COMPOSITION FOR CLEAVING A TARGET DNA COMPRISING A GUIDE RNA SPECIFIC FOR THE TARGET DNA AND CAS PROTEIN-ENCODING NUCLEIC ACID OR CAS PROTEIN, AND USE THEREOF

• US 20150322457A1
• Filed: 04/13/2015
• Published: 11/12/2015
• Est. Priority Date: 10/23/2012
• Status: Active Grant

• Current Assignee: Toolgen Incorporated
• Original Assignee: Toolgen Incorporated
• Inventors: Kim, Jin-Soo, Cho, Seung Woo, Kim, Sojung
• Granted Patent: US 10,851,380 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/8216 Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/10 Type of nucleic acid
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/21 Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)

Abstract

The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.

Claims

1-57. -57. (canceled)

58. A composition for cleaving a target nucleic acid sequence in a eukaryotic cell, the composition comprising a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system in a eukaryotic cell, wherein the Type II CRISPR/Cas system comprises:

a) a nucleic acid encoding a Cas9 polypeptide, and

b) a nucleic acid encoding a guide RNA that hybridizes with the target nucleic acid sequence,

wherein the Cas9 polypeptide and the guide RNA form a Cas9/RNA complex in the eukaryotic cell and wherein the guide RNA has sufficient sequence complementarity to the target nucleic acid sequence in the eukaryotic cell to allow the Cas9 polypeptide to mediate double stranded cleavage at the target nucleic acid sequence.

59. The composition of claim 58, wherein the Cas9 polypeptide further comprises a nuclear localization signal.

60. The composition of claim 58, wherein the nuclear localization signal is located at the C terminus of the Cas9 polypeptide.

61. The composition of claim 58, wherein the eukaryotic cell is a mammalian cell.

62. The composition of claim 58, wherein the mammalian cell is a human cell.

63. The composition of claim 58, wherein the nucleic acid encoding the Cas9 polypeptide is codon-optimized for expression in eukaryotic cells.

64. The composition of claim 58, wherein the target nucleic acid sequence is a genomic sequence located at its endogenous site in the genome of the eukaryotic cell.

65. The composition of claim 58, wherein the nucleic acid encoding the Cas9 polypeptide is a vector.

66. The composition of claim 65, wherein the nucleic acid encoding the guide RNA is a vector.

67. The composition of claim 58, wherein the nucleic acid encoding the guide RNA is in vitro transcribed RNA.

68. The composition of claim 58, wherein the guide RNA is a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans activating crRNA (tracrRNA) portion.

69. The composition of claim 58, wherein the guide RNA comprises a CRISPR RNA (crRNA) and a trans activating crRNA (tracrRNA), wherein the crRNA comprises:

a) two nucleic acid residues at the 5′

end (“

two 5′

nucleic acid residues”

),

b) a first nucleic acid sequence that is complementary to the target nucleic acid sequence, wherein the first nucleic acid sequence is downstream of the two 5′

nucleic acid residues and there are no additional nucleic acids between the two 5′

nucleic acid residues and the first nucleic acid sequence, and

c) a second nucleic acid sequence that hybridizes to the tracrRNA to form a double stranded RNA complex, wherein the second nucleic acid sequence is downstream of the first nucleic acid sequence.

70. The composition of claim 69, wherein the two 5′

nucleotide residues are both guanine residues.

71. The composition of claim 58, wherein the Cas9 polypeptide is a Streptococcus pyogenes Cas9 polypeptide.

72. A method of introducing a site-specific, double-stranded break at a target nucleic acid sequence in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, wherein the CRISPR/Cas system comprises:

a) a nucleic acid encoding a Cas9 polypeptide, and

b) a nucleic acid encoding a guide RNA that hybridizes to the target nucleic acid,

wherein the Cas9 polypeptide and the guide RNA form a Cas9/RNA complex in the eukaryotic cell and wherein the guide RNA has sufficient sequence complementarity to the target sequence nucleic acid in the eukaryotic cell to allow the Cas9 polypeptide to mediate double stranded cleavage at the target nucleic acid sequence.

73. The method of claim 72, wherein the Cas9 polypeptide further comprises a nuclear localization signal.

74. The method of claim 73, wherein the nuclear localization signal is located at the C terminus of the Cas9 polypeptide.

75. The method of claim 72, wherein the eukaryotic cell is a mammalian cell.

76. The method of claim 75, wherein the mammalian cell is a human cell.

77. The method of claim 72, wherein the nucleic acid encoding the Cas9 polypeptide is codon-optimized for expression in eukaryotic cells.

78. The method of claim 72, wherein the target nucleic acid sequence is a genomic sequence located at its endogenous site in the genome of the eukaryotic cell.

