WO2020117837A1 - Methods and compositions for improving silage - Google Patents

Methods and compositions for improving silage Download PDF

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Publication number
WO2020117837A1
WO2020117837A1 PCT/US2019/064282 US2019064282W WO2020117837A1 WO 2020117837 A1 WO2020117837 A1 WO 2020117837A1 US 2019064282 W US2019064282 W US 2019064282W WO 2020117837 A1 WO2020117837 A1 WO 2020117837A1
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seq
com plant
bmr3
plant
modified
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PCT/US2019/064282
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French (fr)
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Edward Cargill
Sivalinganna Manjunath
Linda RYMARQUIS
Michelle VALENTINE
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Monsanto Technology Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8255—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving lignin biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01068—Caffeate O-methyltransferase (2.1.1.68)

Definitions

  • the present disclosure relates to the field of biotechnology. More specifically, the present disclosure relates to compositions and methods for producing com plants that have improved digestibility. In particular, the present disclosure relates to com plants, com plant tissues and com plant seeds that contain altered Brown midrib 3 (BMR3) genes and proteins.
  • BMR3 Brown midrib 3
  • Brown Midrib 3 (Bmr3) (Morrow, S.L. et al. 1997. Molecular characterization of a brown midrib3 deletion mutation in maize. Molecular Breeding. 3:351-357.) and Brown Midrib 4 (Bmr4) (Burnham, C.R. 1947. Maize Genet Coop News 21:36.) genes produce com plants with reddish-brown pigmentation of the leaf midrib and reduced lignin levels.
  • These brown midrib hybrids produce forage that typically has about 5-10 percentage points higher in fiber digestibility than non-brown midrib com hybrids. Higher fiber digestibility is believed to contribute to higher feed intakes and greater milk production by dairy cows. Farraretto and Shaver. J Dairy Sci 98: 2662.
  • brown midrib hybrids exhibit yield drag compared to non brown midrib hybrids.
  • brown midrib hybrids exhibit defects in plant standability, since lignin contributes to the stability of plant architecture.
  • Negative agronomic traits such as a lack of disease resistance and increased susceptibility to drought have also been reported for the currently available brown midrib hybrids.
  • the present disclosure describes using a gene editing approach to provide brown midrib com plants with improved agronomic traits.
  • a modified com plant comprising an edited allele of Bmr3, wherein the modified com plant exhibits brown pigmentation in the leaf midrib at the v3-vl0 stage.
  • the modified com plant is homozygous for an edited allele of Bmr3.
  • the modified com plant is heterozygous for an edited allele of Bmr3.
  • the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507.
  • the edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507.
  • the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382.
  • the edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367,
  • the edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354.
  • the modified com plant comprises a first edited allele of Bmr3 and a second edited allele of Bmr3, wherein the first and second edited allele of Bmr3 each, independently, comprise a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 348, 350, 352, 354, 358, 361, 364, 367, 370, 373, 376, 379, 382, 401-443 and 500-507.
  • SEQ NOs 21-252, 348, 350, 352, 354, 358, 361, 364, 367, 370, 373, 376, 379, 382, 401-443 and 500-507.
  • the modified com plant comprises a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 253-299.
  • silage produced from the modified com plant has at least 30%, 31%, 32%, 33%. 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%. 44%, 45%, 46%, 47%, 48%, 49%, or 50 % Neutral Detergent Fiber (NDF).
  • silage produced from the modified com plant has at least 12%, 13%. 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% Acid Detergent Fiber (ADF).
  • ADF Acid Detergent Fiber
  • the lignin content of the modified com plant is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% or more as compared to a non-brown midrib com plant.
  • 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 3
  • the lignin content of the modified com plant is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% or more as compared to com plant with a native Bmr3 mutant allele.
  • the modified com plant comprises one or more additional traits.
  • the modified com plant comprises a floury -2 trait.
  • a part of the modified com plant selected from the group consisting of an intact plant cell, a plant protoplast, an embryo, a pollen, an ovule, a flower, a kernel, a seed, an ear, a cob, a leaf, a husk, a stalk, a root, a root tip, a brace root, a lateral tassel branch, an anther, a tassel, a glume, a tiller and a silk.
  • Several embodiments relate to a method of generating a modified com plant with reduced lignin comprising the steps of: (a) introducing a non-natural mutation in a Bmr3 gene of a com plant cell; (b) identifying and selecting one or more plant cells of step (a) comprising said non-natural in Bmr3; and (c) regenerating at least one plant from at least one or more cells selected in step (b).
  • the non-natural mutation is introduced by a site-specific genome modification enzyme.
  • the non natural mutation is introduced by Cpfl complexed with guide RNA comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400.
  • the com plant cell of step (a) is derived from a com plant having one or more traits.
  • the com plant cell of step (a) is derived from a com plant having a floury -2 trait.
  • the com plant cell of step (a) is derived from a com plant having a disease resistance trait.
  • the com plant cell of step (a) is derived from a com plant having an herbicide resistance trait. In some embodiments, the com plant cell of step (a) is derived from a com plant having a native brown midrib trait. In some
  • the non-natural mutation comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507.
  • the non natural mutation comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs:
  • the non-natural mutation comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354.
  • the plant of step c is crossed with to produce progeny that contain the desirable trait.
  • a method of generating a modified plant with reduced lignin levels comprising the steps of: (a) introducing a modification in a Bmr3 gene of a plant cell; (b) identifying and selecting one or more plant cells of step (a) comprising said modification in a Bmr3 gene; and (c) regenerating at least one plant from at least one or more cells selected in step (b).
  • the modification comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507.
  • the modification comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382. In some embodiments, the modification comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354.
  • the modification introduced by Cpfl complexed with guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400.
  • the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 383 and SEQ ID NO: 389 and sequencing the amplified DNA.
  • the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 384 and SEQ ID NO: 390 and sequencing the amplified DNA.
  • the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 385 and SEQ ID NO: 391 and sequencing the amplified DNA.
  • the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 386 and SEQ ID NO: 392 and sequencing the amplified DNA. In some embodiments, the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 387 and SEQ ID NO: 393 and sequencing the amplified DNA. In some embodiments, the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 388 and SEQ ID NO: 394 and sequencing the amplified DNA. In some embodiments, the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 520 and SEQ ID NO: 521 and sequencing the amplified DNA.
  • a modified com plant, com plant part, com cell, or com seed comprising an edited allele of Bmr3 as described herein.
  • a modified plant genome comprising an edited allele of Bmr3 as described herein is further provided.
  • the edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507.
  • the edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382.
  • the edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354.
  • com plant part is selected from the group consisting of an intact plant cell, a plant protoplast, an embryo, a pollen, an ovule, a flower, a kernel, a seed, an ear, a cob, a leaf, a husk, a stalk, a root, a root tip, a brace root, a lateral tassel branch, an anther, a tassel, a glume, a tiller and a silk.
  • the com silage is produced from a modified com plant comprising and edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507.
  • the com silage is produced from a modified com plant comprising and edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367,
  • the com silage is produced from a modified com plant comprising and edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354.
  • the com silage has a lignin content of 1%, 2%, 3%, 4%, or 5%.
  • the com silage has at least 30%, 31%, 32%, 33%. 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%.
  • the com silage has at least 12%, 13%. 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% Acid Detergent Fiber (ADF).
  • ADF Acid Detergent Fiber
  • Several embodiments relate to a method of enhancing milk production efficiency in ruminants, the method comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising an edited allele of Bmr3; and feeding the ruminants with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the ruminants.
  • a method of enhancing milk production efficiency in ruminants comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507; and feeding the ruminants with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the ruminants.
  • a method of enhancing milk production efficiency in ruminants comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382; and feeding the ruminants with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the ruminants.
  • a method of enhancing milk production efficiency in ruminants comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354; and feeding the ruminants with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the ruminants.
  • the com hybrid further comprises a mutant allele of floury -2.
  • the com hybrid has a lignin content of 1%, 2%, 3%, 4%, or 5%.
  • Several embodiments relate to a method of enhancing milk production efficiency in dairy cattle, the method comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising an edited allele of Bmr3; and feeding the dairy cattle with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the dairy cattle.
  • a method of enhancing milk production efficiency in dairy cattle comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507; and feeding the dairy cattle with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the dairy cattle.
  • a method of enhancing milk production efficiency in dairy cattle comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382; and feeding the dairy cattle with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the dairy cattle.
  • a method of enhancing milk production efficiency in dairy cattle comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354; and feeding the dairy cattle with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the dairy cattle.
  • the com hybrid further comprises a mutant allele of floury -2.
  • the com hybrid has a lignin content of 1%, 2%, 3%, 4%, or 5%.
  • a modified protein comprising an amino acid sequence that is at least 95%, at least 96%, at least 96%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 253-299, 444-471, and 508-513.
  • a com plant comprising a modified protein comprising an amino acid sequence that is at least 95%, at least 96%, at least 96%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 253-299, 444-471, and 508- 513.
  • com silage produced from a com plant comprising a modified protein comprising an amino acid sequence that is at least 95%, at least 96%, at least 96%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 253-299, 444-471, and 508-513.
  • a modified com plant produced by: (a) providing a com plant cells wherein the cell comprises a target genomic nucleic acid molecule having at least 90% sequence identity with a sequence selected from the group consisting of:
  • the site-specific genome modification enzyme is selected from the group consisting of a zinc-finger nuclease, an engineered meganuclease, a native meganuclease, a TALE-endonuclease, and an RNA-guided endonuclease.
  • the site-specific genome modification enzyme is a CRISPR associated protein complexed with guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400.
  • the CRISPR associated protein is selected from the group consisting of: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csl3, Csf4, Cpfl, CasX, CasY, and Mad7.
  • the site-specific genome modification enzyme is a Cpfl protein complexed with guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400.
  • the modified com plant lacks a Bmr3 gene product.
  • the modified com plant comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 348, 350, 352, 354, 358, 361, 364, 367, 370, 373, 376, 379, 382, 401-443, and 500-507.
  • the modified com plant comprises a Bmr3 gene product comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 253-299, 444-471, and 508- 513.
  • Several embodiments relate to method of generating a com plant that exhibits a brown midrib phenotype, the method comprising: (a) providing com plant cells wherein the cells comprise a target genomic nucleic acid molecule having at least 90% sequence identity with a sequence selected from the group consisting of: nucleotides 146-168 of SEQ ID NO: 1, nucleotides 283-305 of SEQ ID NO: 1, nucleotides 637-659 of SEQ ID NO: 1, nucleotides 1802-1824 of SEQ ID NO: 1, nucleotides 1862-1884 of SEQ ID NO: 1, nucleotides 2461- 2483 of SEQ ID NO: 1, nucleotides 2940-2962 of SEQ ID NO: 1, nucleotides 3071-3093 of SEQ ID NO: 1, nucleotides 73-95 of SEQ ID NO: 4, nucleotides 1276-1298 of SEQ ID NO:
  • nucleotides 1336-1358 of SEQ ID NO: 4 nucleotides 1935-1957 of SEQ ID NO: 4, nucleotides 2401-2423 of SEQ ID NO: 4, nucleotides 1622-1644 of SEQ ID NO: 522, and nucleotides 2231-2254 of SEQ ID NO: 522;
  • the site-specific genome modification enzyme is a Cpfl protein complexed with guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400.
  • the com plant is an elite com plant.
  • the modified com plant comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 348, 350, 352, 354, 358, 361, 364, 367, 370, 373, 376, 379, 382, 401-443, and 500-507.
  • Several embodiments relate to method of generating a com line with favorable yield that exhibits a brown midrib phenotype, the method comprising: (a) providing elite com plant cells wherein the cells comprise a target genomic nucleic acid molecule having at least 90% sequence identity with a sequence selected from the group consisting of: nucleotides 146-168 of SEQ ID NO: 1, nucleotides 283-305 of SEQ ID NO: 1, nucleotides 637-659 of SEQ ID NO: 1, nucleotides 1802-1824 of SEQ ID NO: 1, nucleotides 1862-1884 of SEQ ID NO: 1, nucleotides 2461-2483 of SEQ ID NO: 1, nucleotides 2940-2962 of SEQ ID NO: 1, nucleotides 3071-3093 of SEQ ID NO: 1, nucleotides 73-95 of SEQ ID NO: 4, nucleotides 1276-1298 of SEQ ID NO: 4, nucleotides 1336-1358 of SEQ
  • the site-specific genome modification enzyme is a Cpfl protein complexed with guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400.
  • the modified com plant comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 348, 350, 352, 354, 358, 361, 364, 367, 370, 373, 376, 379, 382, 401-443, and 500-507.
  • FIG. 1 Shows a schematic illustration of the genomic stmcture of the com Bmr3 gene. The two exons are indicated by a rightward bar arrows. 5’ UTR, 3’ UTR and one intron are indicated by solid lines. The positions and names of 6 sites targeted by the Cpfl guide RNAs are indicated by downward arrows along the Bmr3 gene structure.
  • FIG. 2 Shows a schematic illustration of the sites in com Bmr3 gene targeted for editing by the guide RNAs of five recombinant T-DNA vectors.
  • the deletion in Event A-l is indicated by a bottom curly bracket that removes the entire exon2.
  • the small deletions in Events B-l through 3 are indicated in a downward triangle within exon 2.
  • FIG. 3 Shows Bmr3 RNA expression detected by RT-qPCR Taqman assay using primer set A (panel A) and primer set B (panel B). Lanel: wild-type control, Lane 2:
  • the term“and/or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items.
  • the expression“A and/or B” is intended to mean either or both of A and B, e.g., A alone, B alone, or A and B in combination.
  • the expression“A, B and/or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.
  • references to“plant,”“the plant,” or“a plant” also includes a plurality of plants; also, depending on the context, use of the term“plant” can also include genetically similar or identical progeny of that plant; use of the term“a nucleic acid” optionally includes, as a practical matter, many copies of that nucleic acid molecule; similarly, the term“probe” optionally (and typically) encompasses many similar or identical probe molecules.
  • plant refers to a whole plant, any part thereof, or a cell or tissue culture derived from a plant, comprising any of: whole plants, plant components or organs (e.g., ears, husks, leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and/or progeny of the same.
  • a progeny plant can be from any filial generation, e.g., FI, F2, F3, F4, F5, F6, F7, etc.
  • a plant cell is a biological cell of a plant, taken from a plant or derived through culture from a cell taken from a plant.
  • a "plant part” may refer to any organ or intact tissue of a plant, such as a meristem, shoot organ/structure (e.g., leaf, stem or node), root, flower or floral organ/structure (e.g., bract, sepal, petal, stamen, carpel, anther and ovule), seed (e.g., embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), propagule, or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, and the like), or any portion thereof. Plant parts may be viable, nonviable, regenerable, and/or non-regenerable.
  • a "propagule” may include any plant part that is capable of growing into an entire plant.
  • “com”,“maize”,“com plant” or“maize plant” refers to a plant of species Zea mays L. and includes all plant varieties that can be bred with com, including wild maize species.
  • inbred means a line that has been bred for genetic homogeneity.
  • elite line means any line that has resulted from breeding and selection for superior agronomic performance.
  • An elite plant is any plant from an elite line.
  • hybrid means a progeny of mating between at least two genetically dissimilar parents.
  • examples of mating schemes include single crosses, modified single cross, double modified single cross, three-way cross, modified three- way cross, and double cross wherein at least one parent in a modified cross is the progeny of a cross between sister lines.
  • “Brown midrib 3”,“Bm3”, or“Bmr3” refers to the gene encoding caffeic acid-O-methyltransferase, a key enzyme involved in synthesis of flavonolignan, the mutation of which in com plants results in com plants characterized by a brown pigmentation in the leaf midrib at the v3 to vlO stage and lower lignin content in com plant tissue.
  • the native Bmr3 gene in the 01DKD2 inbred com line comprises the nucleotide sequence of SEQ ID NO: l and encodes a protein of SEQ ID NO: 3.
  • the native Bmr3 gene in the LH244 inbred com line comprises the nucleotide sequence of SEQ ID NO: 4 and encodes a protein of SEQ ID NO: 6.
  • the term“deletion mutation” refers to the removal of one or more nucleotides from the DNA. Like insertion mutations, these mutations can alter the reading frame of the gene.
  • germplasm refers to living sources of genetic material.
  • the germplasm can be part of an organism or cell, or can be separate from the organism or cell.
  • germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture.
  • germplasm includes cells, seed or tissues from which new plants may be grown, or plant parts, such as leaves, stems, pollen, or cells that can be cultured into a whole plant.
  • Insertions in the coding region of a gene may alter splicing of the mRNA (splice site mutation), or cause a shift in the reading frame (frameshift), both of which can significantly alter the gene product.
  • “modified”, in the context of plants, seeds, plant components, plant cells, and plant germplasm, refers to a state containing changes or variations from their natural or native state.
  • a“native transcript” of a gene refers to an RNA transcript that is generated from an unmodified gene
  • a“native protein” refers to a protein that is generated from an unmodified gene.
  • a native transcript is a sense transcript.
  • Modified plants or seeds contain molecular changes in their genetic materials, including either genetic or epigenetic modifications.
  • modified plants or seeds, or a parental or progenitor line thereof have been subjected to mutagenesis, genome editing (e.g., without being limiting, via methods using site-specific nucleases), genetic transformation (e.g., without being limiting, via methods of Agrobacterium transformation or microprojectile bombardment), or a combination thereof.
  • a modified plant provided herein comprises no non-plant genetic material or sequences.
  • a modified plant provided herein comprises no interspecies genetic material or sequences.
  • a modified plant provided herein comprises one or more nucleotide changes, additions, or deletions in a gene compared to a native gene.
  • a“mutation” refers to the permanent alteration of the nucleotide sequence of the genome of an organism, the extrachromosomal DNA, or other genetic elements.
  • a“native copy” of a gene refers to a gene that originates from within a given organism, cell, tissue, genome, or chromosome that was not previously modified by human action.
  • a“native protein” refers to a protein encoded by a native gene.
  • substitution mutation refers to an exchange of a single nucleotide for another.
  • the terms“natural mutation”,“naturally-occurring mutation”, or“native mutation”, refers to a mutation as it occurs spontaneously in nature without any involvement of laboratory or experimental procedures or under the exposure to mutagens. Without being bound by scientific theory, a naturally-occurring mutation can arise from a variety of sources, including errors in DNA replication, spontaneous lesion, and transposable elements (or transposon).
  • the terms“synthetic mutation”,“non-natural mutation” or“non- naturally-occurring mutation” refers to non-spontaneous mutation that occurs as a result of experimental procedures, such as exposure to a mutagen or by a site-specific genome modification enzyme.
  • conversion refers to reversing the orientation of a chromosomal segment.
  • a“missense mutation” refers to a single nucleotide change that results in a codon that codes for a different amino acid.
  • the codon“CGU” encodes an arginine amino acid. If a missense mutation changes the G to a U, producing a“CUU” codon, the codon now encodes a leucine amino acid. Missense mutations can be caused by an insertion, deletion, substitution, or inversion.
  • the term“DNA” or“DNA molecule” refers to a double-stranded DNA molecule of genomic or synthetic origin, i.e. a polymer of deoxyribonucleotide bases or a polynucleotide molecule, read from the 5 ' (upstream) end to the 3 ' (downstream) end.
  • the term“DNA sequence” refers to the nucleotide sequence of a DNA molecule.
  • the term“isolated DNA molecule” refers to a DNA molecule at least partially separated from other molecules normally associated with it in its native or natural state.
  • the term“isolated” refers to a DNA molecule that is at least partially separated from some of the nucleic acids which normally flank the DNA molecule in its native or natural state.
  • DNA molecules fused to regulatory or coding sequences with which they are not normally associated, for example as the result of recombinant techniques are considered isolated herein.
  • Such molecules are considered isolated when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules, in that they are not in their native state.
  • PCR polymerase chain reaction
  • Polynucleotide molecules, or fragment thereof can also be obtained by other techniques, such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer.
  • a polynucleotide can be single-stranded (ss) or double-stranded (ds). “Double-stranded” refers to the base-pairing that occurs between sufficiently complementary, anti-parallel nucleic acid strands to form a double-stranded nucleic acid structure, generally under physiologically relevant conditions.
  • percent sequence identity refers to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of a reference (“query”) sequence (or its complementary strand) as compared to a test (“subject”) sequence (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide or amino acid insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison).
  • Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the Sequence Analysis software package of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA), MEGAlign (DNAStar Inc., Madison, WI), and MUSCLE (version 3.6) (Edgar, “MUSCLE: multiple sequence alignment with high accuracy and high throughput” Nucleic Acids Research 32(5): 1792-7 (2004)) for instance with default parameters.
  • tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA
  • An“identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in the portion of the reference sequence segment being aligned, that is, the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more sequences may be to a full-length sequence or a portion thereof, or to a longer sequence.
  • protein-coding polynucleotide molecule refers to a polynucleotide molecule comprising a nucleotide sequence that encodes a protein.
  • A“protein coding sequence” means a polynucleotide sequence that encodes a protein.
  • a protein-coding molecule may comprise a DNA sequence encoding a protein sequence. In some embodiments, a protein-coding molecule may comprise a RNA sequence encoding a protein sequence.
  • transgene expression means the production of a protein through the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into polypeptide chains, which are ultimately folded into proteins.
