Plant epsp synthases and methods of use

A polynucleotide construct that provides a guide RNA in a plant cell, wherein the guide RNA targets an endogenous EPSP synthase (EPSPS) gene of the plant cell, wherein the guide RNA as part of a CRISPR complex generates a modified endogenous EPSPS gene that encodes a plant EPSPS polypeptide that comprises G102A and at least one amino acid mutation selected from the group consisting of:

• a) A2R,
• b) A4W,
• c) H54M,
• d) A72Q,
• e) K84R,
• f) L98C,
• g) K173R,
• h) I208L,
• i) K243E,
• j) T279A,
• k) E302S,
• l) T361S,
• m) E391P,
• n) E391G,
• o) D402G,
• p) A416G,
• q) V438R,
• r) S440R,
• s) T441Q, and
• t) F442V, wherein each amino acid mutation position corresponds to the amino acid position set forth in SEQ ID NO:1 and wherein the endogenous plant EPSPS gene encodes a polypeptide comprising a sequence that is at least 90% identical to SEQ ID NO:2.

A method for producing a glyphosate tolerant plant, the method comprising:

• a) providing a guide RNA, at least one polynucleotide modification template, and at least one Cas endonuclease to a plant cell, wherein the at least one Cas endonuclease introduces a double strand break at an endogenous EPSP synthase (EPSPS) gene in the plant cell, and wherein said polynucleotide modification templates are used to generate a modified EPSPS gene that encodes a plant EPSPS polypeptide that comprises G102A and at least one amino acid mutation selected from the group consisting of
• i. A2R,
• ii. A4W,
• iii. H54M,
• iv. A72Q,
• v. K84R,
• vi. L98C,
• vii. K173R,
• viii. I208L,
• ix. K243E,
• x. T279A,
• xi. E302S,
• xii. T361S,
• xiii. E391P,
• xiv. E391G,
• xv. D402G,
• xvi. A416G,
• xvii. V438R,
• xviii. S440R,
• xix. T441Q, and
• xx. F442V, wherein each amino acid mutation position corresponds to the amino acid position set forth in SEQ ID NO:1 and wherein the endogenous plant EPSPS gene encodes a polypeptide comprising a sequence that is at least 90% identical to SEQ ID NO:2;
• b) obtaining a plant from the plant cell of (a);
• and
• d) generating a glyphosate tolerant progeny plant that is void of said guide RNA and Cas endonuclease from the plant of (b).

A glyphosate tolerant rice plant expressing a plant EPSPS polypeptide comprising an amino acid mutation that is analogous to G102A and at least one amino acid mutation that is analogous to the amino acid mutation selected from the group consisting of:

• a) A2R,
• b) A4W,
• c) H54M,
• d) A72Q,
• e) K84R,
• f) L98C,
• g) K173R,
• h) I208L,
• i) K243E,
• j) T279A,
• k) E302S,
• l) T361S,
• m) E391P,
• n) E391G,
• o) D402G,
• p) A416G,
• q) V438R,
• r) S440R,
• s) T441Q, and
• t) F442V,
• wherein each amino acid mutation position corresponds to the analogous amino acid position set forth in SEQ ID NO:1 and wherein the plant EPSPS polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:7.