Regulatory motifs for enhancing soybean gene expression

Abstract

The present invention relates to synthetic enhancer elements that can be used to increase the expression of a gene of interest in any plant tissue. The disclosed synthetic enhancer elements can be used in a gene expression cassette comprising nucleic acid further comprising a promoter and a sequence to be expressed, wherein the sequence to be expressed has a transcription start site, and wherein the synthetic enhancer element is operably linked to the promoter and the sequence to be expressed. The present invention also relates to a modified plant or part thereof comprising a synthetic enhancer element, wherein the synthetic enhancer element is operably linked to an endogenous promoter in the modified plant or part thereof and an endogenous gene in the modified plant or part thereof, and wherein the synthetic enhancer element increases expression of a transcript or protein encoded by the endogenous gene.

Description

Docket No.: 165362002440REGULATORY MOTIFS FOR ENHANCING SOYBEAN GENE EXPRESSIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Application No. 63 / 741,771, filed January 3, 2025, the contents of which are incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (165362002440seqlist.xml; Size:178,622 bytes; and Date of Creation: December 31, 2025) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] Aspects of the present disclosure relate to regulatory motifs or synthetic enhancer elements. When inserted into the upstream region of a gene, such as before the transcription start site of the gene, the regulatory motifs or synthetic enhancer elements increase the expression of the gene. The present disclosure provides compositions for gene expression cassettes and expression vectors with regulatory motifs or synthetic enhancer elements that may be used to increase gene expression in plant cells, modified plants, or plant parts thereof.BACKGROUND

[0004] There is a demand to improve agriculturally important traits in plants. Producing superior plants or plants with novel traits is critical to addressing local and global challenges to supply in terms of food, employment, economic value, and fuel. A thorough investigation of the regulation of plant genes can be paramount to ascertaining and manipulating certain complex plant biological processes allowing development of superior plants or plants with novel traits. Ascertaining and manipulating such complex plant biological processes may be performed by determining, for example, the genes and regulatory mechanisms controlling these biological processes. To produce a desired phenotype in a superior plant or a plant with novel traits, genes may be knocked in, knocked out, mutated, or manipulated with respect to expression or transcription.

[0005] In some instances, manipulation of regulatory motifs allows for understanding and controlling of gene expression or transcription, and the production of superior plants or plants1MF-365148955Docket No.: 165362002440with novel traits. Regulatory motifs may be classified in terms of their structure, genomic location, and the tissue context in which they are active. For example, a regulatory motif may be an enhancer element, comprising a linear nucleotide fragment of noncoding DNA adjacent to or within a transcribed DNA strand. Enhancer elements may interact with other regulatory elements, such as promoters, to increase the expression of genes in one or more plant tissues. There is an ongoing need to identify regulatory motifs that are capable of reliably increasing transcription of plant genes, to produce plants with beneficial agronomic traits.BRIEF SUMMARY

[0006] The present disclosure provides synthetic enhancer elements that can be used to increase the expression of a gene of interest in any plant tissue. The disclosed synthetic enhancer elements may be used in gene expression cassettes, expression vectors, plant cells, and modified plants or parts thereof, and in diverse tissues and biological contexts. In some instances, a synthetic enhancer element is operably linked to a plant promoter and a plant gene and increases the expression of a transcript or protein encoded by the plant gene. The synthetic enhancer elements described herein can be used to increase the expression of plant genes involved in various agriculturally important traits in soybean, including yield characteristics, abiotic or biotic stress tolerance, pest tolerance, disease resistance, or quality traits such as nutritional improvements. The disclosed synthetic enhancer elements may also be used to eliminate issues with irreproducible upregulation of gene expression, allowing for reliable control of gene expression in multiple tissues and environmental contexts and the generation of stable plant phenotypes for valuable agronomic traits.

[0007] The present disclosure provides synthetic enhancer elements that may be used to precisely control and finetune increase of gene expression of a gene of interest by altering the number of copies of the enhancer positioned in tandem (either with or without the presence of a linker or spacer sequence in between each copy of the enhancer) as will be described and exemplified further herein. The present disclosure provides synthetic enhancer elements that may be used to precisely control and finetune gene expression of a gene of interest by further altering the distance of the insertions of the one or more copies of the synthetic enhancer element from the transcription start site of the gene of interest as will be described and exemplified further herein.

[0008] In one aspect, provided herein is a gene expression cassette comprising nucleic acid comprising a synthetic enhancer element, a promoter, and a sequence to be expressed,2MF-365148955Docket No.: 165362002440wherein: (a) the sequence to be expressed has a transcription start site; (b) the synthetic enhancer element is operably linked to the promoter and the sequence to be expressed; and (c) the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, 138-140, 149-162, GTAAGC, GCTTAC, CTTA, and CACGTG. In some embodiments, the gene expression cassette comprises a single copy of the synthetic enhancer element. In some embodiments, the gene expression cassette further comprises two or more copies of the synthetic enhancer element. In some embodiments, the gene expression cassette further comprises three copies, four copies, five copies, or six copies of the synthetic enhancer element. In some embodiments, the gene expression cassette further comprises a spacer sequence between each copy of the synthetic enhancer element. In some embodiments, the spacer sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 base pairs in length. In some embodiments, the spacer sequence comprises the nucleotide sequence set forth in SEQ ID NO: 145. In some embodiments, the synthetic enhancer element is in reverse orientation. In some embodiments, the synthetic enhancer element is between about 5 base pairs to about 1000 base pairs upstream of the transcription start site of the sequence to be expressed. In some embodiments, the synthetic enhancer element is about 25 base pairs, about 50 base pairs, about 75 base pairs, about 100 base pairs, about 125 base pairs, about 150 base pairs, about 175 base pairs, or about 200 base pairs upstream of the transcription start site of the sequence to be expressed. In some embodiments, the synthetic enhancer element is within the promoter. In some embodiments, the synthetic enhancer element is upstream of the promoter. In some embodiments, the synthetic enhancer element is downstream of the promoter. In some embodiments, the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 115, and SEQ ID NO: 107. In some embodiments, the sequence to be expressed is a gene that is endogenous to a soybean plant genome. In some embodiments, the gene is selected from the group consisting of AlPlOa, AlPlOb, AML4, CRN, HB-1, RIC1, RIC2, RPF1, JAG1, JAG2, KHZ1, PP2C, TCP5-L, FTla, BS1, BS2, TFLlb, CYP76C-1, CYP76C-2, and NF-YC4. In some embodiments, the promoter is endogenous to a soybean plant genome. In some embodiments, the promoter is a promoter selected from the group consisting of the AlPlOa promoter, the AlPlOb promoter, the AML4 promoter, the CRN promoter, the HB-1 promoter, the RIC1 promoter, the RIC2 promoter, the RPF1 promoter, the JAG1 promoter, the JAG2 promoter, the KHZ1 promoter, the PP2C promoter, the TCP5-L promoter, the FTla promoter, the BS1 promoter, the BS23MF-365148955Docket No.: 165362002440promoter, the TFLlb promoter, the CYP76C-1 promoter, the CYP76C-2 promoter, the NF-YC4 promoter, the MYB promoter, and the Hl_5 promoter. In some embodiments, the synthetic enhancer element increases expression of a transcript or protein encoded by the sequence to be expressed.

[0009] In another aspect, provided herein is an expression vector comprising the gene expression cassette of any one of the preceding embodiments. In some embodiments, the sequence to be expressed comprises a protein coding sequence.

[0010] In another aspect, provided herein is a plant cell comprising the gene expression cassette of any one of the preceding embodiments.

[0011] In another aspect, provided herein is a plant cell comprising the expression vector of any one of the preceding embodiments. In some embodiments, the plant cell is a non-regenerable plant cell which is not capable of being regenerated to produce a plant. In some embodiments, plant cell is a soybean plant cell, a corn plant cell, or a wheat plant cell.

[0012] In another aspect, provided herein is a modified plant or part thereof comprising a synthetic enhancer element, wherein the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, 138-140, 149-162, GTAAGC, GCTTAC, CTTA, and CACGTG, and wherein the synthetic enhancer element is operably linked to an endogenous promoter in the modified plant or part thereof and an endogenous gene in the modified plant or part thereof. In some embodiments, the plant or part thereof comprises a single copy of the synthetic enhancer element. In some embodiments, the modified plant or part thereof further comprises two or more copies of the synthetic enhancer element. In some embodiments, the modified plant or part thereof further comprises three copies, four copies, five copies, or six copies of the synthetic enhancer element. In some embodiments, the modified plant or part thereof further comprises a spacer sequence between each copy of the synthetic enhancer element. In some embodiments, the spacer sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 base pairs in length. In some embodiments, the spacer sequence comprises the nucleotide sequence set forth in SEQ ID NO: 145. In some embodiments, the synthetic enhancer element is in reverse orientation. In some embodiments, the synthetic enhancer element is within 1000 base pairs of the endogenous gene. In some embodiments, the synthetic enhancer element is within a noncoding genomic region. In some embodiments, the synthetic enhancer element is between4MF-365148955Docket No.: 165362002440about 5 base pairs to about 1000 base pairs upstream of a transcription start site of the endogenous gene. In some embodiments, the synthetic enhancer element is about 25 base pairs, about 50 base pairs, about 75 base pairs, about 100 base pairs, about 125 base pairs, about 150 base pairs, or about 200 base pairs upstream of a transcription start site of the endogenous gene. In some embodiments, the synthetic enhancer element is within the endogenous promoter. In some embodiments, the synthetic enhancer element is upstream of the endogenous promoter. In some embodiments, the synthetic enhancer element is downstream of the endogenous promoter. In some embodiments, the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 115, and SEQ ID NO: 107. In some embodiments, the modified plant or part thereof is a soybean plant or part thereof. In some embodiments, the endogenous gene is a gene selected from the group consisting of AlPlOa, AlPlOb, AML4, CRN, HB-1, RIC1, RIC2, RPF1, JAG1, JAG2, KHZ1, PP2C, TCP5-L, FTla, BS1, BS2, TFLlb, CYP76C-1, CYP76C-2, and NF-YC4. In some embodiments, the endogenous promoter is a promoter selected from the group consisting of: the AlPlOa promoter, the AlPlOb promoter, the AML4 promoter, the CRN promoter, the HB-1 promoter, the RIC1 promoter, the RIC2 promoter, the RPF1 promoter, the JAG1 promoter, the JAG2 promoter, the KHZ1 promoter, the PP2C promoter, the TCP5-L promoter, the FTla promoter, the BS1 promoter, the BS2 promoter, the TFLlb promoter, the CYP76C-1 promoter, the CYP76C-2 promoter, the NF-YC4 promoter, the MYB promoter, and the Hl_5 promoter. In some embodiments, the synthetic enhancer element increases expression of a transcript or protein encoded by the endogenous gene. In some embodiments, the increased expression of a transcript or protein encoded by the endogenous gene contributes to improved yield characteristics, improved abiotic or biotic stress tolerance, improved pest tolerance, improved disease resistance, or improved quality traits such as nutritional improvements in the modified plant or part thereof.INCORPORATION BY REFERENCE

[0013] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls.5MF-365148955Docket No.: 165362002440BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 depicts a strategy to increase expression of a transcript or protein encoded by a sequence to be expressed. A synthetic enhancer element is investigated in a single copy, in two or more copies, or in three or more copies, operably linked to a promoter. The synthetic enhancer element alters the expression level of the sequence to be expressed (“Edited line expression level”) relative to a wild-type promoter without a synthetic enhancer element (“WT expression level”).

[0015] FIG. 2A depicts an expression vector for use in a dual luciferase assay to assess the activity of synthetic enhancer elements. FIG. 2B depicts control, 3x octopine synthase (OCS), CaMV 35S enhancer, and experimental enhancer vectors. The relative luminescence ratio of firefly luciferase (FLUC) to Renilla luciferase (RLUC) can be measured from each vector.

[0016] FIG. 3 depicts the relative luminescence ratio of firefly luciferase (FLUC) to Renilla luciferase (RLUC) using the expression vectors of FIGS. 2A-2B, where FLUC expression is driven by a promoter containing one copy of the OCS enhancer (lx OCS), three copies of the OCS enhancer element positioned in tandem (3x OCS), one copy of the CaMV 35S enhancer (35S Enhancer), three copies of the OCS enhancer separated by 10 base pairs (3x OCS w / spacers), three copies of the cCRE enhancer positioned in tandem (3x CCRE), 3 copies of the G-box enhancer positioned in tandem (3x GBOX), or the wild-type TFLlb promoter (WT Promoter).

[0017] FIG. 4 depicts the relative luminescence ratio of firefly luciferase (FLUC) to Renilla luciferase (RLUC) for dual luciferase assays conducted with a vector containing the wildtype TFLlb promoter (“WT TFLlb control”), a vector containing three copies of the OCS enhancer (“3x OCS control”), and a vector containing three copies of candidate Enhancer 100 (SEQ ID NO: 100) (“3x_12bp_2666”).

