EP4580392A1 - Procédés et compositions pour la tolérance à l'herbicide ppo - Google Patents

Procédés et compositions pour la tolérance à l'herbicide ppo

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Publication number
EP4580392A1
EP4580392A1 EP23771936.4A EP23771936A EP4580392A1 EP 4580392 A1 EP4580392 A1 EP 4580392A1 EP 23771936 A EP23771936 A EP 23771936A EP 4580392 A1 EP4580392 A1 EP 4580392A1
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EP
European Patent Office
Prior art keywords
seq
plant
beta vulgaris
position corresponding
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23771936.4A
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German (de)
English (en)
Inventor
Hilde Maurice Anne VAN HOUTTE
Shivegowda Shikaranahalli THAMMANNAGOWDA
Murielle Anne Francoise LOMMEL
Glenda WILLEMS
Per Erik SNELL
Ingo Peter LENK
Elisabeth Rosine Armand VEECKMAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SESVanderHave NV
Dlf Seeds
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SESVanderHave NV
Dlf Seeds
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Application filed by SESVanderHave NV, Dlf Seeds filed Critical SESVanderHave NV
Publication of EP4580392A1 publication Critical patent/EP4580392A1/fr
Pending legal-status Critical Current

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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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • 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 [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y103/00—Oxidoreductases acting on the CH-CH group of donors (1.3)
    • C12Y103/03—Oxidoreductases acting on the CH-CH group of donors (1.3) with oxygen as acceptor (1.3.3)
    • C12Y103/03004—Protoporphyrinogen oxidase (1.3.3.4)
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present disclosure relates to the fields of agriculture, plant biotechnology, and molecular biology. More specifically, the disclosure relates to plants and methods of producing said plants which are tolerant to herbicides that inhibit protoporphyrinogen oxidase and methods of use thereof.
  • PPO herbicides that inhibit protoporphyrinogen oxidase (PPO, EC 1.3.3.4), referred to as PPO herbicides.
  • PPO herbicides provide control of a spectrum of herbicide-resistant weeds, thus making a trait conferring tolerance to these herbicides particularly useful in a cropping system.
  • crops having resistance to PPO herbicides Also needed are methods for making such crops and controlling weed growth in the vicinity of such crops.
  • the disclosure teaches a method of producing a Beta vulgaris plant with increased tolerance to an herbicide that inhibits protoporphyrinogen oxidase comprising the steps of: a) transfecting a protoplast obtained from Beta vulgaris cells with a genome editing system to generate a transfected protoplast, wherein the genome editing system comprises: i) a Cas enzyme; ii) at least one guide RNA (gRNA), wherein the at least one gRNA targets a genomic region corresponding to between position 5457 and 5502 of SEQ ID NO: 1; and iii) at least one singlestranded donor DNA repair template designed to introduce a deletion of glycine at a position corresponding to 208 and/or 209 of SEQ ID NO: 3; b) exposing the transfected protoplast to a selective pressure of at least one herbicide that inhibits protoporphyrinogen oxidase; c) selecting a protoplast comprising a deletion of glycine at a position corresponding to
  • the disclosure relates to a Beta vulgaris plant, or part thereof, comprising an engineered nucleic acid encoding a protoporphyrinogen oxidase 2 (PPO2) amino acid sequence, wherein said PPO2 amino acid sequence comprises a deletion of glycine at a position corresponding to 208 and/or 209 of SEQ ID NO: 3.
  • PPO2 amino acid sequence comprises a deletion of glycine at a position corresponding to 208 and/or 209 of SEQ ID NO: 3.
  • the disclosure further relates to a polynucleotide comprising an engineered nucleic acid sequence encoding a protein comprises a deletion of glycine at a position corresponding to 208 and/or 209.
  • the disclosure further teaches methods for producing a plant, plant part, or plant cell having resistance or tolerance to a PPO herbicide, the method comprising: transforming a plant, plant part, or plant cell with the polynucleotides disclosed herein.
  • the disclosure further teaches methods for producing a Beta vulgaris plant or plant cell having an engineered PPO2 protein comprising: a) providing a guide RNA sequence selected from SEQ ID NOs: 67-80; b) providing a donor template sequence selected from SEQ ID NOs: 48-66, and 90-96; c) providing a DNA nuclease; wherein said guide RNA, donor template, and DNA endonuclease are provided on one or more plasmids, or wherein said guide RNA and said DNA nuclease are provided as a ribonucleoprotein; d) transforming the Beta vulgaris plant or plant cell with said guide RNA, donor template, and DNA nuclease; and e) selecting a plant or plant cell having a deletion of glycine at a position corresponding to 208 and/or 209 of SEQ ID NO: 3.
