Gene upregulation through targeted deletions to increase enhancer proximity

Abstract

The disclosure relates to novel soybean plants, soybean plant parts, soybean plant cells, and nucleotide sequences in soybean plants comprising an endogenous gene or fragment thereof containing a deletion of one or more nucleotides between a transcriptional enhancer and a promoter of endogenous target soybean gene sequences to upregulate expression of the endogenous target soybean genes, along with methods of using and making the same.

Description

Docket No. P14694WO00 TITLE: GENE UPREGULATION THROUGH TARGETED DELETIONS TO INCREASE ENHANCER PROXIMITY CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This international patent application claims the benefit of U.S. provisional patent application serial no.63 / 682,104, filed August 12, 2024, and incorporated herein by reference in its entirety. SEQUENCE LISTING STATEMENT

[0002] The instant application contains a sequence listing, which has been submitted in XML file format by electronic submission and is hereby incorporated by reference in its entirety. The XML file, created on August 5, 2025, is named P14694WO00.xml, is 71,798 bytes in size, and is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] Disclosed herein are novel plants, plant parts, and nucleotide sequences in soybean varieties comprising an endogenous gene containing a deletion of one or more nucleotides located between a transcriptional enhancer and a promoter of the endogenous gene to upregulate expression of the gene, along with methods of making the same, and methods of using the same. BACKGROUND

[0004] Agriculture is an essential industry for the global economy and the United States in particular. Soybean (Glycine max) is an important legume crop worldwide due to its ability to fix atmospheric nitrogen. Soybeans serve as a major source of animal feed protein and soybean oil has uses in a wide variety of industries, including the food and beverage, biodiesel, and other industries.

[0005] Soybean sustainability is a priority for farmers worldwide. Farming practices such as water and nutrient management help farmers improve efficiencies, boost crop productivity, conserve water, enrich soil quality, improve nutrient efficiencies of the soil, and produce sustainable soybean crops. The benefits of bioengineering for soybean farmers include increased yields and extreme weather hardiness.Docket No. P14694WO00 SUMMARY

[0006] Disclosed herein are soybean plant cells comprising an endogenous target soybean gene containing a non-naturally occurring deletion of one or more nucleotides located between a transcriptional enhancer and a promoter of the endogenous target soybean gene, wherein expression of the endogenous target soybean gene coding sequence is increased in comparison to expression of the endogenous target soybean gene coding sequence in a control soybean plant lacking the deletion. Also provided are soybean plant parts comprising the aforementioned soybean plant cell, including stems, roots, leaves, flowers, pods, and seeds. Also provided are soybean seed lots comprising the seed. Also provided are soybean plants comprising the aforementioned soybean plant cells.

[0007] Also provided are biological samples comprising a nucleic acid containing endogenous target soybean genes with the non-naturally occurring deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene. Polynucleotides comprising the transcriptional enhancer through an ATG translational start site of endogenous target soybean gene sequences comprising one or more of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof wherein one or more nucleotides located between the transcriptional enhancer and the promoter are deleted are also provided. In some embodiments, the aforementioned polynucleotides are isolated.

[0008] Also disclosed are methods of producing a soybean seed lot comprising: (i) growing a population of soybean plants comprising the aforementioned soybean plant; and (ii) harvesting seed from the population of soybean plants of step (i) at maturity. Methods of producing a soybean crop comprising planting the aforementioned seed lot are provided.

[0009] Guide RNA molecules comprising a spacer RNA molecule which target the nucleotides between the transcriptional enhancer and the promoter of endogenous target soybean gene sequences of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof are provided. Guide RNA molecules comprising a spacer RNA encoded by SEQ ID NO: 13-36 are also provided. Gene editing systems comprising a CRISPR-Cas effector protein in association with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that hybridizes to the nucleotides between the transcriptional enhancer and the promoter of endogenous target soybean gene sequences of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof are provided. Expression cassettes comprising a polynucleotide encoding CRISPR-Cas effector protein comprising a cleavage domain and a guide RNA molecule of the disclosure are also provided.Docket No. P14694WO00

[0010] Also disclosed are methods for generating the aforementioned soybean plant cells, soybean plant parts, and soybean plants. In some embodiments, the methods comprise introducing a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequence. In some embodiments, the methods comprise isolating the soybean plant and / or the soybean plant cell comprising the endogenous gene containing the deletion.

[0011] Methods for determining whether a soybean plant cell, plant part, or plant comprises a deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene are provided. In certain embodiments, the methods comprise analyzing a polynucleotide comprising a portion of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof from the plant cell, plant part, or plant. In some embodiments, the deletion between the transcriptional enhancer and the promoter of the endogenous soybean genes comprises, consists essentially of, or consists of the deletion located within SEQ ID NO: 2, 4, 6, 8, 10, 12, 37, or an allelic variant thereof.

[0012] Uses of the aforementioned soybean plant cells, soybean plant parts, soybean seed lots, and soybean plants to produce soybean seed or to grow a soybean crop are provided. Uses of the aforementioned soybean plant cells, soybean plant parts, soybean seed lots, and soybean plants to obtain a soybean by-product are also provided.

[0013] Uses of the aforementioned guide RNA molecules, gene editing systems, and expression cassettes to introduce a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequences are provided. DESCRIPTION OF THE DRAWINGS

[0014] Fig.1 shows the Glyma.19G143900 WT genomic DNA sequence (SEQ ID NO: 1) from the genomic DNA located 5´ to one or more enhancers on the sense strand up to and including the ATG translation initiation codon. An example deleted region in a variant upstream region of SEQ ID NO: 2 is marked by a black line under the sequence labeled deletion. Guide RNAs (SEQ ID NO: 13-16) are denoted with a directional arrow under the sequence labeled with the SEQ ID NO. Example cut sites are shown at both ends of the deletion region denoted by a triangle labeled Cut site.

[0015] Fig.2 shows the Glyma.10G186500 WT genomic DNA sequence (SEQ ID NO: 3) from the genomic DNA located 5´ to one or more enhancers on the sense strand up to and includingDocket No. P14694WO00 the ATG translation initiation codon. An example deleted region in a variant upstream region of SEQ ID NO: 4 is marked with a black line under the sequence labeled deletion. Guide RNAs (SEQ ID NO: 17-20) are denoted with a directional arrow under the sequence labeled with the SEQ ID NO. Example cut sites are shown at both ends of the deletion region denoted by a triangle labeled Cut site.

[0016] Fig. 3A and 3B show the Glyma.13G279900 WT genomic DNA sequence (SEQ ID NO: 5) from the genomic DNA located 5´ to one or more enhancers on the sense strand up to and including the ATG translation initiation codon and expression of reporter genes under wild- type and variant Glyma.13G279900 upstream regions. Fig. 3A denotes example deleted regions in a variant upstream region of SEQ ID NO: 6 and 37 marked with a black line under the sequence labeled deletion. Guide RNAs (SEQ ID NO: 21-24) are denoted with a directional arrow under the sequence labeled with the SEQ ID NO. Example cut sites are shown at both ends of the deletion region denoted by a triangle labeled cut site. Fig.3B illustrates expression demonstrated by luminescence of firefly luciferase (FLUC) driven by either the wild-type or variant upstream regions relative to expression of Renilla luciferase (RLUC) driven by a control promoter used for normalization. The left column shows expression driven by an intact Glyma.13G279900 upstream region (SEQ ID NO: 5), and the right column is expression from a Glyma.13G279900 variant upstream (SEQ ID NO: 37).

[0017] Fig.4 shows the Glyma.14G197100 WT genomic DNA sequence (SEQ ID NO: 7) from the genomic DNA located 5´ to one or more enhancers on the sense strand up to and including the ATG translation initiation codon. An example deleted region in a variant upstream region of SEQ ID NO: 8 is marked with a black line under the sequence labeled deletion. Guide RNAs (SEQ ID NO: 25-28) are denoted with a directional arrow under the sequence labeled with the SEQ ID. Example cut sites are shown at both ends of the deletion region denoted by a triangle labeled cut site.

[0018] Fig.5 shows the Glyma.11G250200 WT genomic DNA sequence (SEQ ID NO: 9) from the genomic DNA located 5´ to one or more enhancers on the sense strand up to and including the ATG translation initiation codon. An example deleted region in a variant upstream region of SEQ ID NO: 10 is marked with a black line under the sequence labeled deletion. Guide RNAs (SEQ ID NO: 29-32) are denoted with a directional arrow under the sequence labeled with the SEQ ID. Example cut sites are shown at both ends of the deletion region denoted by a triangle labeled Cut site.Docket No. P14694WO00

[0019] Fig. 6 shows the Glyma.17G192000 WT genomic DNA sequence (SEQ ID NO: 11) from the genomic DNA located 5´ to one or more enhancers on the sense strand up to and including the ATG translation initiation codon. An example deleted region in a variant upstream region of SEQ ID NO: 12 is marked with a black line under the sequence labeled deletion. Guide RNAs (SEQ ID NO: 33-36) are denoted with a directional arrow under the sequence labeled with the SEQ ID. Example cut sites are shown at both ends of the deletion region denoted by a triangle labeled Cut site. DETAILED DESCRIPTION

[0020] The phrase “allelic variant” as used herein refers to a polynucleotide or polypeptide sequence variant that occurs in a particular gene at particular locus in a different strain, variety, or isolate of a given organism.

[0021] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0022] As used herein, the phrase “biological sample” refers to either intact or non-intact (e.g., milled soybean seed or soybean plant tissue, chopped soybean plant tissue, lyophilized tissue) soybean plant tissue. It may also be an extract comprising intact or non-intact seed or soybean plant tissue. The biological sample can comprise flour, meal, syrup, oil, starch, and cereals manufactured in whole or in part to contain soybean plant by-products. In certain embodiments, the biological sample is “non-regenerable” (i.e., incapable of being regenerated into a soybean plant or soybean plant part).

[0023] As used herein, the terms “correspond,” “corresponding,” and the like, when used in the context of an nucleotide position, mutation, and / or substitution in any given polynucleotide (e.g., an allelic variant of SEQ ID NO: 1) with respect to the reference polynucleotide sequence (e.g., SEQ ID NO: 1) all refer to the position of the nucleotide in the given sequence that has identity to the nucleotide in the reference nucleotide sequence when the given polynucleotide is aligned to the reference polynucleotide sequence using a pairwise alignment algorithm (e.g., CLUSTAL O 1.2.4 with default parameters).Docket No. P14694WO00

[0024] As used herein, the terms “Cpf1” and “Cas12a” are used interchangeably to refer to the same RNA dependent DNA endonuclease (RdDe).

[0025] As used herein, a “cultivar” refers to a cultivated variety. A cultivar is generally developed using crossing, selfing, and / or selection and is maintained by any suitable method of propagation, through open pollination, selfing, or the like. Details of cultivar development can be found in “Principles of Cultivar Development” by Fehr, Macmillan Publishing Company (1993), which is incorporated herein by reference in its entirety.

[0026] As used herein, the phrases “elite soybean line” or “elite soybean plant” refer to any line or plant which has undergone breeding to provide one or more trait improvements (e.g., desirable agronomic performance (typically commercial production) or superior grain quality). In some cases, an elite line can be an agronomically or otherwise superior line or variety that has resulted from several or many cycles of breeding and selection for one or more trait improvements (e.g., superior agronomic performance or superior grain quality). Similarly, “elite germplasm” is a germplasm resulting from breeding and selection for desirable agronomic performance (typically commercial production). Such germplasm may be agronomically superior germplasm, derived from and / or capable of giving rise to a plant with superior agronomic performance, such as an existing or newly developed elite line of soybean. Elite crop plant lines include plants which are essentially homozygous, e.g., inbred or doubled haploid. Elite crop plants can include inbred lines used as is or used as pollen donors or pollen recipients in breeding (e.g., used to produce F1 plants). Elite crop plants can include inbred lines which are selfed to produce non-hybrid cultivars or varieties. Elite crop plants can include hybrid F1 progeny of a cross between two distinct elite inbred or doubled haploid plant lines.

[0027] As used herein, the phrase “endogenous gene” refers to the native form of a gene unit in its natural location in the genome of an organism.

[0028] As used herein, the term “enhancer” refers to a cis-acting sequence that increases the transcription from a eukaryotic promoter and that can function at a variable distance from the promoter.

[0029] As used herein, the term “expression” refers to the production of a functional end- product (e.g., an mRNA, guide RNA, or a protein) in either precursor or mature form.

[0030] As used herein, the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.Docket No. P14694WO00

[0031] As used herein, the term “isomorphic allele” refers to an allele of a gene having wild- type gene activity.

[0032] The term “isolated” as used herein means having been removed from its natural environment.

[0033] As used herein, the term “introduced” means providing a nucleic acid (e.g., expression construct) or protein into a cell. Introduced includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid can be incorporated into the genome of the cell and includes reference to the transient provision of a nucleic acid or protein to the cell. Introduced includes reference to stable or transient transformation methods. Thus, “introduced” in the context of inserting a nucleic acid fragment (e.g., a recombinant DNA construct / expression construct) into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid fragment into a eukaryotic or prokaryotic cell where the nucleic acid fragment can be incorporated into the genome of the cell (e.g., nuclear chromosome, plasmid, plastid, chloroplast, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0034] As used herein, a “non-natural” or “non-naturally occurring” mutation refers to a mutation in a gene which is generated via human intervention or descended from the mutation generated via human intervention. Non-limiting examples of human intervention which can be used to generate a non-naturally occurring mutation include mutagenesis (e.g., chemical mutagenesis, ionizing radiation mutagenesis), mutagenesis followed by DNA sequence-based screening and selection (TILLING), and targeted genetic modifications (e.g., CRISPR-based methods, TALEN-based methods, zinc finger-based methods).

[0035] As used herein, the term “plant” includes a whole soybean plant and any descendant, cell, tissue, part, or parts of the plant. The term “plant” thus includes reference to an immature or mature whole soybean plant, including a plant from which seed or grain or anthers have been removed.

[0036] The term “plant part” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); grain; stover; a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., pollen, embryos, pods; flowers, fruits, shoots, leaves, roots, stems, and explants). A plant tissue or plant organ can be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture can be capable of regenerating a plant having theDocket No. P14694WO00 physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. Regenerable cells in a plant cell or tissue culture can be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, or stalks. In contrast, some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non- regenerable” plant cells.

[0037] As used herein, the term “promoter” refers to a regulatory region comprising the transcriptional start site) and binding sites for core transcription factor complexes that induce RNA polymerase II-mediated (RNA PolII-mediated) transcription of RNA PolII promoters.

[0038] As used herein, the phrase “upstream region” refers to genomic DNA or DNA in a vector that extends from a nucleotide located 5´ to one or more enhancers on the sense strand up to and including the ATG translation initiation codon of a given gene.

[0039] As used herein, the term “variety” refers a group of similar plants that by one or more structural features, genetic features, and / or performance can be distinguished from other varieties within the same species. In certain embodiments, the term variety refers to the botanical taxonomic designation whereby variety is ranked below species or subspecies, as well as the legal definition whereby the term “variety” refers to a commercial plant that is protected under the terms outlined in the International Convention for the Protection of New Varieties of Plants.

[0040] To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.

[0041] The present disclosure describes the deletion of one or more nucleotides between a transcriptional enhancer and a promoter of endogenous target soybean gene sequences that provide for upregulating expression of a target soybean gene. Many enhancers have been identified in the soybean genome. In certain embodiments, enhancers are identified by any experimental methods including: (i) mutagenesis of DNA located near a promoter and comparison of promoter activity compared to unmutated control promoters; (ii) placement of regions suspected of containing enhancers near heterologous promoters which are operably linked to reporter genes; and / or analysis of epigenetic modifications of regions expected to contain enhancers. In certain embodiments, enhancers comprise identifiable markers or criteria indicative of an enhancer. In certain embodiments, enhancer regions have unmethylatedDocket No. P14694WO00 nucleotides, have ATAC sequence signals in at least four plant tissues, have overlap with at least one acetylated histone marker, and / or have a conserved non-coding region (CNS) in at least the Fabaceae clade.

[0042] Table 1 provides examples of genome regions comprising transcriptional enhancers in soybean.

