Compositions And Methods Of Nucleic Acid-Targeting Nucleic Acids

Patent metadata

Field	Value
Publication number	US-2017051276-A1
Application number	US-201615344487-A
Country	US
Kind code	A1
Filing date	Nov 4, 2016
Priority date	Mar 14, 2013
Publication date	Feb 23, 2017
Grant date	—

Abstract

This disclosure provides for compositions and methods for the use of nucleic acid-targeting nucleic acids and complexes thereof.

First claim

1 - 26 . (canceled)

27 . An engineered single-guide nucleic acid-targeting nucleic acid capable of forming a complex with a Type II CRISPR Cas9 protein, the engineered single-guide nucleic acid-targeting nucleic acid comprising:
a spacer sequence, having a 5′ end and a 3′ end, that is capable of hybridizing to a target nucleic acid sequence; a minimum CRISPR repeat sequence, having a 5′ end and a 3′ end, wherein (i) the minimum CRISPR repeat sequence is between 6 and 15 nucleotides, and (ii) the 3′ end of the spacer sequence is covalently bound to the 5′ end of the minimum CRISPR repeat sequence; a first bulge sequence, having a 5′ end and a 3′ end, wherein (i) the first bulge sequence is between 1 nucleotide and 5 nucleotides, (ii) the first bulge sequence comprises at least one purine, and (iii) the 5′ end of the first bulge sequence is covalently bound to the 3′ end of the minimum CRISPR repeat sequence; a linker sequence, having a 5′ end and a 3′ end, that is between 4 unpaired nucleotides in length and 40 unpaired nucleotides in length, wherein the 5′ end of the linker sequence is covalently bound to the 3′ end of the first bulge sequence; a second bulge sequence, having a 5′ end and a 3′ end, wherein (i) the second bulge sequence is between 1 nucleotide and 10 nucleotides, (ii) the 5′ end of the second bulge sequence is covalently bound to the 3′ end of the linker sequence, and (iii) the first bulge sequence and the second bulge sequence form a region of unpaired nucleotides and at most one wobble pair; and a minimum CRISPR tracr sequence, having a 5′ end and a 3′ end, between 6 and 100 nucleotides comprising a nucleotide sequence at the 5′ end that is hybridized to the minimum CRISPR repeat sequence to form a duplex, wherein (i) the 5′ end of the minimum CRISPR tracr sequence is covalently bound to the 3′ end of the second bulge sequence, and (ii) the duplex comprises no more than 1 mismatch.

28 . The engineered single-guide nucleic acid-targeting nucleic acid of claim 27 , further comprising a 3′ tracr sequence, having a 5′ end and a 3′ end, comprising at least one hairpin, wherein the 5′ end of the 3′ tracr sequence is covalently bound to the 3′ end of the minimum CRISPR tracr sequence.

29 . The engineered single-guide nucleic acid-targeting nucleic acid of claim 28 , wherein the 3′ tracr sequence comprises at least two hairpins.

30 . The engineered single-guide nucleic acid-targeting nucleic acid of claim 27 , wherein the engineered single-guide nucleic acid-targeting nucleic acid comprises DNA.

31 . The engineered single-guide nucleic acid-targeting nucleic acid of claim 30 , wherein the engineered single-guide nucleic acid-targeting nucleic acid further comprises RNA.

32 . The engineered single-guide nucleic acid-targeting nucleic acid of claim 27 , wherein the engineered single-guide nucleic acid-targeting nucleic acid comprises RNA.

33 . The engineered single-guide nucleic acid-targeting nucleic acid of claim 28 , wherein the engineered single-guide nucleic acid-targeting nucleic acid comprises DNA.

34 . The engineered single-guide nucleic acid-targeting nucleic acid of claim 33 , wherein the engineered single-guide nucleic acid-targeting nucleic acid further comprises RNA.

35 . The engineered single-guide nucleic acid-targeting nucleic acid of claim 28 , wherein the engineered single-guide nucleic acid-targeting nucleic acid comprises RNA.

36 . The engineered single-guide nucleic acid-targeting nucleic acid of claim 27 , wherein the first bulge sequence comprises 5′-XXXY-3′ where X is a purine and the second bulge sequence comprises Y, wherein Y is a nucleotide that can form a wobble pair.

37 . The engineered single-guide nucleic acid-targeting nucleic acid of claim 28 , wherein the first bulge sequence comprises 5′-XXXY-3′ where X is a purine and the second bulge sequence comprises Y, wherein Y is a nucleotide that can form a wobble pair.

38 . A polynucleotide encoding the engineered single-guide nucleic acid-targeting nucleic acid of claim 27 .

39 . The polynucleotide of claim 38 , further comprising a promoter operably linked to the polynucleotide.

40 . The polynucleotide of claim 39 , wherein the promoter is a constitutive promoter.

41 . The polynucleotide of claim 40 , wherein the promoter is a CMV promoter or a UBC promoter.

42 . The polynucleotide of claim 39 , wherein the promoter is an inducible promoter.

43 . The polynucleotide of claim 39 , wherein the promoter is a tissue-specific promoter or a cell-type specific promoter.

44 . A composition comprising:
the engineered single-guide nucleic acid-targeting nucleic acid of claim 27 ; and a Cas9 protein.

45 . The composition of claim 44 , wherein the engineered single-guide nucleic acid-targeting nucleic acid and the Cas9 protein comprise a complex.

46 . The composition of claim 44 , wherein the Cas9 protein is a Streptococcus pyogenes Cas9 protein.

Assignees

• Caribou Biosciences Inc

Inventors

• May Andrew Paul
• Haurwitz Rachel E
• Doudna Jennifer A
• Berger James M
• Carter Matthew Merrill
• Donohoue Paul Daniel

Classifications

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• A61P9/10

  for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis · CPC title

• A61P31/04

  Antibacterial agents · CPC title

• A61P27/02

  Ophthalmic agents · CPC title

• A61P29/00

  Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] · CPC title