  • the disclosure further relates to a guide RNA suitable for use in a CRISPR based genome editing system, wherein said guide RNA is selected from SEQ ID NOs: 67-80.
  • the disclosure further relates to a donor template sequence suitable for use in a CRISPR based genome editing system, wherein said donor template sequence is selected from SEQ ID NOs: 48-66, and 90-96.
  • the disclosure further relates to DNA constructs comprising the guide RNAs and donor templates disclosed herein.
  • the disclosure further relates to an engineered PPO2 protein comprising a deletion of glycine at a position corresponding to 208 and/or 209 in SEQ ID NO: 3.
  • the disclosure further teaches a method of detecting an in-frame deletion of glycine at a position corresponding to 208 and/or 209 of SEQ ID NO: 3 in a Beta vulgaris plant or part thereof, comprising: obtaining a Beta vulgaris plant or part thereof; and analyzing the Beta vulgaris plant or part thereof using at least one of SEQ ID NOs: 112-122 to detect an in-frame deletion of glycine at a position corresponding to 208 and/or 209 of SEQ ID NO: 3.
  • Fig. 1 shows the location of genetic edits in a consensus amino acid sequence for sugar beet gene PPO2 which can confer resistance to PPO herbicides.
  • Fig. 2 is a protein alignment produced by Clustal Omega showing the location of various edits (shaded, bold and underlined font) in SEQ ID NOs: 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 37, 40, and 43 compared to wildtype SEQ ID NO: 3.
  • Fig. 3 shows copies of three different edited sugar beet genotypes, HTH195, HTH240 and HTH251 heterozygous for the G208 deletion on SAF 2.5pM containing media after 20 days.
  • Fig. 4 shows a chromatogram obtained after Sanger sequencing of an edited plant showing the Glycine deletion.
  • Figs. 5A-5B are photographs of sugar beet plants with the expected glycine deletion at position 208/209 vs others, 21 days after spraying with Evolution, Treevix, or water.
  • Fig. 5A shows differences in response between edited plants and non-edited (Ctrl) elite sugar beet plants 21 days after spray with Evolution 0.2X.
  • Fig. 5B shows the phytotoxicity effect of Treevix IX, and 2X on an elite sugar beet genotype, compared to the edited sugar beet plant HTH259 having the glycine deletion at position 208/209. BRIEF DESCRIPTION OF THE SEQUENCE LISTING
  • Labels can also include: a radiolabel (a direct label) (e.g., 3H, 1251, 35S, 14C, or 32P); an enzyme (an indirect label) (e.g., peroxidase, alkaline phosphatase, galactosidase, luciferase, glucose oxidase, and the like); a fluorescent protein (a direct label) (e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, and any convenient derivatives thereof); a metal label (a direct label); a colorimetric label; a binding pair member; and the like.
  • binding pair member is meant one of a first and a second moiety, wherein the first and the second moiety have a specific binding affinity for each other.
  • NCBI Basic Local Alignment Search Tool (BLAST®) (Altschul et al. 1990 J. Mol. Biol. 215: 403-10), which is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed on the internet via the National Library of Medicine (NLM)'s world- wide- web URL. A description of how to determine sequence identity using this program is available at the NLM's website on BLAST tutorial.
  • NLM National Library of Medicine
  • At least 90% of the plants in a resistant line will have a score of 1, 2, or 3. If a more detailed visual evaluation is possible, then one can use a scale from 1 to 10 so as to broaden out the range of scores and thereby hopefully provide a greater scoring spread among the plants being evaluated. Instead of scoring individual plants, one can also provide a score on a group of plants, where the plants in one group would belong to the same line and be clones of each other. Any given component, or combination of components can be unlabeled, or can be detectably labeled with a label moiety. In some embodiments, when two or more components are labeled, they can be labeled with label moieties that are distinguishable from one another.
  • Agricultural crop production often utilizes herbicide tolerance (HT) traits predominantly introduced by conventional plant transformation methods, which results in transgenic crops with the desired traits.
  • HT herbicide tolerance
  • DNA can be modified in a targeted way using genome editing techniques, to develop novel, desired traits in plants of interest.
  • the present disclosure teaches utilization of an emerging technology including the targeted genome editing techniques, such as CRISPR/Cas system, to establish herbicide tolerance traits, thereby producing non-transgenic crops with the desired traits.
