Methods for non-transgenic genome editing in plants

Abstract

To provide materials and methods for creating genome-engineered plants with non-transgenic methods.SOLUTION: Provided is a method for targeted genetic modification of a plant genome without inserting exogenous genetic material comprising the steps of: (i) providing a plant cell that comprises an endogenous gene to be modified; (ii) providing a sequence-specific nuclease comprising a sequence recognition domain and a nuclease domain; (iii) transfecting the plant cell with the sequence-specific nuclease; and (iv) inducing one or more double stranded DNA breaks (DSB) in the genome to produce a plant cell or cells having a detectable targeted genomic modification without the presence of any exogenous genetic material in the plant genome.SELECTED DRAWING: None

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority from U.S. Provisional Application No. 61 / 835,307, filed June 14, 2014.

[0002] [Technical field] This application relates to the field of plant molecular biology, and in particular provides materials and methods for producing genome-engineered plants using non-transgenic methods. [Background technology]

[0003] Traditional plant breeding strategies have been developed over many years to introduce desirable traits, such as increased drought tolerance and crop yield, into plant species. Such strategies have the disadvantage that they typically require many successive crosses and therefore can take many years to successfully alter a particular plant trait. The advent of transgenic technology has made it possible to engineer plants with genomic changes by introducing transgene constructs, thereby avoiding the need for traditional plant breeding. However, these transgenic techniques also have several drawbacks. First, transgene insertion into the genome (e.g., mediated by Agrobacterium tumefaciens) is highly random and can result in multiple insertions, which can make it difficult to track multiple transgenes present on different chromosomes during segregation. Furthermore, transgene expression can be unpredictable due to its chromosomal environment, and in many cases, transgene expression is silenced. In addition, the production of transgenic plants has proven to be a highly controversial topic, with public opinion often opposed to the creation of such varieties - particularly when the varieties in question are crop plants grown over wide geographic regions and used as food for human consumption.

[0004] A method that allows targeted modification of plant genomes can overcome the first two of these problems, allowing transgene insertion to be targeted to a single chromosomal site conducive to gene expression, thus reducing or eliminating the possibility of multiple transgene insertions and silencing events. Targeted genome modification has been demonstrated in many species using engineered zinc finger nucleases (ZFNs), which allow the creation of double-stranded DNA breaks at preselected loci and subsequent transgene insertion in a targeted manner (Lloyd et al. 2005; Wright et al. 2005; Townsend et al. 2009). A variation of this technology is to simply use ZFNs to create breaks at selected loci, and then allow DNA repair by NHEJ (non-homologous end joining). During this process, errors are often introduced into the newly added region (e.g., nucleotide deletions), and this method allows targeted mutagenesis of selected plant genes and the insertion of transgene constructs. Summary of the Invention [Problem to be solved by the invention]

[0005] The above-mentioned advantages in plant genetic engineering have not necessarily allayed public concerns regarding the production and widespread cultivation of engineered plant species. [Means for solving the problem]

[0006] Therefore, a solution to this problem is a method that can precisely alter a plant genome in a targeted manner without using traditional transgenic strategies. The disclosure herein provides such a solution by providing a method for targeted, non-transgenic editing of a plant genome. More specifically, the method relies on the introduction of a sequence-specific nuclease into a plant cell in the form of a protein or mRNA, which translocates to the nucleus and acts to precisely cut DNA at a predetermined locus. Errors that occur during the repair of the cut allow for the introduction of loss-of-function (or gain-of-function) mutations without introducing any exogenous genetic material into the genome.

[0007] In this way, genetically modified plant species can be produced that are free of residual exogenous genetic material.

[0008] Prior to the development of the methods described herein, genetic modification of plant cells required stable genomic integration of a transgene cassette for in vivo expression of a nuclease or DNA-modifying enzyme. Such integration was typically achieved through Agrobacterium-mediated transformation of plant species. However, as described herein, consistent and reproducible genome modification can be achieved through the introduction of either purified nuclease proteins or mRNA encoding such nucleases into plant cells. This is an unexpected effect, as recombinant nucleases or purified mRNA were not thought to be sufficiently active to have significant effects on plant chromosomal or organelle DNA. Furthermore, the present application provides novel protocols for genome modification, and also provides sequences and vectors suitable for carrying out the methods described herein and for producing modified plant cells without the introduction of exogenous DNA.

[0009] This application describes a method for editing plant genomes using non-transgenic strategies.Sequence-specific nucleases (including ZFN, homing endonucleases, TAL-effector nucleases, and CRISPR-associated systems [Cas9]) are introduced into plant cells in the form of purified nuclease protein or as mRNA encoding nuclease protein.In the case of CRISPR-associated systems [Cas9], nucleases can be introduced either as mRNA or purified protein together with guide RNA for target site recognition.

[0010] Functional nucleases target specific sequences and cleave cellular DNA at designated loci. DNA damage triggers plant cells to repair double-strand breaks. Errors (e.g., point mutations or small insertions / deletions) occur during DNA repair, resulting in altered DNA sequences in vivo.

[0011] Unlike traditional DNA transformation, the protein- or RNA-based genome editing strategies described herein specifically modify target nucleic acid sequences and leave no trace behind. Because these methods do not use foreign DNA, this process is considered to be non-transgenic plant genome editing.

[0012] In one aspect, the present application features a method for targeted genetic modification of a plant genome without inserting exogenous genetic material.The method can include the following steps: (i) providing a plant cell containing an endogenous gene to be modified; (ii) obtaining a sequence-specific nuclease containing a sequence recognition domain and a nuclease domain; (iii) transfecting the plant cell with the sequence-specific nuclease; and (iv) inducing one or more double-stranded DNA breaks (DSBs) in the genome to produce a plant cell(s) with detectable targeted genome modification without the presence of any exogenous genetic material in the plant genome.The DSBs can be repaired by non-homologous end joining (NHEJ).

