Crispr enzyme mutations reducing off-target effects
US-2019010471-A1 · Jan 10, 2019 · US
US12173301B2 · US · B2
| Field | Value |
|---|---|
| Publication number | US-12173301-B2 |
| Application number | US-202017771075-A |
| Country | US |
| Kind code | B2 |
| Filing date | Oct 26, 2020 |
| Priority date | Nov 1, 2019 |
| Publication date | Dec 24, 2024 |
| Grant date | Dec 24, 2024 |
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The present disclosure relates to, inter alia, a weed control method. In one example, a method comprises applying to the locus a weed controlling amount of a pesticide composition comprising a FatA acyl-ACP thioesterase-inhibiting herbicide, wherein the crop plants are modified such that they comprise a FatA acyl-ACP thioesterase which provides the crop plant with tolerance against the FatA acyl-ACP thioesterase-inhibiting herbicide. Recombinant polynucleotides and suitable for use in the methods, and edited FatA acyl-ACP thioesterases are also disclosed.
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The invention claimed is: 1. A method of selectively controlling weeds at a locus comprising modified crop plants and weeds, the method comprising: applying to the locus a weed-controlling amount of a pesticide composition comprising a FatA acyl-ACP thioesterase-inhibiting herbicide, wherein the modified crop plants comprise a recombinant polynucleotide encoding a modified FatA acyl-ACP thioesterase that provides the modified crop plant with tolerance against the FatA acyl-ACP thioesterase-inhibiting herbicide, wherein the FatA acyl-ACP thioesterase has at least 70% sequence identity to any one of SEQ ID NOS: 1-7 and comprises at least one amino acid substitution corresponding to R176A of SEQ ID NO: 1. 2. The method according to claim 1 , wherein the modified FatA acyl-ACP thioesterase is derived from a species selected from the group consisting of an Arabidopsis species including Arabidopsis thaliana , a Tritium species including Triticum aestivum (Wheat), a Hordeum species including Hordeum vulgare (Barley), an Oryza species including Oryza sativa (Rice), a Zea species including Zea mays (Maize), and a Glycine species including Glycine max (Soybean). 3. The method according to claim 1 , wherein the modified FatA acyl-ACP thioesterase comprises: (a) an amino acid sequence of SEQ ID NOs: 16, 17, 18 or 19; or (b) an amino acid having at least-90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NOs: 16, 17, 18 or 19, wherein expression of the modified FatA acyl-ACP thioesterase in the modified crop plant provides the modified crop plant with tolerance against the FatA acyl-ACP thioesterase-inhibiting herbicide. 4. The method of claim 1 , wherein the modified FatA acyl-ACP thioesterase further comprise the amino acid substitution R191C, wherein the position of the amino acid substitution is relative to the position of the amino acid sequence provided as SEQ ID NO: 1. 5. The method of claim 1 , wherein the modified FatA acyl-ACP thioesterase is provided by editing an endogenous gene to thereby provide the modified crop plant with tolerance against the FatA acyl-ACP thioesterase-inhibiting herbicide. 6. The method of claim 1 , wherein the FatA acyl-ACP thioesterase-inhibiting herbicide is selected from the group consisting of: (a) a compound having Formula (I) wherein R1 and R2 are each independently H, C1-C6 alkyl, C1-C6 haloalkyl, halogen or cyano, preferably Me, Cl, F or H; (b) cinmethylin; and (c) oxaziclomefone. 7. The method of claim 1 wherein the modified crop plant comprises a recombinant polynucleotide encoding a further herbicide tolerance enzyme, wherein the further herbicide tolerance enzyme is selected from the group consisting of, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), Cytochrome P450, phosphinothricin acetyltransferase (PAT), Acetolactate synthase (ALS), Protoporphyrinogen oxidase (PPO), Hydroxyphenyl pyruvate dioxygenase (HPPD) and dicamba degrading enzymes, and wherein the pesticide composition comprises one or more additional herbicides selected from the group consisting of: (i) glyphosate, including agrochemically acceptable salts thereof; (ii) glufosinate, including agrochemically acceptable salts thereof; (iii) chloroacetanilides including