  • Beta vulgaris (“Beet”), is a root vegetable of the subfamily Betoideae within the family Amaranthaceae. Examples of beet include sugar beet, garden beets (red beet), leafy beets (chard), and fodder beets (forage).
  • Sugar beet (B. vulgaris L. ssp. vulgaris) is grown both as a garden vegetable and, since the mid- 18 th century, for its sugar content. Sugar from sugar beet accounts for approximately 20-30% of the world’s annual production of sugar, the rest being extracted from sugar cane (Yamane, Takeo. "Sugar beet”. Encyclopedia Britannica, 12 Apr.
  • herbicide is any molecule that is used to control, prevent, or interfere with the growth of one or more undesired plants in a cultivated area (e.g. weeds).
  • exemplary herbicides include acetyl-CoA carboxylase (ACCase) inhibitors (for example aryl oxy phenoxy propionates and cyclohexanediones); acetolactate synthase (ALS) inhibitors (for example sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones); 5 enolpyruvylshikimate-3 -phosphate synthase (EPSPS) inhibitors (for example glyphosate), synthetic auxins (for example phenoxys, benzoic acids, carboxylic acids, semicarbazones), photosynthesis (photosystem II) inhibitors (for example triazines, triazinones, nitriles, be
  • ACCase ace
  • PPO herbicides include, but are not limited to, diphenylethers (such as acifluorfen, its salts and esters, aclonifen, bifenox, its salts and esters, ethoxyfen, its salts and esters, fluoronitrofen, furyloxyfen, halosafen, chlomethoxyfen, fluoroglycofen, its salts and esters, lactofen, its salts and esters, oxyfluorfen, and fomesafen, its salts and esters); thiadiazoles (such as fluthiacet-methyl and thidiazimin); pyrimidinediones or phenyluracils (such as benzfendizone, butafenacil, ethyl [3-2-chloro-4- fluoro-5-(l-methyl-6-trifluoromethyl- 2,4-dioxo-l,2,3,4-tetrahydr
  • a deletion of glycine at amino acid position no. 208 or 209 of the wild type Beta vulgaris PPO2 protein sequence may confer resistance to a PPO herbicide.
  • the glycine at position no. 208 is deleted.
  • the glycine at position no. 209 is deleted.
  • deletion of glycine at position no. 208 or 209 is combined with one or more of the genetic alterations described below in Table 3.
  • the disclosure provides novel, engineered proteins and the recombinant DNA molecules that encode them.
  • engineered refers to a non-natural DNA, protein, cell, or organism that would not normally be found in nature and was created by human intervention.
  • an “engineered protein”, “engineered enzyme”, or “engineered PPO,” refers to a protein, enzyme, or PPO whose amino acid sequence was conceived of and created in the laboratory using one or more of the techniques of biotechnology, protein design, or protein engineering, such as molecular biology, protein biochemistry, bacterial transformation, plant transformation, site-directed mutagenesis, directed evolution using random mutagenesis, genome editing, gene editing, gene cloning, DNA ligation, DNA synthesis, protein synthesis, and DNA shuffling.
  • an engineered protein may have one or more deletions, insertions, or substitutions relative to the coding sequence of the wild-type protein and each deletion, insertion, or substitution takes place on one or more amino acids.
  • Genetic engineering can be used to create a DNA molecule encoding an engineered protein, such as an engineered PPO that is herbicide tolerant and comprises at least one amino acid substitution or deletion relative to a wild-type PPO protein as described herein.
  • the engineered proteins are genetically engineered with a targeted genome or gene editing system such as CRISPR-Cas system described below.
  • weed control is provided.
  • novel, engineered proteins that are herbicide-tolerant protoporphyrinogen oxidases (PPOs), as well as the recombinant, engineered DNA molecules encoding the herbicide-tolerant PPOs, compositions comprising the herbicide-tolerant PPO, and methods of using the herbicide-tolerant PPOs for weed control.
  • PPOs herbicide-tolerant protoporphyrinogen oxidases
  • engineered proteins e.g. PPO2
  • PPO2 herbicide-tolerant protoporphyrinogen oxidase activity.
  • herbicide-tolerant protoporphyrinogen oxidase means the ability of a protoporphyrinogen oxidase to maintain at least some of its protoporphyrinogen oxidase activity in the presence of one or more PPO herbicide(s).
