A method for abiotic stress resiliency in crops

Abstract

The present invention discloses a method for producing a modified crop plant with enhanced tolerance to abiotic stress-induced oxidative stress. The method comprises introducing at least one genomic modification in the endogenous ATR7 gene using targeted genome editing techniques. A modified crop plant exhibiting improved resilience to oxidative stress conditions induced by abiotic factors, due to the disruption or knockout of the ATR7 gene is also disclosed.

Description

[0001] A METHOD FOR ABIOTIC STRESS RESILIENCY IN CROPS

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to the field of agricultural biotechnology, specifically to methods for conferring desirable abiotic traits in crop plants. More particularly, the current invention pertains to the production of crop plants with enhanced tolerance to abiotic stresses through the manipulation of genes that control oxidative stress pathways in these plants.

[0004] BACKGROUND OF THE INVENTION

[0005] Crop plants are frequently exposed to various environmental stressors that can lead to oxidative stress, a condition characterized by an imbalance between the production of reactive oxygen species (ROS) and the plant's ability to detoxify these reactive intermediates. Increased levels of reactive oxygen species (ROS) in plants are a consequence of various adverse abiotic conditions such as drought, salinity, extreme temperatures, and pollutants, as well as biotic interactions that trigger the hypersensitive response (HR) to pathogens or programmed cell death (PCD).

[0006] Therefore, the genetic basis for traits such as disease resistance and stress tolerance has been a focus of modem breeding programs, aiming to enhance the plant's resilience to environmental challenges.

[0007] Given these persistent challenges, there remains a significant and unmet need to develop effective methods for regulating oxidative stress pathways in crop plants, thereby improving their ability to withstand environmental stressors.

[0008] SUMMARY OF THE INVENTION

[0009] According to the teachings of the present invention, there is provided a method for producing a modified crop plant with abiotic stress-induced oxidative stress tolerance, the method comprising introducing at least one genomic modification in an endogenous ATR7 gene using targeted genome editing techniques.

[0010] It is another object of the present invention, to provide the method as defined above, wherein the at least one genomic modification is a disruption or knock out mutation of the ATR7 gene in said crop plant. It is another object of the present invention, to provide the method as defined in any of the above, wherein the at least one genomic modification is a mutation conferring reduced to completely loss of function of the ATR7 gene.

[0011] It is another object of the present invention, to provide the method as defined in any of the above, wherein the method comprises steps of: (a) selecting a target crop plant species; (b) identifying the ATR7 gene homolog or ortholog of said target plant species; (c) utilizing CRISPR / Cas9 genome editing technology to disrupt or knock out the ATR7 gene in said target plant species; and (d) optionally, generating edited plant lines from the disrupted or knock out ATR7 gene plants with tolerance to abiotic stress-induced oxidative stress conditions.

[0012] It is another object of the present invention, to provide the method as defined in any of the above, wherein said modified crop plant exhibits enhanced abiotic stress -induced oxidative stress tolerance compared to wild-type or a corresponding crop plant lacking the at least one genomic modification.

[0013] It is another object of the present invention, to provide the method as defined in any of the above, wherein said abiotic stress-induced oxidative stress conditions comprise oxidative and abiotic stresses including hydrogen peroxide, ROS -inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCh), osmotic, drought and salinity stress.

[0014] It is another object of the present invention, to provide the method as defined in any of the above, wherein said modified crop plant is a non-transgenic or non-GMO plant.

[0015] It is another object of the present invention, to provide the method as defined in any of the above, wherein said modified crop plant belongs to a crop family selected, but not limited to, from the group consisting of cereals, legumes, fruits, and vegetables.

[0016] It is another object of the present invention, to provide the method as defined in any of the above, wherein said modified crop plant is a plant species selected from, among others, tomato, cucumber, lettuce, rice, maize, soybean, wheat, and potato.

[0017] It is another object of the present invention, to provide the method as defined in any of the above, wherein said modified crop plant is a Tomato plant (Solarium lycopersicum).

[0018] It is another object of the present invention, to provide the method as defined above, wherein said ATR7 gene is Solarium lycopersicum (SI) ATR7 (S1ATR7) comprising a wild-type genomic sequence comprising at least 80% identity to SEQ ID NO: 153 or a genomic sequence encoding a wild-type amino acid sequence comprising at least 80% identity to SEQ ID NO: 157.

[0019] It is another object of the present invention, to provide the method as defined in any of the above, wherein said method comprising generating the genetic modification in planta via transforming the crop plant with a construct comprising (a) Cas DNA and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3- 152 and any combination thereof.

[0020] It is another object of the present invention, to provide the method as defined in any of the above, wherein said genomically edited ATR7 gene comprises a mutated genomic sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 154-156 or a genomic sequence encoding a mutated amino acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 158-160.

[0021] It is another object of the present invention, to provide the method as defined in any of the above, wherein said method comprises steps of regeneration in a tissue culture positively selected transformed plants.

[0022] It is another object of the present invention, to provide the method as defined in any of the above, wherein said method comprises steps of screening and selecting for plants comprising editing events within ATR7 genomic region targeted for editing, from the regenerated positively selected transformed plants.

[0023] It is another object of the present invention, to provide the method as defined in any of the above, wherein said method comprises steps of selection of transformed crop plants comprising an ATR7 knock-out editing event conferring abiotic stress-induced oxidative stress tolerance.

[0024] It is another object of the present invention, to provide a modified crop plant with abiotic stress- induced oxidative stress tolerance, produced by the method as defined in any of the above.

[0025] It is another object of the present invention, to provide a plant part, plant tissue, plant fruit or vegetable, plant seed or plant cell of the modified crop plant as defined above.

[0026] It is another object of the present invention, to provide a tissue culture of regenerable cells, protoplasts or callus obtained from the modified crop plant as defined above. It is another object of the present invention, to provide the modified crop plant as defined above, wherein said modified crop plant does not comprise a transgene.

[0027] It is another object of the present invention, to provide a method for enhancing tolerance to abiotic stress-induced oxidative stress conditions in a crop plant, the method comprising introducing at least one genomic modification in an endogenous ATR7 gene using targeted genome editing techniques.

[0028] It is another object of the present invention, to provide the method as defined above, wherein said method comprises steps of (a) selecting a target crop plant species; (b) identifying an ATR7 gene homolog or ortholog in said target plant species; (c) utilizing CRISPR / Cas9 genome editing technology to disrupt or knock out the ATR7 gene homolog or ortholog in said target plant species; and (d) generating edited plant lines with enhanced oxidative stress tolerance induced by abiotic stress associated with the disruption or knock out of the ATR7 gene homolog or ortholog.

[0029] It is another object of the present invention, to provide the method as defined in any of the above, wherein the at least one genomic modification is a mutation conferring reduced to completely loss of function of the ATR7 gene.

[0030] It is another object of the present invention, to provide the method as defined in any of the above, wherein said modified crop plant exhibits enhanced abiotic stress -induced oxidative stress compared to wild-type or a corresponding crop plant lacking the at least one genomic modification.

[0031] It is another object of the present invention, to provide the method as defined in any of the above, wherein said abiotic stress-induced oxidative stress conditions comprise oxidative and abiotic stresses including hydrogen peroxide, ROS -inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCh), osmotic, drought and salinity stress.

[0032] It is another object of the present invention, to provide the method as defined in any of the above, wherein said modified crop plant is a non-transgenic or non-GMO plant.

[0033] It is another object of the present invention, to provide the method as defined in any of the above, wherein said modified crop plant belongs to a crop family selected, but not limited to, from the group consisting of cereals, legumes, fruits, and vegetables. It is another object of the present invention, to provide the method as defined in any of the above, wherein said modified crop plant is a plant species selected from, among others, tomato, cucumber, lettuce, rice, maize, soybean, wheat, and potato.

[0034] It is another object of the present invention, to provide the method as defined in any of the above, wherein said modified crop plant is a Tomato plant (Solarium lycopersicum).

[0035] It is another object of the present invention, to provide the method as defined above, wherein said ATR7 gene is Solarium lycopersicum (SI) ATR7 (S1ATR7) comprising a wild-type genomic sequence comprising at least 80% identity to SEQ ID NO: 153 or a genomic sequence encoding a wild-type amino acid sequence comprising at least 80% identity to SEQ ID NO: 157.

[0036] It is another object of the present invention, to provide the method as defined in any of the above, wherein said method comprising generating the genetic modification in planta via transforming the crop plant with a construct comprising (a) Cas DNA and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3- 152 and any combination thereof.

[0037] It is another object of the present invention, to provide the method as defined in any of the above, wherein said genomically edited ATR7 gene comprises a mutated genomic sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 154-156 or a genomic sequence encoding a mutated amino acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 158-160.

[0038] It is another object of the present invention, to provide the method as defined in any of the above, wherein said method comprises steps of regeneration in a tissue culture positively selected transformed plants.

[0039] It is another object of the present invention, to provide the method as defined in any of the above, wherein said method comprises steps of screening and selecting for plants comprising editing events within the ATR7 genomic region targeted for editing, from the regenerated positively selected transformed plants. It is another object of the present invention, to provide the method as defined in any of the above, wherein said method comprises steps of selection of transformed crop plants comprising an ATR7 knock-out editing event conferring abiotic stress-induced oxidative stress tolerance.

[0040] It is another object of the present invention, to provide a modified crop plant exhibiting tolerance to abiotic stress-induced oxidative stress conditions, the genome of said plant comprises a disrupted or knocked out ATR7 gene obtained through CRISPR / Cas9 genome editing.

[0041] It is another object of the present invention, to provide the modified crop plant as defined above, wherein the plant exhibits enhanced abiotic stress-induced oxidative stress tolerance compared to wild-type or a corresponding plant lacking the genome edited ATR7 gene.

[0042] It is another object of the present invention, to provide the modified crop plant as defined in any of the above, wherein the plant is a non-transgenic plant line comprising a disrupted or knocked out ATR7 gene obtained through CRISPR / Cas9 genome editing.

[0043] It is another object of the present invention, to provide the modified crop plant as defined in any of the above, wherein the at least one genomic modification is a mutation conferring reduced to completely loss of function of the ATR7 gene.

[0044] It is another object of the present invention, to provide the modified crop plant as defined in any of the above, wherein said modified crop plant exhibits enhanced abiotic stress-induced oxidative stress compared to wild-type or a corresponding crop plant lacking the at least one genomic modification.

[0045] It is another object of the present invention, to provide the modified crop plant as defined in any of the above, wherein said abiotic stress-induced oxidative stress conditions comprise oxidative and abiotic stresses including hydrogen peroxide, ROS -inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCh), osmotic, drought and salinity stress.

[0046] It is another object of the present invention, to provide the modified crop plant as defined in any of the above, wherein said modified crop plant is a non-transgenic or non-GMO plant.

[0047] It is another object of the present invention, to provide the modified crop plant as defined in any of the above, wherein said modified crop plant belongs to a crop family selected, but not limited to, from the group consisting of cereals, legumes, fruits, and vegetables. It is another object of the present invention, to provide the modified crop plant as defined in any of the above, wherein said modified crop plant is a plant species selected from, among others, tomato, cucumber, lettuce, rice, maize, soybean, wheat, and potato.

[0048] It is another object of the present invention, to provide the modified crop plant as defined in any of the above, wherein said modified crop plant is a Tomato plant (Solanum lycopersicum).

[0049] It is another object of the present invention, to provide the modified crop plant as defined above, wherein said ATR7 gene is Solanum lycopersicum (SI) ATR7 (S1ATR7) comprising a wildtype genomic sequence comprising at least 80% identity to SEQ ID NO: 153 or a genomic sequence encoding a wild-type amino acid sequence comprising at least 80% identity to SEQ ID NO: 157.

[0050] It is another object of the present invention, to provide the modified crop plant as defined in any of the above, wherein said method comprising generating the genetic modification in planta via transforming the crop plant with a construct comprising (a) Cas DNA and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3- 152 and any combination thereof.

[0051] It is another object of the present invention, to provide the modified crop plant as defined in any of the above, wherein said genomically edited ATR7 gene comprises a mutated genomic sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 154-156 or a genomic sequence encoding a mutated amino acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 158-160.

