PROKARYOTIC RNAi-LIKE SYSTEM AND METHODS OF USE

• US 20110189776A1
• Filed: 07/24/2009
• Published: 08/04/2011
• Est. Priority Date: 07/25/2008
• Status: Active Grant

• Current Assignee: The University of Georgia Research Foundation, Inc.
• Original Assignee: The University of Georgia Research Foundation, Inc.
• Inventors: Hale, Caryn, Terns, Michael, Terns, Rebecca
• Granted Patent: US 8,546,553 B2
• Time in Patent Office: Days
• US Class Current: 435/471
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/3517 Marker; Tag
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid

Abstract

Provided herein are methods for inactivating a target polynucleotide. The methods use a psiRNA having a 5′ region and a 3′ region. The 5′ region includes, but is not limited to, 5 to 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer. The 3′ region is substantially complementary to a portion of the target polynucleotide. The methods may be practiced in a prokaryotic microbe or in vitro. Also provided are polypeptides that have endonuclease activity in the presence of a psiRNA and a target polynucleotide, and methods for using the polypeptides.

Claims

1-56. -56. (canceled)

57. An isolated polynucleotide comprising a 5′

region and a 3′

region, wherein the 5′

region comprises a psiRNA-tag of between 5 and 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer present in a microbe comprising a CRISPR locus, and wherein the 3′

region comprises at least 18 nucleotide.

58. A vector encoding the isolated polynucleotide of claim 57.

59. The isolated polynucleotide of claim 57 wherein the isolated polynucleotide is RNA.

60. The isolated polynucleotide of claim 57 wherein the psiRNA-tag has at least 80% sequence similarity with 5′

-ATTGAAAS, wherein S is G or C.

61. The isolated polynucleotide of claim 57 wherein the 3′

region comprises at least 31 nucleotides or at least 37 nucleotides.

62. A method for inactivating a target polynucleotide in a microbe comprising:

introducing into the microbe a psiRNA comprising a 5′

region and a 3′

region, wherein the 5′

region is a psiRNA-tag of between 5 and 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer, wherein the 3′

region comprises between 18 and 75 nucleotides, and wherein the 3′

region is substantially complementary to a portion of the target polynucleotide, wherein the target polynucleotide is inactivated.

63. The method of claim 62 wherein the psiRNA is introduced into the cell as an RNA polynucleotide.

64. The method of claim 62 wherein the psiRNA is introduced into the cell as a DNA polynucleotide encoding the psiRNA.

65. The method of claim 62 wherein the psiRNA-tag has at least 80% sequence similarity with 5′

-AUUGAAAS, wherein S is G or C.

66. The method of claim 62 wherein the target polynucleotide is an endogenous polynucleotide.

67. The method of claim 62 wherein the target polynucleotide is an exogenous polynucleotide.

68. The method of claim 67 wherein the exogenous polynucleotide is a bacteriophage polynucleotide.

69. The method of claim 62 wherein the psiRNA-tag nucleotide sequence is chosen from a CRISPR locus present in the microbe.

70. A method for cleaving a target polynucleotide comprising:

incubating under suitable conditions a composition comprising;

a target polynucleotide;

a psiRNA comprising a 5′

region and a 3′

region, wherein the 5′

region is a psiRNA-tag of between 5 and 10 nucleotides chosen from a repeat from a CRISPR locus immediately upstream of a spacer, wherein the 3′

region comprises 5′

-NNNNNNNNNNNNNNNNN↓

NNNNNNNNNNNNNN (SEQ ID NO;

24) or 5′

-NNNNNNNNNNNNNNNNNNNNNNN↓

NNNNNNNNNNNNNNNNNNNN(SEQ ID NO;

25), wherein the 3′

region is substantially complementary to a portion of the target polynucleotide; and

a Cmr1 polypeptide, a Cmr2 polypeptide, a Cmr3 polypeptide, a Cmr4 polypeptide, and a Cmr6 polypeptide;

wherein the target polynucleotide is cleaved in the region that is substantially complementary to the region of SEQ ID NO;

24 or SEQ ID NO;

25.

71. The method of claim 70 wherein the psiRNA-tag has at least 80% sequence similarity with 5′

-AUUGAAAS, wherein S is G or C.

72. The method of claim 70 wherein the incubating is at a temperature greater than 30°

C.

73. The method of claim 70 wherein the amino acid sequence of Cmr1 and the amino acid sequence of SEQ ID NO:

4 have at least 80% identity, wherein the amino acid sequence of Cmr2 and the amino acid sequence of SEQ ID NO;

8 have at least 80% identity, wherein the amino acid sequence of Cmr3 and the amino acid sequence of SEQ ID NO;

10 have at least 80% identity, wherein the amino acid sequence of Cmr4 and the amino acid sequence of SEQ ID NO;

12 have at least 80% identity, wherein the amino acid sequence of Cmr6 and the amino acid sequence of SEQ ID NO;

16 have at least 80% identity, and wherein the polypeptides have endonuclease activity.

74. The method of claim 70 wherein the method is in vitro.

75. The method of claim 70 wherein the method is in vivo.

76. The method of claim 70 wherein the method occurs within a Pyrococcus furiosus, a Sulfolobus solfataricus, or a S. tokodaii cell.