[0018] FIG. 5A depicts the relative luminescence ratio of firefly luciferase (FLUC) to Renilla luciferase (RLUC) for dual luciferase assays conducted with a vector containing the wild-type TFLlb promoter (“WT TFLlb control”), a vector containing three copies of the OCS enhancer (“3x OCS control”), a vector containing three copies of candidate Enhancer 100 (single copy SEQ ID NO: 100; three copies SEQ ID NO: 138) (“3x_12bp_2666”), a vector containing three copies of candidate Enhancer 115 (single copy SEQ ID NO: 115; three copies SEQ ID NO: 140) (“3x_12bp_2681”), and a vector containing three copies of6MF-365148955Docket No.: 165362002440candidate Enhancer 107 (single copy SEQ ID NO: 107; three copies SEQ ID NO: 139) (“3x_12bp_2673”). FIG. 5B depicts the data of FIG. 5A on a log scale.

[0019] FIGS. 6A-6B depict the relative luminescence ratio of firefly luciferase (FLUC) to Renilla luciferase (RLUC) for dual luciferase assays conducted with a vector containing the wild-type TFLlb promoter (“TFLlb - WT”), a vector containing three copies of the OCS enhancer and the TFLlb promoter (“TFLlb - 3x OCS”), a vector containing the wild-type promoter of a medium-expression control gene (a sample gene known to exhibit medium expression levels within the protoplast assay system as determined by RNA sequencing data) (“MID - WT”), a vector containing three copies of the OCS enhancer and the promoter of the medium-expression control gene (“MID - 3x OCS”), a vector containing the wild-type promoter of a high-expression control gene (a sample gene known to exhibit high expression levels within the protoplast assay system as determined by RNA sequencing data) (“HIGH -WT”), a vector containing three copies of the OCS enhancer and the promoter of the high-expression control gene (“HIGH - 3x OCS”). In FIG. 6A, the dual luciferase assays were conducted with each vector at 55 ng / pL. In FIG. 6B, the dual luciferase assays were conducted with each vector at 110 ng / pL.

[0020] FIGS. 7A-7C depict dual luciferase vectors. FIG. 7A depicts a dual luciferase vector used to test Enhancer 100 (single copy SEQ ID NO: 100; three copies SEQ ID NO: 138). FIG. 7B depicts a dual luciferase vector used to test Enhancer 115 (single copy SEQ ID NO: 115; three copies SEQ ID NO: 140). FIG. 7C depicts a dual luciferase vector used to test Enhancer 107 (single copy SEQ ID NO: 107; three copies SEQ ID NO: 139).

[0021] FIG. 8 depicts dual luciferase assays with each vector at a concentration of 110 ng / pL, illustrating the increase in FLUC / RLUC luminescence for vectors containing Enhancer 100.

[0022] FIG. 9 depicts dual luciferase assays with each vector at a concentration of 110 ng / pL, containing Enhancer 100 in various copies (2x, 3x, 4x, 5x, 6x), illustrating a dependency on copy number for this enhancer element.

[0023] FIG. 10 depicts dual luciferase assays with each vector at a concentration of 110 ng / pL, with 3x_12bp_2666 (SEQ ID NO: 138) and 3x OCS cloned at various distances from the transcription start site of the luciferase reporter gene, illustrating a dependency on distance from the TSS of the enhancer element.7MF-365148955Docket No.: 165362002440DETAILED DESCRIPTION

[0024] The present disclosure provides novel synthetic enhancer elements that can be used in gene expression cassettes, expression vectors, plant cells, and modified plants or parts thereof. The synthetic enhancer elements described herein can be operably linked to a plant gene and increase the expression of a transcript or protein encoded by the plant gene. In some aspects, this regulatory approach is used to increase the expression of plant genes involved in various agriculturally important traits in soybean, including yield characteristics, abiotic or biotic stress tolerance, pest tolerance, disease resistance, or quality traits such as nutritional improvements. The synthetic enhancer elements described herein reproducibly increase gene expression and can be operably linked to diverse promoters and genes in multiple plant tissues. Reproducible increases in gene expression can allow for control of valuable agronomic traits in multiple biological contexts.

[0025] In some instances, for example, synthetic enhancer elements are used in a gene expression cassette comprising nucleic acid further comprising a promoter and a sequence to be expressed, wherein the sequence to be expressed has a transcription start site, and wherein the synthetic enhancer element is operably linked to the promoter and the sequence to be expressed. In some instances, the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, 138-140, 149-162, GTAAGC, GCTTAC, CTTA, and CACGTG. In some instances, the gene expression cassette comprises three copies of the synthetic enhancer element. In some instances, the synthetic enhancer element is between about 5 base pairs to about 1000 base pairs upstream of the transcription start site of the sequence to be expressed. In some instances, the gene that is operably linked to the synthetic enhancer element is endogenous to a soybean plant genome.

[0026] The present invention also relates to a modified plant or part thereof comprising a synthetic enhancer element, wherein the synthetic enhancer element is operably linked to an endogenous promoter in the modified plant or part thereof and an endogenous gene in the modified plant or part thereof, the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, 138-140, 149-162, GTAAGC, GCTTAC, CTTA, and CACGTG, and the synthetic enhancer element increases expression of a transcript or protein encoded by the endogenous gene. In some instances, the modified plant or part thereof comprises three copies of the8MF-365148955Docket No.: 165362002440synthetic enhancer element. In some instances, the synthetic enhancer element is between about 5 base pairs to about 1000 base pairs upstream of the transcription start site of the endogenous gene. In some instances, the synthetic enhancer element increases expression of a transcript or protein encoded by the endogenous gene and the increased expression contributes to improved yield characteristics, improved abiotic or biotic stress tolerance, improved pest tolerance, improved disease resistance, or improved quality traits such as nutritional improvements in the modified plant or part thereof.A. Definitions

[0027] The use of the terms “a” and “an” and “the” and “at least one” and similar language in the context of describing embodiments of the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted.

[0028] The phrase “allelic variant” as used herein refers to a polynucleotide or polypeptide sequence variant that occurs in a different strain, variety, or isolate of a given organism.

[0029] 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).

[0030] As used herein, “automatically” and its derivatives means “without human intervention,” unless expressly indicated otherwise or indicated otherwise by context.

[0031] As used herein, the term “complex” refers to two or more associated components, such as two or more associated nucleic acids and / or proteins. A complex may include two or9MF-365148955Docket No.: 165362002440more covalently linked nucleic acids and / or proteins, two or more non-covalently linked nucleic acids and / or proteins, or a combination thereof.

[0032] As used herein, the term “endogenous” refers to something that can be found in the organism prior to human intervention. An “endogenous sequence” refers to a DNA sequence located in the genome of the unedited organism.

[0033] As used herein, the term “exogenous” refers to something that cannot be found in the organism prior to human intervention. An “exogenous sequence” refers to a DNA sequence that is not located in the genome of the unedited organism. An exogenous sequence can be an edited sequence, a synthetic sequence, or a sequence from a different organism.

[0034] As used herein, the terms “heritable genetic modification”, “heritable edit”, and “heritable modification” refer to any insertion, substitution, or deletion of any number of nucleotides in the genomic sequence of a plant that is at least present in a meristem cell of the plant, such that at least one progeny of the plant possesses the same altered genomic sequence.

[0035] 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.

[0036] As used herein, “transforming,” “transformation,” or to “transform” refer to any method requiring human intervention which results in the transfer of an exogenous nucleic acid sequence, recombinant DNA construct, or vector into a cell, irrespective of the method used for transfer. This includes, but is not limited to, transfection, particle bombardment, biolistic transformation, or Agrobacterium- mediated transformation.

[0037] As used herein, the phrase “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter or enhancer is “operably linked” to a sequence to be expressed if the promoter or enhancer affects the transcription of expression of the sequence to be expressed. As used herein, elements may be “operably linked” irrespective of location.

[0038] As used herein, the terms “orthologous,” “ortholog,” or “orthologue” are used to describe genes or the RNAs or proteins encoded by those genes that are from different species but which have the same function. Orthologous genes will typically encode RNAs or proteins with some degree of sequence identity and can also exhibit conservation of sequence motifs, and / or conservation of structural features including RNA stem loop structures.10MF-365148955Docket No.: 165362002440

[0039] As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant. The term “plant parts” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants). A plant part may 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 may be capable of regenerating a plant having the 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 may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, 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.

[0040] As used herein, the term “polynucleotide” refers to a nucleic acid molecule containing multiple nucleotides and encompasses both “oligonucleotides” (defined here as a polynucleotide molecule of between 2-25 nucleotides in length) and polynucleotides of 26 or more nucleotides. Polynucleotides are generally described as single- or double-stranded. Where a polynucleotide contains double-stranded regions formed by intra- or intermolecular hybridization, the length of each double- stranded region is conveniently described in terms of the number of base pairs. Aspects of this invention include the use of polynucleotides or compositions containing polynucleotides; embodiments include one or more oligonucleotides or polynucleotides or a mixture of both, including single- or double-stranded RNA or single-or double-stranded DNA or double- stranded DNA / RNA hybrids or chemically modified analogues or a mixture thereof. In various embodiments, a polynucleotide includes a combination of ribonucleotides and deoxyribonucleotides (e.g., synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleotides), or includes non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In embodiments, the polynucleotide includes chemically modified nucleotides (see, e.g., Verma and Eckstein Annu. Rev. Biochem. 1998, 67: 99-134); for example, the naturally occurring phosphodiester backbone of an oligonucleotide or11MF-365148955Docket No.: 165362002440polynucleotide can be partially or completely modified with phosphorothioate, phosphorodithioate, or methylphosphonate intemucleotide linkage modifications; modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis; and oligonucleotides or polynucleotides can be labelled with a fluorescent moiety (e.g., fluorescein or rhodamine or a fluorescence resonance energy transfer or FRET pair of chromophore labels) or other label (e.g., biotin or an isotope). Modified nucleic acids, particularly modified RNAs, are disclosed in U.S. Pat. No. 9,464,124, incorporated by reference in its entirety herein.

[0041] As used herein, the phrase “sequence identity” refers to the percent similarity of two polynucleotides or polypeptides. A polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available at ncbi[dot]nlm[dot]nih[dot]gov / BLAST. See, e.g., Altschul et al. Mol. Biol. 1990, 215:403-410. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, Calif., USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith-Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol., 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See Mol. Biol., 48: 443-453 (1970).

[0042] As used herein, “or” is inclusive and not exclusive, unless expressly indicated otherwise or indicated otherwise by context. Therefore, herein, “A or B” means “A, B, or both,” unless expressly indicated otherwise or indicated otherwise by context. Moreover, “and” is both joint and several, unless expressly indicated otherwise or indicated otherwise by context. Therefore, herein, “A and B” means “A and B, jointly or severally,” unless expressly indicated otherwise or indicated otherwise by context.

[0043] Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5' to 3' direction. Nucleic acid sequences may be 12MF-365148955Docket No.: 165362002440provided as DNA or as RNA, as specified; disclosure of one necessarily defines the other, as well as necessarily defines the exact complements, as is known to one of ordinary skill in the art.

[0044] As used herein, “reverse orientation” indicates that a nucleic acid sequence in the text of this specification is read from right to left, in the 3' to 5' direction.

[0045] As used herein, “in tandem” is used to mean that there is no space between multiple copies of an element. For example, copies of an enhancer element that are “positioned in tandem” or that are “in tandem” are inserted adjacently and without space between each copy (i.e., the copies are separated by 0 base pairs).

[0046] Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term.

[0047] 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.B. Gene Expression Cassettes and Expression Vectors

[0048] Provided herein are gene expression cassettes comprising nucleic acid comprising a synthetic enhancer element, a promoter, and a sequence to be expressed, wherein the sequence to be expressed has a transcription start site, and wherein the synthetic enhancer element is operably linked to the promoter and the sequence to be expressed. In some embodiments, the gene expression cassette comprises a synthetic enhancer element comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, 138-140, 149-162, GTAAGC, GCTTAC, CTTA, and CACGTG. In some embodiments, the gene expression cassette comprises a single copy of the synthetic enhancer element. In some embodiments, the gene expression cassette comprises two copies, three copies, four copies, five copies, or six copies of the synthetic enhancer element. In some embodiments, the gene expression cassette comprises a synthetic enhancer element that is about 25 base pairs, about 50 base pairs, about 75 base pairs, about 100 base pairs, about 125 base pairs, about 150 base pairs, about 175 base pairs, or about 200 base pairs upstream of the transcription start site of the sequence to be expressed. In some embodiments, the gene expression cassette comprises a promoter that is endogenous to a13MF-365148955Docket No.: 165362002440soybean plant genome. In some embodiments, the gene expression cassette comprises a sequence to be expressed that is a gene endogenous to a soybean plant genome.

[0049] In some embodiments, the gene expression cassette comprises a synthetic enhancer element that is operably linked to a promoter and a sequence to be expressed. In some embodiments, the operably linked synthetic enhancer element increases expression of a transcript or protein encoded by the sequence to be expressed. In some embodiments, the operably linked synthetic enhancer element is upstream of the promoter in the gene expression cassette, downstream of the promoter in the gene expression cassette, or within the promoter in the gene expression cassette.