[0013] The sequence-specific nuclease may be a TAL effector-nuclease, a homing endonuclease, a zinc finger nuclease (ZFN), or a CRISPR-Cas9 endonuclease. The sequence-specific nuclease can be delivered to plant cells in the form of purified protein or purified RNA (e.g., mRNA).

[0014] The sequence-specific nuclease may further comprise one or more subcellular localization domains. The one or more subcellular localization domains may comprise an SV40 nuclear localization signal, an hnRNPA1 acidic M9 domain, a PY-NLS motif signal, a mitochondrial targeting signal, or a chloroplast targeting signal. The sequence-specific nuclease may further comprise one or more cell-penetrating peptide domains (CPPs). The one or more CPPs may comprise a transactivating transcription activator (Tat) peptide or a Pep-1 CPP domain.

[0015] The sequence-specific nuclease can be co-transfected with one or more plasmids encoding one or more exonucleases, which may include members of the TREX exonuclease family (e.g., TREX2).

[0016] The endogenous gene to be modified can be an acetolactate synthase gene (e.g., ALS1 or ALS2) or a vacuolar invertase gene (e.g., the potato (Solanum tuberosum) vacuolar invertase gene (VInv)).

[0017] The plant cells may be from the following crop species: alfalfa, barley, bean, corn, cotton, flax, pea, rapeseed, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, wheat. The plant cells may be from the genus Nicotiana or from the species Arabidopsis thaliana.

[0018] Transfection can be achieved by delivering a sequence-specific nuclease to isolated plant protoplasts. For example, transfection can be achieved by using polyethylene glycol (PEG)-mediated transfection, electroporation, biolistic transfection, sonication-mediated transfection, or liposome-mediated transfection to deliver a sequence-specific nuclease to isolated plant protoplasts.

[0019] Introduction of one or more double-stranded DNA breaks in the genome can be followed by repair of the break(s) through homologous recombination mechanisms.

[0020] The present application also features transformed plant cells obtainable by the methods provided herein, and transformed plants comprising the plant cells.

[0021] In another aspect, this application features a kit for targeted genetic modification of a plant genome without inserting exogenous genetic material. The kit may include (i) one or more sequence-specific nucleases in protein or mRNA form, (ii) one or more plant protoplasts or cultured whole plant cells, and, optionally, (iii) one or more DNA plasmid vectors encoding one or more exonucleases.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.The present invention can be carried out using methods and materials similar or equivalent to those described herein, and suitable methods and materials are described below.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification (including definitions) will control.In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0023] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0024] [Figure 1] Schematic diagram showing the structural organization of sequence-specific nucleases used in genome engineering. Nucleases contain domains responsible for cell permeability, subcellular protein localization, DNA sequence recognition, and DNA cleavage. [Figure 2] 1 is a photograph showing SDS-PAGE of transcription activator-like effector endonucleases (TALEN™) produced in E. coli. ALS2T1L and ALS2T1R are TALEN™ that target sites within the ALS2 gene of Nicotiana benthamiana. VInv7 is a compact TALEN™ (cT) that targets the potato VInv gene. [Figure 3]Photograph of an agarose gel showing the in vitro activity of purified TALEN™ targeting the ALS2 gene of N. benthamiana. A PCR product containing the target site for the ALS2T1 TALEN™ was produced. The PCR product was incubated in the absence (-) or presence (+) of purified ALS2T1L and ALS2T1R proteins. The PCR product was cleaved only in the presence of the two proteins. As a negative control, purified ALS2T1L and ALS2T1R proteins were incubated with a PCR product from the ALS1 gene; no cleavage was observed. [Figure 4] 1 is a table showing the activity of I-SceI on episomal targets when delivered as a protein to plant cells. [Figure 5]

[0023] Figure 1 shows the activity of I-SceI activity against chromosomal sites when delivered to plant cells as a protein alone or in combination with TreX. The number of total sequencing reads used in this analysis is shown in parentheses in column 2. [Figure 6] Figure 1 is a sequence alignment showing examples of mutations induced by I-SceI in transgenic N. tabacum strains containing an integrated I-SceI recognition site. The top row (SEQ ID NO: 8) shows the DNA sequence of the recognition site for I-SceI (underlined). The other sequences (SEQ ID NOs: 9-18) show representative mutations induced by imprecise non-homologous end joining (NHEJ). [Figure 7] 1 is a graph summarizing the activity of TALEN™ ALS2T1 mutagenesis after transformation into plant cells with different forms (DNA or protein) or combinations of treatments. [Figure 8]1 is a sequence alignment showing examples of mutations induced by TALEN™ ALS2T1 in the ALS2 gene of N. benthamiana. The top row (SEQ ID NO: 19) shows the DNA sequence of the recognition site for ALS2T1 (underlined). The other sequences (SEQ ID NOs: 20-31) show representative mutations induced by imprecise non-homologous end joining (NHEJ). [Figure 9] Schematic diagram showing the structural organization of the sequence-specific nuclease used for in vitro mRNA production. The nuclease construct contains a T7 promoter, a nuclease ORF, and a 121 bp polyA tail. [Figure 10] Figure 1 is a graph plotting the cleavage activity of I-CreI mRNA delivered to plant protoplasts in a YFP-based SSA assay. An SSA targeting plasmid was co-delivered to tobacco protoplasts via PEG-mediated transformation along with p35S-I-CreI or I-CreI mRNA. 24 hours after transformation, protoplasts were subjected to flow cytometry to quantify the number of YFP-positive cells. [Figure 11]