79. The method of claim 72, wherein the nucleic acid encoding the Cas9 polypeptide is a vector.

80. The method of claim 72, wherein the nucleic acid encoding the guide RNA is a vector.

81. The method of claim 72, wherein the nucleic acid encoding the guide RNA is in vitro transcribed RNA.

82. The method of claim 72, wherein the guide RNA is a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans activating crRNA (tracrRNA) portion.

83. The method of claim 72, wherein the guide RNA comprises a CRISPR RNA (crRNA) and a trans activating crRNA (tracrRNA), wherein the crRNA comprises:

a) two nucleic acid residues at the 5′

end (“

two 5′

nucleic acid residues”

),

b) a first nucleic acid sequence that is complementary to the target nucleic acid sequence, wherein the first nucleic acid sequence is downstream of the two 5′

nucleic acid residues and there are no additional nucleic acids between the two 5′

nucleic acid residues and the first nucleic acid sequence, and

c) a second nucleic acid sequence that hybridizes to the tracrRNA to form a double stranded RNA complex, wherein the second nucleic acid sequence is downstream of the first nucleic acid sequence.

84. The method of claim 83, wherein the two 5′

nucleotide residues are both guanine residues.

85. The method of claim 72, wherein the Cas9 polypeptide is a Streptococcus pyogenes Cas9 polypeptide.

86. The method of claim 72, wherein the nucleic acid encoding the Cas9 protein is introduced into the eukaryotic cell before introducing the nucleic acid encoding the guide RNA into the eukaryotic cell.

87. A Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system for site-specific, double stranded cleavage of a target nucleic acid sequence in a eukaryotic cell, the CRISPR/Cas system comprising:

a) a Cas9 polypeptide, and

b) a guide RNA that hybridizes with the target nucleic acid sequence,

wherein the Cas9 polypeptide and the guide RNA form a Cas9/RNA complex in the eukaryotic cell and wherein the guide RNA has sufficient sequence complementarity to the target nucleic acid sequence in the eukaryotic cell to allow the Cas9 polypeptide to mediate double stranded cleavage at the target nucleic acid sequence.

88. The CRISPR/Cas system of claim 87, wherein the Cas9 polypeptide further comprises a nuclear localization signal.

89. The CRISPR/Cas system of claim 88, wherein the nuclear localization signal is located at the C terminus of the Cas9 polypeptide.

90. The CRISPR/Cas system of claim 87, wherein the eukaryotic cell is a mammalian cell.

91. The CRISPR/Cas system of claim 90, wherein the mammalian cell is a human cell.

92. The CRISPR/Cas system of claim 87, wherein the nucleic acid encoding the Cas9 polypeptide is codon-optimized for expression in eukaryotic cells.

93. The CRISPR/Cas system of claim 87, wherein the target nucleic acid sequence is a genomic sequence located at its endogenous site in the genome of the eukaryotic cell.

94. The CRISPR/Cas system of claim 87, wherein the guide RNA is a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans activating crRNA (tracrRNA) portion.

95. The CRISPR/Cas system of claim 87, wherein the guide RNA comprises a CRISPR RNA (crRNA) and a trans activating crRNA (tracrRNA), wherein the crRNA comprises:

a) two nucleic residues at the 5′

end (“

two 5′

nucleic acid residues”

),

b) a first nucleic acid sequence that is complementary to the target nucleic acid sequence, wherein the first nucleic acid sequence is downstream of the two 5′

nucleic acid residues and there are no additional nucleic acids between the two 5′

nucleic acid residues and the first nucleic acid sequence, and

c) a second nucleic acid sequence that hybridizes to the tracrRNA to form a double stranded RNA complex, wherein the second nucleic acid sequence is downstream of the first nucleic acid sequence.

96. The CRISPR/Cas system of claim 95, wherein the two 5′

nucleic acid residues are both guanine residues.

97. The CRISPR/Cas system of claim 87, wherein the Cas9 polypeptide is a Streptococcus pyogenes Cas9 polypeptide.

98. An engineered, programmable, non-naturally occurring Type II CRISPR-Cas system, comprising:

a Cas9 protein, and

a guide RNA that targets and hybridizes to a target sequence of a DNA molecule in a eukaryotic cell,

wherein the DNA molecule encodes and the eukaryotic cell expresses at least one gene product and the Cas9 protein cleaves the DNA molecule, whereby expression of the at least one gene product is altered, and wherein the Cas9 protein and the guide RNA do not naturally occur together.

99. The CRISPR-Cas system of claim 98, wherein the CRISPR-Cas system further comprises a nuclear localization signal.

100. The CRISPR-Cas system of claim 98, wherein the guide RNA comprises a guide sequence fused to a tracr sequence.

101. The CRISPR-Cas system of claim 98, wherein the Cas9 protein is codon optimized for expression in the eukaryotic cell.

102. The CRISPR-Cas system of claim 98, wherein the eukaryotic cell is a mammalian or human cell.

103. The CRISPR-Cas system of claim 98, wherein the guide RNA comprises a tracr sequence.