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1. WO2017138986 - METHODS AND COMPOSITIONS FOR INCREASING EFFICIENCY OF TARGETED GENE MODIFICATION USING OLIGONUCLEOTIDE-MEDIATED GENE REPAIR

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CLAIMS

What is claimed is:

1. A method of causing a genetic change in a plant cell, said method comprising exposing said cell to a DNA cutter and a modified GRON.

2. A plant cell comprising a DNA cutter and a modified GRON.

3. A method of causing a genetic change in a plant cell, said method comprising exposing said cell to a DNA cutter and a GRON that comprises DNA and/or RNA.

4. A plant cell comprising a DNA cutter that comprises DNA and/or RNA and/or protein.

5. The method or cell of any of the preceding claims, wherein said DNA cutter is one or more selected from a CRISPR, a TALEN, a zinc finger, meganuclease, and a DNA-cutting antibiotic.

6. The method or cell of any of the preceding claims, wherein said DNA cutter is a CRISPR or a TALEN.

7. The method or cell of any of the preceding claims, wherein said DNA cutter is a CRISPR.

8. The method or cell of any of the preceding claims, wherein said DNA cutter is a TALEN.

9. The method or cell of any of the preceding claims, wherein said DNA cutter is one or more DNA-cutting antibiotics selected from the group consisting of bleomycin, zeocin, phleomycin, tallysomycin and pepleomycin.

10. The method or cell of any of the preceding claims, wherein said DNA cutter is zeocin.

11. The method or cell of any of the preceding claims, wherein said GRON is single stranded.

12. The method or cell of any of the preceding claims, wherein the GRON is a chemically protected oligonucleotide.

13. The method or cell of any of the preceding claims, wherein the GRON comprises a chemically protected oligonucleotide protected at the 5' end.

14. The method or cell of any of the preceding claims, wherein the GRON comprises a chemically protected oligonucleotide protected at the 3' end.

15. The method or cell of any of the preceding claims, wherein the GRON comprises a chemically protected oligonucleotide protected at the 5' and 3' ends.

16. The method or cell of any of the preceding claims, wherein the GRON comprises one or more selected from a Cy3 group, a 3PS group, and a 2'-O-methyl group.

17. The method or cell of any of the preceding claims, wherein the GRON has a Cy3 group.

18. The method or cell of any of the preceding claims, wherein the GRON comprises two or more Cy3 groups.

19. The method or cell of any of the preceding claims, wherein the GRON comprises a Cy3 group at the first (ultimate) base on the 5' end.

20. The method or cell of any of the preceding claims, wherein the GRON comprises an idC group at the first (ultimate) base on the 5' end.

21. The method or cell of any of the preceding claims, wherein the GRON comprises a Cy3 group at the first (ultimate) base on the 3' end.

22. The method or cell of any of the preceding claims, wherein the GRON comprises an idC group at the first (ultimate) base on the 3' end.

23. The method or cell of any of the preceding claims, wherein the GRON comprises a 3PS group.

24. The method or cell of any of the preceding claims, wherein the GRON comprises two or more 3PS groups

25. The method or cell of any of the preceding claims, wherein the GRON comprises a 3PS group at the first (ultimate) base on the 5' end.

26. The method or cell of any of the preceding claims, wherein the GRON comprises a 3PS group at the second (penultimate) base on the 5' end.

27. The method or cell of any of the preceding claims, wherein the GRON comprises a 3PS group at the third (antepenultimate) base on the 5' end.

28. The method or cell of any of the preceding claims, wherein the GRON comprises a 3PS group at the first (ultimate) base on the 3' end.

29. The method or cell of any of the preceding claims, wherein the GRON comprises a 3PS group at the second (penultimate) base on the 3' end.

30. The method or cell of any of the preceding claims, wherein the GRON comprises a 3PS group at the third (antepenultimate) base on the 3' end.

31. The method or cell of any of the preceding claims, wherein the GRON comprises a 3PS group at the first (ultimate) bases on both the 5' and 3' end.

32. The method or cell of any of the preceding embodiments, wherein the GRON comprises a 3PS group at the first two (ultimate and penultimate) bases on both the 5' and the the 3' end.

33. The method or cell of any of the preceding embodiments, wherein the GRON comprises a 3PS group at the first three (ultimate and penultimate, and antepenultimate) bases on both the 5' and the 3' end.

34. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group.

35. The method or cell of any of the preceding claims, wherein the GRON comprises two or more 2'-O-methyl groups.

36. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group at the first (ultimate) base on the 5' end.