[0049] Figure 10 is the target sequence of XylT_T04 TALEN™ in N. benthamiana (SEQ ID NO: 32). [Figure 12] This table summarizes 454 pyrosequencing data for delivery of Xyl_T04 TALEN™ mRNA into tobacco protoplasts. The numbers in parentheses in column 3 are the total number of sequencing reads obtained. *: The frequency of NHEJ mutagenesis was obtained by normalizing the proportion of 454 reads with NHEJ mutations to the efficiency of protoplast transformation. The total number of 454 sequencing reads used in this analysis is shown in parentheses. **: The negative control was obtained from protoplasts transformed only with a YFP-encoding plasmid. [Figure 13] 1 is a sequence alignment showing examples of mutations induced by XylT_T04 mRNA in the XylT1 (SEQ ID NOs: 33-43) and XylT2 (SEQ ID NOs: 44-54) genes in N. benthamiana. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present application provides a novel strategy for editing plant genomes to produce non-transgenic plant material. The methods provided herein are carried out using nucleases designed to recognize specific sequences at any site in the plant genome.

[0026] In one aspect, the present application relates to a method for targeted genetic modification of a plant genome without inserting exogenous genetic material, comprising one or several of the following steps: i) providing a plant cell containing an endogenous gene to be modified; ii) obtaining a sequence-specific nuclease comprising a sequence recognition domain and a nuclease domain; iii) transformation of plant cells with said sequence-specific nuclease; and iv) introduction of one or more double-stranded DNA breaks in the genome;

[0027] The method is aimed at producing a plant cell(s) with a detectable, targeted genome modification, preferably in the absence of any exogenous genetic material in the plant genome.

[0028] The introduction of a double-strand break in the genome generally results in repair of the break by non-homologous end joining (NHEJ), which favors the deletion, modification or insertion of genomic sequences in the genome of the plant cell obtained by this method.

[0029] After the coding sequence for nuclease is synthesized and cloned into an expression vector, the nuclease protein or mRNA is produced and purified. To improve efficacy, cell-penetrating peptides (CPPs) can be added to improve cell membrane permeability for small molecules (drugs), proteins, and nucleic acids (Mae and Langel, 2006; US2012 / 0135021, US2011 / 0177557, US7,262,267). Subcellular localization peptides can also be added to direct intracellular protein transport, particularly to the nucleus (Gaj et al. 2012; US20050042603).

[0030] In some embodiments, proteins with exonuclease activity, such as Trex (WO 2012 / 058458) and / or Tdt (terminal deoxynucleotidyl transferase) (WO 2012 / 13717), are co-delivered to plant cells to increase the efficiency of sequence-specific nuclease-induced mutagenesis. Trex2 (SEQ ID NO: 6) has been shown to be particularly effective at increasing mutagenesis as described herein. The use of Trex2 expressed as a single polypeptide chain (SEQ ID NO: 7) was even more effective.

[0031] Purified nucleases can be delivered to plant cells by a variety of means. For example, a biolistic particle delivery system can be used to transform plant tissue. Standard PEG and / or electroporation methods can be used for protoplast transformation. After transformation, plant tissue / cells are cultured to allow cell division, differentiation, and regeneration. DNA from individual events can be isolated and screened for mutations.

[0032] In some embodiments, the sequence-specific nuclease is a TAL-effector nuclease (Beurdeley et al., 2013). It is anticipated that any type of sequence-specific nuclease can be used to perform the methods provided herein, as long as it has similar functionality to a TAL-effector nuclease. Therefore, it should be possible to induce double-stranded DNA breaks at one or more targeted loci, resulting in one or more targeted mutations at the loci(s), where the mutations arise through misrepair of the breaks by NHEJ or other mechanisms (Certo et al., 2012). Such sequence-specific nucleases include, but are not limited to, ZFNs, homing endonucleases such as I-SceI and I-CreI, restriction endonucleases, and other homing endonucleases or TALENs. In certain embodiments, the endonuclease used comprises a CRISPR-associated Cas protein, such as Cas9 (Gasiunas et al., 2012).

[0033] The sequence-specific nuclease delivered may be in the form of purified nuclease protein or in the form of an mRNA molecule that can be translated into protein after transfection. Nuclease protein can be prepared by many methods known to those skilled in the art using available protein expression vectors, such as, but not limited to, pQE or pET. Suitable vectors allow for the expression and subsequent purification of nuclease protein in various cell types (E. coli, insects, mammals). Nuclease synthesis in the form of mRNA can be performed by various methods known to those skilled in the art, for example, through the use of a T7 vector (pSF-T7), which allows for the production of capped RNA for transfection into cells.

[0034] In some embodiments, mRNA is modified with optimal 5' untranslated region (UTR) and 3' untranslated region. UTR has been shown to play a crucial role in post-translational control of gene expression through regulating localization, stability, and translation efficiency (Bashirullah, 2001). As mentioned above, mRNA delivery is desirable due to its non-transgenic nature, but mRNA is a very fragile molecule and is easily degraded during the plant transformation process. The use of UTR in plant mRNA transformation allows for increased stability and localization of mRNA molecules, resulting in increased transformation efficiency for non-transgenic genome modification.

[0035] In some embodiments, engineered nucleases contain one or more subcellular localization domains to enable efficient trafficking of the nuclease protein within cells, particularly to the nucleus (Gaj. et al., 2012; US2005 / 0042603). Such localization signals may include, but are not limited to, the SV40 nuclear localization signal (Hicks et al., 1993). Other non-classical types of nuclear localization signals may also be adapted for use in the methods provided herein (e.g., the acidic M9 domain of hnRNP A1 or the PY-NLS motif signal (Dormann et al., 2012)). Localization signals may also be incorporated to enable trafficking of the nuclease to other subcellular compartments, such as mitochondria or chloroplasts. Descriptions of the specific mitochondrial and chloroplast signals used can be found in numerous publications (see Bhushan S. et al., 2006), and techniques for modifying proteins such as nucleases to contain these signals are known to those skilled in the art.

