Methods for optimizing gene expression in plants

Abstract

A method for in planta high throughput evaluation of endogenous gene regulatory elements in crop plants, comprising the steps of obtaining a nucleic acid encoding a native regulatory element of a trait-associated gene, introducing one or more genetic alterations into each of a plurality of copies of the native regulatory element, introducing the genetic library into a model plant, utilizing high throughput transformation, screening the transformed model plants, and identifying one or more genetic alterations in the modified regulatory element of the selected model plant.

Description

[Technical Field]

[0001] The present invention relates generally to the field of improving plant traits, and more particularly to optimizing the expression of natural plant traits by screening libraries of genetic elements in plants to identify desirable expression levels of the traits. [Background technology]

[0002] Crops with improved traits have significant economic impact due to the yield and quality of the product. Therefore, enhanced tolerance to various biotic or abiotic stress factors, such as herbicides, insects, diseases, extreme heat, and drought, is a desirable and advantageous characteristic that is not comprehensively addressed by current methodologies. For example, the time-consuming methods for identifying and improving traits through traditional plant breeding, as well as the highly sophisticated regulatory processes involved in the genetic engineering of genetically modified (GMO) crops, do not provide a complete and comprehensive solution.

[0003] For example, the most common problem is weeds growing alongside crops, which disrupt crop uniformity and yield and require the use of herbicides. Yield and quality losses due to unwanted weed growth, as well as the cost of weed control, have a significant economic impact on crop production. It is estimated that approximately 50% of yield loss occurs when weeds are left unattended in the field during crop growth. Therefore, there is a great need for the use of various herbicides to address this problem. Furthermore, the rapid increase in herbicide-resistant weeds is a significant challenge to global food security, as it can reduce crop production and cause significant losses. Given the lack of novel herbicides, cultivating herbicide-resistant crops is an effective strategy for weed control and expands the spectrum of herbicides, thereby reducing phytotoxicity to crops.

[0004] Herbicide-resistant (HT) crops offer advantages such as reduced use and the potential for the adoption of modern, safer pesticides.

[0005] Some of the most common GMO crops, such as corn, soybean, cotton, and canola, carry HT genes. For example, genes such as bar and pat confer resistance to glutamine synthesis inhibitors or glyphosate-based herbicides such as Roundup. Roundup targets the enzyme 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS). Roundup Ready plants carry a gene encoding a glyphosate-insensitive form, derived from Agrobacterium CP4 EPSPS (Roundup) or other resistance genes.

[0006] However, current solutions for HT involve GMO crops with transformed endogenous genes. This approach requires prior knowledge of the trait-associated genes that can be targeted or harnessed for use. Additionally, with the decline in public acceptance of GMO technology over the past few years, new, more environmentally friendly technologies, such as natural resistance, are being explored, but these are becoming increasingly difficult to find.

[0007] In some cases, natural resistance mechanisms via the plant's native genes have proven effective in overcoming stressors. For example, expressing a plant's native gene, which is the target of a herbicide, at higher than normal levels results in enhanced HT, as the high levels of the native enzyme overcome the herbicide, thereby conferring resistance.

[0008] Such natural resistance can be observed for high expression of native 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in plants, resulting in the generation of weeds resistant to EPSPS-inhibiting herbicides such as glyphosate.

[0009] Thus, there is an unmet need for a comprehensive solution to improve and optimize gene expression levels and / or phenotypes in plants. Summary of the Invention [Problem to be solved by the invention]

[0010] According to some aspects, provided herein are methods for high throughput evaluation of endogenous genetic regulatory elements of crop plants and / or coding regions of crop plants in model plants. Each possibility represents a separate embodiment.

[0011] This evaluation involves screening a plant gene library containing a large collection of modified genetic elements (i.e., regulatory elements or coding regions) functionally linked to a reporter gene that provides a phenotypic trait, thereby advantageously allowing in-planta selection for the desired change in reporter gene expression and identification of the associated genetic changes introduced into the regulatory elements or coding sequences.

[0012] In some embodiments, the methods provide for high-throughput evaluation of large collections of modified endogenous genetic elements derived from genes associated with desired traits in desired crop plants (e.g., soybean, corn, rice).

[0013] In some embodiments, large collections of modified endogenous genetic elements are generated in vitro using standard molecular biology tools for mutagenesis, advantageously (i) achieving complete coverage of the elements through high-precision mutations and (ii) allowing the collection to be transformed into different types of plants (i.e., "model plants" such as Arabidopsis) that are easier to manipulate and screen.

[0014] Advantageously, the mutagenesis step is carried out in vitro, which allows dissociating / decoupling the genetic element from the crop plant from its coding sequence, if desired.

[0015] Even more advantageous is the functional association / coupling of the modified genetic element with a reporter gene (e.g., herbicide resistance, visualization gene such as green fluorescent protein (GFP) etc.), thereby providing an easily detectable phenotypic trait that can be assessed simply by applying a herbicide to the plant according to a predetermined cut-off concentration and / or visualizing marker expression.

[0016] In some exemplary embodiments, this approach can be used to create a chimeric pGmppo1-AtPPOX1 gene (the Glycinemax ppo1 promoter linked to the A. thaliana PPOX1 gene).

[0017] Generating transgenic model plants such as Arabidopsis that host large collections of mutated genetic elements derived from crop plants alongside easily screenable markers such as HT will facilitate large-scale in planta screening and selection of genetic changes that improve trait expression.

[0018] In some embodiments, the methods allow for high-throughput screening of desired changes in traits without screening for mutations in individual crop plants.

[0019] In some embodiments, genetic changes in genetic elements originally derived from a desired crop plant and introduced into a model plant can be identified, followed by generation of genetically modified crop plants using gene editing methods that address the identified associated mutations.

[0020] Thus, advantageously, the disclosed invention avoids the inefficient step of screening crop plants for mutations that improve traits.

[0021] In some embodiments, the Arabidopsis plants of the present disclosure are transgenic for modified genetic elements derived from other important crop plants.

[0022] In some embodiments, the reporters of the present disclosure (e.g., herbicide resistance) allow for assessment of the level of optimization in living / viable plants (i.e., without the need for staining assays or any destructive steps to the plants).

[0023] In some embodiments, the genetic alteration is introduced into a transgenic genetic element that is the subject of screening and is derived from a desired target crop plant.

[0024] Introducing changes into the sequence of a gene's regulatory elements (and to some extent its coding sequence) can result in altered / improved expression patterns of the gene, but which of these changes will result in the desired expression pattern and how to discover these changes is unknown; this combination can be represented as a problem of approximately 10,118,250 different sequences to be constructed and screened for a 1500 bp genetic element.

[0025] To overcome or circumvent these limitations, in some embodiments, the present disclosure provides methods that allow for the advantageous screening of tens of thousands to millions of individually mutated genetic elements derived from crop plants in Arabidopsis thaliana. In some embodiments, the use of Arabidopsis thaliana to host genetic elements modified from crop plants allows for the application of simple mutagenesis assays that are performed in vitro rather than within the cellular system of the crop plant.

[0026] In some embodiments, the disclosed methods involve in vitro mutagenesis, whereby any nucleotide can be mutated. In some embodiments, the methods have no off-target effects. In some embodiments, the methods result in truly complete coverage of genetic elements by mutation.

[0027] The disclosed methods advantageously couple a collection of modified genetic elements to a reporter that provides an easily detectable phenotypic trait, such as a herbicide resistance trait (HT trait). In some embodiments, using the HT trait as a reporter allows for automated, truly large-scale in planta screening by simply applying / spraying the relevant herbicide according to a predetermined cutoff concentration.

[0028] In some embodiments, by combining the HT trait, or similar, with a collection of modified genetic elements of interest, automated large-scale screening can be applied to genes associated with any trait of interest.

[0029] For example, in some embodiments, an HT trait, or a similar trait, is advantageous when evaluation of the trait requires evaluation of the effect on each single individual plant, such as a trait that affects plant yield.

[0030] In some embodiments, the reporter gene is the Arabidopsis herbicide resistance (HT)-associated protoporphyrinogen oxidase (PPOX1) gene (AtPPOX1), which results in expression of a desirable / beneficial phenotypic trait with enhanced resistance to PPO inhibitors (such as, but not limited to, oxadiazon, flumioxazin, or carfentrazone-ethyl), thereby enabling high-throughput screening of model plants that exhibit desired changes in expression from modified regulatory elements of any native gene of any desired crop plant that are operably linked to the trait.

[0031] Thus, the screens disclosed herein provide methods for in planta selection and identification of novel genetic elements with optimized activity, which advantageously provide desired in planta levels of expression, activity, and / or expression patterns of any endogenous gene.

[0032] Advantageously, the screens disclosed herein utilized the expression of a reporter gene (e.g., the HT gene) for high-throughput screening of regulatory elements (preferably regulatory elements derived from crop plants, e.g., promoters of drought-related genes) for the identification of optimized regulatory elements. Thus, screening can be performed in model plants by monitoring changes in reporter gene expression and / or model plant phenotype (e.g., improved herbicide resistance, fluorescence levels, etc.) as a result of functional coupling to specific modified versions of the regulatory element.

[0033] According to some embodiments, the identified genetic elements are optimized to express a trait-associated gene (e.g., a gene that provides herbicide resistance (HT)) at a level that provides an advantageous improvement in the trait (e.g., resistance to herbicides in crop plants).

[0034] Also disclosed herein are crop plants with improved natural herbicide resistance (HT), where the gene-edited plants contain genetic changes to regulatory elements previously identified by the present methods to confer enhanced natural resistance.

[0035] According to some embodiments, there is provided an in planta high-throughput method for evaluating endogenous gene regulatory elements, comprising: obtaining a nucleic acid encoding a native regulatory element of a trait-associated gene and coupling it to a coding sequence of a reporter gene, wherein the regulatory element is in a crop plant; introducing one or more genetic alterations into each of multiple copies of the native regulatory element, thereby obtaining a genetic library containing a large collection of modified regulatory elements; introducing the genetic library into model plants using high-throughput transformation so that, on average, each model plant receives a single modified regulatory element; screening the transformed model plants, wherein the screening comprises selecting model plants having a desired change in reporter gene expression level / phenotype; and identifying one or more genetic alterations in the modified regulatory element in the selected model plants. Each possibility is a separate embodiment.

[0036] According to some embodiments, the method further comprises modifying the native regulatory element of the trait-associated gene of the crop plant based on the one or more identified genetic changes in the modified regulatory element of the selected model plant.

[0037] According to some embodiments, the native regulatory elements are located upstream, downstream, within, or any combination of, the coding sequence of the reporter.

[0038] According to some embodiments, the native regulatory element is derived from a crop plant.

[0039] According to some embodiments, the reporter gene coding sequence is of the same or a different gene as the regulatory element of the trait-associated gene.

[0040] According to some embodiments, the native regulatory element is a promoter or a fragment thereof.

[0041] According to some embodiments, the method further comprises applying an algorithm to predict putative hot-spots in the native regulatory elements, and introducing the one or more genetic alterations comprises targeting the one or more genetic alterations to the hot-spots.

[0042] According to some embodiments, the genetic alterations comprise modifications directed to random regions of regulatory elements, predicted hot-spots, and / or combinations thereof.

[0043] According to some embodiments, the one or more genetic alterations are selected from one or more of point mutations, domain swaps, cis-element rearrangements, enhancer additions and / or silencer deletions.

[0044] According to some embodiments, introducing the gene library into the model plant comprises cloning into an Agrobacterium binary vector.

[0045] According to some embodiments, screening model plants for a desired change in reporter gene expression level comprises meeting a predetermined cutoff.

[0046] According to some embodiments, the desired change in expression level / phenotype of the trait-associated gene / reporter gene comprises a change in the transcriptional activity of the modified regulatory element.

[0047] According to some embodiments, the desired change in expression level / phenotype of the trait-associated gene / reporter gene comprises a change in time, developmental stage, cellular localization, tissue specificity and / or expression intensity of the reporter gene.

[0048] According to some embodiments, the desired change in expression level of the trait-associated gene / reporter gene comprises an increase in expression associated with an increase in gene function and / or activity.

[0049] According to some embodiments, the increased function and / or activity of the trait-associated gene / reporter gene results in increased plant resistance or increased plant yield.

[0050] According to some embodiments, the enhanced tolerance comprises tolerance to herbicides, insects, diseases, heat, drought, biotic stress or abiotic stress.

[0051] According to some embodiments, the enhanced tolerance comprises herbicide resistance (HT).

[0052] According to some embodiments, herbicide resistance (HT) comprises enhanced expression of a native enzyme, which overcomes the concentration of the active ingredient of the herbicide in the plant, thereby enhancing natural resistance.

[0053] According to some embodiments, the native enzyme is protoporphyrinogen oxidase (PPO1), or p-hydroxyphenylpyruvate dioxygenase (HPPD), or 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), or glutamine synthase, or acetolactate synthase (ALS enzyme), or 7,8-dihydropteroate synthase, acetyl-CoA carboxylase (ACCase), or any combination thereof. Each possibility is a separate embodiment.

[0054] According to some embodiments, desired crop plants are produced using gene editing tools.