  • mRNA messenger RNA
  • the term“recombinant” in reference to a polynucleotide (DNA or RNA) molecule, protein, construct, vector, etc. refers to a polynucleotide or protein molecule or sequence that is man-made and not normally found in nature, and/or is present in a context in which it is not normally found in nature, including a polynucleotide (DNA or RNA) molecule, protein, construct, etc., comprising a combination of polynucleotide or protein sequences that would not naturally occur contiguously or in close proximity together without human intervention, and/or a polynucleotide molecule, protein, construct, etc., comprising at least two polynucleotide or protein sequences that are heterologous with respect to each other.
  • a recombinant polynucleotide or protein molecule, construct, etc. may comprise polynucleotide or protein sequence(s) that is/are (i) separated from other polynucleotide or protein sequence(s) that exist in proximity to each other in nature, and/or (ii) adjacent to (or contiguous with) other polynucleotide or protein sequence(s) that are not naturally in proximity with each other.
  • a recombinant polynucleotide molecule, protein, construct, etc. may also refer to a polynucleotide or protein molecule or sequence that has been genetically engineered and/or constructed outside of a cell.
  • a recombinant polynucleotide molecule, protein, construct, etc. may also refer to a polynucleotide or protein molecule or sequence that has been edited inside of a cell.
  • the term recombinant can also refer to an organism that harbors recombinant material, e.g., a plant that comprises a recombinant nucleic acid is considered a recombinant plant.
  • the term“allele” refers to an alternative nucleic acid sequence at a particular locus; the length of an allele can be as small as 1 nucleotide base, but is typically larger. For example, a first allele can occur on one chromosome, while a second allele occurs on a second homologous chromosome, e.g., as occurs for different chromosomes of a heterozygous individual, or between different homozygous or heterozygous individuals in a population.
  • a favorable allele is the allele at a particular locus that confers, or contributes to, an agronomically desirable phenotype, or alternatively, is an allele that allows the identification of susceptible plants that can be removed from a breeding program or planting.
  • a favorable allele of a marker is a marker allele that segregates with the favorable phenotype, or alternatively, segregates with susceptible plant phenotype, therefore providing the benefit of identifying disease prone plants.
  • a favorable allelic form of a chromosome interval is a chromosome interval that includes a nucleotide sequence that contributes to superior agronomic performance at one or more genetic loci physically located on the chromosome interval.
  • an edited allele refers to an alternative nucleic acid sequence at a particular locus where such alternative nucleic acid sequence contains man-made changes or variations from the native nucleic acid, for example by use of a site-specific genome modification enzyme.
  • an edited allele may comprise one or more non natural mutations.
  • genotype is the genetic constitution of an individual (or group of individuals) at one or more genetic loci, as contrasted with the observable trait (the phenotype).
  • Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents.
  • genotype can be used to refer to an individual’s genetic constitution at a single locus, at multiple loci, or, more generally, the term genotype can be used to refer to an individual’s genetic make-up for all the genes in its genome.
  • A“haplotype” is the genotype of an individual at a plurality of genetic loci. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome interval.
  • locus is a chromosome region where a polymorphic nucleic acid, trait determinant, gene, or marker is located.
  • Digestibility refers to percentage of nutrients available in a given volume of food, such as silage or feed-ration. Greater digestibility is associated with higher energy availability. Digestibility may be measured in vitro or in vivo.
  • neutral detergent fiber or“NDF” refers to a measure of the fraction of a feed that is not soluble in neutral detergent solution. NDF levels in forage increase as the plant matures. Com silage NDF concentration ranges from 36 to 50%. A low com silage NDF is desirable. Diets with less than 32 percent NDF may cause problems with acidosis.
  • neutral detergent fiber digestibility refers to percentage of neutral detergent fiber that is digestible. NDFD can be determined in vitro by incubating a ground feed sample in rumen fluid and measuring its disappearance to simulate the amount and rate of digestion that would occur in the rumen.
  • milk production refers to the amount of milk produced by lactating dairy cattle on average during one day period.
  • ruminant refers to a mammal that acquire nutrients from plant-based food by regurgitating partially digested food. Examples of ruminants include bison, cattle, sheep, moose, giraffes, and goats.
  • silage means any crop that is harvested green and preserved in a succulent condition by partial fermentation in a more-or-less airtight container.
  • Com silage consists of era, stalks, and leaves.
  • milk production efficiency refers to the amount of milk produced per one unit of the feed intake.
  • Bmr3 com mutants characterized by a brown pigmentation in the leaf midrib at the V4 to V6 stage and lower lignin content in com plant tissue due to a disruption of the gene encoding caffeic acid-O-methyltransferase, an enzyme involved in synthesis of lignin.
  • Lignin polymers limit the digestibility of the fiber in the com plant.
  • the reduced lignin in brown midrib com results in silage with fiber that is more digestible than non-brown midrib com.
  • Animal feeding trials have shown about 10 percent greater intake and increased milk production with ruminants, such as dairy cows, fed with brown midrib com silage (Bmr silage), as compared to non-Bmr silage.
  • the com Bmr3 gene is located on the short arm of chromosome 4 and, as shown in Figure 1, contains 2 exons and 1 intron.
  • the naturally occurring mutations that have been identified in the com Bmr3 gene are recessive and are associated with negative agronomic traits such as reduced yield, reduced standability, lodging, and reduced disease resistance.
  • a modified com plant comprising a non-naturally occurring mutation in a Bmr3 gene.
  • Several embodiments described herein provide a modified com plant comprising at least one non-natural mutation in the com Bmr3 gene, wherein the com plant exhibits a reduced lignin content compared to a control com plant not comprising the non-natural Bmr3 mutation when grown under comparable conditions.
  • the modified com plant does not comprise a naturally occurring mutation in a Bmr3 gene.
  • the modified com plant does not comprise the naturally- occurring Bm3-1 mutant allele.
  • the modified com plant does not comprise the naturally-occurring Bm3-2 mutant allele.
  • the modified com plant does not comprise the naturally-occurring Bm3-2001PR-1 mutant allele. In some embodiments, the modified com plant does not comprise the naturally-occurring Bm3-91598- 3 mutant allele. In some embodiments, the modified com plant does not comprise the naturally- occurring Bm3-Bumham mutant allele. In some embodiments, the modified com plant does not comprise the naturally-occurring Bm3-MM1266 mutant allele. In some embodiments, the modified com plant does not comprise the naturally-occurring Bm3-MM1818 mutant allele.
  • a modified com plant as described herein comprises a synthetic mutation in the Bmr3 gene introduced via targeted genome editing.
  • a method described herein comprises targeted genome editing of a Bmr3 in a desired inbred background.
  • the present disclosure provides a non-transgenic com plant comprising a synthetic mutation in a Bmr3 gene reducing the activity of the Bmr3 gene.
  • the present disclosure provides a modified com plant comprising a non-transgene or non- transposon mediated mutation in a Bmr3 gene reducing the activity of the Bmr3 gene.
  • the term“transgene” refers to a recombinant DNA molecule, constmct or sequence integrated or inserted into a genome, and thus altering the genome.
  • the modified com plant comprises a non-naturally occurring substitution mutation in the Bmr3 gene.
  • the modified com plant comprises a non-naturally occurring insertion in the Bmr3 gene.
  • the modified com plant comprises a non-naturally occurring inversion in the Bmr3 gene.
  • the modified com plant comprises a non-naturally occurring deletion in the Bmr3 gene.
  • the modified com plant comprises a non-naturally occurring substitution mutation in a genomic sequence of SEQ ID NO: l. In some embodiments, the modified com plant comprises a non- naturally occurring insertion in a genomic sequence of SEQ ID NO: l. In some embodiments, the modified com plant comprises a non-naturally occurring inversion in a genomic sequence of SEQ ID NO: l. In some embodiments, the modified com plant comprises a non-naturally occurring deletion in a genomic sequence of SEQ ID NO: l. In some embodiments, the modified com plant comprises a non-naturally occurring substitution mutation in a genomic sequence of SEQ ID NO:4.
  • the modified com plant comprises a non- naturally occurring insertion in a genomic sequence of SEQ ID NO:4. In some embodiments, the modified com plant comprises a non-naturally occurring inversion in a genomic sequence of SEQ ID NO:4. In some embodiments, the modified com plant comprises a non-naturally occurring deletion in a genomic sequence of SEQ ID NO:4.
  • a modified com plant comprising a non- naturally occurring insertion in a Bmr3 gene, where the insertion causes a truncation of the protein encoded by the Bmr3 gene.
  • the insertion occurs within Exon 1 of the Bmr3 gene, where the insertion introduces premature stop codon.
  • the insertion occurs within Exon 2 of the Bmr3 gene, where the insertion introduces premature stop codon.
  • a modified com plant comprising a non-naturally occurring deletion in a Bmr3 gene, where the deletion causes a truncation of the protein encoded by the Bmr3 gene.
  • the deletion occurs within Exon 1 of the Bmr3 gene. In another aspect, the deletion occurs within Exon 2 of the Bmr3 gene.
  • Several embodiments described herein relate to a modified com plant comprising a non-naturally occurring inversion in a Bmr3 gene, where the inversion causes a reduction in the level of the protein encoded by the Bmr3 gene. In one aspect, the inversion occurs within Exon 1 of the Bmr3 gene. In another aspect, the inversion occurs within Exon 2 of the Bmr3 gene. In one aspect, the inversion creates a dominant negative allele of the Bmr3 gene.
  • modified com plant comprising an edited allele of the Bmr3 gene.
  • the modified com plant is heterozygous for an edited allele of the Bmr3 gene.
  • the modified com plant is homozygous for an edited allele of the Bmr3 gene.
  • the modified com plant comprises an edited allele of the Bmr3 gene, wherein the edited allele comprises a nucleotide sequence selected from SEQ ID NOs: 21-252, 401-443 and 500-507.
  • the modified com plant comprises an edited allele of the Bmr3 gene, wherein the edited allele comprises a nucleotide sequence selected from SEQ ID NOs: 358-382.
  • the modified com plant comprises a first edited allele of the Bmr3 gene and a second edited allele of the Bmr3 gene, wherein the first edited allele and the second edited allele comprise a nucleotide sequence independently selected from SEQ ID NOs: 21-252, 358- 382, 401-443 and 500-507.
  • the modified com plant comprises an edited allele of the Bmr3 gene, wherein the edited allele comprises a nucleotide sequence selected from SEQ ID NOs: 358, 361, 364, 370, 373, 376, 379, and 382.
  • the modified com plant comprises a first edited allele of the Bmr3 gene and a second edited allele of the Bmr3 gene, wherein the first edited allele and the second edited allele comprise a nucleotide sequence independently selected from SEQ ID NOs: 21-252, 358, 361, 364, 370, 373, 376, 379, 382, 401-443, and 500-507.
  • the modified com plant comprises an edited allele of the Bmr3 gene, wherein the edited allele comprises a nucleotide sequence selected from SEQ ID NOs: 246, 348, 350, 352 and 354.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele resulting whole gene deletion between a genomic sequence targeted by g90 or g227 and a genomic sequence targeted by g3279.
  • Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 2313 nucleotide deletion between a genomic sequence targeted by g867 and a genomic sequence targeted by g3170.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 1130 nucleotide deletion between a genomic sequence targeted by g2010 and a genomic sequence targeted by g3170.
  • Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 7, 8, 9, 10, 15 or 19 nucleotide deletion at a genomic sequence targeted by g2010.
  • Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 1332 nucleotide deletion at a genomic sequence targeted by g2070.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 944 nucleotide deletion at a genomic sequence targeted by g2010.
  • Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion at a genomic sequence targeted by g2070. In some embodiments, 1 or more, 10 or more, 50 or more, 100 or more, 500 or more, or 1,000 or more nucleotide are deleted at or around a genomic sequence targeted by g2070.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 358, 326, 304, 238, 190, 144, 130, 109, 108, 105,
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises deletion mutation between a genomic sequence targeted by g2070 and a genomic sequence targeted by g2691.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 1214, 784, 736, 646, 595, 594, 593, 592, 591, or 590 nucleotide deletion between a genomic sequence targeted by g2070 and a genomic sequence targeted by g2691.
  • the edited allele comprises a deletion at a genomic sequence targeted by g2691. In some embodiments, 1 or more, 10 or more, 50 or more, 100 or more, 500 or more, or 1,000 or more nucleotides are deleted at or around a genomic sequence targeted by g2691.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 36, 27, 21, 20, 19, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide deletion at or around a genomic sequence targeted by g2691.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises deletion mutation between a genomic sequence targeted by g2010 and a genomic sequence targeted by g3170.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 1453, 1135, 1129, or 1118 nucleotide deletion between a genomic sequence targeted by g2010 and a genomic sequence targeted by g3170.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion at a genomic sequence targeted by g3170. In some embodiments, 1 or more, 10 or more, 50 or more, 100 or more, 500 or more, or 1,000 or more nucleotides are deleted at or around a genomic sequence targeted by g3170.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 24, 23, 22, 20, 19, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide deletion at or around a genomic sequence targeted by g3170.
  • the edited allele comprises deletion mutation between a genomic sequence targeted by g2070 and a genomic sequence targeted by g3270.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 1243 or 1214 nucleotide deletion between a genomic sequence targeted by g2070 and a genomic sequence targeted by g3270.
  • the edited allele comprises deletion mutation between a genomic sequence targeted by g2070 and a genomic sequence targeted by g2691.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 637 nucleotide deletion between a genomic sequence targeted by g2070 and a genomic sequence targeted by g2691.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion at a genomic sequence targeted by g3279. In some embodiments, 1 or more, 10 or more, 50 or more, 100 or more, 500 or more, or 1,000 or more nucleotides are deleted at or around a genomic sequence targeted by g3279.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 24, 23, 22, 20, 19, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide deletion at or around a genomic sequence targeted by g3279.
  • the edited allele comprises deletion mutation between a genomic sequence targeted by g867 and a genomic sequence targeted by g2010.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 1200 nucleotide deletion between a genomic sequence targeted by g867 and a genomic sequence targeted by g2010.
  • Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion at a genomic sequence targeted by g867. In some embodiments, 1 or more, 10 or more, 50 or more, 100 or more, 500 or more, or 1,000 or more nucleotides are deleted at or around a genomic sequence targeted by g867.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 24, 23, 22, 20, 19, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide deletion at or around a genomic sequence targeted by g867.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion of 1 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more, 220 or more, 230 or more, 240 or more, 250 or more, 260 or more, 270 or more, 280 or more, 290 or more, 300 or more, 310 or more, 320 or more, 330 or more, 340 or more, 350 or more, 360 or more, 370 or more, 380 or more, 390 or more, 400 or more, 410 or more, 420 or more, 430 or more, 440 or more, 450 or more, 460 or more, 470 or
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a complete deletion of exon 1 of the Bmr3 gene.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion of the 5’ end of exon 1 of the Bmr3 gene.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion of the 3’ end of exon 1 of the Bmr3 gene.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion of 1 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more, 220 or more, 230 or more, 240 or more, 250 or more, 260 or more, 270 or more, 280 or more, 290 or more, 300 or more, 310 or more, 320 or more, 330 or more, 340 or more, 350 or more, 360 or more, 370 or more, 380 or more, 390 or more, 400 or more, 410 or more, 420 or more, 430 or more, 440 or more, 450 or more, 460 or more, 470 or
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a complete deletion of exon 2 of the Bmr3 gene.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion of the 5’ end of exon 2 of the Bmr3 gene.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion of the 3’ end of exon 2 of the Bmr3 gene.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises an inversion of the nucleotide between a genomic sequence targeted by g2010 and a genomic sequence targeted by g3170.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises an inversion of the nucleotide between a genomic sequence targeted by g2691 and a genomic sequence targeted by g3279.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises an inversion of the nucleotide between a genomic sequence targeted by g2070 and a genomic sequence targeted by g2691.
  • a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises an inversion of the nucleotide between a genomic sequence targeted by g2070 and a genomic sequence targeted by g3279.
  • Several embodiments relate to a modified com plant exhibiting a brown midrib reduced lignin phenotype and a floury-endosperm phenotype.
  • Mutant alleles of the floury -2 (F12) gene in com plants cause a decrease in the synthesis of zein proteins resulting in a floury endosperm, which is another desirable trait in animal feed because floury-endosperm is digested more rapidly and completely than vitreous endosperm.
  • Zein proteins are prolamin storage proteins in the endosperm of com seeds.
  • the Bmr3 and F12 genes are tightly linked approximately 5 cM genetic-distance apart on maize chromosome 4, with mutant Bmr3 and F12 alleles in trans linkage disequilibrium in com germplasm. Meiotic crossing over between these two loci is rare.
  • Several embodiments described herein relate to a modified com plant that is homozygous for a Bmr3 edited allele as described herein and a mutant F12 allele.
  • Several embodiments relate to a modified com germplasm comprising a Bmr3 edited allele as described herein in cis with a mutant F12 allele.
  • a modified com germplasm comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a nucleotide sequence selected from SEQ ID NOs: 21-252, 358, 361, 364, 370, 373, 376, 379, 382, 401-443, and 500- 507, and wherein the edited allele of the Bmr3 gene is in cis with a mutant F12 gene.
  • a modified com germplasm comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a nucleotide sequence selected from SEQ ID NOs: 246, 348, 350, 352 and 354, and wherein the edited allele of the Bmr3 gene is in cis with a mutant F12 gene.
  • breeding lines comprising the modified com germplasm described herein are crossed together to produce com hybrids that are homozygous for an edited Bmr3 alleles and a mutant F12 allele and thereby express both the brown-midrib and floury-endosperm traits.
  • a method described herein comprises targeted genome editing of a Bmr3 gene in a desired inbred background. Genome modification can be accomplished through targeted genome editing as described herein.
  • one or more synthetic mutations are generated at or around a genomic sequence targeted by g867. In some embodiments, one or more synthetic mutations are generated at or around a genomic sequence targeted by g2010.
  • one or more synthetic mutations are generated at or around a genomic sequence targeted by g2070. In some embodiments, one or more synthetic mutations are generated at or around a genomic sequence targeted by g2691. In some embodiments, one or more synthetic mutations are generated at or around a genomic sequence targeted by g3170. In some embodiments, one or more synthetic mutations are generated at or around a genomic sequence targeted by g3279.
  • a modified Bmr3 polypeptide encoded by an edited allele of Bmr3 as described herein.
  • nucleic acid sequences encoding modified Bmr3 polypeptide as well as compositions and methods of using modified polypeptides.
  • a modified Bmr3 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 252-299.
  • a nucleic acid sequence selected from SEQ ID NOs: 300-345 and 347 encodes a modified BMR3 polypeptide as described herein.
  • the edited allele of Bmr3 is a null allele in which no caffeic acid O-methyltransferase protein is produced.
  • nucleic acid molecules, polynucleotides, polypeptides, proteins, com cells, com seeds, or com plants that are the result of genome editing and as such would not normally be found in nature and are created by human intervention.
  • a com plant genome comprising a DNA sequence that does not naturally occur in such plant genome and as such is the result of human intervention.
  • a DNA sequence provided herein encodes a modified Bmr3 polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 252-299.
  • a DNA sequence provided herein encodes a modified Bmr3 polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 261, 280, 283, and 298.
  • modified Bmr3 alleles comprising an alteration relative to the Bmr3 gene sequence of SEQ ID NO: 1 or 4 are provided.
  • modified Bmr3 alleles comprising a nucleotide sequence selected from SEQ ID NOs: 21-252, 401-443 and 500-507 are provided.
  • modified Bmr3 alleles comprising a nucleotide sequence selected from SEQ ID NOs: 358, 361, 364, 370, 373, 376, 379, and 382 are provided.
  • modified Bmr3 alleles comprising a nucleotide sequence selected from SEQ ID NOs: 348, 350, 352, and 354 are provided.
  • Targeted modification of plant genomes through the use of genome editing methods can be used to create improved plant lines through modification of plant genomic DNA.
  • genome editing methods can enable targeted insertion of one or more nucleic acids of interest into a plant genome.
  • site-specific genome modification enzymes such as zinc-finger nucleases, engineered or native meganucleases, TALE-endonucleases, or an RNA-guided endonucleases (for example, a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)/Cas9 system, a CRISPR/Cpfl system, a CRISPR/CasX system, a CRISPR/CasY system, a CRISPR/Cascade system).
  • CRISPR Clustered Regularly Interspersed Short Palindromic Repeat
  • RNA editing is using single-stranded oligonucleotides to introduce precise base pair modifications in a plant genome, as described by Sauer et al ( Plant Physiol. 2016 Apr; 170(4): 1917-1928). Methods of genome editing to modify, delete, or insert nucleic acid sequences into genomic DNA are known in the art.
  • a com plant comprising in its genome a modified Bmr3 gene sequence, wherein the modified Bmr3 gene sequence encodes a truncated or modified COMT as described herein or where in the modified Bmr3 gene sequence is a null allele that does not produce a protein.
  • genome editing methods are utilized for the modification or replacement of an existing Bmr3 sequence within a plant genome.
  • the native Bmr3 gene sequence is modified to comprise one or more targeted nucleotide changes, additions, deletions, or other modifications at one or more sites targeted by g90, g227, g867, g2010, g2070, g2691, g3170 or g3279.
  • the native Bmr3 gene sequence is modified to comprise one or more targeted nucleotide changes, additions, deletions, or other modifications, such that the modified Bmr3 gene sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 21-252, 401-443, and 500-507.