[0036] In some embodiments, the nuclease contains a cell-penetrating peptide domain (CPP), allowing for easier delivery of the protein across the cell membrane (Mae and Langel, 2006; US 2012 / 0135021, US 2011 / 0177557, US 7,262,267). Such CPP domains include, but are not limited to, the transactivating transcription activator (Tat) cell-penetrating peptide (Lakshmanan et al., 2013, Frankel et al., 1988). It is anticipated that other CPPs may also be used, including the Pep-1 CPP domain, which is particularly suitable for aiding in the delivery of proteins into plant cells (see Chugh et al., 2009).

[0037] In some embodiments, the one or more mutations occur in the coding sequence of one of the acetolactate synthase (ALS) genes ALS1 or ALS2, or the one or more mutations occur in the vacuolar invertase (VInv) gene. In further aspects of these embodiments, the mutation can be any transition or transversion that results in a non-functional or functionally reduced coding sequence at a given genetic locus. It is generally expected that one or more mutations can be generated at any particular genomic locus using the methods described herein.

[0038] In some embodiments, the plant species used in the methods provided herein is N. benthamiana, while in further aspects, the plant species may be any monocotyledonous or dicotyledonous plant, such as (but not limited to) Arabidopsis thaliana; agricultural crops (e.g., alfalfa, barley, beans, corn, cotton, flax, peas, rapeseed, rice, rye, safflower, sorghum, soybeans, sunflowers, tobacco, and wheat); vegetable crops (e.g., asparagus, beets, broccoli, cabbage, carrots, cauliflower, celery, cucumber, eggplant, lettuce, onion, bell peppers, potatoes, pumpkins, radishes, spinach, squash, taro, tomatoes, and zucchini); fruit and nut crops (e.g., almonds, apples, apricots, bananas, blackberries, blueberries, cocoa, cherries, coconuts, cucumbers, and zucchini); ramberry, date palm, feijoa, hazel, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut, and watermelon); and ornamental plants (e.g., alder, ash, aspen, azalea, birch, boxwood, camellia, carnation, chrysanthemum, elm, fir, ivy, jasmine, juniper, oak, palm, poplar, pine, sequoia, rhododendron, rose, and rubber).

[0039] In some embodiments, mRNA encoding proteins or nuclease constructs is delivered to plant cells via PEG-mediated transformation of isolated protoplasts. PEG is typically used in a range of half to an equal volume of the mRNA or protein suspension to be transfected, with 40% PEG being commonly used for this purpose.

[0040] In some cases, nucleases may be delivered via biolistic transformation methods or any other suitable transfection method known in the art (Yoo et al., 2007). In the case of biolistic transformation, nucleases can be introduced into plant tissues using a biolistic device that accelerates microprojectiles to speeds of 300-600 m / s, sufficient to penetrate plant cell walls and membranes (see Klein et al., 1992). Another method of introducing proteins or RNA into plants is via sonication of target cells.

[0041] Alternatively, liposome or spheroplast fusion may be used to introduce exogenous material into plants (see, e.g., Christou et al., 1987). Electroporation of protoplasts and whole cells and tissues has also been described (Laursen et al., 1994).

[0042] Depending on the transfection method used and its efficiency, we have determined that the optimal protein concentration for carrying out the methods described herein (particularly using TALEN™) is 0.01-0.1 μg / μl. When using PEG, the volume of the protein suspension was generally 2-20 μl. RNA concentrations were found to be optimal in the range of 1-5 μg / μl, and it was thought that adding non-coding RNA (e.g., carrier tRNA) up to 10 μg / μl to increase the RNA bulk could sometimes be advantageous. The subsequent addition of this RNA improves transfection and has a protective effect on nuclease-encoding RNA with respect to degradative enzymes encountered in plant cells.

[0043] Plants can be obtained by regenerating plant cells produced by any of the methods described herein. If the function of an endogenous gene is suppressed in a plant cell into which a non-silent mutation has been introduced at a target DNA site, the phenotype of the plant regenerated from such a plant cell may be altered in association with the suppression of the function of the endogenous gene. Thus, the methods described herein enable efficient plant breeding. Plants can be regenerated from plant cells by methods known to those skilled in the art, depending on the type of plant cell. Examples include the method described by Christou et al. (1997) for the transformation of rice varieties.

[0044] In some embodiments, nucleases can be co-delivered with a plasmid encoding one or more exonuclease proteins to increase the efficiency of sequence-specific nuclease-induced mutagenesis. Such exonucleases include, but are not limited to, members of the Trex family of exonucleases (therapeutic erythrocyte replacement exonucleases), such as TREX2 (Shevelev et al. 2002). The inventors surprisingly found that co-delivery of an exonuclease such as TREX with purified I-SceI protein increases the frequency of observed NHEJ events compared to delivery of I-SceI protein alone. It should be noted that other suitable exonucleases may also be used in the methods provided herein.

[0045] The term "identity" as used herein refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity is determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base, the molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Alignment algorithms and programs are used to calculate the identity between two sequences. FASTA and BLAST are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, WI) and are used by default. BLASTP can also be used to identify amino acid sequences with at least 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, or 99% sequence similarity to a reference amino acid sequence using a similarity matrix, such as BLOSUM45, BLOSUM62, or BLOSUM80. Unless otherwise indicated, similarity scores are based on the use of BLOSUM62. When using BLASTP, percent similarity is based on BLASTP positive scores, and percent sequence identity is based on BLASTP identity scores. BLASTP "identity" indicates the number and fraction of total residues in a high-scoring sequence pair that are identical; and BLASTP "positive" indicates the number and fraction of residues that have a positive alignment score and are similar to each other. Amino acid sequences with these degrees of identity or similarity, or any intermediate degree of similarity, to the amino acid sequences disclosed herein are contemplated and encompassed by the present disclosure. The same is true for polynucleotide sequences using BLASTN.

