Use

• US 20100093617A1
• Filed: 08/25/2006
• Published: 04/15/2010
• Est. Priority Date: 08/26/2005
• Status: Active Grant

• Current Assignee: International N&H Denmark APS (International Flavors & Fragrances Incorporated)
• Original Assignee: DuPont Nutrition Biosciences ApS (International Flavors & Fragrances Incorporated)
• Inventors: Fremaux, Christophe, Romero, Dennis, Boyaval, Patrick, Horvath, Philippe, Barrangou, Rodolphe
• Granted Patent: US 10,066,233 B2
• Time in Patent Office: Days
• US Class Current: 514/6.9
• CPC Class Codes: A23K 20/10 Organic substances
• CPC Class Codes: A23L 33/135 Bacteria or derivatives thereof, e.g. probiotics
• CPC Class Codes: A23V 2002/00 Food compositions, function of food ingredients or processes for food or foodstuffs
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• 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 15/746 for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention relates to the use of one or more cas genes for modulating resistance in a cell against a target nucleic acid or a transcription product thereof.

Claims

1. Use of one or more cas genes or proteins for modulating resistance in a cell against a target nucleic acid or a transcription product thereof.

2. Use according to claim 1, wherein the one or more cas genes or proteins are used in combination with two or more CRISPR repeats.

3. Use according to claim 2, wherein the one or more cas genes or proteins and/or the two or more CRISPR repeats are or are derivable (preferably, derived) from the same cell.

4. Use according to claim 2 or claim 3, wherein the one or more cas genes or proteins and the two or more CRISPR repeats naturally co-occur in the same cell.

5. Use according to any of the preceding claims, wherein the one or more cas genes or proteins are used in combination with one or more CRISPR spacers.

6. Use according to claim 5, wherein the CRISPR spacer(s) is or is derivable (preferably, derived) from an organism that is different to the cell from which the one or more cas genes or proteins and/or the two or more CRISPR repeats are or are derivable (preferably, derived).

7. Use according to claim 6, wherein the spacer is obtained from a cell which is resistant to a target nucleic acid.

8. Use according to claim 5, wherein the CRISPR spacer is a synthetic nucleic acid sequence.

9. Use according to any of claims 5-8, wherein the CRISPR spacer(s) have homology to the target nucleic acid.

10. Use according to claim 9, wherein the CRISPR spacer(s) have 100% identity to the target nucleic acid over at least the length of the CRISPR spacer core.

11. Use according to any of the preceding claims, wherein the one or more cas genes or proteins are used in combination with at least one or more CRISPR spacers and at least two or more CRISPR repeats.

12. Use according to any of the preceding claims, wherein the target nucleic acid or transcription product thereof is or is derivable (preferably, derived) from bacteriophage DNA.

13. Use according to any of the preceding claims, wherein the target nucleic acid or transcription product thereof is or is derivable (preferably, derived) from plasmid DNA.

14. Use according to any of the preceding claims, wherein the target nucleic acid or transcription product thereof is or is derivable (preferably, derived) from a mobile genetic element.

15. Use according to claim 14, wherein the target nucleic acid or transcription product thereof is or is derivable (preferably, derived) from a transposable element or an insertion sequence.

16. Use according to any of the preceding claims, wherein the target nucleic acid or transcription product thereof is or is derivable (preferably, derived) from an antibiotic resistance gene.

17. Use according to any of the preceding claims, wherein the target nucleic acid or transcription product thereof is or is derivable (preferably, derived) from a nucleic acid encoding a virulence factor.

18. Use according to claim 17, wherein the virulence factor is selected from the group consisting of a toxin-, an internalin- and a hemolysin-encoding nucleic acid.

19. Use of a recombinant nucleic acid sequence comprising at least one cas gene and at least two CRISPR repeats together with at least one CRISPR spacer, wherein at least one CRISPR spacer is heterologous to at least one cas gene and/or at least two CRISPR repeats to modulate resistance against a target nucleic acid or transcription product thereof.