[0043] Table 1. Soybean Genome Regions with Transcriptional Enhancers Gene Name Transcriptional Figure Enhancer Enhancer to Promoter Nucleotide Positionso e e o e s, uc eo e ego s e ee a a sc p o a e a ce a d a promoter that are targeted for deletion also have various identifying characteristics. In certain embodiments, a nucleotide region that is deleted does not include enhancer elements and / or contains transcriptional repressor elements related to repression of expression. Deleted regions are methylated, contain transposons, are depleted in activating (acetylated) histone markers, and / or are not be conserved across the promoters of orthologous genes from other plants. In certain embodiments, the deleted regions correspond to regions which are not conserved in the promoters of orthologous genes of other Fabaceae plants including Phaseolus (beans), Pisum sativum (pea), Cicer arietinum (chickpeas), Vicia faba (broad bean), Medicago sativa (alfalfa), and / or Arachis hypogaea (peanut). In various embodiments, a deletion between a transcriptional enhancer and a promoter of an endogenous target soybean gene can be about five or more to about 4500 or more nucleotides. For example, the deletion can be five or more, ten or more, 50 or more, 100 or more, 250 or more, 500 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, or 4500 or more nucleotides. In some embodiments, a deletion between a transcriptional enhancer and a promoter of an endogenous target soybean gene can be 500 kb to about 8 kb or more. For example, the deletion can be 500 or more, 1000 or more, 1500 or more, 2000 or more, 2500 orDocket No. P14694WO00 more, 3000 or more, 3500 or more, 4000 or more, 4500 or more, 5000 or more, 5500 or more, 6000 or more, 6500 or more, 7000 or more, 7500 or more, or 8000 or more nucleotides.

[0045] The present disclosure provides for soybean plant cells, plant parts including seed, plants, seed lots, and biological samples comprising an endogenous gene or fragment thereof containing a deletion of one or more nucleotides between a transcriptional enhancer and a promoter of an endogenous target soybean gene sequence for upregulating expression of the endogenous target soybean gene. These soybean plants and parts can be utilized for human food, livestock feed, as a raw material in industry, or as breeding material for development of other soybean varieties.

[0046] One or more nucleotides can be deleted in the region between a transcriptional enhancer and a promoter of any endogenous target soybean gene for which an increase in expression is desired. In some embodiments, the endogenous target soybean gene can be the soybean transcription factor Glyma.19G143900 gene located at nucleotides 40,944,904 to 40,953,824 of chromosome 19 of the Glycine max Williams 82 genome assembly version 4 (Wm82.a4.v1; Glyma.19G143900 on the world wide web internet site “soybase.org”; Grant et al. Nucl. Acids Res. (2010) 38 (suppl 1): D843-D846. doi: 10.1093 / nar / gkp798). Gene Glyma.19G143900 is also referred to as BHLH48 or GmPTF1. A nucleotide sequence from one or more enhancers to an ATG translational start site of the endogenous soybean Glyma.19G143900 gene comprises SEQ ID NO: 1 or an allelic variant thereof (Fig.1). The nucleotide sequence of SEQ ID NO: 1 therefore comprises one or more enhancers, promoter, and ATG translational start site of the endogenous soybean gene Glyma.19G143900. Allelic variants of the nucleotide sequence of one or more enhancers to an ATG translational start site of the Glyma.19G143900 gene include variants having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 1. In certain embodiments, allelic variants of the nucleotide sequence from one or more enhancers to an ATG translational start site of the endogenous soybean Glyma.19G143900gene are isomorphic alleles. In certain embodiments, the deletion in the one or more nucleotides between a transcriptional enhancer and a promoter of the Glyma.19G143900 gene can include a deletion of at least nucleotides in positions 788 to 2792 of SEQ ID NO: 1 or corresponding positions in an allelic variant thereof. In certain embodiments, the deletion in the one or more nucleotides between a transcriptional enhancer and a promoter of the Glyma.19G143900 gene can include a deletion of at least a portion of nucleotides in positions 778 to 2794 of SEQ ID NO: 1 or corresponding positions in an allelic variant thereof. In certain embodiments, the one or more nucleotides between theDocket No. P14694WO00 transcriptional enhancer and the promoter of the Glyma.19G143900 gene or corresponding positions in an allelic variant thereof can contain the deletion in a variant upstream region of the Glyma.19G143900 gene set forth in SEQ ID NO: 2. In certain embodiments, the deletion in the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.19G143900 gene can include a deletion at a position of anywhere between +20 nucleotides and -40 nucleotides from the 5´ and / or 3´ ends of the deletion in a variant upstream region of the Glyma.19G143900 gene set forth in SEQ ID NO: 2. In various embodiments, the deletion in the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.19G143900 gene can include a deletion obtained by use of a Class II Type V Cas nuclease and at least two gRNAs comprising: (i) a first gRNA comprising a spacer encoded by SEQ ID NOs: 13 and / or 14; and (ii) a second gRNA comprising a spacer encoded by SEQ ID NOs: 15 and / or 16.

[0047] In some embodiments, the endogenous target soybean gene can be the inorganic phosphate transporter 1-1 (PHT1-1) gene located at nucleotides 42,044,051 to 42,048,185 of chromosome 10 of the Glycine max Williams 82 genome assembly version 4 (Wm82.a4.v1; Glyma.10G186500 on the world wide web internet site “soybase.org”; Grant et al. Nucl. Acids Res. (2010) 38 (suppl 1): D843-D846. doi: 10.1093 / nar / gkp798). Gene Glyma.10G186500 is also referred to as PHT1-1 or GmPT7. A nucleotide sequence from one or more enhancers to an ATG translational start site of the endogenous soybean Glyma.10G186500 gene comprises to SEQ ID NO: 3 or an allelic variant thereof (Fig.2). The nucleotide sequence of SEQ ID NO: 3 therefore comprises one or more enhancers, promoter, and ATG translational start site of the endogenous soybean Glyma.10G186500 gene. Allelic variants of the nucleotide sequence of one or more enhancers to an ATG translational start site of the Glyma.10G186500 gene include variants having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 3. In certain embodiments, allelic variants of the nucleotide sequence from one or more enhancers to an ATG translational start site of the endogenous soybean Glyma.10G186500 gene are isomorphic alleles. In certain embodiments, the deletion in the one or more nucleotides between a transcriptional enhancer and a promoter of the Glyma.10G186500 gene can include a deletion of at least nucleotides in positions 1641 to 6822 of SEQ ID NO: 3 or corresponding positions in an allelic variant thereof. In certain embodiments, the deletion in the one or more nucleotides between a transcriptional enhancer and a promoter of the Glyma.10G186500 gene can include a deletion of at least a portion of nucleotides in positions 1623 to 6850 of SEQ ID NO: 3 or corresponding positions in an allelicDocket No. P14694WO00 variant thereof. In certain embodiments, the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.10G186500 gene or corresponding positions in an allelic variant thereof can contain the deletion in a variant upstream region of the Glyma.10G186500 gene set forth in SEQ ID NO: 4. In certain embodiments, the deletion in the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.10G186500 gene can include a deletion at a position of anywhere between +20 nucleotides and -40 nucleotides from the 5´ and / or 3´ ends of the deletion in a variant upstream region of the Glyma.10G186500 gene set forth in SEQ ID NO: 4. In various embodiments, the deletion in the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.10G186500 gene can include a deletion obtained by use of a Class II Type V Cas nuclease and at least two gRNAs comprising: (i) a first gRNA comprising a spacer encoded by SEQ ID NOs: 17 and / or 18; and (ii) a second gRNA comprising a spacer encoded by SEQ ID NOs: 19 and / or 20.

[0048] In some embodiments, the endogenous target soybean gene can be the NAC transcription factor NAC27 gene located at nucleotides 37,523,878 to 37,525,662 of chromosome 13 of the Glycine max Williams 82 genome assembly version 4 (Wm82.a4.v1; Glyma.13G279900 on the world wide web internet site “soybase.org”; Grant et al. Nucl. Acids Res. (2010) 38 (suppl 1): D843-D846. doi: 10.1093 / nar / gkp798). Gene Glyma.13G279900 is also referred to as NAC27 or GmNAC19. A nucleotide sequence from one or more enhancers to an ATG translational start site of the endogenous soybean Glyma.13G279900gene comprises to SEQ ID NO: 5 or an allelic variant thereof (Fig. 3). The nucleotide sequence of SEQ ID NO: 5 therefore comprises one or more enhancers, promoter, and ATG translational start site of the endogenous soybean Glyma.13G279900 gene. Allelic variants of the nucleotide sequence of one or more enhancers to an ATG translational start site of the Glyma.13G279900 gene include variants having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 5. In certain embodiments, allelic variants of the nucleotide sequence from one or more enhancers to an ATG translational start site of the endogenous soybean Glyma.13G279900 gene are isomorphic alleles. In certain embodiments, the deletion in the one or more nucleotides between a transcriptional enhancer and a promoter of the Glyma.13G279900 gene can include a deletion of at least nucleotides in positions 615 to 1112 or in positions 612 to 1149 of SEQ ID NO: 5 or corresponding positions in an allelic variant thereof. In certain embodiments, the deletion in the one or more nucleotides between a transcriptional enhancer and a promoter of the Glyma.13G279900 gene can include a deletionDocket No. P14694WO00 of at least a portion of nucleotides in positions 612 to 1121of SEQ ID NO: 5 or corresponding positions in an allelic variant thereof. In certain embodiments, the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.13G279900 gene or corresponding positions in an allelic variant thereof can contain the deletion in a variant upstream region of the Glyma.13G279900 gene set forth in SEQ ID NO: 6 or SEQ ID NO: 37. In certain embodiments, the deletion in the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.13G279900 gene can include a deletion at a position of anywhere between +20 nucleotides and -40 nucleotides from the 5´and / or 3´ ends of the deletion in a variant upstream region of the Glyma.13G279900 gene set forth in SEQ ID NO: 6 or SEQ ID NO: 37. In various embodiments, the deletion in the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.13G279900 gene can include a deletion obtained by use of a Class II Type V Cas nuclease and at least two gRNAs comprising: (i) a first gRNA comprising a spacer encoded by SEQ ID NOs: 21 and / or 22; and (ii) a second gRNA comprising a spacer encoded by SEQ ID NOs: 23 and / or 24.

[0049] In some embodiments, the endogenous target soybean gene can be the ETO1 gene, encoding a negative regulator of 1-aminocyclopropane-1-carboxylic acid synthase5 (ACS5), located at nucleotides 47,076,517 to 47,081,358 of chromosome 14 of the Glycine max Williams 82 genome assembly version 4 (Wm82.a4.v1; Glyma.14G197100 on the world wide web internet site “soybase.org”; Grant et al. Nucl. Acids Res. (2010) 38 (suppl 1): D843-D846. doi: 10.1093 / nar / gkp798). Gene Glyma.14G197100 is also referred to as ETO1 or Gm ETO1. A nucleotide sequence from one or more enhancers to an ATG translational start site of the endogenous soybean Glyma.14G197100 gene comprises SEQ ID NO: 7 or an allelic variant thereof (Fig. 4). The nucleotide sequence of SEQ ID NO: 7 therefore comprises one or more enhancers, promoter, and ATG translational start site of the endogenous soybean Glyma.14G197100 gene. Allelic variants of the nucleotide sequence of one or more enhancers to an ATG translational start site of the Glyma.14G197100gene include variants having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 7. In certain embodiments, allelic variants of the nucleotide sequence from one or more enhancers to an ATG translational start site of the endogenous soybean Glyma.14G197100 gene are isomorphic alleles. In certain embodiments, the deletion in the one or more nucleotides between a transcriptional enhancer and a promoter of the Glyma.14G197100 gene can include a deletion of at least nucleotides in positions 799-23474 of SEQ ID NO: 7 or corresponding positions in an allelic variant thereof. In certain embodiments, the deletion in the one or moreDocket No. P14694WO00 nucleotides between a transcriptional enhancer and a promoter of the Glyma.14G197100 gene can include a deletion of at least a portion of nucleotides in positions 783 to 2352 of SEQ ID NO: 7 or corresponding positions in an allelic variant thereof. In certain embodiments, the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.14G197100 gene or corresponding positions in an allelic variant thereof can contain the deletion in a variant upstream region of the Glyma.14G197100 gene set forth in SEQ ID NO: 8. In certain embodiments, the deletion in the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.14G197100 gene can include a deletion in a variant upstream region of the Glyma.14G197100 gene at a position of anywhere between +20 nucleotides and -40 nucleotides from the 5´ and / or 3´ ends of the deletion in a variant upstream region of the Glyma.14G197100 gene set forth in SEQ ID NO: 8. In various embodiments, the deletion in the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.14G197100 gene can include a deletion obtained by use of a Class II Type V Cas nuclease and at least two gRNAs comprising: (i) a first gRNA comprising a spacer encoded by SEQ ID NOs: 25 and / or 26; and (ii) a second gRNA comprising a spacer encoded by SEQ ID NOs: 27 and / or 28.

[0050] In some embodiments, the endogenous target soybean gene can be the CYP78A5 gene, encoding a cytochrome P450 monooxygenase, located at nucleotides 39,073,499 to 39,075,745 of chromosome 11 of the Glycine max Williams 82 genome assembly version 4 (Wm82.a4.v1; Glyma.11G250200 on the world wide web internet site “soybase.org”; Grant et al. Nucl. Acids Res. (2010) 38 (suppl 1): D843-D846. doi: 10.1093 / nar / gkp798). Gene Glyma.11G250200 is also referred to as CYP78A5 or GmCYP78A71. A nucleotide sequence from one or more enhancers to an ATG translational start site of the endogenous soybean Glyma.11G250200 gene comprises SEQ ID NO: 9 or an allelic variant thereof (Fig. 5). The nucleotide sequence of SEQ ID NO: 9 therefore comprises one or more enhancers, promoter, and ATG translational start site of the endogenous soybean Glyma.11G250200 gene. Allelic variants of the nucleotide sequence of one or more enhancers to an ATG translational start site of the Glyma.11G250200 gene include variants having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 9. In certain embodiments, allelic variants of the nucleotide sequence from one or more enhancers to an ATG translational start site of the endogenous soybean Glyma.11G250200 gene are isomorphic alleles. In certain embodiments, the deletion in the one or more nucleotides between a transcriptional enhancer and a promoter of the Glyma.11G250200 gene can include a deletion of at least nucleotides in positions 1018-Docket No. P14694WO00 6144 of SEQ ID NO: 9 or corresponding positions in an allelic variant thereof. In certain embodiments, the deletion in the one or more nucleotides between a transcriptional enhancer and a promoter of the Glyma.11G250200 gene can include a deletion of at least a portion of nucleotides in positions 1004 to 6148 of SEQ ID NO: 9 or corresponding positions in an allelic variant thereof. In certain embodiments, the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.11G250200 gene or corresponding positions in an allelic variant thereof can contain the deletion in a variant upstream region of the Glyma.11G250200 gene set forth in SEQ ID NO: 10. In certain embodiments, the deletion in the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.11G250200 gene can include a deletion at a position of anywhere between +20 nucleotides and -40 nucleotides from the 5´ and / or 3´ ends of the deletion in a variant upstream region of the Glyma.11G250200 gene set forth in SEQ ID NO: 10. In various embodiments, the deletion in the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.11G250200 gene can include a deletion obtained by use of a Class II Type V Cas nuclease and at least two gRNAs comprising: (i) a first gRNA comprising a spacer encoded by SEQ ID NOs: 29 and / or 30; and (ii) a second gRNA comprising a spacer encoded by SEQ ID NOs: 31 and / or 32.

[0051] In some embodiments, the endogenous target soybean gene can be the AAP6 gene, encoding an amino acid permease 6, located at nucleotides 27,323,000 to 27,330,119 of chromosome 17 of the Glycine max Williams 82 genome assembly version 4 (Wm82.a4.v1; Glyma.17G192000 on the world wide web internet site “soybase.org”; Grant et al. Nucl. Acids Res. (2010) 38 (suppl 1): D843-D846. doi: 10.1093 / nar / gkp798). Gene Glyma.17G192000 is also referred to as AAP6 or GmAAP6a. A nucleotide sequence from one or more enhancers to an ATG translational start site of the endogenous soybean Glyma.17G192000 gene comprises SEQ ID NO: 11 or an allelic variant thereof (Fig.6). The nucleotide sequence of SEQ ID NO: 11 therefore comprises one or more enhancers, promoter, and ATG translational start site of the endogenous soybean Glyma.17G192000 gene. Allelic variants of the nucleotide sequence of one or more enhancers to an ATG translational start site of the Glyma.17G192000 gene include variants having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to SEQ ID NO: 11. In certain embodiments, allelic variants of the nucleotide sequence from one or more enhancers to an ATG translational start site of the endogenous soybean Glyma.17G192000 gene are isomorphic alleles. In certain embodiments, the deletion in the one or more nucleotides between a transcriptional enhancer and a promoterDocket No. P14694WO00 of the Glyma.17G192000 gene can include a deletion of at least nucleotides in positions 550- 1104 of SEQ ID NO: 11 or corresponding positions in an allelic variant thereof. In certain embodiments, the deletion in the one or more nucleotides between a transcriptional enhancer and a promoter of the Glyma.17G192000 gene can include a deletion of at least a portion of nucleotides in positions 547 to 1107 of SEQ ID NO: 11 or corresponding positions in an allelic variant thereof. In certain embodiments, the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.17G192000 gene or corresponding positions in an allelic variant thereof can contain the deletion set forth in SEQ ID NO: 12. In certain embodiments, the deletion in the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.17G192000 gene can include a deletion at a position of anywhere between +20 nucleotides and -40 nucleotides from the 5´ and / or 3´ ends of the deletion set forth in SEQ ID NO: 12. In various embodiments, the deletion in the one or more nucleotides between the transcriptional enhancer and the promoter of the Glyma.17G192000 gene can include a deletion obtained by use of a Class II Type V Cas nuclease and at least two gRNAs comprising: (i) a first gRNA comprising a spacer encoded by SEQ ID NOs: 33 and / or 34; and (ii) a second gRNA comprising a spacer encoded by SEQ ID NOs: 35 and / or 36.