  • protoporphyrinogen oxidase activity means the ability to catalyze the six- electron oxidation (removal of electrons) of protoporphyrinogen IX to form protoporphyrin IX, that is, to catalyze the dehydrogenation of protoporphyrinogen to form protoporphyrin.
  • Enzymatic activity of a protoporphyrinogen oxidase can be measured by any means known in the art, for example, by an enzymatic assay in which the production of the product of protoporphyrinogen oxidase or the consumption of the substrate of protoporphyrinogen oxidase in the presence of one or more PPO herbicide(s) is measured via fluorescence, high performance liquid chromatography (HPLC), or mass spectrometry (MS).
  • HPLC high performance liquid chromatography
  • MS mass spectrometry
  • the disclosure provides engineered proteins having herbicide-tolerant protoporphyrinogen oxidase activity.
  • the disclosure provides methods and compositions for using protein engineering and bioinformatics tools to obtain and improve herbicide-tolerant protoporphyrinogen oxidases.
  • the disclosure further provides methods and compositions for producing plants, parts and cells tolerant to PPO herbicides, and methods of weed control using the cells, plants, and seeds.
  • Examples of engineered proteins provided herein are herbicide-tolerant PPOs comprising (i) one or more amino acid substitution(s) selected from R126A, R126G, R126L, R126I, R126M, L397E, G398A, F420V, F420M, F420I, and F420L, and (ii) one or more amino acid deletion(s) selected from G208 and G209, including all possible combinations thereof, wherein the position of the amino acid substitution(s) and/or deletion(s) are relative to the amino acid position set forth in SEQ ID NO: 3.
  • an engineered protein provided herein comprises one, two, three, four, or more of any combination of such substitutions and/or deletions described herein.
  • DNA sequences encoding PPO enzymes with the amino acid substitutions and deletions described herein can be produced by introducing mutations into the DNA sequence encoding a wild-type PPO enzyme using methods known in the art. It is well within the capability of one of skill in the art to create alternative DNA sequences encoding the same, or essentially the same, altered or engineered proteins as described herein. These variant or alternative DNA sequences are within the scope of the embodiments described herein.
  • references to “essentially the same” sequence refers to sequences which encode amino acid substitutions, deletions, additions, or insertions that do not materially alter the functional activity of the protein encoded by the DNA molecule of the embodiments described herein. Allelic variants of the nucleotide sequences encoding a wild-type or engineered protein are also encompassed within the scope of the embodiments described herein.
  • Exemplary methods include the use of sequence specific nucleases, 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).
  • sequence specific nucleases 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
  • the present disclosure provides modification or replacement of an existing coding sequence, such as a PPO coding sequence or another existing transgenic insert, within a plant genome with a sequence encoding an engineered protein, such as an engineered PPO coding sequence of the present disclosure.
  • RNA-guided endonuclease 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
  • Genome editing by CRISPR which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is based on a natural immune process used by bacteria to defend themselves against invading viruses. Indeed, in bacteria the invading viral DNA will be cut through use of a guide RNA (gRNA), or piece of RNA, and a CRISPR-associated protein (Cas). The last step of the bacterial immune process, when the gRNA is combined with Cas and cleaves the target DNA, has been adopted for genome editing in laboratories.
  • gRNA guide RNA
  • Cas CRISPR-associated protein
  • Type I, II, and III There are at least three main CRISPR system types (Type I, II, and III) and at least 10 distinct subtypes (Makarova, K.S., et.al., Nat Rev Microbiol. 2011 May 9; 9(6):467-477).
  • Type I and III systems use Cas protein complexes and short guide polynucleotide sequences to target selected DNA regions.
  • Type II systems rely on a single protein (e.g. Cas9) and the targeting guide polynucleotide, where a portion of the 5’ end of a guide sequence is complementary to a target nucleic acid.
  • Cas9 a single protein
  • the targeting guide polynucleotide where a portion of the 5’ end of a guide sequence is complementary to a target nucleic acid.
  • Either Cas9 or Casl2a can be used to cleave target DNA, resulting in a Double Strand Break (DSB).
  • Each Cas enzyme is directed by the gRNA to a user-specified cut site in the genome.
  • Casl2al family members contain a RuvC-like endonuclease domain, but lack the second HNH endonuclease domain of Cas9.
  • Casl2a cleaves DNA in a staggered pattern in contrast to Cas9 which produces a blunt-end.
  • Cas 12a requires only one RNA rather than the two tracrRNA and crRNA needed by Cas9.
