COMPOSITIONS AND METHODS OF NUCLEIC ACID-TARGETING NUCLEIC ACIDS

• US 20140315985A1
• Filed: 03/12/2014
• Published: 10/23/2014
• Est. Priority Date: 03/14/2013
• Status: Abandoned Application

• Current Assignee: Caribou Biosciences, Inc.
• Original Assignee: Caribou Biosciences, Inc.
• Inventors: Haurwitz, Rachel E., Doudna, Jennifer A., May, Andrew Paul, Berger, James M., Carter, Matthew Merrill, Donohoue, Paul
• Application Number:
  US14/206,319
• Publication Number:
  US 20140315985A1
• Time in Patent Office: Days
• US Class Current: 514/44.R
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/6455 Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• 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 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• 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 2310/531 : Stem-loop; Hairpin
• CPC Class Codes: C12N 2310/533 : having a mismatch or nick in at least one of the strands
• CPC Class Codes: C12N 2800/80 : Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 : involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/686 : Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 : Methods for sequencing
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/203 : incorporating a composite nucleic acid containing a polypeptide sequence other than PNA

Abstract

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

Claims

1-26. -26. (canceled)

27. A composition comprising:

(a) an engineered nucleic acid-targeting nucleic acid comprising;

(i) a tracrRNA sequence; and

(ii) a 3′

hybridizing extension; and

(b) a donor polynucleotide,

wherein said donor polynucleotide is hybridized to said 3′

hybridizing extension.

28. The composition of claim 27, wherein said 3′

hybridizing extension is located at the 3′

end of said engineered nucleic acid-targeting nucleic acid.

29. The composition of claim 27, wherein said 3′

hybridizing extension is adapted to hybridize to at least 5 nucleotides from the 3′

end of said donor polynucleotide.

30. The composition of claim 27, wherein said 3′

hybridizing extension is adapted to hybridize to at least 5 nucleotides from the 5′

end of said donor polynucleotide.

31. The composition of claim 27, wherein said 3′

hybridizing extension is adapted to hybridize to at least 5 adjacent nucleotides within said donor polynucleotide.

32. The composition of claim 27, wherein said 3′

hybridizing extension is adapted to hybridize to all of said donor polynucleotide.

33. The composition of claim 27, wherein said 3′

hybridizing extension is adapted to hybridize to said donor polynucleotide with one or more mismatches.

34. The composition of claim 27, wherein said 3′

hybridizing extension comprises a reverse transcription template.

35. The composition of claim 34, wherein said reverse transcription template is adapted to be reverse transcribed by a reverse transcriptase.

36. The composition of claim 34, wherein further comprising a reverse transcribed DNA polynucleotide.

37. The composition of claim 36, wherein said reverse transcribed DNA polynucleotide is adapted to hybridize to said reverse transcription template.

38. The composition of claim 27, wherein said donor polynucleotide is DNA.

39. The composition of claim 27, wherein said 3′

hybridizing extension is RNA.

40. The composition of claim 27, wherein said tracrRNA sequence comprises at least 80% identity to a tracrRNA from any of SEQ ID NOs:

431-562 over 6 contiguous nucleotides.

41. The composition of claim 27, wherein said engineered nucleic acid-targeting nucleic acid further comprises a CRISPR RNA sequence.

42. The composition of claim 40, wherein said CRISPR RNA comprises at least 80% identity to a CRISPR RNA from any of SEQ ID NOs:

563-679 over 6 contiguous nucleotides.

43. The composition of claim 27, wherein said engineered nucleic acid-targeting nucleic acid is an isolated engineered nucleic acid-targeting nucleic acid.

44. The composition of claim 41, wherein said engineered nucleic acid-targeting nucleic acid comprises a linker linking said tracrRNA sequence and said CRISPR RNA sequence.

45. The composition of claim 27, wherein said engineered nucleic acid-targeting nucleic acid is a recombinant engineered nucleic acid-targeting nucleic acid.

46. A method for introducing a donor polynucleotide into a target nucleic acid comprising:

(a) contacting said target nucleic acid with the composition of claim 27; and

(b) cleaving said target nucleic acid to produce a cleaved target nucleic acid, wherein said cleaving is performed by a polypeptide comprising at least 75% amino acid identity to a sequence from SEQ ID NOs;

1-256 and 795-1346; and

(c) inserting said donor polynucleotide into said cleaved target nucleic acid.

47. The method of claim 46, wherein said cleaving is performed by a polypeptide comprising at least 50% amino acid identity to a nuclease domain of SEQ ID NOs:

1-256 and 795-1346.

48. The method of claim 46, further comprising producing a therapeutic outcome.

49. The method of claim 48, wherein said therapeutic outcome is modulated by said donor polynucleotide.

50. The method of claim 48, wherein said therapeutic outcome is a modulation selected from the group consisting of:

a decrease in the levels of a protein in a pathway related to a disease, an increase in the levels of a protein in a pathway related to a disease, morphological changes in a cell, metabolic changes in a cell, and structural changes in a cell, or any combination thereof.

51. A pharmaceutical composition comprising the composition of claim 27.

52. A vector comprising a polynucleotide sequence encoding the engineered nucleic acid-targeting nucleic acid comprising a 3′

hybridizing extension of claim 27

53. A genetically modified cell comprising the composition of claim 27.

54. A kit comprising:

(a) the composition of claim 27; and

(b) a buffer.

55. The kit of claim 54, further comprising instructions for use.