[0052] Soybean plant cells, plant parts, and plants comprising an endogenous gene containing a deletion of one or more nucleotides between a transcriptional enhancer and a promoter of endogenous target soybean gene sequences are provided. In certain embodiments, the at least one deletion is a non-natural mutation. In certain embodiments, the at least one deletion increases expression of the endogenous target soybean gene (e.g., by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) relative to a wild-type or control soybean plant cell lacking the at least one deletion. The deletion can be made anywhere between a transcriptional enhancer and a promoter of endogenous target soybean gene. In certain embodiments, the at least one deletion comprises, consists essentially of, or consists of a deletion of at least one nucleotide (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750,Docket No. P14694WO00 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides) between a transcriptional enhancer and a promoter of the endogenous target soybean gene. In certain embodiments, the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.19G143900 comprises SEQ ID NO: 2 or an allelic variant thereof. In certain embodiments, the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.10G186500 comprises SEQ ID NO: 4 or an allelic variant thereof. In certain embodiments, the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.13G279900 comprises SEQ ID NO: 6, SEQ ID NO: 37, or an allelic variant thereof. In certain embodiments, the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.14G197100 comprises´ to one or more enhancers on the sense strand up SEQ ID NO: 8 or an allelic variant thereof. In certain embodiments, the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.11G250200 comprises SEQ ID NO: 10 or an allelic variant thereof. In certain embodiments, the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.17G192000 comprises SEQ ID NO: 12 or an allelic variant thereof.

[0053] In certain embodiments, the non-naturally occurring deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene brings the transcriptional enhancer and the promoter in closer proximity in comparison to a control soybean plant lacking the deletion. The closer proximity of the transcriptional enhancer and the promoter can the increase expression of the endogenous target soybean gene resulting in an increase in yield of at least one of a soybean plant root length, root biomass, lateral root number, and / or nodule number relative to the control plant lacking the deletion.

[0054] Increased yield of the soybean plant can be measured in a number of ways, including pod count per plant, seed count per plant, total harvested seed weight per plant, or total harvested seed weight per unit area (e.g., seed weight per acre or seed weight per hectare). In certain embodiments, the increased nodule number per plant and / or yield can result from an improved response to stress, including an abiotic stress (e.g., drought, heat, cold, salt stress,Docket No. P14694WO00 and / or nutrient deficiency). In certain embodiments, the nodule number per plant and / or yield of the soybean plant comprising an endogenous gene containing a deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene is preserved or further enhanced when the deletion(s) are combined with least one mutation in a distinct soybean gene.

[0055] In certain embodiments, the nodule number per soybean plant comprising an endogenous gene containing a deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene is increased in comparison to the nodule number per plant for a wild-type or control soybean plant lacking the deletion(s). In certain embodiments, the nodule number per plant is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in comparison to the nodule number per plant from the corresponding wild-type or control soybean plant lacking the deletion(s). In certain embodiments, the pod count per soybean plant comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene is increased in comparison to the pod count per plant for a wild-type or control soybean plant lacking the deletion(s). In certain embodiments, the pod count per plant is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in comparison to the pod count per plant from the corresponding wild-type or control soybean plant lacking the deletion(s). In certain embodiments, the seed count per plant comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene is increased in comparison to the seed count per plant for a wild-type or control soybean plant lacking the deletion(s). In certain embodiments, the seed count per plant comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in comparison to the seed count per plant from the corresponding wild-type or control soybean plant lacking the deletion(s). In certain embodiments, the total harvested seed weight per plant comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene is increased in comparison to the total harvested seed weightDocket No. P14694WO00 per plant for a wild-type or control soybean plant lacking the deletion(s). In certain embodiments, the total harvested seed weight per plant is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in comparison to the total harvested seed weight per plant from the corresponding wild-type or control soybean plant lacking the deletion(s). In certain embodiments, the total harvested seed weight per unit area for soybean plants comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene is increased in comparison to the total harvested seed weight per unit area for a wild-type or control soybean plant lacking the deletion(s). In certain embodiments, the total harvested seed weight per unit area for soybean plants comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene is increased by at least about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% in comparison to the total harvested seed weight per unit area from the corresponding wild-type or control soybean plant lacking the deletion(s). In certain embodiments, the average weight of 1000 seeds obtained from the soybean plant is equivalent to or essentially the same as the average weight of 1000 seeds obtained from a wild-type or control soybean plant lacking the deletion(s).

[0056] In certain embodiments, the nodule number per plant, pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area for the soybean plant comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene are increased in comparison to nodule number per plant, pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area for a wild-type or control soybean plant lacking the deletion(s) when grown under stress or specific agronomic practices. Non-limiting examples of stresses include drought, cold, heat, salt, shade, nutrient deficiency (e.g., nitrogen deficiency), high planting density, and the presence of pests or pathogens. In certain embodiments, the stress comprises an abiotic stress. In certain embodiments, the abiotic stress comprises drought, cold, heat, salt stress, or nutrient deficiency (e.g., nitrogen deficiency). In these embodiments, the nodule number per plant, pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area can be increased when the plant comprising the endogenous gene containing the deletion of one or more nucleotides locatedDocket No. P14694WO00 between the transcriptional enhancer and the promoter of the endogenous target soybean gene is grown under abiotic stress in comparison to nodule number per plant, pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area for a wild-type or control soybean plant lacking the deletion(s) grown under abiotic stress. In certain embodiments, the nodule number per plant, pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area can be increased when the plant comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene is grown under nitrogen deficient conditions in comparison to nodule number per plant, pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area for a wild- type or control soybean plant lacking the deletion(s) is grown under nitrogen deficient conditions.

[0057] Soybean seed lots comprising the soybean seeds comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene are provided. In certain embodiments, soybean plants comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene can yield seed lots wherein the average weight of 1000 seeds in the seed lot is equivalent to or essentially the same as the average weight of 1000 seeds in a control seed lot obtained from a wild-type or control plant lacking the deletion(s) (e.g., a wild-type soybean plant homozygous for the nucleic acid sequence of the region between the transcriptional enhancer and the promoter of the endogenous target soybean gene). In certain embodiments, the average number of seeds per kilogram of seeds in the seed lot is equivalent to or essentially the same as the average number of seeds per kilogram of seeds in a control seed lot obtained from a wild-type or control soybean plant lacking the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene. In certain embodiments, the seed lot is packaged in lots comprising about 50 to 60 pounds (i.e., about 22.7 to 27.2 kilograms) of seeds.

[0058] Also provided are polynucleotides comprising any of the aforementioned deletion(s) or fragments thereof. In certain embodiments, polynucleotides comprising the endogenous gene or fragments thereof containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ IDDocket No. P14694WO00 NO: 1, 3, 5, 7, 9, and / or 11 are provided. In certain embodiments, the polynucleotide comprises a sequence having at least 95%, 96, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity across the entire length of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 with the proviso that the sequences are not identical across their entire length to SEQ ID NO: 1, 3, 5, 7, 9, and / or 11. In certain embodiments, the polynucleotide is an isolated polynucleotide. In certain embodiments, fragments containing the deletion will be at least 10, 25, 50, 100, 150, 250, 300, 400, or 500 nucleotides in length. In certain embodiments, fragments containing the deletion will comprise at least 5, 10, 20, 50, and / or 100 nucleotides located both 5´ and 3´ to the deletion.

[0059] Biological samples and soybean by-products comprising any of the aforementioned polynucleotides are also provided. In certain embodiments, the by-products are processed products are made from the soybean plants of the disclosure or their seeds, including: (a) soybean seed meal (defatted or non-defatted); (b) extracted soybean proteins, oils, sugars, syrups, and starches; (c) soy fermentation products; (d) soybean based animal feed or human food products (e.g., feed and food comprising soybean seed meal (defatted or non-defatted) and other ingredients (e.g., other cereal grains, other seed meal, other protein meal, other oil, other starch, other sugar, a binder, a preservative, a humectant, a vitamin, and / or mineral); (e) a pharmaceutical; (f) raw or processed biomass (e.g., cellulosic and / or lignocellulosic material; silage); and (g) various industrial products.

[0060] Methods of using the soybean plants, seeds, and seed lots of the disclosure to produce soybean by-products are also provided. Such methods will typically include at least one processing step of cleaning, cracking, flaking, crushing, macerating, pressing, extracting, expelling, and / or extruding the seed.

[0061] This disclosure is also directed to methods for producing a soybean plant having the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene by crossing a first parent soybean plant with a second parent soybean plant wherein the first or second parent soybean plant comprises the deletion(s). Further, both the first and second parent soybean plants can comprise the deletion(s). Any such methods using a soybean plant comprising the endogenous gene containing the deletion(s) are part of this disclosure: selfing, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using a soybean plant comprising the at least one mutation as a parent are within the scope of this disclosure, including plants derived from a soybean plant having the at least one mutation. Also provided are the F1progenyDocket No. P14694WO00 soybean plants produced from the crossing of a soybean plant comprising the at least one mutation with any other soybean plant, F1seed, and various parts of the F1soybean plant. The following describes breeding methods that can be used with soybean plants of the disclosure in the development of further soybean plants. One such embodiment is a method for developing a progeny soybean plant in a soybean plant breeding program comprising: obtaining the soybean plant, or its parts, comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene and utilizing the plant or plant parts as a source of breeding material; and selecting a progeny plant having the at least one mutation. Breeding steps that can be used in the soybean plant breeding program include pedigree breeding, backcrossing, mutation breeding, and recurrent selection. In conjunction with these steps, techniques such as restriction fragment polymorphism enhanced selection, genetic marker enhanced selection (for example SNP or SSR markers), and the making of double haploids can be utilized.

[0062] Field crops are bred through techniques that take advantage of the plant's method of pollination. A soybean plant of the disclosure can be self-pollinated, sib-pollinated, or cross pollinated to create a pedigree soybean plant. A plant is self-pollinated if pollen from one flower is transferred to the same or another flower of the same plant. A plant is sib-pollinated when individuals within the same family or variety are used for pollination. A plant is cross- pollinated if the pollen comes from a flower on a different plant from a different family or variety. The terms “cross-pollination” and “out-cross” as used herein do not include self- pollination or sib-pollination. Soybean plants (Glycine max) are recognized to be naturally self- pollinated plants which, while capable of undergoing cross-pollination, rarely do so in nature. Insects are reported by some researchers to carry pollen from one soybean plant to another and it generally is estimated that less than one percent of soybean seed formed in an open planting can be traced to cross-pollination, i.e., less than one percent of soybean seed formed in an open planting is capable of producing F1 hybrid soybean plants.

[0063] Any other suitable breeding, selection, or growing methods may be used. Choice of the particular breeding or selection method will vary depending on environmental factors, population size, and the like.

[0064] In certain embodiments, soybean plant cells, plant parts (e.g., seeds), and plants comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof and a transgenic locus areDocket No. P14694WO00 provided. In certain embodiments, the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof is combined with one or more soybean GM events providing tolerance to any one or a combination of glyphosate-based, glufosinate- based, HPPD inhibitor-based, sulfonylurea- or imidazolinone-based, AHAS- or ALS- inhibiting and / or auxin-type (e.g., dicamba, 2,4-D) herbicides and / or an insect resistance trait. GM events that can be combined with the mutations disclosed herein include Event EE-GM3 (aka FG-072, MST-FGØ72-3, described in WO2011063411, USDA-APHIS Petition 09-328- 01p), Event SYHTOH2 (aka 0H2, SYN-ØØØH2-5, described in WO2012 / 082548 and 12-215- 01p), Event DAS-68416-4 (aka Enlist Soybean, described in WO2011 / 066384 and WO2011 / 066360, USDA-APHIS Petition 09-349-01p), Event DAS-44406-6 (aka Enlist E3, DAS-444Ø6-6, described in WO2012 / 075426 and USDA-APHIS 11-234-01p), Event MON87708 (dicamba-tolerant event of Roundup Ready 2 Xtend Soybeans, described in WO2011 / 034704 and USDA-APHIS Petition 10-188-01p, MON-877Ø8-9), Event MON89788 (aka Genuity Roundup Ready 2 Yield, described in WO2006 / 130436 and USDA- APHIS Petition 06-178-01p), Event 40-3-2 (aka Roundup Ready, GTS 40-3-2, MON-Ø4Ø32- 6, described in USDA-APHIS Petition 93-258-01), Event A2704-12 (aka LL27, ACS- GMØØ5-3, described in WO2006108674 and USDA-APHIS Petition 96-068-01p), Event 127 (aka BPS-CV127-9, described in WO2010 / 080829), Event A5547-127 (aka LL55, ACS- GMØØ6-4, described in WO2006108675 and in USDA-APHIS Petition 96-068-01p), event MON87705 (MON-877Ø5-6, Vistive Gold, published PCT patent application WO2010 / 037016, USDA-APHIS Petition 09-201-01p), or event DP305423 (aka DP-3Ø5423- 1, published PCT patent application WO2008 / 054747, USDA-APHIS Petition 06-354-01p), or EE-GM5 is combined with a combination of the following events: Event MON98788×MON87708 (aka Roundup Ready 2 Xtend Soybeans, MON-877Ø8-9×MON- 89788-1), Event HOS×Event 40-3-2 (aka Plenish High Oleic Soybeans×Roundup Ready Soybeans), Event EE-GM3×EE-GM2 (aka FG-072xLL55, described in WO2011063413), Event MON 87701×MON 89788 (aka Intacta RR2 Pro Soybean, MON-877Ø1-2×MON- 89788-1), DAS-81419-2×DAS-44406-6 (aka Conkesta™ Enlist E3™ Soybean, DAS-81419- 2×DAS-444Ø6-6), Event DAS-68416-4×Event MON 89788 (aka Enlist™ RoundUp Ready® 2 Soybean, DAS-68416-4×MON-89788-1), Event MON-87769-7×Event MON-89788-1 (aka Omega-3×Genuity Roundup Ready 2 Yield Soybeans), Event MON 87705×Event MON 89788 (aka Vistive Gold, MON-877Ø5-6×MON-89788-1), or Event MON87769×EventDocket No. P14694WO00 MON89788 (aka Omega-3×Genuity Roundup Ready 2 Yield Soybeans, MON-87769- 7×MON-89788-1), where all published PCT patent applications or US national stages thereof are incorporated herein by reference in there entireties. Representative transgenic events that can be combined with the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof include those set forth in Table 2. Also provided herein are soybean plant cells, plant parts (e.g., seeds), and plants comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof and a modification of any of the aforementioned transgenic events or transgenic events set forth in Table 2 below. Modifications of the transgenic events include those disclosed in: WO2022 / 026375, WO2022 / 026379, WO2022 / 026390, WO2022 / 026395, WO2022 / 026403; US Patent Applic. Pub. Nos. US20220030822 and US20230250441; and U.S. Patent No. 11,242,534, which are each incorporated herein by reference in their entireties.