[0046] As used herein, the term "homologous" is intended to mean a sequence that has sufficient identity to another sequence to result in homologous recombination between the sequences, and more particularly, has at least 95% identity (e.g., at least 97% identity, or at least 99% identity).

[0047] As used herein, the term "endonuclease" refers to an enzyme that is capable of creating double-stranded breaks in DNA molecules at highly specific locations.

[0048] The term "exonuclease," as defined herein, refers to an enzyme that acts by cleaving nucleotides one by one from the end (exo) of a polynucleotide chain, resulting in a hydrolysis reaction that cleaves a phosphodiester bond at either the 3' or 5' end.

[0049] As used herein, the term "sequence-specific nuclease" refers to any nuclease enzyme that is capable of inducing a double-stranded DNA break at a desired and predetermined genomic locus.

[0050] As used herein, the term "meganuclease" refers to a naturally occurring or engineered rare-cutting endonuclease that typically has a polynucleotide recognition site approximately 12-40 bp in length, more preferably 14-40 bp in length. Typical meganucleases effect cleavage within their recognition site, resulting in a 4-nt staggered cut with a 3'OH overhang. The meganuclease is preferably a homing endonuclease, more specifically, a TAL-effector-like endonuclease, a zinc finger nuclease, or any nuclease fused to a modular base-pair-binding domain (MBBBD) that is capable of binding to a predetermined nucleic acid target sequence and inducing cleavage at sequences adjacent to it. These meganucleases are useful for inducing double-strand breaks at specific DNA sequences, thereby facilitating targeted manipulation of genomic sequences and site-specific homologous recombination.

[0051] As used herein, the term "vector" refers to a nucleic acid molecule capable of transferring another nucleic acid to which it is linked into a cell or cellular compartment.

[0052] As used herein, the term " zinc finger nuclease " refers to the artificial restriction enzyme produced by fusing zinc finger DNA binding domain with DNA cleavage domain.Simply put, ZFN is a synthetic protein that comprises engineered zinc finger DNA binding domain fused with the cleavage domain of FokI restriction endonuclease.ZFN can be used to induce double-strand breaks in specific DNA sequences, thereby promoting targeted manipulation of genomic sequences and site-specific homologous recombination.

[0053] As used herein, the term "TAL-effector endonuclease" refers to an artificial restriction enzyme produced by fusing the DNA recognition domain from the Xanthomonas TALE protein to the catalytic domain of a nuclease, as described by Voytas and Bogdanove in WO2011 / 072246. TAL-effector endonucleases are designated TALEN™ by the applicant (Cellectis, 8 rue de la Croix Jarry, 75013 PARIS).

[0054] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0055] Example 1: Design and construction of sequence-specific nucleases for protein expression. Sequence-specific nucleases typically contain the following components (Figure 1): 1. A DNA-binding domain that recognizes specific DNA sequences within the plant genome. 2. A nuclease domain that generates a DNA double-strand break at a recognition site in the plant genome. Imprecise repair of the break through non-homologous end joining introduces a mutation at the break site. 3. Subcellular localization signals that direct the nuclease to the nucleus, mitochondria, or chloroplasts. 4. A cell-permeable motif that helps sequence-specific nucleases penetrate the cell membrane during transformation.

[0056] The custom nuclease-encoding sequence may be synthesized and cloned into a protein expression vector, such as pQE or pET. Thus, functional proteins can be expressed in E. coli and purified using standard protocols or commercially available kits. Alternatively, other protein expression systems, including yeast, insect, or mammalian cells, can be used to produce proteins that are difficult to express and purify in E. coli.

[0057] Here, pQE-80L-Kan was used as the protein expression vector. The SV40 nuclear localization signal was added, as was the Tat cell-penetrating peptide (Frankel and Pabo, 1988; Schwarze et al., 1999). The sequence-specific nuclease included a TALEN™ pair targeting a site within the ALS2 gene of N. benthamiana. Additionally, a compact TALEN™ targeting a site within the VInv7 gene of S. tuberosum was also used. E. coli strain BL21 was used for protein expression (Beurdeley et al., 2013). The Qiagen Ni-NTA Spin Kit was used for protein purification. High yields of recombinant protein were obtained in E. coli for all three TALEN™ (Figure 2). Plasmids for producing recombinant TALEN™ were provided by Cellectis Bioresearch (8, rue de la Croix Jarry, 75013 PARIS).

[0058] Example 2: In vitro sequence-specific nuclease activity of purified TALEN™. To test the enzymatic activity of purified TALEN™, equal amounts of ALS2T1L (SEQ ID NO: 2) and ALS2T1R (SEQ ID NO: 3) proteins were mixed and incubated with a PCR fragment derived from the N. benthamiana ALS2 gene (the PCR product contains a TALEN recognition site). The reaction was carried out at 25°C and contained the following buffer system: 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, and 1 mM dithiothreitol, pH 7.9. A PCR fragment derived from the N. benthamiana ALS1 gene (lacking the TALEN recognition site) was used as a negative control. The two TALENs clearly cleaved the ALS2 gene fragment in vitro; no activity was observed with the ALS1 fragment (Figure 3). The data indicate that purified TALEN™ possesses sequence-specific nuclease activity.