[0052] It is another object of the present invention, to provide harvestable parts of a modified crop plant as defined in any of the above, wherein said harvestable parts are preferably fruits, vegetables, shoot biomass and / or seeds.

[0053] It is another object of the present invention, to provide products derived from a modified crop plant as defined in any of the above and / or from harvestable parts of a modified crop plant as defined above.

[0054] It is another object of the present invention, to provide an isolated polynucleotide sequence comprising at least 80% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3-156 and any combination thereof. It is another object of the present invention, to provide an isolated polypeptide sequence comprising at least 80% identity to a polypeptide sequence selected from the group consisting of SEQ ID NOs:2, 157-160 and any combination thereof.

[0055] It is another object of the present invention, to provide use of a nucleic acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 3-152 for targeted genome modification of a Tomato plant (Solanum ly coper sicum), for generating and / or producing a modified crop plant exhibiting tolerance to abiotic stress-induced oxidative stress conditions, as defined in any of the above.

[0056] It is another object of the present invention, to provide a method for identifying and / or selecting for a modified crop plant exhibiting abiotic stress-induced oxidative stress tolerance, said method comprises steps of screening the genome of said plant for a disrupted or knocked out ATR7 gene.

[0057] It is another object of the present invention, to provide a method for identifying and / or selecting for a modified Tomato plant (Solarium lycopersicum) exhibiting tolerance to abiotic stress- induced oxidative stress conditions, said method comprises steps of (a) screening the genome of said Tomato plant (Solarium lycopersicum) plant for a disrupted or knocked out ATR7 gene homolog or ortholog comprising a polynucleotide sequence selected from SEQ ID NO: 154- 156 or a polynucleotide sequence encoding a amino acid sequence selected from SEQ ID NO: 158-160; (b) optionally, selecting a modified Tomato plant (Solarium lycopersicum) plant carrying said genetic modification; and (c) optionally, screening said selected plants for a plant exhibiting oxidative stress tolerance induced by abiotic stress conditions.

[0058] It is another object of the present invention, to provide a method as defined in any of the above, wherein the plant exhibits enhanced abiotic stress-induced oxidative stress tolerance compared to wild-type or a corresponding plant lacking the genome edited ATR7 gene.

[0059] It is another object of the present invention, to provide a method as defined in any of the above, wherein the plant is a non-transgenic plant line comprising a disrupted or knocked out ATR7 gene obtained through CRISPR / Cas9 genome editing.

[0060] It is another object of the present invention, to provide a method as defined in any of the above, wherein the at least one genomic modification is a mutation conferring reduced to completely loss of function of the ATR7 gene. It is another object of the present invention, to provide a method as defined in any of the above, wherein said abiotic stress-induced oxidative stress conditions comprise oxidative and abiotic stresses including hydrogen peroxide, ROS-inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCh), osmotic, drought and salinity stress.

[0061] It is another object of the present invention, to provide a method as defined above, wherein said modified crop plant belongs to a crop family selected, but not limited to, from the group consisting of cereals, legumes, fruits, and vegetables.

[0062] It is another object of the present invention, to provide a method as defined in any of the above, wherein said modified crop plant is a plant species selected from, among others, tomato, cucumber, lettuce, rice, maize, soybean, wheat, and potato.

[0063] It is another object of the present invention, to provide a method as defined in any of the above, wherein said ATR7 gene is Solarium lycopersicum (SI) ATR7 (S1ATR7) comprising a wildtype genomic sequence comprising at least 80% identity to SEQ ID NO: 153 or a genomic sequence encoding a wild-type amino acid sequence comprising at least 80% identity to SEQ ID NO: 157.

[0064] It is another object of the present invention, to provide a method as defined in any of the above, wherein said method comprising generating the genetic modification in planta via transforming the crop plant with a construct comprising (a) Cas DNA and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof.

[0065] It is another object of the present invention, to provide a method as defined in any of the above, wherein said genomically edited ATR7 gene comprises a mutated genomic sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 154-156 or a genomic sequence encoding a mutated amino acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 158-160.

[0066] BRIEF DESCRIPTION OF THE FIGURES

[0067] Exemplary non-limited embodiments of the disclosed subject matter will be described, with reference to the following description of the embodiments, in conjunction with the figures. The figures are generally not shown to scale, and any sizes are only meant to be exemplary and not necessarily limiting. Corresponding or like elements are optionally designated by the same numerals or letters.

[0068] Fig. 1 is presenting an agarose gel following a PCR using ATR7 specific primers to identify editing events. 23_53_P1_P2 refers to a first editing event having a deletion of 168 base pairs (dl68). 23_53_P3_P2 refers to a second editing event having a deletion of 1 base pair in two locations in the ATR7 gene (d-1, -1); and

[0069] Fig. 2 is presenting an agarose gel following a PCR using Cas9 specific primers. 23_53_P1_P2 refers to a first editing event having a deletion of 168 base pairs (dl68). 23_53_P3_P2 refers to a second editing event having a deletion of 1 base pair in two locations in the ATR7 gene (d-1, -1).

[0070] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] In the following detailed description of the preferred embodiments, reference is made to the accompanying drawings that form a part hereof, and in which are shown by way of illustration specific embodiments in which the invention may be practiced. It is understood that other embodiments may be utilized, and structural changes may be made without departing from the scope of the present invention. The present invention may be practiced according to the claims without some or all of these specific details. For the purpose of clarity, technical material that is known in the technical fields related to the invention has not been described in detail so that the present invention is not unnecessarily obscured.

[0072] The present invention offers a solution to enhance oxidative stress tolerance in crop plants, thereby improving crop production. By addressing the challenges posed by environmental stressors, this invention aims to increase the resilience and yield of crop plants under adverse conditions.

[0073] The present invention presents a novel approach to enhancing oxidative stress tolerance in plants, based on the gene ATR7 (AT5G21280) from Arabidopsis thaliana. It is herein acknowledged that the gene ATR7 plays a pivotal role in the plant's response to oxidative stress. Oxidative stress arises when there is an imbalance between the production of reactive oxygen species (ROS) and the plant's antioxidant defense mechanisms. This imbalance can result from various environmental factors, including exposure to high light intensity, drought, salinity, and extreme temperatures, collectively known as abiotic stresses. It is within the scope that abiotic stressors induce the accumulation of reactive oxygen species (ROS) within plant cells, leading to cellular damage and ultimately affecting plant growth, development, and yield. It is shown that the ATR7 gene has been found to regulate oxidative stress tolerance by modulating ROS levels and antioxidant enzyme activities, thereby enhancing the plant's ability to withstand adverse environmental conditions. Knocking out the ATR7 gene using CRISPR / Cas9 genome editing technology, disclosed by the present invention, presents a promising strategy to develop abiotic stress-tolerant crops across various agricultural species.

[0074] Thus, according to one embodiment, the current invention aims to utilize CRISPR / Cas9 technology to precisely target and disrupt the ATR7 gene, thereby mimicking the natural variation observed in stress-tolerant plant varieties. By doing so, this approach offers a non- transgenic and precise method to enhance oxidative stress tolerance in crops, heritably transferred to the next generation, without introducing foreign DNA. Consequently, edited plants with improved stress resilience will not be classified as genetically modified organisms (GMOs) and will not be subjected to the regulatory burdens associated with GMOs.

[0075] This innovative approach has significant implications for global agriculture, offering a sustainable solution to mitigate the adverse effects of abiotic stresses, such as heat and cold, on crop productivity and food security. By harnessing the natural genetic diversity present in plant populations, this technology can be adapted to essentially all crop families, including but not limited to, cereals, legumes, fruits, and vegetables, thereby providing farmers with resilient crop varieties capable of thriving in challenging environments.

[0076] The present invention provides a method for producing a modified crop plant with tolerance to oxidative related stress conditions, particularly, abiotic stress-induced oxidative stress tolerance, the method comprising introducing at least one genomic modification in an endogenous ATR7 gene using targeted genome editing techniques.

[0077] Climate change poses significant challenges to plant growth and development, with extreme temperatures and drought being among the primary environmental constraints on crop productivity. Understanding the impact of these extreme conditions is crucial for developing future strategies to mitigate crop damage. Tomato plants (Solarium lycopersicum), which are among the most widely cultivated crops globally, are valued for their organoleptic qualities and nutritional value. Tomato plants are known as sensitive to temperatures below 12 °C and above 32 °C. The present invention offers enhanced tolerance of crop plants modified to exhibit a loss of function in the ATR7 gene to abiotic stresses, such as drought stress. The presence of ATR7 homologs in agriculturally important species underscores the potential for crop improvement through the modulation of ATR7 levels, specifically targeting loss of function through gene editing techniques. This approach could lead to the development of crop varieties with increased resilience to environmental stressors, thereby enhancing agricultural productivity and sustainability.

[0078] Definitions:

[0079] As used herein the term "about" denotes ± 25% of the defined amount or measure or value.

[0080] As used herein the term "similar" denotes a correspondence or resemblance range of about ± 20%, particularly ± 15%, more particularly about ± 10% and even more particularly about ± 5%.

[0081] As used herein the term "corresponding" generally means similar, analogous, like, alike, akin, parallel, identical, resembling or comparable. In further aspects it means having or participating in the same relationship (such as type or species, kind, degree, position, correspondence, or function). It further means related or accompanying. In some embodiments of the present invention, it refers to plants of the same species or strain or variety or to sibling plant, or one or more individuals having one or both parents in common. The term "corresponding" further encompasses a wild type plant or a plant lacking a genetic modification conferring knockout or loss of function of the ATR7 gene.

[0082] According to further aspects of the current invention, the term "corresponding" or “corresponding to position” as used herein, refers in the context of the present invention to sequence homology or sequence identity. These terms relate to two or more nucleic acid or protein sequences, that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the available sequence comparison algorithms or by visual inspection. If two sequences, which are to be compared with each other, differ in length, sequence identity preferably relates to the percentage of the nucleotide residues of the shorter sequence, which are identical with the nucleotide residues of the longer sequence. As used herein, the percentage of identity or homology between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of identity percent between two sequences can be accomplished using a mathematical algorithm as known in the relevant art. According to further aspects of the invention, the term “corresponding to the nucleotide sequence” or “corresponding to position”, refers to variants, homologues and fragments of the indicated nucleotide sequence, which possess or perform the same biological function or correlates with the same phenotypic characteristic of the indicated nucleotide sequence.

[0083] Another indication that two nucleic acid sequences are substantially identical or that a sequence is “corresponding to the nucleotide sequence” is that the two molecules hybridize to each other under stringent conditions. High stringency conditions, such as high hybridization temperature and low salt in hybridization buffers, permits only hybridization between nucleic acid sequences that are highly similar, whereas low stringency conditions, such as lower temperature and high salt, allows hybridization when the sequences are less similar.

[0084] In other embodiments of the invention, such substantially identical sequences refer to polynucleotide or amino acid sequences that share at least about 80% similarity or identity, preferably at least about 90% similarity or identity, alternatively, about 95%, 96%, 97%, 98% or 99% similarity or identity to the indicated polynucleotide or amino acid sequences.

[0085] According to other aspects of the invention, the term "corresponding" refers also to complementary sequences or base pairing such that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequences will be complementary. The degree of complementarity between two nucleic acid strands may vary.

[0086] A "plant" as used herein refers to any plant at any stage of development, particularly a seed plant. The term "plant" includes the whole plant or any parts or derivatives thereof, such as plant cells, seeds, plant protoplasts, plant cell tissue culture from which tomato plants can be regenerated, plant callus or calli, meristematic cells, microspores, embryos, immature embryos, pollen, ovules, anthers, fruit (e.g. tomato fruit), flowers, leaves, cotyledons, pistil, seeds, seed coat, roots, root tips and the like.

[0087] The term "plant cell" used herein refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.

[0088] The term "plant cell culture" as used herein means cultures of plant units such as, for example, protoplasts, regenerable cells, cell culture, cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development, leaves, roots, root tips, anthers, meristematic cells, microspores, flowers, cotyledons, pistil, fruit, seeds, seed coat or any combination thereof.

