MODIFIED CASCADE RIBONUCLEOPROTEINS AND USES THEREOF

• US 20160186214A1
• Filed: 01/15/2016
• Published: 06/30/2016
• Est. Priority Date: 12/30/2011
• Status: Active Grant

• Current Assignee: Caribou Biosciences, Inc.
• Original Assignee: Caribou Biosciences, Inc.
• Inventors: BROUNS, STAN JOHAN JOZEF, VAN DER OOST, JOHN
• Granted Patent: US 9,885,026 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 of modifying a target nucleic acid comprising:

contacting the target nucleic acid with a ribonucleoprotein complex comprising a designed fusion of (i) at least one clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein subunit, and (ii) a nuclease, or a mutant or an active portion thereof, and a CRISPR RNA (crRNA) molecule;

wherein the modifying is binding and/or cleaving the target nucleic acid.

48. The method of claim 47, wherein the target nucleic acid is a double-stranded DNA (dsDNA).

49. The method of claim 47, wherein the nuclease, or the mutant or the active portion thereof is a ribonuclease.

50. The method of claim 49, wherein the ribonuclease is an endonuclease, a 3′

exonuclease or a 5′

exonuclease.

51. The method of claim 47, wherein the CRISPR-associated protein subunit is selected from the group consisting of Cas6, Cas5, Cse1, Cse2 and Cas7.

52. The method of claim 51, wherein the CRISPR-associated protein subunit is Cse1.

53. The method of claim 47, wherein the nuclease is an endonuclease.

54. The method of claim 53, wherein the nuclease comprises FokI or a modified FokI.

55. The method of claim 54, wherein the modified FokI is KKR Sharkey or ELD Sharkey or a combination thereof.

56. The method of claim 51, wherein the nuclease comprises FokI or a modified FokI.

57. The method of claim 56, wherein the modified FokI is KKR Sharkey or ELD Sharkey or a combination thereof.

58. The method of claim 52, wherein the nuclease comprises FokI or a modified FokI.

59. The method of claim 58, wherein the modified FokI is KKR Sharkey or ELD Sharkey or a combination thereof.

60. The method of claim 47, wherein the designed fusion further comprises a linker polypeptide between (i) the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein subunit, and (ii) the nuclease, or the mutant or the active portion thereof.

61. The method of claim 47, wherein the clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein subunit and (ii) the nuclease, or the mutant or the active portion thereof, are covalently linked by chemical synthesis.

62. The method of claim 47, wherein the method is an in vitro method.

63. The method of claim 47, wherein the method is an in vivo method.

64. The method of claim 47, wherein the method is an ex vivo method.

65. A method of modifying a target nucleic acid comprising:

contacting the target nucleic acid with a first ribonucleoprotein complex comprising a first designed fusion of (i) at least one clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein subunit, and (ii) a FokI or a modified FokI, and a first CRISPR RNA (crRNA) molecule; and

a second ribonucleoprotein complex comprising a second designed fusion of (i) at least one clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein subunit, and (ii) a FokI or a modified FokI, and a second CRISPR RNA (crRNA) molecule;

wherein the modifying is binding and/or cleaving the target nucleic acid.

66. The method of claim 65, wherein (i) the first designed fusion comprises a FokI KKR Sharkey, and the second designed fusion comprises a FokI ELD Sharkey, and (ii) the modifying is cleaving the target nucleic acid.