[0050] Provided herein are expression vectors comprising gene expression cassettes comprising nucleic acid comprising a synthetic enhancer element, a promoter, a sequence to be expressed, wherein the sequence to be expressed has a transcription start site, and wherein the synthetic enhancer element is operably linked to the promoter and the sequence to be expressed. In some embodiments, the expression vector comprises a sequence to be expressed that comprises a protein coding sequence.C. Modified Plants or Parts Thereof

[0051] Provided herein are modified plants or parts thereof comprising a synthetic enhancer element, wherein the synthetic enhancer element is operably linked to an endogenous promoter in the modified plant or part thereof and an endogenous gene in the modified plant or part thereof. In some embodiments, the modified plant or part thereof comprises a synthetic enhancer element comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, 138-140, 149-162, GTAAGC, GCTTAC, CTTA, and CACGTG. In some embodiments, the modified plant or part thereof comprises a synthetic enhancer element inserted upstream of a gene encoding a valuable agronomic trait. In some embodiments, the modified plant or part thereof comprises a synthetic enhancer element inserted upstream of a gene selected from the group consisting of AlPlOa, AlPlOb, AML4, CRN, HB-1, RIC1, RIC2, RPF1, JAG1, JAG2, KHZ1, PP2C, TCP5-L, FTla, BS1, BS2, TFElb, CYP76C-1, CYP76C-2, and NF-YC4.

[0052] In some embodiments, the modified plant or part thereof comprising a synthetic enhancer element is a soybean (e.g., Glycine max) plant or part thereof. In some embodiments, the modified plant or part thereof comprising a synthetic enhancer element is a corn (e.g., maize, Zea mays) plant or part thereof. In some embodiments, the modified plant 14MF-365148955Docket No.: 165362002440or part thereof comprising a synthetic enhancer element is a wheat (e.g., common wheat, spelt, durum, einkom, emmer, kamut, Triticum aestivum, Triticum spelta, Triticum durum, Triticum urartu, Triticum monococcum, Triticum turanicum, Triticum spp.) plant or part thereof.

[0053] In some embodiments, the modified plant or part thereof comprising a synthetic enhancer element has increased expression of a transcript or protein encoded by the endogenous gene to which the synthetic enhancer element is linked. In some embodiments, the modified plant or part thereof comprising a synthetic enhancer element has one or more valuable agronomic traits, such as, but not limited to, improved yield characteristics, improved abiotic or biotic stress tolerance, improved pest tolerance, improved disease resistance, or improved quality traits such as nutritional improvements.D. Synthetic Enhancer Elements

[0054] Provided herein are synthetic enhancer elements that increase gene expression in plants. Enhancer elements are understood to include, but are not limited to, polynucleotide sequences that increase the expression of one or more genes. In some instances, transcription factors may bind to enhancer elements, and those transcription factors may interact with endogenous transcription machinery to ultimately increase gene expression. Endogenous enhancer elements are well-known in the art to exist in genomes and may be present in a variety of genomic locations (see lores et al., Identification of Plant Enhancers and Their Constituent Elements by STARR-seq in Tobacco Leaves, 32 Plant Cell 2120 (2020)). In some instances, specific patterns of gene expression may be driven by combinations of transcription factors that activate specific enhancer elements (see Taskiran et al., Cell-type-directed design of synthetic enhancer, Nature, vol. 626, pp. 212-220 (2024)).

[0055] Provided herein are synthetic enhancer elements that are non-naturally occurring. The disclosed synthetic enhancer elements may be exogenous to plants. As used herein, an exogenous enhancer element is understood as one that is not found in a species, cell, or genome prior to human intervention. In some embodiments, the synthetic enhancer element is a randomly generated polynucleotide sequence comprising a uniform distribution of adenine, guanine, cytosine, and thymine nucleotides. In some embodiments, the synthetic enhancer element comprises a non-uniform distribution of adenine, guanine, cytosine, and thymine nucleotides.15MF-365148955Docket No.: 165362002440

[0056] In some embodiments, the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, 138-140, 149-162, GTAAGC, GCTTAC, CTTA, and CACGTG.

[0057] In some embodiments, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 100. In some embodiments, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 100, and as used herein the synthetic enhancer element is present in one copy. In some embodiments, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 100, and as used herein the synthetic enhancer element is present in two copies, three copies, four copies, five copies, or six copies. In some embodiments, the synthetic enhancer element is present in two copies and comprises the nucleotide sequence set forth in SEQ ID NO: 149. In some embodiments, the synthetic enhancer element is present in three copies and comprises the nucleotide sequence set forth in SEQ ID NO: 138. In some embodiments, the synthetic enhancer element is present in four copies and comprises the nucleotide sequence set forth in SEQ ID NO: 150. In some embodiments, the synthetic enhancer element is present in five copies and comprises the nucleotide sequence set forth in SEQ ID NO: 151. In some embodiments, the synthetic enhancer element is present in six copies and comprises the nucleotide sequence set forth in SEQ ID NO: 152.

[0058] In some embodiments, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 115. In some embodiments, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 115, and as used herein the synthetic enhancer element is present in one copy. In some embodiments, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 115, and as used herein the synthetic enhancer element is present in two copies, three copies, four copies, five copies, or six copies. In some embodiments, the synthetic enhancer element is present in two copies and comprises the nucleotide sequence set forth in SEQ ID NO: 153. In some embodiments, the synthetic enhancer element is present in three copies and comprises the nucleotide sequence set forth in SEQ ID NO: 154 or SEQ ID NO: 140. In some embodiments, the synthetic enhancer element is present in four copies and comprises the nucleotide sequence set forth in SEQ ID NO: 155. In some embodiments, the synthetic enhancer element is present in five copies and comprises the nucleotide sequence set forth in SEQ ID NO: 156. In some embodiments, the synthetic enhancer element is present in six copies and comprises the nucleotide sequence set forth in SEQ ID NO: 157.16MF-365148955Docket No.: 165362002440

[0059] In some embodiments, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 107. In some embodiments, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 107, and as used herein the synthetic enhancer element is present in one copy. In some embodiments, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 107, and as used herein the synthetic enhancer element is present in two copies, three copies, four copies, five copies, or six copies. In some embodiments, the synthetic enhancer element is present in two copies and comprises the nucleotide sequence set forth in SEQ ID NO: 158. In some embodiments, the synthetic enhancer element is present in three copies and comprises the nucleotide sequence set forth in SEQ ID NO: 159 or SEQ ID NO: 139. In some embodiments, the synthetic enhancer element is present in four copies and comprises the nucleotide sequence set forth in SEQ ID NO: 160. In some embodiments, the synthetic enhancer element is present in five copies and comprises the nucleotide sequence set forth in SEQ ID NO: 161. In some embodiments, the synthetic enhancer element is present in six copies and comprises the nucleotide sequence set forth in SEQ ID NO: 162.

[0060] In a preferred embodiment, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 100 and wherein preferably the said enhancer element is repeated in two or more copies as will be described further below. In a more preferred embodiment, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 138.

[0061] In another preferred embodiment, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 141 and wherein the said enhancer element is repeated in two or more copies and wherein the copies of the enhancer element are separated by a spacer, preferably the spacer comprising the nucleotide sequence set forth in SEQ ID NO: 145, as will be described further below. In a more preferred embodiment, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 144.I. Motifs in Synthetic Enhancer Elements

[0062] It is well understood that some enhancer elements contain short nucleotide sequences that are important for enhancer element activity. These short nucleotide sequences may also be referred to as motifs, regulatory motifs, or k-mers where k represents the number of nucleotides in the sequence or motif. These short nucleotide sequences may, in some instances, be palindromic. For example, the G-box is one type of regulatory motif that is 17MF-365148955Docket No.: 165362002440known to modulate transcription of many plant genes (see Ishige et al., A G-box motif (GCCACGTGCC) tetramer confers high-level constitutive expression in dicot and monocot plants, 18 The Plant Journal 443-448 (1999)). The G-box is understood to contain the sequence set forth in Enhancer 99 (SEQ ID NO: 99) and may be flanked by different nucleotides. The octopine synthase (OCS) element is another type of regulatory motif that is known to modulate transcription of plant genes see Bouchez et al., The ocs-element is a component of the promoters of several T-DNA and plant viral genes, 20 The EMBO Journal 4197-4204 (1989)).

[0063] In some embodiments, the synthetic enhancer element comprises, at least in part, one or more known regulatory motifs. A regulatory motif may be repeated 1, 2, 3, or 4 times. Multiple copies of a regulatory motif may be positioned in tandem, or may be separated by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 base pairs. In some embodiments, the synthetic enhancer element comprises, at least in part, one or more k-mers. A k-mer may be repeated 1, 2, 3, or 4 times. Multiple copies of a k-mer may be positioned in tandem, or may be separated by 1, 2, 3, 4, 5, or 6 base pairs.

[0064] In some embodiments, the synthetic enhancer element comprises a G-box element. In some embodiments, the synthetic enhancer element comprises a G-box element flanked by 1, 2, 3, 4, or 5 nucleotides on either side of the G-box element. In some embodiments, the G-box element may be present in about 1, about 2, or about 3 copies. Multiple copies of a G-box element may be positioned in tandem, or may be separated by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 base pairs.

[0065] In some embodiments, the synthetic enhancer element comprises an OCS element. In some embodiments, the synthetic enhancer element comprises an OCS element flanked by 1, 2, 3, 4, or 5 nucleotides on either side of the OCS element. In some embodiments, the OCS element may be present in 1, 2, or 3 copies. Multiple copies of an OCS element may be positioned in tandem, or may be separated by at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 base pairs. In some embodiments, multiple copies of an OCS element are separated by a spacer sequence. In some embodiments, the spacer sequence comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 base pairs. In some embodiments, the spacer sequence comprises about 10 base pairs. In some 18MF-365148955Docket No.: 165362002440embodiments, the spacer sequence comprises the nucleotide sequence set forth in SEQ ID NO: 145.

[0066] In some embodiments, the synthetic enhancer element comprises an OCS element comprising the nucleotide sequence set forth in SEQ ID NO: 141. In some embodiments, the synthetic enhancer element comprises an OCS element comprising the nucleotide sequence set forth in SEQ ID NO: 141, and as used herein the OCS element is present in one copy. In some embodiments, the synthetic enhancer element comprises an OCS element comprising the nucleotide sequence set forth in SEQ ID NO: 141, and as used herein the OCS element is present in two copies, three copies, four copies, five copies, or six copies. In some embodiments, the synthetic enhancer element comprises three copies of an OCS element and comprises the nucleotide sequence set forth in SEQ ID NO: 142, wherein each copy of the OCS element is present in tandem. In some embodiments, the synthetic enhancer element comprises three copies of an OCS element and comprises the nucleotide sequence set forth in SEQ ID NO: 144. In some embodiments, the three copies of the OCS element are separated by a spacer sequence. In some embodiments, the spacer sequence is about 10 base pairs in length. In some embodiments, the spacer sequence comprises the nucleotide sequence set forth in SEQ ID NO: 145.IL Copies of Synthetic Enhancer Elements

[0067] In some embodiments, a gene expression cassette, expression vector, or modified plant or part thereof comprises a synthetic enhancer element in a single copy, that is, the synthetic enhancer element is repeated only one time. In some embodiments, the synthetic enhancer element is present in two copies, that is, the synthetic enhancer element is repeated two times. The two copies of the synthetic enhancer element may be positioned in tandem, or may be separated by a spacer sequence (which may also be referred to as a linker) comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 base pairs. In some embodiments, the two copies of the synthetic enhancer element are separated by a spacer sequence that is about 10 base pairs in length. In some embodiments, the synthetic enhancer element is present in three copies (i.e., repeated three times), in four copies (i.e., repeated four times), in five copies (i.e., repeated five times), or in six copies (i.e., repeated six times). Each copy of the synthetic enhancer element may be positioned in tandem, or each copy may be separated by a spacer sequence comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8,19MF-365148955Docket No.: 165362002440at least 9, at least 10, at least 11, or at least 12 base pairs. In some embodiments, each copy of the synthetic enhancer element is separated by a spacer sequence that is about 10 base pairs in length. In some embodiments, the spacer sequence comprises the nucleotide sequence set forth in SEQ ID NO: 145. In some embodiments, the presence of a spacer sequence between copies of a regulatory motif or synthetic enhancer element improves the ability of the regulatory motif or synthetic enhancer element to increase gene expression. For example, the inclusion of a spacer sequence may remove the palindromic nature of a regulatory motif, which increases genomic stability.