[0065] Table 2. Transgenic Soybean Events Event Name Patent or Patent ATCC;3NCIMB4Trait (traits)1Application Deposit Number; expression ,Docket No. P14694WO00 MST-FGØ72-3 US 8592650 NCIMB 41659 hppdPF W336, (HT)92mepsps, , . ., s), and plants comprising the endogenous gene containing the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof and at least one mutation in a distinct soybean gene are provided. Non-limiting examples of mutations in distinct soybean genes that may be combined with the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of the disclosure include mutations in any one or a combination of the soybean AML4 gene, AIP10a gene, AIP10b gene, RIC1 gene, RIC2 gene, FT1a gene, JAG1 gene, BS1 gene, BS2 gene, Tfl1b gene, and / or NF-YC4 gene. In certain embodiments, the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof is combined with a mutation (e.g., loss-of-function mutation) in an endogenous soybean AML4 gene is located at nucleotides 23,215,077 to 23,222,035 of chromosome 8 of the Glycine max Williams 82 genome assembly version 4. In certain embodiments, the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof is combined with a mutation in the soybean AIP10a gene. The AIP10a gene (Glyma.07G021400) is located at nucleotides 1,664,710 to 1,668,114 of chromosome 7 of the Glycine max Wm82.a4.v1 genome assembly. In certain embodiments, the mutation (e.g., a deletion) in the AIP10a gene can be in the AIP10a gene promoter and / or 5´ UTR. In certain embodiments, the deletion ofDocket No. P14694WO00 one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof is combined with a mutation in the soybean AIP10b gene. The AIP10b gene (Glyma.08G220400) is located at nucleotides 17,985,027 to 17,988,197 of chromosome 8 of the Glycine max Wm82.a4.v1 genome assembly. In certain embodiments, the mutation (e.g., a deletion) in the AIP10b gene can be in the AIP10b gene promoter and / or 5´ UTR. In certain embodiments the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof is combined with a mutation (e.g., loss-of-function mutation) in the soybean RIC1 gene. The RIC1 gene (Glyma.13g292300) is located at nucleotides 38,587,351 to 38,588,133 of chromosome 13 of the Glycine max Wm82.a4.v1 genome assembly. In certain embodiments, the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof is combined with a mutation in the soybean FT1a gene. The FT1a gene (Glyma.18G298900) is located at nucleotides 57,922,912 to 57,928,648 of chromosome 18 of the Glycine max Wm82.a4.v1 genome assembly. In certain embodiments, the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof is combined with a mutation in the soybean JAG1 gene. The JAG1 gene (Glyma.20G116200) is located at nucleotides 35,791,056 to 35,793,868 of chromosome 20 of the Glycine max Wm82.a4.v1 genome assembly. In certain embodiments, the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof is combined with a mutation in the soybean BS1 gene. The BS1 gene (Glyma.10g244400) is located at nucleotides 47,330,160 to 47,335,971 of chromosome 10 of the Glycine max Wm82.a4.v1 genome assembly. In certain embodiments, the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof is combined with a mutation in the soybean BS2 gene. The BS2 gene (Glyma.20g150000) is located at nucleotides 38,879,463 to 38,885,344 of chromosome 20 of the Glycine max Wm82.a4.v1 genome assembly. In certain embodiments, the at least one mutation (e.g., a deletion) in the Tfl1b gene (Glyma.19g194300) can be a promoter deletion as described in WO2023086765, which is incorporated herein by reference in its entirety. InDocket No. P14694WO00 certain embodiments, the at least one mutation in the NF-YC4 gene (Glyma06g17780 and / or Glyma04g37291) can be a promoter element deletion as described in US20230139093, which is incorporated herein by reference in its entirety.

[0067] Methods of producing a soybean seed lot comprising: (i) growing a population of soybean plants comprising an endogenous gene containing a deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene to maturity; and (ii) harvesting seed from the population of soybean plants of step (i) at maturity, thereby producing the soybean seed lot, wherein the soybean plants are homozygous for the deletion(s). In certain embodiments, the seed lot is packaged in lots comprising about 50 to 60 pounds (i.e., about 22.7 to 27.2 kilograms).

[0068] Also provided herein are methods of treating the soybean seeds and seed lots of the disclosure and the resultant treated seeds and seed lots. Seeds can be treated with such fertilizers, biological agents, nematicides, insecticides, and fungicides by methods including in-furrow applications or by coating (e.g., with a drum coater, rotary coater, tumbling drum, fluidized bed, and / or spouted bed apparatus). Methods and compositions including various binders, fillers, film coats, and active ingredients such as fertilizers, surfactants, plant growth regulators, crop desiccants, fungicides, bacteriocides, bacteriostats, insecticides, and insect repellants for coating seeds that can be adapted for use with seeds provided herein are disclosed in US Patent No.10745578, which is incorporated herein by reference in its entirety.

[0069] The disclosure also provides a method of making a soybean plant comprising an endogenous gene containing a deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene. Gene editing molecules of use in methods provided herein include molecules capable of introducing a double-strand break (“DSB”) or single-strand break (“SSB”) at a specific site or sequence in a double-stranded DNA, such as in genomic DNA or in a target gene located within the genomic DNA as well as accompanying guide RNA. In certain embodiments, the deletion(s) result from introduction of a DSB at a target site in the nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene (e.g., SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof) to induce non-homologous end joining (NHEJ) at the site of the break followed by recovery of the desired deletion(s). In certain embodiments, the deletion(s) results from introduction of a DSB at a target site in the nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene (e.g., SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof) followed by homology-Docket No. P14694WO00 directed repair (HDR), microhomology-mediated end joining (MMEJ), or NHEJ to introduce a desired donor or other DNA template polynucleotide at the DSB, followed by recovery of the desired mutation. Examples of such gene editing molecules include: (a) a nuclease comprising an RNA-guided nuclease, an RNA-guided DNA endonuclease or RNA directed DNA endonuclease (RdDe), a class 1 CRISPR type nuclease system, a class 2 type II Cas nuclease, a Cas9, a nCas9 nickase, a class 2 type V Cas nuclease, a Cas12a nuclease, a nCas12a nickase, a Cas12d (CasY), a Cas12e (CasX), a Cas12b (C2c1), a Cas12c (C2c3), a Cas12i, a Cas12j, a Cas14, an engineered nuclease, a codon-optimized nuclease, a zinc-finger nuclease (ZFN) or nickase, a transcription activator-like effector nuclease (TAL-effector nuclease or TALEN) or nickase (TALE-nickase), an Argonaute, and a meganuclease or engineered meganuclease; (b) a polynucleotide encoding one or more nucleases capable of effectuating site-specific alteration (including introduction of a DSB or SSB) of a target nucleotide sequence; (c) a guide RNA (gRNA) for use with an RNA-guided nuclease, or a DNA encoding a gRNA for use with an RNA-guided nuclease; (d) optionally donor DNA template polynucleotides suitable for insertion at a break in genomic DNA by homology-directed repair (HDR) or microhomology- mediated end joining (MMEJ); and (e) optionally other DNA templates (e.g., dsDNA, ssDNA, or combinations thereof) suitable for insertion at a break in genomic DNA (e.g., by non- homologous end joining (NHEJ). In certain embodiments, the deletion(s) are made with a cytosine and / or adenine base editor, or by a PRIME editing system.

[0070] In certain embodiments, the deletion(s) in the nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene and plant cells, parts including seeds, and plants comprising an endogenous gene containing a deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene are generated by CRISPR technology. CRISPR technology for editing the genes of eukaryotes is disclosed in US Patent Application Publications 2016 / 0138008A1 and US2015 / 0344912A1, and in US Patents 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. Cpf1 endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016 / 0208243 A1. Plant RNA promoters for expressing CRISPR guide RNA and plant codon-optimized CRISPR Cas9 endonuclease are disclosed in International Patent Application PCT / US2015 / 018104 (published as WO 2015 / 131101 and claiming priority to US Provisional Patent Application 61 / 945,700). Methods of using CRISPR technology for genome editing in plants are disclosedDocket No. P14694WO00 in US Patent Application Publications US 2015 / 0082478A1 and US 2015 / 0059010A1 and in International Patent Application PCT / US2015 / 038767 A1 (published as WO 2016 / 007347 and claiming priority to US Provisional Patent Application 62 / 023,246). All of the patent publications referenced in this paragraph are incorporated herein by reference in their entirety. In certain embodiments, an RNA-guided endonuclease that leaves a blunt end following cleavage of the target site is used. Blunt-end cutting RNA-guided endonucleases include Cas9. In certain embodiments, an RNA-guided endonuclease that leaves a staggered single stranded DNA overhanging end following cleavage of the target site following cleavage of the target site is used. Staggered-end cutting RNA-guided endonucleases include Cas12a, Cas12b, Cas12d, Cas12e, and Cas12i.

[0071] Guide RNA molecules comprising a spacer RNA molecule which targets the one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof are provided. In certain embodiments, the spacer RNA molecule targets a portion of the nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof. In certain embodiments, the spacer RNA molecule comprises the RNA encoded by SEQ ID NO: 13-36. Guide RNAs comprising a spacer RNA molecule encoded by SEQ ID NO: 13-36 can be used in conjunction with a Cas12a or Cas12i nuclease to generate a deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 which: (i) comprise, consist essentially of, or consist of a deletion of one or more nucleotides (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 50, 45, 50, 65, 70, 75, 80, 85, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 3500, 4000, or 4500 nucleotides) of the nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof.

[0072] CRISPR-type genome editing can be adapted for use in the plant cells and methods provided herein in several ways. CRISPR elements, e.g., gene editing molecules comprising CRISPR endonucleases and CRISPR guide RNAs including single guide RNAs or guide RNAs in combination with tracrRNAs or scoutRNA, or polynucleotides encoding the same, are useful in effectuating genome editing without remnants of the CRISPR elements or selective genetic markers occurring in progeny. In certain embodiments, the CRISPR elements are providedDocket No. P14694WO00 directly to the eukaryotic cell (e.g., soybean plant cells), systems, methods, and compositions as isolated molecules, as isolated or semi-purified products of a cell free synthetic process (e.g., in vitro translation), or as isolated or semi-purified products of in a cell-based synthetic process (e.g., such as in a bacterial or other cell lysate). In certain embodiments, soybean plants or soybean plant cells used in the systems, methods, and compositions provided herein can comprise a transgene that expresses a CRISPR endonuclease (e.g., a Cas9, a Cpf1-type or other CRISPR endonuclease). In certain embodiments, one or more CRISPR endonucleases with unique PAM recognition sites can be used. Guide RNAs (sgRNAs or crRNAs and a tracrRNA or scoutRNA) to form an RNA-guided endonuclease / guide RNA complex which can specifically bind sequences in the gDNA target site that are adjacent to a protospacer adjacent motif (PAM) sequence. The type of RNA-guided endonuclease typically informs the location of suitable PAM sites and design of crRNAs or sgRNAs. G-rich PAM sites, e.g., 5´-NGG are typically targeted for design of crRNAs or sgRNAs used with Cas9 proteins. Examples of PAM sequences include 5´-NGG (Streptococcus pyogenes), 5´-NNAGAA (Streptococcus thermophilus CRISPR1), 5´-NGGNG (Streptococcus thermophilus CRISPR3), 5´-NNGRRT or 5´-NNGRR (Staphylococcus aureus Cas9, SaCas9), and 5´-NNNGATT (Neisseria meningitidis). T-rich PAM sites (e.g., 5´-TTN or 5´-TTTV, where “V” is A, C, or G) are typically targeted for design of crRNAs or sgRNAs used with Cas12a proteins. In some instances, Cas12a can also recognize a 5´-CTA PAM motif. Other examples of potential Cas12a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN (wherein N is defined as any nucleotide). Cpf1 endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016 / 0208243 A1, which is incorporated herein by reference for its disclosure of DNA encoding Cpf1 endonucleases and guide RNAs and PAM sites. Engineered endonucleases with altered or eliminated PAM recognition sites can also be used.

[0073] Identification of a suitable PAM site is only one factor in selecting an efficacious gRNA. Different guides can also have different cutting efficiencies and specificities, which depend on a number of factors. In some embodiments, identification of effective gRNA target sequences in the genome is approached through a combination of in silico selection and experimental evaluation.

[0074] In some cases, a target sequence that perfectly hybridizes with the gRNA spacer sequence occurs only once in a given plant genome. In some embodiments, the genomeDocket No. P14694WO00 comprises additional sequences that imperfectly hybridize with the gRNA spacer sequence, for example, sequences having one or more mismatches (e.g., 1, 2, 3, 4, or 5 mismatches) and / or bulges, relative to the gRNA spacer sequence. In some embodiments, the genome comprises sequences that hybridize the gRNA spacer sequence that are adjacent to a PAM sequence having at least one mismatch relative to the canonical PAM sequence. Such genomic sequences (e.g., target sequences that imperfectly hybridize the gRNA spacer sequence and / or target sequences comprising a non-canonical PAM sequences) are called off-target sites. A favorable off-target profile is typically one that minimizes or eliminates the number of off-target sites and / or the frequency of cutting at these sites.

[0075] The nuclease efficiency and occurrence of off-target activity for a given gRNA / endonuclease combination can be influenced by a number of factors including similarities and dissimilarities between the target site and various off-target sites, as well as the particular endonuclease used. For example, the ability of a given gRNA to promote cleavage at a target sequence in a genomic DNA molecule may relate to the accessibility of the target sequence, which depends on one or more factors that include the chromatin structure of the genomic DNA molecule and / or proximity to transcription factor binding sites. For example, target sequences located within a region of the genomic DNA molecule having a high condensed chromatin structure are less accessible than target sequences located within a region of the genomic DNA molecule having an open chromatin structure. As a further example, target sequences proximal to a region of the genomic DNA molecule bound by a transcription factor or other regulatory protein may be less accessible than target sequences proximal a region of the genomic DNA molecule that is unbound by regulatory proteins. Moreover, the cell state and type of cell may influence the accessibility of target sequences, for example, by influencing the chromatin structure of genomic DNA.

[0076] In certain embodiments, the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene (e.g. SEQ ID NO: 1, 3, 5, 7, 9, and / or 11) and plant cells, parts including seeds, and plants comprising an endogenous gene containing a deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene are generated by use of zinc finger nucleases or zinc finger nickases. Zinc-finger nucleases are site-specific endonucleases comprising two protein domains: a DNA-binding domain, comprising a plurality of individual zinc finger repeats that each recognize between 9 and 18 base pairs, and a DNA-cleavage domain that comprises a nuclease domain (typically Fokl). The cleavageDocket No. P14694WO00 domain dimerizes in order to cleave DNA; therefore, a pair of ZFNs are required to target non- palindromic target polynucleotides. In certain embodiments, zinc finger nuclease and zinc finger nickase design methods which have been described (Urnov et al. (2010) Nature Rev. Genet., 11:636 – 646; Mohanta et al. (2017) Genes vol.8,12: 399; Ramirez et al. Nucleic Acids Res. (2012); 40(12): 5560–5568; Liu et al. (2013) Nature Communications, 4: 2565) can be adapted for use in the methods set forth herein. The zinc finger binding domains of the zinc finger nuclease or nickase provide specificity and can be engineered to specifically recognize any desired target DNA sequence. The zinc finger DNA binding domains are derived from the DNA-binding domain of a large class of eukaryotic transcription factors called zinc finger proteins (ZFPs). The DNA-binding domain of ZFPs typically contains a tandem array of at least three zinc “fingers” each recognizing a specific triplet of DNA. A number of strategies can be used to design the binding specificity of the zinc finger binding domain. One approach, termed “modular assembly”, relies on the functional autonomy of individual zinc fingers with DNA. In this approach, a given sequence is targeted by identifying zinc fingers for each component triplet in the sequence and linking them into a multifinger peptide. Several alternative strategies for designing zinc finger DNA binding domains have also been developed. These methods are designed to accommodate the ability of zinc fingers to contact neighboring fingers as well as nucleotide bases outside their target triplet. Typically, the engineered zinc finger DNA binding domain has a novel binding specificity, compared to a naturally occurring zinc finger protein. Engineering methods include, for example, rational design and various types of selection. Rational design includes, for example, the use of databases of triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, e.g., US Patents 6,453,242 and 6,534,261, both incorporated herein by reference in their entirety. Exemplary selection methods (e.g., phage display and yeast two-hybrid systems) can be adapted for use in the methods described herein. In addition, enhancement of binding specificity for zinc finger binding domains has been described in US Patent 6,794,136, incorporated herein by reference in its entirety. In addition, individual zinc finger domains may be linked together using any suitable linker sequences. Examples of linker sequences are publicly known, e.g., see US Patents 6,479,626; 6,903,185; and 7,153,949, incorporated herein by reference in their entirety. The nucleic acid cleavage domain is non-specific and is typically a restriction endonuclease, such as Fokl. This endonuclease must dimerize to cleave DNA.Docket No. P14694WO00 Thus, cleavage by Fokl as part of a ZFN requires two adjacent and independent binding events, which must occur in both the correct orientation and with appropriate spacing to permit dimer formation. The requirement for two DNA binding events enables more specific targeting of long and potentially unique recognition sites. Fokl variants with enhanced activities have been described and can be adapted for use in the methods described herein; see, e.g., Guo et al. (2010) J. Mol. Biol., 400:96 - 107.

[0077] In certain embodiments, the deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene and plant cells, parts including seeds, and plants comprising an endogenous gene containing a deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene are generated by use of TAL-effector nucleases or TALENs. Transcription activator like effectors (TALEs) are proteins secreted by certain Xanthomonas species to modulate gene expression in host plants and to facilitate the colonization by and survival of the bacterium. TALEs act as transcription factors and modulate expression of resistance genes in the plants. Recent studies of TALEs have revealed the code linking the repetitive region of TALEs with their target DNA-binding sites. TALEs comprise a highly conserved and repetitive region consisting of tandem repeats of mostly 33 or 34 amino acid segments. The repeat monomers differ from each other mainly at amino acid positions 12 and 13. A strong correlation between unique pairs of amino acids at positions 12 and 13 and the corresponding nucleotide in the TALE-binding site has been found. The simple relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for the design of DNA binding domains of any desired specificity. TALEs can be linked to a non- specific DNA cleavage domain to prepare genome editing proteins, referred to as TAL-effector nucleases or TALENs. As in the case of ZFNs, a restriction endonuclease, such as Fokl, can be conveniently used. Methods for use of TALENs in plants have been described and can be adapted for use in the methods described herein, see Mahfouz et al. (2011) Proc. Natl. Acad. Sci. USA, 108:2623 – 2628; Mahfouz (2011) GM Crops, 2:99 – 103; and Mohanta et al. (2017) Genes vol.8,12: 399). TALE nickases have also been described and can be adapted for use in methods described herein (Wu et al.; Biochem Biophys Res Commun. (2014);446(1):261-6; Luo et al; Scientific Reports 6, Article number: 20657 (2016)).

