Method for obtaining mutant plants by targeted mutagenesis

Abstract

The present invention relates to a method for introducing and selecting specific heritable mutations in plants, comprising the following steps: a step of transfecting a plant cell with exogenous DNA, wherein the exogenous DNA codes for an RNA-guided DNA endonuclease, a guide RNA suitable for directing the RNA-guided DNA endonuclease to induce the specific heritable mutation, and a selection marker; a step of regenerating plants from the transfected cells to provide a plurality of T0 plants and cross the T0 plants with a homogeneous plant not containing the exogenous DNA to provide a plurality of offspring plants; and a step of selecting one or more plants having heritable mutations from the offspring plants.

Description

Technology Field

[0001] The present invention relates to the field of plant breeding. A method for introducing and selecting a specific heritable mutation in a plant is provided, comprising the following steps: a step of transfecting a plant cell with exogenous DNA, wherein the exogenous DNA encodes an RNA-guided DNA endonuclease, a guide RNA suitable for directing the RNA-guided DNA endonuclease to induce the specific heritable mutation, and a selection marker; a step of regenerating a plant from the transfected cell to provide a plurality of T0 plants and crossing the T0 plants with an isogenic plant that does not contain the exogenous DNA to provide a plurality of offspring plants; and a step of selecting one or more plants having the heritable mutation from the offspring plants. Background Technology

[0002] Mutagenication techniques allow for the generation of additional genetic diversity useful for plant breeding. This generation of additional genetic diversity is particularly important for most crops, as the genetic diversity available in the germplasm pool is limited by genetic bottleneck effects created by rearing and subsequent selective breeding processes. Induced mutagenesis methods, such as chemical mutagenesis and radiation mutagenesis, have therefore been used in plant breeding for decades in research on new and improved plant traits useful for plant breeding.

[0003] The development of targeted mutagenic methods combined with rapidly advancing knowledge of the gene base of traits in crops has opened up additional possibilities for generating new alleles useful for plant breeding. One particularly promising targeted mutagenic method is genome editing using engineered nucleases, where site-specific double-strand breaks are induced in the genomic DNA of target cells using engineered nucleases. Engineered nucleases useful for genome editing methods include meganucleases, zinc finger nucleases (ZFNs), transcription factor-like effector-based nucleases (TALENs), and clustered regular-interval short palindromic repeat (CRISPR)-associated nucleases. Particularly useful genome editing methods include CRISPR / Cas9-based targeted mutagenic methods and CRISPR / Cpf1-based targeted mutagenic methods; for example, in the literature [Brooks et al. See (2014) Plant Physiol 166, 1292-1297] and WO2016 / 205711 A1.

[0004] Double-strand breaks induced by engineered nucleases can be repaired by the cell's endogenous DNA double-strand break repair mechanisms, such as homologous designated repair mechanisms (HDR) or non-homologous end junctions (NHEJ). In particular, HDR allows for the relatively efficient site-specific introduction of specific mutations into genomic DNA in target cells in the presence of a repair template, which is a DNA molecule used as a template in the host cell's DNA repair process. By introducing a uniquely designed repair template into target cells, specific deletions, modifications, insertions, or substitutions of DNA in target cells can be achieved with good efficiency and reliability. DNA repair of double-strand breaks by NHEJ can result in less predictable and different heritable mutations, such as the substitution and / or deletion and / or insertion of one or more nucleotides at the site of the double-strand break.

[0005] However, specific mutations that may occur after inducing one or more double-strand breaks in the genomic DNA of target plant cells can be expected to occur only at a relatively low frequency. This is particularly true when targeting highly specific modifications, such as highly specific nucleotide deletions, substitutions, or insertions. Other modifications that may occur at a low frequency include deletions of larger DNA fragments, or reversals in the case of DNA fragments or translocations. When these low-frequency mutations represent the target mutation, multiple mutant plants need to be generated and screened to identify and select mutant plants containing the specifically targeted mutation. In particular, when the target mutation occurs only at a very low frequency, a very large number of mutant plants need to be generated for screening, which is costly and time-consuming.

[0006] Therefore, there is a need for a method to introduce and select specific heritable mutations in plants that allows for more time- and cost-effective screening of plants with desired heritable mutations.

[0007] The present invention provides a method for introducing and selecting a specific heritable mutation in a plant, comprising the following steps: (a) transfecting a plant cell with exogenous DNA, wherein the exogenous DNA encodes an RNA-guided DNA endonuclease, a guide RNA (gRNA) suitable for directing the RNA-guided DNA endonuclease to induce the specific heritable mutation, and a selection marker; (b) regenerating a plant from the transfected cell to provide a plurality of T0 plants; (c) crossing the T0 plants with a homogeneous plant not containing the exogenous DNA to provide a plurality of offspring plants; and (d) selecting one or more plants having the heritable mutation from the offspring plants. Brief explanation of the drawing

[0008] Fig. 1: Overview of the approach of Example 1. A. Schematic representation of the location of associated genes on Ch02 of a tomato. MS refers to the male sterility gene, and AA refers to the anthocyanin absence gene. The size and location of the genes are not drawn in a constant ratio. B. The location of the CRISPR-Cas-induced double-strand break in these two genes is presented as a lightning flash. A small portion of the excised chromosomal fragment is restored in the opposite orientation, which causes an induced inversion. Furthermore, both genes (MS and AA) are knocked out. This causes male sterility and anthocyanin absence. In the hybrid, the gene distance between ms and aa is reduced to 0 cM due to recombination inhibition by the induced inversion. C. Primers checking for the presence of the induced inversion are presented as small arrows. Fig. 2: Indication of targeted induced reversal. The CRISPR-Cas9 constructs used consist of two gRNAs per construct, e.g., each MS - Genes and AA It contained gMS1 and gAA1, which target genes. When double-strand breaks are induced at two sites on the same chromosome, DNA fragment inversion can occur between them. Inversion will cause the inactivation of both genes because a portion of one gene is fused inversely to the remaining portion of the other gene. Widely designed PCR primers have been used to clearly detect inversion events. In this example, the two primer sites (MS-R and AA-R) on the same DNA strand at the boundary of the inversion are oriented in a way that prevents random amplification in PCR in the wild-type genome. However, the new positions of the primer binding sites after inversion are close to each other and oriented in opposite directions, which enables the amplification of the DNA fragment. Fig. 3: The top portion of the figure represents the wild-type reference genome. After inversion, the sequence on the gMS side is inverted and connected to the gAA side, as indicated by the arrow. The gRNA sequences located approximately 1.1 Mbp apart on the reference genome were connected together as indicated by the sequence at the bottom. The alignment at the bottom portion of the figure indicates that the DNA was cleaved at the predicted location of the gRNA binding site. The fractions of the gRNA sequence in bold correspond to the sequences on the other side of the inversion. DNA sequencing of one end of the inversion induced after transfection with construct 1. The Sanger sequence is part of the PCR product generated from genomic DNA from protoplasts transfected with construct 1 containing primers MS-R and AA-R and gRNA sequences gMS1 and gAA1. The Sanger sequence refers to the right side of the inversion and the flanking DNA on that side. Fig. 4: The top part of the figure represents the wild-type reference genome. After inversion, the sequence on the gMS side is inverted and connected to the gAA side, which is indicated by the arrow. The gRNA sequences located approximately 1.1 Mbp apart on the reference genome were connected together as indicated by the sequence at the bottom. The alignment at the bottom part of the figure indicates that the DNA was cleaved at the predicted locations of the gRNA binding sites. The fractions of the gRNA sequence in bold correspond to the sequences on the other side of the inversion. DNA sequencing of one end of the inversion induced after transfection with construct 3. The Sanger sequence is part of the PCR product generated from genomic DNA from protoplasts transfected with construct 3 containing primers MS-F and AA-F and gRNA sequences gMS3 and gAA3. The Sanger sequence refers to the left side of the inversion and the flanking DNA on that side. Fig. 5: The top part of the figure represents the wild-type reference genome. After inversion, the sequence on the gMS side is inverted and connected to the gAA side, which is indicated by the arrow. The gRNA sequences located approximately 1.1 Mbp apart on the reference genome were connected together as indicated by the sequence at the bottom. The alignment at the bottom part of the figure indicates that the DNA was cleaved at the predicted location of the gRNA binding site. The fractions of the gRNA sequence in bold correspond to the sequences on the other side of the inversion. DNA sequencing of one end of the inversion induced after transfection with construct 4. The Sanger sequence is part of the PCR product generated from genomic DNA from protoplasts transfected with construct 4 containing primers MS-R and AA-R and gRNA sequences gMS4 and gAA4. The Sanger sequence refers to the right side of the inversion and the flanking DNA on that side. Specific details for implementing the invention

[0009] General definition

[0010] It should be understood that the present invention is not limited to any specific methodology or protocol. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the invention, which is limited only by the appended claims. It should be noted that, as used in the present and appended claims, the singular form includes the plural form unless the context clearly indicates otherwise. Thus, for example, a reference to "vector" refers to one or more vectors and includes equivalents thereof known to those skilled in the art. The term "about" is used herein to mean approximately, nearly, about, or to that extent. When the term "about" is used with a numerical range, it modifies the range by extending the upper and lower limits of the presented numerical value. Generally, the term "about" is used herein to modify the numerical values ​​above and below the specified value by varying them up or down (higher or lower) by 20 percent, preferably 10 percent. As used herein, the word “or” means any one member of a specific list and also includes any combination of members of said list. As used in this specification and the following claims, the words “comprising” and “comprising” are intended to specify the presence of one or more specified features, integers, components, or steps, but do not exclude the presence or addition of one or more other features, integers, components, steps, or groups thereof. For clarity, specific terms used in this specification are defined and used as follows:

[0011] The term "genome" refers to the genetic material of an organism. It consists of DNA. A genome includes both genes and non-coding sequences of DNA.

[0012] The term “gene” means a (genomic) DNA sequence comprising a region that is transcribed into a messenger RNA molecule (mRNA) in a cell (transcriptional region) and an operably linked region (also described herein as a regulatory sequence, e.g., a promoter). Accordingly, a gene may comprise several operably linked sequences, e.g., a promoter, a 5’ leader sequence including a sequence accompanying translation initiation, e.g., a (protein) coding region (cDNA or genomic DNA), and a 3’ non-translation sequence including a transcription termination site, e.g., a transcription termination site. Accordingly, different alleles of a gene are different alternative forms of the gene, which may differ, for example, in one or more nucleotides of the genomic DNA sequence (e.g., promoter sequence, exon sequence, intron sequence, etc.), or in the amino acid sequence of the mRNA and / or coded protein. A gene may be an endogenous gene, which is defined herein as a locus comprising a DNA sequence derived from the originating species.