[0059] Example 3: Delivery of sequence-specific nucleases into plant cells as proteins. The enzyme I-SceI was purchased from New England Biolabs and dialyzed to remove the buffer provided by the manufacturer. Briefly, 20 μl (100 U) of I-SceI was placed on a Millipore 0.025 μm VSWP filter (catalog no. VSWP02500). The filter was floated on MMG buffer (0.4 M mannitol, 4 mM MES, 15 mM MgCl2, pH 5.8) for 1 h at 4°C. After the enzyme solution was fully equilibrated with the MMG buffer, it was transferred to a new tube and kept on ice until further use. Proteins were delivered into plant cells by PEG-mediated protoplast transformation. Tobacco protoplast preparation was performed as previously described (Zhang et al. 2013). Briefly, seeds from transgenic tobacco lines carrying an integrated I-SceI recognition site were planted in moist vermiculite and cultured for 3–5 weeks under low-light conditions ( Pacher et al., 2007 ). Young, fully expanded leaves were collected and surface sterilized to isolate protoplasts.

[0060] Purified I-SceI protein was introduced into N. tabacum protoplasts by PEG-mediated transformation as described elsewhere (Yoo et al., Nature Protocols 2:1565-1572, 2007). Briefly, 20–200 U of I-SceI protein was mixed with 200,000 protoplasts at room temperature in 200 μl of 0.4 M mannitol, 4 mM MES, 15 mM MgCl2, pH 5.8. Other treatments included transforming protoplasts with DNA encoding I-SceI (SEQ ID NO: 1), DNA encoding Trex2 protein, or both. Trex2 is an endonuclease that increases the frequency of imprecise DNA repair by NHEJ (Certo et al. 2012). In addition, in some samples, I-SceI protein was co-delivered with DNA encoding Trex2 (SEQ ID NO: 6). After transformation, an equal volume of 40% PEG-4000, 0.2 M mannitol, 100 mM CaCl2, pH 5.8 was added to the protoplasts and immediately mixed well. The mixture was incubated in the dark for 30 minutes before being washed once with 0.45 M mannitol, 10 mM CaCl2. The protoplasts were then washed twice with K3G1 medium before transferring the cells to 1 ml of K3G1 in a Petri dish for long-term culture.

[0061] Example 4: Activity of I-SceI against episomal target sites in N. tabacum. To assess the protein activity of I-SceI targeting episomal sites in plant cells, the SSA construct was co-delivered with I-SceI protein. For this assay, a target plasmid was constructed with an I-Sce recognition site cloned into a nonfunctional YFP reporter gene. Direct repeats of the YFP coding sequence were placed next to the target site so that when the reporter gene was cleaved by I-Sce, recombination would occur between the direct repeats, restoring function to the YFP gene. Thus, YFP expression served as a measure of I-SceI cleavage activity.

[0062] The activity of I-SceI protein and its control treatment against episomal target sequences is summarized in Figure 4. Delivery of I-SceI as DNA resulted in 0.49% YFP expression; whereas, I-SceI protein resulted in 4.4% expression of YFP. When the SSA construct and I-SceI protein were delivered sequentially, the SSA efficiency of YFP was 2.4%. Transformation efficiency was shown by the YFP expression of the control, 35S:YFP DNA delivery.

[0063] Example 5: Activity of I-SceI against their endogenous target sites in N. tabacum. The transgenic tobacco line contains a single I-SceI recognition site in its genome, as previously shown (Pacher et al., 2007). Transformed protoplasts isolated from this transgenic line were harvested 24–48 h after treatment, and genomic DNA was prepared. Using this genomic DNA as a template, a 301-bp fragment encompassing the I-SceI recognition site was amplified by PCR. The PCR product was then subjected to 454 pyrosequencing. Sequencing reads containing insertion / deletion (indel) mutations within the recognition site were considered to result from imprecise repair of the excised I-SceI recognition site by NHEJ. The frequency of mutagenesis was calculated as the number of sequencing reads containing NHEJ mutations among all sequencing reads.

[0064] The activity of I-SceI protein against its target sequence and its control treatment is summarized in Figure 5. Delivery of I-SceI (SEQ ID NO: 1) as DNA resulted in a 15% mutagenesis frequency. When combined with DNA encoding the exonuclease Trex2, the mutagenesis frequency increased to 59.2%. When I-SceI was delivered as a protein, no mutagenesis activity was detectable; however, when I-SceI protein was co-delivered with DNA encoding Trex2 (SEQ ID NO: 6), a 7.7% mutagenesis frequency was observed. Examples of mutations induced by I-SceI protein are shown in Figure 6. Collectively, the data indicate that I-SceI protein, when delivered as a protein to plant cells, results in targeted chromosomal breaks. Furthermore, imprecise repair of these breaks leads to the introduction of targeted mutations.

[0065] Example 6: Delivery of TALEN proteins into plant cells. Purified TALEN™ proteins were introduced into N. benthamiana protoplasts by PEG-mediated transformation, as described in Example 4. Briefly, 2–20 μl of ALS2T1 protein was mixed with 200,000 protoplasts in 200 μl of 0.4 M mannitol, 4 mM MES, 15 mM MgCl2, pH 5.8 at room temperature. Other treatments included transforming protoplasts with Trex2 protein, DNA encoding ALS2T1, DNA encoding Trex2 protein, or a DNA construct for YFP expression. Trex2 is an endonuclease that increases the frequency of imprecise DNA repair by NHEJ. After transformation, an equal volume of 40% PEG-4000, 0.2 M mannitol, 100 mM CaCl2, pH 5.8 was added to the protoplasts and immediately mixed well. The mixture was incubated in the dark for 30 minutes before being washed once with 0.45 M mannitol, 10 mM CaCl. The protoplasts were then washed twice with K3G1 medium before transferring the cells to 1 ml of K3G1 in a Petri dish for long-term culture.

