RNA. Alternatively, by counting the percentage of colonies that did not sequence and did not emit Venus fluorescence, the genome editing efficiency in the target cell or sequence could be evaluated using fluorescence as an index.

Claims (7)

(1) A plant genome editing method comprising a step of introducing an introduction containing a target-specific nuclease expression cassette containing an RPS5A promoter or a UBQ10 promoter into a pollen by a particle bombardment method. The plant genome editing according to claim 1 , wherein the target-specific nuclease expression cassette is a Cas protein expression cassette, and the introduction further comprises at least one selected from the group consisting of a guide RNA and a guide RNA expression cassette. Method. Introduced substance further comprises a reporter expression cassette, the plant genome editing method according to claim 1 or 2. The plant genome editing method according to claim 3 , wherein the reporter expression cassette is a fluorescent protein expression cassette. (A) For the pollen, using the particle bombardment method to introduce an introduction containing a target-specific nuclease expression cassette containing the RPS5A promoter or UBQ10 promoter , and (B) using the pollen obtained in step A A process for obtaining a next-generation plant,
A method for producing a genome-edited plant comprising: (A) An introduction containing the reporter-specific nuclease expression cassette is introduced by particle bombardment method into at least one target selected from the group consisting of a plant containing a reporter expression cassette and a part thereof. And mutating a reporter gene in the reporter expression cassette, and (b) evaluating the efficiency of the mutation using the reporter signal as an indicator,
Only including,
Step b is
(B1) after step a, obtaining a reporter gene from the nucleic acid obtained from the object by PCR and introducing the reporter gene into a cell in a state in which the reporter gene can be expressed; and
(B2) A step of evaluating genome editing efficiency using the reporter signal in the cell population obtained in step b1 as an index
including,
Evaluation method of plant genome editing efficiency. The evaluation method according to claim 6 , wherein the object is a leaf.