37. The method or cell of any of the preceding claims, wherein the GRON has a 2'-O-methyl group at the first base on the 5' end and does not have any other 2'-O-methyl groups.

38. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first two or more bases at the 5' end.

39. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first three or more bases at the 5' end.

40. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first four or more bases at the 5' end.

41. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first five or more bases at the 5' end.

42. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first six or more bases at the 5' end.

43. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first seven or more bases at the 5' end.

44. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first eight or more bases at the 5' end.

45. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first nine or more bases at the 5' end.

46. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first ten or more bases at the 5' end.

47. The method or cell of any of the preceding claims, wherein the GRON comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA base at the 5' end.

48. The method or cell of any of the preceding claims, wherein said GRON has a wobble base pair relative to the target sequence for the genetic change.

49. The method or cell of any of the preceding claims, wherein said GRON is between 15 and 60 nucleotides in length.

50. The method or cell of any of the preceding claims, wherein said GRON is 41 nucleotides in length.

51. The method or cell of any of the preceding claims, wherein said GRON is between 50 and 110 nucleotides in length.

52. The method or cell of any of the preceding claims, wherein said GRON is 101 nucleotides in length.

53. The method or cell of any of the preceding claims, wherein said GRON is between 150 and 210 nucleotides in length.

54. The method or cell of any of the preceding claims, wherein said GRON is 201 nucleotides in length.

55. The method or cell of any of the preceding claims, wherein said GRON is between 70 and 210 nucleotides in length.

56. The method or cell of any of the preceding claims, wherein said GRON is longer than 70 nucleotides in length.

57. The method or cell of any of the preceding claims, wherein said GRON is longer than 165 nucleotides in length.

58. The method or cell of any of the preceding claims, wherein said GRON is longer than 175 nucleotides in length.

59. The method or cell of any of the preceding claims, wherein said GRON is longer than 185 nucleotides in length.

60. The method or cell of any of the preceding claims, wherein said GRON is longer than 195 nucleotides in length.

61. The method or cell of any of the preceding claims, wherein said GRON is longer than 200 nucleotides in length.

62. The method or cell of any of the preceding claims, wherein said GRON is longer than 210 nucleotides in length.

63. The method or cell of any of the preceding claims, wherein said GRON is longer than 220 nucleotides in length.

64. The method or cell of any of the preceding claims, wherein said GRON is longer than 230 nucleotides in length.

65. The method or cell of any of the preceding claims, wherein said GRON is longer than 240 nucleotides in length.

66. The method or cell of any of the preceding claims, wherein said GRON is longer than 250 nucleotides in length.

67. The method or cell of any of the preceding claims, wherein said GRON is longer than 260 nucleotides in length.

68. The method or cell of any of the preceding claims, wherein said GRON is longer than 270 nucleotides in length.

69. The method or cell of any of the preceding claims, wherein said GRON is longer than 280 nucleotides in length.

70. The method or cell of any of the preceding claims, wherein said GRON is longer than 290 nucleotides in length.

71. The method or cell of any of the preceding claims, wherein said GRON is longer than 300 nucleotides in length.

72. The method or cell of any of the preceding claims, wherein said GRON is longer than 400 nucleotides in length.

73. The method or cell of any of the preceding claims, wherein said GRON is longer than 500 nucleotides in length.

74. The method or cell of any of the preceding claims, wherein said GRON is longer than 600 nucleotides in length.

75. The method or cell of any of the preceding claims, wherein said GRON is longer than 700 nucleotides in length.

76. The method or cell of any of the preceding claims, wherein said GRON is longer than 800 nucleotides in length.

77. The method or cell of any of the preceding claims, wherein said GRON is longer than 900 nucleotides in length.

78. The method or cell of any of the preceding claims, wherein said GRON is longer than 1000 nucleotides in length.

79. The method or cell of any of the preceding claims wherein said plant is selected from the group consisting of canola, sunflower, corn, tobacco, sugar beet, cotton, maize, wheat, barley, rice, alfalfa, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, cassava, potato, carrot, lettuce, onion, soy bean, soya spp, sugar cane, pea, chickpea, field pea, fava bean, lentils, turnip, rutabaga, brussel sprouts, lupin, cauliflower, kale, field beans, poplar, pine, eucalyptus, grape, citrus, triticale. alfalfa, rye, oats, turf and forage grasses, flax, oilseed rape, mustard, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, tulip, iris, cassava, and lily.