[0055] According to some embodiments, there is provided a gene-edited crop plant or crop plant cell comprising a modified regulatory element of a trait-related gene, wherein the modification of the regulatory element of the trait-related gene of the crop plant is based on one or more genetic changes identified in the modified regulatory element of a selected model plant associated with a desired change in expression levels of a reporter gene, as described in the methods disclosed herein.

[0056] According to some embodiments, there is provided a gene-edited crop plant or crop plant cell comprising a modified genetic element, wherein the genetic element comprises a coding sequence of a trait-associated gene, wherein the modification of the genetic element comprising the coding sequence of a trait-associated gene of the crop plant as identified in the methods disclosed herein is based on one or more genetic changes, wherein the changes are identified in the modified genetic element comprising the coding sequence of a selected model plant associated with a desired change in expression level of a reporter gene.

[0057] According to some embodiments, the gene editing results in increased expression of a native enzyme that enhances the plant's natural tolerance to the herbicide.

[0058] According to some embodiments, the native enzyme is protoporphyrinogen oxidase (PPO1), or p-hydroxyphenylpyruvate dioxygenase (HPPD), or 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), or glutamine synthase, or acetolactate synthase (ALS enzyme), or 7,8-dihydropteroate synthase, acetyl-CoA carboxylase (ACCase), or any combination thereof. Each possibility is a separate embodiment.

[0059] According to some embodiments, a construct is provided that includes a genetically modified promoter of a plant trait-associated gene coupled to and functionally associated with a herbicide resistance (HT) gene.

[0060] According to some embodiments, the genetically modified promoter is derived from the endogenous promoter of the trait-associated gene of the plant.

[0061] According to some embodiments, the genetically modified promoter is derived from an endogenous promoter of the crop plant.

[0062] According to some embodiments, the herbicide resistance (HT) gene is exogenous to the crop plant.

[0063] According to some embodiments, there is provided a gene-edited crop plant or crop plant cell comprising a ppo1 promoter genetically modified to include one or more mutations at one or more positions corresponding to any one or more of positions -1229A, -1105T, -668A, -481T, -189A, -70T, and -61T of the soybean promoter set forth in SEQ ID NO:2.

[0064] In some embodiments, a gene-edited crop plant or crop plant cell is provided, wherein the crop plant is soybean.

[0065] In some embodiments, in the gene edited crop plant or crop plant cell, the one or more mutations comprise a substitution, addition, and / or deletion.

[0066] In some embodiments, the gene edited crop plant or crop plant cell, wherein the one or more mutations comprise one or more of: -1229A>C, -1105T>C, -668A deletion, -668A>G, -481T>G, -189 insertion A, -189A>C, -189A deletion, -70T>C, and -61T>G.

[0067] In some embodiments, the gene-edited crop plant or crop plant cell, wherein the one or more mutations comprise at least two mutation positions.

[0068] In some embodiments, the gene-edited crop plant or crop plant cell, wherein the ppo1 promoter has at least 90% sequence identity to SEQ ID NO:2.

[0069] In some embodiments, the gene-edited crop plant or crop plant cell, wherein the ppo1 promoter comprises a sequence set forth in any one of SEQ ID NOs: 4-13.

[0070] Further embodiments, features, advantages, and the full scope of applicability of the present invention will become apparent from the detailed description and drawings given below. It should be understood, however, that the detailed description, while indicating preferred embodiments of the invention, is given by way of example only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0071] The present invention will now be described in connection with specific examples and embodiments with reference to the following illustrative drawings so that the invention may be more fully understood.

[0072] [Figure 1A]Figures 1A and 1B show a diagram of the different components of the binary vector backbone (pPA35H) used to clone the regulatory elements and / or coding sequence of the protoporphyrinogen oxidase (PPO1) gene. RB and LB are the right and left border recombination sites, respectively. BlpR is a bialaphos resistance gene used as a positive selection marker for plant transformation. Pro is the promoter site. Ter is the terminator site. Figure 1A shows the cloning of the Arabidopsis (A. thaliana) protoporphyrinogen oxidase (AtPPOX1) coding sequence downstream of the soybean (Glycine max) protoporphyrinogen oxidase promoter sequence (pGmppo1), thereby creating the chimeric pGmppo1-AtPPOX1 sequence. [Figure 1B] FIG. 1B shows the cloning of the soybean protoporphyrinogen oxidase (GmPPO1) coding sequence together with its promoter pGmppo1 to create the native sequence of the soybean PPO1 gene pGmppo1-GmPPO1. [Figure 2A] Figure 2A shows the general structure of a plant gene, with regulatory regions / elements and coding regions / sequences. Regulatory elements may be located upstream of the proximal and distal promoter regions and may act in cis or trans to affect the expression level of the gene. Regulatory elements may also be located within the coding region / sequence of a gene in its untranslated regions (5'-UTR and 3'-UTR). Genetic modifications may be introduced into the regulatory regions / elements and / or the coding region / sequence. [Figure 2B] Figures 2B-2C show genetic elements of trait-associated genes that are cloned into a binary vector and result in mutated sequences (marked as X) in the promoter regulatory element (Figure 2B) or the protein translation region within the coding sequence (Figure 2C). [Figure 2C]When optimizing the expression level of a trait-associated gene by targeting its translation product, mutations can be introduced into the coding region and the translated protein product can be expressed as a fusion protein fused to a reporter gene, as shown in Figure 2C. [Figure 3A] Figure 3A shows steps I to V of the process of cloning and randomly mutagenizing the native promoter regulatory element of the soybean PPO gene (pGmPPO1). [Figure 3B] Figure 3B shows a histogram of the distribution of the number of mutations per position along the pGmppo1 promoter, forming the mutant library amplified from random colonies and sequenced after error-prone PCR mutagenesis in the chimera pGmppo1-AtPPOX1 (upper panel) or pGmppo1-GmPPO1 (lower panel). [Figure 3C] Figure 3C shows representative photographs from an in planta functional screening assay for PPO herbicide-resistant plants. To identify transgenic plants containing the genetic alteration in the PPO1 promoter (pGmppo1), T1 transformed plants were first selected using glufosinate ammonium. Functional selection was performed by spraying the plants with 5 x 10-3% oxadiazon (Star). The open circle indicates a transgenic PPO-resistant plant containing the desired genetic alteration in the PPO1 promoter (pGmppo1) surrounded by multiple susceptible plants containing other non-resistant enhanced PPO promoters. [Figure 3D] Figure 3D shows an analysis of mutation distribution revealed by the functional herbicide selection assay. Each line represents a genetic change (mutation) found in the sequence of the pGmppo1 promoter region in an individual selected event. Each dot indicates the position of a mutation. A solid dot indicates a mutation detected in more than one event. An open dot indicates a substitution mutation, an X indicates a deletion mutation, and a V indicates an insertion mutation. [Figure 4]Figure 4 outlines the process for optimizing trait-related gene expression in plants. Following selection and cloning of the native regulatory and / or coding sequence elements of the trait-related gene, screening assays are calibrated to predetermine the selection conditions. In parallel, genetic libraries containing a large collection of 10-10 modified gene regulatory and / or modified coding sequence elements are constructed, transformed, and screened in a high-throughput manner in model plants. Selected genetic changes that result in less than the desired change in gene expression of the trait-related gene / reporter are identified and retransformed. The endogenous promoter / coding sequence of the crop plant may then be gene-edited to replicate the identified changes resulting in optimized expression. DETAILED DESCRIPTION OF THE INVENTION

[0073] In the following description, various aspects of the present disclosure are described. For purposes of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the present disclosure. However, it will also be apparent to those skilled in the art that the present disclosure can be practiced without the specific details presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the present disclosure.

[0074] For convenience, certain terms used in the specification, examples, and appended claims are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which they belong.

[0075] [Definition] To facilitate understanding of the present invention, several terms and phrases are defined below. It is to be understood that these terms and phrases are for purposes of description and not of limitation, and that a term or phrase herein should be interpreted by one of ordinary skill in the art in light of the teaching and guidance presented herein, in combination with the knowledge of such an artisan.

[0076] As used herein, "plant" refers to any plant at any stage of development, including plant seeds. The term "plant" includes the entire plant or any part or derivative thereof, such as a plant cell, plant protoplast, plant cell tissue culture (from which a plant can be regenerated), plant callus or callus, meristematic cell, microspore, embryo, immature embryo, pollen, ovule, anther, fruit, flower, leaf, branch, stem, cutting, cotyledon, stamen, pistil, seed, seed coat, root, root tip, etc.

[0077] As used herein, the terms "crop plant" and "crop" can be used interchangeably and refer to any plant that can be grown and harvested extensively for profit or subsistence. Crop can refer to either the harvested part or a more refined harvest. Most crops are grown agriculturally or in aquaculture. Crops can include macroscopic fungi (e.g., mushrooms) or macroscopic marine algae (e.g., seaweed). Most crops are harvested for human food or livestock feed. Some crops are collected from the wild, including intensive harvests of, for example, ginseng, yohimbe, and eucommia. Important non-food crops include horticultural crops, floricultural crops, ornamental plants, forest trees and plants, and industrial crops. Horticultural crops include plants used for other crops (e.g., fruit trees). Floriculture crops include bedding plants, greenhouse plants, flower gardens, and potted plants, cut cultivated greens, and cut flowers. Industrial crops are produced for clothing (fiber crops such as cotton), biofuels (energy crops, algae fuels), or medicine (medicinal plants).

[0078] As used herein, the term "model plant" may refer to any plant, typically derived from an extensively studied plant species, selected to facilitate the investigation of a particular biological phenomenon. Model plants provide biological insights relevant to other plants, such as crop plants. According to some embodiments, crop plants may be model plants.

[0079] As used herein, the term "native" refers to endogenous sequences found naturally in a plant. The terms "native," "naturally," and "endogenously" may be used interchangeably.

[0080] For example, as used herein, in some embodiments, a native sequence is any endogenous gene or segment of DNA naturally found in a plant. In some embodiments, a native sequence is an endogenous regulatory element naturally found in a plant. In some embodiments, a native sequence is an endogenous coding region naturally found in a plant.

[0081] As used herein, the terms DNA "region," DNA "segment," DNA "sequence," and DNA "element" may be used interchangeably and refer to a portion of DNA.

[0082] As used herein, the term "portion of DNA" refers to any portion of DNA, whether a non-coding segment of DNA or a coding segment of DNA.

[0083] As used herein, the term "non-coding sequence" may refer to regulatory elements such as, but not limited to, promoters and / or enhancers. According to some embodiments, the non-coding sequence may be the 5'UTR (untranslated region) or 3'UTR of a gene. According to some embodiments, the non-coding sequence may be an intron.

[0084] As used herein, the term "coding sequence" may refer to a portion of a gene that encodes a functional transcript and / or a protein. A coding sequence includes untranslated regions (UTRs) and protein translation regions (reading frames). The terms "coding sequence" and "coding region" may be used interchangeably.

[0085] As used herein, the term "trait" refers to a phenotype that originates from a particular sequence or group of sequences. A phenotypic trait has a detectable characteristic that can be measured. As used herein, the terms "trait" or "phenotypic trait" can be used interchangeably. A trait may or may not originate from a native sequence.

[0086] As used herein, a trait confers an improved characteristic, such as a desirable / beneficial attribute, on a plant.

[0087] As used herein, the term "trait-related sequence" or "trait-related gene" refers to the gene / genotype from which a trait is derived or by which a trait is affected. The terms "trait-associated sequence" and "trait-associated gene" can be used interchangeably and can refer to the DNA coding sequence of a gene or the functional product of that gene that is produced (i.e., transcribed and / or translated) based on that coding sequence. A trait-associated gene may or may not be native.

[0088] According to some embodiments, the trait-associated gene is in a crop plant.

[0089] For example, as used in the context of the present invention, in some embodiments, a trait confers improved characteristics that are industrially desirable / beneficial attributes, where desirable / beneficial attributes include, but are not limited to, tolerance to herbicides, insects, and diseases or other biotic stresses, resistance to heat, drought, or other abiotic stresses, reduced time to crop maturity, increased yield, improved fertilizer uptake, improved industrial processing such as conversion of starch or biomass to fermentable sugars, and improved agronomic quality such as increased oil content and increased protein content. Each possibility is a separate embodiment.

[0090] As used herein, the term "reporter gene" may refer to a coding sequence to which a regulatory element is functionally coupled and whose expression can be monitored, thereby allowing for evaluation of the effectiveness of the regulatory element in causing expression of the reporter gene. In some embodiments, the reporter gene may be a trait-related gene, such as a herbicide resistance (HT) gene. In some embodiments, the reporter gene may be an exogenous gene that is not normally expressed in plants, such as, but not limited to, GFP.

[0091] According to some embodiments, the reporter gene and the trait-associated gene are of the same or different genes.

[0092] Non-limiting examples of reporter genes include herbicide resistance genes, GFP, and RUBY (red betalain).

[0093] According to some embodiments, the reporter gene allows for evaluation in living and / or viable plants.

[0094] According to some embodiments, the reporter gene provides a color, and according to some embodiments, the intensity of the color correlates with the level of improvement / optimization / enhancement of the trait or natural resistance conferred by the trait.

[0095] As used herein, the terms "resistance" and "tolerance" may be used interchangeably.