[0068] In some embodiments, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 100, is present in more than one copy (e.g., SEQ ID NO: 149, SEQ ID NO: 138, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152), and each copy is separated by a spacer sequence comprising the sequence set forth in SEQ ID NO: 145. In some embodiments, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 115, is present in more than one copy (e.g., SEQ ID NO: 153, SEQ ID NO: 140 or SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157), and each copy is separated by a spacer sequence comprising the sequence set forth in SEQ ID NO: 145. In some embodiments, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 107, is present in more than one copy (e.g., SEQ ID NO: 158, SEQ ID NO: 139 or SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162), and each copy is separated by a spacer sequence comprising the sequence set forth in SEQ ID NO: 145. In a preferred embodiment, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 144 (which consists of 3 copies of SEQ ID NO: 141 separated by the spacer sequence set forth in SEQ ID NO: 145).E. Promoters and Transcription Start Sites

[0069] Synthetic enhancer elements provided herein are useful in conjunction with promoters to drive transcription of a target gene and increase gene expression. The synthetic enhancer element may be located upstream of, downstream of, or within a promoter. The synthetic enhancer element may be operably linked to a promoter regardless of its location. The synthetic enhancer may be operably linked to a heterologous promoter.

[0070] Promoters are well-understood in the art as nucleotide sequences that participate in gene function, regulation, and expression. The interaction of a promoter with a gene to facilitate these processes is commonly referred to as the promoter being operably linked to a 20MF-365148955Docket No.: 165362002440gene. In many instances, a promoter is a nucleotide sequence located near the 5’ end of the coding region of a gene see Villao-Uzho et al., Plant Promoters: Their Identification, Characterization, and Role in Gene Regulation, 14 Genes 1226 (2023)). A promoter may be located upstream or downstream of a transcription start site in a gene. A transcription start site (i.e., TSS) is well-known in the art as the location where transcription begins within a gene. In many instances, the transcription start site is located at the 5’ end of the gene, preceding the coding region, and the promoter is located upstream of the transcription start site. A transcription start site can be identified using a method known in the art. By way of example, a publicly available genome annotation can be examined to identify transcription start sites, for example, a publicly available genome annotation for soybean. As another example, an RNA-based sequencing experiment (e.g., STRIPEseq, CAGEseq) can be performed, wherein transcriptional data is generated that can be subsequently examined to identify or verify a transcription start site of interest, e.g., a transcription start site for a particular gene of interest. See, e.g., Fang et al., Genome level identification of transcription start sites by nanocage sequence in soybean, Scientific Data 12: 1723 (2025); Wang et al., Noncanonical transcription initiation is primarily tissue specific and epigenetically tuned in paleopolyploid plants, Plant Cell 37(l):koae288 (2024); Maruyama et al., Metabolite / phytohormone-gene regulatory networks in soybean organs under dehydration conditions revealed by integration analysis, Plant J 103(1): 197-211 (2020). In some instances, the promoter itself may include or overlap with the transcription start site of the gene. In some embodiments, the promoter is a region about 5 base pairs to about 1000 base pairs upstream of a transcription start site in a plant genome.

[0071] In some instances, a promoter or promoter sequence may be described as a regulatory region that includes binding sites for transcription factors and transcription factor complexes. Transcription factors and transcription factor complexes may interact with core transcription machinery at promoters to initiate transcription and participate in gene regulation and expression. In some embodiments, RNA polymerase binds to the promoter for the purposes of initiating gene transcription. In some embodiments, the promoter is a constitutive promoter, a conditional promoter, an inducible promoter, or a temporally or spatially specific promoter (e.g., a tissue specific promoter, a developmentally regulated promoter, or a cell cycle regulated promoter).

[0072] A promoter or promoter sequence may be exogenous or endogenous. As used herein, an exogenous promoter or promoter sequence is understood as one that is not found in21MF-365148955Docket No.: 165362002440a species, cell, or genome prior to human intervention. As used herein, an endogenous promoter or promoter sequence is understood as one that can be found in a species, cell, or genome prior to human intervention. An endogenous promoter can include a promoter that is naturally found in a species, cell, or genome prior to human intervention, and is then moved to a different location, or genetically modified.

[0073] In some embodiments, the synthetic enhancer element is operably linked to an endogenous promoter. In some embodiments, the synthetic enhancer element is operably linked to an endogenous plant promoter for a target gene. In some embodiments, the synthetic enhancer element is operably linked to a minimal 35S promoter. In some embodiments, the synthetic enhancer element is operably linked to a promoter that is endogenous to a soybean plant genome. In some embodiments, the synthetic enhancer element is operably linked to a promoter that is selected from the group consisting of the AlPlOa promoter, the AlPlOb promoter, the AML4 promoter, the CRN promoter, the HB-1 promoter, the RIC1 promoter, the RIC2 promoter, the RPF1 promoter, the JAG1 promoter, the JAG2 promoter, the KHZ1 promoter, the PP2C promoter, the TCP5-L promoter, the FT la promoter, the BS1 promoter, the BS2 promoter, the TFLlb promoter, the CYP76C-1 promoter, the CYP76C-2 promoter, the NF-YC4 promoter, the MYB promoter, and the Hl_5 promoter.

[0074] In some embodiments, the synthetic enhancer element is linked to a promoter comprising a polynucleotide sequence. In some embodiments, the synthetic enhancer element is operably linked to a promoter comprising a polynucleotide sequence with a length of about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325 or 350 base pairs. In some embodiments, the synthetic enhancer element is operably linked to a promoter comprising a polynucleotide sequence with a length of between about 25 and 350, about 50 and 350, about 100 and 350, about 125 and 350, about 150 and 350, about 200 and 350, about 250 and 350, about 275 and 350, about 300 and 350 or about 325 and 350 base pairs. In some embodiments, the synthetic enhancer element is operably linked to a promoter comprising a polynucleotide sequence with a length of between about 25 and 350, about 25 and 325, about 25 and 300, about 25 and 275, about 25 and 250, about 25 and 225, about 25 and 200, about 25 and 175, about 25 and 150, about 25 and 125, about 25 and 100, about 25 and 75 or about 25 and 50 base pairs. In some embodiments, the promoter comprises a polynucleotide sequence with a length of between about 500 base pairs and 2000 base pairs. In some embodiments, the promoter comprises a polynucleotide sequence with a length of about 500, about 600, about, about 800,22MF-365148955Docket No.: 165362002440about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, or about 2000 base pairs. In some embodiments, the promoter comprises a polynucleotide sequence with a length of between about 500 and 1750, about 500 and 1500, about 500 and 1250, about 500 and 1000, or about 500 and 750 base pairs.F. Locations of Synthetic Enhancer Elements

[0075] The synthetic enhancer elements disclosed herein may be examined, validated, and used in a variety of locations, including but not limited to: within a coding genomic region, within a noncoding genomic region, within a 5’ untranslated region of a gene, within a 3’ untranslated region of a gene, within an exon, within an intron, upstream of a promoter, within a promoter, downstream of a promoter, upstream of a gene, within a gene, downstream of a gene, upstream of a transcription start site, or downstream of a transcription start site. In some embodiments, the synthetic enhancer element is located within about 1,000 base pairs of a gene. In some embodiments, the enhancer is located within about 1,000 base pairs of a transcription start site.

[0076] In some embodiments, the synthetic enhancer element is upstream of a promoter. In some embodiments, the synthetic enhancer element is downstream of a promoter. In some embodiments, the synthetic enhancer element is within a promoter. In some embodiments, the enhancer is located within any one of the promoters for AlPlOa, AlPlOb, AML4, CRN, HB-1, RIC1, RIC2, RPF1, JAG1, JAG2, KHZ1, PP2C, TCP5-L, FTla, BS1, BS2, TFLlb, CYP76C-1, CYP76C-2, and NF-YC4. In some embodiments, the synthetic enhancer element is located upstream of a promoter, where the promoter is any one of the promoters for AlPlOa, AlPlOb, AML4, CRN, HB-1, RIC1, RIC2, RPF1, JAG1, JAG2, KHZ1, PP2C, TCP5-L, FTla, BS1, BS2, TFLlb, CYP76C-1, CYP76C-2, and NF-YC4.

[0077] A synthetic enhancer element as disclosed herein can be at a distance from a transcription start site where one of skill in the art would expect the greatest impact on expression of the gene associated with the transcription start site. In some embodiments, the synthetic enhancer element has a greater impact on gene expression at shorter distances from the transcription start site, i.e., the closer the synthetic enhancer element is to the transcription start site, the greater the impact on gene expression. In some embodiments, the synthetic enhancer element is positionally located such that it does not overlap with known promoter motifs or conserved noncoding sequences.23MF-365148955Docket No.: 165362002440

[0078] In some embodiments, the synthetic enhancer element is located upstream of a transcription start site. In some embodiments, the synthetic enhancer element is located between about 5 base pairs and 1000 base pairs upstream of a transcription start site. In some embodiments, the synthetic enhancer element is located between about 5 base pairs and about 100 base pairs, between about 100 base pairs and about 200 base pairs, between about 200 base pairs and about 300 base pairs, between about 300 base pairs and about 400 base pairs, between about 400 base pairs and about 500 base pairs, between about 500 base pairs and about 600 base pairs, between about 600 base pairs and about 700 base pairs, between about 700 and about 800 base pairs, between about 800 base pairs and about 900 base pairs, or between about 900 base pairs and about 1000 base pairs. In some embodiments, the synthetic enhancer element is located between about 5 base pairs and about 200 base pairs upstream of a transcription start site. In some embodiments, the synthetic enhancer element is located between about 25 base pairs and about 100 base pairs upstream of a transcription start site. In some embodiments, the synthetic enhancer element is located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, or about 200 base pairs upstream of a transcription start site. In some embodiments, the synthetic enhancer element is located about 25 base pairs, about 50 base pairs, about 75 base pairs, about 100 base pairs, about 125 base pairs, about 150 base pairs, about 175 base pairs, or about 200 base pairs upstream of a transcription start site. In some embodiments, the synthetic enhancer element is located about 25 base pairs, about 50 base pairs, about 75 base pairs or about 100 base pairs upstream of a transcription start site.

[0079] In some embodiments, the synthetic enhancer element is intended to be inserted at a target distance upstream of a transcription start site, and is ultimately inserted at an actual distance that is exactly at the intended distance (i.e.., a perfect insertion). In some embodiments, the synthetic enhancer element is intended to be inserted at a target distance upstream of a transcription start site, and is ultimately inserted at an actual distance that differs by one of more base pairs (i.e., an imperfect insertion). Whether the insertion of the synthetic enhancer element at a genomic position is perfect or imperfect may depend on the method used for insertion, which will be understood by one of skill in the art. A synthetic enhancer element as disclosed herein can be inserted at a specific position by, for example,24MF-365148955Docket No.: 165362002440homology-directed repair or non-homologous end joining. In some embodiments, a synthetic enhancer element is inserted at a specific position by homology-directed repair, producing a clean insertion at a cut site. In some embodiments, a synthetic enhancer element is inserted at a specific position by non-homologous end joining, producing an imperfect insertion with additional base pairs included upstream and downstream of the synthetic enhancer element. See, e.g., Claeys et al., Coordinated gene regulation in maize though CRISPR / Cas-mediated enhancer insertion, Plant Biotechnol J. 22( 1): 16- 18 (2023).

[0080] In some embodiments, the synthetic enhancer element is within a gene expression cassette and is operably linked to a promoter and a sequence to be expressed. In some embodiments, the synthetic enhancer element is within an expression vector. In some embodiments, the synthetic enhancer element is in the genome of a modified plant or part thereof.

[0081] In a preferred embodiment, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 100 and wherein preferably the said enhancer element is repeated in two or more copies and is located about 5 base pairs to about 200 base pairs upstream of a transcription start site, even more preferred of about 25 base pairs to about 100 base pairs upstream of a transcription start site. In a more preferred embodiment, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 138 and is located about 5 base pairs to about 200 base pairs upstream of a transcription start site, even more preferred of about 25 base pairs to about 100 base pairs upstream of a transcription start site.

[0082] In another preferred embodiment, the synthetic enhancer element comprises the nucleotide sequence set forth in SEQ ID NO: 144 and is located about 5 base pairs to about 200 base pairs upstream of a transcription start site, even more preferred of about 25 base pairs to about 100 base pairs upstream of a transcription start site.G. Function of Synthetic Enhancer Elements

[0083] The disclosed synthetic enhancer elements may be operably linked to a promoter or operably linked to a promoter and a gene, where the synthetic enhancer element participates in the regulation and expression of the gene along with the promoter. The synthetic enhancer elements described herein may be used to increase the expression of one or more genes. Gene expression may be increased when the synthetic enhancer element is located in a variety of locations, including but not limited to: within a coding genomic region, within a noncoding 25MF-365148955Docket No.: 165362002440genomic region, within a 5’ untranslated region of a gene, within a 3’ untranslated region of a gene, within an exon, within an intron, upstream of a promoter, within a promoter, downstream of a promoter, upstream of a gene, within a gene, downstream of a gene, upstream of a transcription start site, or downstream of a transcription start site.

[0084] In some embodiments, the synthetic enhancer element increases the expression of a gene that is endogenous to a soybean plant genome. In some embodiments, the gene is a gene selected from the group consisting of AlPlOa, AlPlOb, AML4, CRN, HB-1, RIC1, RIC2, RPF1, JAG1, JAG2, KHZ1, PP2C, TCP5-L, FTla, BS1, BS2, TFLlb, CYP76C-1, CYP76C-2, and NF-YC4. In some embodiments, the gene is a gene relating to an agriculturally important trait in soybean. In some embodiments, the gene relates improved yield characteristics, improved abiotic or biotic stress tolerance, improved pest tolerance, improved disease resistance, or improved quality traits such as nutritional improvements.

