Targeted chromosomal mutagenasis using zinc finger nucleases

• US 20050208489A1
• Filed: 01/22/2003
• Published: 09/22/2005
• Est. Priority Date: 01/23/2002
• Status: Active Grant

• Current Assignee: Dana Carroll, Gary N. Drews, Kent Gregory Golic, Maria Bibikova, Mary M. Golic
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Golic, Kent Gregory, Drews, Gary N., Golic, Mary M., Carroll, Dana, Bibikova, Maria
• Granted Patent: US 8,106,255 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 67/0339 Genetically modified insects, e.g. Drosophila melanogaster, medfly
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8257 for the production of primary gene products, e.g. pharmaceutical products, interferon
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention provides for a method or methods of targeted genetic recombination or mutagenesis in a host cell or organism, and compositions useful for carrying out the method. The targeting method of the present invention exploits endogenous cellular mechanisms for homologous recombination and repair of double stranded breaks in genetic material. The present invention provides numerous improvements over previous mutagenesis methods, such advantages include that the method is generally applicable to a wide variety of organisms, the method is targeted so that the disadvantages associated with random insertion of DNA into host genetic material are eliminated, and certain embodiments require relatively little manipulation of the host genetic material for success. Additionally, it provides a method that produces organisms with specific gene modifications in a short period of time.

Claims

1. A method of targeted genetic recombination in a host cell comprising:

introducing into the host cell a nucleic acid molecule encoding a Zinc Finger Nuclease (ZFN) targeted to a chosen host target locus;

inducing expression of the ZFN within the host cell; and

identifying a recombinant host cell in which the selected host DNA sequence exhibits a mutation at the host target locus.

2. The method of claim 1 wherein the mutation is selected from the group consisting of a deletion of genetic material, an insertion of genetic material, and both a deletion and an insertion of genetic material.

5. The method of claim 1 further comprising introducing donor DNA into the host cell.

6. The method of claim 5 wherein the donor DNA provides a gene sequence that encodes a product to be produced in the host cell.

7. The method of claim 6 wherein the donor DNA provides a gene sequence that encodes a product selected from the group consisting of pharmaceuticals, hormones, proteins, nutriceuticals and chemicals.

8. The method of claim 1 wherein the host cell is selected from the group consisting of a single celled organism, a cell from a multicellular organism, and an oocyte.

11. The method of claim 8 wherein the host cell is an insect cell.

12. The method of claim 11 wherein the insect is a member of an order selected from the group Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera, Lepidoptera, or Orthoptera.

13. The method of claim 12 wherein the insect is selected from the group consisting of a fruit fly, a mosquito, and a medfly.

15. The method of claim 8 wherein the multicellular organism is a plant.

16. The method of claim 15 wherein the plant is selected from the group consisting of a monocotyledon and a dicotyledon.

17. The method of claim 16 wherein the plant is selected from the group consisting of maize, rice, wheat, potato, soybean, tomato, tobacco, members of the Brassica family, and Arabidopsis.

20. The method of claim 15 wherein the plant is a tree.

21. The method of claim 8 wherein the multicellular organism is a mammal.

22. The method of claim 21 wherein the mammal is selected from the group consisting of mouse, rat, pig, sheep, cow, dog and cat.

23. The method of claim 8 wherein the multicellular organism is a bird.

24. The method of claim 23 wherein the bird is selected from the group consisting of chicken, turkey, duck and goose.

25. The method of claim 8 wherein the multicellular organism is a fish.

26. The method of claim 25 wherein the fish is selected from the group consisting of a zebrafish, trout and salmon.

27. The method of claim 8 wherein the mutation occurs in a cell selected from the group consisting of a germ line cell of a single celled organism, a germ line cell of a multicellular organism, a somatic cell of a single celled organism and a somatic cell of a multicellular organism.

90. The method of claim 8 wherein the multicellular organism is a fungus.

3. (canceled)

4. (canceled)

9. (canceled)

10. (canceled)

14. (canceled)

18. (canceled)

19. (canceled)

28. (canceled)

29. A method of targeted genetic recombination in a host cell comprising:

selecting a zinc finger DNA binding domain that preferentially binds to a specific host target locus;

selecting a DNA cleavage domain that cleaves double-stranded DNA when operatively linked to said binding domain and introduced into the host cell;

selecting a control element that induces expression in the host cell;

operatively linking the DNA encoding the binding domain and the cleavage domain and said control element to produce a DNA construct;

introducing said DNA construct into a target host cell; and

identifying at least one host cell exhibiting recombination at the target locus in the host DNA.