[0013] Induced genetic modification in plants may be cisgenic modification, which, in the context of the present invention, refers to modification of the plant genome through the integration of whole (undamaged) genes, including coding sequences and their original regulatory elements (e.g., promoters and terminators), derived from the same or sexually compatible species. Induced genetic modification in plants may also be intragenic modification, which, in the context of the present invention, refers to modification of the plant genome through the integration of recombinant genes, including coding sequences and their regulatory elements (e.g., promoters and terminators), derived from the same or sexually compatible species. Intragenesis differs from cisgenesis in that different combinations of genetic elements, e.g., promoters from different genes, may be used. Plants in which any DNA sequence from a sexually incompatible organism or an organism from a different genus has been integrated into the genome are referred to as "transgenic plants." "Mixable species" include species within the taxonomic family of the organism. "Non-intermingling species" are taxonomically outside of the species.

[0014] A "promoter" of a gene sequence is defined as a region of DNA that initiates the transcription of a specific gene. Promoters are located near the gene being transcribed, on the same strand, and upstream of the DNA. Promoters can be about 100 to 1,000 base pairs long. In one aspect, a promoter is defined as a region about 1,000 or more base pairs upstream of the start codon (i.e., ATG) of the protein encoded by the gene, for example, about 1,500 or 2,000 base pairs.

[0015] "Gene expression" refers to the process in which a DNA region operably linked to an appropriate regulatory region, particularly a promoter, can be translated into a biologically active, i.e., a biologically active protein or peptide (or active peptide fragment), or transcribed into an active RNA itself (e.g., in post-transcriptional gene silencing or RNAi). The coding sequence may be sense-oriented and codes for the desired biologically active protein or peptide, or active peptide fragment.

[0016] A "quantitative trait locus," or "QTL," is a chromosomal locus that codes for one or more alleles that influence the expression of a sequentially distributed (quantitative) phenotype.

[0017] The "physical distance" between loci on the same chromosome (e.g., between genes and / or between molecular markers and / or between phenotypic markers) is the actual physical distance expressed in bases or base pairs (bp), kilobases or kilobase pairs (kb), or megabases or megabase pairs (Mb).

[0018] The "genetic distance" between loci on the same chromosome (e.g., between molecular markers and / or phenotypic markers) is measured by the frequency of crossing over, or recombination frequency (RF), and is expressed in centimorgans (cM). 1 cM corresponds to a recombination frequency of 1%. If no recombines can be found, the RF is 0, and the loci are physically extremely close to each other or identical. The further two loci are from each other, the higher the RF.

[0019] The term "meiotic recombination" refers to genetic recombination involving the pairing of homologous chromosomes that occurs in eukaryotes during meiosis. The pairing of homologous chromosomes can lead to the transfer of information between said chromosomes. This transfer of information can occur without physical exchange (where a fragment of genetic material is copied from one chromosome to another without altering the donating chromosome) or through the breakage and rejoining of DNA strands to form newly recombined DNA molecules.

[0020] The term "RNA-guided DNA endonuclease" refers to an enzyme capable of cleaving phosphodiester bonds within a target nucleic acid (e.g., target DNA), the cleavage site being specifically determined by a guide RNA that associates with the RNA-guided DNA endonuclease to form a complex. Such RNA-guided DNA endonucleases are typically associated with clustered, regularly spaced, short palindromic repeats (CRISPR) adaptive immune systems in bacteria, where different RNA-guided DNA endonucleases originate from different bacterial species. RNA-guided DNA endonucleases are widely known in the art and include, but are not limited to, Cas9, Cas12a, Cas12b, Cas12c, and Cas13; see, for example, [Schindele 2018 doi:10.1002 / 1873-3468.13073].

[0021] The term "guide RNA" (gRNA) refers to a short, non-coding RNA capable of associating with an RNA-guided DNA endonuclease and specifically binding to a target nucleic acid sequence, thereby directing the RNA-guided DNA endonuclease to said target nucleic acid. The gRNA comprises a "spacer nucleic acid," which is a nucleic acid containing a sufficient number of bases complementary to the target nucleic acid to specifically bind to the target nucleic acid, thereby directing the RNA-guided DNA endonuclease to a region of interest of the target nucleic acid.

[0022] The term "CRISPR RNA" (crRNA) refers to a component of gRNA, wherein crRNA is an element of gRNA containing a spacer nucleic acid that locates the correct segment of host DNA. In the CRISPR-Cas9 system, crRNA contains a region that binds to tracrRNA, which forms the active gRNA. The active gRNA included in the CRISPR-Cas9 system may include distinct crRNAs and distinct tracrRNAs that form a complex, or may include an active sgRNA comprising crRNAs and tracrRNAs covalently linked, for example, by a linker. The term "trans-activated crRNA" (tracrRNA) generally refers to a component of gRNA included in the CRISPR-Cas9 system that contains a hairpin loop and binds to a crRNA that forms the active gRNA.

[0023] The term "selection marker" refers to a gene that confers traits suitable for artificial selection to indicate the success of a transfection intended to introduce foreign DNA into a cell when introduced. Particularly suitable selection markers are antibiotic resistance genes, for example. ntp II (Neomycin phosphotransferase II for kanamycin resistance), hpt (Hygromycin phosphotransferase against hygromycin B resistance) and aad A It includes (aminoglycoside-3 adenyltransferase for streptomycin resistance).

[0024] The terms "transfection," "transformation," or "transduction" refer to the process of intentionally introducing nucleic acids into a cell.

[0025] "Wild-type allele" (WT) refers to the version of the gene encoding a fully functional protein (wild-type protein) in this context.

[0026] For example, the term "wild type" MS10 "Allele" or " MS10 "Allele" or " MS10 The "wild-type allele" of a gene is wild-type MS10 Allowing normal protein function (i.e., normal protein expression combined with normal enzymatic activity of the expressed protein) compared to the allele, MS10 It refers to a fully functional allele of a gene. MS10 The gene codes for a basic helix-loop-helix transcription factor. The species Solanum lycopersicum ( Solanum lycopersicum Wild type in ) MS10 An example of an allele is the wild type shown, for example, in SEQ ID NO: 2. MS10 It is wild-type genomic DNA encoding a cDNA (mRNA) sequence. This wild type MS10 The protein sequence encoded by the cDNA has 209 amino acid residues and is illustrated in Sequence Identification Number: 1, corresponding to the NCBI reference sequence XM_026029418.1. Wild-type solanum Lycopersicum MS10 The allele is the wild type as described herein. MS10 It further includes functional variants of wild-type genomic DNA encoding cDNA and amino acid sequences. The wild-type specifically described herein MS10 Whether a specific variant of an allele represents a "functional variant" can be determined by using routine methods, including, but not limited to, phenotypic testing for normal growth pollen production and in silico prediction of amino acid changes affecting protein function. For example, the web-based computer program SIFT (Sorting Intolerant from Tolerant) is a program that predicts whether amino acid substitutions affect protein function; see World Wide Web at sift.bii.a-star.edu.sg / . Functionally important amino acids are expected to be conserved in protein families, and therefore changes at well-conserved positions tend to be predicted as unacceptable or harmful; see also [Ng and Henikoff (2003) Nucleic Acids Res 31(13): 3812-3814]. For example, if a position within an alignment of a protein family contains only the amino acid isoleucine, any substitution with any other amino acid is selected against it, and isoleucine is presumed to be necessary for protein function. Therefore, any change to any other amino acid will be predicted to be detrimental to protein function. If a position within an alignment contains the hydrophobic amino acids isoleucine, valine, and leucine, SIFT actually assumes that this position can only contain amino acids with hydrophobic characteristics. At this position, a change to another hydrophobic amino acid is generally predicted to be permissible, but a change to other residues (e.g., charged or polar) will be predicted to affect protein function. An alternative tool useful for predicting protein function is Probean; see World Wide Web at provean.jcvi.org / index.php. Also, Solanum lycopersicum, particularly in wild-type relatives of the species Solanum lycopersicum MS10 The gene ortholog is wild type if the aforementioned variant allows normal protein function. MS10 It can be a functional variant of the allele.

[0027] As an additional example, "wild type AA "Allele" or " AA "Allele" or " AA The "wild-type allele" of a gene is wild-type AA Allowing normal protein function (i.e., normal protein expression combined with normal enzymatic activity of the expressed protein) compared to the allele, AA It refers to a fully functional allele of a gene. AA The gene codes for the glutathione S-transferase enzyme. Wild type in the species Solanum lycopersicum. AA An example of an allele is the wild type shown, for example, in sequence identification number 4. AA It is wild-type genomic DNA encoding a cDNA (mRNA) sequence. This wild type AA The protein sequence encoded by the cDNA has 230 amino acid residues and is shown in Sequence Identification Number: 3, corresponding to the NCBI reference sequence XM_004232621.4. Wild-type solanum Lycopersicum AA The allele is the wild type as described herein. AA It further includes functional variants of wild-type genomic DNA encoding cDNA and amino acid sequences. The wild-type specifically described herein AA Whether a specific variant of an allele represents a "functional variant" can be determined by using routine methods, including but not limited to testing enzymatic activity, phenotypic testing for hypocotyl coloration, and in silico prediction of amino acid changes affecting protein function as further described herein. Additionally, particularly Solanum lycopersicum in wild-type relatives of the species Solanum lycopersicum AA The gene ortholog is wild type if the aforementioned variant allows normal protein function. AA It can be a functional variant of the allele.

[0028] "Mutant allele" refers herein to an allele containing one or more mutations compared to a wild-type allele that gives rise to the trait of the present invention. One or more mutations may be located within a coding sequence (mRNA, cDNA, or genomic sequence) or an associated non-coding sequence and / or a regulatory sequence that regulates the expression level of the coding sequence. Such mutation(s) (e.g., insertion, inversion, deletion, and / or substitution of one or more nucleotides) may result in a coding protein having reduced in vitro and / or in vivo functionality (reduced function) or having no in vitro and / or in vivo functionality (loss of function), for example, due to the protein being truncated or having an amino acid sequence in which one or more amino acids are deleted, inserted, or substituted. Such changes may result in the protein having a different 3D shape, being targeted to a different subcellular compartment, having one or more modified catalytic domains, having modified binding activity to nucleic acids or proteins, etc. Preferably, the mutant allele of the present invention codes for a truncated protein having reduced function or loss of function compared to the wild-type protein. Furthermore, mutation(s) (e.g., insertion, reversal, deletion and / or replacement of one or more nucleotides) may result in the coded protein having reduced expression or no protein expression.