  • the native Bmr3 gene sequence is modified to comprise one or more targeted nucleotide changes, additions, deletions, or other modifications, such that the modified Bmr3 gene sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382.
  • the native Bmr3 gene sequence is modified to comprise one or more targeted nucleotide changes, additions, deletions, or other modifications, such that the modified Bmr3 gene sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 348, 350, 352, and 354.
  • a CRISPR/Cas9 system used to modify or replace an existing coding sequence within a plant genome, such as a sequence encoding caffeic acid-O- methyltransferase (COMT).
  • a CRISPR/Cpfl system used to modify or replace an existing coding sequence within a plant genome, such as a sequence encoding caffeic acid-O-methyltransferase (COMT).
  • a Cas9- cytidine deaminase used to modify an existing coding sequence within a plant genome, such as a sequence encoding caffeic acid-O-methyltransferase (COMT).
  • a Cpfl-cytidine deaminase used to modify an existing coding sequence within a plant genome, such as a sequence encoding caffeic acid-O-methyltransferase (COMT).
  • transcription activator-like effectors TALEs
  • TALEs transcription activator-like effectors
  • an existing caffeic acid-O-methyltransferase (COMT) polypeptide coding sequence within a plant genome is modified by non-templated genome editing with a site-specific genome modification enzyme.
  • an existing COMT polypeptide coding sequence within a plant genome is modified by templated genome editing with a site-specific genome modification enzyme.
  • a site-specific genome modification enzyme and a donor nucleic acid comprising a nucleotide sequence selected from SEQ ID NOs: 21-252, 358, 361, 364, 367, 370, 373, 376, 379, 382, 401-443 and 500-507 is introduced into a plant cell.
  • the modified Bmr3 gene is capable of conferring to reduced lignin levels to a plant.
  • modification or replacement of an endogenous COMT-encoding sequence according to the methods provided herein results in a reduction in lignin.
  • a site-specific genome modification enzyme capable of recognizing a specific nucleotide sequence of interest, such as a sequence targeted by g90, g227, g867, g2010, g2070, g2691, g3170 or g3279, within a genome of a plant to allow for alteration of the Bmr3 gene sequence by non-templated editing or integration of a donor nucleic acid at that site.
  • Modifications to a COMT-encoding sequence may result in a sequence encoding a truncated or altered polypeptide as described herein at SEQ ID NOs: 261, 280, 283, and 298, capable of conferring to a plant improved digestibility and lower lignin content.
  • a“modification” comprises the insertion of 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 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides.
  • a“modification” comprises the deletion of 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 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides.
  • a“modification” comprises the inversion of 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 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides.
  • a“modification” comprises the substitution of 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 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides.
  • a “modification” comprises the substitution of an“A” for a“C”,“G” or“T” in a nucleic acid sequence.
  • a“modification” comprises the substitution of an“C” for a “A”,“G” or“T” in a nucleic acid sequence. In some embodiments, a“modification” comprises the substitution of an“G” for a“A”,“C” or“T” in a nucleic acid sequence. In some embodiments, a“modification” comprises the substitution of an“T” for a“A”,“C” or“G” in a nucleic acid sequence. In some embodiments, a“modification” comprises the substitution of an“C” for a“U” in a nucleic acid sequence. In some embodiments, a“modification” comprises the substitution of an“G” for a“A” in a nucleic acid sequence.
  • a “modification” comprises the substitution of an“A” for a“G” in a nucleic acid sequence. In some embodiments, a“modification” comprises the substitution of an“T” for a“C” in a nucleic acid sequence.
  • a recombinant DNA construct comprising an expression cassette(s) encoding a site-specific genome modification enzyme and/or any associated guide RNAs(s) to carry out genome editing.
  • These expressing cassette(s) may be present in the same molecule or vector as a donor template for templated editing or on a separate molecule or vector.
  • Several methods for genome editing are known in the art involving different site- specific genome modification enzymes (or complexes of proteins and/or guide RNAs) that cut the genomic DNA to produce a double strand break (DSB) or nick at a desired genomic site or locus.
  • a donor template DNA may become integrated into the genome at the site of the DSB or nick.
  • the presence of the homology arm(s) in the donor template DNA may promote the adoption and targeting of the insertion sequence into the plant genome during the repair process through homologous recombination, although an insertion of the donor template may occur through non-homologous end joining (NHEJ).
  • NHEJ non-homologous end joining
  • one or more of an insertion mutation, a deletion mutation or an inversion mutation may occur at or around the target site during the process of repairing the DSB or nick introduced by the site-specific genome modification enzyme.
  • site-specific genome modification enzymes examples include zinc-finger nucleases, engineered or native meganucleases, TALE-endonucl eases, and RNA-guided endonucleases (e.g, Cas9, CasX, CasY or Cpfl).
  • RNA-guided endonucleases e.g., Cas9, CasX, CasY or Cpfl
  • the recombinant DNA construct(s) may also comprise a sequence encoding one or more guide RNAs to direct the nuclease to the desired site within the plant genome.
  • one or more guide RNAs may be provided on a separate molecule or vector (in trans).
  • double-strand break inducing agent refers to any agent that can induce a double-strand break (DSB) in a DNA molecule.
  • the double-strand break inducing agent is a site-specific genome modification enzyme.
  • a site-specific genome modification enzyme refers to any enzyme that can modify a nucleotide sequence in a sequence-specific manner.
  • a site-specific genome modification enzyme modifies the genome by inducing a single-strand break.
  • a site-specific genome modification enzyme modifies the genome by inducing a double-strand break.
  • a site-specific genome modification enzyme comprises a cytidine deaminase.
  • a site- specific genome modification enzyme comprises an adenine deaminase.
  • site-specific genome modification enzymes include endonucleases, recombinases, transposases, deaminases, hebcases and any combination thereof.
  • the site-specific genome modification enzyme is a sequence-specific nuclease.
  • the site-specific genome modification enzyme is selected from an endonuclease, a recombinase, a transposase, a deaminase, a helicase or any combination thereof.
  • recombination occurs between A chromosomes.
  • recombination occurs between B chromosomes.
  • recombination occurs between a B chromosome and an A chromosome.
  • the site-specific genome modification enzyme is selected from a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nucleases (TALEN), an Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), an RNA-guided nuclease, such as a CRISPR associated nuclease (non-limiting examples of CRISPR associated nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Cas 12a (also known as Cpfl), Csyl, Csy2,
  • the site-specific genome modification enzyme is a dCas9-Fokl fusion protein.
  • the site-specific genome modification enzyme is a dCas9- recombinase fusion protein.
  • a“dCas9” refers to a Cas9 endonuclease protein with one or more amino acid mutations that result in a Cas9 protein without endonuclease activity, but retaining RNA-guided site-specific DNA binding.
  • a“dCas9- recombinase fusion protein” is a dCas9 with a protein fused to the dCas9 in such a manner that the recombinase is catalytically active on the DNA.
  • the site-specific genome modification enzyme comprises a DNA binding domain operably linked to a deaminase. In some embodiments, the site-specific genome modification enzyme further comprises uracil DNA glycosylase (UGI). In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an adenine deaminase. In some embodiments, the deaminase is an APOPEC deaminase. In some embodiments, the deaminase is an activation-induced cytidine deaminase (AID).
  • UBI uracil DNA glycosylase
  • the DNA binding domain is a zinc-finger DNA-binding domain, a TALE DNA- binding domain, a Cas9 nuclease, a Cas 12a nuclease, a catalytically inactive Cas9 nuclease, a catalytically inactive Cas 12a nuclease, a Cas9 nickase, or a Cpfl nikase.
  • the site-specific genome modification enzyme is a dCas9- cytosine deaminase fusion protein. In another aspect, the site-specific genome modification enzyme is a dCas9-adenine deaminase fusion protein. In some embodiments, one or more of a dCas9-cytosine deaminase fusion protein and a dCas9-adenine deaminase fusion protein are utilized to modify a Bmr3 gene sequence. In some embodiments, a Bmr3 gene sequence is modified to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 21-252, 401-443, and 500-507.
  • a Bmr3 gene sequence is modified to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382. In some embodiments, a Bmr3 gene sequence is modified to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 348, 350, 352, and 354.
  • the site-specific genome modification enzyme is a dCpfl - cytosine deaminase fusion protein. In another aspect, the site-specific genome modification enzyme is a dCpfl -adenine deaminase fusion protein. In some embodiments, one or more of a dCpfl -cytosine deaminase fusion protein and a dCpfl -adenine deaminase fusion protein are utilized to modify a Bmr3 gene sequence. In some embodiments, a Bmr3 gene sequence is modified to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 21-252, 401-443 and 500-507.
  • a Bmr3 gene sequence is modified to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382. In some embodiments, a Bmr3 gene sequence is modified to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 348, 350, 352, and 354.
  • a site-specific genome modification enzymes such as meganucleases, ZFNs, TALENs, Argonaute proteins
  • Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), homologs thereof, or modified versions thereol
  • RNA-guided nucleases include the CRISPR associated nucleases, such as Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Cas 12a (also known as Cpfl), Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csc2, Csc2, Cs
  • breaks or nicks in the target DNA sequence are repaired by the natural processes of homologous recombination (HR) or non-homologous end-joining (NHEJ).
  • sequence modifications occur at or near the cleaved or nicked sites, which can include deletions or insertions that result in modification of the nucleic acid sequence, or integration of exogenous nucleic acids by homologous recombination or NHEJ.
  • a targeted genome modification as described herein comprises the use of a zinc-finger nuclease (ZFN).
  • ZFNs are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to the cleavage domain of the Fokl restriction nuclease.
  • ZFNs can be designed to cleave almost any long stretch of double-stranded DNA for modification of the zinc finger DNA-binding domain.
  • ZFNs form dimers from monomers composed of anon-specific DNA cleavage domain of Fokl nuclease fused to a zinc finger array engineered to bind a target DNA sequence.
  • the DNA-binding domain of a ZFN is typically composed of 3-4 zinc-finger arrays.
  • the other amino acids form the consensus backbone to generate ZFNs with different sequence specificities. Rules for selecting target sequences for ZFNs are known in the art.
  • the Fokl nuclease domain requires dimerization to cleave DNA and therefore two ZFNs with their C-terminal regions are needed to bind opposite DNA strands of the cleavage site (separated by 5-7 nt).
  • the ZFN monomer can cut the target site if the two-ZF-binding sites are palindromic.
  • ZFN is broad and includes a monomeric ZFN that can cleave double stranded DNA without assistance from another ZFN.
  • ZFN is also used to refer to one or both members of a pair of ZFNs that are engineered to work together to cleave DNA at the same site.
  • a ZFN provided herein is capable of generating a targeted DSB.
  • a ZFN provided herein is capable of generating a targeted DSB in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, or g3279.
  • a ZFN provided herein is capable of generating a targeted single-strand break (SSB).
  • SSB single-strand break
  • a ZFN provided herein is capable of generating a targeted SSB in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, or g3279.
  • vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more ZFNs are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation).
  • meganucleases which are commonly identified in microbes, are unique enzymes with high activity and long recognition sequences (> 14 nt) resulting in site-specific digestion of target DNA.
  • Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (for example, 14 to 40 nt).
  • the engineering of meganucleases can be more challenging than that of ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain.
  • Specialized methods of mutagenesis and high-throughput screening have been used to create novel meganuclease variants that recognize unique sequences and possess improved nuclease activity.
  • a method and/or composition provided herein comprises one or more, two or more, three or more, four or more, or five or more meganucleases directed to a target sequence in a Bmr3 gene.
  • a meganuclease provided herein is capable of generating a targeted DSB.
  • a meganuclease provided herein is capable of generating a targeted DSB in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, org3279.
  • a meganuclease provided herein is capable of generating a targeted SSB. In some embodiments, a meganuclease provided herein is capable of generating a targeted SSB in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, or g3279.
  • vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more meganucleases are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation).
  • a targeted genome modification as described herein comprises the use of a transcription activator-like effector nuclease (TALEN).
  • TALENs are artificial restriction enzymes generated by fusing the transcription activator-like effector (TALE) DNA binding domain to a nuclease domain.
  • the nuclease is selected from a group consisting of PvuII, MutH, Tevl and Fokl, Alwl, Mlyl, Sbfl, Sdal, Stsl, CleDORF, Clo051, Pept071.
  • TALEN as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN.
  • TALEN is also used to refer to one or both members of a pair of TALENs that work together to cleave DNA at the same site.
  • Transcription activator-like effectors TALEs
  • TALEs can be engineered to bind practically any DNA sequence, such as a target sequence in a Bmr3 gene.
  • TALE proteins are DNA-binding domains derived from various plant bacterial pathogens of the genus Xanthomonas.
  • the X pathogens secrete TALEs into the host plant cell during infection.
  • the TALE moves to the nucleus, where it recognizes and binds to a specific DNA sequence in the promoter region of a specific DNA sequence in the promoter region of a specific gene in the host genome.
  • TALE has a central DNA-binding domain composed of 13-28 repeat monomers of 33-34 amino acids.
  • the amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13.
  • the two variable amino acids are called repeat-variable diresidues (RVDs).
  • the amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine/adenine, respectively, and modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.
  • a method and/or composition provided herein comprises one or more, two or more, three or more, four or more, or five or more TALENs.
  • a TALEN provided herein is capable of generating a targeted DSB in a target sequence in Bmr3 gene.
  • a TALEN provided herein is capable of generating a targeted DSB in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, or g3279.
  • a TALEN provided herein is capable of generating a targeted SSB in a target sequence in Bmr3 gene.
  • a TALEN provided herein is capable of generating a targeted SSB in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, or g3279.
  • vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more TALENs are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation).
  • a targeted genome modification as described herein comprises the use of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten RNA-guided nucleases.
  • a CRISPR/Cas9 system, a CRISPR/Cpfl system, a CRISPR/CasX system, or a CRISPR/CasY system are alternatives that may be used to generate modifications to a nucleic acid encoding Bmr3 gene.
  • the CRISPR systems are comprised of a CRISPR associated protein that binds to a guide RNA that uses complementary base pairing to recognize DNA sequences at target sites.
  • CRISPR systems are an alternative to synthetic proteins whose DNA-binding domains enable them to modify genomic DNA at specific sequences (e.g.,
  • RNA-guide that uses complementary base pairing to recognize target DNA sequences.
  • the RNA-guide comprises a sequence as set forth in SEQ ID NO: 13 and a sequence selected from the group consisting of SEQ ID NOs: 7-12, 399 and 400.
  • the site-specific genome modification enzyme is a
  • a site-specific genome modification enzyme provided herein can comprise any RNA-guided Cas nuclease (non-limiting examples of RNA-guided nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Cs
  • Expression constructs generally include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements.
  • expression construct refers to a combination of nucleic acid sequences that provides for transcription of an operably linked nucleic acid sequence.
  • operably linked means two DNA molecules linked in manner so that one may affect the function of the other. Operably-linked DNA molecules may be part of a single contiguous molecule and may or may not be adjacent.
  • a promoter is operably linked with a polypeptide-encoding DNA molecule in a DNA construct where the two DNA molecules are so arranged that the promoter may affect the expression of the DNA molecule.
  • heterologous refers to the relationship between two or more items derived from different sources and thus not normally associated in nature.
  • a protein-coding recombinant DNA molecule is heterologous with respect to an operably linked promoter if such a combination is not normally found in nature.
  • a particular recombinant DNA molecule may be heterologous with respect to a cell, seed, or organism into which it is inserted when it would not naturally occur in that particular cell, seed, or organism.
  • An expression construct can comprise a promoter sequence operably linked to a polynucleotide sequence encoding a site-specific genome modification enzyme as described herein. Promoters can be incorporated into a polynucleotide using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression construct as described herein. In some embodiments, a promoter can be positioned about the same distance from the transcription start site in the expression construct as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity. A transcription start site is typically included in the expression construct.
  • plant viral promoters such as, for example, a cauliflower mosaic virus (CaMV) 35 S (including the enhanced CaMV 35S promoter (see, for example U.S. Pat. No. 5,106,739)) or a CaMV 19S promoter or a cassava vein mosaic can be used.
  • CaMV cauliflower mosaic virus
  • Other promoters that can be used for expression constructs in plants include, for example, zein promoters including maize zein promoters, prolifera promoter, Ap3 promoter, heat shock promoters, T-DNA - or 2'-promoter of A.
  • tumefaciens polygalacturonase promoter, chalcone synthase A (CHS-A) promoter from petunia, tobacco PR- la promoter, ubiquitin promoter, actin promoter, alcA gene promoter, pin2 promoter (Xu et al, 1993), maize Wipl promoter, maize trpA gene promoter (U.S. Pat. No. 5,625,136), maize CDPK gene promoter, and RUBISCO SSU promoter (U.S. Pat. No. 5,034,322) can also be used.
  • CHS-A chalcone synthase A
  • Constitutive promoters such as the CaMV, ubiquitin, actin, or NOS promoter
  • developmentally -regulated promoters such as those promoters than can be induced by heat, light, hormones, or chemicals
  • inducible promoters such as those promoters than can be induced by heat, light, hormones, or chemicals
  • Expression constructs may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and/or enhancer elements.
  • Transcription termination regions can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination.
  • a signal peptide sequence is a short amino acid sequence typically present at the amino terminus of a protein that is responsible for the relocation of an operably linked mature polypeptide to a wide range of post-translational cellular destinations, ranging from a specific organelle compartment to sites of protein action and the extracellular environment.
  • Classical enhancers are cis- acting elements that increase gene transcription and can also be included in the expression construct.
  • Classical enhancer elements are known in the art, and include, but are not limited to, the CaMV 35S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element.
  • CMV cytomegalovirus
  • Intron-mediated enhancer elements that enhance gene expression are also known in the art. These elements must be present within the transcribed region and are orientation dependent. Examples include the maize shrunken- 1 enhancer element (Clancy and Hannah, 2002). Transformation Methods
  • telomeres are recombinant DNA encoding a site-specific genome modification enzyme as described herein.
  • the recombinant DNA encoding a site-specific genome modification enzyme is bred out of plants containing a modified Bmr3 gene.
  • Suitable methods for transformation of host plant cells include virtually any method by which DNA or RNA can be introduced into a cell (for example, where a recombinant DNA construct is stably integrated into a plant chromosome or where a recombinant DNA construct or an RNA is transiently provided to a plant cell) and are well known in the art.
  • Two effective methods for cell transformation are Agrobacterium- mediated transformation and microprojectile bombardment-mediated transformation. Microprojectile bombardment methods are illustrated, for example, in US Patent Nos. US 5,550,318; US 5,538,880; US 6,160,208; and US 6,399,861.
  • Agrobacterium-modiated transformation methods are described, for example in US Patent No.
  • Transformation of plant material is practiced in tissue culture on nutrient media, for example a mixture of nutrients that allow cells to grow in vitro.
  • Recipient cell targets include, but are not limited to, meristem cells, shoot tips, hypocotyls, calli, immature or mature embryos, and gametic cells such as microspores and pollen. Callus can be initiated from tissue sources including, but not limited to, immature or mature embryos, hypocotyls, seedling apical meristems, microspores and the like. Cells containing a transgenic nucleus are grown into transgenic plants.
  • DNA is typically introduced into only a small percentage of target plant cells in any one transformation experiment.
  • Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a recombinant DNA molecule into their genomes.
  • Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or an herbicide. Any of the herbicides to which plants of this disclosure can be resistant is an agent for selective markers.
  • Potentially transformed cells are exposed to the selective agent. In the population of surviving cells are those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells can be tested further to confirm stable integration of the exogenous DNA.
  • Select marker genes include those conferring resistance to antibiotics such as kanamycin and paromomycin ( nptll ), hygromycin B ( aph IV), spectinomycin (aaclA) and gentamycin (aac3 and aacCA) or resistance to herbicides such as glufosinate ( bar or pat), dicamba (DMO) and glyphosate ( aroA or EPSPS). Examples of such selectable markers are illustrated in US Patent Nos. US 5,550,318; US 5,633,435; US 5,780,708 and US 6,118,047.
  • Markers which provide an ability to visually screen transformants can also be employed, for example, a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a ieto-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.
  • a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a ieto-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.
  • Several embodiments relate to methods and constructs for regenerating a plant from a cell with modified Bmr3 gene sequence resulting from genome editing. The regenerated plant can then be used to propagate additional plants.
  • Several embodiments relate to a method of introducing an edited allelic variant of a Bmr3 gene comprising: (a) introducing at least one modification to an endogenous Bmr3 gene in at least one plant cell, thereby generating said edited allelic variant; (b) identifying and selecting at least one plant cell of step (a) comprising said edited allelic variant that exhibits one or more of reduced lignin content, reddish-brown pigmentation of the leaf midrib, reduction of RNA expression from the Bmr3 gene, reduction of COMT levels, and absence of COMT; and (c) regenerating a plant from the at least one plant cell selected in step (b).
  • the method comprises introducing a modification in a genomic sequence targeted by g90, g227, g867, g2010, g2070, g2691, g3170 or g3279.
  • the edited allelic variant of a Bmr3 comprises a sequence selected from SEQ ID NOs: 21-252, 401-443, and 500-507.
  • the edited allelic variant of a Bmr3 comprises a sequence selected from SEQ ID NOs: 358, 361, 364, 370, 373, 376, 379, and 382.
  • the edited allelic variant of a Bmr3 comprises a sequence selected from SEQ ID NOs: 348, 350, 352, and 354.