30. The method of claim 29 further comprising introducing donor DNA into the host cell.

31. The method of claim 30 wherein the donor DNA provides a gene sequence that encodes a product to be produced in the host cell.

32. The method of claim 31 wherein the donor DNA provides a gene sequence that encodes a product selected from the group consisting of pharmaceuticals, hormones, proteins, nutriceuticals and chemicals.

33. The method of claim 29 wherein the DNA binding domain is comprised of three zinc fingers.

34. The method of claim 29 wherein the zinc fingers are selected from the group consisting of Cys₂His₂ zinc fingers.

35. The method of claim 29 wherein the cleavage domain is selected from the group consisting of Type II restriction endonucleases.

36. The method of claim 29 wherein the Type II restriction endonuclease is FokI.

37. The method of claim 29 wherein the control element is selected from the group consisting of heat-shock inducible control elements.

39. The method of claim 29 wherein the host cell is a single celled organism, a cell from a multicellular organism, a gamete cell, or an oocyte.

41. The method of claim 30 wherein the target cell is a gamete cell of a host organism.

42. The method of claim 29 wherein the host cell is an insect cell.

45. The method of claim 42 wherein the insect is a member of an order selected from the group consisting of Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera, Lepidoptera, and Orthoptera.

46. The method of claim 45 wherein the insect is selected from the group consisting of a fruit fly, a mosquito, and a medfly.

48. The method of claim 39 wherein the multicellular organism is a plant.

49. The method of claim 48 wherein the plant is selected from the group consisting of a monocotyledon and a dicotyledon.

50. The method of claim 49 wherein the plant is selected from the group consisting of maize, rice, wheat, potato, soybean, tomato, tobacco, members of the Brassica family, and Arabidopsis.

53. The method of claim 48 wherein the plant is a tree.

54. The method of claim 39 wherein the multicellular organism is a mammal.

55. The method of claim 54 wherein the mammal is selected from the group consisting of mouse, rat, pig, sheep, cow, dog and cat.

56. The method of claim 39 wherein the multicellular organism is a bird.

57. The method of claim 56 wherein the bird is selected from the group consisting of chicken, turkey, duck and goose.

58. The method of claim 39 wherein the multicellular organism is a fish.

59. The method of claim 58 wherein the fish is selected from the group consisting of a zebrafish, trout and salmon.

60. The method of claim 39 wherein the multicellular organism is a fungus.

61. The method of claim 39 wherein the recombination occurs in a cell selected from the group consisting of a germ line cell of a single celled organism, a germ line cell of a multicellular organism, a somatic cell of a single celled organism and a somatic cell of a multicellular organism.

62. The method of claim 30 wherein the DNA binding domain is comprised of three zinc fingers.

63. The method of claim 30 wherein the cleavage domain is selected from the group consisting of Type II restriction endonucleases.

64. The method of claim 30 wherein the control element is selected from the group consisting of heat-shock inducible control elements.

66. The method of claim 30 wherein the host cell is selected from the group consisting of a single celled organism, a cell from a multicellular organism, and an oocyte.

68. The method of claim 66 wherein the host cell is an insect cell.

69. The method of claim 68 wherein the insect is a member of an order selected from the group consisting of Coleoptera, Diptera, Hemiptera, Homoptera, Hymenoptera, Lepidoptera, and Orthoptera.

70. The method of claim 69 wherein the insect is selected from the group consisting of a fruit fly, a mosquito, and a medfly.

72. The method of claim 66 wherein the multicellular organism is a plant.

73. The method of claim 72 wherein the plant is selected from the group consisting of a monocotyledon and a dicotyledon.

77. The method of claim 72 wherein the plant is a tree.

78. The method of claim 66 wherein the multicellular organism is a mammal.

79. The method of claim 78 wherein the mammal is selected from the group consisting of mouse, rat, pig, sheep, cow, dog and cat.

80. The method of claim 66 wherein the multicellular organism is a bird.

81. The method of claim 80 wherein the bird is selected from the group consisting of chicken, turkey, duck and goose.

82. The method of claim 66 wherein the multicellular organism is a fish.

83. The method of claim 82 wherein the fish is selected from the group consisting of a zebrafish, trout and salmon.

84. The method of claim 66 wherein the multicellular organism is a fungus.

85. The method of claim 66 wherein the recombination occurs in a cell selected from the group consisting of a germ line cell of a single celled organism, a germ line cell of a multicellular organism, a somatic cell of a single celled organism and a somatic cell of a multicellular organism.