[0085] In some embodiments, the synthetic enhancer element increases gene expression by enhancing chromatin accessibility in the promoter region upstream of the gene. In some embodiments, the synthetic enhancer element facilitates interactions with transcription factors and other regulatory elements to enhance gene expression. In some embodiments, the synthetic enhancer element constitutively activates gene transcription, enabling gene expression under conditions where it would otherwise be inactive. In some embodiments, the synthetic enhancer element elevates gene expression to levels exceeding expression levels observed in the absence of the synthetic enhancer element. In some embodiments, the synthetic enhancer element increases gene expression about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, or about 80-fold above levels of expression in the absence of the synthetic enhancer element.H. Validating an Enhancer Element in a Plant or Part Thereof

[0086] As described herein, synthetic enhancer elements may be operably linked to a promoter or operably linked to a promoter and a gene, where the synthetic enhancer element participates in the regulation and expression of the gene along with the promoter. Enhancer elements may increase the expression or transcription of genes. It is well-understood in the art that reporter assays may be used to measure gene expression in biological systems, and to interrogate the impact of certain perturbations on gene expression. In many instances, the expression of a reporter gene can indicate the activity of an enhancer or other genetic26MF-365148955Docket No.: 165362002440modification. For example, the increased expression of a reporter gene in the presence of an enhancer element, compared to in the absence of an enhancer element, may indicate that the synthetic enhancer element increases gene expression.

[0087] In some embodiments, the synthetic enhancer element is validated using a reporter assay, wherein expression of a reporter gene indicates validation of the synthetic enhancer element. The synthetic enhancer element may be validated in reporter assays involving a promoter, wherein the synthetic enhancer element is operably linked to the promoter. In some embodiments, validating the enhancer comprises two or more reporter assays. In some embodiments, the two or more reporter assays are reporter assays as described herein, using different promoters.

[0088] In some aspects, the reporter assay comprises transforming a vector comprising the synthetic enhancer element operably linked to an endogenous promoter, a transcription start site, and a reporter gene into a plant or part thereof, wherein the reporter gene is a luciferase gene, and testing for expression of the luciferase gene. The reporter assay may comprise transformation of one or more vectors into a plant or part thereof and a comparison of the relative expression of the reporter genes in the one or more vectors.

[0089] In some aspects, the reporter assay comprises transforming a first vector comprising, the synthetic enhancer element operably linked to an endogenous promoter, a transcription start site, and a reporter gene into a first plant or part thereof from the plant; transforming a second vector comprising, a control sequence element operably linked to the endogenous promoter, the transcription start site, and the reporter gene into a second plant or part thereof from the plant; and measuring the expression of the reporter gene in the first plant or part thereof and in the second plant or part thereof, wherein increased expression of the reporter gene in the first plant or part thereof compared to the second plant or part thereof indicates that the synthetic enhancer element is effective for increasing expression.I. Reporter Genes

[0090] In some embodiments, a reporter assay is used to experimentally validate an enhancer element. In some embodiments, the reporter assay uses at least one reporter gene. The reporter gene may include, but is not limited to, a luciferase gene, a fluorescence-related gene, or a gene for another detectable marker. The reporter gene may encode a luciferase protein, a fluorescent protein, or any protein that may produce a detectable readout. In some embodiments, the luciferase is a firefly luciferase, a Renilla luciferase, or a nanoluciferase. In 27MF-365148955Docket No.: 165362002440some embodiments, the fluorescent protein is green fluorescent protein, red fluorescent protein, or yellow fluorescent protein. In some embodiments, the reporter assay uses two reporter genes. In some aspects, the vector contains two reporter genes. Each of the reporter genes may be a luciferase gene, a fluorescence gene, or another gene that produces a discernable readout that may be measured. In some aspects, the vector contains two reporter genes that are both luminescence genes. In some aspects, the two reporter genes are a firefly luciferase gene and a Renilla luciferase gene.IL Vectors

[0091] Reporter assays may involve a vector, which may also be referred to as a plasmid, a construct, an expression construct, or an expression vector. In some embodiments, the vector contains one or more genes may be capable of being expressed under certain conditions. In some embodiments, the vector comprises one or more promoters. In some embodiments, the vector comprises one or more enhancer elements, motifs, regulatory motifs, or k-mers. In some embodiments, the one or more genes, one or more promoters, and one or more enhancer elements, motifs, regulatory motifs, or k-mers are operably linked within the vector.III. Transformation

[0092] In some instances, the reporter assay may involve transformation of a plant or part thereof with the vector. In some instances, the plant or part thereof is an isolated protoplast. The vector can be delivered to the plant, meristem cells of the plant, or plant protoplasts by particle mediated delivery, and any other direct method of delivery, such as but not limiting to, Ag ZwctenM -mediated transformation, polyethylene glycol (PEG) -mediated transfection to protoplasts, whiskers mediated transformation, electroporation, particle bombardment, microprojectile-mediated delivery with a biolistic device, DNA injection, and / or by use of cell-penetrating peptides.

[0093] In some embodiments, the vector may be transformed into a plant or part thereof via at least one viral vector selected from the group consisting of adenoviruses, lentiviruses, adeno-associated viruses, retroviruses, gemini viruses, begomoviruses, tobamoviruses, potex viruses, comoviruses, wheat streak mosaic virus, barley stripe mosaic virus, bean yellow dwarf virus, bean pod mottle virus, cabbage leaf curl virus, beet curly top virus, tobacco yellow dwarf virus, tobacco rattle virus, potato virus X, and cowpea mosaic virus. In some embodiments, the vector may be transformed into a cell via at least one bacterial vector28MF-365148955Docket No.: 165362002440capable of transforming a plant cell and selected from the group consisting of Agrobacterium sp., Rhizobium sp., Sinorhizobium (Ensifer) sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., and Phyllobacterium sp. In some embodiments, a viral vector may be delivered to a plant by transformation with Agrobacterium.

[0094] In some embodiments, a T-DNA vector may be used to deliver at least one expression vector to a plant or part thereof. In some embodiments, a T-DNA binary vector is used. In some embodiments, a T-DNA superbinary vector system is used. In other embodiments, a T-DNA ternary vector system is used. In some embodiments, the T-DNA system further comprises an additional virulence gene cluster. In some embodiments, the T-DNA system further comprises an accessory plasmid or virulence helper plasmid. In some embodiments, the T-DNA vector is an Agrobacterium vector.IV. Analysis of the Reporter Assay

[0095] In some aspects, the reporter gene or reporter genes contained within the vectors produce detectable readouts that may be used to analyze results of the reporter assay. In some aspects, the reporter assay readout is the amount of luminescence or fluorescence produced from a single luciferase or fluorescence reporter gene. In some aspects, the reporter assay readout is the amount of luminescence or fluorescence produced from two luciferase or fluorescence reporter genes. In some aspects, the reporter assay readout is the comparison between one reporter gene and a second reporter gene, wherein the first reporter gene is a luciferase gene, and the second reporter gene is a different luciferase gene. In some aspects, the reporter assay readout is the comparison between a firefly luciferase reporter gene and a Renilla luciferase reporter gene.V. Additional Validation Methods

[0096] In some aspects, validating an enhancer element may comprise inserting the synthetic enhancer element upstream of a gene in two or more plant tissues and measuring expression of the gene in the two or more plant tissues, wherein increased expression of the gene compared to endogenous expression of the gene in at least two or the two or more plant tissues indicates validation of the synthetic enhancer element. In some aspects, validating the enhancer may comprise measuring expression of the gene without inserting the enhancer. In some embodiments, an enhancer is validated if the expression of the gene is higher in two or29MF-365148955Docket No.: 165362002440more plant tissues wherein the enhancer has been inserted compared to exogenous expression of the gene without the inserted enhancer.

[0097] In some aspects, validating an enhancer elements according to the methods described herein may comprise the use of gene editing techniques to insert the enhancer into the genome or a plant or part thereof and measuring the expression (mRNA or protein) of a gene. The gene may be selected based on knowledge that the up regulation of the gene in a plant or part thereof confers a favorable phenotype.

[0098] In some aspects, measuring expression comprises measuring expression of mRNA transcribed from a gene. In some aspects, measuring expression comprises qPCR or RNA sequencing. The RNA sequencing may be whole genome or targeted to measure expression of the gene. In some aspects, measuring expression comprises measuring expression of a protein product of the gene. Measuring protein expression may comprise a western blot, ELISA, mass spectrometry, immunohistochemistry or flow cytometry.I. Gene Editing Techniques

[0099] The disclosed synthetic enhancer elements may be introduced into the genome of a plant or part thereof. In some embodiments, a modified plant or part thereof comprises a synthetic enhancer element that is operably linked to an endogenous promoter in the modified plant or part thereof and an endogenous gene in the modified plant or part thereof. In some embodiments, the synthetic enhancer element is introduced into the genome of a plant or part thereof via a gene editing approach. In some embodiments, a gene editing approach involves the introduction of gene editing machinery (e.g., a genome editing reagent) into a modified plant or part thereof. This may include the introduction of a Cas nuclease, a TALEN, or a zinc-finger nuclease. In some embodiments, the synthetic enhancer element is delivered to a plant or part thereof along with gene editing machinery.

[0100] In some embodiments, the synthetic enhancer element or the synthetic enhancer element and gene editing machinery are delivered into a soybean plant or part thereof. In some embodiments, the synthetic enhancer element or the synthetic enhancer element and gene editing machinery are delivered into a maize plant or part thereof. In some embodiments, the synthetic enhancer element and gene editing machinery are delivered into a plant cell of a rootstock, of a grafted scion, of a seed (including mature seed and immature seed), of a plant cutting, of a plant cell culture, of a plant organ (e.g., intact nodal bud, shoot apex or shoot apical meristem, root apex or root apical meristem, lateral meristem, intercalary 30MF-365148955Docket No.: 165362002440meristem, zygotic embryo, somatic embryo, ovule, pollen, microspore, anther, hypocotyl, cotyledon, leaf, petiole, stem, tuber, root, flowers, fruits, shoots, and explants). In some embodiments, the synthetic enhancer element and gene editing machinery are introduced to one or more crop seeds (e.g., com crop seeds, soybean crop seeds, rice crop seeds, wheat crop seeds, tomato crop seeds, citrus fruit crop seeds, cacao crop seeds, potato crop seeds, cotton crop seeds, cabbage crop seeds, mushroom crop seeds, canola crop seeds, papaya crop seeds, and so forth) to germinate one or more crops (e.g., com crop, soybean crop, rice crop, wheat crop, tomato crop, citms fmit crop, cacao crop, potato crop, cotton crop, cabbage crop, mushroom crop, canola crop, papaya crop, and so forth).

[0101] In some embodiments, one or more one chemical, enzymatic, or physical agent, separately or in combination with gene editing machinery, is provided / applied at a location in the plant or plant part other than the plant location, part, or tissue from which the plant cell or plant protoplast is obtained or isolated. In some embodiments, gene editing machinery is applied to adjacent or distal cells or tissues and is transported (e.g., through the vascular system or by cell-to-cell movement) to the meristem from which plant cells or plant protoplasts are subsequently isolated. In some embodiments, a gene editing machinerycontaining composition is applied by soaking a seed or seed fragment or zygotic or somatic embryo in the gene editing machinery-containing composition, whereby the gene editing machinery is delivered to the seed or seed fragment or zygotic or somatic embryo from which plant cells or plant protoplasts are subsequently isolated. In embodiments, a flower bud or shoot tip is contacted with a gene editing machinery-containing composition, whereby the gene editing machinery is delivered to cells in the flower bud or shoot tip from which plant cells or plant protoplasts are subsequently isolated. In embodiments, a gene editing machinery-containing composition is applied to the surface of a plant or of a part of a plant (e.g., a leaf surface), whereby the gene editing machinery is delivered to tissues of the plant from which plant cells or plant protoplasts are subsequently isolated. In embodiments a whole plant or plant tissue is subjected to particle- or nanoparticle-mediated delivery (e.g., Biolistics or carbon nanotube or nanoparticle delivery) of a gene editing machinery-containing composition, whereby the gene editing machinery is delivered to cells or tissues from which plant cells or plant protoplasts are subsequently isolated.

[0102] Gene editing machinery and compositions comprising synthetic enhancer elements can be delivered to the plant and / or meristem cells of the plant by any method of delivery, by particle mediated delivery, and any other direct method of delivery, such as but not limiting31MF-365148955Docket No.: 165362002440to, Agrobacterium-mediated transformation, polyethylene glycol (PEG) -mediated transfection to protoplasts, whiskers mediated transformation, electroporation, particle bombardment, and / or by use of cell-penetrating peptides. In some embodiments, the synthetic enhancer element may be delivered to the plant or part thereof by treatment with pressure, centrifugation, Biolistics or particle bombardment, microinjection, infiltration (e.g., with a syringe), or by direct application to the surface of the plant tissue.