[0096] For example, as used in the context of the present invention, in some embodiments, high expression levels of the plant native trait-associated gene protoporphyrinogen oxidase (PPO1) result in the expression of the desirable / beneficial crop plant trait of herbicide resistance (HT) because the enzymatic activity of the gene exceeds its herbicidal activity, thereby conferring tolerance to PPO-inhibiting herbicides (e.g., Group 14, including active groups: diphenyl ether, N-phenylphthalimide, thiadiazole, triazolinone, N-phenyloxadiazolone, phenylpyrazole, such as flumioxazin, oxadiazon, fomesafen, fluthiacet, carfentrazone, sulfentrazone, etc.).

[0097] As another example used herein in the context of the present invention, in some embodiments, the trait being evaluated may be that of a reporter, such as a fluorescent, luminescent, or other state-of-the-art molecular reporter used for screening purposes during research and development. Once a desired expression level of the reporter is achieved, changes to the regulatory elements identified as responsible for the desired expression level can be implemented with respect to the endogenous gene.

[0098] Another example, as used herein in the context of the present invention, in some embodiments, expression of a non-native trait-associated sequence of green fluorescent protein (GFP) results in the expression of a phenotypic trait having a detectable fluorescent signal.

[0099] As referred to herein, affecting or altering the "expression level" of a trait or trait-associated gene includes a decrease or increase in the intensity of expression, efficiency of activity / function, and changes in the expression pattern of the trait. As referred to herein, affecting or altering the "expression pattern" of a gene includes changes in time, developmental stage, cellular localization, tissue specificity, and / or intensity of expression.

[0100] As used herein, the terms "desired change in expression of a trait" or "desired change in expression of a trait-associated gene" may be used interchangeably and refer to optimizing the level, expression pattern and / or activity / function of a trait-associated gene or trait in a plant.

[0101] As used herein, in some embodiments, an optimized level includes an increase or decrease in the intensity of expression of a trait or trait-associated gene. In some embodiments, an optimized pattern includes a change in the pattern of expression of a trait or trait-associated gene, including a change in time, developmental stage, cellular localization, tissue specificity, and / or expression intensity. Each possibility is a separate embodiment. In some embodiments, an optimized function / activity includes a change in enzymatic activity, structural activity, signal transduction, ligand binding function, or reporter activity. Each possibility is a separate embodiment.

[0102] According to some embodiments, the term "desired change in trait expression" or "desired change in trait-associated gene / reporter gene expression" refers to optimizing the transcriptional activity of endogenous gene regulatory elements, including changes in transcriptional activity due to modified regulatory elements.

[0103] According to some embodiments, the term "desired change in trait expression" or "desired change in trait-associated gene / reporter gene expression" refers to optimization of the coding sequence, including alteration of the function / activity of the gene product due to modified coding sequence elements.

[0104] The desired change in expression level, pattern, and / or activity / function of the trait-associated gene involves screening and selecting plants into which genetic changes have been introduced into native regulatory elements and / or coding sequences that meet a predetermined cutoff, where the cutoff for selection of modified genetic elements (regulatory elements or coding sequences) is predetermined with respect to the elements from which they are derived.

[0105] As used herein, the term "predetermined cutoff" refers to the screening process for individual plants that exhibit a desired change in the expression level of a trait. The predetermined cutoff is determined by exposing plants transformed with a genetic element to selection conditions, including gradual changes in the strength and / or intensity of the "selection condition," and selecting based on "selection criteria." The cutoff may be predetermined, e.g., tolerance to a predetermined level of herbicide, or may be experimentally established (e.g., following lethal dose experiments). The cutoff is then established for screening and selecting plants carrying a modified genetic element (modified regulatory element or modified coding sequence) to exhibit a desired expression level of the trait. Thus, the cutoff for selection of a regulatory element is preferably predetermined relative to the element from which it is derived. According to some embodiments, meeting the predetermined cutoff represents a comparison of the expression levels obtained between plants with modified genetic elements and plants with the native genetic elements from which they are derived.

[0106] As used herein, the term "selective conditions" refers to conditions that induce stress for the purpose of selection. In some embodiments, the selection is selection for a desired expression of a trait. Non-limiting examples of selective conditions include plant growth conditions, including herbicides for herbicide resistance, insecticides for insecticide tolerance, higher than normal salt concentrations for salt tolerance selection, higher than normal osmotic stress for drought tolerance, elevated temperatures for extreme heat tolerance, sources of stimulus or induction of activity for molecular reporters, such as substrate molecules or sources of excitation, etc. Each possibility is a separate embodiment.

[0107] As used herein, in some embodiments, when screening for herbicide resistance (HT) traits, selection conditions can be established by inducing increased stress conditions with increasing concentrations of herbicide or using predetermined cutoff concentrations of PPO-inhibiting herbicides such as flumioxazin, oxadiazon, fomesafen, fluthiacet, carfentrazone, and sulfentrazone (14 groups include the active groups: diphenyl ether, N-phenylphthalimide, thiadiazole, triazolinone, N-phenyloxadiazolone, and phenylpyrazole).

[0108] As used herein, "selection criteria" refers to a set of conditions for detecting and / or measuring a phenotypic trait. Non-limiting examples of selection criteria include any signal derived from a molecular reporter, turgor pressure, plant death, leaf area, plant shoot fresh weight, leaf number, chlorophyll content, sugar content, protein content, organ color, branch fresh weight, main branch length, flower yield, pod or fruit yield, chlorosis, leaf damage, plant condition or performance, and any combination thereof. Each possibility is a separate embodiment.

[0109] As used herein, in some embodiments, when screening for a trait of a fluorescent reporter signal, the selection criteria are set to detect a gradual change in the fluorescent signal or a predetermined cutoff in the fluorescent signal.

[0110] As used herein, the term "transcriptional activity" refers to any event in the initiation of transcription that can be modified or regulated by DNA-binding proteins such as RNA polymerases, transcription factors and other transcription-associated proteins or protein complexes.

[0111] As used herein, the term "other transcription-related proteins or protein complexes" refers to any proteins or protein complexes that have an activity or function that affects the transcriptional activity of genes and that directly or indirectly bind or interact with DNA regulatory elements. Non-limiting examples of transcription-related proteins or protein complexes include RNA polymerases, general and specific transcription factors and transcription cofactors, cis-acting factors, trans-acting factors, transcription activators, transcription repressors, mediators, modulators, and transcription preinitiation complexes (PICs). Each possibility is a separate embodiment.

[0112] As used herein, the term "hot-spot" refers to specific sites within regulatory elements that are particularly important for regulating the binding of DNA-binding proteins involved in the process of transcription. These include transcription factors and other transcription-associated proteins that interact with DNA in a sequence-specific manner. According to some embodiments, hot-spots can be identified by applying dedicated hot-spot identification algorithms. In some embodiments, hot-spots can be identified by in planta functional screening of randomly mutated genetic elements and identifying resistance events that exhibit, for example, improved HT resistance, as exemplified in Example 5.

[0113] As used herein, the term "genetic element" refers to a segment of a polynucleotide DNA molecule and includes both non-coding (i.e., regulatory) and coding sequences.

[0114] As used herein, the term "gene regulatory element" refers to a segment of a polynucleotide DNA molecule that can change the expression level and / or expression pattern of a specific gene, thereby controlling gene expression. As referred to herein, regulatory elements are sequences of non-coding DNA that bind to proteins such as transcription factors and other transcription-related proteins and protein complexes. These proteins regulate the signals transmitted to the promoter associated with the gene, thereby affecting the transcriptional activity of the gene and changing the associated expression of the gene product.

[0115] According to some embodiments, the native regulatory element is of a trait-associated gene of the crop plant. According to some embodiments, the native coding sequence is of a trait-associated gene of the crop plant.

[0116] As used herein, in some embodiments, a regulatory element is up to about 5 base pairs (bp) in length, up to about 10 base pairs (bp) in length, up to about 50 base pairs (bp) in length, up to about 100 base pairs (bp) in length, or up to about 500 base pairs (bp) in length, or up to about 1000 base pairs (bp) in length, or up to about 5000 base pairs (bp) in length. Each possibility is a separate embodiment.

[0117] In some embodiments, the regulatory element is located within the promoter region of the gene upstream of the transcription region of the affected gene. In some embodiments, the regulatory element is located downstream of the transcription region of the affected gene. In some embodiments, the regulatory element is located within the transcription region of the affected gene. In some embodiments, the regulatory element is located within the 5' or 3' UTR of the gene. In some embodiments, the regulatory element is located within an intron of the gene.

[0118] As used herein, in some embodiments, a regulatory element is located at a length of up to about 10 base pairs (bp) from the transcription start site (TSS), up to about 100 base pairs (bp) from the TSS, up to about 500 base pairs (bp) from the TSS, up to about 1,000 base pairs (bp) from the TSS, up to about 5,000 base pairs (bp) from the TSS, or up to about 10,000 base pairs (bp) or up to about 50,000 base pairs (bp) from the TSS. In some embodiments, the regulatory element is located on a different molecule from the TSS. Each possibility is a separate embodiment.

[0119] Regulatory elements act in cis or trans to regulate the signals transmitted to the promoter associated with a gene.

[0120] As used herein, the term "trans-regulatory element" (TRE) refers to a regulatory element that modifies or regulates the expression of a non-adjacent gene. In some embodiments, the trans-regulatory element is located on a different molecule from the gene whose transcription it regulates.

[0121] As used herein, the term "cis-regulatory element" (CRE) refers to a regulatory element that modifies or regulates the expression of genes in its vicinity. In some embodiments, cis-regulatory elements are located within the core, proximal, or distal promoter. In some embodiments, cis-regulatory elements are located within the transcribed region of the genes they regulate, and in some embodiments, cis-regulatory elements are located downstream of the genes they regulate.

[0122] As used herein, the term "promoter" refers to a non-coding sequence of DNA to which proteins bind to initiate transcription and further regulate the degree and / or manner of expression of an RNA transcript from the DNA downstream of the promoter. A promoter is located upstream of the transcription start site (TSS) on the DNA toward the 5' region of the transcribed strand.

[0123] As used herein, the terms "promoter" and "regulatory element" may be used interchangeably in reference to a DNA sequence or fragment that controls the transcription process and expression level and / or pattern of a gene in a sequence-dependent manner by binding proteins involved in transcription (e.g., transcription factors and other transcription-associated proteins). According to some embodiments, the regulatory element may be a naturally occurring regulatory element, e.g., a regulatory element of a plant or other organism. According to some embodiments, the regulatory element may be a synthetic regulatory element not found in nature.

[0124] As used herein, in some embodiments, a typical structure of a plant gene includes a regulatory element including a promoter. In some embodiments, a typical structure of a plant gene includes a promoter including a regulatory element.

[0125] In some embodiments, the promoter consists of a proximal promoter (including a core promoter) and a distal promoter.

[0126] As used herein, the term "proximal promoter" refers to a regulatory element located near the transcription start site of a gene. In some embodiments, the proximal promoter is up to about 100 base pairs (bp) long, up to about 250 base pairs (bp) long, up to about 500 base pairs (bp) long, up to about 1000 base pairs (bp), or up to about 1500 base pairs (bp), or up to about 2000 base pairs (bp). Each possibility is a separate embodiment.

[0127] The proximal promoter includes the "core promoter" of a gene, a term used herein to describe the minimum portion of the proximal promoter required to properly initiate transcription. In some embodiments, the core promoter may be located around the TSS and extend into the transcribed region. In some embodiments, the core promoter may include regulatory elements extending into the transcribed region by up to about several tens of base pairs, and non-limiting examples of such regulatory elements extending into the transcribed region include downstream promoter elements (DPEs).

[0128] Thus, as used herein, the terms "core promoter" and "proximal promoter" can be used interchangeably in reference to regulatory elements located near the TSS.

[0129] Non-limiting examples of regulatory elements in proximal and core promoters include cis-regulatory elements, TATA boxes, initiator sequences (INRs), downstream promoter elements (DPEs), E boxes, upstream activator sequences (UASs), and response elements (REs). Each possibility is a separate embodiment.

[0130] As used herein, the term "distal promoter" refers to a regulatory element located up to about several kilobases or up to about tens of kilobases away from the transcription start site. In some embodiments, the distal promoter contains a cis-regulatory element (CRE), which can function as a transcription enhancer and also play an auxiliary role in increasing the transcription activity of the enhancer. In some embodiments, domain swapping, rearrangement of cis-elements, and combination of cis-regulatory elements from different promoters have significant effects on transcription levels. Non-limiting examples of regulatory elements in distal promoters include cis-regulatory elements, enhancers, silencers, and insulators.

[0131] In some embodiments, the regulatory element is an enhancer. As used herein, the term "enhancer" refers to a regulatory element that upregulates the overall expression of a gene by binding a transcriptional activator that promotes the formation of a transcription preinitiation complex at the core promoter region. In some embodiments, an enhancer can also bind to a transcriptional repressor that reduces the overall expression of a gene by competing with the binding of an activator to the enhancer, thereby preventing the formation of a preinitiation complex. In some embodiments, an enhancer regulates the expression level and / or expression pattern.

[0132] In some embodiments, the regulatory element is a silencer. As used herein, the term "silencer" refers to a regulatory element (for example, but not limited to, an insulator) that prevents enhancers from acting on the promoter of a gene, thereby downregulating the overall transcription of the gene. In some embodiments, the silencer regulates expression level and / or expression pattern.