  • the modified plants, seeds, plant cells, and plant parts as described herein, and any progeny thereof may also contain one or more additional traits. Additional traits may be introduced by crossing a plant containing an edited Bmr3 allele as described herein with another plant containing one or more additional trait(s). As used herein,“crossing” means breeding two individual plants to produce a progeny plant. Two plants may be crossed to produce progeny that contain the desirable traits from each parent. As used herein“progeny” means the offspring of any generation of a parent plant, and progeny comprise an edited Bmr3 allele as described herein and inherited from at least one parent plant. Additional trait(s) also may be introduced by any means known in the art.
  • Such additional traits include, but are not limited to, increased insect resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, floury-endosperm, short stature, and herbicide-tolerance, in which the trait is measured with respect to a wild-type plant.
  • Examples of additional herbicide tolerance traits may include transgenic or non-transgenic tolerance to one or more herbicides such as ACCase inhibitors (for example, aryloxyphenoxy propionates and cyclohexanediones), ALS inhibitors (for example, sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones) EPSPS inhibitors (for example, glyphosate), synthetic auxins (for example, phenoxys, benzoic acids, carboxylic acids, semicarbazones), photosynthesis inhibitors (for example, triazines, triazinones, nitriles, benzothiadiazoles, and ureas), glutamine synthesis inhibitors (for example, glufosinate), HPPD inhibitors (for example, isoxazoles, pyrazolones, and triketones), PPO inhibitors (for example, diphenylethers, N-phenylphthalimide,
  • insect resistance traits may include resistance to one or more insect members within one or more of the orders of Lepidoptera, Coleoptera, Hemiptera, Thysanoptera, Diptera, Hymenoptera, and Orthoptera, among others.
  • additional traits are well-known to one of skill in the art; for example, and a list of such transgenic traits is provided by the United States Department of Agriculture’s (USDA) Animal and Plant Health Inspection Service (APHIS).
  • USDA United States Department of Agriculture
  • API Animal and Plant Health Inspection Service
  • Examples of short stature traits are disclosed in U.S. Patent Application No. 15/679,699, incorporated herein by reference.
  • the modified plants, seeds, plant cells, and plant parts comprising an edited allele of Bmr3 as described herein, and any progeny thereof may also contain one or more additional brown midrib traits.
  • an additional brown midrib trait may be conferred by a native mutation in the brown midrib 1 gene (bml).
  • an additional brown midrib trait may be conferred by a native mutation in the brown midrib 2 gene (bm2 or bmr2).
  • an additional brown midrib trait may be conferred by a native mutation in the brown midrib 4 gene (bm4).
  • an additional brown midrib trait may be conferred by a native mutation in the brown midrib 5 gene (bm5).
  • an additional brown midrib trait may be conferred by a native mutation in the brown midrib 6 gene (bmr6). In some embodiments, an additional brown midrib trait may be conferred by a native mutation in the brown midrib 12 gene (bml2). In some embodiments, an additional brown midrib trait may be conferred by a native mutation in the brown midrib 19 gene (bmrl9). Additional brown midrib traits may be introduced by crossing a plant containing an edited Bmr3 allele as described herein with another plant containing one or more additional trait(s). In some embodiments, an edited Bmr3 allele may be introduced by genome editing methods as described herein in a germplasm comprising one or more additional brown midrib traits.
  • Plants and progeny that comprise an edited Bmr3 allele as described herein may be used with any breeding methods that are commonly known in the art.
  • the traits may be independently segregating, linked, or a combination of both in plant lines comprising three or more traits.
  • Backcrossing to a parental plant and outcrossing with a non-traited plant are also contemplated, as is vegetative propagation. Descriptions of breeding methods that are commonly used for different traits and crops are well-known to those of skill in the art.
  • Such assays include, for example, molecular biology assays, such as Southern and northern blotting, PCR, and DNA sequencing; biochemical assays, such as detecting the presence of a protein product, for example, by immunological means (ELISAs and western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and, by analyzing the phenotype of the whole plant.
  • molecular biology assays such as Southern and northern blotting, PCR, and DNA sequencing
  • biochemical assays such as detecting the presence of a protein product, for example, by immunological means (ELISAs and western blots) or by enzymatic function
  • plant part assays such as leaf or root assays
  • analyzing the phenotype of the whole plant include, for example, molecular biology assays, such as Southern and northern blotting, PCR, and DNA sequencing; biochemical assays, such as detecting the presence of a protein product, for example, by
  • Introgression of a trait into a plant genotype is achieved as the result of the process of backcross conversion.
  • a plant genotype into which a trait has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid.
  • a plant genotype lacking the desired trait may be referred to as an unconverted genotype, line, inbred, or hybrid.
  • Example 1 Brown midrib (Bmr3) gene structure and design of gRNAs
  • the Brown midrib 3 (Bmr3) gene encodes caffeic acid-O-methyltransferase (COMT), a key enzyme involved in synthesis of flavonolignan.
  • the com Bmr3 gene is located on chromosome 4 and has two exons and one intron as shown in Figure 1.
  • the genomic sequences of the Bmr3 gene from 01DKD2 and LH244 inbred com lines are set forth in SEQ ID NO: 1 and SEQ ID NO: 4, respectively.
  • Eight guide RNA targeting sequences (SEQ ID NOs: 7 through 12, and SEQ ID NO: 399 and 400) were identified in the com Bmr3 gene with their positions relative to the Bmr3 genomic DNA sequence as shown in Table 1.
  • Each gRNA comprises a Cpfl crRNA sequence as set forth in SEQ ID NO: 13 and one of gRNA targeting sequences listed in Table 1.
  • Example 2 Constructs to express cpfl and gRNA in corn plants
  • T-DNA vectors (A through E as illustrated in Figure 2) having a Cpfl expression cassette, a gRNA expression cassette, and an expression cassette for a selectable marker conferring resistance to glyphosate were generated to deliver Cpfl and one or more gRNAs as described in Example 1 into plant cells.
  • a codon optimized version of Lachnospiraceae bacterium ND2006 Cpfl (SEQ ID NO: 19) was cloned into an expression cassette operably linked to a Zea mays Ubiquitin Ml promoter as set forth in SEQ ID NO: 15 followed by its leader sequence as set forth in SEQ ID NOs: 16 and intron sequence as set forth in SEQ ID NO: 17.
  • the Cpfl coding sequence was flanked by a nuclear localization sequence (NLS) as set forth in SEQ ID NO: 18.
  • the 5’ end copy of NLS further comprises ATGGCG for translation start and the 3’ end copy of NLS further comprises TGA for translation stop.
  • the Cpfl expression cassette further has a transcription terminator sequence from rice LTP as set forth in SEQ ID NO: 20.
  • the recombinant T-DNA vectors each contain a gRNA expression cassette having a com U6 promoter as set forth in SEQ ID NO: 14 operably linked to one or more gRNAs described in Example 1.
  • the gRNA expression cassette for recombinant T-DNA vector A was generated with three guide RNAs, g867, g2010 and g3170, positioned in tandem.
  • the gRNA expression cassette for recombinant T-DNA vector B was generated with the gRNA expression cassette for three guide RNAs, g2070, g2691 and g3279, positioned in tandem.
  • the recombinant T- DNA vector C was designed to have g2691 as the single guide RNA.
  • the recombinant T-DNA vector D and E were designed to generate whole gene deletion of Bmr3 gene using g90 and g227 respectively in combination with g3279.
  • Com embryos were transformed with recombinant T-DNA vectors disclosed hereinby agrobacterium-mediated transformation. Transformed plants were selected on glyphosate.
  • Leaf samples were collected from selected plants transformed with the recombinant DNA vectors as described in Example 2 for DNA extraction. PCR reactions were carried out using primers as show in Table 2 to amplify the segments of the Bmr3 gene targeted by the Cpfl editing systems. The PCR products were sequenced and aligned with sequences of the unedited Bmr3 gene from inbred com lines to identify the edited Bmr3 alleles listed in Table 3.
  • Example 4 Gene expression, physiology and characterization of corn plants having edited Bmr3 alleles
  • Com plants with mutations in Brown midrib genes exhibit a characteristic brown coloration of the leaf mid veins that is visibly scorable at v3-vl0 stages.
  • Table 5 and Figure 2 three events generated by recombinant T-DNA vector B (described in Example 2) were bred to R1 stage.
  • One of the events, B-l is biallelic at the site targeted by g2691.
  • Plants that are homozygous for Bmr3 alleles comprising SEQ ID NO 191, 194 and 172 exhibited the brown midrib phenotype.
  • plants with one Bmr3 allele comprising SEQ ID NO: 191 and one Bmr3 allele comprising 209 were also exhibited brown midrib phenotype.
  • D-l was generated by recombinant T-DNA vector D whereas two events, E-l and E-2, were generated by recombinant T-DNA vector E as shown in Table 7. These three events have whole gene deletion for Bmr3 and their progenies are homozygous for the deletion exhibited brown midrib phenotype.
  • Leaf samples were collected from plants that were homozygous for one of the Bmr 3 alleles comprising SEQ ID NO: 172, 191, or 194. In addition, leaf samples were collected from plants that were heterozygous for the Bmr3 allele comprising SEQ ID NO: 22.
  • RNA was extracted from the leaf samples using a standard RNA extraction protocol. Two primers sets were used to detect Bmr3 RNA levels in wild type (control) and the edited plants. Primer set 1, which comprised primers of SEQ ID NO: 395 and 396 spans exon 2 to detect only the wild type Bmr3 mRNA, but not the mRNA from the edited Bmr3 alleles.
  • Primer set 2 which comprised primers of SEQ ID NOs: 397 and 398 spans exon-1 of Bmr3 gene and this primer set amplifies and detects RNA from both the wild-type and the edited alleles, since the deletions in the edited lines are in exon2.
  • Bmr3 gene along with two reference genes used for internal normalization were detected by RT-qPCR using Taqman assays. Expression (also referred to as Relative Quantity) in arbitrary units was calculated as per standard protocols.
  • Bmr3 mRNA was detected only in the wild-type control and Bmr3 edited event that is heterozygous for the exon2 deletion (SEQ ID NO: 22), but not in the three edited events that are homozygous for 8bp (SEQ ID NO: 194), 9bp (SEQ ID NO: 191), and 16bp deletions (SEQ ID NO: 172) ( Figure 3A).
  • primer set 2 which spans exon-1, RNA of both the wild-type control and the edited lines were detected ( Figure 3B).
  • Brown midrib visible phenotype is associated with reduced lignin content and altered lignin composition, which are useful to improve forage digestibility for livestock.

Abstract

Provided herein are novel methods and compositions for producing corn plants that have improved digestibility. Also provided are corn plants, corn plant tissues and corn plant seeds that contain novel edited Brown midrib 3 (Bmr3) genes and proteins, as well as methods of producing the same.

Description

METHODS AND COMPOSITIONS FOR IMPROVING SILAGE
FIELD
[0001] The present disclosure relates to the field of biotechnology. More specifically, the present disclosure relates to compositions and methods for producing com plants that have improved digestibility. In particular, the present disclosure relates to com plants, com plant tissues and com plant seeds that contain altered Brown midrib 3 (BMR3) genes and proteins.
INCORPORATION OF SEQUENCE LISTING
[0002] A sequence listing contained in the file named“P34704WO00_SL.txt” which is 547,772 bytes measured in MS-Windows®) and created on December 3, 2019, comprises 530 sequences, is filed electronically herewith and incorporated by reference in its entirety.
BACKGROUND
[0003] Dairy farmers rely on silage from com and other grains to feed their herds. One mechanism for improving the digestibility of silage com is to reduce the lignin level in the plant through breeding, since lignin is indigestible by ruminants. Naturally occurring mutations in the Brown Midrib 1 (Bmrl) (Jorgenson, L.R. 1931. Brown midrib in maize and its lignage relations. J. Am. Soc. Agron. 23:549-557.), Brown Midrib 2 (Bmr2) (Burnham, C.R., and R.A. Brink. 1932. Linkage relations of a second brown midrib gene (bm2) in maize. J. Am. Soc. Agron. 24:960-963.), Brown Midrib 3 (Bmr3) (Morrow, S.L. et al. 1997. Molecular characterization of a brown midrib3 deletion mutation in maize. Molecular Breeding. 3:351-357.) and Brown Midrib 4 (Bmr4) (Burnham, C.R. 1947. Maize Genet Coop News 21:36.) genes produce com plants with reddish-brown pigmentation of the leaf midrib and reduced lignin levels. These brown midrib hybrids produce forage that typically has about 5-10 percentage points higher in fiber digestibility than non-brown midrib com hybrids. Higher fiber digestibility is believed to contribute to higher feed intakes and greater milk production by dairy cows. Farraretto and Shaver. J Dairy Sci 98: 2662.
[0004] The currently available brown midrib hybrids exhibit yield drag compared to non brown midrib hybrids. In addition to impacted yield, brown midrib hybrids exhibit defects in plant standability, since lignin contributes to the stability of plant architecture. Negative agronomic traits such as a lack of disease resistance and increased susceptibility to drought have also been reported for the currently available brown midrib hybrids. There is therefore a need to develop brown midrib com lines that lack the yield drag and other negative agronomic traits associated with the currently available brown midrib hybrids. The present disclosure describes using a gene editing approach to provide brown midrib com plants with improved agronomic traits.
SUMMARY
[0005] Several embodiments relate to a modified com plant comprising an edited allele of Bmr3, wherein the modified com plant exhibits brown pigmentation in the leaf midrib at the v3-vl0 stage. In some embodiments, the modified com plant is homozygous for an edited allele of Bmr3. In some embodiments, the modified com plant is heterozygous for an edited allele of Bmr3. In some embodiments, the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507. In some embodiments, the edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507. In some embodiments, the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382. In some embodiments, the edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367,
370, 373, 376, 379, and 382. In some embodiments, the edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354. In some embodiments, the modified com plant comprises a first edited allele of Bmr3 and a second edited allele of Bmr3, wherein the first and second edited allele of Bmr3 each, independently, comprise a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 348, 350, 352, 354, 358, 361, 364, 367, 370, 373, 376, 379, 382, 401-443 and 500-507. In some
embodiments, the modified com plant comprises a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 253-299. In some embodiments, silage produced from the modified com plant has at least 30%, 31%, 32%, 33%. 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%. 44%, 45%, 46%, 47%, 48%, 49%, or 50 % Neutral Detergent Fiber (NDF). In some embodiments, silage produced from the modified com plant has at least 12%, 13%. 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% Acid Detergent Fiber (ADF). In some
embodiments, the lignin content of the modified com plant is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% or more as compared to a non-brown midrib com plant. In some
embodiments, the lignin content of the modified com plant is reduced by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% or more as compared to com plant with a native Bmr3 mutant allele. Several embodiments relate to a seed produced from the modified com plant. In some embodiments, the modified com plant comprises one or more additional traits. In some embodiments, the modified com plant comprises a floury -2 trait. Several embodiments relate to a part of the modified com plant selected from the group consisting of an intact plant cell, a plant protoplast, an embryo, a pollen, an ovule, a flower, a kernel, a seed, an ear, a cob, a leaf, a husk, a stalk, a root, a root tip, a brace root, a lateral tassel branch, an anther, a tassel, a glume, a tiller and a silk.
[0006] Several embodiments relate to a method of generating a modified com plant with reduced lignin comprising the steps of: (a) introducing a non-natural mutation in a Bmr3 gene of a com plant cell; (b) identifying and selecting one or more plant cells of step (a) comprising said non-natural in Bmr3; and (c) regenerating at least one plant from at least one or more cells selected in step (b). In some embodiments, the non-natural mutation is introduced by a site-specific genome modification enzyme. In some embodiments, the non natural mutation is introduced by Cpfl complexed with guide RNA comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400. In some embodiments, the com plant cell of step (a) is derived from a com plant having one or more traits. In some embodiments, the com plant cell of step (a) is derived from a com plant having a floury -2 trait. In some embodiments, the com plant cell of step (a) is derived from a com plant having a disease resistance trait. In some embodiments, the com plant cell of step (a) is derived from a com plant having an herbicide resistance trait. In some embodiments, the com plant cell of step (a) is derived from a com plant having a native brown midrib trait. In some
embodiments, the non-natural mutation comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507. In some embodiments, the non natural mutation comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs:
358, 361, 364, 367, 370, 373, 376, 379, and 382. In some embodiments, the non-natural mutation comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354. In some embodiments, the plant of step c is crossed with to produce progeny that contain the desirable trait.
[0007] In another aspect, a method of generating a modified plant with reduced lignin levels is provided, comprising the steps of: (a) introducing a modification in a Bmr3 gene of a plant cell; (b) identifying and selecting one or more plant cells of step (a) comprising said modification in a Bmr3 gene; and (c) regenerating at least one plant from at least one or more cells selected in step (b). In some embodiments, the modification comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507. In some embodiments, the modification comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382. In some embodiments, the modification comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354. In some embodiments, the modification introduced by Cpfl complexed with guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400. In some embodiments, the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 383 and SEQ ID NO: 389 and sequencing the amplified DNA. In some embodiments, the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 384 and SEQ ID NO: 390 and sequencing the amplified DNA. In some embodiments, the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 385 and SEQ ID NO: 391 and sequencing the amplified DNA. In some embodiments, the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 386 and SEQ ID NO: 392 and sequencing the amplified DNA. In some embodiments, the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 387 and SEQ ID NO: 393 and sequencing the amplified DNA. In some embodiments, the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 388 and SEQ ID NO: 394 and sequencing the amplified DNA. In some embodiments, the identifying of step (b) comprises amplifying a DNA sequence with primers of SEQ ID NO: 520 and SEQ ID NO: 521 and sequencing the amplified DNA.
[0008] In another aspect, a modified com plant, com plant part, com cell, or com seed comprising an edited allele of Bmr3 as described herein is provided. A modified plant genome comprising an edited allele of Bmr3 as described herein is further provided. In some embodiments, the edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507. In some embodiments, the edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382. In some embodiments, the edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354. In some embodiments, com plant part is selected from the group consisting of an intact plant cell, a plant protoplast, an embryo, a pollen, an ovule, a flower, a kernel, a seed, an ear, a cob, a leaf, a husk, a stalk, a root, a root tip, a brace root, a lateral tassel branch, an anther, a tassel, a glume, a tiller and a silk.
[0009] Several embodiments relate to com silage produced from a modified com plant comprising an edited allele of Bmr3. In some embodiments, the com silage is produced from a modified com plant comprising and edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507. In some embodiments, the com silage is produced from a modified com plant comprising and edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367,
370, 373, 376, 379, and 382. In some embodiments, the com silage is produced from a modified com plant comprising and edited allele of Bmr3 comprises a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354. In some embodiments, the com silage has a lignin content of 1%, 2%, 3%, 4%, or 5%. In some embodiments, the com silage has at least 30%, 31%, 32%, 33%. 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%. 44%, 45%, 46%, 47%, 48%, 49%, or 50 % Neutral Detergent Fiber (NDF). In some embodiments, the com silage has at least 12%, 13%. 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% Acid Detergent Fiber (ADF).
[0010] Several embodiments relate to a method of enhancing milk production efficiency in ruminants, the method comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising an edited allele of Bmr3; and feeding the ruminants with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the ruminants. Several embodiments relate to a method of enhancing milk production efficiency in ruminants, the method comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507; and feeding the ruminants with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the ruminants. Several embodiments relate to a method of enhancing milk production efficiency in ruminants, the method comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382; and feeding the ruminants with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the ruminants. Several embodiments relate to a method of enhancing milk production efficiency in ruminants, the method comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354; and feeding the ruminants with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the ruminants. In some embodiments, the com hybrid further comprises a mutant allele of floury -2. In some embodiments, the com hybrid has a lignin content of 1%, 2%, 3%, 4%, or 5%.
[0011] Several embodiments relate to a method of enhancing milk production efficiency in dairy cattle, the method comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising an edited allele of Bmr3; and feeding the dairy cattle with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the dairy cattle. Several embodiments relate to a method of enhancing milk production efficiency in dairy cattle, the method comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507; and feeding the dairy cattle with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the dairy cattle. Several embodiments relate to a method of enhancing milk production efficiency in dairy cattle, the method comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382; and feeding the dairy cattle with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the dairy cattle. Several embodiments relate to a method of enhancing milk production efficiency in dairy cattle, the method comprising: preparing a feed ration comprising a com silage made from a com hybrid comprising a nucleotide sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354; and feeding the dairy cattle with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the dairy cattle. In some embodiments, the com hybrid further comprises a mutant allele of floury -2. In some embodiments, the com hybrid has a lignin content of 1%, 2%, 3%, 4%, or 5%. [0012] Several embodiments relate to a modified protein comprising an amino acid sequence that is at least 95%, at least 96%, at least 96%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 253-299, 444-471, and 508-513. Several embodiments relate to a com plant comprising a modified protein comprising an amino acid sequence that is at least 95%, at least 96%, at least 96%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 253-299, 444-471, and 508- 513. Several embodiments relate to com silage produced from a com plant comprising a modified protein comprising an amino acid sequence that is at least 95%, at least 96%, at least 96%, at least 98%, at least 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 253-299, 444-471, and 508-513.