[0103] CRISPR technology for editing the genes of eukaryotes is disclosed in U.S. Patent Application Publications 2016 / 0138008A1 and US2015 / 0344912A1, and in U.S. Pat. Nos.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. Cpfl endonuclease and corresponding guide RNAs and PAM sites are disclosed in U. S. Patent Application Publication 2016 / 0208243 Al. Other CRISPR nucleases useful for editing genomes include C2cl and C2c3 (see Shmakov et al. (2015) Mol. Cell, 60:385-397) and CasX and CasY (see Burstein et al. (2016) Nature, doi:10.1038 / nature21059). 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 disclosed in in U. S. Patent Application Publications US 2015 / 0082478A1 and US 2015 / 0059010A1 and in International Patent Application PCT / US2015 / 038767 Al (published as WO 2016 / 007347 and claiming priority to U.S. Provisional Patent Application 62 / 023,246).

[0104] In some embodiments, the synthetic enhancer element may be transformed into a plant or part thereof via at least one viral vector selected from the group consisting of adenoviruses, lentiviruses, adeno-associated viruses, retroviruses, gemini viruses, begomoviruses, tobamoviruses, potex viruses, comoviruses, wheat streak mosaic virus, barley stripe mosaic virus, bean yellow dwarf virus, bean pod mottle virus, cabbage leaf curl virus, beet curly top virus, tobacco yellow dwarf virus, tobacco rattle virus, potato virus X, and cowpea mosaic virus. In some embodiments, the synthetic enhancer element may be transformed into a plant or part thereof via at least one bacterial vector capable of transforming a plant cell and selected from the group consisting of Agrobacterium sp., Rhizobium sp., Sinorhizobium (Ensifer) sp., Mesorhizobium sp., Bradyrhizobium sp., Azobacter sp., and Phyllobacterium sp. In some embodiments, a viral vector may be delivered to a plant by transformation with Agrobacterium.32MF-365148955Docket No.: 165362002440

[0105] In some embodiments, a T-DNA vector may be used to deliver at least one synthetic enhancer element to a plant or part thereof. In some embodiments, a T-DNA binary vector is used. In some embodiments, a T-DNA superbinary vector system is used. In other embodiments, a T-DNA ternary vector system is used. In some embodiments, the T-DNA system further comprises an additional virulence gene cluster. In some embodiments, the T-DNA system further comprises an accessory plasmid or virulence helper plasmid. In some embodiments, the T-DNA vector is an Agrobacterium vector.SEQUENCES33MF-365148955Docket No.: 16536200244034 MF-365148955Docket No.: 16536200244035MF-365148955Docket No.: 16536200244036MF-365148955Docket No.: 16536200244037MF-365148955Docket No.: 16536200244038MF-365148955Docket No.: 16536200244039MF-365148955Docket No.: 16536200244040MF-365148955Docket No.: 16536200244041MF-365148955Docket No.: 16536200244042MF-365148955Docket No.: 16536200244043MF-365148955Docket No.: 165362002440EXEMPLARY EMBODIMENTS

[0106] Exemplary embodiments provided herein include:1. A gene expression cassette comprising nucleic acid comprising a synthetic enhancer element, a promoter, a sequence to be expressed, whereinthe sequence to be expressed has a transcription start site, and wherein the synthetic enhancer element is operably linked to the promoter and the sequence to be expressed, and wherein the synthetic enhancer element comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, GTAAGC, GCTTAC, CTTA, and CACGTG.2. The gene expression cassette of embodiment 1, further comprising a single copy of the synthetic enhancer element.44MF-365148955Docket No.: 1653620024403. The gene expression cassette of embodiment 1, further comprising two or more copies of the synthetic enhancer element.4. The gene expression cassette of embodiment 1, further comprising three copies of the synthetic enhancer element.5. The gene expression cassette of embodiment 3, wherein the two or more copies of the synthetic enhancer element are separated by 1, 2, 3, 4, 5, or 6 base pairs.6. The gene expression cassette of embodiment 4, wherein the three copies of the synthetic enhancer element are separated by 1, 2, 3, 4, 5, or 6 base pairs.7. The gene expression cassette of any of embodiments 1-6, wherein the synthetic enhancer element is in reverse orientation.8. The gene expression cassette of any of embodiments 1-7, wherein the synthetic enhancer element is between about 40 base pairs to about 1000 base pairs upstream of the transcription start site of the sequence to be expressed.9. The gene expression cassette of any of embodiments 1-8, wherein the synthetic enhancer element is about 75 base pairs, about 100 base pairs, about 150 base pairs, about 225 base pairs, or about 350 base pairs upstream of the transcription start site of the sequence to be expressed.10. The gene expression cassette of any of embodiments 1-9, wherein the synthetic enhancer element is within the promoter.11. The gene expression cassette of any of embodiments 1-9, wherein the synthetic enhancer element is upstream of the promoter.12. The gene expression cassette of any of embodiments 1-9, wherein the synthetic enhancer element is downstream of the promoter.45MF-365148955Docket No.: 16536200244013. The gene expression cassette of any of embodiments 1-12, wherein the synthetic enhancer element comprises the nucleotide sequence set forth in any one of SEQ ID NO: 100, SEQ ID NO: 115, or SEQ ID NO: 107.14. The gene expression cassette of any of embodiments 1-13, wherein the sequence to be expressed is a gene that is endogenous to a soybean plant genome.15. The gene expression cassette of embodiment 14, wherein the gene is a gene selected from the group consisting of AlPlOa, AlPlOb, AML4, CRN, HB-1, RIC1, RIC2, RPF1, JAG1, JAG2, KHZ1, PP2C, TCP5-L, FTla, BS1, BS2, TFLlb, CYP76C-1, CYP76C-2, and NF-YC4.16. The gene expression cassette of any of embodiments 1-15, wherein the promoter is endogenous to a soybean plant genome.17. The gene expression cassette of embodiment 16, wherein the promoter is a promoter selected from the group consisting of the AlPlOa promoter, the AlPlOb promoter, the AML4 promoter, the CRN promoter, the HB-1 promoter, the RIC1 promoter, the RIC2 promoter, the RPF1 promoter, the JAG1 promoter, the JAG2 promoter, the KHZ1 promoter, the PP2C promoter, the TCP5-L promoter, the FTla promoter, the BS1 promoter, the BS2 promoter, the TFLlb promoter, the CYP76C-1 promoter, the CYP76C-2 promoter, the NF-YC4 promoter, the MYB promoter, and the Hl_5 promoter.18. The gene expression cassette of any of embodiments 1-17, wherein the synthetic enhancer element increases expression of a transcript or protein encoded by the sequence to be expressed.19. An expression vector comprising the gene expression cassette of any of embodiments 1-18.20. The expression vector of embodiment 19, wherein the sequence to be expressed comprises a protein coding sequence.21. A plant cell comprising the gene expression cassette of any of embodiments 1-18.46MF-365148955Docket No.: 16536200244022. A plant cell comprising the expression vector of embodiment 19 or embodiment 20.23. The plant cell of embodiment 21 or embodiment 22, wherein the plant cell is a non-regenerable plant cell which is not capable of being regenerated to produce a plant.24. The plant cell of any of embodiments 21-23, wherein the plant cell is a soybean plant cell.25. A modified plant or part thereof comprising a synthetic enhancer element, wherein the synthetic enhancer element comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, GTAAGC, GCTTAC, CTTA, and CACGTG, and wherein the synthetic enhancer element is operably linked to an endogenous promoter in the modified plant or part thereof and an endogenous gene in the modified plant or part thereof.26. The modified plant or part thereof of embodiment 25, further comprising a single copy of the synthetic enhancer element.27. The modified plant or part thereof of embodiment 25, further comprising two or more copies of the synthetic enhancer element.28. The modified plant or part thereof of embodiment 25, further comprising three copies of the synthetic enhancer element.29. The modified plant or part thereof of embodiment 27, wherein the two or more copies of the synthetic enhancer element are separated by 1, 2, 3, 4, 5, or 6 base pairs.30. The modified plant or part thereof of embodiment 28, wherein the three copies of the synthetic enhancer element are separated by 1, 2, 3, 4, 5, or 6 base pairs.31. The modified plant or part thereof of any of embodiments 25-30, wherein the synthetic enhancer element is in reverse orientation.47MF-365148955Docket No.: 16536200244032. The modified plant or part thereof of any of embodiments 25-31, wherein the synthetic enhancer element is within 1000 base pairs of the endogenous gene.33. The modified plant or part thereof of any of embodiments 25-32, wherein the synthetic enhancer element is within a noncoding genomic region.34. The modified plant or part thereof of any of embodiments 25-33, wherein the synthetic enhancer element is between about 40 base pairs to about 1000 base pairs upstream of a transcription start site of the endogenous gene.35. The modified plant or part thereof of any of embodiments 25-34, wherein the synthetic enhancer element is about 75 base pairs, about 100 base pairs, about 150 base pairs, about 225 base pairs, or about 350 base pairs upstream of a transcription start site of the endogenous gene.36. The modified plant or part thereof of any of embodiments 25-35, wherein the synthetic enhancer element is within the endogenous promoter.37. The modified plant or part thereof of any of embodiments 25-35, wherein the synthetic enhancer element is upstream of the endogenous promoter.38. The modified plant or part thereof of any of embodiments 25-35, wherein the synthetic enhancer element is downstream of the endogenous promoter.39. The modified plant or part thereof of any of embodiments 25-38, wherein the synthetic enhancer element comprises the nucleotide sequence set forth in any one of SEQ ID NO: 100, SEQ ID NO: 115, or SEQ ID NO: 107.40. The modified plant or part thereof of any of embodiments 25-39, wherein the modified plant or part thereof is a soybean plant or part thereof.41. The modified plant or part thereof of embodiment 40, wherein the endogenous gene is a gene selected from the group consisting of AlPlOa, AlPlOb, AML4, CRN, HB-1, RIC1,48MF-365148955Docket No.: 165362002440RIC2, RPF1, JAG1, JAG2, KHZ1, PP2C, TCP5-L, FTla, BS1, BS2, TFLlb, CYP76C-1, CYP76C-2, and NF-YC4.42. The modified plant or part thereof of embodiment 40 or 41, wherein the endogenous promoter is a promoter selected from the group consisting of: the AlPlOa promoter, the AlPlOb promoter, the AML4 promoter, the CRN promoter, the HB-1 promoter, the RIC1 promoter, the RIC2 promoter, the RPF1 promoter, the JAG1 promoter, the JAG2 promoter, the KHZ1 promoter, the PP2C promoter, the TCP5-L promoter, the FTla promoter, the BS1 promoter, the BS2 promoter, the TFLlb promoter, the CYP76C-1 promoter, the CYP76C-2 promoter, the NF-YC4 promoter, the MYB promoter, and the Hl_5 promoter.43. The modified plant or part thereof of any of embodiments 25-42, wherein the synthetic enhancer element increases expression of a transcript or protein encoded by the endogenous gene.44. The modified plant or part thereof of embodiment 43, wherein the increased expression of a transcript or protein encoded by the endogenous gene contributes to improved yield characteristics, improved abiotic or biotic stress tolerance, improved pest tolerance, improved disease resistance, or improved quality traits such as nutritional improvements in the modified plant or part thereof.