[0133] As used herein, the term "native regulatory element" refers to an endogenous regulatory sequence found naturally in a plant. The terms "native regulatory element," "natural regulatory element," and "endogenous regulatory element" may be used interchangeably.

[0134] As used herein, the term "native genetic elements" refers to the endogenous regulatory sequences or coding sequences thereof found naturally in a plant.

[0135] Reference is now made to Figure 2A, which shows the plant gene structure.

[0136] In some embodiments, native gene regulatory elements from one plant species can be coupled upstream to trait-associated genes and / or reporter genes from a different plant species. In some embodiments, native gene regulatory elements from, for example, soybean (Glycine max) can be linked to trait-associated / reporter sequences from Arabidopsis (A. thaliana). In some embodiments, for example, a native soybean promoter (pGmX) can be cloned upstream of a native herbicide resistance (HT)-associated gene (e.g., protoporphyrinogen oxidase (PPOX1), Arabidopsis protoporphyrinogen oxidase (AtPPOX1)), etc., to generate a soybean-Arabidopsis chimera (pGmX-AtPPOX1) clone. Alternatively, in some embodiments, a native soybean promoter (pGmX) can be cloned upstream of a sequence encoding a reporter gene, such as the coding sequence for GFP, to generate a non-native (pGmX-GFP) clone.

[0137] As used herein, the term "modified genetic element(s)" refers to a polynucleotide sequence that has been manipulated by any method known in the art that is commonly used to introduce genetic changes into nucleic acid sequences to produce a new sequence that contains modifications relative to the native genetic element from which it was derived. As used herein, a modified nucleic acid contains at least one or more genetic changes to the polynucleotide relative to the copy of the native genetic element from which it originated.

[0138] As used herein, in some embodiments, modified genetic regulatory element(s) refers to a genetic change introduced into a regulatory element, thereby generating a new sequence that contains a modification relative to the original copy of the native regulatory element from which it was derived.

[0139] As used herein, in some embodiments, modified genetic element(s) refers to a genetic element that contains an alteration introduced into a coding sequence region, thereby generating a new coding sequence / region that contains an alteration relative to the original copy of the native coding element from which it was derived.

[0140] In some embodiments, the desired change in gene expression of the trait-associated gene / reporter gene comprises a genetic change introduced into a regulatory region / element and / or a coding region / sequence. Each possibility is a separate embodiment.

[0141] In some embodiments, one or more genetic alterations are introduced into each of multiple copies of a native coding sequence, thereby generating a genetic library containing a large collection of modified coding regions.

[0142] In some embodiments, the genetic alteration comprises an alteration to a randomized region of the coding sequence, hi some embodiments, the genetic alteration comprises an alteration to a randomized region of the protein translation sequence / protein reading frame.

[0143] Reference is now made to the figures: Figures 2B-2C show possible regions for the introduction of genetic alterations.

[0144] As used herein, the terms "changed," "altered," "manipulated," and "edited" may be used interchangeably.

[0145] Non-limiting examples of genetic alterations include one or more point mutations (deletions, substitutions and / or additions), domain swaps, rearrangements of cis elements, enhancer additions and / or silencer deletions, changes that may affect cis or trans elements. Each possibility is a separate embodiment.

[0146] Non-limiting examples of methods for introducing genetic changes into nucleic acid sequences include site-specific mutagenesis (site-directed mutagenesis), chemically or biologically induced random mutagenesis, gene editing methods, recombination, restriction enzyme-ligation, and solid-phase synthesis. Each possibility is a separate embodiment.

[0147] As used herein, the term "editing" or "gene editing" refers to any method known in the art that is commonly used to direct and introduce specific changes at specific loci into nucleic acid sequences using engineered nucleases, such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector system nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPRs). Each possibility is a separate embodiment.

[0148] The terms "genetically modified" (GM), "genetic modification" (GM) or "genetically modified organism" (GMO) may be used interchangeably.

[0149] The terms "gene function" and "gene activity" may be used interchangeably and refer to any cellular function and / or activity that a functional gene product or protein may participate in. Non-limiting examples of cellular functions of a protein include enzymatic activity, structural function, cell signaling, and ligand binding.

[0150] As used herein, the terms "massively parallel," "high throughput," and "large scale" may be used interchangeably and refer to the simultaneous transformation and screening of at least 50, at least 100, at least 500, at least 1000, at least 10,000, or at least 100,000 altered gene regulatory elements or coding sequences. Each possibility is a separate embodiment.

[0151] As used herein, the term "evaluation" refers to the process of screening and identifying regulatory elements for the desired transcriptional changes associated with the desired change in expression and / or activity of the trait-associated gene product to which they are coupled, by any one of the methods described herein by the present invention.

[0152] As used herein, the terms "polynucleotide molecule," "oligonucleotide," "polynucleotide," "nucleic acid," and "nucleotide" sequence may be used interchangeably. This term refers to polymers of deoxyribonucleotides (DNA), ribonucleotides (RNA), and modified forms thereof, which exist as separate fragments or as components of larger constructs, linear or branched, single-stranded (ss), double-stranded (ds), triple-stranded (ts), or hybrids thereof. Thus, as used herein, the terms "polynucleotide molecule," "oligonucleotide," "polynucleotide," "nucleic acid," and "nucleotide" sequence refer to both DNA and RNA molecules. As used herein, "nucleotide" includes a nitrogenous base, a sugar molecule, and a phosphate group. Nucleic acids may include naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine). The term further includes oligonucleotides composed of naturally occurring bases, sugars, and oligonucleotides having non-naturally occurring portions (but which function similarly to the naturally occurring portions). As used herein, nucleotides (A, G, C, or T) and nucleotide sequences are written in lowercase (a, g, c, or t). As used herein, the term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides. The term "DNA" or "DNA molecule" or deoxyribonucleic acid molecule refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA or RNA, respectively). DNA and RNA can also be synthesized chemically.

[0153] As used herein, the term "approximately" or "about" in reference to a number is generally interpreted as including numbers that fall within a 5% range or a 1% range (larger or smaller) in either direction of the number, unless otherwise stated or clear from the context (except where such number exceeds 100% of its possible values). Where a range is stated, the endpoints are included in the range unless otherwise stated or clear from the context.

[0154] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0155] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances when it does not occur.

[0156] As used herein, the term "average" refers to an average value obtained by measuring a predetermined parameter and calculating an average value, wherein the measurement of said parameter is measuring the predetermined parameter in the measurement data of a particular plant population or any group of individual plants, and the calculation of the average value is done according to the number of plants in said population.

[0157] According to some aspects, provided herein are methods for in planta high-throughput evaluation of endogenous gene regulatory elements.

[0158] In some embodiments, the plant is a crop plant and / or a model plant.

[0159] In some embodiments, evaluation of endogenous gene regulatory elements involves screening and identifying desired changes in the expression levels and / or activity of trait-associated genes and / or reporter genes functionally associated with the regulatory elements.

[0160] In some embodiments, the desired change in expression level and / or activity of the trait-associated gene and / or reporter gene comprises identifying a change in the transcriptional activity of an altered regulatory element functionally associated with the gene.

[0161] According to some embodiments, provided herein are methods for in planta high-throughput evaluation of endogenous genetic coding elements.

[0162] In some embodiments, the desired change in expression level and / or activity of the trait-associated gene comprises identifying a change in the activity of a functional product of the modified coding element functionally associated with the regulatory element.

[0163] In some embodiments, the activity of the functional product of the modified coding element includes, but is not limited to, enzymatic activity, structural function, activity in cell signaling, and / or ligand binding.

[0164] In some embodiments, the change in expression level and / or activity comprises a change in the level of intensity of expression and / or the pattern of expression.

[0165] According to some embodiments, the method comprises obtaining a nucleic acid encoding a native regulatory element linked to a coding sequence of the trait-associated gene / reporter gene.

[0166] In some embodiments, the coupling comprises a functional association.

[0167] In some embodiments, the regulatory elements comprise native regulatory elements.

[0168] In some embodiments, the regulatory elements include a core promoter, a proximal promoter, a distal promoter, an enhancer, a silencer, an insulator, a cis-acting element, a trans-acting element, a TATA box, an initiator sequence (INR), a downstream promoter element (DPE), an E box, an upstream activator sequence (UAS), and / or a response element (RE), or any combination thereof. Each option is its own embodiment. Each possibility is a separate embodiment.

[0169] In some embodiments, the native regulatory element is a promoter or a fragment thereof.

[0170] In some embodiments, the native regulatory element is derived from a crop plant. In some embodiments, the native regulatory element is of any native gene and the coding sequence is of a trait-related gene / reporter gene; in some embodiments, the native regulatory element is of a trait-related gene and the coding sequence is of a trait-related gene / reporter gene.

[0171] In some embodiments, the reporter gene coding sequence is of the same or different origin as the trait-associated gene regulatory element.

[0172] In some embodiments, the encoded native or modified regulatory element is located upstream of the coding sequence of the trait-associated gene and / or reporter gene; in some embodiments, the encoded native or modified regulatory element is located within the transcribed region of the trait-associated gene and / or reporter gene; in some embodiments, the encoded native or modified regulatory element is located downstream of the coding sequence of the trait-associated gene and / or reporter gene.

[0173] In some embodiments, the nucleic acid encodes a regulatory element upstream of the trait-associated gene and / or reporter gene, and further encodes a regulatory element within the coding sequence and / or downstream of the trait-associated gene and / or reporter gene.

[0174] In some embodiments, the trait confers improved properties to the plant.

[0175] In some embodiments, improving a property includes inducing / causing an attribute that is desirable / beneficial; in some embodiments, the desirable / beneficial attribute is an industrially desirable / beneficial attribute.

[0176] In some embodiments, commercially desirable / beneficial attributes include, but are not limited to, improved plant resistance or tolerance and increased plant yield.

[0177] In some embodiments, enhanced resistance or tolerance traits include, but are not limited to, tolerance to herbicides, insects, and diseases or other biotic stresses, resistance to heat, radiation, drought, salt, cold, hypoxia, or other abiotic stresses; in some embodiments, increased yield traits include, but are not limited to, shortening the time to maturity of crops, altering plant architecture, reducing size, increasing size, improving photosynthetic activity, improving fertilizer uptake, improving fertilizer use efficiency, improving health components, improving industrial processing such as converting starch or biomass to fermentable sugars, and improving agronomic quality such as increasing oil content and increasing protein content. Each option is its own embodiment. Each possibility is a separate embodiment.

[0178] In some embodiments, the trait-associated gene is a reporter gene. In some embodiments, the trait-associated gene / reporter gene is a native gene from the plant.

[0179] In some embodiments, the trait-associated gene / reporter gene is derived from other sources (including but not limited to animals, bacteria, fungi, and viruses).

[0180] In some embodiments, the trait-associated gene is a reporter gene, including, but not limited to, a fluorescent protein, a luminescent protein, or any biological or agronomic screenable trait (e.g., but not limited to, fertility, color, shape, plant organ width, plant organ or tissue size, or number of plant organs or tissues).

[0181] In some embodiments, enhancing the function and / or activity of a trait-associated gene results in enhanced plant resistance or increased plant yield.

[0182] In some embodiments, the enhanced tolerance comprises resistance to herbicides, insects, diseases, heat, drought, biotic stress, or abiotic stress.

[0183] In some embodiments, the enhanced tolerance comprises herbicide resistance (HT).

[0184] In some embodiments, herbicide resistance (HT) involves increased expression of a native enzyme that overcomes the concentration of the active ingredient of the herbicide in the plant, thereby increasing natural resistance.

[0185] In some embodiments, the native enzyme that overcomes the herbicide active ingredient concentration in the plant, thereby increasing natural resistance, is protoporphyrinogen oxidase 1 (PPO1), protoporphyrinogen oxidase 2 (PPO2), p-hydroxyphenylpyruvate dioxygenase (HPPD), 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), glutamine synthase, acetolactate synthase (ALS enzyme), 7,8-dihydropteroate synthase, or acetyl-CoA carboxylase (ACCase). Each possibility is a separate embodiment.

[0186] According to some embodiments, the method comprises introducing one or more genetic alterations into each of multiple copies of a native regulatory element, thereby obtaining a genetic library comprising a large collection of modified regulatory elements.

[0187] In some embodiments, the modified regulatory element comprises one or more genetic alterations.

[0188] In some embodiments, the genetic alteration comprises at least one or more modifications to the polynucleotide relative to the copy of the endogenous regulatory element from which it was derived.

[0189] In some embodiments, genetic modifications are introduced into the original / native regulatory elements using at least one or more methods for altering nucleic acids, including, but not limited to, site-directed mutagenesis (site-directed mutagenesis), chemically or biologically induced random mutagenesis, gene editing methods, recombination, restriction enzyme-ligation, and / or solid phase synthesis. Each option is its own embodiment.

[0190] In some embodiments, modified regulatory elements include, but are not limited to, core promoters, proximal promoters, distal promoters, cis elements, and / or trans elements, with each option being an embodiment in itself.

[0191] In some embodiments, regulatory elements include, but are not limited to, enhancers, silencers, insulators, TATA boxes, initiator sequences (INRs), downstream promoter elements (DPEs), E boxes, upstream activator sequences (UASs), and / or response elements (REs), or any combination thereof, with each option being its own embodiment.