[0089] The term "plant material" or "plant part" used herein refers to leaves, stems, roots, root tips, flowers or flower parts, fruits (e.g. tomato fruit, particularly modified tomato fruit as disclosed by the current invention), vegetables, pollen, egg cells, zygotes, seeds, seed coat, cuttings, cell or tissue cultures, or any other part or product of a plant or a combination thereof.

[0090] A "plant organ" as used herein means a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower, flower bud, or embryo.

[0091] The term "plant tissue" as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture, protoplasts, meristematic cells, calli and any group of plant cells organized into structural and / or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

[0092] As used herein, the term "progeny" or "progenies" refers in a non-limiting manner to offspring or descendant plants. According to certain embodiments, the term "progeny" or "progenies" refers to plants developed or grown or produced from the disclosed or deposited seeds as detailed inter alia. The grown plants and progeny thereof preferably have the desired traits of the disclosed or deposited seeds, i.e. knockout or loss of function mutation in the ATR7 gene.

[0093] The term "crop plant" or "crop producing plant" refers, without limitation, to any plant or plant product that can be grown and harvested for profit or subsistence. By use, crops fall into six categories: food crops, feed crops, fiber crops, oil crops, ornamental crops, and industrial crops. Food crops, such as fruit and vegetables are cultured for enhanced physiological characteristics and / or crop yield. A non-limiting list of examples includes plants that produce fruits, vegetables, seeds, nuts, grains, oil, wood, and fibers as well as cereals, and legumes. In the context of the present invention, the crop plant is selected from seed plant, for example tomato. The term “Tomato” refers hereinafter to a genus of flowering plants in the family Solanaceae. In certain aspects of the present invention, it refers to a plant species within the genus Solarium, such as the species S. lycopersicum.

[0094] The term "stress" refers, without limitation, to any external factor that has a negative influence on plant growth, function and / or reproduction. Stress in plants can be caused by abiotic or biotic factors.

[0095] The term “biotic stress” in plants refers, without limitation, to the stress caused by living organisms, specifically viruses, bacteria, fungi, nematodes, insects, arachnids, and weeds. In contrast to abiotic stress caused by environmental factors such as drought and heat, biotic stress agents directly deprive their host of its nutrients leading to reduced plant vigor and, in extreme cases, death of the host plant. In agriculture, biotic stress is a major cause of pre- and postharvest losses. In the context of the present invention, the abiotic and biotic stresses can be monitored by plants on biosensing technology.

[0096] The term "abiotic stress" refers, without limitation, to the negative impact of non-living factors on the plant in a specific environment. The non-living variable must influence the environment beyond its normal range of variation to adversely affect the plant or plant population's performance or physiology in a significant way. Non-limiting examples of abiotic stress factors, stressors, or environmental factors may encompass factors such as sunlight, wind, temperature (cold, heat), salinity, over watering (flooding), drought and factors such as fertilizer uptake and fertilizer usage efficiency as well as hydrogen peroxide, ROS -inducing agents such as paraquat (PQ), heavy metals (e.g., CdCl2) osmotic stress and any combination thereof. Abiotic stress resistance or tolerance may enhance the growth and productivity of plants, specifically crops. In the context of the present invention, the stress is caused by oxidative, drought, salinity and heat. In one embodiment, the stress is oxidative stress induced by abiotic stress factors or conditions. Many organisms have physiological and genetic adaptations conferring drought tolerance.

[0097] The term “oxidative stress” in plants refers to a condition where there is an imbalance between the production of reactive oxygen species (ROS) and the plant's ability to detoxify or neutralize them. ROS are highly reactive molecules, such as superoxide anion (O2 -) hydrogen peroxide (H2O2), and hydroxyl radicals (OH ), which are generated naturally in plant cells during processes like photosynthesis and respiration. When ROS accumulate to levels higher than the plant’s antioxidant defence system can handle, it leads to oxidative damage to cellular components like lipids, proteins, and DNA. This can result in impaired cellular functions, stunted growth, reduced photosynthesis, and even cell death. Various environmental stress factors, such as drought, salinity, extreme temperatures, and pollution, can increase ROS production in plants, leading to oxidative stress.

[0098] The term “abiotic stress-induced oxidative stress” refers to the oxidative stress condition in plants that is specifically triggered by abiotic stresses (namely, non-living environmental factors). Abiotic stresses such as drought, salinity, extreme temperatures, and pollution can lead to an overproduction of reactive oxygen species (ROS) in plant cells. These ROS are highly reactive molecules that can cause damage to cellular components like lipids, proteins, and DNA when they accumulate beyond the plant's antioxidant defense capacity. The imbalance between ROS production and the plant's ability to detoxify them results in oxidative stress, which can impair cellular functions, reduce photosynthesis, stunt growth, and even lead to cell death. According to one embodiment, the plants of the present invention are resilient to abiotic stresses and thus are characterized by enhanced yield.

[0099] The term “extreme temperatures” for plants, refer to temperatures that are significantly higher or lower than what is optimal for their growth and development. Both excessively high and low temperatures can cause stress and affect a plant's physiological functions. Above 30-35°C (86- 95°F): Plants can experience heat stress when exposed to high temperatures, especially during the day. High temperatures can cause enzymes involved in photosynthesis to break down, limiting the plant's ability to produce energy. Excessive heat can increase transpiration rates, leading to water loss. If water is not replenished, plants may wilt or even die. High temperatures can impair cell division and elongation, leading to stunted growth and poor fruit or flower development. In some cases, prolonged heat stress can lead to leaf scorch, sunburn, or tissue damage. Below 0-5°C (32-41°F): When temperatures fall too low, plants can suffer from frost damage or chilling injury, particularly if exposed to freezing conditions. Freezing temperatures can cause water within plant cells to freeze, rupturing cell walls and causing irreversible damage. Eow temperatures can slow or halt metabolic processes, preventing plants from growing or even causing them to go dormant. Frost can cause leaf discoloration, wilting, or even death of plant tissue, particularly for non-hardy species. Cold soil temperatures can prevent seeds from germinating or lead to weak, poor seedling development.

[0100] The term “drought” refers to a prolonged period of abnormally low rainfall, leading to a shortage of water. In the context of agriculture and plant biology, drought is considered an abiotic stress that can severely impact plant growth, development, and productivity. During drought conditions, plants experience water scarcity, which can lead to reduced photosynthesis, stunted growth, wilting, and even plant death if the stress is prolonged and severe. Plants have developed various adaptive mechanisms to cope with drought, such as closing stomata to reduce water loss, altering root architecture to access deeper water sources, and producing stress- related proteins and metabolites to protect cellular structures.

[0101] The term "phenotype" refers, without limitation, to distinguishable characteristics from a genetically controlled trait, which can be a physical feature. "Phenotypic traits" refer to the appearance or other detectable characteristic of an individual, resulting from the interaction of its genome, proteome and / or metabolome with the environment. It is within the scope of the present invention that the term phenotype refers to the characteristics of a plant resulting from the expression of genes. As used herein, the term “phenotype” is interchangeable with “characteristic” or “phenotypic feature” or trait. A non-limiting list of phenotypes including physical features, within the context of the present invention, include tolerance (or enhanced tolerance) to oxidative related stress conditions, such as drought, salinity, extreme temperatures, pollutants etc. It further refers to tolerance to abiotic stress -induced oxidative stress conditions comprising oxidative and abiotic stresses including hydrogen peroxide, ROS-inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCl2) osmotic, drought and salinity stress.

[0102] The term "resistance" refers, without limitation, to the ability of a plant to normally grow and reproduce when compared to susceptible plants under similar environmental conditions, such as oxidative stress conditions, drought conditions, salinity or heat or cold condition and any combination thereof.

[0103] The term "tolerance" refers hereinafter to the characteristic of a plant that allows a plant to avoid, tolerate or recover from biotic or abiotic stressors, under conditions that would typically cause a greater amount of injury to other plants of the same species. These inheritable characteristics influence the degree of damage caused to the plant. In terms of agricultural production, tolerance means that the plant can be under stress (such as physiologically challenged) but the extent of loss does not exceed the economic threshold level (an extent of loss which does not hamper the economic potential of the produce). According to further aspects of the present invention, tolerance is a relative term. In general, "resistance" and "tolerance" are the terms used to denote the ability of the plant to manage stress, be it biotic or abiotic. A non-limiting list of tolerance includes drought tolerance, heat tolerance, cold tolerance and similar. According to the present invention, a non-limiting list of produced stress-tolerant plant species by the disclosed method includes Solarium lycopersicum (Tomato), Cucumis sativus (cucumber), Lactuca sativa (lettuce), Oryza sativa (rice), Zea mays (maise), Glycine max (soybean), Triticum aestivum (wheat), and Solanum tuberosum (potato).

[0104] In the context of the present invention, stress resistance is characterized by normal growth under stress, and stress tolerance by higher growth relative to a corresponding stress-sensitive plant or by reduced growth (relative to growth at normal conditions) but not as much as a corresponding stress-sensitive plant. A stress sensitive plant refers herein to a wild-type corresponding plant (of the same species) or a corresponding plant (of the same species) lacking the at least one genomic modification in ATR7 gene.

[0105] The term "drought tolerance or resistance phenotype" refers to the observable characteristics or traits of a plant that enable it to survive, grow, and reproduce under conditions of limited water availability. Plants exhibiting a drought tolerance or resistance phenotype typically possess a range of adaptive features that help them cope with water scarcity. A drought resistance phenotype enables plants to maintain growth and productivity despite experiencing periods of drought, thereby contributing to their survival and reproductive success in arid or semi-arid environments.

[0106] The term "salinity" refers, without limitation, to a measure of soluble salts in soil or water. The salinity of soil (or another growth medium) can be expressed: (a) as the salt concentration of the medium in terms of grams per liter (g / L) or (b) in terms of electric conductivity (EC, in deciSiemens per meter — dSm1, or in equivalent units milliMhos per centimeter — mmhos / cm or milliSiemens per centimeter — mScm1).

[0107] The term “salt” refers, without limitation, to any molecule comprised of a cation, such as sodium (Na+), potassium (K+), magnesium (Mg2+), or calcium (Ca2+), and an anion, such as chloride (Cl -) bicarbonate (HCO3), carbonate (CO32), or sulfate (SO42). Sodium chloride (NaCl) is the most common salt in groundwater and soils.

[0108] The term “saline” refers, without limitation, to a medium having an electrical conductivity of the saturated paste extract (ECe) of 4 dSm1or greater; “slightly saline” (or medium) can be defined as having an electrical conductivity of the saturated paste extract (Ece) of between 4 and 8; moderately saline can be defined as having an electrical conductivity of the saturated paste extract (ECe) of between 8 and 16, and severely saline can be defined as having an electrical conductivity of the saturated paste extract (ECe) of greater than 16. For instance, sea water may have a salt concentration of 30 g / L and an EC of 50 dSm1. However, effects can occur on crops at salinity levels lower than 4 dSm1. Salt-sensitive crops can exhibit growth problems at 0.75-1.5 dSm1and many crops can nonetheless experience growth rate decreases at 1.5-3.0 dSm1. In the context of the present invention, the salt tolerance is characterized as reduced growth but not as much as or less than a stress-sensitive plant, e.g. in a solution presenting an electrical conductivity (EC) of at least about 3 dSm1, at least about 5 dSm1, at least about 7 dSm1, at least about 9 dSm1, at least about 11 dSm1, at least about 13 dSm1, at least about 15 dSm1, at least about 17 dSm1, at least about 19 dSm1, at least about 21 dSm1, at least about 23 dSm1, at least about 25 dSm1, at least about 27 dSm1, at least about 29 dSm’x, at least about 31 dSm1, at least about 33 dSm1, at least about 35 dSm1, at least about 37 dSm1, at least about 39 dSm1, at least about 41 dSm1, at least about 43 dSm1, at least about 45 dSm1, at least about 47 dSm1, at least about 49 dSm1, or at least about 51 dSm1.

[0109] According to further aspects, the gene-edited plant lines of the present invention are capable of growing under a range of NaCl concentrations e.g. 50 mM to 500 mM NaCl, for example, 100 mM to 500 mM NaCl or at higher salinity levels.