[0029] For example, the term "mutant" ms10 "Allele" or " ms10 "Allele" or " MS10 "Mutant allele" or "wild type" of a gene MS10 "Mutant alleles of a gene" include, among them, one or more mutations in the coding sequence MS10 Refers to alleles of a gene, wherein one or more mutations cause reduced function or loss of function of the coded gene product, and when the mutant allele is homozygous, cause the plant to possess the male sterility trait. The terms "male sterility" or "male sterility trait" refer to a plant trait that results in the failure of a plant to produce functional anthers, pollen, or male gametes. The term mutant ms10 Alleles are also knockout ms10 Alleles and Knock-Down ms10 Not only the alleles but also mutants encoding the ms10 protein with reduced function or no function ms10 Includes alleles. As used herein, the term "knock-out allele" refers to an allele in which the expression of each (wild-type) gene is no longer detectable. "Knock-down" alleles have reduced expression of each (wild-type) gene compared to the wild-type allele.

[0030] As an additional example, the term "mutant" aa "Allele" or " aa "Allele" or " AA "Mutant allele" or "wild type" of a gene AA "Mutant alleles of a gene" include, among them, one or more mutations in the coding sequence AA Refers to alleles of a gene, wherein one or more mutations cause reduced function or loss of function of the coded gene product, and when the mutant allele is homozygous, cause the plant to possess an anthocyanin-absence trait. The terms "anthocyanin absence" or "anthocyanin-absence trait" refer to a plant trait resulting in the absence of anthocyanin coloration in the hypocotyl of the said plant. The term mutant aa Alleles are also knockout aa Alleles and Knock-Down aa Not only alleles but also mutant aa proteins coding for reduced or non-functional variants aa It includes alleles.

[0031] The term "induced mutant allele" as used herein refers to any allele of the wild-type gene that results in the trait of the present invention, produced by human intervention, e.g., mutagenicity. Preferably, the induced mutant allele cannot be found in plants of natural or breeding populations.

[0032] The term "natural mutant allele" as used herein refers to any allele of a wild-type gene that results in the trait of the present invention, which has evolved without direct human intervention. Preferably, the natural mutant allele may be found in plants of a natural population or a breeding population.

[0033] The term "osololog gene" or "osololog" is defined as a gene in a different species that has evolved through speciation events. Generally, osolologs are assumed to have the same biological function in different species. Therefore, species solanum Wild-type solanum in the wild-type relatives of Lycopersicum Lycopersicum MS10 The protein encoded by the gene's ortholog is wild-type solanum Lycopersicum It is particularly desirable to have the same biological function as the MS10 protein. Furthermore, the species Solanum Wild-type solanum in the wild-type relatives of Lycopersicum Lycopersicum AA The protein encoded by the gene's ortholog is wild-type solanum Lycopersicum It is particularly desirable to have the same biological function as the AA protein. Methods for identifying orthologs are very widely known in the relevant technical field as they achieve two goals: to describe the phylogeny of genes to investigate the forces and mechanisms of evolutionary processes and to generate a group of genes with the same biological function (Fang G, et al (2010) Getting Started in Gene Orthology and Functional Analysis. PLoS Comput Biol 6(3): e1000703. doi:10.1371 / journal.pcbi.1000703). For example, the ortholog of a specific gene or protein can be identified using sequence alignment or sequence identity of the gene sequence of the protein of interest with the gene sequence of another species. Gene alignment or determination of gene sequence identity can be performed according to methods known in the relevant technical field, for example, by verifying nucleic acid or protein sequences in existing nucleic acid or protein databases (e.g., GENBANK, SWISSPROT, TrEMBL) and using standard sequence analysis software, such as sequence similarity search tools (BLASTN, BLASTP, BLASTX, TBLAST, FASTA, etc.).

[0034] "Transferred fragment," "transferred segment," or "transferred region" refers to a chromosomal fragment (or part or region of a chromosome) introduced into another plant of the same or related species by crossbreeding or traditional breeding techniques, such as backcrossing; that is, the transferred fragment is the result of the breeding method (e.g., backcrossing) referred to by the verb "to transfer." The term "transferred fragment" is understood to never include an entire chromosome, but only a part of a chromosome. The gene transfer fragment can be large, for example, even three-quarters or half of a chromosome, but preferably smaller, for example, about 15 Mb or less, for example, about 10 Mb or less, about 9 Mb or less, about 8 Mb or less, about 7 Mb or less, about 6 Mb or less, about 5 Mb or less, about 4 Mb or less, about 3 Mb or less, about 2.5 Mb or 2 Mb or less, about 1 Mb (equivalent to 1,000,000 base pairs) or less, or about 0.5 Mb (equivalent to 500,000 base pairs) or less, for example, about 200,000 bp (equivalent to 200 kilobase pairs) or less, about 100,000 bp (100 kb) or less, about 50,000 bp (50 kb) or less, about 25,000 bp (25 kb) or less.

[0035] The term "homogenetic plant" refers to two plants that are genetically identical except for the mutant allele of interest. Subsequently, the effect of the mutant allele of interest on the phenotype of, for example, the mutant plant, can be compared between a plant line (variety) containing the mutation and its homogenetic line that does not contain the mutation.

[0036] The terms “nucleic acid sequence,” “nucleic acid molecule,” or “polynucleotide” are used interchangeably and refer to DNA or RNA molecules in single or double strands, in particular DNA encoding a protein or protein fragment according to the present invention. “Isolated nucleic acid sequence” refers to a nucleic acid sequence that is no longer in the natural environment from which the sequence was isolated, for example, a nucleic acid sequence in a bacterial host cell or a plant nucleus or chloroplast genome.

[0037] The terms "protein," "peptide sequence," "amino acid sequence," or "polypeptide" are used interchangeably and refer to a molecule composed of a chain of amino acids, regardless of its specific mode of action, size, three-dimensional structure, or origin. Thus, a "fragment" or "part" of a protein may still be referred to as a "protein." "Isolated protein" is used to refer to a protein that is no longer in its natural environment, for example, in vitro or in recombinant bacteria or plant host cells.

[0038] "Active protein" or "functional protein" is a protein having protein activity that can be measured in vitro, e.g., by an in vitro activity assay, and / or in vivo, e.g., by the phenotype conferred by the protein. "Wild-type" protein is a fully functional protein as it exists in wild-type plants. "Mutant protein" is a protein comprising one or more mutations in the nucleic acid sequence encoding the protein as described herein, thereby resulting in a protein having altered activity, preferably a protein having reduced activity, most preferably a protein having no activity (a mutant nucleic acid molecule encoding the protein).

[0039] "Functional derivatives" of a protein as described herein are fragments, variants, analogs, or chemical derivatives of a protein that retain at least some of the immunological cross-reactivity with antibodies specific to the active or mutant protein.

[0040] A fragment of a mutant protein refers to any subset of a molecule.

[0041] Variant peptides can be produced by direct chemical synthesis, for example, using methods widely known in the relevant technical field.

[0042] Analogues of mutant proteins refer to non-natural proteins that are substantially similar to the whole protein or its fragments.

[0043] A "mutation" in a nucleic acid molecule is a change of one or more nucleotides compared to a wild-type sequence, for example, by the replacement, deletion, or insertion of one or more nucleotides.

[0044] A "mutation" in an amino acid molecule that makes up a protein is a change in one or more amino acids compared to the wild-type sequence, for example, by the substitution, deletion, or insertion of one or more amino acids. Subsequently, such a protein is also referred to as a "mutant protein."

[0045] A "point mutation" is the replacement of a single nucleotide, or the insertion or deletion of a single nucleotide.

[0046] A "nonsense mutation" is a (point) mutation in a nucleic acid sequence coding for a protein, in which a codon in the nucleic acid molecule is changed into a stop codon. This results in the presence of an early stop codon in the mRNA and leads to the translation of a truncated protein. A truncated protein may have reduced function or loss of function.

[0047] "Missense or non-synonymous mutations" are (point) mutations in the nucleic acid sequence coding for a protein, which cause a codon to change to code for a different amino acid. The resulting protein may have reduced function or loss of function.

[0048] "Splice-site mutations" are mutations in the nucleic acid sequence encoding a protein that alter the RNA splicing of the precursor mRNA, resulting in mRNA with a different nucleotide sequence and a protein with a different amino acid sequence compared to the wild type. The resulting protein may have reduced function or loss of function.

[0049] "Frameshift mutations" are mutations in the nucleic acid sequence coding for a protein that alter the reading frame of the mRNA, resulting in a different amino acid sequence. The resulting protein may have reduced function or loss of function.

[0050] In the context of the present invention, "deletion" will mean that at least one nucleotide is lost at any point in the provided nucleic acid sequence compared to the corresponding wild-type sequence, or at least one amino acid is lost at any point in the provided amino acid sequence compared to the corresponding (wild-type) sequence.

[0051] In the context of the present invention, "reversal" will mean a mutation in which the nucleotide sequence of at least three nucleotide fragments in the provided nucleic acid sequence is reversed compared to the wild-type nucleotide sequence.

[0052] In the context of the present invention, "duplication" will mean a mutation in which the nucleotide sequence of a fragment of at least three nucleotides overlaps with the provided nucleic acid sequence compared to the wild-type nucleotide sequence. The duplicated fragment may include one or more whole genes. The duplicated fragment may additionally include one or more regulatory regions. Furthermore, the fragment may be duplicated within the same chromosome of the genome or on different chromosomes within the genome.

[0053] In the context of the present invention, "translocation" will mean a mutation in which the nucleotide sequence of a fragment of at least three nucleotides in the provided nucleic acid sequence is deleted from one locus in the genome and inserted into a different locus in the genome compared to the wild-type nucleotide sequence. The translocated fragment of at least three nucleotides may contain one or more whole genes. The translocated fragment of at least three nucleotides may additionally contain one or more regulatory regions. Furthermore, the fragment may be translocated within the same chromosome of the genome or on different chromosomes within the genome.