[0192] In some embodiments, the genetic alteration comprises, but is not limited to, at least one or more point mutations; in some embodiments, the one or more point mutations are selected from the group consisting of a nucleotide deletion, a nucleotide substitution, and / or a nucleotide addition. In some embodiments, the genetic alteration comprises one or more alterations. Each possibility is a separate embodiment.

[0193] In some embodiments, the genetic alteration includes, but is not limited to, the addition of an enhancer and / or the deletion of a silencer.

[0194] In some embodiments, genetic alterations include, but are not limited to, domain swapping and / or rearrangement of cis elements, and alterations can affect elements in cis or trans.

[0195] In some embodiments, mutations are targeted to previously known hotspots and / or their vicinity within regulatory elements.

[0196] In some embodiments, an algorithm is applied to predict putative hotspots in native regulatory elements. In some embodiments, introducing one or more genetic alterations comprises targeting one or more genetic alterations to the predicted hotspots.

[0197] In some embodiments, the genetic alterations comprise random mutagenesis, genetic alterations to predicted hotspots, and / or a combination thereof.

[0198] According to some embodiments, the method comprises using high throughput transformation to introduce the gene library into model plants, so that, on average, each model plant receives a single modified regulatory element or coding region, which may be altered at a single or multiple positions.

[0199] In some embodiments, model plants include, but are not limited to, Arabidopsis thaliana, Camelina sativa, or Nicotiana tabacum.

[0200] In some embodiments, introducing the gene library into the model plant comprises cloning into an Agrobacterium binary vector. In some embodiments, introducing the gene library into the model plant comprises using a virus and viral library with modified genetic elements to inoculate the plant. In some embodiments, introducing the gene library into the model plant comprises using "gene gun" technology or other transformation methods known in the art.

[0201] According to some embodiments, the method includes screening the transformed model plants, wherein the screening includes selecting, from the plurality of transformed plants, a model plant having a desired change in expression level of the trait-associated gene.

[0202] In some embodiments, the desired change in the expression level of a trait or the expression level of a trait-associated gene comprises optimizing the level of intensity and / or pattern of expression in the plant.

[0203] In some embodiments, the optimized expression intensity level comprises an increase or decrease in the expression intensity of a trait, and in some embodiments, the optimized expression intensity level comprises an increase or decrease in the expression intensity of a trait-associated gene / reporter gene or its activity.

[0204] In some embodiments, the optimized expression pattern comprises a change in the expression pattern of the trait or trait-associated gene / reporter gene, including changes in time, developmental stage, cellular localization, tissue specificity, and / or expression intensity. Each possibility is its own embodiment.

[0205] In some embodiments, the desired change in expression level of the trait-associated gene / reporter gene comprises an increase in expression associated with an increase in gene function and / or activity, and in some embodiments, the desired change in expression level of the trait-associated gene / reporter gene comprises a decrease in expression associated with a decrease in gene function and / or activity.

[0206] In some embodiments, the desired change in the expression level of a trait or the expression level of a trait-associated gene / reporter gene comprises optimizing the transcriptional activity of a functionally related endogenous gene regulatory element.

[0207] In some embodiments, the desired change in expression level of the trait-associated gene / reporter gene comprises a change in the transcriptional activity of an altered regulatory element functionally associated with the gene.

[0208] In some embodiments, the desired change in expression levels of a trait or trait-associated gene / reporter gene involves screening and selecting plants into which genetic changes have been introduced into native regulatory elements and / or coding sequences that meet a predetermined cutoff.

[0209] In some embodiments, screening for model plants with a desired change in expression level of the trait-associated gene / reporter gene comprises meeting a predetermined cutoff.

[0210] In some embodiments, the predetermined cutoff is determined by exposing plants transformed with the native genetic elements to selection conditions that include incremental changes in the intensity or intensity of the selection conditions and selecting based on the selection criteria.

[0211] In some embodiments, a predetermined cutoff is utilized to screen and select plants harboring modified regulatory elements to exhibit a desired expression level of a trait.

[0212] Thus, in some embodiments, a cutoff for selection of modified regulatory elements is first predetermined for the derived elements.

[0213] Thus, in some embodiments, meeting a predetermined cutoff represents a comparison between the modified regulatory elements and the native gene elements from which they are derived.

[0214] In some embodiments, the desired change in expression level and / or activity of the trait-associated gene / reporter gene comprises a change in the level of intensity of expression; in some embodiments, the desired change in the level of intensity of expression comprises a decrease in expression; in some embodiments, the desired change in the level of intensity of expression comprises an increase in expression.

[0215] In some embodiments, the desired change in expression level and / or activity of the trait-associated gene / reporter gene comprises a change in expression pattern. In some embodiments, the desired change in the expression pattern of the trait-associated gene includes, but is not limited to, a change in time, developmental stage, cellular localization, tissue specificity, and / or expression intensity. Each option is an embodiment in itself.

[0216] According to some embodiments, the method comprises identifying one or more genetic alterations in the modified regulatory element of the selected model plant.

[0217] In some embodiments, identification of one or more genetic alterations in the modified regulatory element is carried out using DNA sequencing.

[0218] According to some embodiments, the method comprises modifying a regulatory element of a crop plant based on one or more identified genetic changes in the modified regulatory element of the selected model plant.

[0219] In some embodiments, the modification of the regulatory element of a crop plant is based on the modified regulatory element from which it is derived; in some embodiments, the modification of the regulatory element of a crop plant is based on the modified regulatory element of a different plant crop.

[0220] In some embodiments, crop plants include, but are not limited to, corn, soybean, cotton, and rapeseed, each option being its own embodiment.

[0221] The method can be applied to any plant species, including but not limited to monocotyledons and dicotyledons. Examples of plant species include corn (Zeamays), Brassica species (e.g., Brassica napus, Brassica juncea), particularly Brassica species useful as sources of seed oils, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Panicum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis hypogaea), cotton (Gossypium badense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), palms from the palm family (Arecaceae) [e.g., coconut palm (Cocos mucifera), oil palm (Elaeis guineensis), date palm (Phoenix spp.)], pineapple (Ananas comosus), citrus trees (Citrus spp.), eucalyptus (Eucalyptus sp.), pine (Pinus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (occidentals), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, grasses (Poaceae), and conifers. Each possibility is a separate embodiment.

[0222] In some embodiments, desired crop plants are generated using gene editing tools.

[0223] In some embodiments, methods for gene editing include, but are not limited to, using engineered nucleases such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPRs), or any combination thereof, each option being its own embodiment.

[0224] According to some aspects, provided herein is a non-naturally occurring crop plant or crop plant cell comprising a gene-edited regulatory element.

[0225] In some embodiments, gene editing of regulatory elements of crop plants is based on one or more identified genetic changes in the modified regulatory elements of a selected model plant that are associated with a desired change in the expression level of a trait-associated gene.

[0226] In some embodiments, the gene-edited plant or plant cell comprises increased expression of a native enzyme that increases the plant's natural tolerance to the herbicide.

[0227] In some embodiments, the gene-edited plant or plant cell comprises enhanced expression of an enzyme selected from the group consisting of protoporphyrinogen oxidase 1 (PPO1), or protoporphyrinogen oxidase 2 (PPO2), or p-hydroxyphenylpyruvate dioxygenase (HPPD), or 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), or glutamine synthase, or acetolactate synthase (ALS enzyme), or 7,8-dihydropteroate synthase, or acetyl-CoA carboxylase (ACCase).

[0228] According to some embodiments, provided herein are constructs comprising a genetically modified promoter of a plant trait-associated gene coupled to and functionally associated with a herbicide resistance (HT) gene.

[0229] In some embodiments, the genetically modified promoter is derived from the endogenous promoter of the trait-associated gene in the plant.

[0230] In some embodiments, the genetically modified promoter is derived from an endogenous promoter of the crop plant.

[0231] In some embodiments, the herbicide resistance (HT) gene is exogenous to the crop plant.

[0232] According to some embodiments, disclosed herein are methods for in planta high-throughput optimization of trait-associated gene function / activity.

[0233] According to some embodiments, the method comprises obtaining a nucleic acid encoding a coding sequence of the trait-associated gene.

[0234] According to some embodiments, the method comprises introducing one or more genetic alterations into each of a plurality of copies of the coding sequence of the native trait-associated gene, thereby obtaining a genetic library comprising a large collection of modified copies of the trait-associated gene coding sequence.

[0235] According to some embodiments, the method comprises using high-throughput transformation to introduce a gene library into model plants, whereby each model plant receives, on average, a single modified trait-associated gene.

[0236] According to some embodiments, the method comprises screening the transformed model plants, wherein said screening comprises selecting model plants having a desired change in function / activity of the trait-associated gene.

[0237] According to some embodiments, the method comprises identifying one or more genetic alterations in an altered trait-associated gene coding sequence in a selected model plant.

[0238] According to some embodiments, the method comprises modifying a trait gene of a crop plant based on one or more identified genetic changes in the modified trait-associated gene of the selected model plant.

[0239] In some embodiments, the desired change in trait-associated gene function / activity comprises optimized enzyme activity.

[0240] In some embodiments, the desired change is an increase in enzyme activity.

[0241] According to some exemplary embodiments, the soybean (Glycinemax) ppo1 promoter is cloned into the Arabidopsis (Athaliana) PPOX1 gene.

[0242] In some embodiments, optimizing the expression level of a plant's native protoporphyrinogen oxidase (PPO1) gene can result in improved herbicide resistance (HT).

[0243] In some embodiments, the activity of protoporphyrinogen oxidase 1 (PPOX1) exceeds concentrations of the active ingredient of the herbicide in plants, but similar concentrations severely affect or kill WT Arabidopsis plants.

[0244] In some embodiments, it is desirable to enhance expression of protoporphyrinogen oxidase 1 (PPO1) to provide improved natural tolerance to PPO-inhibiting herbicides, such as flumioxazin.

[0245] According to some embodiments, the promoter may have the sequence set forth in SEQ ID NO: 2, although other native promoters (of the same or different trait-associated genes) are also contemplated and as such are within the scope of the present disclosure. In some embodiments, the native soybean (Glycine max) ppo1 promoter (pGmppo1) (SEQ ID NO: 2) is cloned and coupled to the Arabidopsis (A. thaliana) PPOX1 gene (AtPPOX1) (SEQ ID NO: 1) to create pGmppo1-AtPPOX1.

[0246] In some embodiments, the soybean native ppo1 promoter (pGmppo1) is amplified from soybean gDNA and cloned upstream of the native AtPPOX1 coding sequence.

[0247] In some embodiments, the native AtPPOX1 coding sequence is cloned with a specific sequence marker (barcode) that distinguishes it from the native copy of the gene.

[0248] In some embodiments, stable T1 or T2 pGmppo1-AtPPOX1 transgenic plants are used for calibration of chemically predetermined cutoff concentrations of PPO inhibitors.

[0249] In some embodiments, chemically predetermined cutoff concentrations of PPO inhibitors are used for screening and selection of mutant plant libraries.

[0250] In some embodiments, a pGmppo1-AtPPOX1-containing binary vector commonly used for library construction is introduced into Agrobacterium.

[0251] In some embodiments, Agrobacterium carrying the pGmppo1-AtPPOX1 gene is then used to transform Arabidopsis plants to produce seeds with stable T1 or T2 generation events.

[0252] In some embodiments, transformed Arabidopsis seeds are germinated and treated with a selective herbicide to detect transformed plants.

[0253] In some embodiments, T1 or T2 Arabidopsis seeds are germinated and treated with a selected herbicide.

[0254] In some embodiments, calibration of the selection cutoff value is used to screen transformed model plants for increased expression and / or activity of the AtPPOX1 enzyme and associated enhancement of herbicide resistance (HT) traits.

[0255] In some embodiments, stable T1 or T2 Arabidopsis plants transformed with the pGmppo1-AtPPOX1 gene are treated with a concentration gradient of the PPO-inhibiting herbicides flumioxazin or carfentrazone ethyl to calibrate the cutoff concentration.

[0256] In some embodiments, the stable T1 or T2 event that exhibits the best resistance to the highest concentration of the PPO-inhibiting herbicide flumioxazin or carfentrazone ethyl is selected based on at least one selection criterion.

[0257] In some embodiments, a predetermined cutoff concentration is determined for use in selecting a promoter variant transgenic library.

[0258] In some embodiments, the cutoff concentration is determined based on stable T1 or T2 Arabidopsis plants transformed with the pGmppo1-AtPPOX1 gene and exhibiting the greatest resistance to the highest concentrations of the PPO-inhibiting herbicides flumioxazin or carfentrazone ethyl.

[0259] According to some embodiments, algorithmic analysis of the native soybean ppo promoter (pGmppo1) is utilized for the detection of putative hotspots.

[0260] In some embodiments, unique algorithms are utilized to analyze non-coding regulatory elements and predict putative "hotspots" of DNA-binding proteins involved in the transcription process, which in some embodiments include transcription factors and other transcription-associated proteins that interact with DNA in a sequence-specific manner.

[0261] In some embodiments, the algorithm utilizes available information on plant regulatory element sequences from public databases such as PlantCARE (Plant cis-Acting regulatory elements), PLACE (Plant cis-Acting regulatory DNA elements), and RegSite Site (Plant-oriented Regulatory Regulatory Elements).