[0110] The term "salt-tolerant plant" refers, without limitation, to any genus or species of plant or modified plant that can grow and complete their life cycle on a substrate that contains high concentrations of soluble salt in a hypersaline environment or environment of moderate salinity (a solution of at least 3 dSm1), for example in soil or waters of high salinity. As used herein, the term “salt- tolerant plant” is interchangeable with “salt-resistant plant". In the context of the present invention it includes a modified crop plant exhibiting tolerance to abiotic stress-induced oxidative stress conditions, the genome of said plant comprises a disrupted or knocked out ATR7 gene obtained through CRISPR / Cas9 genome editing.

[0111] It is within the scope of the current invention that an analysis of the ATR7 gene (AT5G21280) from Arabidopsis thaliana revealed that it encodes a nuclear-localized protein with a previously unreported function (see Sujeeth, N., Mehterov, N., Gupta, S. et al. A novel seed plants gene regulates oxidative stress tolerance in Arabidopsis thaliana. Cell. Mol. Life Sci. 77, 705-718 (2020), incorporated herein by its entirety). The gene is specific to seed plants; there are no homologs in lower plants (algae, fems, lycopods, and mosses), fungi, and animals. The atr7 mutant was previously obtained by chemical mutagenesis from its genetic background loh2, obtained by knocking out a gene involved in ceramide synthesis. Additionally, a screening of the TAIR database identified a knockout line (KO, SALK_006796) featuring a T-DNA insertion within the AT5G21280 gene. Furthermore, ATR7 expression was suppressed by creating RNA interference (RNAi) lines in both the loh2 and Wassilewskija backgrounds. Contrary to the available method described in the Sujeeth et al publication, the current invention employs precise genetic editing techniques to achieve targeted modifications in the ATR7 gene, ensuring specific and controlled alterations without affecting other parts of the plant's genome. This approach allows for enhanced precision and predictability in heritably modifying plant traits, without involving a transgene, distinguishing it from traditional mutagenesis and insertion methods resulting in a transgenic, GMO plant. Thus, the present invention provides a non-transgenic or non-GMO plant characterized by heritable abiotic stress-induced oxidative stress tolerance trait.

[0112] In contrast to traditional methods such as chemical mutagenesis, T-DNA insertion, and RNA interference (RNAi), which can lead to random and unintended mutations and involves insertion of a foreign DNA or gene into the plant genome content, the present invention employs precise genetic editing techniques resulting in a non-transgenic or non-GMO plant. These advanced techniques allow for targeted modifications in specific genes, such as the ATR7 gene, ensuring that only the desired traits are altered without affecting other parts of the plant's genome. This precision reduces the risk of unintended phenotypic changes and enhances the stability of the desired traits across generations. Additionally, genetic editing is often more efficient and less time-consuming than traditional methods, as it requires less extensive screening to identify plants with the desired characteristics. Furthermore, this approach offers a non-transgenic and precise method to enhance stress tolerance heritably in crops without introducing foreign DNA. The use of genetic editing may face fewer regulatory and public acceptance challenges compared to chemically mutagenized or T-DNA inserted plants, as it often perceived as a more safe and controlled approach to plant modification and will not be classified as genetically modified organisms (GMOs) and will not be subjected to the regulatory burdens associated with the relevant authorities.

[0113] According to main aspects of the present invention, methods are provided for enhancing oxidative stress tolerance by using targeted genome editing, aiming to introduce modifications in the ATR7 gene that result in reduced function to completely inactive (a loss of function or knockout) gene. This genome editing modification is intended to improve the plant's resilience to environmental (abiotic) stressors such as drought, extreme heat or cold, sub-optimal temperatures such as cold or heat stress and other oxidative and abiotic stresses including hydrogen peroxide, ROS-inducing agents such as paraquat (PQ), heavy metals (e.g. CdCh), osmotic, drought and salinity stress, thereby enhancing agricultural productivity and sustainability. In further aspects, the present invention discloses the ATR7 gene genomic and amino acid sequence in tomato plants (Solarium lycopersicum), namely S1ATR7, that is associated with oxidative stress tolerance, and more precisely with abiotic stress-induced oxidative stress tolerance, as set forth in SEQ ID NO: 1, which encodes for the amino acid sequence as set forth in SEQ ID NO: 2.

[0114] As used herein the term "genetic modification" refers hereinafter to genetic manipulation or modulation, which is the direct manipulation of an organism's genes using biotechnology. It also refers to a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species, targeted mutagenesis and genome editing technologies to produce improved organisms. According to main embodiments of the present invention, modified crop plants (such as tomato plant) with tolerance to abiotic-induced oxidative stress traits are generated using genome editing mechanism. This technique enables to achieve in planta modification of specific genes that relate to and / or control the response to oxidative stress tolerance induced by abiotic stress in the crop plant (such as tomato).

[0115] The term "genome editing", or "genome / genetic modification" or "gene editing" generally refers hereinafter to a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Unlike previous genetic engineering techniques that randomly insert genetic material into a host genome, genome editing targets the insertions to site specific locations. It is emphasized that employing this technique by the present invention results in non-transgenic or non-GMO plants modified in the desired target gene locus.

[0116] It is within the scope of the present invention that the common methods for such editing use engineered nucleases, or "molecular scissors". These nucleases create site-specific doublestrand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations ('edits'). Families of engineered nucleases used by the current invention include, but are not limited to: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR / Cas9) system.

[0117]

[0118] According to specific aspects of the present invention, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes are used for the first time for generating genome modification in the targeted gene ATR7 (e.g. in the Tomato plant). Without wishing to be bound by theory, reference is now made to a type of CRISPR mechanism, in which invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus comprising a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA, namely CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity.

[0119] According to further aspects of the invention, Cas protein, such as Cas9 (also known as Csnl) is required for gene silencing. Cas9 participates in the processing of crRNAs, and is responsible for the destruction of the target DNA. Cas9’s function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNH-like nuclease domain that resides in the mid-region of the protein. To achieve sitespecific DNA recognition and cleavage, Cas9 is complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA. The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9.

[0120] Without wishing to be bound by theory, it is herein acknowledged that during the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double- stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript. The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.

[0121] It is further noted that the double- stranded endonuclease activity of Cas9 also requires that a short conserved sequence, (2-5 bps, preferably 3 bp) known as protospacer-associated motif (PAM), follows immediately 3'- of the crRNA complementary sequence.

[0122] According to some embodiments, the gRNA sequence comprises a 3’ NGG Protospacer Adjacent Motif (PAM) selected from the group consisting of NGG (SpCas), NNNNGATT (NmeCas9), NNAGAAW, (StCas9), NAAAAC (TdCas9), NNGRRT (SaCas9) and TBN (Cas- phi).

[0123] According to further aspects of the invention, a two-component system may be used by the current invention, combining trRNA and crRNA into a single synthetic single guide RNA (sgRNA) for guiding targeted gene alterations.

[0124] It is further within the scope that Cas9 nuclease variants include wild-type Cas9, Cas9D10A and nuclease-deficient Cas9 (dCas9).

[0125] The term “gene editing” further refers, without limitation, to the addition, removal, or alteration of genetic material at a particular desired location in the genome. Gene editing can be used to correct, introduce, or delete almost any DNA sequence in many different types of cells and organisms, such as animals, plants and bacteria. Gene editing is performed using enzymes, particularly nucleases, which have been engineered to target a specific DNA sequence, where they introduce cuts into the DNA strands, enabling the removal of existing DNA and the insertion of replacement DNA. A non-limiting list of targeted gene editing techniques for genome editing includes restriction enzymes, zinc finger nucleases, prime editing, Programmable Addition via Site-specific Targeting Elements (PASTE), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR / Cas9). CRISPR / Cas9 (or Casl2 or Casl3) is an RNA-based system, and it works by cutting a DNA sequence at a specific genetic location and deleting or inserting DNA sequences, which can change a single base pair of DNA, large pieces of chromosomes, or regulation of gene expression levels. As used herein, the term “gene editing” is interchangeable with "editing", “genome modification”, “genetic modification”, “gene modification”, “genome editing”, “genome engineering” or “targeted genome editing techniques”. A non-limiting list of modifications includes deleting, silencing, downregulating, de -regulating, deactivating, knockout, loss of function, reducing expression, at least partially deleting, at least partially silencing, and at least partially deactivating, removing, partially removing, duplicating, inverting, modifying a promoter, modifying a terminator, splice modifying and / or changing a DNA / nucleic acid sequence. Thus, the reduced expression can be achieved by deletion, partial deletion, removal, partial removal, duplication, inversion, modification of at least one promoter, modification of at least one terminator and / or splice modification. In the context of the present invention, the aforementioned gene editing techniques are used to edit a target gene in a desirable crop according to the information obtained from the genes associated with increasing oxidative stress tolerance (i.e. oxidative stress tolerance induced by abiotic stress) identified by the method of the present invention. The term gene editing also includes and encompasses applying base editing techniques as known to a person skilled in the relevant art. In the context of the present invention, genetic modification comprises modification of the expression level of at least one homologue or ortholog nucleic acid sequence to increase oxidative stress tolerance (i.e. oxidative stress tolerance induced by abiotic stress) in the crop plant.

[0126] The term " rotospacer adjacent motif" or "PAM" as used herein refers hereinafter to a 3 base pairs DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. PAM is a component of the invading virus or plasmid, but is not a component of the bacterial CRISPR locus. PAM is an essential targeting component which distingui hes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by nuclease.

[0127] The term gene "knockout" as used herein refers hereinafter to an experimental technique by which a specific gene is deliberately inactivated or "knocked out" in an organism. In the process, the gene of interest is often disrupted or replaced with a modified version, rendering it nonfunctional. A gene knockout caused by deletion refers to a process where a specific segment of DNA, often containing a gene or a portion of a gene, is intentionally removed or deleted from the genome. This deletion results in the loss of function of the targeted gene, meaning that the gene no longer produces its normal protein or produces a non-functional version of the protein. In gene knockout via deletion, a part of the gene or the entire gene is deleted using various molecular techniques, such as CRISPR-Cas9 or homologous recombination. The deleted region may include the gene's coding sequence or regulatory elements that control its expression. As such, the organism can no longer produce the normal protein encoded by that gene. The absence or dysfunction of the protein can disrupt the normal biological process that the gene regulates, leading to observable changes in the organism's phenotype. Types of Deletions:

[0128] Complete deletion: This involves the removal of the entire gene from the genome, leading to a total loss of its function. Such deletions result in the gene no longer being able to produce its corresponding protein, effectively eliminating its role in the organism's biological processes.

[0129] Partial deletion: In this scenario, only a portion of the gene is removed. This can result in the production of a truncated protein exhibiting reduced to entirely nonfunctional protein. While the gene's function is lost, the effects may be milder or more specific, depending on the extent and location of the deletion within the gene. Partial deletions can sometimes allow for residual activity or alter the gene's function in a way that affects only certain pathways or processes. A synonyms of the term “partial deletion” included within the scope of the present invention is “gene disruption” or “gene knock out”.

[0130] The term "gene knockdown" as used herein refers hereinafter to an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur through genetic modification, i.e. targeted genome editing or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript. The reduced expression can be at the level of RNA or at the level of protein. It is within the scope of the present invention that the term gene knockdown also refers to a loss of function mutation and / or gene knockout mutation in which an organism's genes is made inoperative or nonfunctional.

[0131] According to certain aspects of the present invention, gene knockdown is temporary and often incomplete, unlike gene knockout, which is permanent and complete. Gene knockdown is sometimes called gene silencing, but “silencing” can also refer to gene knockout.

[0132] It is within the scope that gene knockdown reduces gene expression temporarily and partially, while gene knockout eliminates gene function permanently and completely.

[0133] The term "gene silencing" as used herein refers hereinafter to the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation. In certain aspects of the invention, gene silencing is considered to have a similar meaning as gene knockdown. When genes are silenced, their expression is reduced. Gene silencing may be considered a gene knockdown mechanism since the methods used to silence genes, such as RNAi, CRISPR, or siRNA, generally reduce the expression of a gene by at least 70% but do not completely eliminate it. The term "loss of function mutation" as used herein refers to a type of mutation in which the altered gene product lacks the function of the wild-type gene. A synonym of the term included within the scope of the present invention is null mutation.