[0066] Example 7: Activity of TALEN™ ALS2T1 against their endogenous target sites in N. benthamiana. Transformed protoplasts were harvested 48 hours after treatment, and genomic DNA was prepared. Using this genomic DNA as a template, a 253-bp fragment encompassing the ALS2T1 recognition site was amplified by PCR. The PCR product was then subjected to 454 pyrosequencing. Sequencing reads containing insertion / deletion (indel) mutations within the recognition site were considered to result from imprecise repair of the excised I-SceI recognition site by NHEJ. The frequency of mutagenesis was calculated as the number of sequencing reads containing NHEJ mutations among all sequencing reads.

[0067] The activity of the ALS2T1 protein against target sequences and its control treatments is summarized in Figure 7. Delivery of ALS2T1 (SEQ ID NOs: 2 and 3) as DNA resulted in a mutagenesis frequency of 18.4%. When combined with DNA encoding the exonuclease Trex2 (SEQ ID NO: 6), the mutagenesis frequency increased to 48%. When ALS2T1 was delivered as a protein, the mutagenesis activity ranged from 0.033% to 0.33%, whereas when the ALS2T1 protein was co-delivered with 35S:YFP DNA, a mutagenesis frequency of 0.72% was observed. Examples of mutations induced by the ALS2T1 protein are shown in Figure 8. Collectively, the data indicate that the ALS2T1 protein, when delivered as a protein into plant cells, results in targeted chromosomal breaks. Furthermore, imprecise repair of these breaks leads to the introduction of targeted mutations.

[0068] Example 8: Preparation of mRNA encoding a sequence-specific nuclease. Sequence-specific nucleases, including meganucleases, zinc finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs), are cloned into T7 expression vectors (Figure 9). A variety of different 5' and 3' UTR pairs were selected based on data from a genome-wide study of transcription decay rates in A. thaliana (Narsai, 2007). The selected sequences were based on the half-lives and functional categories of the various transcripts. These UTR pairs were synthesized to allow convenient cloning into T7-driven plasmid vectors. The resulting nuclease constructs were linearized by SapI digestion; the SapI site is located immediately after the polyA sequence. The linearized plasmid served as a DNA template for in vitro mRNA production using the T7 Ultra kit (Life Technologies Corporation). Alternatively, mRNA encoding the nucleases can be prepared by commercial sources. The synthesized mRNA is dissolved in nuclease-free distilled water and stored at -80°C.

[0069] Example 9: Activity of sequence-specific nucleases delivered as mRNA against episomal targets. A single-strand annealing (SSA) assay was used to measure the activity of nuclease mRNA transformed into tobacco protoplasts (Zhang et al. 2013). As described in Example 4, the SSA assay uses a non-functional YFP reporter that is cleaved by the nuclease. Upon cleavage, recombination between repeat sequences within the reporter reconstitutes a functional YFP gene. As a result, YFP fluorescence can be quantified by flow cytometry.

[0070] To determine whether mRNA can be delivered to plant cells and mediate targeted DNA modification, I-CreI mRNA was introduced into tobacco protoplasts via PEG-mediated transformation along with the SSA targeting plasmid (Golds et al. 1993, Yoo et al. 2007, Zhang et al. 2013). The SSA reporter contains an I-CreI site between repeat sequences within YFP. Methods for tobacco protoplast preparation and transformation were as previously described (Zhang et al. 2013). The SSA targeting plasmid alone served as a negative control. As a positive control, cells were transformed with a DNA construct expressing I-CreI (p35S-I-CreI) and the I-CreI SSA reporter. 24 hours after transformation, YFP fluorescence was measured by flow cytometry (Figure 10). Similar levels of targeted cleavage of the SSA reporter were observed with both p35S-I-CreI DNA and I-CreI mRNA. The data show that functional nucleases can be successfully delivered to protoplasts in the form of mRNA.

[0071] Example 10: Cleavage activity of sequence-specific nucleases delivered as mRNA against chromosomal targets. A TALEN pair (XylT TALEN™) was designed to cleave the endogenous β1,2-xylosyltransferase gene of N. benthamiana (Strasser et al. 2008) (Figure 11). These genes were named XylT1 and XylT2, and the TALEN™ recognizes the same sequence found in both genes. Each XylT TALEN™ was subcloned into a T7-driven expression plasmid (Figure 12). The resulting TALEN™ expression plasmid was linearized by SapI digestion and served as a DNA template for in vitro mRNA production as described in Example 8.

[0072] Next, plasmid DNA or mRNA encoding TALEN™ was introduced into N. benthamiana protoplasts by PEG-mediated transformation (Golds et al., 1993, Yoo et al., 2007, Zhang et al., 2013). Protoplasts were isolated from well-expanded leaves of 1-month-old N. benthamiana. The protoplast density was 5 × 10 5 / ml ~ 1 × 10 6 The cell density was adjusted to 100 / ml, and 200 μl of protoplasts were used for each transformation. For mRNA delivery, an RNA cocktail was prepared by mixing 15 μl of L-TALEN mRNA (2 μg / μl), 15 μl of R-TALEN mRNA (2 μg / μl), and 10 μl of yeast tRNA carrier (10 μg / μl). To minimize potential RNAse degradation, the RNA cocktail was immediately added to 200 μl of protoplasts and gently mixed by finger tapping for only a few seconds. Almost immediately, 210 μl of 40% PEG was added and mixed well by finger tapping for 1 minute. The transformation reaction was incubated at room temperature for 30 minutes. Transformation was stopped by adding 900 μl of wash buffer. After several washes, the transformed protoplasts were cultured at 5 × 10 in K3 / G1 medium. 5 The cells were cultured at a cell density of 100 / ml. Plasmid DNA encoding TALEN was also transfected as a positive control.

