Modified cascade ribonucleoproteins and uses thereof

Patent metadata

Field	Value
Publication number	US-2016186152-A1
Application number	US-201614997467-A
Country	US
Kind code	A1
Filing date	Jan 15, 2016
Priority date	Dec 30, 2011
Publication date	Jun 30, 2016
Grant date	—

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.

First claim

1 - 46 . (canceled)

47 . A composition 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.

48 . The composition of claim 47 , wherein the nuclease is a ribonuclease, or a mutant or an active portion thereof.

49 . The composition of claim 48 , wherein the ribonuclease is an endonuclease, a 3′ exonuclease or a 5′ exonuclease.

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

51 . The composition of claim 50 , wherein the CRISPR-associated protein subunit is Cse1.

52 . The composition of claim 47 , wherein the nuclease is an endonuclease, or mutant or an active portion thereof.

53 . The composition of claim 52 , wherein the endonuclease comprises FokI or a modified FokI.

54 . The composition of claim 53 , wherein the modified FokI is KKR Sharkey or ELD Sharkey or a combination thereof.

55 . The composition of claim 47 , wherein the nuclease is a FokI KKR Sharkey, further comprising a second composition comprising a second 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, wherein the nuclease is a FokI ELD Sharkey.

56 . The composition of claim 47 , further comprising a second composition comprising a second 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.

57 . The composition of claim 50 , wherein the nuclease comprises FokI or a modified FokI.

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

59 . The composition of claim 51 , wherein the nuclease comprises FokI or a modified FokI.

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

61 . The composition 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.

62 . The composition 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.

63 . One or more nucleic acid molecules encoding the composition of claim 47 .

64 . One or more expression vectors comprising the nucleic acid molecules of claim 63 .

Assignees

• Caribou Biosciences Inc

Inventors

• Brouns Stan Johan Jozef
• Van Der Oost John

Classifications

• A61P31/12

  Antivirals · CPC title

• C07K2319/80

  containing a DNA binding domain, e.g. Lacl or Tet-repressor · CPC title

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C07K2319/22

  containing a Strep-tag · CPC title

• C07K2319/85

  containing an RNA binding domain · CPC title