[0134] The term "in planta" means in the context of the present invention within the plant or plant cells. More specifically, it means introducing CRISPR / Cas complex into plant material comprising a tissue culture of several cells, a whole plant, or into a single plant cell, without introducing a foreign gene or a mutated gene. It is also used to describe conditions present in a non-laboratory environment (e.g. in vivo).

[0135] The term "wild type" refers to the typical form of a species as it occurs in nature. In genetics, it denotes the standard or reference genotype or phenotype of an organism, which is considered the norm or baseline against which mutations or variations are compared. The wild type is the naturally occurring, non-mutated version of a gene, allele, or organism, and it typically exhibits the standard characteristics and functions expected for that species in its natural environment. In the context of the present document, a wild type crop plant (e.g. Tomato) is one that does not have any mutant ATR7 alleles.

[0136] The term "functional variant" or "functional variant of a nucleic acid or amino acid sequence" as used herein, refers to a variant of a sequence or part of a sequence which retains the biological function of the full non-variant allele (e.g. S1ATR7 allele) and hence has the activity of ATR7 expressed gene or protein. A functional variant also comprises a variant of the gene of interest encoding a polypeptide which has sequence alterations that do not affect function of the resulting protein, for example, in non-conserved residues. Also encompassed is a variant that is substantially identical (has at least 80% sequence similarity or identity), i.e. has only some sequence variations, for example, in non-conserved residues, to the wild type nucleic acid or amino acid sequences of the alleles as shown herein, and is biologically active.

[0137] The term "variety" or "cultivar" used herein means a group of similar plants that by structural features and performance can be identified from other varieties within the same species.

[0138] The term "allele" used herein means any of one or more alternative or variant forms of a gene or a genetic unit at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus. In a diploid cell of an organism, alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome. Alternative or variant forms of alleles may be the result of single nucleotide polymorphisms, insertions, inversions, translocations or deletions, or the consequence of gene regulation caused by, for example, by chemical or structural modification, transcription regulation or post-translational modification / regulation. An allele associated with a qualitative trait may comprise alternative or variant forms of various genetic units including those that are identical or associated with a single gene or multiple genes or their products or even a gene disrupting or controlled by a genetic factor contributing to the phenotype represented by the locus. According to further embodiments, the term "allele" designates any of one or more alternative forms of a gene at a particular locus. Heterozygous alleles are two different alleles at the same locus. Homozygous alleles are two identical alleles at a particular locus. A wild type allele is a naturally occurring allele. In the context of the current invention, the term allele refers to the identified ATR7 gene, i.e. in Tomato, namely S1ATR7 having the genomic nucleotide sequence as set forth in SEQ ID NO: 1.

[0139] As used herein, the term "locus" (loci plural) means a specific place or places or region or a site on a chromosome where for example a gene or genetic marker element or factor is found. In specific embodiments, such a genetic element is contributing to a trait.

[0140] As used herein, the term "germplasm" refers to the totality of the genotypes of a population or other group of individuals (e.g., a species). The term "germplasm" can also refer to plant material; e.g., a group of plants that act as a repository for various alleles. Such germplasm genotypes or populations include plant materials of proven genetic superiority; e.g., for a given environment or geographical area, and plant materials of unknown or unproven genetic value; that are not part of an established breeding population and that do not have a known relationship to a member of the established breeding population.

[0141] The terms "hybrid", "hybrid plant" and "hybrid progeny" used herein refers to an individual produced from genetically different parents (e.g., a genetically heterozygous or mostly heterozygous individual).

[0142] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. The term further refers hereinafter to the amount of characters which match exactly between two different sequences. Hereby, gaps are not counted, and the measurement is relational to the shorter of the two sequences.

[0143] It is further within the scope that the terms "similarity" and "identity" additionally refer to local homology, identifying domains that are homologous or similar (in nucleotide and / or amino acid sequence). It is acknowledged that bioinformatics tools such as BLAST, SSEARCH, FASTA, and HMMER calculate local sequence alignments which identify the most similar region between two sequences. For domains that are found in different sequence contexts in different proteins, the alignment should be limited to the homologous domain, since the domain homology is providing the sequence similarity captured in the score. According to some aspects the term similarity or identity further includes a sequence motif, which is a nucleotide or amino-acid sequence pattern that is widespread and has, or is conjectured to have, a biological significance. Proteins may have a sequence motif and / or a structural motif, a motif formed by the three- dimensional arrangement of amino acids which may not be adjacent.

[0144] As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene", "allele" or "gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and / or may include cDNAs in combination with regulatory sequences. Thus, according to the various aspects of the invention, genomic DNA, cDNA or coding DNA may be used. In one embodiment, the nucleic acid is cDNA or coding DNA.

[0145] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

[0146] According to other aspects of the invention, a "modified" or a "mutant" plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant. Specifically, the endogenous nucleic acid sequences of the ATR7 homolog (e.g. in Tomato nucleic acid sequences of S1ATR7) has been altered compared to wild type sequences using genome editing methods as described herein. This causes inactivation of the endogenous ATR7 gene and thus disables ATR7 function. Such plants have an altered phenotype and show enhanced abiotic stress-induced oxidative stress tolerance comprising tolerance to oxidative and abiotic stresses including hydrogen peroxide, ROS-inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCh), osmotic, drought and salinity stress compared to wild type plants. Therefore, the improved or enhanced oxidative stress tolerance induced by abiotic stress phenotype is conferred by the presence of at least one mutated endogenous ATR7 gene (e.g. in the Tomato plant genome) which has been specifically targeted using genome editing technique.

[0147] It is further noted that a wild type crop plant (e.g. Tomato) is a plant that does not have any mutant ATR7 alleles.

[0148] The term “genotype” refers, without limitation, to the genetic constitution of a cell or organism. It is a scoring of the type of variant present at a given location (i.e. a locus) in the genome. An individual’s genotype includes the specific alleles, for one or more genetic marker loci, present in the individual’s haplotype. As is known in the art, a genotype can relate to a single locus or multiple loci, whether the loci are related or unrelated and / or are linked or unlinked. In some embodiments, an individual’s genotype relates to one or more genes that are related in that one or more of the genes are involved in the expression of a phenotype of interest. Some genotypes contribute to the phenotype.

[0149] The term “genome” refers, without limitation, to the entire set of DNA instructions found in a cell. Plants comprise the genome located in the cell’s nucleus, as well as a small chromosome in the cell’s mitochondria and chloroplasts. A genome in a plant contains all the information needed for a plant to develop and function. In the context of the present invention, the genome can be assembled and annotated by genomics, transcriptomics, or proteomics techniques.

[0150] The term “gene” refers, without limitation, to the basic unit of inheritance. Genes are passed from parents to offspring and contain the information needed to specify physical and biological traits. Most genes code for specific proteins, or segments of proteins, which have differing functions within the body. In the context of the present invention, the method allows identification of a nucleic acid sequence associated with increasing salt tolerance in a crop plant, wherein the nucleic acid sequence can be a Cis-regulatory element (CREs), a Cis -regulatory module (CRM) or a Trans- regulatory element (TRE). A non-limiting list of nucleic acid sequences includes a Multiple Stress Regulatory (MSTR) gene, a regulatory gene, a promoter, an operator, an enhancer, a silencer, a regulatory sequence, a transcription co-regulator, a transcription factor, an RNA processing factor, activator gene, a facultative gene, or an inducible gene.

[0151] The term “gene encoding” sequence refers, without limitation, to the information encoded in a gene that is used to either make RNA molecules that code for proteins or to make non-coding RNA molecules that serve other functions.

[0152] The term “mutagenesis” refers, without limitation, to the process by which mutations are induced in the cells of organisms. It may occur spontaneously in nature, essential to produce genetic variation necessary for natural selection, or because of exposure to mutagens. It can also be achieved experimentally using laboratory procedures. There are a variety of mutations that can occur in DNA, such as changes in the DNA sequence or rearrangement of the chromosomes. Genome editing techniques utilize the activity of sequence- specific engineered nucleases and make use of the intrinsic DNA repair system of the cell. The designed nucleases cut specific DNA sequences and lead to the occurrence of double-stranded breaks; these are repaired either by nonhomologous end joining or homology-directed repair. This results in either site-directed mutagenesis, gene replacement, nucleotide insertions or deletions.

[0153] The term "genomic" or "genomics" refers, without limitation, to the study of genes and their function. Genomics emphasizes the integration of basic and applied research in comparative gene mapping, molecular cloning, large-scale restriction mapping, and DNA sequencing and computational analysis. Genetic information is extracted using fundamental techniques, such as DNA sequencing, protein sequencing and PCR. The gene function is determined (1) by analyzing the effects of DNA mutations in genes on the normal development and health of the cell, tissue, organ or organism; (2) by analyzing a variety of signals encoded in the DNA sequence; and (3) by studying the proteins produced by a gene or system of related genes.

[0154] Main aspects of the invention involve targeted mutagenesis methods, specifically genome editing, and exclude embodiments that are solely based on generating plants by traditional breeding methods. In a further embodiment of the current invention, as explained herein, the enhanced oxidative stress tolerance is not due to the presence of a transgene.

[0155] The term "homology" or "sequence homology" refers, without limitation, to the degree of resemblance between two sequences when they are compared. This is dependent on their identity, and it shows the extent to which residues are aligned. Sequence homology refers to an optimal matching problem (i.e. for sequence alignments). The optimal matching algorithm finds the minimal number of edit operations (inserts, deletes, and substitutions) to align one sequence to another sequence. Sequence homology searches can identify "homologous" proteins or genes by detecting excess similarity, meaning, a statistically significant similarity that reflects common ancestry. Usually, homologue fragments, such as homologue DNA sequences, possess or perform the same biological function or correlate with the same phenotypic characteristic of the indicated DNA sequence. Another indication that two nucleic acid sequences are substantially similar is that the two molecules hybridize with each other under stringent conditions. High stringency conditions, such as high hybridization temperature and low salt in hybridization buffers, permit only hybridization between nucleic acid sequences that are highly similar, whereas low stringency conditions, such as lower temperature and high salt, allow hybridization when the sequences are less similar. As used herein, the term “sequence homology” is interchangeable with “sequence similarity" or "similarity" or "identity" or "sequence identity".

[0156] In the context of the present invention, similarity or identity is understood to refer to a sequence similarity of at least 80%, particularly a similarity of at least 85%, preferably more than 90% and still preferably more than 95%. In some embodiments of the invention, such similar sequences refer to polynucleotide or amino acid sequences that share at least about 80% similarity, preferably at least about 90% similarity, alternatively, about 95%, 96%, 97%, 98% or 99% similarity to the indicated polynucleotide or amino acid sequence / s. In the context of the present invention, the modified gene locus sequence comprises at least 80% identity / homology with a sequence in at least one plant species adapted to environments characterized by abiotic stress-induced oxidative stress.

[0157] The term "homologue" refers, without limitation, to a DNA or amino acid sequence having a degree of sequence similarity in terms of shared amino acid or nucleotide sequences. There may be partial similarity or complete similarity (i.e. identity). For protein sequences, amino acid similarity matrices may be used as are known in different bioinformatics programs (e.g. BLAST, FASTA, Bestfit program- Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, WI 53711, Smith Waterman). Different results may be obtained when performing a particular search with a different matrix. Degrees of similarity for nucleotide sequences are based upon identity matches with penalties made for gaps or insertions required to optimize the alignment, as is well known in the art (e.g. Altschul S. F. et al, 1990, J Mol Biol 215(3):403-10; Altschul S. F. et al, 1997, Nucleic Acids Res. 25:3389-3402). Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNASTAR software. The comparison of sequences and determination of identity percent between two sequences can be accomplished using a mathematical algorithm as known in the relevant art.

[0158] The term "ortholog" or "orthologs" refers herein to a gene in different species that evolved from a common ancestral gene through speciation. Orthologs typically retain the same function across species, making them useful for studying gene function and evolutionary relationships. These are genes in different species that evolved from a common ancestral gene by speciation and normally retain the same function. It further encompasses the relationship between two homologous genes that originate from the same gene in the most recent common ancestor of the species that are compared. In aspects of the present invention, it refers to homologous genes where a gene diverges after a speciation event, but the gene and its main function are conserved. The present invention discloses a method for identifying the ATR7 homolog or ortholog in a desirable crop plant, such as tomato, and employing targeted genome editing technology to disrupt or knock out the ATR7 gene homolog or ortholog in the target plant species. This approach aims to enhance the plant's tolerance to abiotic stress-induced oxidative stress conditions, thereby improving its resilience and productivity.