  • the target sequence of the gRNAs must be next to a PAM sequence.
  • the PAM sequence corresponds to NGG, where N is any base.
  • the gRNA will recognize and bind to 20 nucleotides on the DNA strand opposite from the NGG PAM site.
  • the PAM sequence is TTTV, where V can represent A, C, or G.
  • a TTTT PAM sequence may also work.
  • the “V” of the TTTV is immediately adjacent to the base at the 5’ end of the nontargeted strand side of the protospacer element.
  • the guide RNA for Casl2a is relatively short and is approximately 40 to 44 bases long.
  • the present disclosure relates to a recombinant DNA construct comprising an expression cassette(s) encoding a site-specific nuclease and, optionally, any associated protein(s) to carry out genome modification.
  • These nuclease-expressing cassette(s) may be present in the same molecule or vector as a donor template for templated editing or an expression cassette comprising nucleic acid sequence encoding a PPO protein as described herein or on a separate molecule or vector.
  • Several methods for site-directed integration are known in the art involving different sequence-specific nucleases (or complexes of proteins or guide RNA or both) that cut the genomic DNA to produce a double strand break (DSB) or nick at a desired genomic site or locus.
  • DSB double strand break
  • the site-specific genome modification enzyme is a recombinase.
  • recombinases include a tyrosine recombinase attached to a DNA recognition motif provided herein is selected from the group consisting of a Cre recombinase, a Gin recombinase, a Flp recombinase, and a Tnpl recombinase.
  • a Cre recombinase or a Gin recombinase provided herein is tethered to a zinc-finger DNA-binding domain, or a TALE DNA-binding domain, or a Cas9 nuclease.
  • plants comprising one or more of the genetic alterations described herein may be selfed or crossed to produce lines that are homozygous for one or more of the genetic alterations described herein.
  • the genetic alterations described herein may be transferred or introgressed to other beet varieties through conventional breeding schemes.
  • the disclosure provides a guide RNA suitable for use in the CRISPR-Cas based genome editing system taught herein, wherein said guide RNA comprises a nucleic acid sequence selected from SEQ ID NOs: 67-80.
  • the disclosure provides a donor DNA suitable for use in the CRISPR-Cas based genome editing system taught herein, wherein said donor DNA comprises a nucleic acid sequence selected from SEQ ID NOs: 48-66 and 90-96.
  • the guide RNA and donor template are provided in a ribonucleoprotein (RNP) complex. In some embodiments, the guide RNA and donor template are provided in a plasmid.
  • RNP ribonucleoprotein
  • a CRISPR-Cas genome editing system comprising; (a) a first expression construct comprising a target locus-specific guide RNA (gRNA) and a donor template, wherein said guide RNA comprises a nucleic acid sequence selected from SEQ ID NOs: 67-80, and wherein said donor template is selected from SEQ ID NOs: 48-66 and 90-96; and (b) a second expression construct comprising a polynucleotide encoding a CRISPR- associated protein nuclease.
  • gRNA target locus-specific guide RNA
  • the CRISPR-Cas based genome editing system comprises at least one gRNA, a donor template, PAM sequence, and CRISPR-associated nuclease selected from the group consisting of Cas9, Casl2, Casl3, CasX, and CasY.
  • RNP or the first expression construct comprises a target locusspecific guide RNA (gRNA) selected from the group consisting of SEQ ID NOs: 77 and 78 and a donor template selected from the group consisting of SEQ ID NOs: 48-50, 57, 65, and 96.
  • gRNA target locusspecific guide RNA
  • the genome editing method comprises the steps of: a) transfecting a protoplast obtained from Beta vulgaris cells with a genome editing system to generate a transfected protoplast, wherein the genome editing system comprises: i) a Cas enzyme; ii) at least one guide RNA (gRNA), wherein the at least one gRNA targets a genomic region corresponding to between position 5457 and 5502 of SEQ ID NO: 1; and iii) at least one singlestranded donor DNA repair template designed to introduce a deletion of glycine at a position corresponding to 208 and/or 209 of SEQ ID NO: 3; b) exposing the transfected protoplast to a selective pressure of at least one herbicide that inhibits protoporphyrinogen oxidase; c) selecting a protoplast comprising a deletion of glycine at a position corresponding to 208 and/or 209 of SEQ ID NO: 3; and d) regenerating a plant from said selected protoplast
  • the at least one gRNA targets a genomic region corresponding to between position 5483 and 5502, or between 5457 and 5479 of SEQ ID NO: 1.