[0073] Three days after treatment, the transformed protoplasts were harvested and genomic DNA was prepared. Using the genomic DNA prepared from the protoplasts as a template, an approximately 300 bp fragment encompassing the TALEN™ recognition site was amplified by PCR. The PCR product was then subjected to 454 pyrosequencing. Sequencing reads with insertion / deletion (indel) mutations within the spacer region were considered to result from imprecise repair of the cleaved TALEN™ recognition site by non-homologous end joining (NHEJ). The frequency of mutagenesis was calculated as the number of sequencing reads with NHEJ mutations out of the total number of sequencing reads.

[0074] Xyl_T04 TALEN™ DNA and mRNA were tested on their targets, namely, the XylT1 and XylT2 genes in N. benthamiana. As shown above, TALEN™ recognition sites are present in both the XylT1 and XylT2 genes. As summarized in Figure 12, Xyl_T04 TALEN™ plasmid DNA induced very high frequencies of NHEJ mutations in both genes, ranging from 31.2% to 54.9%. Concurrently, Xyl_T04 TALEN™ mRNA also induced high frequencies of NHEJ mutations in both genes, ranging from 22.9% to 44.2%. Examples of TALEN™-induced mutations at the XylT1 and XylT2 loci are shown in Figure 13.

[0075] [Other embodiments] While the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is for illustrative purposes only and does not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. [References] JPEG2025122000000001.jpg212153 JPEG2025122000000002.jpg217153 JPEG2025122000000003.jpg85153

Claims

1. 1. A method for targeted genetic modification of a plant genome without inserting exogenous genetic material, comprising: (i) providing a plant cell containing an endogenous gene to be modified; (ii) providing a sequence-specific nuclease comprising a sequence recognition domain and a nuclease domain; (iii) transfecting the plant cell with the sequence-specific nuclease; and (iv) inducing one or more double-stranded DNA breaks (DSBs) in the plant genome to produce plant cell(s) having a detectable targeted genome modification in the absence of any exogenous genetic material in the plant genome; Including, method.

2. 10. The method of claim 1, The DSB is repaired by non-homologous end joining (NHEJ). method.

3. 3. The method of claim 2, Induction of one or more double-stranded DNA breaks in the genome followed by repair of the break(s) through a homologous recombination mechanism. method.

4. 10. The method of claim 1, The sequence-specific nuclease is a TAL effector-nuclease. method.

5. 10. The method of claim 1, The sequence-specific nuclease is a homing endonuclease. method.

6. 10. The method of claim 1, The sequence-specific nuclease is a zinc finger nuclease (ZFN). method.

7. 10. The method of claim 1, The sequence-specific nuclease is a CRISPR-Cas9 endonuclease. method.

8. 8. The method of any one of claims 1 to 7, The sequence-specific nuclease is in the form of a purified protein. method.

9. 8. The method of any one of claims 1 to 7, The sequence-specific nuclease is in the form of RNA, such as purified mRNA. method.

10. 10. The method of any one of claims 1 to 9, The sequence-specific nuclease further comprises one or more subcellular localization domains. method.

11. 11. The method of claim 10, the one or more subcellular localization domains comprise an SV40 nuclear localization signal; method.

12. 11. The method of claim 10, the one or more subcellular localization domains comprise the acidic M9 domain of hnRNPA1; method.

13. 11. The method of claim 10, the one or more subcellular localization domains comprise a PY-NLS motif signal; method.

14. 11. The method of claim 10, the one or more subcellular localization domains comprise a mitochondrial targeting signal; method.

15. 11. The method of claim 10, the one or more subcellular localization domains comprise a chloroplast targeting signal; method.

16. 16. The method of any one of claims 1 to 15, The sequence-specific nuclease further comprises one or more cell-penetrating peptide domains (CPPs). method.

17. 17. The method of claim 16, the one or more CPPs comprise a transactivating transcription activator (Tat) peptide; method.

18. 17. The method of claim 16, the one or more CPPs comprise a Pep-1 CPP domain; method.

19. 19. The method of any one of claims 1 to 18, The sequence-specific nuclease protein is co-transfected with one or more plasmids encoding one or more exonucleases. method.

20. 20. The method of claim 19, The one or more exonucleases include members of the TREX exonuclease family, such as TREX2. method.

21. 21. The method of any one of claims 1 to 20, The endogenous gene to be modified is an acetolactate synthase gene, such as ALS1 or ALS2. method.

22. 21. The method of any one of claims 1 to 20, The endogenous gene to be modified is a vacuolar invertase gene, such as the potato (Solanum tuberosum) vacuolar invertase gene (VInv). method.

23. 23. The method of any one of claims 1 to 22, The plant cells are derived from the following crop species: alfalfa, barley, bean, corn, cotton, flax, pea, rapeseed, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, wheat; method.

24. 24. The method of claim 23, The plant cell is from the genus Nicotiana. method.

25. 24. The method of claim 23, The species is Arabidopsis thaliana. method.

26. 26. The method of any one of claims 1 to 25, Transfection is achieved through delivery of the sequence-specific nuclease to isolated plant protoplasts. method.

27. 27. The method of claim 26, The delivery comprises polyethylene glycol (PEG)-mediated transfection. method.

28. 27. The method of claim 26, The delivery comprises electroporation. method.

29. 27. The method of claim 26, The delivery comprises biolistic-mediated transfection. method.

30. 27. The method of claim 26, The delivery comprises sonication-mediated transfection. method.

31. 27. The method of claim 26, The delivery comprises liposome-mediated transfection. method.

32. 32. A method according to claim 1 , Transformed plant cells.

33. 33. The plant cell of claim 32. Transformed plants.

34. 1. A kit for targeted genetic modification of a plant genome without inserting exogenous genetic material, comprising: The kit comprises: (i) one or more sequence-specific nucleases in the form of proteins or mRNA; (ii) one or more plant protoplasts or cultured whole plant cells; and, in some cases, (iii) one or more DNA plasmid vectors encoding one or more exonucleases; Including, kit.

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