[0107] Further exemplary embodiments provided herein include:1. A gene expression cassette comprising nucleic acid comprising a synthetic enhancer element, a promoter, and a sequence to be expressed, wherein:(a) the sequence to be expressed has a transcription start site;(b) the synthetic enhancer element is operably linked to the promoter and the sequence to be expressed; and(c) the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, 138-140, 149-162, GTAAGC, GCTTAC, CTTA, and CACGTG.2. The gene expression cassette of embodiment 1, wherein the cassette comprises a single copy of the synthetic enhancer element.49MF-365148955Docket No.: 1653620024403. The gene expression cassette of embodiment 1, further comprising two or more copies of the synthetic enhancer element.4. The gene expression cassette of embodiment 1, further comprising three copies, four copies, five copies, or six copies of the synthetic enhancer element.5. The gene expression cassette of embodiment 3 or 4, further comprising a spacer sequence between each copy of the synthetic enhancer element.6. The gene expression cassette of embodiment 5, wherein the spacer sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 base pairs in length.7. The gene expression cassette of embodiment 5, wherein the spacer sequence comprises the nucleotide sequence set forth in SEQ ID NO: 145.8. The gene expression cassette of any one of embodiments 1-7, wherein the synthetic enhancer element is in reverse orientation.9. The gene expression cassette of any one of embodiments 1-8, wherein the synthetic enhancer element is between about 5 base pairs to about 1000 base pairs upstream of the transcription start site of the sequence to be expressed.10. The gene expression cassette of any one of embodiments 1-9, wherein the synthetic enhancer element is about 25 base pairs, about 50 base pairs, about 75 base pairs, about 100 base pairs, about 125 base pairs, about 150 base pairs, about 175 base pairs, or about 200 base pairs upstream of the transcription start site of the sequence to be expressed.11. The gene expression cassette of any one of embodiments 1-10, wherein the synthetic enhancer element is within the promoter.12. The gene expression cassette of any one of embodiments 1-10, wherein the synthetic enhancer element is upstream of the promoter.50MF-365148955Docket No.: 16536200244013. The gene expression cassette of any one of embodiments 1-10, wherein the synthetic enhancer element is downstream of the promoter.14. The gene expression cassette of any one of embodiments 1-13, wherein the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 115, and SEQ ID NO: 107.15. The gene expression cassette of any one of embodiments 1-14, wherein the sequence to be expressed is a gene that is endogenous to a soybean plant genome.16. The gene expression cassette of embodiment 15, wherein the gene is selected from the group consisting of AlPlOa, AlPlOb, AML4, CRN, HB-1, RIC1, RIC2, RPF1, JAG1, JAG2, KHZ1, PP2C, TCP5-L, FTla, BS1, BS2, TFLlb, CYP76C-1, CYP76C-2, and NF-YC4.17. The gene expression cassette of any one of embodiments 1-16, wherein the promoter is endogenous to a soybean plant genome.18. The gene expression cassette of embodiment 17, wherein the promoter is a promoter selected from the group consisting of the AlPlOa promoter, the AlPlOb promoter, the AML4 promoter, the CRN promoter, the HB-1 promoter, the RIC1 promoter, the RIC2 promoter, the RPF1 promoter, the JAG1 promoter, the JAG2 promoter, the KHZ1 promoter, the PP2C promoter, the TCP5-L promoter, the FTla promoter, the BS1 promoter, the BS2 promoter, the TFLlb promoter, the CYP76C-1 promoter, the CYP76C-2 promoter, the NF-YC4 promoter, the MYB promoter, and the Hl_5 promoter.19. The gene expression cassette of any one of embodiments 1-18, wherein the synthetic enhancer element increases expression of a transcript or protein encoded by the sequence to be expressed.20. An expression vector comprising the gene expression cassette of any one of embodiments 1-19.51MF-365148955Docket No.: 16536200244021. The expression vector of embodiment 20, wherein the sequence to be expressed comprises a protein coding sequence.22. A plant cell comprising the gene expression cassette of any one of embodiments 1-19.23. A plant cell comprising the expression vector of embodiment 20 or 21.24. The plant cell of embodiment 22 or 23, wherein the plant cell is a non-regenerable plant cell which is not capable of being regenerated to produce a plant.25. The plant cell of any one of embodiments 22-24, wherein the plant cell is a soybean plant cell, a corn plant cell, or a wheat plant cell.26. A modified plant or part thereof comprising a synthetic enhancer element, wherein the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, 138-140, 149-162, GTAAGC, GCTTAC, CTTA, and CACGTG, and wherein the synthetic enhancer element is operably linked to an endogenous promoter in the modified plant or part thereof and an endogenous gene in the modified plant or part thereof.27. The modified plant or part thereof of embodiment 26, wherein the plant or part thereof comprises a single copy of the synthetic enhancer element.28. The modified plant or part thereof of embodiment 26, further comprising two or more copies of the synthetic enhancer element.29. The modified plant or part thereof of embodiment 26, further comprising three copies, four copies, five copies, or six copies of the synthetic enhancer element.30. The modified plant or part thereof of embodiment 28 or 29, further comprising a spacer sequence between each copy of the synthetic enhancer element.52MF-365148955Docket No.: 16536200244031. The modified plant or part thereof of embodiment 30, wherein the spacer sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 base pairs in length.32. The modified plant or part thereof of embodiment 30, wherein the spacer sequence comprises the nucleotide sequence set forth in SEQ ID NO: 145.33. The modified plant or part thereof of any one of embodiments 26-32, wherein the synthetic enhancer element is in reverse orientation.34. The modified plant or part thereof of any one of embodiments 26-33, wherein the synthetic enhancer element is within 1000 base pairs of the endogenous gene.35. The modified plant or part thereof of any one of embodiments 26-34, wherein the synthetic enhancer element is within a noncoding genomic region.36. The modified plant or part thereof of any one of embodiments 26-35, wherein the synthetic enhancer element is between about 5 base pairs to about 1000 base pairs upstream of a transcription start site of the endogenous gene.37. The modified plant or part thereof of any one of embodiments 26-36, wherein the synthetic enhancer element is about 25 base pairs, about 50 base pairs, about 75 base pairs, about 100 base pairs, about 125 base pairs, about 150 base pairs, or about 200 base pairs upstream of a transcription start site of the endogenous gene.38. The modified plant or part thereof of any one of embodiments 26-37, wherein the synthetic enhancer element is within the endogenous promoter.39. The modified plant or part thereof of any one of embodiments 26-37, wherein the synthetic enhancer element is upstream of the endogenous promoter.40. The modified plant or part thereof of any one of embodiments 26-37, wherein the synthetic enhancer element is downstream of the endogenous promoter.53MF-365148955Docket No.: 16536200244041. The modified plant or part thereof of any one of embodiments 26-40, wherein the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 115, and SEQ ID NO: 107.42. The modified plant or part thereof of any one of embodiments 26-41, wherein the modified plant or part thereof is a soybean plant or part thereof.43. The modified plant or part thereof of embodiment 42, wherein the endogenous gene is a gene selected from the group consisting of AlPlOa, AlPlOb, AML4, CRN, HB-1, RIC1, RIC2, RPF1, JAG1, JAG2, KHZ1, PP2C, TCP5-L, FTla, BS1, BS2, TFLlb, CYP76C-1, CYP76C-2, and NF-YC4.44. The modified plant or part thereof of embodiment 42 or 43, wherein the endogenous promoter is a promoter selected from the group consisting of: the AlPlOa promoter, the AlPlOb promoter, the AML4 promoter, the CRN promoter, the HB-1 promoter, the RIC1 promoter, the RIC2 promoter, the RPF1 promoter, the JAG1 promoter, the JAG2 promoter, the KHZ1 promoter, the PP2C promoter, the TCP5-L promoter, the FTla promoter, the BS1 promoter, the BS2 promoter, the TFLlb promoter, the CYP76C-1 promoter, the CYP76C-2 promoter, the NF-YC4 promoter, the MYB promoter, and the Hl_5 promoter.45. The modified plant or part thereof of any one of embodiments 26-44, wherein the synthetic enhancer element increases expression of a transcript or protein encoded by the endogenous gene.46. The modified plant or part thereof of embodiment 45, wherein the increased expression of a transcript or protein encoded by the endogenous gene contributes to improved yield characteristics, improved abiotic or biotic stress tolerance, improved pest tolerance, improved disease resistance, or improved quality traits such as nutritional improvements in the modified plant or part thereof.54MF-365148955Docket No.: 165362002440EXAMPLESExample 1: Synthetic enhancer elements show activity in dual luciferase assays

[0108] To investigate the activity of synthetic enhancer elements that may increase the expression of a sequence to be expressed, or of a transcript or protein encoded by a sequence to be expressed, synthetic enhancer elements were investigated in one or more copies while operably linked to a promoter and a sequence to be expressed (FIG. 1). A series of expression vectors were generated for use in dual luciferase assays to synthetic enhancer element activity.

[0109] All dual luciferase vectors contained two cassettes: firefly luciferase (FLUC) for testing experimental conditions and Renilla luciferase (RLUC) for transfection efficiency control (FIG. 2A). Known or candidate enhancer elements were evaluated in dual luciferase assays by measuring the increase in gene expression of FLUC, measured by the ratio of luminescence signal from FLUC over RLUC. The dual luciferase vectors were used to test known enhancer elements (“3x OCS”, “35S enhancer”) and candidate enhancer elements (“Experimental enhancer”) (FIG. 2B).

[0110] To generate the FLUC cassette in each dual luciferase vector, a low expressing promoter, TFLlb, was amplified from the soybean genome and cloned upstream of the FLUC gene, followed by the CaMV 35S terminator. To generate the RLUC cassette in each dual luciferase vector, RLUC was cloned with the CaMV 35S promoter, RLUC gene, and CaMV 35S terminator. To screen enhancer candidates, a LacZ cassette was cloned via Gibson assembly into the TFLlb promoter sequence of the dual luciferase vector 75 base pairs upstream of the FLUC transcription start site. The LacZ cassette enables blue-white screening to identify vectors with correctly inserted enhancers. Known enhancer elements and candidate enhancer elements were synthesized with 4 base pair overhangs and ligated into the screening vector digested with Esp3I.

[0111] Protoplasts were isolated from 10 to 11-day old soy epicotyl tissue from Glycine max strains (e.g., cultivar Williams 82) and transfected with dual luciferase vectors containing either known or candidate enhancer elements via polyethylene glycol (PEG)-mediated transfection. Then, the dual luciferase assays were run using the Dual-Glo kit. Transfected protoplasts were incubated for 48 hours prior to dual luciferase readout. The assay readout was FLUC / RLUC luminescence, which measures changes in FLUC expression based on the experimental condition while normalizing to RLUC expression to account for 55MF-365148955Docket No.: 165362002440sample-to-sample variation. FLUC values were divided by RLUC values to analyze expression via relative change from control FLUC / RLUC values. Candidate enhancer elements were validated when the FLUC / RLUC ratio was increased compared to the TFLlb control (wildtype) vector. The following vectors were tested in an initial experiment to validate the dual luciferase assays:Table 1: Expression vectors for dual luciferase assays.

[0112] The insertion of the OCS enhancer three times in tandem (FIG. 3, “3x OCS”) into the promoter region upstream of FLUC produced a 28-fold increase in luminescence, indicating a high degree of enhancer activity. The insertion of the CaMV 35S enhancer (FIG.3, “35S Enhancer”) into the promoter region upstream of FLUC produced a 59-fold increase in luminescence, indicating a high degree of enhancer activity.

[0113] Candidate enhancers were also tested in dual luciferase vectors. A vector (SEQ ID NO: 124, comprising SEQ ID NOs: 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, FIG. 7A) containing three copies of Enhancer 100 (single copy SEQ ID NO: 100; three copies SEQ ID NO: 138) increased FLUC / RLUC luminescence over a control vector containing the wild-type TFLlb promoter, and increased FLUC / RLUC luminescence over a control vector containing three copies of the OCS element (FIG. 4). A vector (SEQ ID NO: 126, comprising SEQ ID NOs: 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, FIG.7B) containing three copies of Enhancer 115 (single copy SEQ ID NO: 115; three copies SEQ ID NO: 140) increased FLUC / RLUC luminescence over a control vector containing the wild-type TFLlb promoter and increased FLUC / RLUC luminescence over a control vector56MF-365148955Docket No.: 165362002440containing three copies of the OCS element (FIGS. 5A-5B). A vector (SEQ ID NO: 125, comprising SEQ ID NOs: 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, FIG. 7C) containing three copies of Enhancer 107 (single copy SEQ ID NO: 107; three copies SEQ ID NO: 140) increased FLUC / RLUC luminescence over a control vector containing the wildtype TFLlb promoter and increased FLUC / RLUC luminescence over a control vector containing three copies of the OCS element (FIGS. 5A-5B).

[0114] Enhancers were also tested in dual luciferase vectors containing promoters from control genes that were randomly selected based on their expression levels in the protoplast assay system and specifically, expression as determined by RNA sequencing data taken at various timepoints during the protoplast lifespan (FIGS. 6A-6B). The vectors were constructed following the steps described above, but with a promoter from a mediumexpression control gene (a sample gene known to exhibit medium expression levels within the protoplast assay system) and a promoter from a high-expression control gene (a sample gene known to exhibit high expression levels within the protoplast assay system).

[0115] Dual luciferase assays, as described above, were conducted for the following constructs: a vector containing the wild-type TFLlb promoter (“TFLlb - WT”), a vector containing three copies of the OCS enhancer and the TFLlb promoter (“TFLlb - 3x OCS”, SEQ ID NO: 163), a vector containing the wild-type promoter of the medium-expression control gene (“MID - WT”, SEQ ID NO: 165), a vector containing three copies of the OCS enhancer and the promoter of the medium-expression control gene (“MID - 3x OCS”, SEQ ID NO: 166), a vector containing the wild-type promoter of the high-expression control gene (“HIGH - WT”, SEQ ID NO: 167), and a vector containing three copies of the OCS enhancer and the promoter of the high-expression control gene (“HIGH - 3x OCS”, SEQ ID NO: 168).

[0116] The dual luciferase assays were performed with vectors at 55 ng / L (FIG. 6A) or 110 ng / pL (FIG. 6B). Irrespective of the promoter (TFLlb, medium-expression control, high-expression control) or vector concentration (55 ng / pL, 110 ng / pL), the vectors containing three copies of the OCS enhancer consistently increased FLUC / RLUC luminescence over control vectors containing only the wild- type promoters.