[0013] Several embodiments relate to a modified com plant produced by: (a) providing a com plant cells wherein the cell comprises a target genomic nucleic acid molecule having at least 90% sequence identity with a sequence selected from the group consisting of:
nucleotides 146-168 of SEQ ID NO: 1, nucleotides 283-305 of SEQ ID NO: 1, nucleotides 637-659 of SEQ ID NO: 1, nucleotides 1802-1824 of SEQ ID NO: 1, nucleotides 1862-1884 of SEQ ID NO: 1, nucleotides 2461-2483 of SEQ ID NO: 1, nucleotides 2940-2962 of SEQ ID NO: 1, nucleotides 3071-3093 of SEQ ID NO: 1, nucleotides 73-95 of SEQ ID NO: 4, nucleotides 1276-1298 of SEQ ID NO: 4, nucleotides 1336-1358 of SEQ ID NO: 4, nucleotides 1935-1957 of SEQ ID NO: 4, nucleotides 2401-2423 of SEQ ID NO: 4, nucleotides 1622-1644 of SEQ ID NO: 522, and nucleotides 2231-2254 of SEQ ID NO: 522; (b) selecting a site-specific genome modification enzyme that specifically binds and cleaves the target genomic nucleic acid molecule; (c) introducing the site-specific genome modification enzyme into the com plant cells, wherein the site-specific nuclease cleaves the target genomic nucleic acid molecule; (d) generating com plants from the com plant cells; and (e) selecting a com plant exhibiting brown coloration of the leaf mid veins at v3-vl0 stages. In some embodiments, the site-specific genome modification enzyme is selected from the group consisting of a zinc-finger nuclease, an engineered meganuclease, a native meganuclease, a TALE-endonuclease, and an RNA-guided endonuclease. In some embodiments, the site-specific genome modification enzyme is a CRISPR associated protein complexed with guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400. In some embodiments, the CRISPR associated protein is selected from the group consisting of: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csl3, Csf4, Cpfl, CasX, CasY, and Mad7. In some embodiments, the site-specific genome modification enzyme is a Cpfl protein complexed with guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400. In some embodiments, the modified com plant lacks a Bmr3 gene product. In some embodiments, the modified com plant comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 348, 350, 352, 354, 358, 361, 364, 367, 370, 373, 376, 379, 382, 401-443, and 500-507. In some embodiments, the modified com plant comprises a Bmr3 gene product comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 253-299, 444-471, and 508- 513.
[0014] Several embodiments relate to method of generating a com plant that exhibits a brown midrib phenotype, the method comprising: (a) providing com plant cells wherein the cells comprise a target genomic nucleic acid molecule having at least 90% sequence identity with a sequence selected from the group consisting of: nucleotides 146-168 of SEQ ID NO: 1, nucleotides 283-305 of SEQ ID NO: 1, nucleotides 637-659 of SEQ ID NO: 1, nucleotides 1802-1824 of SEQ ID NO: 1, nucleotides 1862-1884 of SEQ ID NO: 1, nucleotides 2461- 2483 of SEQ ID NO: 1, nucleotides 2940-2962 of SEQ ID NO: 1, nucleotides 3071-3093 of SEQ ID NO: 1, nucleotides 73-95 of SEQ ID NO: 4, nucleotides 1276-1298 of SEQ ID NO:
4, nucleotides 1336-1358 of SEQ ID NO: 4, nucleotides 1935-1957 of SEQ ID NO: 4, nucleotides 2401-2423 of SEQ ID NO: 4, nucleotides 1622-1644 of SEQ ID NO: 522, and nucleotides 2231-2254 of SEQ ID NO: 522; (b) introducing a site-specific genome modification enzyme that specifically binds and cleaves the target genomic nucleic acid molecule, wherein the site-specific nuclease cleaves the target genomic nucleic acid molecule; (d) generating com plants from the com plant cells; and (e) selecting a com plant exhibiting brown coloration of the leaf mid veins at v3-vl0 stages. In some embodiments, the site-specific genome modification enzyme is a Cpfl protein complexed with guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400. In some embodiments, the com plant is an elite com plant. In some embodiments, the modified com plant comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 348, 350, 352, 354, 358, 361, 364, 367, 370, 373, 376, 379, 382, 401-443, and 500-507.
[0015] Several embodiments relate to method of generating a com line with favorable yield that exhibits a brown midrib phenotype, the method comprising: (a) providing elite com plant cells wherein the cells comprise a target genomic nucleic acid molecule having at least 90% sequence identity with a sequence selected from the group consisting of: nucleotides 146-168 of SEQ ID NO: 1, nucleotides 283-305 of SEQ ID NO: 1, nucleotides 637-659 of SEQ ID NO: 1, nucleotides 1802-1824 of SEQ ID NO: 1, nucleotides 1862-1884 of SEQ ID NO: 1, nucleotides 2461-2483 of SEQ ID NO: 1, nucleotides 2940-2962 of SEQ ID NO: 1, nucleotides 3071-3093 of SEQ ID NO: 1, nucleotides 73-95 of SEQ ID NO: 4, nucleotides 1276-1298 of SEQ ID NO: 4, nucleotides 1336-1358 of SEQ ID NO: 4, nucleotides 1935- 1957 of SEQ ID NO: 4, nucleotides 2401-2423 of SEQ ID NO: 4, nucleotides 1622-1644 of SEQ ID NO: 522, and nucleotides 2231-2254 of SEQ ID NO: 522; (b) introducing a site- specific genome modification enzyme that specifically binds and cleaves the target genomic nucleic acid molecule, wherein the site-specific nuclease cleaves the target genomic nucleic acid molecule; (d) generating elite com plants from the elite com plant cells; and (e) selecting an elite com plant exhibiting brown coloration of the leaf mid veins at v3-vl0 stages. In some embodiments, the site-specific genome modification enzyme is a Cpfl protein complexed with guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400. In some embodiments, the modified com plant comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 348, 350, 352, 354, 358, 361, 364, 367, 370, 373, 376, 379, 382, 401-443, and 500-507.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1: Shows a schematic illustration of the genomic stmcture of the com Bmr3 gene. The two exons are indicated by a rightward bar arrows. 5’ UTR, 3’ UTR and one intron are indicated by solid lines. The positions and names of 6 sites targeted by the Cpfl guide RNAs are indicated by downward arrows along the Bmr3 gene structure.
[0017] FIG. 2: Shows a schematic illustration of the sites in com Bmr3 gene targeted for editing by the guide RNAs of five recombinant T-DNA vectors. The deletion in Event A-l is indicated by a bottom curly bracket that removes the entire exon2. The small deletions in Events B-l through 3 are indicated in a downward triangle within exon 2.
[0018] FIG. 3: Shows Bmr3 RNA expression detected by RT-qPCR Taqman assay using primer set A (panel A) and primer set B (panel B). Lanel: wild-type control, Lane 2:
homozygous plant comprising SEQ ID NO: 194, Lane3: homozygous plant comprising SEQ ID NO: 172, and Lane 4: heterozygous plant comprising SEQ ID NO: 22 and the wild-type Bmr3 allele.
DETAILED DESCRIPTION
[0019] Unless defined otherwise herein, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Examples of resources describing many of the terms related to molecular biology used herein can be found in Alberts et al, Molecular Biology of The Cell, 5th Edition, Garland Science Publishing, Inc.: New York, 2007; Rieger et al, Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; King et al, A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, Genes IX, Oxford University Press: New York, 2007. The nomenclature for DNA bases as set forth at 37 C.F.R. § 1.822 is used.
[0020] Any references cited herein, including, e.g., all patents, published patent applications, and non-patent publications, are incorporated by reference in their entirety.
[0021] The term“and/or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression“A and/or B” is intended to mean either or both of A and B, e.g., A alone, B alone, or A and B in combination. Also, for example, the expression“A, B and/or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.
[0022] As used herein, terms in the singular and the singular forms“a,”“an,” and“the,” for example, include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to“plant,”“the plant,” or“a plant” also includes a plurality of plants; also, depending on the context, use of the term“plant” can also include genetically similar or identical progeny of that plant; use of the term“a nucleic acid” optionally includes, as a practical matter, many copies of that nucleic acid molecule; similarly, the term“probe” optionally (and typically) encompasses many similar or identical probe molecules.
[0023] The term“about” as used herein, is intended to qualify the numerical values that it modifies, denoting such a value as variable within a margin of error. When no particular margin of error, such as a standard deviation to a mean value, is recited, the term“about” should be understood to mean that range which would encompass the recited value and the range which would be included by rounding up or down to that figure, taking into account significant figures.
[0024] As used herein,“plant” refers to a whole plant, any part thereof, or a cell or tissue culture derived from a plant, comprising any of: whole plants, plant components or organs (e.g., ears, husks, leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and/or progeny of the same. A progeny plant can be from any filial generation, e.g., FI, F2, F3, F4, F5, F6, F7, etc. A plant cell is a biological cell of a plant, taken from a plant or derived through culture from a cell taken from a plant. As used herein, a "plant part" may refer to any organ or intact tissue of a plant, such as a meristem, shoot organ/structure (e.g., leaf, stem or node), root, flower or floral organ/structure (e.g., bract, sepal, petal, stamen, carpel, anther and ovule), seed (e.g., embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), propagule, or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, and the like), or any portion thereof. Plant parts may be viable, nonviable, regenerable, and/or non-regenerable. A "propagule" may include any plant part that is capable of growing into an entire plant.
[0025] As used herein,“com”,“maize”,“com plant” or“maize plant” refers to a plant of species Zea mays L. and includes all plant varieties that can be bred with com, including wild maize species.
[0026] As used herein, the term "inbred" means a line that has been bred for genetic homogeneity.
[0027] As used herein, the term "elite line" means any line that has resulted from breeding and selection for superior agronomic performance. An elite plant is any plant from an elite line.
[0028] As used herein, the term "hybrid" means a progeny of mating between at least two genetically dissimilar parents. Without limitation, examples of mating schemes include single crosses, modified single cross, double modified single cross, three-way cross, modified three- way cross, and double cross wherein at least one parent in a modified cross is the progeny of a cross between sister lines.
[0029] As used herein,“Brown midrib 3”,“Bm3”, or“Bmr3” refers to the gene encoding caffeic acid-O-methyltransferase, a key enzyme involved in synthesis of flavonolignan, the mutation of which in com plants results in com plants characterized by a brown pigmentation in the leaf midrib at the v3 to vlO stage and lower lignin content in com plant tissue. In one aspect, the native Bmr3 gene in the 01DKD2 inbred com line comprises the nucleotide sequence of SEQ ID NO: l and encodes a protein of SEQ ID NO: 3. In another aspect, the native Bmr3 gene in the LH244 inbred com line comprises the nucleotide sequence of SEQ ID NO: 4 and encodes a protein of SEQ ID NO: 6.
[0030] As used herein, the term“deletion mutation” refers to the removal of one or more nucleotides from the DNA. Like insertion mutations, these mutations can alter the reading frame of the gene.
[0031] As used herein, “germplasm” refers to living sources of genetic material. The germplasm can be part of an organism or cell, or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed or tissues from which new plants may be grown, or plant parts, such as leaves, stems, pollen, or cells that can be cultured into a whole plant.
[0032] As used herein, the term“insertion mutation” refers to the addition of one or more extra nucleotides into the DNA. Insertions in the coding region of a gene may alter splicing of the mRNA (splice site mutation), or cause a shift in the reading frame (frameshift), both of which can significantly alter the gene product.
[0033] As used herein,“modified”, in the context of plants, seeds, plant components, plant cells, and plant germplasm, refers to a state containing changes or variations from their natural or native state. For instance, a“native transcript” of a gene refers to an RNA transcript that is generated from an unmodified gene and a“native protein” refers to a protein that is generated from an unmodified gene. Typically, a native transcript is a sense transcript. Modified plants or seeds contain molecular changes in their genetic materials, including either genetic or epigenetic modifications. Typically, modified plants or seeds, or a parental or progenitor line thereof, have been subjected to mutagenesis, genome editing (e.g., without being limiting, via methods using site-specific nucleases), genetic transformation (e.g., without being limiting, via methods of Agrobacterium transformation or microprojectile bombardment), or a combination thereof. In one aspect, a modified plant provided herein comprises no non-plant genetic material or sequences. In yet another aspect, a modified plant provided herein comprises no interspecies genetic material or sequences. In one aspect, a modified plant provided herein comprises one or more nucleotide changes, additions, or deletions in a gene compared to a native gene.
[0034] As used herein, a“mutation” refers to the permanent alteration of the nucleotide sequence of the genome of an organism, the extrachromosomal DNA, or other genetic elements.
[0035] As used herein, a“native copy” of a gene refers to a gene that originates from within a given organism, cell, tissue, genome, or chromosome that was not previously modified by human action. Similarly, a“native protein” refers to a protein encoded by a native gene.
[0036] As used herein, the term“substitution mutation” refers to an exchange of a single nucleotide for another.
[0037] As used herein, the terms“natural mutation”,“naturally-occurring mutation”, or“native mutation”, refers to a mutation as it occurs spontaneously in nature without any involvement of laboratory or experimental procedures or under the exposure to mutagens. Without being bound by scientific theory, a naturally-occurring mutation can arise from a variety of sources, including errors in DNA replication, spontaneous lesion, and transposable elements (or transposon).
[0038] As used herein, the terms“synthetic mutation”,“non-natural mutation” or“non- naturally-occurring mutation” refers to non-spontaneous mutation that occurs as a result of experimental procedures, such as exposure to a mutagen or by a site-specific genome modification enzyme.
[0039] As used herein, the term “inversion” refers to reversing the orientation of a chromosomal segment.
[0040] As used herein, a“missense mutation” refers to a single nucleotide change that results in a codon that codes for a different amino acid. For example, the codon“CGU” encodes an arginine amino acid. If a missense mutation changes the G to a U, producing a“CUU” codon, the codon now encodes a leucine amino acid. Missense mutations can be caused by an insertion, deletion, substitution, or inversion.
[0041] As used herein, the term“DNA” or“DNA molecule” refers to a double-stranded DNA molecule of genomic or synthetic origin, i.e. a polymer of deoxyribonucleotide bases or a polynucleotide molecule, read from the 5 ' (upstream) end to the 3 ' (downstream) end. As used herein, the term“DNA sequence” refers to the nucleotide sequence of a DNA molecule.
[0042] As used herein, the term“isolated DNA molecule” refers to a DNA molecule at least partially separated from other molecules normally associated with it in its native or natural state. In one embodiment, the term“isolated” refers to a DNA molecule that is at least partially separated from some of the nucleic acids which normally flank the DNA molecule in its native or natural state. Thus, DNA molecules fused to regulatory or coding sequences with which they are not normally associated, for example as the result of recombinant techniques, are considered isolated herein. Such molecules are considered isolated when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules, in that they are not in their native state.
[0043] Any number of methods well known to those skilled in the art can be used to isolate and manipulate a polynucleotide, or fragment thereof, as disclosed herein. For example, polymerase chain reaction (PCR) technology can be used to amplify a particular starting polynucleotide molecule and/or to produce variants of the original molecule. Polynucleotide molecules, or fragment thereof, can also be obtained by other techniques, such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer. A polynucleotide can be single-stranded (ss) or double-stranded (ds). “Double-stranded” refers to the base-pairing that occurs between sufficiently complementary, anti-parallel nucleic acid strands to form a double-stranded nucleic acid structure, generally under physiologically relevant conditions.
[0044] As used herein, the term“percent sequence identity” or“% sequence identity” refers to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of a reference (“query”) sequence (or its complementary strand) as compared to a test (“subject”) sequence (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide or amino acid insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the Sequence Analysis software package of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA), MEGAlign (DNAStar Inc., Madison, WI), and MUSCLE (version 3.6) (Edgar, “MUSCLE: multiple sequence alignment with high accuracy and high throughput” Nucleic Acids Research 32(5): 1792-7 (2004)) for instance with default parameters. An“identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in the portion of the reference sequence segment being aligned, that is, the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more sequences may be to a full-length sequence or a portion thereof, or to a longer sequence.
[0045] As used herein, the term “protein-coding polynucleotide molecule” refers to a polynucleotide molecule comprising a nucleotide sequence that encodes a protein. A“protein coding sequence” means a polynucleotide sequence that encodes a protein.
[0046] As used herein, the term“sequence” means a sequential arrangement of nucleotides or amino acids. The boundaries of a protein-coding sequence may be determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. In some embodiments, a protein-coding molecule may comprise a DNA sequence encoding a protein sequence. In some embodiments, a protein-coding molecule may comprise a RNA sequence encoding a protein sequence.
[0047] As used herein,“transgene expression”,“expressing a transgene”,“protein expression”, and“expressing a protein” mean the production of a protein through the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into polypeptide chains, which are ultimately folded into proteins.
[0048] As used herein, the term“recombinant” in reference to a polynucleotide (DNA or RNA) molecule, protein, construct, vector, etc., refers to a polynucleotide or protein molecule or sequence that is man-made and not normally found in nature, and/or is present in a context in which it is not normally found in nature, including a polynucleotide (DNA or RNA) molecule, protein, construct, etc., comprising a combination of polynucleotide or protein sequences that would not naturally occur contiguously or in close proximity together without human intervention, and/or a polynucleotide molecule, protein, construct, etc., comprising at least two polynucleotide or protein sequences that are heterologous with respect to each other. A recombinant polynucleotide or protein molecule, construct, etc., may comprise polynucleotide or protein sequence(s) that is/are (i) separated from other polynucleotide or protein sequence(s) that exist in proximity to each other in nature, and/or (ii) adjacent to (or contiguous with) other polynucleotide or protein sequence(s) that are not naturally in proximity with each other. In some embodiments, a recombinant polynucleotide molecule, protein, construct, etc., may also refer to a polynucleotide or protein molecule or sequence that has been genetically engineered and/or constructed outside of a cell. In other embodiments, a recombinant polynucleotide molecule, protein, construct, etc., may also refer to a polynucleotide or protein molecule or sequence that has been edited inside of a cell. The term recombinant can also refer to an organism that harbors recombinant material, e.g., a plant that comprises a recombinant nucleic acid is considered a recombinant plant.
[0049] As used herein, the term“allele” refers to an alternative nucleic acid sequence at a particular locus; the length of an allele can be as small as 1 nucleotide base, but is typically larger. For example, a first allele can occur on one chromosome, while a second allele occurs on a second homologous chromosome, e.g., as occurs for different chromosomes of a heterozygous individual, or between different homozygous or heterozygous individuals in a population. A favorable allele is the allele at a particular locus that confers, or contributes to, an agronomically desirable phenotype, or alternatively, is an allele that allows the identification of susceptible plants that can be removed from a breeding program or planting. A favorable allele of a marker is a marker allele that segregates with the favorable phenotype, or alternatively, segregates with susceptible plant phenotype, therefore providing the benefit of identifying disease prone plants. A favorable allelic form of a chromosome interval is a chromosome interval that includes a nucleotide sequence that contributes to superior agronomic performance at one or more genetic loci physically located on the chromosome interval. [0050] As used herein, the term“edited allele” refers to an alternative nucleic acid sequence at a particular locus where such alternative nucleic acid sequence contains man-made changes or variations from the native nucleic acid, for example by use of a site-specific genome modification enzyme. In some embodiments, an edited allele may comprise one or more non natural mutations.
[0051] As used herein,“genotype” is the genetic constitution of an individual (or group of individuals) at one or more genetic loci, as contrasted with the observable trait (the phenotype). Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to an individual’s genetic constitution at a single locus, at multiple loci, or, more generally, the term genotype can be used to refer to an individual’s genetic make-up for all the genes in its genome. A“haplotype” is the genotype of an individual at a plurality of genetic loci. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome interval.
[0052] As used herein,“locus” is a chromosome region where a polymorphic nucleic acid, trait determinant, gene, or marker is located.
[0053] As used herein the term“digestibility” refers to percentage of nutrients available in a given volume of food, such as silage or feed-ration. Greater digestibility is associated with higher energy availability. Digestibility may be measured in vitro or in vivo.
[0054] As used herein, the term“neutral detergent fiber” or“NDF” refers to a measure of the fraction of a feed that is not soluble in neutral detergent solution. NDF levels in forage increase as the plant matures. Com silage NDF concentration ranges from 36 to 50%. A low com silage NDF is desirable. Diets with less than 32 percent NDF may cause problems with acidosis.
[0055] As used herein the term“neutral detergent fiber digestibility” or“NDFD” refers to percentage of neutral detergent fiber that is digestible. NDFD can be determined in vitro by incubating a ground feed sample in rumen fluid and measuring its disappearance to simulate the amount and rate of digestion that would occur in the rumen.
[0056] As used herein the term“milk production” refers to the amount of milk produced by lactating dairy cattle on average during one day period. [0057] As used herein, the term“ruminant” refers to a mammal that acquire nutrients from plant-based food by regurgitating partially digested food. Examples of ruminants include bison, cattle, sheep, moose, giraffes, and goats.
[0058] As used herein, the term“silage” means any crop that is harvested green and preserved in a succulent condition by partial fermentation in a more-or-less airtight container. Com silage consists of era, stalks, and leaves.
[0059] As used herein the term“milk production efficiency” refers to the amount of milk produced per one unit of the feed intake.