20. A nucleic acid sequence consisting essentially of at least one cas gene.

25. The nucleic acid sequence according to any of claims 20-24, wherein the one or more cas genes and the two or more CRISPR repeats are or are derivable (preferably, derived) from the same cell.

26. The nucleic acid sequence according to any of claims 20-25, wherein the one or more cas genes and the two or more CRISPR repeats naturally co-occur in the same cell.

27. The nucleic acid sequence according to any of claims 20-26, wherein the CRISPR spacers are or are derivable (preferably, derived) from an organism that is different to the cell from which the one or more cas genes and/or the two or more CRISPR repeats are or are derivable (preferably, derived).

28. A construct comprising one or more of the nucleic acid sequences according to any of claims 20-27.

29. A vector comprising one or more of the nucleic acid sequences according to any of claims 20-27 or one or more of the constructs according to claim 24.

30. A cell comprising the nucleic acid sequence according to any of claims 20-27, the construct according to claim 28 or the vector according to claim 29.

31. The cell according to claim 30, wherein the cell is a recipient cell or a host cell.

21. A nucleic acid sequence consisting essentially of at least one cas gene and at least two CRISPR repeats.

22. A nucleic acid sequence consisting essentially of at least one cas gene and at least one CRISPR spacer.

23. A nucleic acid sequence consisting essentially of at least one cas gene, at least one CRISPR spacer and at least two CRISPR repeats.

24. A recombinant nucleic acid sequence comprising at least one cas gene and at least two CRISPR repeats together with at least one CRISPR spacer, wherein the CRISPR spacer is heterologous to the at least one cas gene and/or the at least two CRISPR repeats.

32. A method for modulating (e.g. conferring or increasing) the resistance of a cell against a target nucleic acid or a transcription product thereof comprising the steps of:

(i) identifying a sequence (eg. a conserved sequence) in an organism (preferably, a sequence essential to the function or survival of the organism);

(ii) preparing a CRISPR spacer which is homologous to the identified sequence;

(iii) preparing a nucleic acid (eg. a recombinant nucleic acid) comprising at least one cas gene and at least two CRISPR repeats together with the CRISPR spacer; and

(iv) introducing said nucleic acid into a cell thus to render the cell resistant to said target nucleic acid or transcription product thereof.

35. The method according to any of claims 32-34, wherein the one or more cas genes or proteins and/or the two or more CRISPR repeats are or are derivable (preferably, derived) from the same cell.

36. The method according to any of claims 32-35, wherein the spacers are or are derivable (preferably, derived) from an organism that is different to the cell comprising the one or more cas genes or proteins and/or the two or more CRISPR repeats.

37. The method according to any of claims 32-35, wherein the one or more cas genes or proteins and the two or more CRISPR repeats naturally co-occur in the same cell.

38. The method according to any of claims 32-37 wherein said modification comprises inserting one or more CRISPR spacers and/or pseudo CRISPR spacers into the cell.

39. The method according to claim 38, wherein the spacer of the cell has 100% homology to the CRISPR spacer or pseudo CRISPR spacer of the organism.

40. The method according to claim 38 or claim 39, wherein said modification comprises genetically engineering the CRISPR spacer of the cell.

41. The method according to claim 40, wherein all or part of the spacer in the cell is modified.

42. The method according to claim 40 or claim 41, wherein said modification comprises the modification of a recombinant spacer.

43. The method according to any of claims 40-42, wherein said modification occurs through spontaneous mutation or mutagenesis.

70. A cell obtained or obtainable by the method of any of claims 32-69.

76. Use of a cell according to claim 70, a CRISPR spacer or pseudo CRISPR spacer according to claim 71, a cas gene according to claim 72, a CRISPR repeat according to claim 73 or a functional combination according to claim 74 for modulating the resistance of a cell against a target nucleic acid or a transcription product thereof.