[0054] "Terminal cleavage" will be understood to mean that at least one nucleotide at the 3'-end or 5'-end of the nucleotide sequence is lost compared to the nucleic acid sequence of the corresponding wild-type sequence, or at least one amino acid at the N-end or C-end of the protein is lost compared to the amino acid sequence of the corresponding wild-type protein, thereby ensuring that at least the first nucleotide of the 5'-end or the first amino acid of the N-end remains present at the 3'-end or C-end cleavage, respectively, and at least the last nucleotide of the 3'-end or the last amino acid of the C-end remains present at the 5'-end or N-end cleavage, respectively. The 5'-end is determined by the ATG codon used as the start codon in the translation of the corresponding wild-type nucleic acid sequence.

[0055] "Substitution" will mean that, due to the exchange of nucleotides in the coding sequence of each protein, at least one nucleotide in the nucleic acid sequence or one amino acid in the protein sequence is different from the corresponding wild-type nucleic acid sequence or the corresponding wild-type amino acid sequence, respectively.

[0056] "Insertion" will mean that the nucleic acid sequence or amino acid sequence of a protein contains at least one additional nucleotide or amino acid compared to the corresponding wild-type nucleic acid sequence or the corresponding wild-type amino acid sequence, respectively.

[0057] In the context of the present invention, "early stop codon" means that the stop codon is located in a coding sequence (cds) that is closer to the start codon at the 5'-end compared to the stop codon of the corresponding wild-type coding sequence.

[0058] "Mutations in regulatory sequences," for example, mutations in the promoter or enhancer of a gene, are changes of one or more nucleotides compared to the wild-type sequence, for example, by the substitution, deletion, or insertion of one or more nucleotides, which cause, for example, the mRNA transcript of the gene to be reduced or not produced. Thus, a "promoter of a gene sequence" is defined as a region of DNA that initiates the transcription of a specific gene. The promoter is located near the gene being transcribed, on the same strand, and upstream of the DNA. The promoter may be about 100 to 1,000 base pairs long. In one aspect, the promoter is defined as a region about 2,000 or more base pairs upstream of the start codon (i.e., ATG) of the protein encoded by the gene, preferably, the promoter is a region about 1,500 base pairs upstream of the start codon, and more preferably, the promoter is a region about 1,000 base pairs upstream of the start codon.

[0059] As used herein, the term "operably linked" refers to the linkage of polynucleotide elements into a functional relationship. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter, or more precisely, a transcription regulatory sequence, is operably linked to a coding sequence when it influences the transcription of the coding sequence. Operability implies that the linked nucleic acid sequences are typically continuous.

[0060] "Sequence identity" and "sequence similarity" can be determined by the alignment of two peptide or two nucleotide sequences using a global or local alignment algorithm. Subsequently, sequences may be referred to as "substantially identical" if they share at least a certain minimum percentage of sequence identity (as further defined below) when optimally aligned by, for example, the programs GAP or BESTFIT or the Emboss program "Needle" (using default parameters, see below). These programs use the Needleman and Wunsch global alignment algorithms to align the two sequences over their full lengths, maximizing the number of matches and minimizing the number of gaps. Typically, default parameters are used with a gap generation penalty of 10 and a gap extension penalty of 0.5 (for both nucleotide and protein alignment). For nucleotides, the default scoring matrix used is DNAFULL, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915-10919). Scores for sequence alignment and percentage sequence identity can be determined, for example, using a computer program, such as EMBOSS (available online by ebi.ac.uk at http: / / www.ebi.ac.uk under / Tools / psa / emboss_needle / ). Alternatively, sequence similarity or identity can be determined by searching databases, such as FASTA, BLAST, etc., but hits must be retrieved and aligned in pairs to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences have a percentage sequence identity of at least 95%, 96%, 97%, 98%, 98.3%, 98.7%, 99.0%, or 99.3%, or more preferably 99.It has "substantial sequence identity" in the case of 7% (as determined by the Emboss "needle" using default parameters, i.e., gap generation penalty = 10, gap extension penalty = 0.5, and using the scoring matrix DNAFULL for nucleic acids and Blosum62 for proteins). Such sequences are also referred to herein as "variants," and other variants of alleles and proteins resulting in the male sterility trait and / or the anthocyanin absence trait of the present invention, other than the specific nucleic acid and amino acid sequences disclosed herein, which have the same effect as the plants of the present invention for male sterility and / or the absence of anthocyanin in the hypocotyl.

[0061] The term "hybridization" as used herein refers to the hybridization of nucleic acids under appropriate severity conditions (strict hybridization conditions) that are generally readily apparent to a person skilled in the art, depending on the nature of the probe sequence and the target sequence. Hybridization and washing conditions are widely known in the art, and adjustment to conditions according to the desired severity is easily achieved by varying the incubation time, temperature, and / or the ionic strength of the solution. For example, refer to [Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, New York, 1989]. The selection of conditions is dictated by the length of the sequence to be hybridized, in particular, the length of the probe sequence, the relative GC content of the nucleic acid, and the amount of mismatch to be allowed. Low severity conditions are preferred when partial hybridization between strands with a lesser degree of complementarity is desired. High severity conditions are preferred when complete or nearly complete complementarity is desired. Under typical high-rigidity conditions, the hybridization solution contains 6X SSC, 0.01 M EDTA, 1X Denhardt solution, and 0.5% SOS. Hybridization is performed at approximately 68°C for about 3 to 4 hours for cloned DNA fragments and for about 12 to 16 hours for whole eukaryotic DNA. For lower rigor, the hybridization temperature is the melting temperature of the duplex (T M ) It decreases to approximately 42℃ below. T M It is known that it is a function of the GC content and duplex length as well as the ionic strength of the solution.

[0062] As used herein, the phrase “hybridizes” to a DNA or RNA molecule means that the molecule to be hybridized, e.g., an oligonucleotide, a polynucleotide, or any nucleotide sequence (sense or antisense orientation), recognizes and hybridizes to the sequence of another nucleic acid molecule of approximately the same size, and has sufficient sequence similarity to it to perform hybridization under appropriate conditions. For example, a molecule of 100 nucleotides in length from the 3' coding or non-coding region of a gene will recognize and hybridize to a portion of the nucleotide sequence of approximately 100 nucleotides within the 3' coding or non-coding region of that gene or any other plant gene, provided there is at least about 70% sequence similarity between the two sequences. It should be understood that the size of the corresponding part will allow for some mismatch within the hybridization, so that the corresponding part may be smaller or larger than the molecule hybridized to it, for example, 20-30% larger or smaller, preferably about 12-15% or less larger or smaller.

[0063] As used herein, the phrases “sequence having at least 95% sequence identity,” “sequence having at least 95% amino acid sequence identity,” or “sequence having at least 95% nucleotide sequence identity” mean a sequence having at least 95%, e.g., at least 96%, 97%, 98%, 98.3%, 98.7%, 99.0%, or 99.3%, or more preferably 99.7% sequence identity compared to the indicated reference sequence. Sequence identity may be determined according to the methods described herein.

[0064] A “fragment” of a gene or DNA sequence refers to any subset of a molecule, for example, a shorter polynucleotide or oligonucleotide. In one aspect, a fragment includes a mutation as defined by the present invention.

[0065] A “variant” of a gene or DNA refers to a nucleotide-substituted variant that has a molecule substantially similar to the whole gene or a fragment thereof, such as having one or more substituted nucleotides, but retains the ability to hybridize with a specific gene or to code for an mRNA transcript that hybridizes with the original DNA. Preferably, the variant comprises a mutant allele as defined by the present invention.

[0066] As used herein, the term “plant” includes the whole plant or any part or derivative thereof, such as plant organs (e.g., harvested or unharvested flowers, leaves, etc.), plant cells, plant protoplasts, plant cell or tissue cultures from which the whole plant can be regenerated, regenerative or non-regenerative plant cells, plant calluses, plant cell masses, and undamaged plant cells from a plant, or parts of a plant, such as embryos, pollen, ovules, ovaries (e.g., harvested tissues or organs), flowers, leaves, seeds, tubers, clonal plants, roots, stems, cotyledons, hypocotyls, root tips, etc. Additionally, any stage of development, such as immature and mature seedlings, etc., is included.

[0067] "Plant line" or "breeding line" refers to a plant and its offspring. As used herein, the term "inbred line" refers to a plant line that is repeatedly self-pollinated and nearly homozygous for all characteristics. Thus, "inbred line" or "parent line" refers to a plant that has undergone inbreeding for several generations (e.g., at least 5, 6, or 7 generations or more) to produce a plant line with high uniformity.

[0068] "Plant variety" is a group of plants within the same botanical taxon of the lowest known grade, which may be defined based on the expression of a specific genotype or a combination of genotypes (regardless of whether the conditions for the approval of the plant breeder's rights have been met), and which may be considered as an independent entity because it can be distinguished from any other group of plants by the expression of at least one of these characteristics and can be propagated without any change. Accordingly, the term "plant variety" cannot be used to refer to a group of plants, even if they are of the same kind, where they all feature the presence of a single locus or gene (or a series of phenotypic characteristics resulting from this single locus or gene), but may differ greatly from one another in relation to other loci or genes. "F1, F2, etc." refers to successive related generations following a cross between two parent plants or parent lines. Plants grown from seeds produced by crossing two plants or lines are referred to as the F1 generation. Self-pollination of F1 plants results in the F2 generation, etc. "F1 hybrid" plants (or F1 seeds, or hybrids) are a generation obtained from the cross of two inbred parent lines. Therefore, "self-pollination" refers to the self-pollination of plants, that is, the association of gametes from the same plant.

[0069] "Backcrossing" refers to a breeding method in which a (single) trait, e.g., male sterility and / or anthocyanin absence trait, can be transferred from one genetic background (also referred to as the "donor," which is, but not necessarily, an inferior genetic background) to another genetic background (also referred to as the "repeating parent"; which is, but not necessarily, a superior genetic background). The offspring of the cross (e.g., obtained by crossing a first plant of a specific plant species containing the mutant allele of the present invention with a second plant of the same plant species or a different plant species that can be crossed with said first plant species, wherein said second plant species is an F1 plant that does not contain the mutant allele of the present invention; or an F2 plant or F3 plant obtained by self-pollination of the F1, etc.) is "backcrossed" to the parent plants of said second plant species. After repeated backcrossing, mutant alleles conferring traits of the donor genetic background, e.g., male sterility traits and / or anthocyanin absence traits as described herein, may have been incorporated into the recurrent parent genetic background. In this context, the terms “genetically converted,” “converted plant,” or “single locus conversion” refer to plants produced by backcrossing in which all of the essentially desired morphological and / or physiological characteristics of the recurrent parent are restored in addition to one or more genes transmitted from the donor parents. Plants grown from seeds produced by backcrossing F1 plants with a second parent plant line are referred to as the “BC1 generation.” Plants from the BC1 population may be self-pollinated to produce the BC1F2 generation, or backcrossed again with the cultivated parent plant line to provide the BC2 generation. The “M1 population” consists of multiple mutated seeds / plants of a specific plant line. "M2, M3, M4, etc." refers to successive generations obtained after self-pollination of the first mutated seed / plant (M1).As used herein, the term "T0 plant" relates to a plant regenerated from one or more transfected cells. "T1, T2, T3, etc." refers to successive generations obtained after self-pollination of the first transfected seed / plant (T0).