[0060] Bmr3 com mutants characterized by a brown pigmentation in the leaf midrib at the V4 to V6 stage and lower lignin content in com plant tissue due to a disruption of the gene encoding caffeic acid-O-methyltransferase, an enzyme involved in synthesis of lignin. Lignin polymers limit the digestibility of the fiber in the com plant. The reduced lignin in brown midrib com results in silage with fiber that is more digestible than non-brown midrib com. Animal feeding trials have shown about 10 percent greater intake and increased milk production with ruminants, such as dairy cows, fed with brown midrib com silage (Bmr silage), as compared to non-Bmr silage. The com Bmr3 gene is located on the short arm of chromosome 4 and, as shown in Figure 1, contains 2 exons and 1 intron. The naturally occurring mutations that have been identified in the com Bmr3 gene are recessive and are associated with negative agronomic traits such as reduced yield, reduced standability, lodging, and reduced disease resistance.
[0061] Several embodiments relate to a modified com plant comprising a non-naturally occurring mutation in a Bmr3 gene. Several embodiments described herein provide a modified com plant comprising at least one non-natural mutation in the com Bmr3 gene, wherein the com plant exhibits a reduced lignin content compared to a control com plant not comprising the non-natural Bmr3 mutation when grown under comparable conditions. In some embodiments, the modified com plant does not comprise a naturally occurring mutation in a Bmr3 gene. In some embodiments, the modified com plant does not comprise the naturally- occurring Bm3-1 mutant allele. In some embodiments, the modified com plant does not comprise the naturally-occurring Bm3-2 mutant allele. In some embodiments, the modified com plant does not comprise the naturally-occurring Bm3-2001PR-1 mutant allele. In some embodiments, the modified com plant does not comprise the naturally-occurring Bm3-91598- 3 mutant allele. In some embodiments, the modified com plant does not comprise the naturally- occurring Bm3-Bumham mutant allele. In some embodiments, the modified com plant does not comprise the naturally-occurring Bm3-MM1266 mutant allele. In some embodiments, the modified com plant does not comprise the naturally-occurring Bm3-MM1818 mutant allele.
[0062] In some embodiments, a modified com plant as described herein comprises a synthetic mutation in the Bmr3 gene introduced via targeted genome editing. In another aspect, a method described herein comprises targeted genome editing of a Bmr3 in a desired inbred background. In one aspect, the present disclosure provides a non-transgenic com plant comprising a synthetic mutation in a Bmr3 gene reducing the activity of the Bmr3 gene. In another aspect, the present disclosure provides a modified com plant comprising a non-transgene or non- transposon mediated mutation in a Bmr3 gene reducing the activity of the Bmr3 gene. As used herein, the term“transgene” refers to a recombinant DNA molecule, constmct or sequence integrated or inserted into a genome, and thus altering the genome. In some embodiments, the modified com plant comprises a non-naturally occurring substitution mutation in the Bmr3 gene. In some embodiments, the modified com plant comprises a non-naturally occurring insertion in the Bmr3 gene. In some embodiments, the modified com plant comprises a non- naturally occurring inversion in the Bmr3 gene. In some embodiments, the modified com plant comprises a non-naturally occurring deletion in the Bmr3 gene. In some embodiments, the modified com plant comprises a non-naturally occurring substitution mutation in a genomic sequence of SEQ ID NO: l. In some embodiments, the modified com plant comprises a non- naturally occurring insertion in a genomic sequence of SEQ ID NO: l. In some embodiments, the modified com plant comprises a non-naturally occurring inversion in a genomic sequence of SEQ ID NO: l. In some embodiments, the modified com plant comprises a non-naturally occurring deletion in a genomic sequence of SEQ ID NO: l. In some embodiments, the modified com plant comprises a non-naturally occurring substitution mutation in a genomic sequence of SEQ ID NO:4. In some embodiments, the modified com plant comprises a non- naturally occurring insertion in a genomic sequence of SEQ ID NO:4. In some embodiments, the modified com plant comprises a non-naturally occurring inversion in a genomic sequence of SEQ ID NO:4. In some embodiments, the modified com plant comprises a non-naturally occurring deletion in a genomic sequence of SEQ ID NO:4.
[0063] Several embodiments described herein relate to a modified com plant comprising a non- naturally occurring insertion in a Bmr3 gene, where the insertion causes a truncation of the protein encoded by the Bmr3 gene. In one aspect, the insertion occurs within Exon 1 of the Bmr3 gene, where the insertion introduces premature stop codon. In another aspect, the insertion occurs within Exon 2 of the Bmr3 gene, where the insertion introduces premature stop codon. Several embodiments described herein relate to a modified com plant comprising a non-naturally occurring deletion in a Bmr3 gene, where the deletion causes a truncation of the protein encoded by the Bmr3 gene. In one aspect, the deletion occurs within Exon 1 of the Bmr3 gene. In another aspect, the deletion occurs within Exon 2 of the Bmr3 gene. Several embodiments described herein relate to a modified com plant comprising a non-naturally occurring inversion in a Bmr3 gene, where the inversion causes a reduction in the level of the protein encoded by the Bmr3 gene. In one aspect, the inversion occurs within Exon 1 of the Bmr3 gene. In another aspect, the inversion occurs within Exon 2 of the Bmr3 gene. In one aspect, the inversion creates a dominant negative allele of the Bmr3 gene.
[0064] Several embodiments described herein relate to a modified com plant comprising an edited allele of the Bmr3 gene. In some embodiments, the modified com plant is heterozygous for an edited allele of the Bmr3 gene. In some embodiments, the modified com plant is homozygous for an edited allele of the Bmr3 gene. In some embodiments, the modified com plant comprises an edited allele of the Bmr3 gene, wherein the edited allele comprises a nucleotide sequence selected from SEQ ID NOs: 21-252, 401-443 and 500-507. In some embodiments, the modified com plant comprises an edited allele of the Bmr3 gene, wherein the edited allele comprises a nucleotide sequence selected from SEQ ID NOs: 358-382. In some embodiments, the modified com plant comprises a first edited allele of the Bmr3 gene and a second edited allele of the Bmr3 gene, wherein the first edited allele and the second edited allele comprise a nucleotide sequence independently selected from SEQ ID NOs: 21-252, 358- 382, 401-443 and 500-507. In some embodiments, the modified com plant comprises an edited allele of the Bmr3 gene, wherein the edited allele comprises a nucleotide sequence selected from SEQ ID NOs: 358, 361, 364, 370, 373, 376, 379, and 382. In some embodiments, the modified com plant comprises a first edited allele of the Bmr3 gene and a second edited allele of the Bmr3 gene, wherein the first edited allele and the second edited allele comprise a nucleotide sequence independently selected from SEQ ID NOs: 21-252, 358, 361, 364, 370, 373, 376, 379, 382, 401-443, and 500-507. [0065] In some embodiments, the modified com plant comprises an edited allele of the Bmr3 gene, wherein the edited allele comprises a nucleotide sequence selected from SEQ ID NOs: 246, 348, 350, 352 and 354.
[0066] Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele resulting whole gene deletion between a genomic sequence targeted by g90 or g227 and a genomic sequence targeted by g3279. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 2313 nucleotide deletion between a genomic sequence targeted by g867 and a genomic sequence targeted by g3170. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 1130 nucleotide deletion between a genomic sequence targeted by g2010 and a genomic sequence targeted by g3170. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 7, 8, 9, 10, 15 or 19 nucleotide deletion at a genomic sequence targeted by g2010. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 1332 nucleotide deletion at a genomic sequence targeted by g2070. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 944 nucleotide deletion at a genomic sequence targeted by g2010. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion at a genomic sequence targeted by g2070. In some embodiments, 1 or more, 10 or more, 50 or more, 100 or more, 500 or more, or 1,000 or more nucleotide are deleted at or around a genomic sequence targeted by g2070. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 358, 326, 304, 238, 190, 144, 130, 109, 108, 105,
76, 68, 67, 63, 56, 53, 51, 49, 48, 37, 36, 34, 33, 29, 28, 27, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide deletion at or around a genomic sequence targeted by g2070. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises deletion mutation between a genomic sequence targeted by g2070 and a genomic sequence targeted by g2691. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 1214, 784, 736, 646, 595, 594, 593, 592, 591, or 590 nucleotide deletion between a genomic sequence targeted by g2070 and a genomic sequence targeted by g2691. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion at a genomic sequence targeted by g2691. In some embodiments, 1 or more, 10 or more, 50 or more, 100 or more, 500 or more, or 1,000 or more nucleotides are deleted at or around a genomic sequence targeted by g2691. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 36, 27, 21, 20, 19, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide deletion at or around a genomic sequence targeted by g2691. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises deletion mutation between a genomic sequence targeted by g2010 and a genomic sequence targeted by g3170. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 1453, 1135, 1129, or 1118 nucleotide deletion between a genomic sequence targeted by g2010 and a genomic sequence targeted by g3170. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion at a genomic sequence targeted by g3170. In some embodiments, 1 or more, 10 or more, 50 or more, 100 or more, 500 or more, or 1,000 or more nucleotides are deleted at or around a genomic sequence targeted by g3170. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 24, 23, 22, 20, 19, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide deletion at or around a genomic sequence targeted by g3170. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises deletion mutation between a genomic sequence targeted by g2070 and a genomic sequence targeted by g3270. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 1243 or 1214 nucleotide deletion between a genomic sequence targeted by g2070 and a genomic sequence targeted by g3270. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises deletion mutation between a genomic sequence targeted by g2070 and a genomic sequence targeted by g2691. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 637 nucleotide deletion between a genomic sequence targeted by g2070 and a genomic sequence targeted by g2691. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion at a genomic sequence targeted by g3279. In some embodiments, 1 or more, 10 or more, 50 or more, 100 or more, 500 or more, or 1,000 or more nucleotides are deleted at or around a genomic sequence targeted by g3279. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 24, 23, 22, 20, 19, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide deletion at or around a genomic sequence targeted by g3279. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises deletion mutation between a genomic sequence targeted by g867 and a genomic sequence targeted by g2010. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 1200 nucleotide deletion between a genomic sequence targeted by g867 and a genomic sequence targeted by g2010. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion at a genomic sequence targeted by g867. In some embodiments, 1 or more, 10 or more, 50 or more, 100 or more, 500 or more, or 1,000 or more nucleotides are deleted at or around a genomic sequence targeted by g867. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a 24, 23, 22, 20, 19, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide deletion at or around a genomic sequence targeted by g867.
[0067] Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion of 1 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more, 220 or more, 230 or more, 240 or more, 250 or more, 260 or more, 270 or more, 280 or more, 290 or more, 300 or more, 310 or more, 320 or more, 330 or more, 340 or more, 350 or more, 360 or more, 370 or more, 380 or more, 390 or more, 400 or more, 410 or more, 420 or more, 430 or more, 440 or more, 450 or more, 460 or more, 470 or more, 480 or more, 490 or more, 500 or more, nucleotides of exon 1 of the Bmr3 gene. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a complete deletion of exon 1 of the Bmr3 gene. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion of the 5’ end of exon 1 of the Bmr3 gene. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion of the 3’ end of exon 1 of the Bmr3 gene.
[0068] Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion of 1 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, 200 or more, 210 or more, 220 or more, 230 or more, 240 or more, 250 or more, 260 or more, 270 or more, 280 or more, 290 or more, 300 or more, 310 or more, 320 or more, 330 or more, 340 or more, 350 or more, 360 or more, 370 or more, 380 or more, 390 or more, 400 or more, 410 or more, 420 or more, 430 or more, 440 or more, 450 or more, 460 or more, 470 or more, 480 or more, 490 or more, 500 or more, 510 or more, 520 or more, 530 or more, 540 or more, 550 or more, 560 or more, 570 or more, 580 or more, 590 or more, 600 or more, 610 or more, 620 or more, 630 or more, 640 or more, 650 or more, 660 or more, 670 or more, 680 or more, 690 or more, 700 or more, 710 or more, 720 or more, 730 or more, 740 or more, 750 or more, 760 or more, 770 or more, 780 or more, 790 or more, 800 or more, 810 or more, 820 or more, 830 or more, 840 or more, 850 or more, 860 or more, 870 or more, 880 or more, 890 or more, 900 or more, 910 or more, 920 or more, 930 or more, 940 or more, 950 or more, 960 or more, 970 or more, 980 or more, 990 or more, 1,000 or more, 1,010 or more, 1,020 or more, 1,030 or more, 1,040 or more, 1,050 or more, 1,060 or more, 1,070 or more, 1,080 or more, 1,090 or more, 1,100 or more, 1,110 or more, 1,120 or more, or 1,130 or more nucleotides of exon 2 of the Bmr3 gene. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a complete deletion of exon 2 of the Bmr3 gene. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion of the 5’ end of exon 2 of the Bmr3 gene. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a deletion of the 3’ end of exon 2 of the Bmr3 gene. [0069] Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises an inversion of the nucleotide between a genomic sequence targeted by g2010 and a genomic sequence targeted by g3170. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises an inversion of the nucleotide between a genomic sequence targeted by g2691 and a genomic sequence targeted by g3279. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises an inversion of the nucleotide between a genomic sequence targeted by g2070 and a genomic sequence targeted by g2691. Several embodiments relate to a modified com plant comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises an inversion of the nucleotide between a genomic sequence targeted by g2070 and a genomic sequence targeted by g3279.
[0070] Several embodiments relate to a modified com plant exhibiting a brown midrib reduced lignin phenotype and a floury-endosperm phenotype. Mutant alleles of the floury -2 (F12) gene in com plants cause a decrease in the synthesis of zein proteins resulting in a floury endosperm, which is another desirable trait in animal feed because floury-endosperm is digested more rapidly and completely than vitreous endosperm. Zein proteins are prolamin storage proteins in the endosperm of com seeds. The Bmr3 and F12 genes are tightly linked approximately 5 cM genetic-distance apart on maize chromosome 4, with mutant Bmr3 and F12 alleles in trans linkage disequilibrium in com germplasm. Meiotic crossing over between these two loci is rare. Several embodiments described herein relate to a modified com plant that is homozygous for a Bmr3 edited allele as described herein and a mutant F12 allele. Several embodiments relate to a modified com germplasm comprising a Bmr3 edited allele as described herein in cis with a mutant F12 allele. Several embodiments relate to a modified com germplasm comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a nucleotide sequence selected from SEQ ID NOs: 21-252, 358, 361, 364, 370, 373, 376, 379, 382, 401-443, and 500- 507, and wherein the edited allele of the Bmr3 gene is in cis with a mutant F12 gene. Several embodiments relate to a modified com germplasm comprising an edited allele of the Bmr3 gene, wherein the edited allele comprises a nucleotide sequence selected from SEQ ID NOs: 246, 348, 350, 352 and 354, and wherein the edited allele of the Bmr3 gene is in cis with a mutant F12 gene. In several embodiments breeding lines comprising the modified com germplasm described herein are crossed together to produce com hybrids that are homozygous for an edited Bmr3 alleles and a mutant F12 allele and thereby express both the brown-midrib and floury-endosperm traits.
[0071] Several embodiments relate to methods of designing and producing com plants that exhibit improved digestibility. In some embodiments, methods of designing com plants that exhibit improved digestibility and/or starch content by modifying the genome of a com cell to alter a Bmr3 gene as described herein is provided. In some embodiments, a method described herein comprises targeted genome editing of a Bmr3 gene in a desired inbred background. Genome modification can be accomplished through targeted genome editing as described herein. In some embodiments, one or more synthetic mutations are generated at or around a genomic sequence targeted by g867. In some embodiments, one or more synthetic mutations are generated at or around a genomic sequence targeted by g2010. In some embodiments, one or more synthetic mutations are generated at or around a genomic sequence targeted by g2070. In some embodiments, one or more synthetic mutations are generated at or around a genomic sequence targeted by g2691. In some embodiments, one or more synthetic mutations are generated at or around a genomic sequence targeted by g3170. In some embodiments, one or more synthetic mutations are generated at or around a genomic sequence targeted by g3279.
[0072] Several embodiments relate to a modified Bmr3 polypeptide encoded by an edited allele of Bmr3 as described herein. Several embodiments relate to nucleic acid sequences encoding modified Bmr3 polypeptide, as well as compositions and methods of using modified polypeptides. In some embodiments, a modified Bmr3 polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 252-299. In some embodiments, a nucleic acid sequence selected from SEQ ID NOs: 300-345 and 347, encodes a modified BMR3 polypeptide as described herein. In some embodiments, the edited allele of Bmr3 is a null allele in which no caffeic acid O-methyltransferase protein is produced.
[0073] Several embodiments relate to nucleic acid molecules, polynucleotides, polypeptides, proteins, com cells, com seeds, or com plants that are the result of genome editing and as such would not normally be found in nature and are created by human intervention. Several embodiments relate to a com plant genome comprising a DNA sequence that does not naturally occur in such plant genome and as such is the result of human intervention. In some embodiments, a DNA sequence provided herein encodes a modified Bmr3 polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 252-299. In some embodiments, a DNA sequence provided herein encodes a modified Bmr3 polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 261, 280, 283, and 298. In certain embodiments, modified Bmr3 alleles comprising an alteration relative to the Bmr3 gene sequence of SEQ ID NO: 1 or 4 are provided. In some embodiments, modified Bmr3 alleles comprising a nucleotide sequence selected from SEQ ID NOs: 21-252, 401-443 and 500-507 are provided. In some embodiments, modified Bmr3 alleles comprising a nucleotide sequence selected from SEQ ID NOs: 358, 361, 364, 370, 373, 376, 379, and 382 are provided. In some embodiments, modified Bmr3 alleles comprising a nucleotide sequence selected from SEQ ID NOs: 348, 350, 352, and 354 are provided.
Genome Editing
[0074] Targeted modification of plant genomes through the use of genome editing methods can be used to create improved plant lines through modification of plant genomic DNA. In addition, genome editing methods can enable targeted insertion of one or more nucleic acids of interest into a plant genome. Examples methods for introducing donor polynucleotides into a plant genome or modifying genomic DNA of a plant include the use of site-specific genome modification enzymes, such as zinc-finger nucleases, engineered or native meganucleases, TALE-endonucleases, or an RNA-guided endonucleases (for example, a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)/Cas9 system, a CRISPR/Cpfl system, a CRISPR/CasX system, a CRISPR/CasY system, a CRISPR/Cascade system). Several embodiments relate to methods of genome editing is using single-stranded oligonucleotides to introduce precise base pair modifications in a plant genome, as described by Sauer et al ( Plant Physiol. 2016 Apr; 170(4): 1917-1928). Methods of genome editing to modify, delete, or insert nucleic acid sequences into genomic DNA are known in the art.
[0075] Several embodiments relate to a com plant comprising in its genome a modified Bmr3 gene sequence, wherein the modified Bmr3 gene sequence encodes a truncated or modified COMT as described herein or where in the modified Bmr3 gene sequence is a null allele that does not produce a protein. In certain embodiments, genome editing methods are utilized for the modification or replacement of an existing Bmr3 sequence within a plant genome. In some embodiments, the native Bmr3 gene sequence is modified to comprise one or more targeted nucleotide changes, additions, deletions, or other modifications at one or more sites targeted by g90, g227, g867, g2010, g2070, g2691, g3170 or g3279. In some embodiments, the native Bmr3 gene sequence is modified to comprise one or more targeted nucleotide changes, additions, deletions, or other modifications, such that the modified Bmr3 gene sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 21-252, 401-443, and 500-507. In some embodiments, the native Bmr3 gene sequence is modified to comprise one or more targeted nucleotide changes, additions, deletions, or other modifications, such that the modified Bmr3 gene sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382. In some embodiments, the native Bmr3 gene sequence is modified to comprise one or more targeted nucleotide changes, additions, deletions, or other modifications, such that the modified Bmr3 gene sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 348, 350, 352, and 354.
[0076] Several embodiments relate to a CRISPR/Cas9 system used to modify or replace an existing coding sequence within a plant genome, such as a sequence encoding caffeic acid-O- methyltransferase (COMT). Several embodiments relate to a CRISPR/Cpfl system used to modify or replace an existing coding sequence within a plant genome, such as a sequence encoding caffeic acid-O-methyltransferase (COMT). Several embodiments relate to a Cas9- cytidine deaminase used to modify an existing coding sequence within a plant genome, such as a sequence encoding caffeic acid-O-methyltransferase (COMT). Several embodiments relate to a Cpfl-cytidine deaminase used to modify an existing coding sequence within a plant genome, such as a sequence encoding caffeic acid-O-methyltransferase (COMT). In further embodiments, transcription activator-like effectors (TALEs) are used for modification or replacement of an existing coding sequence within a plant genome, such as a sequence encoding caffeic acid-O-methyltransferase (COMT). In some embodiments, an existing caffeic acid-O-methyltransferase (COMT) polypeptide coding sequence within a plant genome is modified by non-templated genome editing with a site-specific genome modification enzyme. In some embodiments, an existing COMT polypeptide coding sequence within a plant genome is modified by templated genome editing with a site-specific genome modification enzyme. In some embodiments, a site-specific genome modification enzyme and a donor nucleic acid comprising a nucleotide sequence selected from SEQ ID NOs: 21-252, 358, 361, 364, 367, 370, 373, 376, 379, 382, 401-443 and 500-507 is introduced into a plant cell. In some embodiments, the modified Bmr3 gene is capable of conferring to reduced lignin levels to a plant. In some embodiments, modification or replacement of an endogenous COMT-encoding sequence according to the methods provided herein results in a reduction in lignin. Several embodiments therefore relate to providing a site-specific genome modification enzyme capable of recognizing a specific nucleotide sequence of interest, such as a sequence targeted by g90, g227, g867, g2010, g2070, g2691, g3170 or g3279, within a genome of a plant to allow for alteration of the Bmr3 gene sequence by non-templated editing or integration of a donor nucleic acid at that site.