80. The method or cell of any of the preceding claims wherein said plant is canola.

81. The method or cell of any of the preceding claims wherein said plant is corn

82. The method or cell of any of the preceding claims wherein said plant is maize.

83. The method or cell of any of the preceding claims wherein said plant is rice.

84. The method or cell of any of the preceding claims wherein said plant is sorghum.

85. The method or cell of any of the preceding claims wherein said plant is potato.

86. The method or cell of any of the preceding claims wherein said plant is soy bean.

87. The method or cell of any of the preceding claims wherein said plant is flax.

88. The method or cell of any of the preceding claims wherein said plant is oilseed rape.

89. The method or cell of any of the preceding claims wherein said plant is cassava.

90. The method or cell of any of the preceding claims wherein said plant is sunflower.

91. A method of causing a genetic change in a plant cell, said method comprising exposing said cell to a CRISPR and a modified GRON.

92. The method or cell of any of the preceding claims wherein multiple genetic changes are made.

93. The method or cell of any of the preceding claims wherein two or more guide RNAs are used.

94. The method or cell of any of the preceding claims wherein each of the more than one guide RNAs is complimentary to a different target for genetic change.

95. The method or cell of any of the preceding claims wherein the CRISPR includes a nickase.

96. The method or cell of any of the preceding claims wherein the DNA cutter includes two or more nickases.

97. The method or cell of any of the preceding claims wherein two or more nickases cuts on opposite strands of the target nucleic acid sequence.

98. The method or cell of any of the preceding claims wherein two or more nickases cuts on the same strand of the target nucleic acid sequence.

99. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group at the first (ultimate) base on the 3' end.

100. The method or cell of any of the preceding claims, wherein the GRON comprises a 2' -O-methyl group at the first (ultimate) base on the 3' end and does not have any other 2'-O-methyl groups.

101. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first two or more bases at the 3' end.

102. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first three or more bases at the 3' end.

103. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first four or more bases at the 3' end.

104. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first five or more bases at the 3' end.

105. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first six or more bases at the 3' end.

106. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first seven or more bases at the 3' end.

107. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first eight or more bases at the 3' end.

108. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first nine or more bases at the 3' end.

109. The method or cell of any of the preceding claims, wherein the GRON comprises a 2'-O-methyl group on each of the first ten or more bases at the 3' end.

110. The method or cell of any of the preceding claims, wherein the GRON comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA bases at the 3' end.

111. The method or cell of any of the preceding claims 1-98, wherein the GRON does not comprise a 2'-O-methyl group at the first (ultimate) base on the 3' end.

112. The method or cell of any of the preceding claims 1-98 and 111, wherein the GRON does not comprise a 2'-O-methyl group on any of the first two or more bases at the 3' end.

113. The method or cell of any of the preceding claims 1-98 and 111-112, wherein the GRON does not comprise a 2'-O-methyl group on any of the first three or more bases at the 3' end.

114. The method or cell of any of the preceding claims 1-98 and 111-113, wherein the GRON does not comprise a 2'-O-methyl group on any of the first four or more bases at the 3' end.

115. The method or cell of any of the preceding claims 1-98 and 111-114, wherein the GRON does not comprise a 2'-O-methyl group on any of the first five or more bases at the 3' end.

116. The method or cell of any of the preceding claims 1-98 and 111-115, wherein the GRON does not comprise a 2'-O-methyl group on any of the first six or more bases at the 3' end.

117. The method or cell of any of the preceding claims 1-98 and 111-116, wherein the GRON does not comprise a 2'-O-methyl group on any of the first seven or more bases at the 3' end.

118. The method or cell of any of the preceding claims 1-98 and 111-117, wherein the GRON does not comprise a 2'-O-methyl group on any of the first eight or more bases at the 3' end.

119. The method or cell of any of the preceding claims 1-98 and 111-118, wherein the GRON does not comprise a 2'-O-methyl group on any of the first nine or more bases at the 3' end.

120. The method or cell of any of the preceding claims 1-98 and 111-119, wherein the GRON does not comprise a 2'-O-methyl group on any of the first ten or more bases at the 3' end.

121. The method or cell of any of the preceding claims 1-98 and 111-120, wherein the GRON does not comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 RNA bases at the 3' end.

122. A non-transgenic herbicide resistant or tolerant plant made by the method or from the cell of one any of the preceding claims.

123. The method or cell or plant of any of the preceding claims, wherein said plant cell has a genetic change or mutation in Acetyl-Coenzyme A carboxylase (ACCase) and is selected from the group consisting of barley, maize, millet, oats, rye, rice, sorghum, sugarcane, turf grasses, and wheat.