[0070] The terms “cultivated plant” or “cultivated variety” refer to a provided species that is cultivated by humans and possesses good agronomic characteristics, e.g., a variety, breeding line, or cultivated variety of said species. So-called heirloom varieties or cultivated varieties, namely, neglect-pollinated varieties or cultivated varieties that were typically grown during the early stages of human history and often adapted to specific geographical areas, are included herein as cultivated plants in one aspect of the invention. The term “cultivated plant” does not include wild plants. “Wild plant” includes, for example, wild catches.

[0071] The term "food" refers to any substance consumed to provide nutritional support to the body. It is typically of plant or animal origin and contains essential nutrients, such as carbohydrates, fats, proteins, vitamins, or minerals. The substance is ingested by an organism in an effort to produce energy, sustain life, or stimulate growth, and is assimilated by the organism's cells. The term "food" includes substances consumed to provide nutritional support to both human and animal bodies.

[0072] "Vegetative propagation" or "clonal propagation" refers to the propagation of plants from vegetative tissues, for example, by propagating plants from cuttings or by in vitro propagation. In vitro propagation involves in vitro cell or tissue culture and the regeneration of a whole plant from the in vitro culture. Thus, clones of the original plant (i.e., genetically identical vegetative propagates) can be produced by in vitro culture. "Cell culture" or "tissue culture" refers to the in vitro culture of plant cells or tissues. "Regeneration" refers to the development of a plant from cell culture, tissue culture, or vegetative propagation. "Non-propagating cells" refer to cells that cannot regenerate into a whole plant.

[0073] "Average" here refers to the arithmetic mean.

[0074] It is understood that comparison between different plant lines involves growing a number of plants of a line (or variety) (e.g., at least 5 plants per line, preferably at least 10 plants) under the same conditions as plants of one or more control plant lines (preferably wild-type plants) and determining the differences between plant lines, preferably statistically significant differences, when grown under the same environmental conditions. Preferably, the plants are of the same line or variety.

[0075] Methods for introducing and selecting specific heritable mutations in plants

[0076] The present invention provides a method for introducing and selecting a specific heritable mutation in a plant, comprising the following steps: (a) transfecting a plant cell with exogenous DNA, wherein the exogenous DNA encodes an RNA-guided DNA endonuclease, a guide RNA (gRNA) suitable for directing the RNA-guided DNA endonuclease to induce the specific heritable mutation, and a selection marker; (b) regenerating a plant from the transfected cell to provide a plurality of T0 plants; (c) crossing the T0 plants with a homogeneous plant not containing the exogenous DNA to provide a plurality of offspring plants; and (d) selecting one or more plants having the heritable mutation from the offspring plants.

[0077] By crossing T0 plants with wild-type plants, the need to generate and screen a very high number of T0 plants can be avoided when specific heritable mutations occur only at a low frequency. Without being bound by theory, it is believed that the DNA endonuclease / gRNA complex is active in at least a subset of T0 plants. By crossing T0 plants with wild-type plants (i.e., plants that do not contain the active construct), T0 plants possessing the active DNA endonuclease / gRNA complex will induce the desired mutation in the F1 generation (the offspring of the cross between T0 plants with the active construct and wild-type plants). As a result, it is possible to generate a very high number of F1 plants required to identify the specific heritable mutation of interest. Such a high number of F1 plants can be obtained more easily than a very high number of T0 plants. For example, when the target hereditary mutation is a reversal in the genomic DNA of a target plant intended to reduce the gene distance between two traits to zero, the most critical problem to overcome is that the actual reversal occurs only at a very low frequency. It can even be very difficult to generate enough transgenic events to identify plants with the target reversal. Instead, the inventors have discovered that they can select transfected plants with a surprisingly active construct and cross these plants with pollen from homogenotype wild-type plants having a damage-free gRNA target site. This homogenotype wild-type pollen provides a new template for post-fertilization. Consequently, a much larger quantity of seedlings derived from seeds from these crosses can be easily screened by cotyledon sampling, DNA isolation, and PCR-based screening. Thus, by using the method of the present invention, many new individual plants can be newly ( de novo It can be easily generated without the need for in vitro generations of transfected or transformed individuals. In fact, plants with active CRISPR-Cas constructs can be used to induce crosses from any pollen donor as "mutagenic inducers." Only a small number of backcrosses will be required to produce a donor genotype with the desired mutation.

[0078] Therefore, the step of crossing a T0 plant with a homogeneous plant that does not contain exogenous DNA provides an active DNA endonuclease / gRNA complex having genomic DNA with an undamaged gRNA target site, thereby inducing a desired mutation by the active DNA endonuclease / gRNA. Accordingly, the process step of crossing a T0 plant with the homogeneous plant that does not contain exogenous DNA, as included in the present invention, represents a technical step of introducing a trait into the genome or modifying a trait in the genome of the produced plant. The introduction or modification of the trait in the process step of crossing the T0 plant with the homogeneous plant that does not contain exogenous DNA is not the result of mixing genes of plants selected for sexual crosses.

[0079] Accordingly, in one step of the method of the present invention, a plant cell is transfected with exogenous DNA. Means and methods for introducing exogenous DNA into plant cells are widely known in the art, including but not limited to bacterial-mediated transformation (e.g., Agrobacterium-mediated transformation), virus-mediated gene transfer, particle-based transfection methods, e.g., gene gun methods using particle impact, non-chemical methods, e.g., electroporation, sonication and microinjection, and chemical methods, e.g., liposomal transfection; see, for example, the literature [Keith Lindsey and Wenbin Wei. In: Arabidopsis, A Practical Approach. Ed. Za Wilson. Oxford University Press, 2000. New York, USA]. The introduction of exogenous DNA into plant cells may be performed in plant cell cultures, plant tissue cultures, or even in plant cells contained within a whole plant. In the latter case, the transfected cells are isolated or separated from non-transfected plant cells before regenerating plants from the transfected cells. When a new mutation event needs to be generated and verified in the offspring of a transfected plant requiring an active heritable DNA construct, stable transformation by, for example, Agrobacterium is desirable.

[0080] In the method of the present invention, the exogenous DNA introduced into the plant cell encodes an RNA-guided DNA endonuclease, a guide RNA (gRNA) suitable for directing the RNA-guided DNA endonuclease to induce the specific heritable mutation, and a selection marker. In the method of the present invention, the exogenous DNA introduced into the plant cell may further encode elements, such as a repair template, but is not limited thereto. A repair template is a sequence of nucleic acid molecules that functions as a template in the DNA repair process of a host cell, particularly in the homologous designation repair mechanism (HDR). However, in the context of the present invention, it is preferable that the exogenous DNA introduced into the plant cell does not contain a repair template.

[0081] Accordingly, the exogenous DNA introduced into the plant cell in the method of the present invention encodes an RNA-guided DNA endonuclease. Various different RNA-guided DNA endonucleases that can be used in the method of the present invention have been identified. In one aspect, the RNA-guided DNA endonuclease encoded by the exogenous DNA introduced into the plant cell in the method of the present invention is selected from the group consisting of Cas9, Cas12a, Cas12b, Cas12c, and Cas13. In one aspect, the RNA-guided DNA endonuclease encoded by the exogenous DNA introduced into the plant cell in the method of the present invention is preferably Cas12a (previously also named Cpf1). Cas12a cleaves DNA while leaving sticky ends, which can lead to more efficient subsequent DNA repair.

[0082] In the method of the present invention, the exogenous DNA introduced into a plant cell further encodes a guide RNA (gRNA) suitable for directing an RNA-guided DNA endonuclease to induce a desired specific hereditary mutation. The gRNA used in the context of the present invention may consist of multiple RNAs forming a complex or a single gRNA (sgRNA), wherein different components of the gRNA are covalently linked, for example, by one or more linkers. The design of the gRNA may vary significantly and is partially determined by the selection of the specific RNA-guided DNA endonuclease used in the method of the present invention. For example, the selection of Cas9 as an RNA-guided DNA endonuclease requires that the gRNA comprises a trans-activated crRNA (tracrRNA) and at least one CRISPR RNA (crRNA). Alternatively, the gRNA used in combination with Cas9 may also be a single guide RNA (sgRNA) comprising a tracrRNA combined with one RNA and at least one crRNA. In one aspect, therefore, the RNA-guided DNA endonuclease in the process of the present invention is Cas9, wherein the gRNA comprises tracrRNA and at least crRNA, or the gRNA is sgRNA comprising tracrRNA combined with one RNA and at least one crRNA. As an additional example, the selection of Cas12a as the RNA-guided DNA does not require that the gRNA comprises tracrRNA. Instead, Cas12a requires that the gRNA comprises a T-rich protospacer-adjacent motif (PAM).

[0083] The method according to the present invention can be used to induce any heritable mutation in plants. Such heritable mutation may be one or more mutations selected from the group consisting of point mutations, nonsense mutations, missense mutations, splice-site mutations, frameshift mutations, early stop codon mutations, deletions, end cuts, replacements, insertions, inversions, duplications, and translocations. The mutation may be in one or more selected from the group consisting of coding regions, non-coding regions, and regulatory sequences, such as promoter sequences or enhancer sequences. The method according to the present invention is particularly useful for the introduction and selection of heritable mutations expected to occur only at a relatively low frequency. Such low-frequency mutations may be, for example, mutations obtained by non-homologous end linkages (NHEJs). Accordingly, in a further aspect, the present invention provides a method for introducing and selecting specific heritable mutations in plants as described herein, wherein the specific heritable mutation is an inversion, duplication, or translocation. These mutations are particularly useful when the purpose is to use the method of the present invention to obtain plants that are not transgenic.