[0077] Modifications to a COMT-encoding sequence, for example a sequence provided herein as one of SEQ ID NOs: 348, 250, 352, and 354, may result in a sequence encoding a truncated or altered polypeptide as described herein at SEQ ID NOs: 261, 280, 283, and 298, capable of conferring to a plant improved digestibility and lower lignin content.
[0078] In an aspect, a“modification” comprises the insertion of 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 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides. In another aspect, a“modification” comprises the deletion of 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 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides. In a further aspect, a“modification” comprises the inversion of 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 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides. In still another aspect, a“modification” comprises the substitution of 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 15, at least 25, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000, at least 5000, or at least 10,000 nucleotides. In some embodiments, a “modification” comprises the substitution of an“A” for a“C”,“G” or“T” in a nucleic acid sequence. In some embodiments, a“modification” comprises the substitution of an“C” for a “A”,“G” or“T” in a nucleic acid sequence. In some embodiments, a“modification” comprises the substitution of an“G” for a“A”,“C” or“T” in a nucleic acid sequence. In some embodiments, a“modification” comprises the substitution of an“T” for a“A”,“C” or“G” in a nucleic acid sequence. In some embodiments, a“modification” comprises the substitution of an“C” for a“U” in a nucleic acid sequence. In some embodiments, a“modification” comprises the substitution of an“G” for a“A” in a nucleic acid sequence. In some embodiments, a “modification” comprises the substitution of an“A” for a“G” in a nucleic acid sequence. In some embodiments, a“modification” comprises the substitution of an“T” for a“C” in a nucleic acid sequence.
[0079] Several embodiments relate to a recombinant DNA construct comprising an expression cassette(s) encoding a site-specific genome modification enzyme and/or any associated guide RNAs(s) to carry out genome editing. These expressing cassette(s) may be present in the same molecule or vector as a donor template for templated editing or on a separate molecule or vector. Several methods for genome editing are known in the art involving different site- specific genome modification enzymes (or complexes of proteins and/or guide RNAs) that cut the genomic DNA to produce a double strand break (DSB) or nick at a desired genomic site or locus. As understood in the art, during the process of repairing the DSB or nick introduced by the site-specific genome modification enzyme, a donor template DNA may become integrated into the genome at the site of the DSB or nick. The presence of the homology arm(s) in the donor template DNA may promote the adoption and targeting of the insertion sequence into the plant genome during the repair process through homologous recombination, although an insertion of the donor template may occur through non-homologous end joining (NHEJ). In other embodiments where a donor template DNA is not provided, one or more of an insertion mutation, a deletion mutation or an inversion mutation may occur at or around the target site during the process of repairing the DSB or nick introduced by the site-specific genome modification enzyme. Examples of site-specific genome modification enzymes that may be used include zinc-finger nucleases, engineered or native meganucleases, TALE-endonucl eases, and RNA-guided endonucleases (e.g, Cas9, CasX, CasY or Cpfl). For methods using RNA- guided site-specific nucleases (e.g., Cas9, CasX, CasY or Cpfl), the recombinant DNA construct(s) may also comprise a sequence encoding one or more guide RNAs to direct the nuclease to the desired site within the plant genome. In some embodiments, one or more guide RNAs may be provided on a separate molecule or vector (in trans).
Site-specific genome modification enzymes
[0080] As used herein, the term“double-strand break inducing agent” refers to any agent that can induce a double-strand break (DSB) in a DNA molecule. In some embodiments, the double-strand break inducing agent is a site-specific genome modification enzyme.
[0081] As used herein, the term“site-specific genome modification enzyme” refers to any enzyme that can modify a nucleotide sequence in a sequence-specific manner. In some embodiments, a site-specific genome modification enzyme modifies the genome by inducing a single-strand break. In some embodiments, a site-specific genome modification enzyme modifies the genome by inducing a double-strand break. In some embodiments, a site-specific genome modification enzyme comprises a cytidine deaminase. In some embodiments, a site- specific genome modification enzyme comprises an adenine deaminase. In the present disclosure, site-specific genome modification enzymes include endonucleases, recombinases, transposases, deaminases, hebcases and any combination thereof. In some embodiments, the site-specific genome modification enzyme is a sequence-specific nuclease.
[0082] Several embodiments relate to promoting recombination between the Bmr3 and floury- 2 loci. Several embodiments relate to promoting recombination by providing a site-specific genome modification enzyme, for example, a sequence-specific nuclease, to a plant cell. In some embodiments, recombination is promoted by providing a single-strand break inducing agent. In some embodiments, recombination is promoted by providing a double-strand break inducing agent. In some embodiments, recombination is promoted by providing a strand separation inducing reagent. In one aspect, the site-specific genome modification enzyme is selected from an endonuclease, a recombinase, a transposase, a deaminase, a helicase or any combination thereof. In some embodiments, recombination occurs between A chromosomes. In some embodiments, recombination occurs between B chromosomes. In some embodiments, recombination occurs between a B chromosome and an A chromosome.
[0083] In some embodiments of the genome editing methods described herein, the site-specific genome modification enzyme is selected from a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nucleases (TALEN), an Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), an RNA-guided nuclease, such as a CRISPR associated nuclease (non-limiting examples of CRISPR associated nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Cas 12a (also known as Cpfl), Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csl3, Csf4, CasX, CasY, Mad7, homologs thereof, or modified versions thereol).
[0084] In some embodiments, the site-specific genome modification enzyme is a dCas9-Fokl fusion protein. In another aspect, the site-specific genome modification enzyme is a dCas9- recombinase fusion protein. As used herein, a“dCas9” refers to a Cas9 endonuclease protein with one or more amino acid mutations that result in a Cas9 protein without endonuclease activity, but retaining RNA-guided site-specific DNA binding. As used herein, a“dCas9- recombinase fusion protein” is a dCas9 with a protein fused to the dCas9 in such a manner that the recombinase is catalytically active on the DNA.
[0085] In some embodiments, the site-specific genome modification enzyme comprises a DNA binding domain operably linked to a deaminase. In some embodiments, the site-specific genome modification enzyme further comprises uracil DNA glycosylase (UGI). In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an adenine deaminase. In some embodiments, the deaminase is an APOPEC deaminase. In some embodiments, the deaminase is an activation-induced cytidine deaminase (AID). In some embodiments, the DNA binding domain is a zinc-finger DNA-binding domain, a TALE DNA- binding domain, a Cas9 nuclease, a Cas 12a nuclease, a catalytically inactive Cas9 nuclease, a catalytically inactive Cas 12a nuclease, a Cas9 nickase, or a Cpfl nikase.
[0086] In some embodiments, the site-specific genome modification enzyme is a dCas9- cytosine deaminase fusion protein. In another aspect, the site-specific genome modification enzyme is a dCas9-adenine deaminase fusion protein. In some embodiments, one or more of a dCas9-cytosine deaminase fusion protein and a dCas9-adenine deaminase fusion protein are utilized to modify a Bmr3 gene sequence. In some embodiments, a Bmr3 gene sequence is modified to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 21-252, 401-443, and 500-507. In some embodiments, a Bmr3 gene sequence is modified to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382. In some embodiments, a Bmr3 gene sequence is modified to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 348, 350, 352, and 354.
[0087] In some embodiments, the site-specific genome modification enzyme is a dCpfl - cytosine deaminase fusion protein. In another aspect, the site-specific genome modification enzyme is a dCpfl -adenine deaminase fusion protein. In some embodiments, one or more of a dCpfl -cytosine deaminase fusion protein and a dCpfl -adenine deaminase fusion protein are utilized to modify a Bmr3 gene sequence. In some embodiments, a Bmr3 gene sequence is modified to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 21-252, 401-443 and 500-507. In some embodiments, a Bmr3 gene sequence is modified to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382. In some embodiments, a Bmr3 gene sequence is modified to comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 348, 350, 352, and 354.
[0088] In some embodiments, a site-specific genome modification enzymes, such as meganucleases, ZFNs, TALENs, Argonaute proteins (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), homologs thereof, or modified versions thereol), RNA-guided nucleases (non-limiting examples of RNA-guided nucleases include the CRISPR associated nucleases, such as Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Cas 12a (also known as Cpfl), Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csl3, Csf4, CasX, CasY, Mad7, homologs thereof, or modified versions thereol) and engineered RNA-guided nucleases (RGNs), induce a genome modification such as a double-stranded DNA break (DSB) or single-strand DNA break in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, or g3279. In some embodiments, breaks or nicks in the target DNA sequence are repaired by the natural processes of homologous recombination (HR) or non-homologous end-joining (NHEJ). In some embodiments, sequence modifications occur at or near the cleaved or nicked sites, which can include deletions or insertions that result in modification of the nucleic acid sequence, or integration of exogenous nucleic acids by homologous recombination or NHEJ.
[0089] In some embodiments, a targeted genome modification as described herein comprises the use of a zinc-finger nuclease (ZFN). ZFNs are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to the cleavage domain of the Fokl restriction nuclease. ZFNs can be designed to cleave almost any long stretch of double-stranded DNA for modification of the zinc finger DNA-binding domain. ZFNs form dimers from monomers composed of anon-specific DNA cleavage domain of Fokl nuclease fused to a zinc finger array engineered to bind a target DNA sequence. The DNA-binding domain of a ZFN is typically composed of 3-4 zinc-finger arrays. The amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger ¥-helix, which contribute to site-specific binding to the target DNA, can be changed and customized to fit specific target sequences. The other amino acids form the consensus backbone to generate ZFNs with different sequence specificities. Rules for selecting target sequences for ZFNs are known in the art. The Fokl nuclease domain requires dimerization to cleave DNA and therefore two ZFNs with their C-terminal regions are needed to bind opposite DNA strands of the cleavage site (separated by 5-7 nt). The ZFN monomer can cut the target site if the two-ZF-binding sites are palindromic. The term ZFN, as used herein, is broad and includes a monomeric ZFN that can cleave double stranded DNA without assistance from another ZFN. The term ZFN is also used to refer to one or both members of a pair of ZFNs that are engineered to work together to cleave DNA at the same site.
[0090] Without being limited by any scientific theory, because the DNA-binding specificities of zinc finger domains can in principle be re-engineered using one of various methods, customized ZFNs can theoretically be constructed to target nearly any gene sequence. Publicly available methods for engineering zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly.
[0091] Several embodiments relate to a method and/or composition provided herein comprising one or more, two or more, three or more, four or more, or five or more ZFNs directed to a target sequence in a Bmr3 gene. In one aspect, a ZFN provided herein is capable of generating a targeted DSB. In one aspect, a ZFN provided herein is capable of generating a targeted DSB in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, or g3279. In one aspect, a ZFN provided herein is capable of generating a targeted single-strand break (SSB). In one aspect, a ZFN provided herein is capable of generating a targeted SSB in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, or g3279. In one aspect, vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more ZFNs are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation).
[0092] Several embodiments relate to generating a modification as described herein using a meganuclease. Meganucleases, which are commonly identified in microbes, are unique enzymes with high activity and long recognition sequences (> 14 nt) resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (for example, 14 to 40 nt). The engineering of meganucleases can be more challenging than that of ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain. Specialized methods of mutagenesis and high-throughput screening have been used to create novel meganuclease variants that recognize unique sequences and possess improved nuclease activity.
[0093] In one aspect, a method and/or composition provided herein comprises one or more, two or more, three or more, four or more, or five or more meganucleases directed to a target sequence in a Bmr3 gene. In some embodiments, a meganuclease provided herein is capable of generating a targeted DSB. In some embodiments, a meganuclease provided herein is capable of generating a targeted DSB in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, org3279. In some embodiments, a meganuclease provided herein is capable of generating a targeted SSB. In some embodiments, a meganuclease provided herein is capable of generating a targeted SSB in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, or g3279. In some embodiments, vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more meganucleases are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation). [0094] In some embodiments, a targeted genome modification as described herein comprises the use of a transcription activator-like effector nuclease (TALEN). TALENs are artificial restriction enzymes generated by fusing the transcription activator-like effector (TALE) DNA binding domain to a nuclease domain. In one aspect, the nuclease is selected from a group consisting of PvuII, MutH, Tevl and Fokl, Alwl, Mlyl, Sbfl, Sdal, Stsl, CleDORF, Clo051, Pept071. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that work together to cleave DNA at the same site. Transcription activator-like effectors (TALEs) can be engineered to bind practically any DNA sequence, such as a target sequence in a Bmr3 gene. TALE proteins are DNA-binding domains derived from various plant bacterial pathogens of the genus Xanthomonas. The X pathogens secrete TALEs into the host plant cell during infection. The TALE moves to the nucleus, where it recognizes and binds to a specific DNA sequence in the promoter region of a specific DNA sequence in the promoter region of a specific gene in the host genome. TALE has a central DNA-binding domain composed of 13-28 repeat monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeat-variable diresidues (RVDs). The amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine/adenine, respectively, and modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.
[0095] In one aspect, a method and/or composition provided herein comprises one or more, two or more, three or more, four or more, or five or more TALENs. In another aspect, a TALEN provided herein is capable of generating a targeted DSB in a target sequence in Bmr3 gene. In some embodiments, a TALEN provided herein is capable of generating a targeted DSB in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, or g3279. In another aspect, a TALEN provided herein is capable of generating a targeted SSB in a target sequence in Bmr3 gene. In some embodiments, a TALEN provided herein is capable of generating a targeted SSB in a com genome at a site targeted by g90, g227, g867, g2010, g2070, g2691, g3170, or g3279. In one aspect, vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more TALENs are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation).
[0096] In some embodiments, a targeted genome modification as described herein comprises the use of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten RNA-guided nucleases. In some
embodiments, a CRISPR/Cas9 system, a CRISPR/Cpfl system, a CRISPR/CasX system, or a CRISPR/CasY system are alternatives that may be used to generate modifications to a nucleic acid encoding Bmr3 gene. The CRISPR systems are comprised of a CRISPR associated protein that binds to a guide RNA that uses complementary base pairing to recognize DNA sequences at target sites. CRISPR systems are an alternative to synthetic proteins whose DNA-binding domains enable them to modify genomic DNA at specific sequences (e.g.,
ZFN and TALEN). Specificity of the CRISPR/Cas system is based on an RNA-guide that uses complementary base pairing to recognize target DNA sequences. In some embodiments, the RNA-guide comprises a sequence as set forth in SEQ ID NO: 13 and a sequence selected from the group consisting of SEQ ID NOs: 7-12, 399 and 400.
[0097] In some embodiments, the site-specific genome modification enzyme is a
CRISPR/Cas system. In an aspect, a site-specific genome modification enzyme provided herein can comprise any RNA-guided Cas nuclease (non-limiting examples of RNA-guided nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csl3, Csf4, Cpfl, CasX, CasY, Mad7, homologs thereof, or modified versions thereol); and, optionally, the guide RNA necessary for targeting the respective nucleases. In some embodiments, the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 7-12, 399 and 400.
[0098] Several embodiments relate to plant cells, plant tissue, plant seed and plants produced by the methods disclosed herein. Expression Constructs
[0099] Polynucleotides encoding a site-specific genome modification enzyme as described herein can be provided in an expression construct. Expression constructs generally include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements. As used herein, the term“expression construct” refers to a combination of nucleic acid sequences that provides for transcription of an operably linked nucleic acid sequence. As used herein, “operably linked” means two DNA molecules linked in manner so that one may affect the function of the other. Operably-linked DNA molecules may be part of a single contiguous molecule and may or may not be adjacent. For example, a promoter is operably linked with a polypeptide-encoding DNA molecule in a DNA construct where the two DNA molecules are so arranged that the promoter may affect the expression of the DNA molecule.
[00100] As used herein, the term“heterologous” refers to the relationship between two or more items derived from different sources and thus not normally associated in nature. For example, a protein-coding recombinant DNA molecule is heterologous with respect to an operably linked promoter if such a combination is not normally found in nature. In addition, a particular recombinant DNA molecule may be heterologous with respect to a cell, seed, or organism into which it is inserted when it would not naturally occur in that particular cell, seed, or organism.
[00101] An expression construct can comprise a promoter sequence operably linked to a polynucleotide sequence encoding a site-specific genome modification enzyme as described herein. Promoters can be incorporated into a polynucleotide using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression construct as described herein. In some embodiments, a promoter can be positioned about the same distance from the transcription start site in the expression construct as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity. A transcription start site is typically included in the expression construct.
[00102] If the expression construct is to be provided in or introduced into a plant cell, then plant viral promoters, such as, for example, a cauliflower mosaic virus (CaMV) 35 S (including the enhanced CaMV 35S promoter (see, for example U.S. Pat. No. 5,106,739)) or a CaMV 19S promoter or a cassava vein mosaic can be used. Other promoters that can be used for expression constructs in plants include, for example, zein promoters including maize zein promoters, prolifera promoter, Ap3 promoter, heat shock promoters, T-DNA - or 2'-promoter of A. tumefaciens, polygalacturonase promoter, chalcone synthase A (CHS-A) promoter from petunia, tobacco PR- la promoter, ubiquitin promoter, actin promoter, alcA gene promoter, pin2 promoter (Xu et al, 1993), maize Wipl promoter, maize trpA gene promoter (U.S. Pat. No. 5,625,136), maize CDPK gene promoter, and RUBISCO SSU promoter (U.S. Pat. No. 5,034,322) can also be used. Constitutive promoters (such as the CaMV, ubiquitin, actin, or NOS promoter), developmentally -regulated promoters, and inducible promoters (such as those promoters than can be induced by heat, light, hormones, or chemicals) are also contemplated for use with polynucleotide expression constructs described herein.
[00103] Expression constructs may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and/or enhancer elements. Transcription termination regions can typically be obtained from the 3' untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination. A signal peptide sequence is a short amino acid sequence typically present at the amino terminus of a protein that is responsible for the relocation of an operably linked mature polypeptide to a wide range of post-translational cellular destinations, ranging from a specific organelle compartment to sites of protein action and the extracellular environment. Targeting gene products to an intended cellular and/or extracellular destination through the use of an operably linked signal peptide sequence is contemplated for use with the polypeptides described herein. Classical enhancers are cis- acting elements that increase gene transcription and can also be included in the expression construct. Classical enhancer elements are known in the art, and include, but are not limited to, the CaMV 35S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element. Intron-mediated enhancer elements that enhance gene expression are also known in the art. These elements must be present within the transcribed region and are orientation dependent. Examples include the maize shrunken- 1 enhancer element (Clancy and Hannah, 2002). Transformation Methods
[00104] Several embodiments relate to plant cells, plant tissues, plants, and seeds that comprise a recombinant DNA encoding a site-specific genome modification enzyme as described herein. In some embodiments, the recombinant DNA encoding a site-specific genome modification enzyme is bred out of plants containing a modified Bmr3 gene.
[00105] Suitable methods for transformation of host plant cells include virtually any method by which DNA or RNA can be introduced into a cell (for example, where a recombinant DNA construct is stably integrated into a plant chromosome or where a recombinant DNA construct or an RNA is transiently provided to a plant cell) and are well known in the art. Two effective methods for cell transformation are Agrobacterium- mediated transformation and microprojectile bombardment-mediated transformation. Microprojectile bombardment methods are illustrated, for example, in US Patent Nos. US 5,550,318; US 5,538,880; US 6,160,208; and US 6,399,861. Agrobacterium-modiated transformation methods are described, for example in US Patent No. US 5,591,616, which is incorporated herein by reference in its entirety. Transformation of plant material is practiced in tissue culture on nutrient media, for example a mixture of nutrients that allow cells to grow in vitro. Recipient cell targets include, but are not limited to, meristem cells, shoot tips, hypocotyls, calli, immature or mature embryos, and gametic cells such as microspores and pollen. Callus can be initiated from tissue sources including, but not limited to, immature or mature embryos, hypocotyls, seedling apical meristems, microspores and the like. Cells containing a transgenic nucleus are grown into transgenic plants.
[00106] In transformation, DNA is typically introduced into only a small percentage of target plant cells in any one transformation experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a recombinant DNA molecule into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or an herbicide. Any of the herbicides to which plants of this disclosure can be resistant is an agent for selective markers. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells are those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells can be tested further to confirm stable integration of the exogenous DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin and paromomycin ( nptll ), hygromycin B ( aph IV), spectinomycin (aaclA) and gentamycin (aac3 and aacCA) or resistance to herbicides such as glufosinate ( bar or pat), dicamba (DMO) and glyphosate ( aroA or EPSPS). Examples of such selectable markers are illustrated in US Patent Nos. US 5,550,318; US 5,633,435; US 5,780,708 and US 6,118,047. Markers which provide an ability to visually screen transformants can also be employed, for example, a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a ieto-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known.
[00107] Several embodiments relate to methods and constructs for regenerating a plant from a cell with modified Bmr3 gene sequence resulting from genome editing. The regenerated plant can then be used to propagate additional plants.