  • the present disclosure provides a Beta vulgaris plant, or part thereof comprising a nucleic acid encoding a protoporphyrinogen oxidase 2 (PPO2) amino acid sequence.
  • said PPO2 amino acid sequence comprises a deletion of glycine at a position corresponding to 208 or 209 of SEQ ID NO: 3.
  • the deletion of glycine is at a position corresponding to 208 of SEQ ID NO: 3.
  • the deletion of glycine is at a position corresponding to 209 of SEQ ID NO: 3.
  • the Beta vulgaris plant or part thereof further comprises a nucleic acid encoding a protoporphyrinogen oxidase 2 (PPO2) amino acid sequence comprising at least one of: (a) a substitution of arginine at a position corresponding to 126 of SEQ ID NO: 3; (b) a substitution of leucine at a position corresponding to 397 of SEQ ID NO: 3; (c) a substitution of glycine at a position corresponding to 398 of SEQ ID NO: 3; and (d) a substitution of phenylalanine at a position corresponding to 420 of SEQ ID NO: 3.
  • PPO2 protoporphyrinogen oxidase 2
  • leucine at position 397 is replaced with glutamic acid.
  • glycine at position 398 is replaced with alanine.
  • phenylalanine at position 420 is replaced with valine, methionine, isoleucine, or leucine.
  • the disclosure relates to a Beta vulgaris plant, or part thereof, comprising an engineered PPO2 protein having a deletion of glycine corresponding to position number 208 or 209 of SEQ ID NO: 3, and at least 90% identical to SEQ ID NO: 6.
  • the sequence is 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6.
  • the disclosure relates to a Beta vulgaris plant, or part thereof, comprising a nucleic acid encoding a deletion of glycine corresponding to position number 208 or 209 of SEQ ID NO: 3, and a nucleic acid at least 90% identical to SEQ ID NO: 4, 97, or
  • the disclosure relates to a Beta vulgaris plant, or part thereof, comprising a nucleic acid encoding a deletion of glycine corresponding to position number 208 or 209 of SEQ ID NO: 3, and a nucleic acid at least 90% identical to SEQ ID NO: 5, 98, or
  • the Beta vulgaris plant, or part thereof is resistant or tolerant to a PPO herbicide selected from the group consisting of: acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, azafenidin, carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl, oxadiazon, pyraflufen-ethyl, saflufenacil, trifludimoxazin, and S-3100.
  • a PPO herbicide selected from the group consisting of: acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, azafenidin, carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, ox
  • plants carrying the mutation G208/209 deletion described below in Table 6 were acclimatized in soil for 3-5 weeks and screened for PPO resistance with a spray test of the PPO herbicides Saflufenacil (Brand name TREEVIX at a recommended dose of 25g a.i./ha), and Pyraflufen-ethyl (Brand name EVOLUTION at a recommended dose of 0.81/ha, or a.i. 26.5g/l).
  • the spray was performed at a concentration of 2X and IX of the recommended dose for Saflufenacil), and 0.2X for Pyraflufen-ethyl). Water treatment was included as a control.
  • Plants may further comprise additional edits as described in Table 7 below at positions 126, 397/420, 398, and 420 in the PPO2 gene (Butterrez chromosome 9) of sugar beet (Beta vulgaris L.).
  • co-cultivation buffer 5 mM 4- Morpholineethanesulfonic acid (Sigma-Aldrich, Saint Louis, MO, U.S.), 5 mM MgSO4 (Sigma-Aldrich, Saint Louis, MO, U.S.), pH 5.7, 100 pM acetosyringone (Sigma-Aldrich, Saint Louis, MO, U.S.)) to a final ODeoo of 0.2 to form the co-cultivation media.
  • co-cultivation buffer 5 mM 4- Morpholineethanesulfonic acid (Sigma-Aldrich, Saint Louis, MO, U.S.), 5 mM MgSO4 (Sigma-Aldrich, Saint Louis, MO, U.S.), pH 5.7, 100 pM acetosyringone (Sigma-Aldrich, Saint Louis, MO, U.S.)) to a final ODeoo of 0.2 to form the co-cultivation media.
  • the suspension is then spread unto a solid agar medium containing 40 mM of CaCh. After one hour at room temperature the solidified discs containing the protoplasts will be transferred to a protoplast regeneration media (PRGM). After several weeks, friable microcalluses will be transferred to shoot inducing media.