[0084] The exogenous DNA introduced into a plant cell in the method of the present invention further codes for a selection marker that allows for the selection of a plant cell in which the introduction of the exogenous DNA was successful and which allows for the expression of the coded RNA-guided DNA endonuclease and gRNA.

[0085] In an additional step of the method of the present invention, a plant is regenerated from a transfected cell to provide a plurality of T0 plants. Means and methods for regenerating a plant from a plant cell are widely known in the relevant art. For example, a cell or tissue culture can be treated with a budding and / or rooting medium to regenerate an entire plant.

[0086] In one aspect of the present invention, a transfected cell from which a T0 plant is regenerated is selected based on the activity of a selection marker, wherein the selection marker is preferably selected from the group consisting of kanamycin resistance; chloramphenicol resistance, tetracycline resistance, and ampicillin resistance.

[0087] In a further step of the method of the present invention, the T0 plant obtained is crossed with a homogeneous plant that does not contain the exogenous DNA to provide multiple offspring plants. Surprisingly, it was discovered that a much higher number of mutant plants can be obtained by crossing the T0 plant with a homogeneous plant that does not contain exogenous DNA. Consequently, a much higher number of seedlings derived from seeds from these crosses can be easily screened.

[0088] In a further step of the method of the present invention, one or more plants having hereditary mutations are selected from the offspring plants. Means and methods for screening for specific mutations in a large number of plants may include screening at the DNA, RNA (or cDNA) or protein level using methods widely known in the art and known to detect the presence of the desired mutation(s). For example, seedlings of the offspring plants obtained in the process of the present invention may be grown and then samples may be taken from plant parts, such as cotyledons. Subsequently, DNA may be isolated from the cotyledon sample and then PCR-based screening for the desired mutation(s) may be performed.

[0089] DNA samples can be pooled prior to PCR-based screening to reduce the number of PCRs. For example, 1,000 DNA samples can be pooled into 100 pools, each containing DNA from 10 plants. After PCR-based screening of these 100 pools and identification of mutations in one or more of these pools, in the next PCR, individual DNA samples constituting the identified pools can be screened to identify DNA samples containing mutations (one-dimensional pooling).

[0090] Alternatively, for example, 900 DNA samples can be organized into a hypothetical two-dimensional grid with 30 rows (x1..x30) and 30 columns (y1..y30), where each sample has its own unique coordinates starting at x1y1 and ending at x30y30. Pooling DNA from all rows results in 30 x-pools, and pooling DNA from all columns results in 30 y-pools. PCR-based screening is performed on these 30 x-pools and 30 y-pools and may result in the identification of mutations in two pools; one in the x-pool and one in the y-pool. The number of x-pools and y-pools results in the identification of a specific DNA sample containing a mutation, e.g., x20y10.

[0091] Alternatively, for example, 1,000 DNA samples can be organized into a virtual 3-dimensional grid with 10 x-coordinates, 10 y-coordinates, and 10 z-coordinates, where each DNA sample has its own unique coordinates starting at x1, y1, z1 and ending at x10, y10, z10. Pooling of DNA samples in each dimension results in 10 x-pools, 10 y-pools, and 10 z-pools, each containing DNA from 100 individuals, with each individual represented by one pool in each dimension. PCR-based analysis of 30 pools can result in the identification of mutations in one of the pools in each dimension, for example, x-pool 3, y-pool 5, and z-pool 4. This will lead to the identification of a DNA sample from one individual plant with coordinates x3,y5,z4 (Tsai et al., 2011; https: / / doi.org / 10.1104 / pp.110.169748).

[0092] PCR-based screening can be performed using primers specific to expected or target mutations. Primers can be designed to amplify DNA fragments only when a specific inversion occurs. For example, two primers designed to be oriented in the same direction on target DNA will not amplify the product. However, when an inversion occurs, it causes a reversal of one of the primer binding sites, and the new orientation of one primer toward the other can allow the amplification of a specific DNA fragment that can be used as a marker for that specific event.

[0093] Alternatively, the primer set can also be designed to amplify DNA fragments only when a deletion occurs. For example, the positions of forward-oriented and reverse primers may be too far apart to amplify the product under selected reaction conditions. Certain DNA polymerases can transcribe 1 kbp per minute. In PCR using a 10-second extension time, products larger than approximately 1 kbp cannot be amplified. Mutations, such as deletions, cause reduced physical distance between primers and may allow the production of amplicons in PCR using a 10-second extension time, which can subsequently serve as markers for the deletion.

[0094] Alternatively, digital PCR systems, such as droplet digital PCR (ddPCR), can be used to identify mutations in pooled DNA samples. Droplet digital PCR sorts DNA samples into thousands of droplets. PCR amplification of the template subsequently occurs in each individual droplet, and counting of positive droplets provides accurate, absolute target quantification. Digital PCR allows for the detection and quantification of rare sequences, such as SNPs, allelic variants, and edited DNA.

[0095] Accordingly, in an additional aspect, the present invention provides a method for selecting one or more plants having hereditary mutations by isolating genomic DNA from a portion of a progeny plant and determining whether said genomic DNA contains hereditary mutations.

[0096] Preferably, DNA sequencing is used to determine whether the genomic DNA of a plant obtained by the method of the present invention contains a desired heritable mutation. Means and methods for DNA sequencing are widely known in the art and include, for example, chain termination sequencing (Sanger sequencing), sequencing by synthesis (Illumina sequencing), nanopore DNA sequencing, polony sequencing, ion semiconductor sequencing, and DNA nanoball sequencing.

[0097] Alternatively, SNP genotyping assays can be used to select plants with a target heritable mutation, particularly when it is sufficient to detect single nucleotide differences (single nucleotide polymorphisms, SNPs) between plants containing the target mutation and plants not containing the target mutation. For example, SNPs can be easily detected using the KASP assay (see World Wide Web at kpbioscience.co.uk) or other SNP genotyping assays. To develop the KASP assay, for example, 70 base pairs upstream and 70 base pairs downstream of the SNP may be selected, and two allele-specific forward primers and one allele-specific reverse primer may be designed. For example, for the KASP assay method, refer to the literature [Allen et al. 2011, Plant Biotechnology J. 9, 1086-1099], specifically pp. 097-1098. Equally different genotyping assays, such as TaqMan SNP genotyping assays, high-resolution melting (HRM) assays, and SNP-genotyping arrays (e.g., Fluidigm, Illumina, etc.) may be used.

[0098] In a further aspect, the method of the present invention comprises, prior to crossbreeding a T0 plant and a wild-type plant, selecting a T0 plant expressing an RNA-guided DNA endonuclease and providing one or more T0 plants having an active construct, and crossbreeding only the one or more T0 plants having the active construct with the wild-type plant to provide a plurality of offspring plants. Using the term "active construct" as used herein, it means that a DNA endonuclease / gRNA complex encoded by previously introduced exogenous DNA is active, that is, the DNA endonuclease / gRNA complex is capable of inducing a desired mutation in the F1 generation (the offspring of a cross between a T0 plant having the active construct and a wild-type plant).

[0099] Screening of offspring plants is more efficient by selecting T0 plants with active constructs and crossing them with plants without active constructs. When T0 plants with only one or a relatively low number of active constructs are crossed with wild-type plants (which lack active constructs), a high number of distinct F1 plants are produced and can be screened for low-frequency events. It is much more efficient to generate a very high number of F1 plants capable of carrying specific heritable mutations when T0 plants with non-active DNA endonuclease / gRNA complexes are removed from the population crossing with wild-type plants.

[0100] In one aspect, the method of the present invention comprises the step of a selected T0 plant expressing an RNA-guided DNA endonuclease being a T0 plant in which the RNA of said RNA-guided DNA endonuclease is detected. One indirect method of identifying a T0 plant having an active CRISPR-Cas construct is the presence of mutations caused by the active construct. For example, a CRISPR-Cas construct may encode two or more guide RNAs that target the flank of a genomic DNA fragment to be reversed. The desired reversal may be a rare event and may not be present in the T0 generation. However, mutations causing small deletions may occur at gRNA target sites depending on the activity of the construct. The presence of these mutations in a heterozygous state indicates an active construct. Mutations in a homozygous state may be much better evidence that the CRISPR-Cas construct is active.

[0101] In an additional aspect, the method of the present invention comprises the step of self-pollinating offspring plants and then selecting one or more plants having hereditary mutations from the offspring plants.

[0102] First, F1 offspring plants are self-pollinated to obtain an F2 offspring population, and by selecting plants having heritable mutations from said F2 offspring population, plant homozygosity with respect to the presence of an active construct is obtained. F1 offspring plants containing the active construct are always heterozygous with respect to the active construct. Alternatively, T0 plants can be self-pollinated to obtain homozygous T1 plants, which can be crossed with homogeneous plants that do not contain exogenous DNA to provide multiple offspring plants. Accordingly, the method of the present invention may include the step of self-pollinating T0 plants to obtain T1 plants, which are crossed with homogeneous plants that do not contain exogenous DNA to provide multiple offspring plants.

[0103] In a further aspect, the method of the present invention further comprises a subset of a plurality of T0 plants (as obtained in method step (a) as described herein) undergoing at least one self-pollination step, and the offspring plants obtained by said at least one self-pollination step are pooled (i.e. combined) with offspring plants (as obtained in method step (c) as described herein) from which one or more plants having hereditary mutations are selected. Thus, a portion of the T0 plants regenerated from a transfected cell undergoes at least one self-pollination step instead of being crossed with a homogeneous plant that does not contain exogenous DNA. The plants obtained from said at least one self-pollination step are pooled with offspring plants obtained by crossing the T0 plants with said homogeneous plant that does not contain exogenous DNA, followed by the step of selecting one or more plants having hereditary mutations.

[0104] In a further aspect, the method of the present invention further comprises backcrossing one or more selected plants having hereditary mutations to obtain offspring plants containing specific hereditary mutations and not containing any exogenous DNA.

[0105] The following non-limiting examples describe a method for efficiently obtaining mutant tomato plants using a method according to the present invention, wherein the tomato plants are wild-type male sterile 10 in their genomes ( MS10 ) mutant alleles of the gene and absence of wild-type anthocyanin ( AA ) comprising at least one chromosome containing a mutant allele of a gene, wherein meiotic recombination in the plant is wild type MS10 The aforementioned mutant allele of the gene and the wild type AA The above mutant alleles of the gene are repressed. Unless otherwise specified in the examples, all recombinant DNA techniques are performed according to standard protocols as described in the literature [Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA]. Standard materials and methods for plant molecular work are described in the literature [Plant Molecular Biology Labfax (1993) by RDD Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK]. Standard breeding methods are described in the literature ['Principles of Plant breeding', Second Edition, Robert W. Allard (ISBN 0-471-02309-4)].