77. A cell culture comprising a cell according to claim 70, a CRISPR spacer or pseudo CRISPR spacer according to claim 71, a cas gene according to claim 72, a CRISPR repeat according to claim 73 or a functional combination according to claim 74 for modulating the resistance of a cell against a target nucleic acid or a transcription product thereof.

78. The cell culture according to claim 77, wherein said culture is a starter culture or a probiotic culture.

79. A food product or feed comprising the culture according to 77 or claim 78.

80. A process for preparing a food product or feed comprising the use of the culture according to claim 77 or claim 78.

81. A food product of feed obtained or obtainable by the process of claim 80.

82. Use of the culture according to claim 77 or claim 78 for preparing a food product.

33. A method for modulating (eg. conferring or increasing) the resistance of a cell against a target nucleic acid or a transcription product thereof comprising the steps of

(i) identifying one or more CRISPR spacers or pseudo CRISPR spacers in an organism resistant to the target nucleic acid or transcription product thereof;

(ii) preparing a recombinant nucleic acid comprising at least one cas gene or protein and at least two CRISPR repeats together with said identified one or more spacers; and

(iii) introducing said recombinant nucleic acid into a cell thus to render the cell resistant to said target nucleic acid or transcription product thereof.

34. A method for modulating (eg. conferring or increasing) the resistance of a cell comprising at least one or more cas genes or proteins and two or more CRISPR repeats against a target nucleic acid or a transcription product thereof comprising the steps of

(i) identifying one or more CRISPR spacers in an organism resistant to the target nucleic acid or transcription product thereof;

and

(ii) modifying the sequence of one or more CRISPR spacer(s) in the cell such that the CRISPR spacer(s) has homology to the CRISPR spacer(s) in the organism.

44. A method for modulating (eg. reducing or decreasing) the resistance of a cell comprising at least one or more cas genes or proteins and two or more CRISPR repeats against a target nucleic acid or a transcription product thereof comprising the steps of

(i) identifying one or more CRISPR spacers in an organism that is substantially resistant to the target nucleic acid or transcription product thereof;

and

(ii) modifying the sequence of at least one or more CRISPR spacer(s) in the cell such that the CRISPR spacer(s) has a reduced degree of homology to the spacer(s) in the organism.

45. The method according to claim 44, wherein the at least one or more CRISPR spacer(s) in the cell are deleted.

46. The method according to claim 44 or claim 45, wherein at least one or more CRISPR repeat(s) in the cell are deleted.

47. The method according to any of claims 44-46, wherein one or more cas genes are deleted,

48. The method according to any of claims 44-47, wherein CRISPR and/or one or more cas genes are deleted.

49. A method for modulating (eg. reducing or decreasing) the resistance of a cell comprising at least one or more cas genes or proteins and two or more CRISPR repeats against a target nucleic acid or a transcription product thereof comprising modifying the one or more cas genes or proteins and/or two or more CRISPR repeats in the cell.

50. The method according to claim 49, wherein the one or more cas genes or proteins and/or two or more CRISPR repeats in the cell are deleted.

51. A method for identifying a CRISPR spacer or pseudo CRISPR spacer for use in modulating the resistance of a cell against a target nucleic acid or a transcription product thereof comprising the steps of

(i) preparing a cell comprising at least two CRISPR repeats and at least one cas gene or protein;

(ii) identifying at least one CRISPR spacer or pseudo CRISPR spacers in an organism that is substantially resistant to the target nucleic acid or transcription product thereof;

(iii) modifying the sequence of the CRISPR spacer in the cell such that the CRISPR spacer has homology to the spacer of the organism; and

(iv) determining if the cell modulates resistance against the target nucleic acid or transcription product thereof,

wherein modulation of the resistance of the cell against the target nucleic acid or transcription product thereof is indicative that the CRISPR spacer modulates the resistance of the cell.