[0078] Various treatments can be used for delivery of gene editing molecules and / or other molecules to a plant cell. In certain embodiments, one or more treatments is employed to deliver the gene editing or other molecules (e.g., comprising a polynucleotide, polypeptide orDocket No. P14694WO00 combination thereof) into a plant cell, e.g., through barriers such as a cell wall, a plasma membrane, a nuclear envelope, and / or other lipid bilayer. In certain embodiments, a polynucleotide-, polypeptide-, or RNP (ribonucleoprotein) -containing composition comprising the molecules are delivered directly, for example by direct contact of the composition with a plant cell. Aforementioned compositions can be provided in the form of a liquid, a solution, a suspension, an emulsion, a reverse emulsion, a colloid, a dispersion, a gel, liposomes, micelles, an injectable material, an aerosol, a solid, a powder, a particulate, a nanoparticle, or a combination thereof can be applied directly to a plant, plant part, plant cell, or plant explant (e.g., through abrasion or puncture or otherwise disruption of the cell wall or cell membrane, by spraying or dipping or soaking or otherwise directly contacting, by microinjection). For example, a plant cell or plant protoplast is soaked in a liquid genome editing molecule-containing composition. In certain embodiments, the composition is delivered using negative or positive pressure, for example, using vacuum infiltration or application of hydrodynamic or fluid pressure. In certain embodiments, the composition is introduced into a plant cell or plant protoplast, e.g., by microinjection or by disruption or deformation of the cell wall or cell membrane, for example by physical treatments such as by application of negative or positive pressure, shear forces, or treatment with a chemical or physical delivery agent such as surfactants, liposomes, or nanoparticles; see, e.g., delivery of materials to cells employing microfluidic flow through a cell-deforming constriction as described in US Published Patent Application 2014 / 0287509, incorporated by reference in its entirety herein. Other techniques useful for delivering the composition to a eukaryotic cell, plant cell or plant protoplast include: ultrasound or sonication; vibration, friction, shear stress, vortexing, cavitation; centrifugation or application of mechanical force; mechanical cell wall or cell membrane deformation or breakage; enzymatic cell wall or cell membrane breakage or permeabilization; abrasion or mechanical scarification (e.g., abrasion with carborundum or other particulate abrasive or scarification with a file or sandpaper) or chemical scarification (e.g., treatment with an acid or caustic agent); and electroporation. In certain embodiments, the composition is provided by bacterially mediated (e.g., Agrobacterium sp., Rhizobium sp., Sinorhizobium sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., Phyllobacterium sp.) transfection of the plant cell or plant protoplast with a polynucleotide encoding the genome editing molecules (e.g., RNA dependent DNA endonuclease, RNA dependent DNA binding protein, RNA dependent nickase, ABE, or CBE, and / or guide RNA); see, e.g., Broothaerts et al. (2005) Nature, 433:629 – 633). Any of these techniques or a combination thereof areDocket No. P14694WO00 alternatively employed on a plant explant, plant part or tissue or intact plant (or seed) from which a plant cell is optionally subsequently obtained or isolated; in certain embodiments, the composition is delivered in a separate step after the plant cell has been isolated.

[0079] In certain embodiments, the methods for upregulating expression of a target soybean gene in a soybean plant and / or a soybean plant cell comprise: (i) identifying a transcriptional enhancer and a promoter in an endogenous target soybean gene in a soybean genome, wherein the transcriptional enhancer is 5´ to the target soybean gene sequence; (ii) contacting the soybean genome with gene editing agents to produce a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequence; and (iii) isolating the soybean plant and / or the soybean plant cell comprising an endogenous gene containing a deletion.

[0080] In certain embodiments, the population of soybean plant cells, parts, or plants which are screened for the presence of a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequence (e.g., the variant upstream region deletions provided in SEQ ID NO: 2, 4, 6, 8, 10,12, 37, or an allelic variant thereof) are first pre-screened by screening of phenotypic characteristics plants having a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequence. In certain embodiments, such phenotypic characteristics include increased nodule number per plant and / or yield (e.g., pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area) in comparison to nodule number per plant and / or yield for a wild-type or control soybean plant lacking the deletion(s). In certain embodiments, such phenotypic characteristics include increased nodule number per plant and / or yield (e.g., pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area) in comparison to increased nodule number per plant and / or yield per plant for a wild-type or control soybean plant lacking the deletion(s) where the screened and wild-type or control plants are grown under nitrogen deficient conditions. In certain embodiments, plants exhibiting one or more of the aforementioned phenotypic characteristics are then subjected to screening for the presence of a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequence and soybean plants comprising an endogenous gene containing the deletion(s) are identified and / or selected.Docket No. P14694WO00

[0081] In certain embodiments, the population of soybean plant cells, parts, or plants which are screened for the presence of a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequence have been subjected to one or more mutagenesis treatments. A deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequence can be generated by mutagenesis methods known in the art, such as chemical mutagenesis or radiation mutagenesis. Suitable chemical mutagens include ethyl methanesulfonate (EMS), sodium azide, methylnitrosourea (MNU), and diepoxybutane (DEB). Suitable radiation includes x-rays, fast neutron radiation, and gamma radiation.

[0082] Soybean plant cells, parts, or plants comprising an endogenous gene containing a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequence can be generated using mutagenesis and identified by TILLING (Targeting Induced Local Lesions IN Genomes) or identified using EcoTILLING. TILLING is a general reverse genetics technique that uses mutagenesis methods to create libraries of mutagenized individuals that are later subjected to high throughput screens for the discovery of mutations. In addition to allowing efficient detection of induced mutations, high-throughput TILLING technology is ideal for the detection of natural mutations. EcoTILLING is a method that uses TILLING techniques to look for natural mutations in individuals (Barkley and Wang. Current genomics vol. 9,4 (2008): 212-26. doi:10.2174 / 138920208784533656). Identified deletion(s) can then be introduced into desirable genetic backgrounds by crossing the mutant with a plant of the desired genetic background and performing a suitable number of backcrosses to cross out the originally undesired parent background. A more detailed description of methods and compositions for TILLING are disclosed in US Patent Application Publication 2004 / 0053236 A1, which is incorporated herein by reference in its entirety and can be adapted for use in the methods provided herein for identifying soybean plant cells, parts, or plants comprising an endogenous gene or fragment thereof containing a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequence.

[0083] In certain embodiments, the screening comprises analyzing pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area in one or more candidate plants or one or more candidate plant populations. In these embodiments, an increase in pod count per plant, seed count per plant, total harvested seed weight per plant, and / or total harvested seed weight per unit area in comparison to a wild-typeDocket No. P14694WO00 or control soybean plant lacking the deletion(s) is indicative of a soybean plant cell, soybean plant part, or soybean plant comprising an endogenous gene containing the deletion(s). In certain embodiments, the screening is conducted on a population of plants grown under stress. Suitable examples of stress conditions include drought, salt, cold, heat, salt, shade, nutrient deficiency, high planting density, and the presence of pests or pathogens.

[0084] Methods for determining whether a soybean plant cell, plant part, or plant comprises a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequence are provided. Methods for determining the presence or absence of the deletion(s) can be used in, for example, breeding programs for identification, selection, introgression, and the like.

[0085] In certain embodiments, the methods comprise analyzing a polynucleotide comprising a portion of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof from the plant cell, plant part, or plant. Detection of the deletion(s) in a nucleic acid sample (e.g., DNA) can be achieved by any combination of nucleic acid amplification (e.g., PCR amplification), hybridization, sequencing, and / or mass-spectrometry based techniques. In certain embodiments, such detection is achieved by amplification and / or hybridization-based detection methods using a primer (e.g., selective amplification primers) and / or probe (e.g., capable of selective hybridization or generation of a specific primer extension product) which specifically recognizes the deletion(s) (e.g., a portion of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof). Such primers and / or probes can comprise or consist of about 15, 20, 25, 30, 40, 45 or 50 more contiguous nucleotides of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof. In certain embodiments, the primers or probes can comprise or consist of about 10 to 50 contiguous nucleotides, about 10 to 40 contiguous nucleotides, about 10 to 30 contiguous nucleotides or about 15 to 30 contiguous nucleotides of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof. In certain embodiments, the hybridization probes (e.g., polynucleotides comprising at least about 15 to 30 base pairs of SEQ ID NO: 1 or an allelic variant thereof) can comprise detectable labels (e.g., fluorescent, radioactive, epitope, and chemiluminescent labels). In certain embodiments, SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof can be directly sequenced using nucleic acid sequencing technologies, including whole genome sequencing.

[0086] In certain optional embodiments, the soybean plant cells disclosed herein are non- regenerable soybean plant cells. In certain optional embodiments provided herein, the soybean plant cells, soybean plant propagules (e.g., a seed, seedling, ovule, embryo, pollen, root, stem,Docket No. P14694WO00 leaf, shoot, explant, or callus), and soybean plants provided herein are not produced by an exclusively biological process. In certain optional embodiments provided herein, the methods for producing soybean plant cells, soybean plant propagules (e.g., a seed, seedling, ovule, embryo, pollen, root, stem, leaf, shoot, explant, or callus), and soybean plants provided herein are not exclusively biological processes.