[0159] According to one embodiment, the present invention provides a method for producing a modified crop plant with abiotic stress-induced oxidative stress tolerance, the method comprising introducing at least one genomic modification in an endogenous ATR7 gene using targeted genome editing techniques.

[0160] According to a further embodiment, the present invention provides the method as defined above, wherein the at least one genomic modification is a disruption or knock out mutation of the ATR7 gene in said crop plant.

[0161] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the at least one genomic modification is a mutation conferring reduced to completely loss of function of the ATR7 gene.

[0162] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the method comprises steps of: (a) selecting a target crop plant species; (b) identifying the ATR7 gene homolog or ortholog of said target plant species; (c) utilizing CRISPR / Cas9 genome editing technology to disrupt or knock out the ATR7 gene in said target plant species; and (d) optionally, generating edited plant lines from the disrupted or knock out ATR7 gene plants with tolerance to abiotic stress-induced oxidative stress conditions. It is another object of the present invention, to provide the method as defined in any of the above, wherein said modified crop plant exhibits enhanced abiotic stress-induced oxidative stress tolerance compared to wild-type or a corresponding crop plant lacking the at least one genomic modification.

[0163] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said abiotic stress-induced oxidative stress conditions comprise oxidative and abiotic stresses including hydrogen peroxide, ROS -inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCh), osmotic, drought and salinity stress.

[0164] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said modified crop plant is a non-transgenic or non-GMO plant.

[0165] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said modified crop plant belongs to a crop family selected, but not limited to, from the group consisting of cereals, legumes, fruits, and vegetables.

[0166] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said modified crop plant is a plant species selected from, among others, tomato, cucumber, lettuce, rice, maize, soybean, wheat, and potato.

[0167] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said modified crop plant is a Tomato plant (Solarium lycopersicum).

[0168] According to a further embodiment, the present invention provides the method as defined above, wherein said ATR7 gene is Solarium lycopersicum (SI) ATR7 (S1ATR7) comprising a wild-type genomic sequence comprising at least 80% identity to SEQ ID NO: 153 or a genomic sequence encoding a wild-type amino acid sequence comprising at least 80% identity to SEQ ID NO: 157.

[0169] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said method comprising generating the genetic modification in planta via transforming the crop plant with a construct comprising (a) Cas DNA and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3- 152 and any combination thereof. According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said genomically edited ATR7 gene comprises a mutated genomic sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 154-156 or a genomic sequence encoding a mutated amino acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 158-160.

[0170] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said method comprises steps of regeneration in a tissue culture positively selected transformed plants.

[0171] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said method comprises steps of screening and selecting for plants comprising editing events within ATR7 genomic region targeted for editing, from the regenerated positively selected transformed plants.

[0172] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said method comprises steps of selection of transformed crop plants comprising an ATR7 knock-out editing event conferring abiotic stress-induced oxidative stress tolerance.

[0173] According to a further embodiment, the present invention provides a modified crop plant with abiotic stress-induced oxidative stress tolerance, produced by the method as defined in any of the above.

[0174] According to a further embodiment, the present invention provides a plant part, plant tissue, plant fruit or vegetable, plant seed or plant cell of the modified crop plant as defined above.

[0175] According to a further embodiment, the present invention provides a tissue culture of regenerable cells, protoplasts or callus obtained from the modified crop plant as defined above.

[0176] According to a further embodiment, the present invention provides the modified crop plant as defined above, wherein said modified crop plant does not comprise a transgene.

[0177] According to a further embodiment, the present invention provides a method for enhancing tolerance to abiotic stress-induced oxidative stress conditions in a crop plant, the method comprising introducing at least one genomic modification in an endogenous ATR7 gene using targeted genome editing techniques.

[0178] According to a further embodiment, the present invention provides the method as defined above, wherein said method comprises steps of (a) selecting a target crop plant species; (b) identifying an ATR7 gene homolog or ortholog in said target plant species; (c) utilizing CRISPR / Cas9 genome editing technology to disrupt or knock out the ATR7 gene homolog or ortholog in said target plant species; and (d) generating edited plant lines with enhanced oxidative stress tolerance induced by abiotic stress associated with the disruption or knock out of the ATR7 gene homolog or ortholog.

[0179] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein the at least one genomic modification is a mutation conferring reduced to completely loss of function of the ATR7 gene.

[0180] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said modified crop plant exhibits enhanced abiotic stress-induced oxidative stress compared to wild-type or a corresponding crop plant lacking the at least one genomic modification.

[0181] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said abiotic stress-induced oxidative stress conditions comprise oxidative and abiotic stresses including hydrogen peroxide, ROS -inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCl2) osmotic, drought and salinity stress.

[0182] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said modified crop plant is a non-transgenic or non-GMO plant.

[0183] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said modified crop plant belongs to a crop family selected, but not limited to, from the group consisting of cereals, legumes, fruits, and vegetables.

[0184] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said modified crop plant is a plant species selected from, among others, tomato, cucumber, lettuce, rice, maize, soybean, wheat, and potato.

[0185] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said modified crop plant is a Tomato plant (Solanum lycopersicum).

[0186] According to a further embodiment, the present invention provides the method as defined above, wherein said ATR7 gene is Solanum lycopersicum (SI) ATR7 (S1ATR7) comprising a wild-type genomic sequence comprising at least 80% identity to SEQ ID NO: 153 or a genomic sequence encoding a wild-type amino acid sequence comprising at least 80% identity to SEQ ID NO: 157. According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said method comprising generating the genetic modification in planta via transforming the crop plant with a construct comprising (a) Cas DNA and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3- 152 and any combination thereof.

[0187] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said genomically edited ATR7 gene comprises a mutated genomic sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 154-156 or a genomic sequence encoding a mutated amino acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 158-160.

[0188] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said method comprises steps of regeneration in a tissue culture positively selected transformed plants.

[0189] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said method comprises steps of screening and selecting for plants comprising editing events within the ATR7 genomic region targeted for editing, from the regenerated positively selected transformed plants.

[0190] According to a further embodiment, the present invention provides the method as defined in any of the above, wherein said method comprises steps of selection of transformed crop plants comprising an ATR7 knock-out editing event conferring abiotic stress-induced oxidative stress tolerance.

[0191] According to a further embodiment, the present invention provides a modified crop plant exhibiting tolerance to abiotic stress-induced oxidative stress conditions, the genome of said plant comprises a disrupted or knocked out ATR7 gene obtained through CRISPR / Cas9 genome editing.

[0192] According to a further embodiment, the present invention provides the modified crop plant as defined above, wherein the plant exhibits enhanced abiotic stress-induced oxidative stress tolerance compared to wild-type or a corresponding plant lacking the genome edited ATR7 gene. According to a further embodiment, the present invention provides the modified crop plant as defined in any of the above, wherein the plant is a non-transgenic plant line comprising a disrupted or knocked out ATR7 gene obtained through CRISPR / Cas9 genome editing.

[0193] According to a further embodiment, the present invention provides the modified crop plant as defined in any of the above, wherein the at least one genomic modification is a mutation conferring reduced to completely loss of function of the ATR7 gene.

[0194] According to a further embodiment, the present invention provides the modified crop plant as defined in any of the above, wherein said modified crop plant exhibits enhanced abiotic stress- induced oxidative stress compared to wild-type or a corresponding crop plant lacking the at least one genomic modification.

[0195] According to a further embodiment, the present invention provides the modified crop plant as defined in any of the above, wherein said abiotic stress-induced oxidative stress conditions comprise oxidative and abiotic stresses including hydrogen peroxide, ROS -inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCh), osmotic, drought and salinity stress.

[0196] According to a further embodiment, the present invention provides the modified crop plant as defined in any of the above, wherein said modified crop plant is a non-transgenic or non-GMO plant.

[0197] According to a further embodiment, the present invention provides the modified crop plant as defined in any of the above, wherein said modified crop plant belongs to a crop family selected, but not limited to, from the group consisting of cereals, legumes, fruits, and vegetables.

[0198] According to a further embodiment, the present invention provides the modified crop plant as defined in any of the above, wherein said modified crop plant is a plant species selected from, among others, tomato, cucumber, lettuce, rice, maize, soybean, wheat, and potato.

[0199] According to a further embodiment, the present invention provides the modified crop plant as defined in any of the above, wherein said modified crop plant is a Tomato plant (Solarium lycopersicum).

[0200] According to a further embodiment, the present invention provides the modified crop plant as defined above, wherein said ATR7 gene is Solarium lycopersicum (SI) ATR7 (S1ATR7) comprising a wild-type genomic sequence comprising at least 80% identity to SEQ ID NO: 153 or a genomic sequence encoding a wild-type amino acid sequence comprising at least 80% identity to SEQ ID NO: 157.

[0201] According to a further embodiment, the present invention provides the modified crop plant as defined in any of the above, wherein said method comprising generating the genetic modification in planta via transforming the crop plantwith a construct comprising (a) Cas DNA and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof.

[0202] According to a further embodiment, the present invention provides the modified crop plant as defined in any of the above, wherein said genomically edited ATR7 gene comprises a mutated genomic sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 154-156 or a genomic sequence encoding a mutated amino acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 158-160.

[0203] According to a further embodiment, the present invention provides harvestable parts of a modified crop plant as defined in any of the above, wherein said harvestable parts are preferably fruits, vegetables, shoot biomass and / or seeds.

[0204] According to a further embodiment, the present invention provides products derived from a modified crop plant as defined in any of the above and / or from harvestable parts of a modified crop plant as defined above.

[0205] According to a further embodiment, the present invention provides an isolated polynucleotide sequence comprising at least 80% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs:l, 3-156 and any combination thereof.

[0206] According to a further embodiment, the present invention provides an isolated polypeptide sequence comprising at least 80% identity to a polypeptide sequence selected from the group consisting of SEQ ID NOs:2, 157-160 and any combination thereof.

[0207] According to a further embodiment, the present invention provides use of a nucleic acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 3-152 for targeted genome modification of a Tomato plant (Solanum ly coper sicum), for generating and / or producing a modified crop plant exhibiting tolerance to abiotic stress-induced oxidative stress conditions, as defined in any of the above. According to a further embodiment, the present invention provides a method for identifying and / or selecting for a modified crop plant exhibiting abiotic stress-induced oxidative stress tolerance, said method comprises steps of screening the genome of said plant for a disrupted or knocked out ATR7 gene.

[0208] According to a further embodiment, the present invention provides a method for identifying and / or selecting for a modified Tomato plant (Solarium lycopersicum) exhibiting tolerance to abiotic stress-induced oxidative stress conditions, said method comprises steps of (a) screening the genome of said Tomato plant (Solarium lycopersicum) plant for a disrupted or knocked out ATR7 gene homolog or ortholog comprising a polynucleotide sequence selected from SEQ ID NO: 154-156 or a polynucleotide sequence encoding a amino acid sequence selected from SEQ ID NO: 158-160; (b) optionally, selecting a modified Tomato plant (Solarium lycopersicum) plant carrying said genetic modification; and (c) optionally, screening said selected plants for a plant exhibiting oxidative stress tolerance induced by abiotic stress conditions.

[0209] According to a further embodiment, the present invention provides a method as defined in any of the above, wherein the plant exhibits enhanced abiotic stress-induced oxidative stress tolerance compared to wild-type or a corresponding plant lacking the genome edited ATR7 gene.

[0210] According to a further embodiment, the present invention provides a method as defined in any of the above, wherein the plant is a non-transgenic plant line comprising a disrupted or knocked out ATR7 gene obtained through CRISPR / Cas9 genome editing.

[0211] According to a further embodiment, the present invention provides a method as defined in any of the above, wherein the at least one genomic modification is a mutation conferring reduced to completely loss of function of the ATR7 gene.

[0212] According to a further embodiment, the present invention provides a method as defined in any of the above, wherein said abiotic stress-induced oxidative stress conditions comprise oxidative and abiotic stresses including hydrogen peroxide, ROS-inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCh), osmotic, drought and salinity stress.