86. The method of claim 30 wherein the DNA construct further comprises DNA encoding one or more selectable markers.

87. The method of claim 86 wherein the selectable marker provides positive selection for cells expressing the marker selected from the group consisting of positive selection, negative selection and both positive and negative selection.

38. (canceled)

40. (canceled)

43. (canceled)

44. (canceled)

47. (canceled)

51. (canceled)

52. (canceled)

65. (canceled)

67. (canceled)

71. (canceled)

74. (canceled)

75. (canceled)

76. The method of claim 75 wherein the plant is selected from the group consisting of maize, rice, wheat, potato, soybean, tomato, tobacco, members of the Brassica family, and Arabidopsis.

88. (canceled)

89. (canceled)

91. A method of targeted genetic recombination in a plant cell comprising:

introducing into the plant cell a nucleic acid molecule encoding a Zinc Finger Nuclease (ZFN) targeted to a chosen host target locus;

inducing expression of the ZFN within the plant cell; and

identifying a recombinant plant cell in which the selected plant DNA sequence exhibits a mutation at the plant target locus.

92. The method of claim 91 wherein the mutation is selected from the group consisting of a deletion of genetic material, an insertion of genetic material, and both a deletion and an insertion of genetic material.

93. The method of claim 91 further comprising introducing donor DNA into the plant cell.

94. The method of claim 93 wherein the donor DNA provides a gene sequence that encodes a product to be produced in the plant cell.

95. The method of claim 94 wherein the donor DNA provides a gene sequence that encodes a product selected from the group consisting of pharmaceuticals, hormones, proteins, nutriceuticals or chemicals.

96. The method of claim 91 wherein the plant is selected from the group consisting of a monocotyledon and a dicotyledon.

97. The method of claim 96 wherein the plant is selected from the group consisting of maize, rice, wheat, potato, soybean, tomato, tobacco, members of the Brassica family, and Arabidopsis.

98. The method of claim 91 wherein the plant is a tree.

99. A method of targeted genetic recombination in a plant cell comprising:

selecting a zinc finger DNA binding domain that preferentially binds to a specific plant target locus;

selecting a DNA cleavage domain that cleaves double-stranded DNA when operatively linked to said binding domain and introduced into the plant cell;

selecting a control element that induces expression in the plant cell;

operatively linking the DNA encoding the binding domain and the cleavage domain and the control element to produce a DNA construct;

introducing said DNA construct into a target plant cell; and

identifying at least one plant cell exhibiting recombination at the target locus in the plant DNA.

100. The method of claim 99 further comprising introducing donor DNA into the plant cell.

101. The method of claim 100 wherein the donor DNA provides a gene sequence that encodes a product to be produced in the plant cell.

102. The method of claim 101 wherein the donor DNA provides a gene sequence that encodes a product selected from the group consisting of pharmaceuticals, hormones, proteins, nutriceuticals and chemicals.

103. The method of claim 99 wherein the DNA binding domain is comprised of three zinc fingers.

104. The method of claim 103 wherein the zinc fingers are selected from the group consisting of Cys₂His₂ zinc fingers.

105. The method of claim 99 wherein the cleavage domain is selected from the group consisting of Type II restriction endonucleases.

106. The method of claim 105 wherein the Type II restriction endonuclease is FokI.

107. The method of claim 99 wherein the control element is selected from the group consisting of heat-shock inducible control elements.

108. The method of claim 99 wherein the plant is selected from the group consisting of a monocotyledon and a dicotyledon.

109. The method of claim 108 wherein the plant is selected from the group consisting of maize, rice, wheat, potato, soybean, tomato, tobacco, members of the Brassica family, and Arabidopsis.

110. The method of claim 99 wherein the plant is a tree.

111. The method of claim 99 wherein the DNA binding domain is comprised of three zinc fingers.

112. The method of claim 99 wherein the DNA construct further comprises DNA encoding one or more selectable markers.

113. The method of claim 112 wherein the selectable marker provides positive selection for cells expressing the marker selected from the group consisting of positive selection, negative selection and both positive and negative selection.