[0117] Next, the ability of Enhancer 100 (SEQ ID NO: 100) to improve expression of different promoters was tested. Enhancer 100 was tested in three copies with no linker between each copy (SEQ ID NO: 138, “3x_12bp_2666”). Dual luciferase assays, as described above, were conducted for the following constructs: a vector containing the wild-57MF-365148955Docket No.: 165362002440type TFLlb promoter (“TFLlb - WT”), a vector containing three copies of the OCS enhancer and the TFLlb promoter (“TFLlb - 3x OCS”), a vector containing 3x_12bp_2666 and the TFLlb promoter (“TFLlb - 3x_12bp_2666”, SEQ ID NO: 164), a vector containing the wild-type promoter of the high-expression control gene (“HIGH - WT”), a vector containing three copies of the OCS enhancer and the promoter of the high-expression control gene (“HIGH - 3x OCS”), and a vector containing 3x_12bp_2666 and the promoter of the high-expression control gene (“HIGH - 3x_12bp_2666”).

[0118] The dual luciferase assays were performed with these vectors at 110 ng / pL (FIG. 8).The results of these experiments surprisingly showed that irrespective of the promoter (TFLlb or high expression promoter control) the vectors containing three copies of Enhancer 100 (SEQ ID NO: 138) consistently increased FLUC / RLUC luminescence over control vectors containing only the wild-type promoters.

[0119] To investigate the copy number effect of enhancer elements, Enhancer 100 (SEQ ID NO: 100) was repeated in different enhancer constructs from 2x to 6x (i.e., in two copies, three copies, four copies, five copies, or six copies) without linker or spacer sequencers present between each copy (2x_12bp_2666 (SEQ ID NO: 149), 3x_12bp_2666 (SEQ ID NO: 138), 4x_12bp_2666 (SEQ ID NO: 150), 5x_12bp_2666 (SEQ ID NO: 151), 6x_12bp_2666 (SEQ ID NO: 152)). The ability of the different enhancer constructs to enhance the TFL1B promoter was tested in the dual luciferase assay as described above.

[0120] The dual luciferase assays were performed with the vectors comprising Enhancer 100 in different copy numbers and the TFL1B promoter at 110 ng / pL and results are displayed in FIG. 9. The results illustrated a dependency on copy number of Enhancer 100 (i.e., increased copy number was associated with an increase in expression), with a plateau reached between five and six copies of the enhancer.

[0121] Next, the distance of the enhancers to the transcription start site (TSS) was investigated further. The construct with three copies of Enhancer 100 without spacers (3x_12bp_2666; SEQ ID NO: 138) and 3xOCS were cloned at various distances from the TSS of the luciferase reporter gene of the dual luciferase assay as described herein.

[0122] The enhancers were cloned at 75 base pairs, 100 base pairs, 150 base pairs, 225 base pairs and 350 base pairs from the TSS, using the TFL1B promoter. The dual luciferase assay was run as described above with 110 ng / pL of each vector. FIG. 10 illustrates the dependence on distance from the TSS of the enhancer element (i.e., expression was most58MF-365148955Docket No.: 165362002440enhanced at 75 base pairs from the TSS). This was observed for both the OCS element and Enhancer 100.Example 2: Genomic insertion of synthetic enhancer elements

[0123] The efficiency of the various synthetic enhancer elements as described herein is tested using the soybean transient transgenic hairy root system as is performed essentially as described in the literature (Toth et al., Curr Protoc Plant Biol 2016 May; 1(1): 1- 13. Doi: 10.1002 / cppb.20017; Song et al., Curr Protoc 2021 July; l(7):el95. doi: 10.1002 / cpzl.l95). Specifically, the ability of Enhancer 100 (SEQ ID NO: 100) to improve expression of different promoters is tested using the hairy root system. Therefore, the Enhancer 100 is tested in three copies with no linker between each copy (SEQ ID NO: 138, “3x_12bp_2666”) and is inserted approximately 50 base pairs upstream of a gene of interest and its effect on gene expression of the gene of interest is evaluated. In a separate experiment the synthetic enhancer element according to SEQ ID NO: 144 is inserted approximately 50 base pairs upstream of a gene of interest and its effect on gene expression of the gene of interest is evaluated in the soybean transgenic hairy root system.

[0124] Various other methods exist in the art for the insertion of DNA elements such as synthetic enhancer elements into the genome of a plant such as a soy plant, maize plant or wheat plant. Examples of obtaining a stably transformed plant by precision editing an insertion of a synthetic enhancer element may be found in PCT / US2020 / 039410 “Improved homology dependent repair genome editing”, US 17 / 456465 “Genetic regulatory element”, Chen et al., An update on precision genome editing by homology-directed repair in plants. Plant Physiol. 2022 Mar 28;188(4):1780-1794, Dong et al., Targeted DNA insertion in plants, Proc. Natl. Acad. Sci. U.S.A. 118 (22) e2004834117 (2021), Zhongsen et al., Cas9-Guide RNA Directed Genome Editing in Soybean, Plant Physiology, 169(2): 960-970 (2015), and Claeys et al., Coordinated gene regulation in maize though CRISPR / Cas-mediated enhancer insertion, Plant Biotechnol J. 22(1): 16-18 (2023).59MF-365148955

Claims

Docket No.: 165362002440CLAIMSWhat is claimed is:

1. A gene expression cassette comprising nucleic acid comprising a synthetic enhancer element, a promoter, and a sequence to be expressed, wherein:(a) the sequence to be expressed has a transcription start site;(b) the synthetic enhancer element is operably linked to the promoter and the sequence to be expressed; and(c) the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, 138-140, 149-162, GTAAGC, GCTTAC, CTTA, and CACGTG.

2. The gene expression cassette of claim 1, wherein the gene expression cassette comprises a single copy of the synthetic enhancer element.

3. The gene expression cassette of claim 1, further comprising two or more copies of the synthetic enhancer element.

4. The gene expression cassette of claim 1, further comprising three copies, four copies, five copies, or six copies of the synthetic enhancer element.

5. The gene expression cassette of claim 3, further comprising a spacer sequence between each copy of the synthetic enhancer element.

6. The gene expression cassette of claim 5, wherein the spacer sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 base pairs in length.

7. The gene expression cassette of claim 5, wherein the spacer sequence comprises the nucleotide sequence set forth in SEQ ID NO: 145.

8. The gene expression cassette of claim 1, wherein the synthetic enhancer element is in reverse orientation.60MF-365148955Docket No.: 1653620024409. The gene expression cassette of claim 1, wherein the synthetic enhancer element is between about 5 base pairs to about 1000 base pairs upstream of the transcription start site of the sequence to be expressed.

10. The gene expression cassette of claim 1, wherein the synthetic enhancer element is about 25 base pairs, about 50 base pairs, about 75 base pairs, about 100 base pairs, about 125 base pairs, about 150 base pairs, about 175 base pairs, or about 200 base pairs upstream of the transcription start site of the sequence to be expressed.

11. The gene expression cassette of claim 1, wherein the synthetic enhancer element is within the promoter.

12. The gene expression cassette of claim 1, wherein the synthetic enhancer element is upstream of the promoter.

13. The gene expression cassette of claim 1, wherein the synthetic enhancer element is downstream of the promoter.

14. The gene expression cassette of claim 1, wherein the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 115, and SEQ ID NO: 107.

15. The gene expression cassette of claim 1, wherein the sequence to be expressed is a gene that is endogenous to a soybean plant genome.

16. The gene expression cassette of claim 15, wherein the gene is selected from the group consisting of AlPlOa, AlPlOb, AML4, CRN, HB-1, RIC1, RIC2, RPF1, JAG1, JAG2, KHZ1, PP2C, TCP5-L, FTla, BS1, BS2, TFLlb, CYP76C-1, CYP76C-2, and NF-YC4.

17. The gene expression cassette of claim 1, wherein the promoter is endogenous to a soybean plant genome.61MF-365148955Docket No.: 16536200244018. The gene expression cassette of claim 17, wherein the promoter is a promoter selected from the group consisting of the AlPlOa promoter, the AlPlOb promoter, the AML4 promoter, the CRN promoter, the HB-1 promoter, the RIC1 promoter, the RIC2 promoter, the RPF1 promoter, the JAG1 promoter, the JAG2 promoter, the KHZ1 promoter, the PP2C promoter, the TCP5-L promoter, the FTla promoter, the BS1 promoter, the BS2 promoter, the TFLlb promoter, the CYP76C-1 promoter, the CYP76C-2 promoter, the NF-YC4 promoter, the MYB promoter, and the Hl_5 promoter.

19. The gene expression cassette of claim 1, wherein the synthetic enhancer element increases expression of a transcript or protein encoded by the sequence to be expressed.

20. An expression vector comprising the gene expression cassette of any one of claims 1-19.

21. The expression vector of claim 20, wherein the sequence to be expressed comprises a protein coding sequence.

22. A plant cell comprising the gene expression cassette of any one of claims 1-19.

23. A plant cell comprising the expression vector of claim 20.

24. The plant cell of claim 22, wherein the plant cell is a non-regenerable plant cell which is not capable of being regenerated to produce a plant.

25. The plant cell of claim 22, wherein the plant cell is a soybean plant cell, a com plant cell, or a wheat plant cell.

26. A modified plant or part thereof comprising a synthetic enhancer element, wherein the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3-38, 40-96, 98, 100-115, 117-123, 138-140, 149-162, GTAAGC, GCTTAC, CTTA, and CACGTG, and wherein the synthetic enhancer element is operably linked to an endogenous promoter in the modified plant or part thereof and an endogenous gene in the modified plant or part thereof.62MF-365148955Docket No.: 16536200244027. The modified plant or part thereof of claim 26, wherein the plant or part thereof comprises a single copy of the synthetic enhancer element.

28. The modified plant or part thereof of claim 26, further comprising two or more copies of the synthetic enhancer element.

29. The modified plant or part thereof of claim 26, further comprising three copies, four copies, five copies, or six copies of the synthetic enhancer element.

30. The modified plant or part thereof of claim 28, further comprising a spacer sequence between each copy of the synthetic enhancer element.

31. The modified plant or part thereof of claim 30, wherein the spacer sequence is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 base pairs in length.

32. The modified plant or part thereof of claim 30, wherein the spacer sequence comprises the nucleotide sequence set forth in SEQ ID NO: 145.

33. The modified plant or part thereof of claim 26, wherein the synthetic enhancer element is in reverse orientation.

34. The modified plant or part thereof of claim 26-33, wherein the synthetic enhancer element is within 1000 base pairs of the endogenous gene.

35. The modified plant or part thereof of claim 26, wherein the synthetic enhancer element is within a noncoding genomic region.

36. The modified plant or part thereof of claim 26, wherein the synthetic enhancer element is between about 5 base pairs to about 1000 base pairs upstream of a transcription start site of the endogenous gene.

37. The modified plant or part thereof of claim 26, wherein the synthetic enhancer element is about 25 base pairs, about 50 base pairs, about 75 base pairs, about 100 base pairs,63MF-365148955Docket No.: 165362002440about 125 base pairs, about 150 base pairs, or about 200 base pairs upstream of a transcription start site of the endogenous gene.

38. The modified plant or part thereof of claim 26, wherein the synthetic enhancer element is within the endogenous promoter.

39. The modified plant or part thereof of claim 26, wherein the synthetic enhancer element is upstream of the endogenous promoter.

40. The modified plant or part thereof of claim 26, wherein the synthetic enhancer element is downstream of the endogenous promoter.

41. The modified plant or part thereof of claim 26, wherein the synthetic enhancer element comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 115, and SEQ ID NO: 107.

42. The modified plant or part thereof of claim 26, wherein the modified plant or part thereof is a soybean plant or part thereof.

43. The modified plant or part thereof of claim 42, wherein the endogenous gene is a gene selected from the group consisting of AlPlOa, AlPlOb, AML4, CRN, HB-1, RIC1, RIC2, RPF1, JAG1, JAG2, KHZ1, PP2C, TCP5-L, FTla, BS1, BS2, TFLlb, CYP76C-1, CYP76C-2, and NF-YC4.

44. The modified plant or part thereof of claim 42 or 43, wherein the endogenous promoter is a promoter selected from the group consisting of: the AlPlOa promoter, the AlPlOb promoter, the AML4 promoter, the CRN promoter, the HB-1 promoter, the RIC1 promoter, the RIC2 promoter, the RPF1 promoter, the JAG1 promoter, the JAG2 promoter, the KHZ1 promoter, the PP2C promoter, the TCP5-L promoter, the FTla promoter, the BS1 promoter, the BS2 promoter, the TFLlb promoter, the CYP76C-1 promoter, the CYP76C-2 promoter, the NF-YC4 promoter, the MYB promoter, and the Hl_5 promoter.64MF-365148955Docket No.: 16536200244045. The modified plant or part thereof of any one of claims 26-43, wherein the synthetic enhancer element increases expression of a transcript or protein encoded by the endogenous gene.

46. The modified plant or part thereof of claim 45, wherein the increased expression of a transcript or protein encoded by the endogenous gene contributes to improved yield characteristics, improved abiotic or biotic stress tolerance, improved pest tolerance, improved disease resistance, or improved quality traits such as nutritional improvements in the modified plant or part thereof.65MF-365148955

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