[0262] In some embodiments, the algorithm is applied to analyze the native soybean ppo1 gene and predict hotspots that may facilitate the desired change of enhanced transcriptional activity from pGmppo1.

[0263] In some embodiments, the algorithm receives SEQ ID NO: 1 as an input sequence (the sequence of pGmppo1), analyzes the typical structure of the pGmppo1 promoter, and recognizes specific regulatory elements therein; in some embodiments, this includes analysis of the proximal promoter (including the core promoter) and the distal promoter.

[0264] In some embodiments, promoter regions are identified by analyzing several hundred nucleotides surrounding the TSS, considered the core / proximal promoter, e.g., to identify elements that regulate transcription start site (TSS) selection, and several thousand more nucleotides upstream of the TSS, considered the distal promoter, e.g., for the presence of regulatory cis-elements that can function as transcription enhancers and play ancillary roles in increasing transcriptional activity.

[0265] In some embodiments, an algorithm identifies hotspots of putative regulatory elements in pGmppo1, processes and integrates the data, and then predicts sites on the promoter sequence that are suitable for gene manipulation / editing, thereby predicting potentially new regulatory elements with new attributes for gene expression patterns.

[0266] In some embodiments, in the case of the AtPPOX1 or GmPPO1 gene product, the desired change is an increase in enzyme expression and activity; in some embodiments, this can be achieved, for example, by introducing hotspot genetic changes that force the binding activity of a transcriptional activator or prevent the binding activity of a suppressor.

[0267] In some embodiments, a desired increase in transcriptional activity of a gene during a specific developmental growth stage of a plant can be achieved by targeting a hotspot on the pGmppo1 promoter sequence.

[0268] In some embodiments, by targeting hotspots on the pGmppo1 promoter sequence, one can achieve the desired increase in transcriptional activity of a gene in specific tissues where expression of the gene can be increased.

[0269] In some embodiments, by targeting hotspots on the pGmppo1 promoter sequence, the number of transcription events within plant cells can be enhanced to achieve the desired increase in transcriptional activity of a gene.

[0270] According to some embodiments, an in planta genetic library of a large collection of modified soybean ppo1 promoter (pGmppo1) elements is generated for high-throughput assessment of enhanced herbicide resistance (HT) in Arabidopsis.

[0271] In some embodiments, several gene libraries containing large collections of modified regulatory elements are generated by introducing genetic changes into pGmppo1.

[0272] In some embodiments, genetic modifications to pGmppo1 can be directed to specific regions (e.g., hotspots). In some embodiments, modifications can be introduced into random regions of regulatory elements.

[0273] In some embodiments, modifications to the pGmppo1 regulatory elements can be introduced using site-directed mutagenesis, chemically or biologically induced random mutagenesis, recombination, shuffling, error-prone PCR, restriction enzyme-ligation, solid-phase synthesis, synthetic libraries, or any combination thereof. Each possibility is a separate embodiment.

[0274] In some embodiments, the pGmppo1 gene mutant library is validated by random sequencing of the library to confirm the quality of the library, followed by statistical analysis of the nucleotide distribution at the mutation sites.

[0275] In some embodiments, the resulting pGmppo1-AtPPOX1 or pGmppo1-GmPPO1 nucleic acid is introduced into Agrobacterium and transformed into Arabidopsis plants using high-throughput transformation methods to generate an in planta promoter library.

[0276] In some embodiments, each in planta promoter library comprises about 10 3 ~10 6 A collection of transgenic pGmppo1-AtPPOX1 or pGmppo1-GmPPO1 plants containing

[0277] In some embodiments, each transgenic pGmppo1-AtPPOX1 or pGmppo1-GmPPO1 plant in an in planta promoter library carries, on average, a single genetically modified regulatory element.

[0278] In some embodiments, each single genetically modified regulatory element has at least one or more genetic changes in pGmppo1.

[0279] According to some embodiments, Arabidopsis plants are screened and selected for enhanced herbicide resistance (HT).

[0280] In some embodiments, evaluation of improved herbicide resistance (HT) traits in model plants is performed by a high-throughput screening process using PPO inhibitors.

[0281] In some embodiments, transgenic pGmppo1-AtPPOX1 seeds or pGmppo1-GmPPO1 seeds are germinated and the plants are treated with flumioxazin, the active ingredient in Strike, or carfentrazone ethyl, the active ingredient in Aurora, according to a predetermined cutoff concentration.

[0282] In some embodiments, plants that exhibit high tolerance to the cut-off concentration of flumioxazin or carfentrazone ethyl are selected for their potential tolerance to the herbicide.

[0283] In some embodiments, the level of resistance is determined based on at least one selection criterion that is the same as that used in preliminary experiments with the native promoter to establish the cutoff concentration.

[0284] In some embodiments, specific genetic mutations in the pGmppo1 promoter or in selected pGmppo1-AtPPOX1- or pGmppo1-GmPPO1-containing plants are identified by DNA sequencing.

[0285] In some embodiments, the best putative promoters are used to re-transform Arabidopsis plants, which are again tested for resistance to flumioxazin or carfentrazone ethyl.

[0286] According to some embodiments, enhanced herbicide resistance (HT) gene edited soybean crop plants are generated.

[0287] In some embodiments, genetic alterations identified in model plants that express a desired level of resiliency are introduced into desired soybean crop plants.

[0288] In some embodiments, the identified genetic alterations can be introduced into desired soybean crop plants using gene editing methods, thereby creating genetically modified, non-transgenic soybean plants with enhanced herbicide resistance HT.

[0289] According to some embodiments, the soybean (Glycinemax) ppo1 promoter is cloned with GFP to allow for enhanced fluorescent expression.

[0290] In some embodiments, expression of a non-native trait-associated sequence of a reporter protein, such as a fluorescent protein, results in the expression of a desirable / beneficial phenotypic trait having a detectable fluorescent signal.

[0291] In some embodiments, the native promoter of soybean (pGmX) is cloned upstream of the sequence encoding the GFP coding sequence to produce the non-native (pGmX-GFP).

[0292] In some embodiments, evaluation of enhanced fluorescence in model plants is performed by a high-throughput screening process using detection of fluorescence levels according to a predetermined green fluorescence cutoff.

[0293] In some embodiments, plants that exhibit fluorescence levels higher than the green fluorescence cutoff are selected for their potential desired changes in expression associated with the soybean modified promoter (pGmX).

[0294] In some embodiments, pGmX-GFP is stably expressed in a model plant. In some embodiments, pGmX-GFP is transiently expressed in a soybean plant.

[0295] In some embodiments, the level of fluorescence is determined based on at least one selection criterion that is the same as or different from that used in preliminary experiments with native pGmX-GFP to establish a green fluorescence cutoff concentration.

[0296] According to some embodiments, native regulatory elements are cloned using the herbicide resistance (HT)-associated protoporphyrinogen oxidase (PPOX1) gene of Arabidopsis thaliana (AtPPOX1) for assessment of desired expression changes.

[0297] According to some embodiments, native regulatory elements are cloned using the soybean herbicide resistance (HT)-associated protoporphyrinogen oxidase (PPO1) gene (GmPPO1) for evaluation of desired expression changes.

[0298] In some embodiments, expression of the coding sequence of the herbicide resistance (HT)-associated protoporphyrinogen oxidase (PPOX1) gene from Arabidopsis thaliana (AtPPOX1) results in the expression of a desirable / beneficial phenotypic trait with enhanced tolerance to PPO inhibitors, e.g., oxadiazon, flumioxazin, or carfentrazone ethyl, thereby enabling high-throughput screening of model plants that exhibit desired changes in expression from modified regulatory elements of any native gene of any desired crop plant operably linked to the trait.

[0299] In some embodiments, expression of the coding sequence of the herbicide resistance (HT)-associated protoporphyrinogen oxidase (PPO1) gene in soybean (Glycinemax) (GmPPO1) results in the expression of a desirable / beneficial phenotypic trait with enhanced tolerance to PPO inhibitors, e.g., oxadiazon, flumioxazin, or carfentrazone ethyl, thereby enabling high-throughput screening of model plants that exhibit desired changes in expression from modified regulatory elements of any native gene of any desired crop plant operably linked to the trait.

[0300] In some embodiments, the coding sequence for Arabidopsis protoporphyrinogen oxidase (PPOX1) (AtPPOX1) is cloned downstream of the promoter of the native gene to assess desired changes in expression associated with improved activity of protoporphyrinogen oxidase (PPOX1).

[0301] In some embodiments, the coding sequence for soybean protoporphyrinogen oxidase (PPO1) (GmPPO1) is cloned downstream of the promoter of the native gene to assess desired changes in expression associated with improved enzymatic activity of protoporphyrinogen oxidase (PPO1).

[0302] In some embodiments, expression of a reporter gene (e.g., an HT gene) can be used in high-throughput screening to identify optimized regulatory elements (regulatory elements, preferably regulatory elements derived from crop plants, e.g., promoters of salt tolerance-related genes). Thus, screening may be performed in model plants by monitoring reporter gene expression and / or phenotypic changes of the model plants (e.g., increased herbicide resistance, fluorescence levels, etc.) as a result of functionally coupling mutated regulatory elements.

[0303] In some embodiments, expression of a trait (such as, but not limited to, HT) is utilized for high-throughput screening of model plants that exhibit desired changes in expression of trait-associated genes / reporter genes operably coupled to modified regulatory elements, which may be derived from any native gene of any desired crop plant.

[0304] In some embodiments, expression of a trait is based on meeting a predetermined cutoff for the corresponding selection criterion, utilized in high-throughput screening of model plants harboring a library of modified regulatory elements by applying selection conditions corresponding to that trait.

[0305] In some embodiments, high-throughput screening of model plants is performed using traits for resistance or increased yield, including, but not limited to, resistance to herbicides, insects, and diseases or other biotic stresses, resistance to heat, drought, or other abiotic stresses, reduced time to crop maturity, increased yield, improved fertilizer uptake, improved industrial processing such as conversion of starch or biomass to fermentable sugars, and improved agronomic quality such as increased oil content and increased protein content. Each possibility is a separate embodiment. Each possibility is a different embodiment.

[0306] In some embodiments, evaluation and selection of improved herbicide resistance (HT) traits in model plants involves high-throughput in planta functional screening assays using PPO inhibitor herbicide sprays with the goal of readily identifying low-frequency mutants.

[0307] Now, reference is made to Figure 3C.

[0308] In some embodiments, determining the enhanced resistance comprises assessing damage to the leaves at least at a predetermined cutoff concentration.

[0309] In some embodiments, mutant sequences of pGmppo1 generated by random mutagenesis, screened in planta, and identified by sequencing the transgenes of selected resistance events are represented by any one of SEQ ID NOS: 4-63. In some embodiments, mutations and their locations are summarized in Tables 1 and 2. Table 1 lists identified hotspots in the soybean ppo1 promoter (pGmppo1) of different resistance events, represented by any one of SEQ ID NOS: 4-13, and Table 2 lists the remaining mutations identified in the soybean ppo1 promoter (pGmppo1) of different resistance events, represented by any one of SEQ ID NOS: 14-63. Each possibility is a separate embodiment. Now, see FIG. 3D.

[0310] [Table 1]

[0311] [Table 2]

[0312] [Table 3]

[0313] In some embodiments, the soybean ppo1 promoter (pGmppo1) contains seven "hot" loci that, when mutated, confer enhanced / improved herbicide resistance (HT) traits.

[0314] In some embodiments, the disclosed ppo1 promoter (ppo1) of the present invention contains genetic alterations at any one or more of seven "hot" positions that, when mutated, confer enhanced / improved herbicide resistance (HT) traits to plants.

[0315] In some embodiments, the disclosed ppo1 promoter (ppo1) of the present invention contains one or more mutations at one or more positions corresponding to any one or more mutation positions in the soybean ppo1 promoter set forth in SEQ ID NOs: 4-63 (i.e., as detailed in Table 1 and Table 2).

[0316] In some embodiments, the disclosed ppo1 promoter (ppo1) of the present invention comprises one or more mutations at one or more positions corresponding to any one or more mutation positions in the soybean ppo1 promoter set forth in SEQ ID NOs: 4-13 (i.e., as detailed in Table 1).

[0317] In some embodiments, the disclosed ppo1 promoter (ppo1) of the present invention contains one or more mutations at one or more positions corresponding to any one or more mutation positions in the soybean ppo1 promoter set forth in SEQ ID NOs: 14-63 (i.e., as detailed in Table 2).

[0318] In some embodiments, the disclosed ppo1 promoter (ppo1) of the present invention comprises a sequence set forth in any one of SEQ ID NOs: 4-63, or any combination thereof. In some embodiments, the disclosed ppo1 promoter (ppo1) of the present invention comprises a sequence set forth in any one of SEQ ID NOs: 4-13, or any combination thereof. In some embodiments, the disclosed ppo1 promoter (ppo1) of the present invention comprises a sequence set forth in any one of SEQ ID NOs: 14-63, or any combination thereof.

[0319] In some embodiments, seven "hot" positions in the native soybean ppo1 promoter that, when mutated, confer enhanced / improved herbicide resistance (HT) traits include the following positions with respect to SEQ ID NO:2: [-1229A], [-1105T], [-668A], [-481T], [-189A], [-70T], and [-61T] (where (-) indicates an upstream position relative to the transcription start site (TSS)). Referring now to Table 1 and Figure 3D (hot positions are marked within rectangles).