[0087] The following numbered embodiments also form part of the present disclosure: 1. A method for upregulating expression of a target soybean gene in a soybean plant and / or a soybean plant cell, the method comprising: identifying a transcriptional enhancer and a promoter in an endogenous target soybean gene in a soybean genome, wherein the transcriptional enhancer is 5´ to the target soybean gene; contacting the soybean genome with gene editing agents to produce a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene; and isolating the soybean plant and / or the soybean plant cell comprising endogenous target soybean gene containing the deletion. 2. The method of embodiment 1, wherein the gene editing agents comprise: (a) one or more guide RNA (gRNA) molecules that can hybridize to a nucleotide sequence that is adjacent to a protospacer adjacent motif (PAM) and that is located between the transcriptional enhancer and the promoter, and (b) an RNA dependent endonuclease (RDE) which recognizes the gRNA molecule. 3. The method of embodiment 2, wherein one or more nucleotides between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.19G143900 is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 13, 14, 15, and / or 16. 4. The method of embodiments 2 or 3, wherein a nucleotide sequence of one or more enhancers to an ATG transcriptional start site of the endogenous soybean gene Glyma.19G143900 comprises SEQ ID NO: 1 or an allelic variant thereof.Docket No. P14694WO00 5. The method of embodiment 2, wherein one or more nucleotides between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.10G186500 is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 17, 18, 19, and / or 20. 6. The method of embodiments 2 or 5, wherein the nucleotide sequence of one or more enhancers to an ATG translational start site of the endogenous soybean gene Glyma.10G186500 comprises SEQ ID NO: 3 or an allelic variant thereof. 7. The method of embodiment 2, wherein one or more nucleotides between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.13G279900 is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 21, 22, 23, and / or 24. 8. The method of embodiments 2 or 7, wherein the nucleotide sequence of one or more enhancers to an ATG translational start site of the endogenous soybean gene Glyma.13G279900 comprises SEQ ID NO: 5 or an allelic variant thereof. 9. The method of embodiment 2, wherein one or more nucleotides between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.14G197100 is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 25, 26, 27, and / or 28. 10. The method of embodiments 2 or 9, wherein the nucleotide sequence of one or more enhancers to an ATG translational start site of the endogenous soybean gene Glyma.14G197100 comprises SEQ ID NO: 7 or an allelic variant thereof. 11. The method of embodiment 2, wherein one or more nucleotides between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.11G250200 is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 29, 30, 31, and / or 32.Docket No. P14694WO00 12. The method of embodiments 2 or 11, wherein the nucleotide sequence of one or more enhancers to an ATG translational start site of the endogenous soybean gene Glyma.11G250200 comprises SEQ ID NO: 9 or an allelic variant thereof. 13. The method of embodiment 2, wherein one or more nucleotides between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.17G192000 is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 33, 34, 35, and / or 36. 14. The method of embodiments 2 or 13, wherein the nucleotide sequence of one or more enhancers to an ATG translational start site of the endogenous soybean gene Glyma.17G192000 comprises SEQ ID NO: 11. 15. The method of any one of embodiments 1 to 4, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.19G143900 comprises SEQ ID NO: 2 or an allelic variant thereof. 16. The method of any one of embodiments 1, 2, 5, or 6, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.10G186500 comprises SEQ ID NO: 4 or an allelic variant thereof. 17. The method of any one of embodiments 1, 2, 7, or 8, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.13G279900 comprises SEQ ID NO: 6, SEQ ID NO: 37, or an allelic variant thereof. 18. The method of any one of embodiments 1, 2, 9, or 10, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.14G197100 comprises SEQ ID NO: 8 or an allelic variant thereof.Docket No. P14694WO00 19. The method of any one of embodiments 1, 2, 11, or 12, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.11G250200 comprises SEQ ID NO: 10 or an allelic variant thereof. 20. The method of any one of embodiments 1, 2, 13, or 14, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.17G192000 comprises SEQ ID NO: 12 or an allelic variant thereof. 21. The method of embodiment 2, wherein the gene editing agents comprise one or more transcription activator-like effector nucleases (TALENs) that cleave a DNA sequence located between the transcriptional enhancer and the promoter. 22. The method of embodiment 2, wherein the gene editing agents comprise one or more artificial zinc finger nucleases (aZFN) that cleave a DNA sequence located between the transcriptional enhancer and the promoter. 23. The method of embodiments 1 or 2, wherein the deletion of one or more nucleotides between the transcriptional enhancer and the promoter is five or more, ten or more, 50 or more, 100 or more, 250 or more, 500 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, or 4500 or more nucleotides. 24. The method of any one of embodiments 1 to 23, wherein the contacting of the gRNA and the RDE with the genome of the target soybean plant cell comprises introducing the gRNA, the RDE, a gRNA / RDE complex, a nucleic acid encoding the gRNA, and / or a nucleic acid encoding the RDE into the target soybean plant cell. 25. A modified soybean plant comprising an endogenous target soybean gene containing a non-naturally occurring deletion of one or more nucleotides located between a transcriptional enhancer and the promoter of the endogenous target soybean gene, wherein expression of the endogenous target soybean gene coding sequence is increased inDocket No. P14694WO00 comparison to expression of the endogenous target soybean gene coding sequence in a control soybean plant lacking the deletion. 26. The modified soybean plant of embodiment 25, wherein: (i) a nucleic acid sequence of the transcriptional enhancer through an ATG translational start site of the endogenous target soybean gene sequence comprises one or more of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof; (ii) the deletion of one or more nucleotides in SEQ ID NO: 1 comprises at least a 2010 bp deletion of nucleotides at positions 788 to 2792 and optionally wherein a phenotype associated with the at least 2010 bp deletion comprises root elongation and increased soybean yield relative to a wild-type or control soybean plant without the at least 2010 bp deletion; (iii) the deletion of one or more nucleotides in SEQ ID NO: 3 comprises at least a 5188 bp deletion of nucleotides at positions 1638-6825 and optionally wherein a phenotype associated with the at least 5188 bp deletion comprises an increased number of nodules, nodule weight, and / or increased soybean yield relative to a wild-type or control soybean plant without the at least 5188 bp deletion; (iv) the deletion of one or more nucleotides in SEQ ID NO: 5 comprises at least a 505 bp deletion of nucleotides at positions 615 to 1112 or at least a 538 bp deletion of nucleotides at positions 612 to 1149 and optionally wherein a phenotype associated with the at least 505 bp or 538 bp deletion comprises increased root growth, water-deficient stress tolerance, increases in the number of pods, seeds, and / or seed weight in soybean plants relative to a wild-type or control soybean plant without the at least 505 bp or 538 bp deletion; (v) the deletion of one or more nucleotides in SEQ ID NO: 7 comprises at least a 1549 bp deletion of nucleotides at positions 799-2347 and optionally wherein a phenotype associated with the at least 1549 bp deletion comprises root elongation and / or increased soybean yield relative to a wild-type or control soybean plant without the at least 1549 bp deletion; (vi) the deletion of one or more nucleotides in SEQ ID NO: 9 comprises at least a 5127 bp deletion of nucleotides at positions 1018-6144 and optionally wherein a phenotype associated with the at least 5127 bp deletion comprises an increase in plant biomass when the soybean plant is grown in water-deficient conditions and / or with herbicides relative to a wild-type or control soybean plant without the at least 5127 bp deletion; or (vii) the deletion of one or more nucleotides in SEQ ID NO: 11 comprises at least a 555 bp deletion of nucleotides at positions 550-1104 and optionally wherein a phenotype associatedDocket No. P14694WO00 with the at least 555 bp deletion comprises an increase in nitrogen deficiency tolerance, root development, and / or biomass relative to a wild-type or control soybean plant without the at least 555 bp deletion. 27. The modified soybean plant of embodiments 25 or 26, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 37, or an allelic variant thereof. 28. The modified soybean plant of embodiments 25 or 26, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene comprises at least a portion of SEQ ID NO: 2, 4, 6, 8, 10, 12, 37, or an allelic variant thereof. 29. The modified soybean plant of embodiment 25, wherein the one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene comprises, consists essentially of, or consists of five or more, ten or more, 50 or more, 100 or more, 250 or more, 500 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, or 4500 or more nucleotides. 30. The modified soybean plant of any one of embodiments 25 to 29, wherein the non- naturally occurring deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene brings the transcriptional enhancer and a promoter in closer proximity in comparison to a control soybean plant lacking the deletion. 31. The modified soybean plant of any one of embodiments 25 to 30, wherein the increase of expression of the endogenous target soybean gene results in an increase in yield of at least one of a soybean plant root length, root biomass, lateral root number, and / or nodule number relative to the control plant lacking the deletion.Docket No. P14694WO00 32. The modified soybean plant of any one of embodiments 25 to 31, wherein the increase of expression of the endogenous target soybean gene is about 2-fold to about 50-fold relative to the control plant lacking the deletion. 33. The modified soybean plant of any one of embodiments 25 to 32, wherein the increase of expression in the endogenous target soybean gene is at least about 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 22-fold, 24-fold, 26-fold, 28-fold, 30-fold, 32-fold, 34-fold, 36-fold, 38-fold, 40-fold, 42-fold, 44-fold, 46-fold, 48- fold, or 50-fold. 34. The modified soybean plant of any one of embodiments 25 to 33, with the proviso that the soybean plant is not exclusively produced by an essentially natural biological method. 35. A modified soybean plant cell containing a chromosome comprising the endogenous target soybean gene containing the non-naturally occurring deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene set forth in any one of embodiments 25 to 33. 36. The modified soybean plant cell of embodiment 35, wherein the soybean plant cell is homozygous for the endogenous target soybean gene containing the non-naturally occurring deletion. 37. A tissue culture of regenerable cells comprising the modified soybean plant cell of embodiments 35 or 36. 38. A modified soybean plant part containing a chromosome comprising the endogenous target soybean gene containing the non-naturally occurring deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene set forth in any one of embodiments 25 to 34. 39. The modified soybean plant part of embodiment 38, wherein the plant part is a leaf, stem, root, pod, or seed.Docket No. P14694WO00 40. The modified soybean plant part of embodiment 39, wherein the plant part is a soybean seed that comprises an elite soybean germplasm, a soybean cultivar, a soybean variety, and / or is homozygous for the endogenous target soybean gene containing deletion of the one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene. 41. The modified soybean plant part of embodiments 39 or 40, wherein the soybean seed is heterozygous for the endogenous target soybean gene containing the deletion of the one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene. 42. A modified soybean seed lot comprising the seed of any one of embodiments 39 to 41. 43. The modified soybean plant of any one of embodiments 25 to 34, soybean plant cell of embodiments 35 or 36, or the soybean plant part of any of embodiments 38 to 41, further comprising at least one mutation in the soybean RIC1 gene, RIC2 gene, FT1a gene, JAG1 gene, BS1 gene, BS2 gene, Tfl1b gene, NF-YC4 gene, endogenous AIP10a gene promoter, and / or endogenous AIP10b gene promoter. 44. The modified soybean plant of any one of embodiments 25 to 34, soybean plant cell of any of embodiments 35 or 36, or the soybean plant part of any of embodiments 38 to 41, further comprising one or more transgenes, optionally wherein the transgenes encode proteins or RNAs conferring herbicide tolerance or pest tolerance. 45. The modified soybean plant of any one of embodiments 25 to 34, soybean plant cell of any of embodiments 35 or 36, or the soybean plant part of any of embodiments 38 to 41, further comprising an A2704-12, A5547-127, BPS-CV127-9, DAS44406-6, DAS68416-4, DAS81419-2, DP305423, GTS 40- 3-2, HOS, A5547-127, MON87701, MON87705, MON87708, MON87769, MON89788, MON98788, MST-FG072-3, or SYHT0H210 transgenic event or modification thereof.Docket No. P14694WO00 46. A method of soybean seed production comprising crossing the modified soybean plant of any one of embodiments 25 to 34 with a second soybean plant to produce soybean seed and optionally harvesting the seed. 47. A method of soybean seed production comprising allowing the modified soybean plant of any one of embodiments 25 to 34 to self-pollinate to produce plant seed and optionally harvesting the seed. 48. A method of producing a soybean seed lot comprising: (i) growing a population of soybean plants comprising the soybean plant of any one of embodiments 25 to 34; and (ii) harvesting seed from the population of soybean plants of step (i) at maturity, thereby producing the soybean seed lot. 49. A method for producing a soybean by-product comprising at least one processing step of cleaning, cracking, flaking, crushing, macerating, pressing, extracting, expelling, and / or extruding the seed lot of embodiment 42. 50. The method of embodiment 49, wherein the by-product is soybean protein and wherein the soybean seed lot is subjected to processing steps comprising: (i) at least one of a cracking, flaking, crushing, pressing, and / or macerating step; (ii) extracting the cracked, flaked, crushed, pressed, and / or macerated soybean seed product from step (i) with an organic solvent to produce defatted soymeal; and (iii) extracting the defatted soymeal from step (ii) with an aqueous solvent to produce an aqueous fraction comprising soybean protein. 51. The method of embodiment 50, wherein the by-product is soybean oil and wherein the soybean seed lot is subjected to processing steps comprising: (i) at least one of a cracking, flaking, crushing, pressing, and / or macerating step; and (ii) solvent extracting, expelling, and / or extruding step the cracked, flaked, crushed, pressed, and / or macerated soybean seed product from step (i) to produce the oil. 52. A method of producing a commodity soybean plant product, the method comprising processing a modified soybean seed obtained from the modified soybean plant of any one ofDocket No. P14694WO00 embodiments 25 to 34 and recovering the commodity plant product from the processed plant or seed. 53. The method of embodiment 52, wherein the commodity plant product is seed meal, starch, silage, oil, or protein. 54. The method of embodiments 52 or 53, wherein the commodity plant product comprises a detectable amount of a DNA molecule comprising the endogenous target soybean gene or a fragment thereof containing the deletion of the one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene. 55. A method of producing soybean plant material, the method comprising: (a) providing the modified soybean plant of any one of embodiments 25 to 34; and, (b) growing the modified soybean plant under conditions that allow for expression of the endogenous target soybean gene at levels that are increased compared to expression levels of the endogenous target soybean gene in a reference soybean plant which lacks the deletions. 56. The method of embodiment 55, wherein growing the modified soybean plant further comprises at least one of sowing a soybean seed which germinates and forms the soybean plant, irrigating the soybean seed or plant, and / or treating the soybean plant or the soybean seed with a biological agent, herbicide, insecticide, or fungicide. 57. The method of embodiments 55 or 56, wherein the soybean plant material comprises a seed, optionally wherein the method further comprises harvesting the seed from the plant. 58. A method for generating a modified soybean plant, plant cell, or plant part comprising: (i) directing both: (a) a guide RNA (gRNA) molecule which targets the nucleic acid sequence of the transcriptional enhancer through the ATG translational start site of the endogenous target soybean gene sequence comprising one or more of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof and (b) an RNA dependent endonuclease (RDE) which recognizes the gRNA molecule, to the genome of a target soybean plant cell; andDocket No. P14694WO00 (ii) isolating a modified soybean plant cell, modified soybean plant part, or modified soybean plant comprising at a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequence of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof. 59. The method of embodiment 58, wherein nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by any of SEQ ID NO: 13-36. 60. The method of embodiments 58 or 59, wherein the directing of the gRNA and the RDE to the genome of the target soybean plant cell comprises introducing the gRNA, the RDE, a gRNA / RDE complex, a nucleic acid encoding the gRNA, and / or a nucleic acid encoding the RDE into the target soybean plant cell. 61. The method of any one of embodiments 25 to 41, wherein the soybean plant cell, soybean plant part, or soybean plant comprising the endogenous gene containing the deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene is identified by: (i) analyzing a polynucleotide comprising at least a portion of SEQ ID NO: 1, 3, 5, 7, 9 and / or 11 or an allelic variant thereof in one or more candidate plant cells, plant parts, or plants; and / or (ii) analyzing yield of the soybean plant part, soybean plant, or soybean plant grown from the soybean plant cell, wherein the yield comprises least one of a soybean plant root length, root biomass, lateral root number, and / or nodule number in comparison to a wild-type or control soybean plant lacking the deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene, and wherein an increase in the yield indicates that the soybean plant cell, soybean plant part, or soybean plant comprises the at least one deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene. 62. A soybean plant made by the method of any one of embodiments 1 to 24.Docket No. P14694WO00 63. Use of the modified soybean plant of any one of embodiments 25 to 34 or 62, seed obtained therefrom, or a seed of any of embodiments 38 to 41 to: (a) grow a soybean plant with improved yield of at least one of a soybean plant root length, root biomass, lateral root number, and / or nodule number in comparison to a soybean plant lacking the deletion; (b) harvest soybean plant seed comprising the endogenous gene containing the deletion; (c) produce a commodity product; or (d) breed soybean plants with improved yield of at least one of a soybean plant root length, root biomass, lateral root number, and / or nodule number in comparison to a soybean plant lacking the deletion. 64. Use of the modified soybean plant cell of any one of embodiments 35 to 37, the modified soybean plant part of any of embodiments 38 to 41, the modified soybean seed lot of embodiment 42, or the modified soybean plant of any of embodiments 25 to 34 or 62 to produce a modified soybean seed. 65. Use of the modified soybean plant cell of any one of embodiments 35 to 37, the modified soybean plant part of any of embodiments 38 to 41, the modified soybean seed lot of embodiment 42, or the modified soybean plant of any of embodiments 25 to 34 or 62 to grow a modified soybean crop. 66. Use of the soybean plant cell of any one of embodiments 35 to 37, the modified soybean plant part of any of embodiments 38 to 41, the modified soybean seed lot of embodiment 42, or the modified soybean plant of any of embodiments 25 to 34 or 62 to obtain a soybean by-product. 67. The use of embodiment 66, wherein the soybean by-product comprises soybean flour, meal, protein, oil, syrup, or starch. 68. A DNA molecule comprising at least a portion of an endogenous target soybean gene containing the deletion of the one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene set forth in any one of embodiments 1 to 24.Docket No. P14694WO00 69. The DNA molecule of embodiment 68, wherein the DNA molecule comprises, consists essentially of, or consists of at least the portion of the nucleic acid sequence of the transcriptional enhancer through an ATG translational start site of the endogenous target soybean gene sequence of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof. 70. The DNA molecule of embodiment 69, wherein the at least a portion of the nucleic acid sequence of the transcriptional enhancer through an ATG translational start site of the endogenous target soybean gene sequence of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11, or an allelic variant thereof, has one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene. 71. The DNA molecule of embodiment 70, wherein the DNA molecule comprises, consists essentially of, or consists of at least the portion of the nucleic acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 37, or an allelic variant thereof containing the deletion of the one or more nucleotides. 72. Soybean seed meal comprising the DNA molecule of any one of embodiments 68 to 71, optionally wherein the seed meal is non-regenerable. 73. A biological sample comprising the DNA molecule of any one of embodiments 68 to 71. 74. A method of identifying a biological sample comprising the DNA molecule containing the deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene set forth in any one of embodiments 68 to 71, comprising the step of detecting the presence of the DNA molecule in the biological sample. 75. A guide RNA molecule comprising a spacer RNA molecule which hybridizes to nucleotides located between a transcriptional enhancer and a promoter of an endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11, or an allelic variant thereof, optionally wherein the spacer RNA molecule comprises the RNA encoded by any one of SEQ ID NO: 13-36.Docket No. P14694WO00 76. The guide RNA molecule of embodiment 75, further comprising a Cas12 direct repeat element which is operably linked to the spacer RNA. 77. A gene editing system comprising a CRISPR-Cas effector protein and a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that hybridizes to a nucleic acid sequence of a transcriptional enhancer through an ATG translational start site of an endogenous target soybean gene sequence comprising one or more of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof. 78. An expression cassette comprising a polynucleotide encoding the guide RNA molecule of embodiment 75 or 76. 79. A soybean genome characterized by the fact that it comprises the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 which contains a non-natural mutation. 80. The soybean genome of embodiment 79, wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 optionally wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least five or more, ten or more, 50 or more, 100 or more, 250 or more, 500 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, or 4500 or more nucleotides SEQ ID NO: 1, 3, 5, 7, 9, and / or 11. 81. A soybean plant cell obtainable by the process comprising introducing a deletion in an endogenous target soybean gene with a Type V Cas nuclease and at least two guide RNA (gRNA) molecules comprising a direct repeat RNA molecule bound by the Cas nuclease and a spacer RNA molecule which hybridizes to nucleotides located between a transcriptional enhancer and a promoter of the endogenous target soybean gene, wherein: (a) the endogenous soybean gene Glyma.19G143900 is targeted with one or more gRNA(s) comprising a spacer is encoded by SEQ ID NOs: 13 and / or 14; and one or more gRNA(s) comprising a spacer encoded by SEQ ID NOs: 15 and / or 16;Docket No. P14694WO00 (b) the endogenous soybean gene Glyma.10G186500 is targeted with one or more gRNA(s) comprising a spacer is encoded by SEQ ID NOs: 17 and / or 18; and one or more gRNA(s) comprising a spacer encoded by SEQ ID NOs: 19 and / or 20; (c) the endogenous soybean gene Glyma.13G279900 is targeted with one or more gRNA(s) comprising a spacer is encoded by SEQ ID NOs: 21 and / or 22; and one or more gRNA(s) comprising a spacer encoded by SEQ ID NOs: 23 and / or 24; (d) the endogenous soybean gene Glyma.14G197100 is targeted with one or more gRNA(s) comprising a spacer is encoded by SEQ ID NOs: 25 and / or 26; and one or more gRNA(s) comprising a spacer encoded by SEQ ID NOs: 27 and / or 28; (e) the endogenous soybean gene Glyma.11G250200 is targeted with one or more gRNA(s) comprising a spacer is encoded by SEQ ID NOs: 29 and / or 30; and one or more gRNA(s) comprising a spacer encoded by SEQ ID NOs: 31 and / or 32; or (f) the endogenous soybean gene Glyma.17G192000 is targeted with one or more gRNA(s) comprising a spacer is encoded by SEQ ID NOs: 33 and / or 34; and one or more gRNA(s) comprising a spacer encoded by SEQ ID NOs: 35 and / or 36. 82. The soybean plant cell of embodiment 81, wherein the soybean call containing the deletion is selected. 83. A soybean plant or plant part obtained from the soybean plant cell of embodiments 81 or 82. EXAMPLES Example 1. Generation of soybean with mutated GmPTF1 upstream region

[0088] Using genomics features, a putative transcriptional enhancer was identified in the upstream region of Glyma.19G143900, ending about 3.6 kb upstream of the predicted start codon of the coding sequence (SEQ ID NO: 1, Fig.1). gRNAs were designed to make a large deletion between the end of one or more enhancers and the core promoter (SEQ ID NO: 13- 16).

[0089] A vector encoding Cas nuclease, four gRNAs (two on either side of the expected deletion), and the visible fluorescent marker mScarlet is constructed and used to generate transgenic soybean hairy roots on composite plants. In brief, the root of five-day old soybean seedlings is removed, and the resulting explants (shoot) are dipped a bacterial suspension ofDocket No. P14694WO00 Agrobacterium rhizogenes carrying the vector described above. Seedlings are then placed into rolls of germination paper a sterile bucket with sterile nutrient solution, and plants are grown in a Conviron plant growth cabinet (26°C / 16h and 22°C / 8h). After a week, the explants are transferred into new germination paper rolls and supplied with fresh medium. Transgenic roots, identified through fluorescence, are then collected around 3 weeks post-transformation for DNA and RNA extraction. After confirmation of the presence of the intended deletion (Fig.1) by PCR, expression of Glyma.19G143900 is measured by qRT-PCR, and compared to expression in transgenic roots of control plants transformed with a construct encoding mScarlet, Cas and an unrelated gRNA, and to expression in non-transgenic hairy roots.

[0090] Identification of soybean plants containing the deletion between the transcriptional enhancer and the promoter of the Glyma.19G143900 gene can include identifying plants with phenotypic changes consistent with upregulation of the Glyma.19G143900 gene. For example, it is predicted that upregulation of the Glyma.19G143900 gene by the deletions disclosed herein will result in phenotype changes in root architecture such as root elongation and / or increased soybean yield in the field (e.g., through promotion of root growth and phosphorus uptake) relative to wild-type or control soybean plants lacking the deletions. Example 2. Generation of soybean with mutated GmPT7 upstream region

[0091] Using genomics features, a putative transcriptional enhancer was identified in the upstream region of Glyma.10G186500 WT, ending about 7.6 kb upstream of the predicted start codon of the coding sequence (SEQ ID NO: 3, Fig.2). gRNAs were designed to make a large deletion between the end of one or more enhancers and the core promoter (SEQ ID NO: 17- 20).