[0213] According to a further embodiment, the present invention provides a method as defined above, wherein said modified crop plant belongs to a crop family selected, but not limited to, from the group consisting of cereals, legumes, fruits, and vegetables. According to a further embodiment, the present invention provides a method as defined in any of the above, wherein said modified crop plant is a plant species selected from, among others, tomato, cucumber, lettuce, rice, maize, soybean, wheat, and potato.

[0214] According to a further embodiment, the present invention provides a method as defined in any of the above, wherein said ATR7 gene is Solarium lycopersicum (SI) ATR7 (S1ATR7) comprising a wild-type genomic sequence comprising at least 80% identity to SEQ ID NO: 153 or a genomic sequence encoding a wild-type amino acid sequence comprising at least 80% identity to SEQ ID NO: 157.

[0215] According to a further embodiment, the present invention provides a method as defined in any of the above, wherein said method comprising generating the genetic modification in planta via transforming the crop plant with a construct comprising (a) Cas DNA and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3- 152 and any combination thereof.

[0216] According to a further embodiment, the present invention provides a method as defined in any of the above, wherein said genomically edited ATR7 gene comprises a mutated genomic sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 154-156 or a genomic sequence encoding a mutated amino acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 158-160.

[0217] Each of the subcomponents of the system can be implemented in various ways and perform different functions. The method of application will be better understood with the following examples.

[0218] EXAMPLE

[0219] Production of a modified crop plant with enhanced oxidative stress tolerance induced by abiotic stress through targeted genome editing

[0220] This example describes a generalized scheme of the process for generating the genome edited crop plants of the present invention. The process comprises the following steps:

[0221] 1. Design and synthesize gRNAs targeting a specific sequence for editing. The gRNAs are designed to be flanked by a unique restriction site sequence, so that if editing is successful, the restriction site will be disrupted, enabling easier screening. 2. Performing transformation using Agrobacterium or bioli sties. For Agrobacterium and bioloistics transformation using a DNA plasmid, a vector containing a selection marker, Cas9 gene and relevant gRNAs are constructed. For biolistics using Ribonucleoprotein (RNP) complexes, RNP complexes are created by mixing the Cas9 protein with relevant gRNAs.

[0222] 3. Regeneration in tissue culture. For transformation performed with plasmid DNA, antibiotics are used for selection of positive transformants.

[0223] 4. Selecting positive transformants. Once regenerated plants appear in the culture, obtaining leaf, or any other selected tissue samples, extracting DNA from the obtained sample and performing PCR using primers flanking the genomic region targeted for editing. The resulting PCR products are digested with enzymes recognizing the restriction site near the original gRNA sequence. If an editing event occurred, the restriction site will be disrupted, and PCR product will not be cleaved. Absence of an editing event will result in a cleaved PCR product.

[0224] It is within the scope of the current invention that genetic markers specific to the above- mentioned editing events and / or phenotypes, for various crops are developed and provided by the current invention.

[0225] It is further within the scope of the current invention that allele and genetic variation is analyzed for the crop strains used.

[0226] Reference is now made to optional stages that have been used to produce Tomato plants exhibiting a loss of function, or a knock-out of the ATR7 gene by genome editing:

[0227] Stage 1. Identifying the ATR7 gene in the target crop Solarium lycopersicum (SI) Tomato species (S1ATR7).

[0228] The ATR7 orthologue gene has been identified in Solarium lycopersicum (SI) namely S1ATR7. The S1ATR7 gene has a genomic DNA sequence as set forth in SEQ ID NO:1 (Solarium lycopersicum cv. M82 genome), encoding amino acid sequence as set forth in SEQ ID NO: 2.

[0229] Stage 2. Designing and synthesizing gRNA molecules corresponding to (or complementary to) the sequence targeted for editing, i.e. S1ATR7 genomic sequence.

[0230] It is noted that the editing event is preferably targeted to a unique restriction site sequence to allow easier screening for plants carrying an editing event within their genome. According to some aspects of the invention, the nucleotide sequence of the gRNAs should be compatible with the genomic sequence of the target gene. Therefore, for example, suitable gRNA molecules should be constructed for different ATR7 homologues of different crop strains.

[0231] Reference is made to Table 1 presenting a summary of the sequences within the scope of the current invention for the ATR7 gene of the Solarium lycopersicum cv. M82 genome species (S1ATR7).

[0232] Table 1: Summary of WT and mutated ATR7 sequences within the scope of the present invention

[0233] Reference is now made to Table 2 presenting gRNA sequences constructed for knocking down the ATR7 gene in the Tomato plant. The term 'PAM' refers hereinafter to Protospacer Adjacent Motif, which is a 3 base pair DNA sequence immediately following the DNA sequence targeted by the Cas nuclease in the CRISPR bacterial adaptive immune system. Table 2: gRNA and PAM sequences targeted for S1ATR7

[0234] The above gRNA molecules have been cloned into suitable vectors and their sequence has been verified.

[0235] Stage 3. Utilizing CRISPR / Cas9 genome editing technology to disrupt or knock out the S1ATR7 gene.

[0236] Transforming crop plants using Agrobacterium or biolistics (gene gun) methods. For Agrobacterium and bioloistics, a DNA plasmid carrying (Cas9 + gene specific gRNAs) can be used. For biolistics, Ribonucleoprotein (RNP) complexes carrying (Cas9 protein + gene specific gRNAs) are used. RNP complexes are created by mixing the Cas9 protein with relevant gene specific gRNAs.

[0237] According to some embodiments of the present invention, transformation of the Tomato crop plant tissues was performed using particle bombardment of:

[0238] • DNA vectors

[0239] • Ribonucleoprotein complex (RNP’s)

[0240] According to further embodiments of the present invention, transformation of Tomato crop plant tissues was performed using Agrobacterium (Agrobacterium tumefaciens) by:

[0241] • Regeneration-based transformation

[0242] According to further embodiments of the present invention, additional transformation tools were used in crops, including, but not limited to:

[0243] Protoplast PEG transformation Extend RNP use

[0244] • Directed editing screening using fluorescent tags

[0245] Stage 4: Regeneration in tissue-culture. When transforming DNA constructs into the plant, antibiotics is used for selection of positive transformed plants.

[0246] Stage 5: Selection of positive transformants. Once regenerated plants appear in tissue culture, DNA is extracted from leaf samples of the transformed plant and PCR is performed using primers flanking the genomic region targeted for editing. PCR products are then digested with enzymes recognizing the restriction site near the original gRNA sequence. If an editing event occurred, the restriction site will be disrupted, and the PCR product will not be cleaved. No editing event will result in a cleaved PCR product.

[0247] Screening for CRISPR / Cas9 gene editing events has been performed by at least one of the following analysis methods:

[0248] • Restriction Fragment Length Polymorphism (RFLP)

[0249] • Next Generation Sequencing (NGS)

[0250] • PCR fragment analysis

[0251] • Fluorescent-tag based screening

[0252] • High resolution melting curve analysis (HRMA)

[0253] Reference is now made to Table 3 presenting editing events resulting from the use of a first gRNA as set forth in SEQ ID NO: 45 (in bold) and a second gRNA reverse complementary to the sequence as set forth in SEQ ID NO: 60 (in bold and italics). PAM sequence is underlined. The following DNA sequences present a fragment or section of the S1ATR7 WT or editing events genomic sequence as set forth in Table 3.

[0254] Table 3: S1ATR7 genomic DNA Sequences: Wild Type (WT) and edited variants

[0255] The first editing event of Table 3 refers to dl68 as set forth in SEQ ID NO: 154, presenting a mutated S1ATR7 fragment having a deletion of 168 base pairs. dl68 encodes an amino acid sequence as set forth in SEQ ID NO: 158.

[0256] The second editing event of Table 3 refers to dldl as set forth in SEQ ID NO: 155, presenting a mutated S1ATR7 fragment having a deletion of two base pairs (one base pair positioned 3 base pairs upstream to the PAM sequence for the first gRNA shown in bold, and another deletion of the forth base pair of the second gRNA shown in bold and italic), dldl encodes an amino acid sequence as set forth in SEQ ID NO: 159.

[0257] The third editing event of Table 3 refers to d378 as set forth in SEQ ID NO: 156, presenting a mutated S1ATR7 fragment having a deletion of 378 base pairs. d378 encodes an amino acid sequence as set forth in SEQ ID NO: 160.

[0258] The following protein sequences present a fragment or section of the S1ATR7 WT or editing events amino acid sequences as set forth in Table 4.

[0259] Table 4: S1ATR7 Protein Sequences: Wild Type (WT) and edited variants

[0260] As can be seen, Table 4 presents Wild Type (WT) and edited amino acid sequences as set forth in SEQ ID NO: 157-160, which correspond to or encoded by genomic sequences as set forth in SEQ ID NO: 153-156, respectively.

[0261] DNA was extracted from T1 transformed plants. PCR was performed using ATR7 specific primers to identify editing events (large deletions are visible on the gel and small deletions are sent for sequencing). In addition, PCR using Cas9 specific primers was performed to identify transgenic plants. Based on this analysis, edited, non-transgenic plants were selected for further analysis.

[0262] Reference is now made to Fig. 1 presenting an agarose gel showing PCR products. PCR was performed using S1ATR7 specific primers to identify editing events, wherein editing events having large deletions were identified by gel separation (dl68 and d378), while editing events with small deletions were identified by sequencing (d -1, -1).

[0263] The WT target amplification size is 727 base pairs. In Fig. 1, the dl68 editing event showing deletion of 168 bp (23_53_P1_P2 in Fig. 1) is migrating on the agarose gel as a 559 base pairs band (samples marked as #117-131 in Fig. 1). The d-1, -1 editing event is a deletion of 1 bp at two locations in the S1ATR7 gene (23_53_P3_P2 in Fig. 1) and is migrating on the agarose gel as a 725 base pairs band (samples marked as #132-153 in Fig.l).

[0264] Reference is now made to Fig. 2 presenting an agarose gel showing PCR products. PCR was performed using Cas9 specific primers identifying transgenic plants for Cas9, for the dl68 editing event transformed plants (23_53_P1_P2, samples #117-131 in Fig. 2) and for the d-1, - 1 editing event transformed plants (23_53_P3_P2, samples #132-153 in Fig. 2). Based on the analysis of both agarose gels and / or sequencing results, edited, non-transgenic plants were selected for further analysis. The selected tested plants were those that had the deletion (editing event) and were negative for Cas9 presence, namely were non-transgenic (see d-1, -1 samples # 139, 140, 142, 144, 147, & 148 of Figs 1 and 2).

[0265] Stage 6: Selection of transformed crop plants presenting enhanced abiotic stress- induced oxidative stress tolerance resulting from knocking out the ATR7 gene.

[0266] The selected plants were grown to produce T2 seeds. T2 seeds are collected and used to conduct further experiments under abiotic stress conditions (inducing oxidative stress). Plants that express an enhanced oxidative stress tolerance phenotype are selected for further development.

[0267] Abiotic stress challenges can include drought stress, salt stress, heat stress etc.

[0268] To evaluate the enhanced tolerance phenotype, response to drought stress and several ROS- inducing agents such as paraquat (PQ) treatment, were tested (the protocol of Sujeeth et al. Cellular and Molecular Life Sciences (2020) 77:705-718) is adapted, e.g. with several modifications). Plants exhibiting abiotic stress- induced oxidative stress tolerance phenotype are selected for further development, ensuring that the best editing events conferring stress resilience are advanced for subsequent cultivation and testing.

[0269] The genome editing events herein described introduce mutations that knock-out ATR7 function in the plant. Since the gene is edited, we assume reduced to completely non active gene function.

[0270] By knocking out the function of ATR7 using gene editing technique, crops with enhanced abiotic stress-induced oxidative stress tolerance are provided. Such resilient crop varieties can thrive in challenging environments (high oxidative stress, drought, salinity, heat etc).

Claims

CLAIMS1. A method for producing a modified crop plant with abiotic stress-induced oxidative stress tolerance, the method comprising introducing at least one genomic modification in an endogenous ATR7 gene using targeted genome editing techniques.

2. The method according to claim 1, wherein the at least one genomic modification is a disruption or knock out mutation of the ATR7 gene in said crop plant.