[0320] In some embodiments, the identified hotspot in pGmppo1 comprises a mutation represented by any one of SEQ ID NOs: 4-13.

[0321] According to some embodiments, a gene-edited crop plant or crop plant cell is provided that comprises a ppo1 promoter that has been genetically modified to include one or more mutations at one or more positions corresponding to any one or more of positions -1229A, -1105T, -668A, -481T, -189A, -70T, and -61T of the soybean promoter set forth in SEQ ID NO:2.

[0322] As used herein, the term "corresponding to" refers to a position in the ppo1 promoter of any other crop plant with respect to the homologous position in the soybean ppo1 promoter (pGmppo1) as determined by aligning the sequences together using a sequence alignment tool.

[0323] In some embodiments, the genetically edited crop plant or crop plant cell is a genetically edited crop plant or crop plant cell, wherein the one or more mutations in the genetically modified ppo1 promoter comprise substitutions, additions, and / or deletions.

[0324] In some embodiments, the genetically edited crop plant or crop plant cell comprises one or more mutations in the genetically modified ppo1 promoter comprising one or more of: -1229A>C, -1105T>C, -668A deletion, -668A>G, -481T>G, -189 insertion A, -189A>C, -189A deletion, -70T>C, and -61T>G, where the positions correspond to the positions of the soybean promoter set forth in SEQ ID NO:2.

[0325] In some embodiments, the genetically edited crop plant or crop plant cell comprises one or more mutations in the genetically modified ppo1 promoter, including at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more mutation positions. Each possibility is a separate embodiment.

[0326] In some embodiments, the gene-edited crop plant or crop plant cell is a gene-edited crop plant or crop plant cell, wherein the ppo1 promoter comprises at least 90% sequence identity to SEQ ID NO:2.

[0327] In some embodiments, the ppo1 promoter has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 2. Each possibility is a separate embodiment.

[0328] In some embodiments, the gene-edited crop plant or crop plant cell is a gene-edited crop plant or crop plant cell, wherein the ppo1 promoter comprises a sequence set forth in any one of SEQ ID NOs: 4-13.

[0329] In some embodiments, the gene-edited crop plant or crop plant cell is a gene-edited crop plant or crop plant cell, wherein the ppo1 promoter comprises a sequence set forth in any one of SEQ ID NOs: 4-13, or any combination thereof.

[0330] In some embodiments, the gene-edited crop plant or crop plant cell is a gene-edited crop plant or crop plant cell, wherein the ppo1 promoter comprises a sequence set forth in any one of SEQ ID NOs: 14-63.

[0331] In some embodiments, the gene-edited crop plant or crop plant cell is a gene-edited crop plant or crop plant cell, wherein the ppo1 promoter comprises a sequence set forth in any one of SEQ ID NOs: 14-63, or any combination thereof.

[0332] In some embodiments, a gene-edited crop plant or crop plant cell is provided, wherein the crop plant is soybean.

[0333] According to some embodiments, a nucleic acid construct is provided that comprises a genetically modified promoter of a plant trait-associated gene coupled to and functionally associated with a herbicide resistance (HT) gene.

[0334] According to some embodiments, a nucleic acid construct is provided that includes a ppo1 promoter that has been genetically modified to include one or more mutations at one or more positions corresponding to any one or more of positions -1229A, -1105T, -668A, -481T, -189A, -70T, and -61T of the soybean promoter set forth in SEQ ID NO:2.

[0335] In some embodiments, a nucleic acid construct is provided in which a genetically modified ppo1 promoter is coupled to and operably associated with a herbicide resistance (HT) gene.

[0336] In some embodiments, provided are nucleic acid constructs comprising one or more mutations in a genetically modified ppo1 promoter, comprising one or more of: -1229A>C, -1105T>C, -668A deletion, -668A>G, -481T>G, -189 insertion A, -189A>C, -189A deletion, -70T>C, and -61T>G.

[0337] In some embodiments, a nucleic acid construct is provided, wherein the one or more mutations in the genetically modified ppo1 promoter comprise substitutions, additions, and / or deletions.

[0338] In some embodiments, provided are nucleic acid constructs wherein the one or more mutations in the genetically modified ppo1 promoter include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more mutation positions. Each possibility is a separate embodiment.

[0339] In some embodiments, the nucleic acid construct provided comprises a genetically modified ppo1 promoter comprising at least 90% sequence identity to SEQ ID NO:2.

[0340] In some embodiments, the ppo1 promoter has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or at least 99.9% sequence identity to SEQ ID NO: 2. Each possibility is a separate embodiment.

[0341] In some embodiments, a nucleic acid construct is provided, wherein the genetically modified ppo1 promoter comprises a sequence set forth in any one of SEQ ID NOs: 4-13, or any combination thereof.

[0342] In some embodiments, a nucleic acid construct is provided, wherein the genetically modified ppo1 promoter comprises a sequence set forth in any one of SEQ ID NOs: 14-63, or any combination thereof.

[0343] In some embodiments, a reporter gene is cloned downstream of the promoter of a native trait-associated gene to assess desired changes in reporter gene expression associated with genetic alterations introduced into the promoter of the native gene. See Figure 2B.

[0344] In some embodiments, the reporter gene is cloned as a fusion protein downstream of the coding sequence of the native trait-associated gene to assess desired changes in reporter expression associated with genetic alterations introduced into the coding sequence of the native gene. See Figure 2C.

[0345] The following examples are presented to provide a more complete understanding of the present invention. The specific techniques, conditions, materials, proportions, and reported data set forth to illustrate the principles of the invention are exemplary and should not be construed as limiting the scope of the invention. [Example]

[0346] The following examples illustrate the use of the methods disclosed herein to enhance gene function / activity associated with improved traits in plants in planta. However, it will be understood by those skilled in the art that herbicide resistance (HT) serves as an example only, and the platform can be used to optimize expression of any endogenous trait.

[0347] Example 1: Cloning of the soybean (Glycine max) ppo1 promoter with the Arabidopsis (A. thaliana) PPOX1 gene to determine the selection cutoff for transformed and enhanced native herbicide resistance (HT). Optimizing the expression level of the native plant protoporphyrinogen oxidase (PPOX1) gene can improve herbicide resistance (HT) when the enzyme's activity exceeds the concentration of the herbicide's active ingredient in the plant. Therefore, it is desirable to improve natural resistance to PPO-inhibiting herbicides, such as oxadiazon, flumioxazin, or carfentrazone-ethyl (the active ingredients in Strike and Aurora, respectively), by enhancing the expression of protoporphyrinogen oxidase (PPOX1).

[0348] First, the Arabidopsis (Athaliana) PPOX1 mRNA sequence (SEQ ID NO: 1) (encoding AtPPOX1) was cloned into the backbone of the Ppa35H binary vector, which is commonly used for library construction. This was then cloned downstream of the soybean (Glycine max) ppo1 promoter (pGmppo1) (SEQ ID NO: 2), creating the chimera pGmppo1-AtPPOX1 (Figure 1A). The soybean native ppo1 promoter (pGmppo1) was amplified from soybean gDNA by standard PCR and cloned upstream of the native AtPPOX1 mRNA coding sequence with a specific sequence marker. This sequence marker allows for subsequent molecular identification by quantitative PCR from the native copy of the Arabidopsis promoter.

[0349] [Table 4]

[0350] [Table 5]

[0351] The pGmppo1-AtPPOX1 vector was used to transform Arabidopsis model plants to generate stable T2 generation events, which were then used for calibration of predetermined cutoff concentrations of PPO used in subsequent steps of plant library screening and selection.

[0352] The production of Arabidopsis seeds with stable T2 generation events was initiated by first introducing a binary vector into Agrobacterium. The Agrobacterium carrying the pGmppo1-AtPPOX1 gene was then used to transform Arabidopsis plants. The transformed Arabidopsis seeds were germinated and treated with a selective herbicide to detect transformed plants. Seeds were harvested, and the T2 Arabidopsis seeds were germinated again and treated with a selective herbicide to detect stable T2 events.

[0353] A selection cutoff value was calibrated for later use in screening transformed model plants for increased expression and / or activity of the AtPPOX1 enzyme and the associated enhancement of herbicide resistance (HT) traits. Stable T2 Arabidopsis plants transformed with the chimeric pGmppo1-AtPPOX1 gene were treated with a gradient of the PPO-inhibiting herbicide flumioxazin to calibrate the cutoff concentration. Stable T2 events exhibiting the strongest resistance to the highest concentration of the PPO-inhibiting herbicide flumioxazin were selected. Selection was based on at least one selection criterion. A predetermined cutoff concentration was then determined for later use in selecting promoter mutant transgenic libraries. The cutoff concentration was determined based on stable T2 Arabidopsis plants transformed with the chimeric pGmppo1-AtPPOX1 gene and exhibiting the strongest resistance to the highest concentration of the PPO-inhibiting herbicide flumioxazin.

[0354] Example 2: Cloning of the complete native soybean (Glycine max) PPO1 gene for transformation and determination of selection cutoffs for enhanced, native herbicide resistance (HT). The soybean (Glycinemax) PPO1 mRNA sequence (encoding GmPPO1) (SEQ ID NO: 3) was cloned into a binary vector downstream of the promoter pGmppo1 to generate pGmppo-GmPPO1 (FIG. 1B).

[0355] [Table 6]

[0356] The pGmppo-GmPPO1 vector was used to transform Arabidopsis thaliana model plants to generate stable T2 events, which were then used to calibrate a predetermined cutoff concentration of PPO. This concentration was then used in subsequent steps of plant library screening and selection.

[0357] The production of Arabidopsis seeds with stable T2 generation events and the calibration of selection cutoff values ​​(for later use in screening transformed model plants for increased expression and / or activity of the GmPPO1 enzyme) were performed according to the same guidelines as described above in Example 1.

[0358] Example 3: Algorithmic analysis of the native soybean ppo1 promoter (pGmppo1) for detection of putative hotspots A proprietary algorithm is utilized to analyze the non-coding regulatory elements of pGmppo1 and predict putative "hot spots" for DNA-binding proteins involved in the process of transcription. These include transcription factors and other transcription-related proteins that interact with DNA in a sequence-specific manner. Figure 2 shows the general structure of a plant gene, including regulatory regions and downstream coding sequences.

[0359] This algorithm utilizes available information on plant regulatory element sequences derived from public databases such as PlantCARE (Plant cis-acting Regulatory Elements), PLACE (Plant cis-acting Regulatory DNA Elements), and Regulatory Site (Plant-Directed Transcriptional Regulatory Element Collection). The algorithm is then applied to the analysis of the native soybean PPO1 gene and prediction of hotspots that could promote the desired changes in transcriptional activity enhanced by pGmppo1.

[0360] The algorithm receives SEQ ID NO: 2 as the input sequence (the sequence of pGmppo1). The typical structure of the pGmppo1 promoter is analyzed to identify specific regulatory elements within it. This includes analyzing the proximal promoter (including the core promoter) and the distal promoter. Promoter regions are identified by analyzing several hundred nucleotides surrounding the TSS, which is considered the core / proximal promoter (e.g., to identify elements that regulate transcription start site (TSS) selection), and by analyzing several thousand or more nucleotides further upstream of the TSS, which is considered the distal promoter (e.g., for the presence of regulatory cis-elements that may function as transcription enhancers and play ancillary roles in increasing transcription activity).

[0361] This algorithm identifies putative regulatory hotspots in pGmppo1, processes and integrates the data, and then predicts sites on the promoter sequence suitable for genetic manipulation / editing, thereby predicting potentially new regulatory elements with novel attributes for gene expression patterns. In the case of the PPO1 gene product, the desired change is an increase in enzyme expression and activity. This can be achieved, for example, by introducing a hotspot genetic alteration that forces the binding activity of a transcriptional activator or prevents the binding activity of a suppressor. By targeting hotspots on the pGmppo1 promoter sequence, it is possible to achieve a desired increase in the transcriptional activity of a gene during specific developmental growth periods in a plant, as well as in specific tissues where gene expression is normally low, in addition to enhancing the number and strength of transcription events of already active promoters in plant cells.

[0362] Example 4: Generation of an in planta gene library of a large collection of modified soybean ppo1 promoter (pGmppo1) elements for high-throughput evaluation of enhanced herbicide resistance (HT) in Arabidopsis. Construction of a mutagenesis library containing a large collection of modified regulatory elements was performed by randomly altering the soybean ppo1 promoter pGmppo1, thereby modifying random regions of the regulatory elements.

[0363] The regulatory region (-1 bp to -1273 bp) upstream of the transcription start site (TSS) of the GmPPO1 gene was amplified from soybean genomic DNA shown in SEQ ID NO: 2. The PCR fragment was then subjected to random mutagenesis using error-prone PCR according to the manufacturer's manual. 2+ Ion concentration and Mg 2+ Varying the ion concentration or imbalance of the available base pool increased the mutation rate. The mutagenesis procedure was monitored by adjusting the number of amplification cycles, which allowed selection for the degree of mutation.