124. The method or cell or plant of any of the preceding claims, wherein said plant cell has a genetic change or mutation in Acetyl-Coenzyme A carboxylase (ACCase) and is resistant or tolerant to one or more herbicides.

125. The method or cell or plant of any of the preceding claims, wherein said plant cell has a genetic change or mutation in Acetyl-Coenzyme A carboxylase (ACCase), is resistant to one or more ACCase-inhibiting herbicides.

126. The method or cell or plant of any of the preceding claims, wherein said plant cell has a genetic change or mutation in Acetyl-Coenzyme A carboxylase (ACCase), is resistant to one or more herbicides selected from the group consisting of alloxydim, butroxydim, clethodim, cloproxydim, cycloxydim, sethoxydim, tepraloxydim, tralkoxydim, chlorazifop, clodinafop, clofop, diclofop, fenoxaprop, fenoxaprop-P, fenthiaprop, fluazifop, fluazifop-P, haloxyfop, haloxyfop-P, isoxapyrifop, propaquizafop, quizalofop, quizalofop-P, trifop, pinoxaden, agronomically acceptable salts and esters of any of these herbicides, and combinations thereof.

127. The method or cell or plant of any of the preceding claims, wherein said plant cell has a genetic change or mutation in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein said plant cell is selected from the group consisting of corn, wheat, rice, barley, sorghum, oats, rye, sugarcane, soybean, cotton, sugarbeet, oilseed rape, canola, flax, cassava, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grape and turf grasses.

128. The method or cell or plant of any of the preceding claims, wherein said plant or plant cell has a genetic change or mutation in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein plant or plant cell is resistant to at least one herbicide.

129. The method or cell or plant of any of the preceding claims, wherein said plant or plant cell has a genetic change or mutation in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein plant or plant cell is resistant to a herbicide of the phosphonomethylglycine family.

130. The method or cell or plant of any of the preceding claims, wherein said plant or plant cell has a genetic change or mutation in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein plant or plant cell is resistant to glyphosate.

131. The method or cell or plant of any of the preceding claims, wherein said plant or plant cell has a genetic change or mutation in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), and wherein plant or plant cell is selected from the group consisting of corn, wheat, rice, barley, sorghum, oats, rye, sugarcane, soybean, cotton, sugarbeet, oilseed rape, canola, flax, cassava, sunflower, potato, tobacco, tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grape and turf grasses.

132. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell occurs at one allele of the gene.

133. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell occurs at two alleles of the gene.

134. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell occurs at three alleles of the gene.

135. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell occurs at four alleles of the gene.

136. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell occurs at one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve alleles of the gene.

137. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell comprises a deletion or insertion resulting in a knockout of one allele of the gene.

138. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell comprises a deletion or insertion resulting in a knockout of two alleles of the gene.

139. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell comprises a deletion or insertion resulting in a knockout of three alleles of the gene.

140. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell comprises a deletion or insertion resulting in a knockout of four alleles of the gene.

141. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell comprises a deletion or insertion resulting in a knockout of one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve alleles of the gene.

142. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell occurs at one allele of the gene and a second allele of the gene comprises a deletion or insertion resulting in a knockout of said second allele.

143. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell occurs at one allele of the gene and a second allele and third allele of the gene comprises a deletion or insertion resulting in a knockout of said second allele and said third allele.

144. The method or cell or plant of any of the preceding claims, wherein the genetic change or mutation in the cell occurs at one allele of the gene and a second allele, third allele, and fourth allele of the gene comprises a deletion or insertion resulting in a knockout of said second allele, said third allele and said fourth allele.

145. The method or cell or plant of any of the preceding claims, wherein the genetic change in the cell comprises at least one mutation at one allele and at least one knockout in another allele.

146. The method or cell or plant of any of the preceding claims, wherein the genetic change in the cell comprises at least one mutation at one allele and at least one knockout in at least one other allele.

147. The method or cell or plant of any of the preceding claims, wherein the genetic change in the cell comprises at least one mutation at one allele and at least one knockout in at least two other alleles.

148. The method or cell or plant of any of the preceding claims, wherein the genetic change in the cell comprises at least one mutation at one allele and at least one knockout in at least three other alleles.

149. The method or cell or plant of any of the preceding claims, wherein the genetic change in the cell comprises at least one mutation at one allele and a knockout in all other alleles.