[0106] Examples

[0107] Example 1

[0108] Design and cloning of structures

[0109] To design the gRNA for MS, the inventors used the first exon of MS. To design the gRNA for AA, the inventors used the first and second exons of this gene. For each gene, the inventors designed two gRNAs (Table 1) using the software CRISPOR (http: / / crispor.tefor.net).

[0110] Table 1. gRNA for inducing double-stranded DNA breaks in both Ch02-phase male sterility gene MS and anthocyanin absence gene AA.

[0111]

[0112] Two CRISPR-Cas constructs were prepared by combining one gRNA for AA with one for MS. Construct 1 contained gMS1 and gAA1, and construct 2 contained gMS2 and gAA2 (Table 1).

[0113] CRISPR-Cas constructs were prepared by cloning using the Golden Gate MoClo Toolkit (https: / / www.addgene.org / kits / marillonnet-moclo / ). The designed gRNAs were ordered as oligos and each was separately inserted following the U6-26 promoter from Arabidopsis thaliana (pICSL90002 plasmid, https: / / www.addgene.org / 68261 / ). The gRNAs containing the promoters were combined with Arabidopsis codon-optimized SpCas9 sequences under the Petroselinum crispum ubiquitin 4-2 promoter and NOS terminator (pDe-CAS9, https: / / www.addgene.org / 61433 / ). The inventors included a fluorescent GFP gene (p35S-fGFP-ter35S) driven by a CaMV-35S promoter and terminator to estimate the proportion of successfully transfected protoplasts in a CRISPR-Cas construct.

[0114] Isolation of a plasmid containing CRISPR-Cas construct 1 or 2

[0115] Plasmids containing two CRISPR-Cas constructs were bred in Escherichia coli (25 ml LB) and isolated using the QIAGEN Plasmid Midi Kit (Catalog No. / ID: 12143) as described in the manual.

[0116] Plasmid DNA was eluted in 200 μl of EB buffer and quantified using Nanodrop One (Thermofisher). For transfection, 10 μg of plasmid was pipetted into a 2 ml tube, and water was added until the volume reached 20 μl.

[0117] plant material

[0118] Tomato seeds of the cultivated Moneymaker variety were sterilized with 1% hypochlorite and 0.01% Tween 20. Subsequently, the seeds were washed three times with sterile water and placed in a germination medium. The germination medium consisted of 2.2 grams of MS medium (Duchefa) supplemented with MS vitamin (Duchefa) and 5 grams of sucrose per liter. The pH was adjusted to 5.8. The pots were kept at 24°C under a 16-hour light and 8-hour dark regime.

[0119] Young seedlings were transplanted into pots equipped with air filters, with two seedlings per pot. The pots contained 4.4 g / l MS medium (Ducheffa) containing vitamins and 30 g sucrose per liter, and the pH was adjusted to 5.8. The plants were grown at 24°C for 4 weeks under 16 hours of light and 8 hours of darkness until several fully developed leaves were present.

[0120] Transfection

[0121] For each transfection, healthy, expanded leaves were harvested from two pots, cut into small strips in enzyme digestion buffer, and incubated overnight. The protoplasts were filtered, washed, and treated with PEG and plasmids for transfection using standard methods. Subsequently, the protoplasts were washed, placed in recovery medium, and incubated in a dark room at 24°C for 24 hours. Transfection efficiency was measured by estimating the proportion of protoplasts exhibiting green fluorescence due to the presence of the GFP gene. For all transfection reactions, there were approximately 200,000 protoplasts in a final volume of 150 μl. This is equivalent to a concentration of approximately 1,300 protoplasts per μl.

[0122] Check for the existence of induced reversal

[0123] To check for the presence of induced mutations, the inventors used targeted PCR as shown in FIGS. 1B and C. Forward and reverse primers were designed around MS and AA guides using a reference genome for sequences flanking gRNA loci, and primer design software Primer3 (http: / / bioinfo.ut.ee / primer3-0.4.0 / ). The primer pairs are (AA-F) 5'-TGGTTGCTGCTCATCTTCAC-3' (Sequence ID: 9) and (AA-R) 5'-GCAAAGCCACCTTCATTCAT-3' (Sequence ID: 10) and (MS-F) 5'-TAGGGGATTTTCATGCTGGT-3' (Sequence ID: 11) and (MS-R) 5'-GCCAAAAATGAGTCCTTCCA-3' (Sequence ID: 12).

[0124] Transfected protoplasts were processed in the following manner: per sample, 2 μl of isolated protoplasts were diluted 1:1 with 20 mM KOH, 1% casein solution, boiled for 5 minutes, and placed on ice for 5 minutes.

[0125] The 'left flank' and 'right flank' of the induced inversion were amplified by PCR using Phire polymerase (Thermo Fisher). For the left end, forward MS primers (MS-F) and forward AA primers (AA-F) were used, but for the right flank, the inventors used reverse MS-R and reverse AA-R primers (Fig. 1C). The reaction mixture contained 5 μl 5x buffer, 1 μl 5 mM DNTP, 1.25 μl 10 pmol / μl F primer, 10 pmol / μl R primer, 0.35 μl Phire polymerase, 4 μl protoplast solution (~2700 protoplasts), and 12.5 μl water. A water control was also included. 80 PCR cycles (10 seconds at 98°C, 20 seconds at 61.5°C, and 40 seconds extension at 72°C) were performed to also detect rare events. Non-transfected protoplasts were used as negative controls. PCR products were placed on an agarose gel and visualized.

[0126] qPCR

[0127] To estimate the frequency of reversal events, qPCR was performed on processed protoplast samples. This was carried out using iQ SYBR Green Supermix (Biorad) on a qPCR machine (Biorad). The reaction conditions were as follows: 12.5 μl iQ SYBR Green Supermix, 1.25 μl 10 pmol / μl F primer, 10 pmol / μl R primer, 2 μl protoplast solution (~1300 protoplasts), and up to 25 μl of water.

[0128] For reference, actin genes from tomatoes were included using the sequences 5'-ACTGTCCCTATCTATGAAGGTTATGC-3' (sequence identification number: 13) and 5'-GAAACAGACAGGACACTCGCACT-3' (sequence identification number: 14) as forward and reverse primers, respectively.

[0129] Plant transformation

[0130] A transgenic plant (T0) containing CRISPR-Cas, Agrobacterium tumefaciens ( Agrobacterium tumefaciens It was produced by )-mediated transformation. Therefore, the following transformation method may be used. Cotyledons of 10-day-old seedlings were treated with 2% MSO (100 mg L⁻¹) at an absorbance of 0.5 at 600 nm. -1 Myo-inositol, 400μg L -1 Thiamine HCl and 20g L -1 8 ml of Agrobacterium cells suspended in liquid MS medium containing sucrose were incubated. After 30 minutes, cotyledons were dry-blotted onto sterile filter paper and inoculated with 1x Nitsch and Nitsch vitamin mixture, 3% w / v sucrose, and 1 mg L⁻¹. -1 NAA, 1 mg L -1 Place in MS culture medium containing 6-benzylaminopurine and 0.7% w / v agar, pH 5.7. After 2 days of co-culture, 200 mg L of cotyledons -1 Wash in liquid MS medium containing carbenicillin and 1x Niche and Niche vitamin mixture, 3% w / v sucrose, 2 mg L -1 Zeatin, 200mg L -1 Carbenicillin, 0.7% w / v agar, pH 5.7 and 100 mg L for selection -1 Transfer to budding-inducing MS culture medium containing kanamycin. Cotyledons that have begun to develop callus are treated with half the concentration of zeatin and 1 mg L⁻¹. -1 Transfer to fresh culture medium containing GA3. Transfer cotyledons to fresh medium every two weeks. When the initial callus forms, excise the budding primordia and administer 200 mg L -1 Carbenicillin and 100 mg L -1Transfer to Budding-Elongation MS Culture Medium, a germination medium containing kanamycin. Excise a 2-4 cm elongated bud from the callus and place it in Rooting MS Culture Medium (1x Niche and Niche Vitamin Mixture, 1.5% w / v sucrose, 5 mg L⁻¹). -1 IAA, 200mg L -1 Carbenicillin, 50 mg L -1 The seedlings were transferred to kanamycin and 0.7% w / v agar (pH 5.7). Rooted (T0) seedlings were transferred to soil for further analysis. The culture medium components and antibiotics were obtained from Duchefa Biochemie.

[0131] Selection of T0 transgenic plants containing active CRISPR-Cas constructs

[0132] The activity of CRISPR-Cas constructs depends on the location of integration in the genome. To identify T0 plants possessing the active construct, plants containing the mutation of the T0 plants MS 및 AA The targeted regions in the genes were identified by sequencing. These domains were amplified by PCR using pyrepolymerase (Thermo Fisher). MS or AA A primer set specific to the targeted region in the gene was used. The reaction mixture contained 5 μl 5x buffer, 1 μl 5 mM DNTP, 1.25 μl 10 pmol / μl forward primer, 10 pmol / μl reverse primer, 0.35 μl pyrene polymerase, 4 μl genomic DNA solution (4 ng), and 12.5 μl water. The target region was amplified by performing 30 PCR cycles (10 seconds at 98°C, 20 seconds at 60°C, and a 5-second extension at 72°C). The PCR products were sequenced by a service provider, and the presence of mutations was determined using a computer program for DNA sequencing analysis.

[0133] Self-pollination of T0 plants to provide multiple offspring plants and crossbreeding with plants not containing the exogenous DNA

[0134] Selected T0 plants with an active CRISPR-Cas construct were grown and flowers were self-pollinated to produce fruits containing T1 seeds. The fruits were grown and T1 seeds were collected.

[0135] Alternatively, selected T0 plants containing an active CRISPR-Cas construct were grown, and the flowers were decimated before the anthers opened to prevent self-pollination. Pollen isolated from wild-type plants was collected and used to pollinate the selected T0 plants, and fruits were grown to collect F1 seeds. F1 plants containing an active CRISPR-Cas construct can be grown to produce F2 seeds.