[0364] The resulting population of molecules with random point mutations was cloned into a binary vector (Ppa35H) upstream of the AtPPOx1 coding region shown in SEQ ID NO: 1, and 5 A large pool of clones was generated.

[0365] Next, we verified the pGmppo1 gene mutant library. The quality of the library was tested by random sequencing, and then the nucleotide distribution at the mutation sites was statistically analyzed. The results of the analysis are shown in Figure 3B, which demonstrates the uniform distribution and randomization of mutagenesis without bias toward specific positions. These two libraries were cloned upstream of the GmPPO1 or AtPPOX1 coding sequence to generate chimeric pGmppo1-AtPPOX1 or pGmppo1-GmPPO1, which were then mixed and used for high-throughput transformation into Arabidopsis plants.

[0366] Following the preparation and quality verification of the pGmppo1 mutant library, the resulting chimeric pGmppo1-AtPPOX1 (or pGmppo1-GmPPO1) nucleic acid was introduced into Agrobacterium and transformed into Arabidopsis plants using a high-throughput transformation method (floral dip), yielding approximately 10 3 ~10 5 We generated an in planta promoter library containing a collection of transgenic pGmppo1-AtPPOX1 (or pGmppo1-GmPPO1) plants, each containing, on average, a single genetically modified regulatory element with at least one or more genetic changes in pGmppo1. The process described above is illustrated in steps I, II, and III of Figure 3A.

[0367] Example 5 - Screening and selection of Arabidopsis plants for enhanced herbicide resistance (HT) and identification of the responsible genetic changes. Evaluation and selection of improved herbicide resistance (HT) traits in model plants was performed by a high-throughput in planta functional screening assay using spraying with PPO-inhibiting herbicides, allowing for easy identification of low-frequency mutants. First, transgenic pGmppo1-AtPPOX1 (or pGmppo1-GmPPO1) T1 generation seedlings were subjected to glufosinate ammonium to select for transformed T1 events. Subsequently, 14-day-old seedlings were cultured at 1 × 10 sucrose, according to the previously determined cutoff concentration. -3 The plants were subjected to 50% oxadiazon (Star) or flumioxazin (Strike) treatment. Plants that showed high resistance to the cutoff concentration of oxadiazon (Star) or flumioxazin (Strike) were selected for their possible tolerance to the herbicides. The level of resistance was determined based on damage to the leaves. This is the same selection criterion used in preliminary experiments using the native promoter to establish the cutoff concentration. The above process is shown in Step IV of Figure 3A.

[0368] Advantageously, Figure 3C shows a representative result of the functional screen, showing a non-damaged PPO-resistant plant (i.e., a "healthy" plant, white dashed circle) containing a "positive mutation" (i.e., enhancing HT) in the PPO promoter, surrounded by multiple susceptible plants (with damaged leaves) containing other non-resistance-enhancing mutations in the PPO promoter.

[0369] The transgenes in the selected plants were then PCR amplified and sequenced to identify the location and forward mutation in pGmppo1 that enhanced HT. The process described is shown in step V of Figure 3A.

[0370] The pGmppo1 mutant sequences identified by sequencing the transgene are represented by any one of SEQ ID NOS: 4 to 63. The mutations and their locations are summarized in Table 1 (which lists hotspots in the soybean ppo1 promoter (pGmppo1)) and Table 2 (which lists mutations identified in the soybean ppo1 promoter (pGmppo1)).

[0371] The results of the sequencing analysis are shown in Figure 3D, which shows the distribution of mutations that occurred along the nucleic acid sequence of the soybean PPO1 promoter (pGmppo1) in 14 different resistance events discovered by functional herbicide selection assays. Advantageously, the analysis revealed that among the 14 independent resistance events, each independent resistance event shared at least one identical mutation at the same position with another resistance event, suggesting that these seven positions may be major hotspot positions where mutations confer enhanced / improved herbicide resistance (HT) traits.

[0372] These seven major hotspots in the soybean PPO1 promoter, identified in 14 events, are shown in Figure 3D (marked by rectangles) and include the following positions relative to SEQ ID NO:2, where (-) indicates an upstream position relative to the transcription start site (TSS): [-1229A], [-1105T], [-668A], [-481T], [-189A], [-70T], and [-61T]. The mutations identified at these seven hotspot positions in pGmppo1 and their occurrence in resistance events selected in functional in planta screening are summarized in Table 1 of the detailed description of this application.

[0373] Example 6 - Differential Resistance Assays To assess the resistance of promoters carrying selected mutations to additional PPO herbicides, the selected putative promoters are used to again transform Arabidopsis plants, which are again tested for resistance to different PPO herbicides, as described below.

[0374] T2 plants that exhibit good tolerance to oxadiazon (Star) or flumioxazin (Strike) PPO herbicides and have the desired genetic changes demonstrated in one of the sequenced transgenes from the previously identified resistance events are grown under glufosinate ammonium to confirm the presence of the transgene. These plants are then exposed to a variety of herbicides, including oxadiazon (Star), flumioxazin (Strike), and other PPO herbicides, for up to 10 days. -3 , 5×10 -3 , 10 -2 , 5×10 -2 , 10 -1 , 5×10 -1 The plants are then subjected to the active ingredient at a concentration of 1000 mg / kg / day to evaluate their different tolerances. The plants that show the best tolerance are then re-evaluated for genetic changes in the pGmppo1 promoter region.

[0375] A schematic overview of the process is shown in Figure 4, illustrating the general workflow utilized to improve traits such as herbicide resistance (HT) in plants.

[0376] Example 7 - Generation of enhanced herbicide resistance (HT) gene-edited soybean crop plants Genetic changes identified in the model plants that express the desired level of herbicide resistance are introduced into the desired soybean crop plants using gene editing techniques to generate soybean plants with enhanced herbicide resistance HT modified by gene editing methods.

[0377] Example 8 - Cloning of the soybean (Glycinemax) ppo1 promoter (pGmppo1) with GFP for evaluation of fluorescence enhancement in stably transformed Arabidopsis plants. Expression of a non-native trait-associated sequence for a reporter protein, such as a fluorescent protein, results in the expression of a phenotypic trait with a detectable fluorescent signal. The native promoter of soybean ppo1 promoter (pGmppo1) was cloned upstream of the coding sequence for GFP to produce pGmppo1-GFP.

[0378] Evaluation of improved fluorescence in model plants is performed through a high-throughput screening process using detection of fluorescence levels according to a predetermined green fluorescence cutoff (criteria value). Plants exhibiting fluorescence levels higher than the green fluorescence cutoff are selected for potential desired changes in expression associated with the modified soybean promoter (pGmX). The level of fluorescence is determined based on at least one of the same selection criteria used in preliminary experiments with the native promoter to establish the green fluorescence cutoff concentration.

[0379] Example 9 - Cloning of a soybean (Glycinemax) promoter (pGmX) using GFP for evaluation of fluorescence enhancement during transient expression in soybean plants. Evaluation of improved fluorescence in target crop plants is performed through a screening process that detects fluorescence levels at a predetermined green fluorescence cutoff. A soybean native promoter (pGmX) is cloned into a binary vector upstream of a sequence encoding GFP, producing pGmX-GFP. Transient expression of GFP in soybean leaves is achieved by infecting the leaves with Agrobacterium tumefaciens carrying the binary vector. Leaves that exhibit fluorescence levels higher than the green fluorescence cutoff are selected for their potential desired changes in expression associated with the modified regulatory elements in the soybean promoter (pGmX). The level of fluorescence is determined based on at least one of the same selection criteria used in the preliminary cutoff experiments using the native promoter.

[0380] Example 10 - Cloning of native regulatory elements and evaluation of desired expression changes using the protoporphyrinogen oxidase (PPOX1) gene associated with herbicide resistance (HT) in Arabidopsis thaliana (AtPPOX1). Expression of the coding sequence of the Arabidopsis (AtPPOX1) or soybean (GmPPO1) protoporphyrinogen oxidase (PPOX1) gene associated with herbicide resistance (HT) results in the expression of a desirable / beneficial phenotypic trait, such as enhanced tolerance to the PPO-inhibitors oxadiazon, flumioxazin, or carfentrazone-ethyl, or other inhibitors, thereby enabling its use as a reporter gene for high-throughput screening of model plants that exhibit the desired change in expression from modified regulatory elements and / or coding sequences of any native gene of any desired crop plant that is operably linked to the trait.

[0381] Arabidopsis PPOX1 (AtPPOX1) or soybean PPO1 (GmPPO1) are used as reporter genes cloned downstream of the promoter of a native trait-related gene to assess desired changes in the expression of protoporphyrinogen oxidase associated with genetic changes introduced into the promoter of the native gene (see Figure 2B).

[0382] Furthermore, the Arabidopsis PPOX1 (AtPPOX1) or soybean PPO1 (GmPPO1) reporter gene is cloned as a fusion protein downstream of the coding sequence of the native trait-related gene for assessment of desired changes in the expression of protoporphyrinogen oxidase associated with genetic changes introduced into the coding sequence of the native gene (see Figure 2C).

Claims

1. 1. A method for in planta high-throughput evaluation of endogenous gene regulatory elements in crop plants, comprising: (a) obtaining a nucleic acid of a native regulatory element of a trait-associated gene and coupling it to a coding sequence of a reporter gene that has a native function in said crop plant, wherein said regulatory element is in the crop plant; (b) introducing one or more random genetic alterations into each of the multiple copies of the native regulatory element, thereby obtaining a genetic library comprising a large collection of modified regulatory elements; (c) introducing the gene library into model plants using high-throughput stable transformation such that, on average, each model plant receives a single modified regulatory element; (d) screening the transformed model plants, wherein the screening comprises selecting model plants having a desired change in phenotype, the change being related to the expression level of the reporter gene; (e) identifying one or more genetic alterations in the modified regulatory element of the selected model plant.

2. 10. The method of claim 1, further comprising modifying the native regulatory element of the trait-associated gene of the crop plant based on the one or more identified genetic changes in the modified regulatory element of the selected model plant to generate a modified desired crop plant.

3. 2. The method of claim 1, wherein the native regulatory elements are located upstream, downstream, within, or any combination of, the coding sequence of the reporter gene.

4. The method of claim 1 , wherein the coding sequence of the reporter gene is of the same or different origin as the regulatory element of the trait-associated gene.

5. The method of claim 1 , wherein the native regulatory element is a promoter or a fragment thereof.

6. 2. The method of claim 1, wherein the one or more genetic alterations are selected from one or more of point mutations, domain swaps, cis-element rearrangements, enhancer additions and / or silencer deletions.

7. The method of claim 1 , wherein the step of introducing the gene library into a model plant comprises a step of cloning the gene library into a binary vector of Agrobacterium.

8. The method described in claim 1, wherein the desired change in phenotype is the expression level of a reporter gene and the screening of the model plant includes meeting a predetermined cutoff.

9. The method described in claim 1, wherein the desired change in phenotype comprises a change in the transcriptional activity of the modified regulatory element.

10. The method described in claim 1, wherein the desired change in phenotype includes one or more changes in time, developmental stage, cellular localization, tissue specificity or expression intensity.

11. The method of claim 1, wherein the desired change in phenotype comprises an increase in expression associated with an increase in gene function and / or activity.

12. The method of claim 1 , wherein an increase in the function or activity of the trait-associated gene results in enhanced plant resistance or increased plant yield.

13. 13. The method of claim 12, wherein the enhanced tolerance comprises tolerance to herbicides, insects, diseases, heat, cold, drought, biotic stress, or abiotic stress.

14. 13. The method of claim 12, wherein the enhanced tolerance comprises herbicide resistance (HT).

15. 15. The method of claim 14, wherein the herbicide resistance (HT) comprises increased expression of a native enzyme, which exceeds the concentration of the active ingredient of the herbicide in the plant, thereby enhancing natural resistance.

16. 16. The method of claim 15, wherein the native enzyme is protoporphyrinogen oxidase (PPO1), or p-hydroxyphenylpyruvate dioxygenase (HPPD), or 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), or glutamine synthase, or acetolactate synthase (ALS enzyme), or 7,8-dihydropteroate synthase, or acetyl-CoA carboxylase (ACCase).

17. 3. The method of claim 2, wherein the desired crop plant is generated using gene editing tools.

18. 1. A gene-edited soybean crop plant or crop plant cell comprising a ppo1 promoter that has been genetically modified to include one or more mutations at one or more positions corresponding to any one or more of positions -1229A, -1105T, -668A, -481T, -189A, -70T, and -61T of the soybean promoter set forth in SEQ ID NO:2, wherein the ppo1 promoter comprises a sequence as set forth in any one of SEQ ID NOs:4-13.

19. 19. The gene-edited crop plant or crop plant cell of claim 18, wherein the one or more mutations comprise a substitution, addition and / or deletion.

20. 19. The gene edited crop plant or crop plant cell of claim 18, wherein the one or more mutations comprise one or more of: -1229A>C, -1105T>C, -668A deletion, -668A>G, -481T>G, -189 insertion A, -189A>C, -189A deletion, -70T>C, and -61T>G.

21. 19. The gene-edited crop plant or crop plant cell of claim 18, wherein the one or more mutations comprise at least two mutation positions.

22. 19. The gene-edited crop plant or crop plant cell of claim 18, wherein the ppo1 promoter has at least 90% sequence identity to SEQ ID NO:2.

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