  • PRGM protoplast regeneration media
  • sgRNA sequences comprising SEQ ID NOs: 70, 74, 75, 76, 77 and 78 can be cloned into a plant Cpfl-sgRNA expression vector downstream a CaMV 35S promoter.
  • Two sgRNAs (SEQ ID NOs: 70 and 74) are used for substitutions of Arg (R) to Ala (A), Gly (G), Leu (L), He (I), or Met (M) at position 126.
  • Two sgRNAs (SEQ ID NOs: 75 and 76) are used for deletion of Gly (G) at position 208 or 209.
  • the vector may also contain a codon-optimized Cpfl, SEQ ID NO: 34, under regulatory control of a CaMV 35S promoter.
  • the donor sequence comprising of SEQ ID NOs: 51 (for deletion of G at 209), 52-56 and 58 (for substitutions of R to A, G, L, I and M at 126), and 57, 48-50 (for substitutions of F to V, M, I and L at 420) are individually cloned into a donor vector.
  • the Cpfl-sgRNA vector and one of the donor vectors are then transformed into sugar beet protoplasts isolated from each cultivar in the same way as described above using 10 pg of each plasmid. An aliquot of the transformed protoplasts is taken immediately before fixation in alginate and analyzed for efficiency using NGS. >1% of correct sequences is required for continued work. Once shoots have been formed, a tissue sample is taken and analyzed using Sanger sequencing for targeted mutations and for PCR-evaluation of transgene insertions. Only plants showing positive results for the targeted mutation and no transgene insert are transferred to the rooting step described above.
  • the cells are harvested using centrifugation, resuspended in infection buffer (5 mM 4-Morpholineethanesulfonic acid, 5 mM MgSO4, pH 5.7, 100 pM acetosyringone) for a final ODeoo of 0.2 and then infilitrated into the lower part of the hypocotyl in young sugar beet seedlings. Hairy roots forming from the infected sites are then collected and placed onto solid media containing ’ MS supplemented with 200 mg/1 claforan and 50 mg/1 kanamycin. After two weeks, surviving tissue is screened for presence of target mutation and transgene inserts using sanger sequencing and fragment length PCR. Once positive tissue has been determined, it is transferred to shoot inducing media followed by root inducing media, using 0.7872 mg/1 lactofen as selective agent for PPO herbicide resistance.
  • infection buffer 5 mM 4-Morpholineethanesulfonic acid, 5 mM MgSO4, pH 5.7, 100
  • Another way of achieving the targeted edits shown in Table 7 is through biolistic transformation of sugar beet calli.
  • particle bombardment can be performed using a particle bombardment system (e.g. a BioRad PDSIOOO/He at a target distance of 60 mm and at helium pressure 1100 psi) to introduce the plasmids into 1 month old calli.
  • a particle bombardment system e.g. a BioRad PDSIOOO/He at a target distance of 60 mm and at helium pressure 1100 psi
  • After 48 h aliquots can be taken to verify the efficiency of the method using PCR or NGS-based methods.
  • protoplasts are transferred to solid cultivation media that may or may not contain a PPO targeting herbicide. Regenerated plants are screened for the relevant edit using PCR or NGS- based approaches.
  • Table 8 below lists the expected genomic, cDNA, and protein sequences of lines generated carrying various mutations disclosed herein. Table 8: Summary of Sequence Information
  • the herbicide is selected from the group consisting of: acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, azafenidin, carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl,
  • Beta vulgaris plant or part thereof of any one of embodiments 1-22, further comprising an additional desired trait.
  • a DNA construct comprising the polynucleotide of any one of embodiments 27-29.
  • a method for producing a Beta vulgaris plant or plant cell having an engineered PPO2 protein comprising: introducing a nucleic acid mutation by targeted genome editing that results in an in-frame amino acid deletion corresponding to position 208 and/or 209 of SEQ ID NO: 3.
  • the CRISPR/Cas system comprises: a) a guide RNA sequence comprising SEQ ID NO: 79, or an expression construct encoding said guide RNA; b) a donor template sequence selected from SEQ ID NO: 60 and 90; and c) a Cas9 DNA nuclease, or an expression construct encoding said nuclease.
  • Beta vulgaris plant or plant cell further comprises a nucleic acid encoding a protoporphyrinogen oxidase 2 (PPO2) amino acid sequence having at least one of: a) a substitution of arginine at a position corresponding to 126 of SEQ ID NO: 3; b) a substitution of leucine at a position corresponding to 397 of SEQ ID NO: 3; c) a substitution of glycine at a position corresponding to 398 of SEQ ID NO: 3; and d) a substitution of phenylalanine at a position corresponding to 420 of SEQ ID NO: 3.