[0136] T1, F1, and F2 seeds were germinated, and genomic DNA was isolated from the first leaf. PCR was designed to detect target mutations, inversions, or other structural variations. By using a pooling strategy (e.g., Tsai et al., 2011; https: / / doi.org / 10.1104 / pp.110.169748), a large number of seedlings can be analyzed with only a limited number of PCRs.

[0137] Example 2

[0138] CRISPR-Cas9 constructs were prepared as described in Example 1. Three CRISPR-Cas9 constructs were prepared by combining one gRNA for AA with one for MS. Construct 1 contained gMS1 and gAA1, construct 3 contained gMS3 and gAA3, and construct 4 contained gMS4 and gAA4.

[0139] Table 2. gRNA target sites for producing double-stranded DNA breaks in both tomato Ch02-phase male sterility gene MS and anthocyanin absence gene AA. The symbol is placed at the location where double-strand breakage is expected to occur. The PAM region is shown in italics.

[0140]

[0141] The construct was propagated on Escherichia coli, the plasmid was isolated, and purified for transfection of tomato protoplasts. Protoplasts were isolated from the leaves of plants grown in vitro on 'MoneyMaker'. After transfection and incubation, the cell cultures were screened for reversal by PCR. To perform this, a portion of the transfected protoplasts (~2,700 cells) were transferred to PCR tubes and denatured in KOH as described in Example 1.

[0142] PCR was performed on cell lysates using primer pairs AA-F and MS-F or AA-R and MS-R in separate PCRs. The annealing strands of the primer pairs are identical, which prevents amplification of wild-type DNA and allows amplification only when inducing inversion (Fig. 2). Additionally, the distance between the primer pairs is ~1.1 Mbp on the wild-type genome, which is too large to amplify the PCR product.

[0143] Table 3. PCR primers used to amplify the inversion boundary.

[0144]

[0145] However, the inversion of the region between the gRNA targets will bind the primer sites together in an orientation that enables the amplification of the PCR product, and this includes the boundary of the inversion as shown in Fig. 2.

[0146] PCR products from protoplasts transfected with constructs 1, 3, and 4 were isolated on an agarose gel, fragments of expected size were excised from the gel, and the DNA was Sanger sequenced. It was surprising that most PCR DNA fragments produced the expected size, which would imply that reverse transfection occurs in more than one protoplast per reaction. One reaction contained DNA from approximately 2,700 protoplasts, and the transfection efficiency was approximately 70% (also present in the constructs GFP Considering that it was indicated from gene fluorescence (data not presented), this would mean that the inversion frequency is >1 / (0.7*2700) cells. The expected PCR amplicon size was based on the genomic locations of the primers and gRNA binding sites, which is approximately 1.3 kb. The locations of the primers and gRNA binding sites and their sequences are shown in Tables 2 and 3.

[0147] Figure 3 shows a portion of the DNA sequence of a PCR product generated from genomic DNA from a protoplast culture transfected with Construct 1, which contains primers MS-R and AA-R, and gRNAs gMS1 and gAA1. The sequence is from the downstream right end of the inversion. The alignment at the bottom of the figure indicates that the DNA was cleaved at the gMS1 binding site and ligated upstream to the gAA1 binding site. Evidently, the Cas enzyme generated double-strand breaks (DSBs) at the two gRNA binding sites, which caused the inversion of a ~1.1 Mbp chromosomal fragment between them. The DSB was generated precisely at the predicted location of the gRNA binding site, between 3 to 4 base pairs upstream of the PAM site. Ligation of the end of the inverted chromosomal fragment occurred without any additional sequence modification.

[0148] Figure 4 shows a portion of the DNA sequence of a PCR product generated from genomic DNA from protoplasts transfected with primers MS-F and AA-F and construct 3. The sequence is from the upstream left end of the inversion. The alignment at the bottom of the figure indicates that the DNA was cleaved at the gMS3 binding site and the upstream portion was connected to the gAA3 binding site.

[0149] The Cas9 enzyme also generated double-strand breaks (DSBs) at the two gRNA binding sites here, which caused the inversion of a ~1.1 Mbp chromosomal fragment between them. The DSBs were generated precisely at the predicted locations of the gRNA binding sites, and ligation of the ends of the inverted chromosomal fragments occurred without any additional sequence modifications.

[0150] Figure 5 shows a portion of the DNA sequence of the PCR product generated from genomic DNA from protoplasts transfected with primers MS-R and AA-R and construct 4. The sequence is the downstream right end of the inversion. The alignment at the bottom of the figure indicates that the DNA was cleaved at the gMS4 binding site and ligated at the gAA4 binding site. The Cas enzyme also generated DSBs at the predicted locations of the gRNA binding sites on both sides of the inversion induced here, and the ligation at the end occurred with the deletion of one adenosine at the ligation site.

[0151] The three sequences in Figures 3 to 5 indicate that gRNA targets Cas9 to the predicted location and that DNA cleavage occurs at the expected location. Additionally, the sequenced transpositions demonstrate that DSBs were generated at two locations on the chromosome and, in some cases, the chromosomal fragment between them was reversed after repair. The sequences (transpositions) of the ligated ends demonstrate that DSBs were generated at the predicted location.

[0152] Therefore, Example 2 indicates that the inversion can be detected by boundary amplification from the inversion in most PCRs containing DNA from approximately 2,700 protoplasts. As described above, this would mean that the inversion occurs in about 1 out of 2,700 * 0.7 (~transfection efficiency) = approximately 1,900 protoplasts. Obtaining plants with the desired mutation would require a large number (>1,900) of regenerated buds to have a chance of detecting those with the desired mutation. In many species, including tomatoes, the regeneration of buds from protoplasts is technically very difficult and has not been successfully applied in some species.

[0153] To address this technical problem, the present invention provides a seed-based screening approach for identifying such mutations. Generally, CRISPR-Cas constructs result in double-strand breaks in DNA that are repaired in a number of events. The repair system often modifies the guide-RNA (gRNA) binding site, meaning that after repair, the binding site for gRNA disappears and new double-strand breaks cannot be generated. However, when plants containing an active CRISPR-Cas construct are crossed with wild-type plants, at least half of the resulting seeds will contain the active CRISPR-Cas construct in addition to wild-type DNA with unmodified gRNA binding sites. This means that each seed from these plants provides a new opportunity to induce and identify rare mutation events. Thus, if an event, e.g., a reversal, occurs once every 1,900 events, screening this number of multiplicity of seedlings can be performed to identify the target mutation. The screening may be PCR using boundary-specific primers as described above for protoplasts. To reduce the number of individual PCRs, 1-, 2-, or 3-dimensional pooling strategies can be designed.

[0154] Therefore, the method described herein makes it possible for the first time to provide a tomato plant containing three desirable traits simultaneously:

[0155] 1. Inducing male sterility in homozygous plants to facilitate the production of hybrid seeds MS Knockout;

[0156] 2. Absence of anthocyanin, and consequently causing loss of purple hypocotyl color AA Knockout.

[0157] 3. Mutant alleles induced by reversal ms and aa Genetic linkage of these two traits resulting from inhibition of meiotic recombination between them, and thus loss of sequence homology between the two genes.

Claims

Claim 1 A method for introducing and selecting specific heritable mutations in plants, comprising the following steps: (a) transfecting plant cells with exogenous DNA, wherein the exogenous DNA codes for an RNA-guided DNA endonuclease, a guide RNA (gRNA) for directing the RNA-guided DNA endonuclease to induce the specific heritable mutation, and a selection marker; (b) regenerating plants from the transfected cells to provide a plurality of T0 plants; (c) crossing the T0 plants with a homogeneous plant not containing the exogenous DNA to provide a plurality of offspring plants; and (d) selecting one or more plants having specific heritable mutations from the offspring plants, wherein at least one subset of T0 plants crossed with the homogeneous plant not containing the exogenous DNA comprises a DNA endonuclease / gRNA complex to induce the specific heritable mutation in the F1 generation. Claim 2 A method according to claim 1, wherein, prior to crossing a T0 plant with a homogeneous plant not containing the exogenous DNA, a T0 plant expressing an RNA-guided DNA endonuclease is selected, and one or more T0 plants expressing an RNA-guided DNA endonuclease are provided, and only the one or more T0 plants expressing the RNA-guided DNA endonuclease are crossed with the homogeneous plant not containing the exogenous DNA to provide multiple offspring plants. Claim 3 A method according to claim 2, wherein a selected T0 plant expressing an RNA-guided DNA endonuclease is a T0 plant in which RNA encoding the RNA-guided DNA endonuclease is detected. Claim 4 A method according to any one of claims 1 to 3, wherein the offspring plant is self-pollinated and then one or more plants having a specific hereditary mutation are selected from the offspring plant. Claim 5 A method according to any one of claims 1 to 3, wherein a subset of a plurality of T0 plants undergoes at least one self-pollination step and is pooled with a progeny plant obtained in step (c), wherein one or more plants having a specific heritable mutation are selected from the progeny plants obtained by said at least one self-pollination step. Claim 6 A method according to any one of claims 1 to 3, wherein the specific heritable mutation is an inversion, duplication, or translocation. Claim 7 A method according to any one of claims 1 to 3, wherein the RNA-guided DNA endonuclease is selected from the group consisting of Cas9, Cas12a, Cas12b, Cas12c, and Cas13. Claim 8 The method of claim 7, wherein the RNA-guided DNA endonuclease is Cas9 and: the gRNA comprises a trans-activated crRNA (tracrRNA) and at least one CRISPR RNA (crRNA); or the gRNA is a single guide RNA (sgRNA) comprising a tracrRNA combined with at least one crRNA. Claim 9 A method according to any one of claims 1 to 3, wherein one or more selected plants having specific heritable mutations are backcrossed to obtain offspring plants containing specific heritable mutations and not containing any exogenous DNA. Claim 10 A method according to any one of claims 1 to 3, wherein the transfected cell from which a T0 plant is regenerated is selected based on the activity of a selection marker. Claim 11 A method according to claim 10, wherein the selection marker is selected from the group consisting of kanamycin resistance and ampicillin resistance. Claim 12 A method according to any one of claims 1 to 3, wherein one or more plants having specific heritable mutations are selected by isolating genomic DNA from a portion of a progeny plant and determining whether said genomic DNA contains specific heritable mutations. Claim 13 In paragraph 12, the method in which the part of the offspring plant from which the genomic DNA is isolated is the cotyledon.

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