alachlor, acetochlor, metolachlor, S-metholachlor; (iv) photo system II inhibitors including: (iv-i) triazines including ametryn, atrazine, cyanazineterbuthylazine; (iv-ii) triazinones including hexazinone and metribuzin; and (iv-iii) ureas including chlorotoluron, diuron, isoproturon, linuron and terbuthiuron; (v) ALS-inhibitors including: (v-i) sulfonyl ureas including amidosulfuron, chlorsulfuron, flupyrsulfuron, halosulfuron, nicosulfuron, primisulfuron, prosulfuron, rimsulfuron, triasulfuron, trifloxysulfuron and tritosulfuron; (v-ii) diphenyl ethers including aciflurofen and fomesafen; (vi) HPPD-inhibiting herbicides including mesotrione and bicyclopyrone; (vii) dicamba; and (viii) 2,4D. 8. A recombinant polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a modified FatA acyl-ACP thioesterase polypeptide operably linked to a plant operable promoter, wherein the modified FatA acyl-ACP thioesterase polypeptide has at least 70% sequence identity to any one of SEQ ID NOS: 1-7 and at least one amino acid substitution corresponding to R1764 of SEQ ID NO: 1, wherein expression of said polypeptide in a crop plant provide the crop plant with tolerance against a FatA acyl-ACP thioesterase-inhibiting herbicide. 9. The recombinant polynucleotide of claim 8 , wherein the modified FatA acyl-ACP thioesterase polypeptide comprises the pair of amino acid substitutions R176A and R191C, wherein the position of the pair of amino acid substitutions is relative to the position of the amino acid sequence provided as SEQ ID NO: 1. 10. The recombinant polynucleotide of claim 8 , further comprising: at least one additional nucleotide sequence encoding a herbicide tolerance enzyme selected from the group consisting of hydroxyphenyl pyruvate dioxygenase (HPPD), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), Glyphosate acetyl transferase (GAT), Cytochrome P450, phosphinothricin acetyltransferase (PAT), Acetolactate synthase (ALS), Protoporphyrinogen oxidase (PPO), hydroxyphenyl pyruvate dioxygenase (HPPD) and dicamba degrading enzymes, operably linked to a second plant operable promoter. 11. A plant cell which is tolerant to a FatA acyl-ACP thioesterase-inhibiting herbicide, said plant cell comprising a recombinant polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising a modified FatA acyl-ACP thioesterase having at least 70% sequence identity to any one of SEQ ID NOS: 1-7 and comprising at least one amino acid substitution corresponding to R176A of SEQ ID NO: 1 , operably linked to a plant operable promotor, wherein expression of the polypeptide in the plant cell provides tolerance to the FatA acyl-ACP thioesterase-inhibiting herbicide. 12. A modified FatA acyl-ACP thioesterase polypeptide comprising: an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOS: 1-7 and comprising at least a first amino acid substitution corresponding to R176A, wherein the position of the amino acid substitution is relative to the position of the amino acid sequence provided as SEQ ID NO: 1, and wherein expression of the modified FatA acyl-ACP thioesterase in a plant provides the plant with tolerance to a FatA acyl-ACP thioesterase-inhibiting herbicide. 13. A recombinant polynucleotide comprising: a nucleic acid that encodes a DNA modification enzyme; and a nucleic acid that encodes at least one guide RNA (gRNA), wherein the at least one guide RNA mediates, via the DNA modification enzyme, at least one modification to a polynucleotide encoding a FatA acyl-ACP thioesterase polypeptide having at least 70% sequence identity to any one of SEQ ID NOS: 1-7, wherein the at least one modification in said polynucleotide results in amino acid substitution in said polypeptide comprising R176A, wherein the position of the amino acid substitution is relative to the position of the amino acid sequence provided as SEQ ID NO: 1. 14. A method for producing plants having tolerance to a FatA acyl-ACP thioesterase-inhibiting herbicide, the method comprising: a) transforming at least one recombinant polynucleotide into a plant cell, wherein the at least one recombinant polynucle
Brassica juncea · CPC title
Glycine max [soybean] · CPC title
Panicum [switchgrass] · CPC title
Oryza sp. [rice] · CPC title
Hordeum vulgarus [barley] · CPC title
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