[0092] A vector encoding Cas nuclease, four gRNAs (two on either side of the expected deletion), and the visible fluorescent marker mScarlet is constructed and used to generate transgenic soybean hairy roots on composite plants. In brief, the root of five-day old soybean seedlings is removed, and the resulting explants (shoot) are dipped a bacterial suspension of Agrobacterium rhizogenes carrying the vector described above. Seedlings are then placed into rolls of germination paper a sterile bucket with sterile nutrient solution, and plants are grown in a Conviron plant growth cabinet (26°C / 16h and 22°C / 8h). After a week, the explants are transferred into new germination paper rolls and supplied with fresh medium. Transgenic roots, identified through fluorescence, are then collected around 3 weeks post-transformation for DNA and RNA extraction. After confirmation of the presence of the intended deletion (Fig.2)Docket No. P14694WO00 by PCR, expression of Glyma.10G186500 is measured by qRT-PCR, and compared to expression in transgenic roots of control plants transformed with a construct encoding mScarlet, Cas and an unrelated gRNA, and to expression in non-transgenic hairy roots.

[0093] Identification of soybean plants containing the deletion between the transcriptional enhancer and the promoter of the Glyma.10G186500 gene can include identifying plants with phenotypic changes consistent with upregulation of the Glyma.10G186500 gene. For example, it is predicted that upregulation of the Glyma.10G186500 gene by the deletions disclosed herein will result in phenotype changes in nodules including increases in the number of nodules, nodule weight, nitrogenase activity of nodules and / or increased soybean yield in the field through increased nitrogen fixation in nodules relative to wild-type or control soybean plants lacking the deletions. Example 3. Generation of soybean with mutated GmNAC19 upstream region

[0094] Using genomics features, a putative transcriptional enhancer was identified in the upstream region of Glyma.13G279900, ending about 1.1 kb upstream of the predicted start codon of the coding sequence (SEQ ID NO: 5, Fig.3A). gRNAs were designed to make a large deletion between the end of one or more enhancers and the core promoter (SEQ ID NO: 21- 24).

[0095] A vector encoding Cas nuclease, four gRNAs (two on either side of the expected deletion), and the visible fluorescent marker mScarlet is constructed and used to generate transgenic soybean hairy roots on composite plants. In brief, the root of five-day old soybean seedlings is removed, and the resulting explants (shoot) are dipped a bacterial suspension of Agrobacterium rhizogenes carrying the vector described above. Seedlings are then placed into rolls of germination paper a sterile bucket with sterile nutrient solution, and plants are grown in a Conviron plant growth cabinet (26°C / 16h and 22°C / 8h). After a week, the explants are transferred into new germination paper rolls and supplied with fresh medium. Transgenic roots, identified through fluorescence, are then collected around 3 weeks post-transformation for DNA and RNA extraction. After confirmation of the presence of the intended deletion (Fig. 3A) by PCR, expression of Glyma.13G279900 is measured by qRT-PCR, and compared to expression in transgenic roots of control plants transformed with a construct encoding mScarlet, Cas and an unrelated gRNA, and to expression in non-transgenic hairy roots.

[0096] Identification of soybean plants containing the deletion between the transcriptional enhancer and the promoter of the Glyma.13G279900 gene can include identifying plants withDocket No. P14694WO00 phenotypic changes consistent with upregulation of the Glyma.13G279900 gene. For example, it is predicted that upregulation of the Glyma.13G279900 gene by the deletions disclosed herein will result in phenotype changes in root growth including increased root growth, water- deficient stress tolerance and increases in the number of pods, seeds, and / or seed weight in soybean plants relative to wild-type or control soybean plants lacking the deletions. Example 4. Generation of soybean with mutated GmETO1 upstream region

[0097] Using genomics features, a putative transcriptional enhancer was identified in the upstream region of Glyma.14G197100, ending about 1.9kb upstream of the predicted start codon of the coding sequence (SEQ ID NO: 7, Fig.4). gRNAs were designed to make a large deletion between the end of one or more enhancers and the core promoter (SEQ ID NO: 25- 28).

[0098] A vector encoding Cas nuclease, four gRNAs (two on either side of the expected deletion), and the visible fluorescent marker mScarlet is constructed and used to generate transgenic soybean hairy roots on composite plants. In brief, the root of five-day old soybean seedlings is removed, and the resulting explants (shoot) are dipped a bacterial suspension of Agrobacterium rhizogenes carrying the vector described above. Seedlings are then placed into rolls of germination paper a sterile bucket with sterile nutrient solution, and plants are grown in a Conviron plant growth cabinet (26°C / 16h and 22°C / 8h). After a week, the explants are transferred into new germination paper rolls and supplied with fresh medium. Transgenic roots, identified through fluorescence, are then collected around 3 weeks post-transformation for DNA and RNA extraction. After confirmation of the presence of the intended deletion (Fig.4) by PCR, expression of Glyma.14G197100 is measured by qRT-PCR, and compared to expression in transgenic roots of control plants transformed with a construct encoding mScarlet, Cas and an unrelated gRNA, and to expression in non-transgenic hairy roots.

[0099] Identification of soybean plants containing the deletion between the transcriptional enhancer and the promoter of the Glyma.14G197100 gene can include identifying plants with phenotypic changes consistent with upregulation of the Glyma.14G197100 gene. For example, it is predicted that upregulation of the Glyma.14G197100 gene by the deletions disclosed herein will result in significantly enhanced inorganic phosphorus (Pi) deficiency tolerance, Pi uptake, Piuse efficiency, and / or increased root proliferation relative to wild-type or control soybean plants lacking the deletions.Docket No. P14694WO00 Example 5. Generation of soybean with mutated GmCYP78A71 upstream region

[0100] Using genomics features, a putative transcriptional enhancer was identified in the upstream region of Glyma.11G250200, ending about 5.5 kb upstream of the predicted start codon of the coding sequence (SEQ ID NO: 9, Fig.5). gRNAs were designed to make a large deletion between the end of one or more enhancers and the core promoter (SEQ ID NO: 29- 32).

[0101] A vector encoding Cas nuclease, four gRNAs (two on either side of the expected deletion), and the visible fluorescent marker mScarlet is constructed and used to generate transgenic soybean hairy roots on composite plants. In brief, the root of five-day old soybean seedlings is removed, and the resulting explants (shoot) are dipped a bacterial suspension of Agrobacterium rhizogenes carrying the vector described above. Seedlings are then placed into rolls of germination paper a sterile bucket with sterile nutrient solution, and plants are grown in a Conviron plant growth cabinet (26°C / 16h and 22°C / 8h). After a week, the explants are transferred into new germination paper rolls and supplied with fresh medium. Transgenic roots, identified through fluorescence, are then collected around 3 weeks post-transformation for DNA and RNA extraction. After confirmation of the presence of the intended deletion (Fig.5) by PCR, expression of Glyma.11G250200 is measured by qRT-PCR, and compared to expression in transgenic roots of control plants transformed with a construct encoding mScarlet, Cas and an unrelated gRNA, and to expression in non-transgenic hairy roots.

[0102] Identification of soybean plants containing the deletion between the transcriptional enhancer and the promoter of the Glyma.11G250200 gene can include identifying plants with phenotypic changes consistent with upregulation of the Glyma.11G250200 gene. For example, it is predicted that upregulation of the Glyma.11G250200 gene by the deletions disclosed herein will result in: (i) improved plant stress responses including detoxification of xenobiotics and drought stress tolerance; and / or (ii)an increase in plant biomass may be observed in plants overexpressing Glyma.11G250200 when grown in water-deficient conditions and / or with herbicides. Example 6. Generation of soybean with mutated GmAAP6a upstream region

[0103] Using genomics features, a putative transcriptional enhancer was identified in the upstream region of Glyma.17G192000, ending about 1.0kb upstream of the predicted start codon of the coding sequence (SEQ ID NO: 11, Fig.6). gRNAs were designed to make a largeDocket No. P14694WO00 deletion between the end of one or more enhancers and the core promoter (SEQ ID NO: 33- 36).

[0104] A vector encoding Cas nuclease, four gRNAs (two on either side of the expected deletion), and the visible fluorescent marker mScarlet is constructed and used to generate transgenic soybean hairy roots on composite plants. In brief, the root of five-day old soybean seedlings is removed, and the resulting explants (shoot) are dipped a bacterial suspension of Agrobacterium rhizogenes carrying the vector described above. Seedlings are then placed into rolls of germination paper a sterile bucket with sterile nutrient solution, and plants are grown in a Conviron plant growth cabinet (26°C / 16h and 22°C / 8h). After a week, the explants are transferred into new germination paper rolls and supplied with fresh medium. Transgenic roots, identified through fluorescence, are then collected around 3 weeks post-transformation for DNA and RNA extraction. After confirmation of the presence of the intended deletion (Fig.6) by PCR, expression of Glyma.17G192000 is measured by qRT-PCR, and compared to expression in transgenic roots of control plants transformed with a construct encoding mScarlet, Cas and an unrelated gRNA, and to expression in non-transgenic hairy roots.

[0105] Identification of soybean plants containing the deletion between the transcriptional enhancer and the promoter of the Glyma.17G192000 gene can include identifying plants with phenotypic changes consistent with upregulation of the Glyma.17G192000 gene. For example, it is predicted that upregulation of the Glyma.17G192000 gene in soybean by the deletions disclosed herein will result in significantly enhanced nitrogen deficiency tolerance, more developed roots, and / or a higher biomass than wild-type or control soybean plants. Example 7. Enhanced reporter expression in vectors with a deletion between a GmNAC19 transcriptional enhancer and the GmNAC19 promoter

[0106] A dual luciferase assay was designed to measure the effect of deletions in the upstream region of the GmNAC19 (Glyma.13G279900) soybean gene on expression of fused genes. In this assay plasmid vector DNA molecules containing expression cassettes for both firefly luciferase (FLUC) and Renilla luciferase (RLUC) were delivered to soybean protoplasts. A variant GmNAC19 gene upstream region set forth in SEQ ID NO: 37 and Fig.3A comprising a 538 bp deletion between one or more enhancers and the promoter was fused to the FLUC reporter gene in one vector. A wild-type GmNAC19 upstream region was fused to the FLUC reporter gene in another vector. A control promoter was fused to the RLUC promoter in the other expression cassette. FLUC was thus used to measure differences in the experimentalDocket No. P14694WO00 conditions (i.e., the wild-type versus the variant GmNAC19 gene upstream region) and RLUC was used to normalize FLUC accounting for transfection efficiency, cell number, and other variables in the reaction (FLUC / RLUC). The Glyma.13G279900 upstream region (SEQ ID NO: 5) and the variant Glyma.13G279900 upstream region (SEQ ID NO: 37) were both cloned upstream of FLUC while RLUC remained constant (i.e., was fused to the same control promoter). The vectors were transfected into the soybean protoplasts and after 2 days, the Promega Dual-Glo kit was used to read out the magnitude of luminescence caused by FLUC and RLUC interacting with their respective substrates. After readout, the Glyma.13G279900 variant upstream region (Fig.3B right column, SEQ ID NO: 37) comprising a 538 bp deletion between one or more enhancers and the promoter had a higher FLUC / RLUC value, meaning it increased expression of FLUC compared to the wild-type upstream region (Fig.3B left column, SEQ ID NO: 5).

[0107] All cited patents and patent publications referred to in this application are incorporated herein by reference in their entirety. All of the materials and methods disclosed and claimed herein can be made and used without undue experimentation as instructed by the above disclosure and illustrated by the examples. Although the materials and methods of this disclosure have been described in terms of embodiments and illustrative examples, it will be apparent to those of skill in the art that substitutions and variations can be applied to the materials and methods described herein without departing from the concept, spirit, and scope of the disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the disclosure as encompassed by the embodiments of the disclosures recited herein and the specification and appended claims.

Claims

Docket No. P14694WO00 CLAIMS What is claimed is:

1. A method for upregulating expression of a target soybean gene in a soybean plant and / or a soybean plant cell, the method comprising: identifying a transcriptional enhancer and a promoter in an endogenous target soybean gene in a soybean genome, wherein the transcriptional enhancer is 5´ to the target soybean gene; contacting the soybean genome with gene editing agents to produce a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene; and isolating the soybean plant and / or the soybean plant cell comprising endogenous target soybean gene containing the deletion.

2. The method of claim 1, wherein the gene editing agents comprise: (a) one or more guide RNA (gRNA) molecules that can hybridize to a nucleotide sequence that is adjacent to a protospacer adjacent motif (PAM) and that is located between the transcriptional enhancer and the promoter, and (b) an RNA dependent endonuclease (RDE) which recognizes the gRNA molecule.

3. The method of claim 2, wherein one or more nucleotides between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.19G143900 is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 13, 14, 15, and / or 16.

4. The method of claims 2 or 3, wherein a nucleotide sequence of one or more enhancers to an ATG transcriptional start site of the endogenous soybean gene Glyma.19G143900 comprises SEQ ID NO: 1 or an allelic variant thereof.

5. The method of claim 2, wherein one or more nucleotides between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.10G186500 is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 17, 18, 19, and / or 20.Docket No. P14694WO00 6. The method of claims 2 or 5, wherein the nucleotide sequence of one or more enhancers to an ATG translational start site of the endogenous soybean gene Glyma.10G186500 comprises SEQ ID NO: 3 or an allelic variant thereof.

7. The method of claim 2, wherein one or more nucleotides between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.13G279900 is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 21, 22, 23, and / or 24.

8. The method of claims 2 or 7, wherein the nucleotide sequence of one or more enhancers to an ATG translational start site of the endogenous soybean gene Glyma.13G279900 comprises SEQ ID NO: 5 or an allelic variant thereof.

9. The method of claim 2, wherein one or more nucleotides between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.14G197100 is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 25, 26, 27, and / or 28.

10. The method of claims 2 or 9, wherein the nucleotide sequence of one or more enhancers to an ATG translational start site of the endogenous soybean gene Glyma.14G197100 comprises SEQ ID NO: 7 or an allelic variant thereof.

11. The method of claim 2, wherein one or more nucleotides between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.11G250200 is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 29, 30, 31, and / or 32.

12. The method of claims 2 or 11, wherein the nucleotide sequence of one or more enhancers to an ATG translational start site of the endogenous soybean gene Glyma.11G250200 comprises SEQ ID NO: 9 or an allelic variant thereof.Docket No. P14694WO00 13. The method of claim 2, wherein one or more nucleotides between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.17G192000 is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by SEQ ID NO: 33, 34, 35, and / or 36.

14. The method of claims 2 or 13, wherein the nucleotide sequence of one or more enhancers to an ATG translational start site of the endogenous soybean gene Glyma.17G192000 comprises SEQ ID NO:

11.

15. The method of claims 3 or 4, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of an endogenous soybean gene Glyma.19G143900 comprises SEQ ID NO: 2 or an allelic variant thereof.

16. The method of claims 5 or 6, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.10G186500 comprises SEQ ID NO: 4 or an allelic variant thereof.

17. The method of claims 7 or 8, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.13G279900 comprises SEQ ID NO: 6, SEQ ID NO: 37, or an allelic variant thereof.

18. The method of claims 9 or 10, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.14G197100 comprises SEQ ID NO: 8 or an allelic variant thereof.

19. The method of claims 11 or 12, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.11G250200 comprises SEQ ID NO: 10 or an allelic variant thereof.Docket No. P14694WO00 20. The method of claims 13 or 14, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene Glyma.17G192000 comprises SEQ ID NO: 12 or an allelic variant thereof.

21. The method of claim 2, wherein the gene editing agents comprise one or more transcription activator-like effector nucleases (TALENs) that cleave a DNA sequence located between the transcriptional enhancer and the promoter.

22. The method of claim 2, wherein the gene editing agents comprise one or more artificial zinc finger nucleases (aZFN) that cleave a DNA sequence located between the transcriptional enhancer and the promoter.

23. The method of claims 1 or 2, wherein the deletion of one or more nucleotides between the transcriptional enhancer and the promoter is five or more, ten or more, 50 or more, 100 or more, 250 or more, 500 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, or 4500 or more nucleotides.

24. The method of claims 1 or 2, wherein the contacting of the gRNA and the RDE with the genome of the target soybean plant cell comprises introducing the gRNA, the RDE, a gRNA / RDE complex, a nucleic acid encoding the gRNA, and / or a nucleic acid encoding the RDE into the target soybean plant cell.

