MODIFIED CASCADE RIBONUCLEOPROTEINS AND USES THEREOF

• US 20150024499A1
• Filed: 07/08/2014
• Published: 01/22/2015
• Est. Priority Date: 12/30/2011
• Status: Abandoned Application

• Current Assignee: Caribou Biosciences, Inc.
• Original Assignee: Wageningen University
• Inventors: Brouns, Stan Johan Jozef, Van Der Oost, John
• Application Number:
  US14/326,099
• Publication Number:
  US 20150024499A1
• Time in Patent Office: Days
• US Class Current: 435/462
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: C07K 14/245 Escherichia (G)
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/22 containing a Strep-tag
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C07K 2319/85 containing an RNA binding domain
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/82 for plant cells , e.g. plant artificial chromosomes (PACs)
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/20 : involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• 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 301/21004 : Type II site-specific deoxyribonuclease (3.1.21.4)

Abstract

A clustered regularly interspaced short palindromic repeat (CRISPR)-associated complex for adaptive antiviral defence (Cascade); the Cascade protein complex comprising at least CRISPR-associated protein subunits Cas7, Cas5 and Cas6 which includes at least one subunit with an additional amino acid sequence possessing nucleic acid or chromatin modifying, visualising, transcription activating or transcription repressing activity. The Cascade complex with additional activity is combined with an RNA molecule to produce a ribonucleoprotein complex. The RNA molecule is selected to have substantial complementarity to a target sequence. Targeted ribonucleoproteins can be used as genetic engineering tools for precise cutting of nucleic acids in homologous recombination, non-homologous end joining, gene modification, gene integration, mutation repair or for their visualisation, transcriptional activation or repression. A pair of ribonucleotides fused to FokI dimers may be used to generate double-strand breakages in the DNA to facilitate these applications in a sequence-specific manner.

Claims

1-46. -46. (canceled)

47. A method for modifying a genomic target nucleic acid in a eukaryotic cell comprising:

(a) contacting a nucleic acid-targeting nucleic acid with a target nucleic acid, wherein said nucleic acid-targeting nucleic acid comprises a designed clustered regularly interspaced short palindromic repeat RNA; and

(b) modifying said target nucleic acid in said eukaryotic cell.

48. The method of claim 47, wherein said target nucleic acid is adjacent to a protospacer adjacent motif.

49. The composition of claim 48, wherein said protospacer adjacent motif marks a point in said target nucleic acid at which base-pairing with said clustered regularly interspaced short palindromic repeat RNA occurs.

50. The method of claim 48, wherein said protospacer adjacent motif comprises a triplet selected from the group consisting of:

5′

-CCT-3′

,5′

-CTT-3′

,5′

-CAT-3′

, and 5′

-CTC-3′

.

51. The method of claim 48, wherein said protospacer adjacent motif comprises 5′

-CCT-3′

.

52. The method of claim 48, wherein said protospacer adjacent motif comprises a CC dinucleotide.

53. The method of claim 48, wherein said protospacer adjacent motif comprises a double-stranded triplet of nucleotides, wherein one strand said protospacer adjacent motif comprises a CC dinucleotide, wherein said CC dinucleotide is hybridized with a GG dinucleotide on the other strand of said protospacer adjacent motif.

54. The method of claim 47, wherein said target nucleic acid is single stranded DNA.

55. The method of claim 47, wherein said target nucleic acid is double stranded DNA.

56. The method of claim 47, wherein said target nucleic acid is genomic DNA.

57. The method of claim 47, wherein said nucleic acid-targeting nucleic acid is introduced into said eukaryotic cell by a delivery vehicle selected from the group consisting of:

a cationic polymer and lipid delivery vehicle, a cell-penetrating peptide, a virus, an adenovirus, and an agrobacterium.

58. The method of claim 47, wherein said contacting comprises hybridizing a designed spacer portion of said clustered regularly interspaced short palindromic repeat RNA to a sequence of said target nucleic acid, wherein said designed spacer portion comprises at least 80% identity to said target nucleic acid.

59. The method of claim 47, wherein said contacting comprises hybridizing a designed spacer portion of said clustered regularly interspaced short palindromic repeat RNA to a sequence of said target nucleic acid, wherein said designed spacer portion comprises at least 50% identity over 32 nucleotides of said target nucleic acid.

60. The method of claim 47, wherein said nucleic acid-targeting nucleic acid comprises a seed region, a 3′

hairpin region, or both a seed region and a 3′

hairpin region.

61. The method of claim 47, wherein said nucleic acid-targeting nucleic acid comprises a spacer region.

62. The method of claim 47, wherein said modifying comprises introducing a single-stranded break in said target nucleic acid.

63. The method of claim 47, wherein said modifying comprises introducing a double-stranded break in said target nucleic acid.

64. The method of claim 47, wherein said modifying comprises introducing a modification selected from the group consisting of:

an organic dye, a radiolabel, and a spin label.

65. The method of claim 47, wherein said modifying comprises deleting a portion of said target nucleic acid.

66. The method of claim 47, wherein said modifying is selected from the group consisting of:

activating transcription of said target nucleic acid, repressing transcription of said target nucleic acid.

67. The method of claim 47, wherein said modifying comprises inserting a nucleic acid into said target nucleic acid.

68. The method of claim 47, wherein said modifying is performed by a Cas polypeptide.

69. The method of claim 68, wherein said Cas polypeptide is a nuclease.

70. The method of claim 47, wherein said cell is a cell selected from the group consisting of:

an insect cell, a yeast cell, and a fungal cell.

71. The method of claim 47, wherein said cell is a cell of a multicellular organism.

72. The method of claim 47, wherein said cell is a mammalian cell.

73. The method of claim 47, wherein said cell is a stem cell that is not a human embryonic stem cell.

74. The method of claim 47, wherein said cell is a human cell.

75. The method of claim 47, wherein said cell is a plant cell.

76. A method of altering expression of a target nucleic acid comprising introducing into a eukaryotic cell containing a DNA molecule encoding said target nucleic acid a designed clustered regularly interspaced short palindromic repeat system comprising one or more vectors comprising:

a) a first nucleotide sequence encoding a designed clustered regularly interspaced short palindromic repeat RNA that hybridizes with the target nucleic acid, and b) a second nucleotide sequence encoding a clustered regularly interspaced short palindromic repeat associated polypeptide, wherein components (a) and (b) are located on same or different vectors of the system, whereby said RNA targets said target nucleic acid and said clustered regularly interspaced short palindromic repeat associated polypeptide protein cleaves said DNA molecule.