[00108] Several embodiments relate to a method of introducing an edited allelic variant of a Bmr3 gene comprising: (a) introducing at least one modification to an endogenous Bmr3 gene in at least one plant cell, thereby generating said edited allelic variant; (b) identifying and selecting at least one plant cell of step (a) comprising said edited allelic variant that exhibits one or more of reduced lignin content, reddish-brown pigmentation of the leaf midrib, reduction of RNA expression from the Bmr3 gene, reduction of COMT levels, and absence of COMT; and (c) regenerating a plant from the at least one plant cell selected in step (b). In an aspect, the method comprises introducing a modification in a genomic sequence targeted by g90, g227, g867, g2010, g2070, g2691, g3170 or g3279. In some embodiments, the edited allelic variant of a Bmr3 comprises a sequence selected from SEQ ID NOs: 21-252, 401-443, and 500-507. In some embodiments, the edited allelic variant of a Bmr3 comprises a sequence selected from SEQ ID NOs: 358, 361, 364, 370, 373, 376, 379, and 382. In some embodiments, the edited allelic variant of a Bmr3 comprises a sequence selected from SEQ ID NOs: 348, 350, 352, and 354.
[00109] The modified plants, seeds, plant cells, and plant parts as described herein, and any progeny thereof may also contain one or more additional traits. Additional traits may be introduced by crossing a plant containing an edited Bmr3 allele as described herein with another plant containing one or more additional trait(s). As used herein,“crossing” means breeding two individual plants to produce a progeny plant. Two plants may be crossed to produce progeny that contain the desirable traits from each parent. As used herein“progeny” means the offspring of any generation of a parent plant, and progeny comprise an edited Bmr3 allele as described herein and inherited from at least one parent plant. Additional trait(s) also may be introduced by any means known in the art. Such additional traits include, but are not limited to, increased insect resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, floury-endosperm, short stature, and herbicide-tolerance, in which the trait is measured with respect to a wild-type plant. Examples of additional herbicide tolerance traits may include transgenic or non-transgenic tolerance to one or more herbicides such as ACCase inhibitors (for example, aryloxyphenoxy propionates and cyclohexanediones), ALS inhibitors (for example, sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones) EPSPS inhibitors (for example, glyphosate), synthetic auxins (for example, phenoxys, benzoic acids, carboxylic acids, semicarbazones), photosynthesis inhibitors (for example, triazines, triazinones, nitriles, benzothiadiazoles, and ureas), glutamine synthesis inhibitors (for example, glufosinate), HPPD inhibitors (for example, isoxazoles, pyrazolones, and triketones), PPO inhibitors (for example, diphenylethers, N-phenylphthalimide, aryl triazinones, and pyrimidinediones), and long-chain fatty acid inhibitors (for example, chloroacetamindes, oxyacetamides, and pyrazoles), among others. Examples of insect resistance traits may include resistance to one or more insect members within one or more of the orders of Lepidoptera, Coleoptera, Hemiptera, Thysanoptera, Diptera, Hymenoptera, and Orthoptera, among others. Such additional traits are well-known to one of skill in the art; for example, and a list of such transgenic traits is provided by the United States Department of Agriculture’s (USDA) Animal and Plant Health Inspection Service (APHIS). Examples of short stature traits are disclosed in U.S. Patent Application No. 15/679,699, incorporated herein by reference.
[00110] The modified plants, seeds, plant cells, and plant parts comprising an edited allele of Bmr3 as described herein, and any progeny thereof may also contain one or more additional brown midrib traits. In some embodiments, an additional brown midrib trait may be conferred by a native mutation in the brown midrib 1 gene (bml). In some embodiments, an additional brown midrib trait may be conferred by a native mutation in the brown midrib 2 gene (bm2 or bmr2). In some embodiments, an additional brown midrib trait may be conferred by a native mutation in the brown midrib 4 gene (bm4). In some embodiments, an additional brown midrib trait may be conferred by a native mutation in the brown midrib 5 gene (bm5). In some embodiments, an additional brown midrib trait may be conferred by a native mutation in the brown midrib 6 gene (bmr6). In some embodiments, an additional brown midrib trait may be conferred by a native mutation in the brown midrib 12 gene (bml2). In some embodiments, an additional brown midrib trait may be conferred by a native mutation in the brown midrib 19 gene (bmrl9). Additional brown midrib traits may be introduced by crossing a plant containing an edited Bmr3 allele as described herein with another plant containing one or more additional trait(s). In some embodiments, an edited Bmr3 allele may be introduced by genome editing methods as described herein in a germplasm comprising one or more additional brown midrib traits.
[00111] Plants and progeny that comprise an edited Bmr3 allele as described herein may be used with any breeding methods that are commonly known in the art. In plant lines comprising two or more traits, the traits may be independently segregating, linked, or a combination of both in plant lines comprising three or more traits. Backcrossing to a parental plant and outcrossing with a non-traited plant are also contemplated, as is vegetative propagation. Descriptions of breeding methods that are commonly used for different traits and crops are well-known to those of skill in the art. To confirm the presence of the transgene(s) in a plant or seed, a variety of assays may be performed. Such assays include, for example, molecular biology assays, such as Southern and northern blotting, PCR, and DNA sequencing; biochemical assays, such as detecting the presence of a protein product, for example, by immunological means (ELISAs and western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and, by analyzing the phenotype of the whole plant.
[00112] Introgression of a trait into a plant genotype is achieved as the result of the process of backcross conversion. A plant genotype into which a trait has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid. Similarly, a plant genotype lacking the desired trait may be referred to as an unconverted genotype, line, inbred, or hybrid.
[00113] Having described several embodiments in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible. Furthermore, it should be appreciated that the examples in the present disclosure are provided as non-limiting examples. EXAMPLES
Example 1: Brown midrib (Bmr3) gene structure and design of gRNAs
[00114] The Brown midrib 3 (Bmr3) gene encodes caffeic acid-O-methyltransferase (COMT), a key enzyme involved in synthesis of flavonolignan. The com Bmr3 gene is located on chromosome 4 and has two exons and one intron as shown in Figure 1. The genomic sequences of the Bmr3 gene from 01DKD2 and LH244 inbred com lines are set forth in SEQ ID NO: 1 and SEQ ID NO: 4, respectively. Eight guide RNA targeting sequences (SEQ ID NOs: 7 through 12, and SEQ ID NO: 399 and 400) were identified in the com Bmr3 gene with their positions relative to the Bmr3 genomic DNA sequence as shown in Table 1. Each gRNA comprises a Cpfl crRNA sequence as set forth in SEQ ID NO: 13 and one of gRNA targeting sequences listed in Table 1.
Table 1.
Figure imgf000046_0001
Example 2: Constructs to express cpfl and gRNA in corn plants
[00115] Five recombinant T-DNA vectors (A through E as illustrated in Figure 2) having a Cpfl expression cassette, a gRNA expression cassette, and an expression cassette for a selectable marker conferring resistance to glyphosate were generated to deliver Cpfl and one or more gRNAs as described in Example 1 into plant cells. To produce the T-DNA vectors, a codon optimized version of Lachnospiraceae bacterium ND2006 Cpfl (SEQ ID NO: 19) was cloned into an expression cassette operably linked to a Zea mays Ubiquitin Ml promoter as set forth in SEQ ID NO: 15 followed by its leader sequence as set forth in SEQ ID NOs: 16 and intron sequence as set forth in SEQ ID NO: 17. The Cpfl coding sequence was flanked by a nuclear localization sequence (NLS) as set forth in SEQ ID NO: 18. The 5’ end copy of NLS further comprises ATGGCG for translation start and the 3’ end copy of NLS further comprises TGA for translation stop. The Cpfl expression cassette further has a transcription terminator sequence from rice LTP as set forth in SEQ ID NO: 20. In addition the recombinant T-DNA vectors each contain a gRNA expression cassette having a com U6 promoter as set forth in SEQ ID NO: 14 operably linked to one or more gRNAs described in Example 1.
[00116] The gRNA expression cassette for recombinant T-DNA vector A was generated with three guide RNAs, g867, g2010 and g3170, positioned in tandem. The gRNA expression cassette for recombinant T-DNA vector B was generated with the gRNA expression cassette for three guide RNAs, g2070, g2691 and g3279, positioned in tandem. The recombinant T- DNA vector C was designed to have g2691 as the single guide RNA. The recombinant T-DNA vector D and E were designed to generate whole gene deletion of Bmr3 gene using g90 and g227 respectively in combination with g3279. Com embryos were transformed with recombinant T-DNA vectors disclosed hereinby agrobacterium-mediated transformation. Transformed plants were selected on glyphosate.
Example 3: Select corn plants with edited Bmr3 gene
[00117] Leaf samples were collected from selected plants transformed with the recombinant DNA vectors as described in Example 2 for DNA extraction. PCR reactions were carried out using primers as show in Table 2 to amplify the segments of the Bmr3 gene targeted by the Cpfl editing systems. The PCR products were sequenced and aligned with sequences of the unedited Bmr3 gene from inbred com lines to identify the edited Bmr3 alleles listed in Table 3.
Table 2. Primers used to identify edits in Bmr3
Figure imgf000048_0001
Table 3. Description of edited alleles with deletions in the Bmr3 gene
Figure imgf000048_0002
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000058_0001
[00118] In addition to the edited alleles having deletions within the Bmr3 gene, several edited alleles were identified with inversions. These inversions occurred between two sites targeted by the Cpfl guide RNAs, see Table 4.
Table 4. Description of edited alleles with inversions in the Bmr3 gene
Figure imgf000059_0001
Example 4: Gene expression, physiology and characterization of corn plants having edited Bmr3 alleles
1. Phenotype Characterization.
[00119] Com plants with mutations in Brown midrib genes exhibit a characteristic brown coloration of the leaf mid veins that is visibly scorable at v3-vl0 stages. As shown in Table 5 and Figure 2, three events generated by recombinant T-DNA vector B (described in Example 2) were bred to R1 stage. One of the events, B-l, is biallelic at the site targeted by g2691. Plants that are homozygous for Bmr3 alleles comprising SEQ ID NO 191, 194 and 172 exhibited the brown midrib phenotype. In addition, plants with one Bmr3 allele comprising SEQ ID NO: 191 and one Bmr3 allele comprising 209 were also exhibited brown midrib phenotype.
[00120] One event, A-l, generated by recombinant T-DNA vector A was bred to R1 stage. This edited Bmr3 allele, comprising SEQ ID NO:22, has the entirety of exon2 deleted. Plant homozygous for SEQ ID NO: 22 exhibited the brown midrib phenotype. The genomic sequence of the Bmr3 gene of event A-l is set forth in SEQ ID NO: 246.
[00121] Two events, C-l and C-2 were generated by recombinant T-DNA vector C as shown in Table 6. Cl line is biallelic and the two alleles were segregating in Rl. Homozygous plants comprising SEQ ID NO: 166 or SEQ ID: 191 derived from event Cl exhibited the brown midrib phenotype. Homozygous plants comprising SEQ ID NO: 191 derived from event C2 also exhibited the brown midrib phenotype.
[00122] One event, D-l was generated by recombinant T-DNA vector D whereas two events, E-l and E-2, were generated by recombinant T-DNA vector E as shown in Table 7. These three events have whole gene deletion for Bmr3 and their progenies are homozygous for the deletion exhibited brown midrib phenotype.
Table 5. Edited corn events generated by Construct B
Figure imgf000061_0001
Table 6. Edited corn events by construct C
Figure imgf000061_0002
Table 7. Edited corn events generated by constructs D and E
Figure imgf000062_0001
2. RNA expression
[00123] Leaf samples were collected from plants that were homozygous for one of the Bmr 3 alleles comprising SEQ ID NO: 172, 191, or 194. In addition, leaf samples were collected from plants that were heterozygous for the Bmr3 allele comprising SEQ ID NO: 22. RNA was extracted from the leaf samples using a standard RNA extraction protocol. Two primers sets were used to detect Bmr3 RNA levels in wild type (control) and the edited plants. Primer set 1, which comprised primers of SEQ ID NO: 395 and 396 spans exon 2 to detect only the wild type Bmr3 mRNA, but not the mRNA from the edited Bmr3 alleles. Primer set 2, which comprised primers of SEQ ID NOs: 397 and 398 spans exon-1 of Bmr3 gene and this primer set amplifies and detects RNA from both the wild-type and the edited alleles, since the deletions in the edited lines are in exon2.
[00124] Bmr3 gene along with two reference genes used for internal normalization were detected by RT-qPCR using Taqman assays. Expression (also referred to as Relative Quantity) in arbitrary units was calculated as per standard protocols. Using primer set 1, Bmr3 mRNA was detected only in the wild-type control and Bmr3 edited event that is heterozygous for the exon2 deletion (SEQ ID NO: 22), but not in the three edited events that are homozygous for 8bp (SEQ ID NO: 194), 9bp (SEQ ID NO: 191), and 16bp deletions (SEQ ID NO: 172) (Figure 3A). Using primer set 2, which spans exon-1, RNA of both the wild-type control and the edited lines were detected (Figure 3B). 3. Silage characterization
[00125] The Brown midrib visible phenotype is associated with reduced lignin content and altered lignin composition, which are useful to improve forage digestibility for livestock.
[00126] Edited com plants described in Example 4 and control plants were grown in a field at Jerseyville, IL and were harvested at R3 stage. Up to 12 plants per plot were harvested, not including ears, as samples for silage characterization. The samples were processed using a single row silage chopper, vacuum sealed, frozen and shipped overnight to Dairyland labs in Arcadia, WI. The samples were dried in an oven at a degree lower than 60 °C and ground to ~lmm in size. Homogenized samples were run on Dairyland Labs’ NIR instrument. LIGP is the percentage of Lignin. ADF is the percentage of acid detergent fiber. STAS is the percentage of silage starch. NDF30 is 30hr in vitro NDF digestibility. LIGP, ADF, STAS, and NDF30 data shown in Tables 9-12 were direct measurements of the Dairyland Labs’ NIR instrument. MPT is an estimation of the pounds of milk per ton of dry silage. The total digestible nutrients are calculated and then converted into energy of lactation, and then MPT is calculated. This calculation is heavily influenced by NDF30 and STAS. Since the silage samples did not include the cob and kernels, the starch numbers were generally lower and had less impact on the calculations. In these silage characterization, wildtype com plants and MGCSC-408E plants (Molecular characterization of a brown midrib3 deletion mutation in maize. Morrow et. al. Molecular Breeding 3: 351-357, 1997), a publicly available Bmr3 com line acquired from MaizeGDB stock were grown and processed under the same conditions to be used as controls.
Table 8. Silage characterization of Event B-2 in comparison to wildtype plants
Figure imgf000063_0001
Table 9. Silage characterization of Event B-3 in comparison to wildtype plants
Figure imgf000064_0001
Table 10. Silage characterization of Event B-2 in comparison to MGCSC-408E
Figure imgf000064_0002
Table 11. Silage characterization of Event B-2 in comparison to MGCSC-408E
Figure imgf000064_0003
Table 12. Silage characterization of MGCSC-408E in comparison to wildtype plants
Figure imgf000064_0004

Claims

What Is Claimed Is:
1. A modified com plant comprising an edited allele of Bmr3, wherein the modified com plant exhibits brown pigmentation in the leaf midrib at the v3-vl0 stage.
2. The modified com plant of claim 1, wherein the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443 and 500-507.
3. The modified com plant of claim 1, wherein the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382.
4. The modified com plant of claim 1, wherein the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354.
5. The modified com plant of claim 1, wherein the edited allele of Bmr3 encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 253-299, 444-471, and 508-513.
6. The modified com plant of claims 1 -5, wherein Neutral Detergent Fiber (NDF) of silage produced from the modified com plant is about 30-50 %.
7. The modified com plant of claims 1-5, wherein Acid Detergent Fiber (ADF) of silage produced from the modified com plant is about 15-26 %.
8. The modified com plant of claims 1-5, wherein lignin content of silage produced from the modified com plant is about 2-4 %.
9. The modified com plant of claims 1-5, wherein lignin content of the modified com plant is reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or more as compared to com plant with a wild-type Bmr3 gene.
10. A seed produced from the modified com plant of claims 1-5.
11. The modified com plant of claims 1-5, wherein the modified com plant further comprises a floury -2 trait.
12. The modified com plant of claims 1-5, wherein the modified com plant is homozygous for the edited allele of Bmr3.
13. The modified com plant of claims 1-5, wherein the modified com plant is inbred.
14. The modified com plant of claim 13, wherein the modified com plant is male.
15. The modified com plant of claim 13, wherein the modified com plant is female.
16. A hybrid com plant, wherein the hybrid com plant is produced by crossing the modified com plant of claim 14 with the modified com plant of claim 15.
17. The modified com plant of claims 1-5, wherein the modified com plant is derived from an elite line.
18. A part of the modified com plant of claims 1-15, selected from the group consisting of an intact plant cell, a plant protoplast, an embryo, a pollen, an ovule, a flower, a kernel, a seed, an ear, a cob, a leaf, a husk, a stalk, a root, a root tip, a brace root, a lateral tassel branch, an anther, a tassel, a glume, a tiller and a silk.
19. A method of generating a modified com plant with reduced lignin comprising the steps of: a. introducing a non-natural mutation in a Bmr3 gene of a com plant cell; b. identifying and selecting one or more plant cells of step (a) comprising said non natural in Bmr3; and c. regenerating at least one plant from at least one or more cells selected in step (b).
20. The method of claim 19, wherein the non-natural mutation is introduced by a site- specific genome modification enzyme.
21. The method of claim 19, wherein the site-specific genome modification enzyme is Cpfl.
22. The method of claim 19, wherein the non-natural mutation is introduced at a site targeted by guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400.
23. The method of claims 19-22, wherein the com plant cell comprises a mutant allele of floury-2.
24. The method of claim 19, wherein the non-natural mutation comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443, and 500-507.
25. The method of claim 19, wherein the non-natural mutation comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382.
26. The method of claim 19, wherein the non-natural mutation comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354.
27. The method of claim 19, wherein the com plant cell is from an elite com plant.
28. Com silage produced from a modified com plant comprising an edited allele of Bmr3.
29. The com silage of claim 28, wherein the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443, and 500-507.
30. The com silage of claim 28, wherein the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382.
31. The com silage of claim 28, wherein the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354.
32. The com silage of claims 28-31, wherein the lignin content is about 2-4%.
33. A method of enhancing milk production efficiency in dairy cattle, the method comprising: preparing a feed ration comprising a com silage made from a com plant comprising an edited allele of Bmr3; and feeding the dairy cattle with the feed ration to provide an increased amount of milk produced per one unit of the feed ration consumed by the dairy cattle.
34. The method of claim 33, wherein the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 401-443, and 500-507.
35. The method of claim 33, wherein the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 358, 361, 364, 367, 370, 373, 376, 379, and 382.
36. The method of claim 33, wherein the edited allele of Bmr3 comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 348, 350, 352, and 354.
37. The method of claims 33-36, wherein the com plant further comprises a mutant allele of floury-2.
38. The method of claim 33, wherein the com plant is a hybrid com plant.
39. The method of claim 33, wherein the com plant is an elite com plant.
40. A modified protein comprising an amino acid sequence selected from SEQ ID NOs:
253-299, 444-471, and 508-513.
41. A com plant comprising a modified com plant of claim 40.
42. A com silage produced from the modified com plant of claim 40.
43. A modified com plant produced by: a. providing a com plant cells wherein the cell comprises a target genomic nucleic acid molecule having at least 90% sequence identity with a sequence selected from the group consisting of: nucleotides 146-168 of SEQ ID NO: 1, nucleotides 283-305 of SEQ ID NO: 1, nucleotides 637-659 of SEQ ID NO: 1, nucleotides 1802-1824 of SEQ ID NO: 1, nucleotides 1862-1884 of SEQ ID NO: 1, nucleotides 2461-2483 of SEQ ID NO: 1, nucleotides 2940-2962 of SEQ ID NO: 1, nucleotides 3071-3093 of SEQ ID NO: 1, nucleotides 73-95 of SEQ ID NO: 4, nucleotides 1276-1298 of SEQ ID NO: 4, nucleotides 1336-1358 of SEQ ID NO: 4, nucleotides 1935-1957 of SEQ ID NO: 4, nucleotides 2401-2423 of SEQ ID NO: 4, nucleotides 1622-1644 of SEQ ID NO: 522, and nucleotides 2231-2254 of SEQ ID NO: 522; b. selecting a site-specific genome modification enzyme that specifically binds and cleaves the target genomic nucleic acid molecule; c. introducing the site-specific genome modification enzyme into the com plant cells, wherein the site-specific nuclease cleaves the target genomic nucleic acid molecule; d. generating com plants from the com plant cells; and e. selecting a com plant exhibiting brown coloration of the leaf mid veins at v3-vl0 stages.
44. The modified com plant of claim 43, wherein the site-specific genome modification enzyme is a CRISPR associated protein complexed with guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400.
45. The modified com plant of claim 43, wherein the CRISPR associated protein is selected from the group consisting of: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, CasX, CasY, and Mad7.
46. The modified com plant of claim 43, wherein the site-specific genome modification enzyme is a Cpfl protein complexed with guide RNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7-12, 399 and 400.
47. The modified com plant of claims 43-46, wherein the modified com plant lacks a Bmr3 gene product.
48. The modified com plant of claims 43-46, wherein the modified com plant comprises a nucleotide sequence selected from the group consisting of SEQ NOs: 21-252, 348, 350, 352, 354, 358, 361, 364, 367, 370, 373, 376, 379, 382, 401-443, and 500-507.
49. The modified com plant of claims 43-46, wherein the modified com plant comprises a Bmr3 gene product comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 253-299, 444-471, and 508-513.
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