25. A modified soybean plant comprising an endogenous target soybean gene containing a non-naturally occurring deletion of one or more nucleotides located between a transcriptional enhancer and the promoter of the endogenous target soybean gene, wherein expression of the endogenous target soybean gene coding sequence is increased in comparison to expression of the endogenous target soybean gene coding sequence in a control soybean plant lacking the deletion.Docket No. P14694WO00 26. The modified soybean plant of claim 25, wherein: (i) a nucleic acid sequence of the transcriptional enhancer through an ATG translational start site of the endogenous target soybean gene sequence comprises one or more of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof; (ii) the deletion of one or more nucleotides in SEQ ID NO: 1 comprises at least a 2010 bp deletion of nucleotides at positions 788 to 2792 and optionally wherein a phenotype associated with the at least 2010 bp deletion comprises root elongation and increased soybean yield relative to a wild-type or control soybean plant without the at least 2010 bp deletion; (iii) the deletion of one or more nucleotides in SEQ ID NO: 3 comprises at least a 5188 bp deletion of nucleotides at positions 1638-6825 and optionally wherein a phenotype associated with the at least 5188 bp deletion comprises an increased number of nodules, nodule weight, and / or increased soybean yield relative to a wild-type or control soybean plant without the at least 5188 bp deletion; (iv) the deletion of one or more nucleotides in SEQ ID NO: 5 comprises at least a 505 bp deletion of nucleotides at positions 615 to 1112 or at least a 538 bp deletion of nucleotides at positions 612 to 1149 and optionally wherein a phenotype associated with the at least 505 bp or 538 bp deletion comprises increased root growth, water-deficient stress tolerance, increases in the number of pods, seeds, and / or seed weight in soybean plants relative to a wild-type or control soybean plant without the at least 505 bp or 538 bp deletion; (v) the deletion of one or more nucleotides in SEQ ID NO: 7 comprises at least a 1549 bp deletion of nucleotides at positions 799-2347 and optionally wherein a phenotype associated with the at least 1549 bp deletion comprises root elongation and / or increased soybean yield relative to a wild-type or control soybean plant without the at least 1549 bp deletion; (vi) the deletion of one or more nucleotides in SEQ ID NO: 9 comprises at least a 5127 bp deletion of nucleotides at positions 1018-6144 and optionally wherein a phenotype associated with the at least 5127 bp deletion comprises an increase in plant biomass when the soybean plant is grown in water-deficient conditions and / or with herbicides relative to a wild-type or control soybean plant without the at least 5127 bp deletion; or (vii) the deletion of one or more nucleotides in SEQ ID NO: 11 comprises at least a 555 bp deletion of nucleotides at positions 550-1104 and optionally wherein a phenotype associated with the at least 555 bp deletion comprises an increase in nitrogen deficiency tolerance, root development, and / or biomass relative to a wild-type or control soybean plant without the at least 555 bp deletion.Docket No. P14694WO00 27. The modified soybean plant of claims 25 or 26, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 37, or an allelic variant thereof.

28. The modified soybean plant of claims 25 or 26, wherein the endogenous target soybean gene containing one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene comprises at least a portion of SEQ ID NO: 2, 4, 6, 8, 10, 12, 37, or an allelic variant thereof.

29. The modified soybean plant of claim 25, wherein the one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene comprises, consists essentially of, or consists of five or more, ten or more, 50 or more, 100 or more, 250 or more, 500 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, or 4500 or more nucleotides.

30. The modified soybean plant of claims 25 or 26, wherein the non-naturally occurring deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene brings the transcriptional enhancer and a promoter in closer proximity in comparison to a control soybean plant lacking the deletion.

31. The modified soybean plant of claims 25 or 26, wherein the increase of expression of the endogenous target soybean gene results in an increase in yield of at least one of a soybean plant root length, root biomass, lateral root number, and / or nodule number relative to the control plant lacking the deletion.

32. The modified soybean plant of claims 25 or 26, wherein the increase of expression of the endogenous target soybean gene is about 2-fold to about 50-fold relative to the control plant lacking the deletion.

33. The modified soybean plant of claims 25 or 32, wherein the increase of expression in the endogenous target soybean gene is at least about 2-fold, 4-fold, 6-fold, 8-fold, 10-fold,Docket No. P14694WO00 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, 22-fold, 24-fold, 26-fold, 28-fold, 30-fold, 32- fold, 34-fold, 36-fold, 38-fold, 40-fold, 42-fold, 44-fold, 46-fold, 48-fold, or 50-fold.

34. The modified soybean plant of claims 25 or 26, with the proviso that the soybean plant is not exclusively produced by an essentially natural biological method.

35. A modified soybean plant cell containing a chromosome comprising the endogenous target soybean gene containing the non-naturally occurring deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene set forth in claims 25 or 26.

36. The modified soybean plant cell of claim 35, wherein the soybean plant cell is homozygous for the endogenous target soybean gene containing the non-naturally occurring deletion.

37. A tissue culture of regenerable cells comprising the modified soybean plant cell of claims 35 or 36.

38. A modified soybean plant part containing a chromosome comprising the endogenous target soybean gene containing the non-naturally occurring deletion of one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene set forth in claims 25 or 26.

39. The modified soybean plant part of claim 38, wherein the plant part is a leaf, stem, root, pod, or seed.

40. The modified soybean plant part of claim 39, wherein the plant part is a soybean seed that comprises an elite soybean germplasm, a soybean cultivar, a soybean variety, and / or is homozygous for the endogenous target soybean gene containing deletion of the one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene.Docket No. P14694WO00 41. The modified soybean plant part of claims 39 or 40, wherein the soybean seed is heterozygous for the endogenous target soybean gene containing the deletion of the one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene.

42. A modified soybean seed lot comprising the seed of claims 40 or 41.

43. The modified soybean plant of claims 25 or 35, further comprising at least one mutation in the soybean RIC1 gene, RIC2 gene, FT1a gene, JAG1 gene, BS1 gene, BS2 gene, Tfl1b gene, NF-YC4 gene, endogenous AIP10a gene promoter, and / or endogenous AIP10b gene promoter.

44. The modified soybean plant of claims 25 or 35, further comprising one or more transgenes, optionally wherein the transgenes encode proteins or RNAs conferring herbicide tolerance or pest tolerance.

45. The modified soybean plant of claims 25 or 35, further comprising an A2704-12, A5547-127, BPS-CV127-9, DAS44406-6, DAS68416-4, DAS81419-2, DP305423, GTS 40- 3-2, HOS, A5547-127, MON87701, MON87705, MON87708, MON87769, MON89788, MON98788, MST-FG072-3, or SYHT0H210 transgenic event or modification thereof.

46. A method of soybean seed production comprising crossing the modified soybean plant of claims 25 or 26, with a second soybean plant to produce soybean seed and optionally harvesting the seed.

47. A method of soybean seed production comprising allowing the modified soybean plant of claims 25 or 26 to self-pollinate to produce plant seed and optionally harvesting the seed.

48. A method of producing a soybean seed lot comprising: (i) growing a population of soybean plants comprising the soybean plant of claims 25 or 26; and (ii) harvesting seed from the population of soybean plants of step (i) at maturity, thereby producing the soybean seed lot.Docket No. P14694WO00 49. A method for producing a soybean by-product comprising at least one processing step of cleaning, cracking, flaking, crushing, macerating, pressing, extracting, expelling, and / or extruding the seed lot of claim 42.

50. The method of claim 49, wherein the by-product is soybean protein and wherein the soybean seed lot is subjected to processing steps comprising: (i) at least one of a cracking, flaking, crushing, pressing, and / or macerating step; (ii) extracting the cracked, flaked, crushed, pressed, and / or macerated soybean seed product from step (i) with an organic solvent to produce defatted soymeal; and (iii) extracting the defatted soymeal from step (ii) with an aqueous solvent to produce an aqueous fraction comprising soybean protein.

51. The method of claim 50, wherein the by-product is soybean oil and wherein the soybean seed lot is subjected to processing steps comprising: (i) at least one of a cracking, flaking, crushing, pressing, and / or macerating step; and (ii) solvent extracting, expelling, and / or extruding step the cracked, flaked, crushed, pressed, and / or macerated soybean seed product from step (i) to produce the oil.

52. A method of producing a commodity soybean plant product, the method comprising processing a modified soybean seed obtained from the modified soybean plant of any one of claims 25 to 34 and recovering the commodity plant product from the processed plant or seed.

53. The method of claim 52, wherein the commodity plant product is seed meal, starch, silage, oil, or protein.

54. The method of claim 52 or 53, wherein the commodity plant product comprises a detectable amount of a DNA molecule comprising the endogenous target soybean gene or a fragment thereof containing the deletion of the one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene.

55. A method of producing soybean plant material, the method comprising: (a) providing the modified soybean plant of claims 25 or 26; and, (b) growing the modified soybean plant under conditions that allow for expression of theDocket No. P14694WO00 endogenous target soybean gene at levels that are increased compared to expression levels of the endogenous target soybean gene in a reference soybean plant which lacks the deletions.

56. The method of claim 55, wherein growing the modified soybean plant further comprises at least one of sowing a soybean seed which germinates and forms the soybean plant, irrigating the soybean seed or plant, and / or treating the soybean plant or the soybean seed with a biological agent, herbicide, insecticide, or fungicide.

57. The method of claims 55 or 56, wherein the soybean plant material comprises a seed, optionally wherein the method further comprises harvesting the seed from the plant.

58. A method for generating a modified soybean plant, plant cell, or plant part comprising: (i) directing both: (a) a guide RNA (gRNA) molecule which targets the nucleic acid sequence of the transcriptional enhancer through the ATG translational start site of the endogenous target soybean gene sequence comprising one or more of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof and (b) an RNA dependent endonuclease (RDE) which recognizes the gRNA molecule, to the genome of a target soybean plant cell; and (ii) isolating a modified soybean plant cell, modified soybean plant part, or modified soybean plant comprising at a deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene sequence of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof.

59. The method of claim 58, wherein nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof is targeted and the gRNA comprises a spacer RNA molecule comprising the RNA encoded by any of SEQ ID NO: 13-36.

60. The method of claims 58 or 59, wherein the directing of the gRNA and the RDE to the genome of the target soybean plant cell comprises introducing the gRNA, the RDE, a gRNA / RDE complex, a nucleic acid encoding the gRNA, and / or a nucleic acid encoding the RDE into the target soybean plant cell.Docket No. P14694WO00 61. The method of claims 25 to 39, wherein the soybean plant cell, soybean plant part, or soybean plant comprising the endogenous gene containing the deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene is identified by: (i) analyzing a polynucleotide comprising at least a portion of SEQ ID NO: 1, 3, 5, 7, 9 and / or 11 or an allelic variant thereof in one or more candidate plant cells, plant parts, or plants; and / or (ii) analyzing yield of the soybean plant part, soybean plant, or soybean plant grown from the soybean plant cell, wherein the yield comprises least one of a soybean plant root length, root biomass, lateral root number, and / or nodule number in comparison to a wild-type or control soybean plant lacking the deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene, and wherein an increase in the yield indicates that the soybean plant cell, soybean plant part, or soybean plant comprises the at least one deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene.

62. A soybean plant made by the method of claims 1 or 39.

63. Use of the modified soybean plant of claims 25 or 62, seed obtained therefrom, or a seed of any of claims 38 to 41 to: (a) grow a soybean plant with improved yield of at least one of a soybean plant root length, root biomass, lateral root number, and / or nodule number in comparison to a soybean plant lacking the deletion; (b) harvest soybean plant seed comprising the endogenous gene containing the deletion; (c) produce a commodity product; or (d) breed soybean plants with improved yield of at least one of a soybean plant root length, root biomass, lateral root number, and / or nodule number in comparison to a soybean plant lacking the deletion.

64. Use of the modified soybean plant cell of claims 25 or 62, or the modified soybean plant of any of claims 25 to 34 or 62 to produce a modified soybean seed.

65. Use of the modified soybean plant cell of claims 25 or 62, to grow a modified soybean crop.

66. Use of the soybean plant cell of claims 25 or 62, to obtain a soybean by-product.Docket No. P14694WO00 67. The use of claim 66, wherein the soybean by-product comprises soybean flour, meal, protein, oil, syrup, or starch.

68. A DNA molecule comprising at least a portion of an endogenous target soybean gene containing the deletion of the one or more nucleotides located between the transcriptional enhancer and the promoter of the endogenous target soybean gene set forth in claims 1 or 2.

69. The DNA molecule of claim 68, wherein the DNA molecule comprises, consists essentially of, or consists of at least the portion of the nucleic acid sequence of the transcriptional enhancer through an ATG translational start site of the endogenous target soybean gene sequence of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof.

70. The DNA molecule of claim 69, wherein the at least a portion of the nucleic acid sequence of the transcriptional enhancer through an ATG translational start site of the endogenous target soybean gene sequence of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11, or an allelic variant thereof, has one or more nucleotides deleted between the transcriptional enhancer and the promoter of the endogenous soybean gene.

71. The DNA molecule of claim 70, wherein the DNA molecule comprises, consists essentially of, or consists of at least the portion of the nucleic acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 37, or an allelic variant thereof containing the deletion of the one or more nucleotides.

72. Soybean seed meal comprising the DNA molecule of claims 68 or 69, optionally wherein the seed meal is non-regenerable.

73. A biological sample comprising the DNA molecule of claims 68 or 69.

74. A method of identifying a biological sample comprising the DNA molecule containing the deletion of one or more nucleotides between the transcriptional enhancer and the promoter of the endogenous target soybean gene set forth in any one of claims 68 to 71, comprising the step of detecting the presence of the DNA molecule in the biological sample.Docket No. P14694WO00 75. A guide RNA molecule comprising a spacer RNA molecule which hybridizes to nucleotides located between a transcriptional enhancer and a promoter of an endogenous target soybean gene of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11, or an allelic variant thereof, optionally wherein the spacer RNA molecule comprises the RNA encoded by any one of SEQ ID NO: 13-36.

76. The guide RNA molecule of claim 75, further comprising a Cas12 direct repeat element which is operably linked to the spacer RNA.

77. A gene editing system comprising a CRISPR-Cas effector protein and a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that hybridizes to a nucleic acid sequence of a transcriptional enhancer through an ATG translational start site of an endogenous target soybean gene sequence comprising one or more of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 or an allelic variant thereof.

78. An expression cassette comprising a polynucleotide encoding the guide RNA molecule of claim 75 or 76.

79. A soybean genome characterized by the fact that it comprises the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 which contains a non-natural mutation.

80. The soybean genome of claim 79, wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least one nucleotide of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11 optionally wherein the non-natural mutation comprises, consists essentially of, or consists of a deletion of at least five or more, ten or more, 50 or more, 100 or more, 250 or more, 500 or more, 1000 or more, 1500 or more, 2000 or more, 2500 or more, 3000 or more, 3500 or more, 4000 or more, or 4500 or more nucleotides of SEQ ID NO: 1, 3, 5, 7, 9, and / or 11.

81. A soybean plant cell obtainable by the process comprising introducing a deletion in an endogenous target soybean gene with a Type V Cas nuclease and at least two guide RNA (gRNA) molecules comprising a direct repeat RNA molecule bound by the Cas nuclease andDocket No. P14694WO00 a spacer RNA molecule which hybridizes to nucleotides located between a transcriptional enhancer and a promoter of the endogenous target soybean gene, wherein: (a) the endogenous soybean gene Glyma.19G143900 is targeted with one or more gRNA(s) comprising a spacer is encoded by SEQ ID NOs: 13 and / or 14; and one or more gRNA(s) comprising a spacer encoded by SEQ ID NOs: 15 and / or 16; (b) the endogenous soybean gene Glyma.10G186500 is targeted with one or more gRNA(s) comprising a spacer is encoded by SEQ ID NOs: 17 and / or 18; and one or more gRNA(s) comprising a spacer encoded by SEQ ID NOs: 19 and / or 20; (c) the endogenous soybean gene Glyma.13G279900 is targeted with one or more gRNA(s) comprising a spacer is encoded by SEQ ID NOs: 21 and / or 22; and one or more gRNA(s) comprising a spacer encoded by SEQ ID NOs: 23 and / or 24; (d) the endogenous soybean gene Glyma.14G197100 is targeted with one or more gRNA(s) comprising a spacer is encoded by SEQ ID NOs: 25 and / or 26; and one or more gRNA(s) comprising a spacer encoded by SEQ ID NOs: 27 and / or 28; (e) the endogenous soybean gene Glyma.11G250200 is targeted with one or more gRNA(s) comprising a spacer is encoded by SEQ ID NOs: 29 and / or 30; and one or more gRNA(s) comprising a spacer encoded by SEQ ID NOs: 31 and / or 32; or (f) the endogenous soybean gene Glyma.17G192000 is targeted with one or more gRNA(s) comprising a spacer is encoded by SEQ ID NOs: 33 and / or 34; and one or more gRNA(s) comprising a spacer encoded by SEQ ID NOs: 35 and / or 36.

82. The soybean plant cell of claim 81, wherein the soybean call containing the deletion is selected.

83. A soybean plant or plant part obtained from the soybean plant cell of claim 81 or 82.

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