3. The method according to any one of claims 1 and 2, wherein the at least one genomic modification is a mutation conferring reduced to completely loss of function of the ATR7 gene.

4. The method according to any one of claims 1-3, wherein the method comprises steps of: a. selecting a target crop plant species; b. identifying the ATR7 gene homolog or ortholog of said target plant species; c. utilizing CRISPR / Cas9 genome editing technology to disrupt or knock out the ATR7 gene in said target plant species; and d. optionally, generating edited plant lines from the disrupted or knock out ATR7 gene plants with tolerance to abiotic stress-induced oxidative stress conditions.

5. The method according to any one of claims 1-4, wherein said modified crop plant exhibits enhanced abiotic stress-induced oxidative stress tolerance compared to wildtype or a corresponding crop plant lacking the at least one genomic modification.

6. The method according to any one of claims 1-5, wherein said abiotic stress-induced oxidative stress conditions comprise oxidative and abiotic stresses including hydrogen peroxide, ROS-inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCh), osmotic, drought and salinity stress.

7. The method according to any one of claims 1-6, wherein said modified crop plant is a non-transgenic or non-GMO plant.

8. The method according to any one of claims 1-7, wherein said modified crop plant belongs to a crop family selected, but not limited to, from the group consisting of cereals, legumes, fruits, and vegetables.

9. The method according to any one of claims 1-8, wherein said modified crop plant is a plant species selected from, among others, tomato, cucumber, lettuce, rice, maize, soybean, wheat, and potato.

10. The method according to any one of claims 1-9, wherein said modified crop plant is a Tomato plant (Solanum lycopersicum).

11. The method according to claim 10, wherein said ATR7 gene is Solarium lycopersicum (SI) ATR7 (S1ATR7) comprising a wild-type genomic sequence comprising at least 80% identity to SEQ ID NO: 153 or a genomic sequence encoding a wild-type amino acid sequence comprising at least 80% identity to SEQ ID NO: 157.

12. The method according to any one of claims 10-11, wherein said method comprising generating the genetic modification in planta via transforming the crop plant with a construct comprising (a) Cas DNA and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof.

13. The method according to any one of claims 10-12, wherein said genomically edited ATR7 gene comprises a mutated genomic sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 154-156 or a genomic sequence encoding a mutated amino acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 158-160.

14. The method according to any one of claims 1-13, wherein said method comprises steps of regeneration in a tissue culture positively selected transformed plants.

15. The method according to any one of claims 1-14, wherein said method comprises steps of screening and selecting for plants comprising editing events within ATR7 genomic region targeted for editing, from the regenerated positively selected transformed plants.

16. The method according to any one of claims 1-15, wherein said method comprises steps of selection of transformed crop plants comprising an ATR7 knock-out editing event conferring abiotic stress-induced oxidative stress tolerance.

17. A modified crop plant with abiotic stress-induced oxidative stress tolerance, produced by the method according to any one of claims 1-16.

18. A plant part, plant tissue, plant fruit or vegetable, plant seed or plant cell of the modified crop plant according to claim 17.

19. A tissue culture of regenerable cells, protoplasts or callus obtained from the modified crop plant according to claiml7.

20. The modified crop plant according to claim 17, wherein said modified crop plant does not comprise a transgene.

21. A method for enhancing tolerance to abiotic stress-induced oxidative stress conditions in a crop plant, the method comprising introducing at least one genomic modification in an endogenous ATR7 gene using targeted genome editing techniques.

22. The method according to claim 21, wherein said method comprises steps of a. selecting a target crop plant species; b. identifying an ATR7 gene homolog or ortholog in said target plant species; c. utilizing CRISPR / Cas9 genome editing technology to disrupt or knock out the ATR7 gene homolog or ortholog in said target plant species; and d. generating edited plant lines with enhanced oxidative stress tolerance induced by abiotic stress associated with the disruption or knock out of the ATR7 gene homolog or ortholog.

23. The method according to any one of claims 21 and 22, wherein the at least one genomic modification is a mutation conferring reduced to completely loss of function of the ATR7 gene.

24. The method according to any one of claims 21-23, wherein said modified crop plant exhibits enhanced abiotic stress-induced oxidative stress compared to wild-type or a corresponding crop plant lacking the at least one genomic modification.

25. The method according to any one of claims 21-24, wherein said abiotic stress-induced oxidative stress conditions comprise oxidative and abiotic stresses including hydrogen peroxide, ROS-inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCh), osmotic, drought and salinity stress.

26. The method according to any one of claims 21-25, wherein said modified crop plant is a non-transgenic or non-GMO plant.

27. The method according to any one of claims 21-26, wherein said modified crop plant belongs to a crop family selected, but not limited to, from the group consisting of cereals, legumes, fruits, and vegetables.

28. The method according to any one of claims 21-27, wherein said modified crop plant is a plant species selected from, among others, tomato, cucumber, lettuce, rice, maize, soybean, wheat, and potato.

29. The method according to any one of claims 21-28, wherein said modified crop plant is a Tomato plant (Solanum lycopersicum).

30. The method according to claim 29, wherein said ATR7 gene is Solarium lycopersicum (SI) ATR7 (S1ATR7) comprising a wild-type genomic sequence comprising at least 80% identity to SEQ ID NO: 153 or a genomic sequence encoding a wild-type amino acid sequence comprising at least 80% identity to SEQ ID NO: 157.

31. The method according to any one of claims 29 and 30, wherein said method comprising generating the genetic modification in planta via transforming the crop plantwith a construct comprising (a) Cas DNA and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof.

32. The method according to any one of claims 29-31, wherein said genomically edited ATR7 gene comprises a mutated genomic sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 154-156 or a genomic sequence encoding a mutated amino acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 158-160.

33. The method according to any one of claims 21-32, wherein said method comprises steps of regeneration in a tissue culture positively selected transformed plants.

34. The method according to any one of claims 21-33, wherein said method comprises steps of screening and selecting for plants comprising editing events within the ATR7genomic region targeted for editing, from the regenerated positively selected transformed plants.

35. The method according to any one of claims 21-34, wherein said method comprises steps of selection of transformed crop plants comprising an ATR7 knock-out editing event conferring abiotic stress-induced oxidative stress tolerance.

36. A modified crop plant exhibiting tolerance to abiotic stress-induced oxidative stress conditions, the genome of said plant comprises a disrupted or knocked out ATR7 gene obtained through CRISPR / Cas9 genome editing.

37. The modified crop plant according to claim 36, wherein the plant exhibits enhanced abiotic stress-induced oxidative stress tolerance compared to wild-type or a corresponding plant lacking the genome edited ATR7 gene.

38. The modified crop plant according to any one of claims 36 and 37, wherein the plant is a non-transgenic plant line comprising a disrupted or knocked out ATR7 gene obtained through CRISPR / Cas9 genome editing.

39. The modified crop plant according to any one of claims 36-38, wherein the at least one genomic modification is a mutation conferring reduced to completely loss of function of the ATR7 gene.

40. The modified crop plant according to any one of claims 36-39, wherein said abiotic stress-induced oxidative stress conditions comprise oxidative and abiotic stresses including hydrogen peroxide, ROS-inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCh), osmotic, drought and salinity stress.

41. The modified crop plant according to any one of claims 36-40, wherein said modified crop plant belongs to a crop family selected, but not limited to, from the group consisting of cereals, legumes, fruits, and vegetables.

42. The modified crop plant according to any one of claims 36-41, wherein said modified crop plant is a plant species selected from, among others, tomato, cucumber, lettuce, rice, maize, soybean, wheat, and potato.

43. The modified crop plant according to any one of claims 36-42, wherein said modified crop plant is a Tomato plant (Solarium lycopersicum).

44. The modified crop plant according to claim 43, wherein said ATR7 gene is Solarium lycopersicum (SI) ATR7 (S1ATR7) comprising a wild-type genomic sequencecomprising at least 80% identity to SEQ ID NO: 153 or a genomic sequence encoding a wild-type amino acid sequence comprising at least 80% identity to SEQ ID NO: 157.

45. The modified crop plant according to any one of claims 43 and 44, wherein said method comprising generating the genetic modification in planta via transforming the crop plantwith a construct comprising (a) Cas DNA and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3- 152 and any combination thereof.

46. The modified crop plant according to any one of claims 43-45, wherein said genomically edited ATR7 gene comprises a mutated genomic sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 154-156 or a genomic sequence encoding a mutated amino acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 158-160.

47. Harvestable parts of a modified crop plant according to any one of claims 36-46, wherein said harvestable parts are preferably fruits, vegetables, shoot biomass and / or seeds.

48. Products derived from a modified crop plant according to any one of claims 36-47 and / or from harvestable parts of a modified crop plant according to claim 47.

49. An isolated polynucleotide sequence comprising at least 80% identity to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: l, 3-156 and any combination thereof.

50. An isolated polypeptide sequence comprising at least 80% identity to a polypeptide sequence selected from the group consisting of SEQ ID NOs:2, 157-160 and any combination thereof.

51. Use of a nucleic acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 3-152 for targeted genome modification of a Tomato plant (Solanum ly coper sicum), for generating and / or producing a modified crop plant exhibiting tolerance to abiotic stress-induced oxidative stress conditions, according to any one of claims 36-46.

52. A method for identifying and / or selecting for a modified crop plant exhibiting abiotic stress-induced oxidative stress tolerance, said method comprises steps of screening the genome of said plant for a disrupted or knocked out ATR7 gene.

53. A method for identifying and / or selecting for a modified Tomato plant (Solarium lycopersicum) exhibiting tolerance to abiotic stress-induced oxidative stress conditions, said method comprises steps of a. screening the genome of said Tomato plant (Solarium lycopersicum) plant for a disrupted or knocked out ATR7 gene homolog or ortholog comprising a polynucleotide sequence selected from SEQ ID NO: 154-156 or a polynucleotide sequence encoding a amino acid sequence selected from SEQ ID NO: 158-160; b. optionally, selecting a modified Tomato plant (Solarium lycopersicum) plant carrying said genetic modification; and c. optionally, screening said selected plants for a plant exhibiting oxidative stress tolerance induced by abiotic stress conditions.

54. The method according to any one of claims 52 and 53, wherein the plant exhibits enhanced abiotic stress-induced oxidative stress tolerance compared to wild-type or a corresponding plant lacking the genome edited ATR7 gene.

55. The method according to any one of claims 52-54, wherein the plant is a non-transgenic plant line comprising a disrupted or knocked out ATR7 gene obtained through CRISPR / Cas9 genome editing.

56. The method according to any one of claims 54-57, wherein the at least one genomic modification is a mutation conferring reduced to completely loss of function of the ATR7 gene.

57. The method according to any one of claims 52-56, wherein said abiotic stress-induced oxidative stress conditions comprise oxidative and abiotic stresses including hydrogen peroxide, ROS-inducing agents such as paraquat (PQ), heat, cold, heavy metals (e.g. CdCh), osmotic, drought and salinity stress.

58. The method according to claim 52, wherein said modified crop plant belongs to a crop family selected, but not limited to, from the group consisting of cereals, legumes, fruits, and vegetables.

59. The method according to any one of claims 52 and 58, wherein said modified crop plant is a plant species selected from, among others, tomato, cucumber, lettuce, rice, maize, soybean, wheat, and potato.

60. The method according to any one of claims 52-59, wherein said ATR7 gene is Solarium lycopersicum (SI) ATR7 (S1ATR7) comprising a wild-type genomic sequence comprising at least 80% identity to SEQ ID NO: 153 or a genomic sequence encoding a wild-type amino acid sequence comprising at least 80% identity to SEQ ID NO: 157.

61. The method according to any one of claims 52-60, wherein said method comprising generating the genetic modification in planta via transforming the crop plant with a construct comprising (a) Cas DNA and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising Cas protein and gRNA molecule comprising a sequence selected from the group consisting of SEQ ID NO: 3-152 and any combination thereof.

62. The method according to any one of claims 52-61, wherein said genomically edited ATR7 gene comprises a mutated genomic sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 154-156 or a genomic sequence encoding a mutated amino acid sequence comprising at least 80% identity to a sequence selected from SEQ ID NO: 158-160.

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