  • PPO2 protoporphyrinogen oxidase 2
  • nucleic acid encoding a protoporphyrinogen oxidase 2 (PPO2) amino acid sequence having at least one of a), b), c), and d) is on a different allele as the deletion corresponding to position number 208 and/or 209 of SEQ ID NO: 3.
  • PPO2 protoporphyrinogen oxidase 2
  • Beta vulgaris plant produced by the method of any one of embodiments 31-38, wherein said plant is resistant or tolerant to one or more herbicides.
  • Beta vulgaris plant of embodiment 39 wherein the plant is resistant to a PPO herbicide selected from the group consisting of: acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, azafenidin, carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadiargyl, oxadiazon, pyraflufen-ethyl, saflufenacil, trifludimoxazin, and S-3100.
  • a PPO herbicide selected from the group consisting of: acifluorfen, fomesafen, lactofen, fluoroglycofen-ethyl, oxyfluorfen, flumioxazin, azafenidin, carfentrazone-ethyl, sulfentrazone, fluthiacet-methyl, oxadia
  • a donor template sequence suitable for use in a CRISPR based genome editing system wherein said donor template sequence is selected from SEQ ID NOs: 48-66 and 90-96.
  • a DNA construct comprising the guide RNA of embodiment 43 and the donor template sequence of embodiment 44.
  • An engineered PPO2 protein comprising an in-frame amino acid deletion corresponding to position number 208 and/or 209 in SEQ ID NO: 3.
  • a method of producing a Beta vulgaris plant with increased tolerance to an herbicide that inhibits protoporphyrinogen oxidase comprising the steps of: a) transfecting a protoplast obtained from Beta vulgaris cells with a genome editing system to generate a transfected protoplast, wherein the genome editing system comprises: i) a Cas enzyme; ii) at least one guide RNA (gRNA), wherein the at least one gRNA targets a genomic region corresponding to between position 5457 and 5502 of SEQ ID NO: 1; and iii) at least one single-stranded donor DNA repair template designed to introduce a deletion of glycine at a position corresponding to 208 and/or 209 of SEQ ID NO: 3; b) exposing the transfected protoplast to a selective pressure of at least one herbicide that inhibits protoporphyrinogen oxidase; c) selecting a protoplast comprising a deletion of glycine at a position corresponding to 208 and/or 20
  • a method of detecting an in-frame deletion of glycine at a position corresponding to 208 and/or 209 of SEQ ID NO: 3 in a Beta vulgaris plant or part thereof comprising: obtaining a Beta vulgaris plant or part thereof; and analyzing the Beta vulgaris plant or part thereof using at least one of SEQ ID NOs: 112- 122 to detect an in-frame deletion of glycine at a position corresponding to 208 and/or 209 of SEQ ID NO: 3.

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Abstract

La présente divulgation concerne des plantes Beta vulgaris possédant une résistance aux herbicides PPO et des procédés de production desdites plantes par édition ciblée du génome. La divulgation concerne également des séquences génétiques à utiliser avec des technologies d'édition du génome ciblé et/ou de génotypage, et des protéines PPO résistantes aux herbicides produites à partir de plantes Beta vulgaris génétiquement modifiées et non transgéniques.
EP23771936.4A 2022-09-01 2023-09-01 Procédés et compositions pour la tolérance à l'herbicide ppo Pending EP4580392A1 (fr)

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GB9321183D0 (en) 1993-10-14 1993-12-01 Zeneca Ltd A method of plant transformation
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US7335816B2 (en) 2003-02-28 2008-02-26 Kws Saat Ag Glyphosate tolerant sugar beet
US7842856B2 (en) * 2005-08-25 2010-11-30 The Board Of Trustees Of The University Of Illinois Herbicide resistance gene, compositions and methods
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
EP2840140B2 (fr) 2012-12-12 2023-02-22 The Broad Institute, Inc. Procédé utilisant de composants CRISPR-cas pour la mutation de cellules procaryotiques
CN113355357B (zh) 2012-12-12 2024-12-03 布罗德研究所有限公司 对用于序列操纵的改进的系统、方法和酶组合物进行的工程化和优化
CA3026528A1 (fr) * 2017-12-15 2019-06-15 Monsanto Technology Llc Methodes et compositions de tolerance a l'herbicide ppo
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