71. A CRISPR spacer or pseudo CRISPR spacer obtained or obtainable by the method of claim 51.

75. A recombinant CRISPR locus comprising a CRISPR spacer or pseudo CRISPR spacer according to claim 71, and/or a cas gene according to claim 72, and/or a CRISPR repeat according to claim 73 and/or a functional combination according to claim 74.

52. A method for identifying a cas gene for use in modulating the resistance of a cell against a target nucleic acid or transcription product thereof comprising the steps of

(i) preparing a cell comprising at least one CRISPR spacer and at least two CRISPR repeats;

(ii) engineering the cell such that it comprises at least one cas gene; and

(iii) determining if the cell modulates resistance against the target nucleic acid or transcription product thereof;

wherein modulation of the resistance of the cell against the target nucleic acid or transcription product thereof is indicative that the cas gene can be used to modulate the resistance of the cell.

72. A cas gene obtained or obtainable by the method of claim 52.

53. A method for identifying a CRISPR repeat for use in modulating the resistance of a cell against a target nucleic acid or transcription product thereof comprising the steps of:

(i) preparing a cell comprising at least one CRISPR spacer and at least one cas gene;

(ii) engineering the cell such that it contains the CRISPR repeat; and

(iii) determining if the cell modulates resistance against the target nucleic acid or transcription product thereof,

wherein modulation of the resistance of the cell against the target nucleic acid or transcription product thereof is indicative that the CRISPR repeat can be used to modulate resistance.

73. A CRISPR repeat obtained or obtainable by the method of claim 53.

54. A method for identifying a functional combination of a cas gene and a CRISPR repeat comprising the steps of

(a) determining the sequences of the cas gene and the CRISPR repeat;

(b) identifying one or more clusters of cas genes as determined by sequence comparison analysis;

(c) identifying one or more clusters of CRISPR repeats; and

(d) combining those cas gene and CRISPR repeat sequences that fall within the same cluster,

wherein the combination of the cas gene and CRISPR repeat sequences within the same cluster is indicative that the combination is a functional combination.

55. The method according to claim 54, wherein the nucleotide sequences of the cas gene and the CRISPR repeat are or are derivable (preferably, derived) from the same or different strains.

56. The method according to claim 54, wherein the nucleotide sequences of the cas gene and the CRISPR repeat are or are derivable (preferably, derived) from the same or different species.

57. The method according to claim 54, wherein the nucleotide sequences of the cas gene and the CRISPR repeat are or are derivable (preferably, derived) from the same or different genera.

58. The method according to claim 54, wherein the nucleotide sequences of the cas gene and the CRISPR repeat are or are derivable (preferably, derived) from the same or different organisms.

74. A functional combination obtained or obtainable by the method of any of claims 54-58.

59. A method for modulating the lysotype of a bacterial cell comprising one or more cas genes or proteins and two or more CRISPR repeats comprising the steps of:

(i) identifying one or more pseudo CRISPR spacers in the genomic sequence of a bacteriophage against which resistance is to be modulated; and

(ii) modifying the sequence of one or more CRISPR spacers of the bacterial cell such that the CRISPR spacer(s) of the bacterial cell has homology to the pseudo CRISPR spacer(s) of the bacteriophage against which resistance is to be modulated.

60. A method for modulating (eg. conferring or increasing) the resistance of a bacterial cell against a bacteriophage comprising the steps of:

(i) identifying a sequence (eg. a conserved sequence) in a bacteriophage (preferably, a sequence essential to the function or survival of the bacteriophage);

(ii) preparing a CRISPR spacer which is homologous to the identified sequence;

(iii) preparing a nucleic acid comprising at least one cas gene and at least two CRISPR repeats together with the CRISPR spacer; and

(iv) introducing said nucleic acid into the bacterial cell thus to render the bacterial cell resistant to said target nucleic acid or transcription product thereof.

61. A method for modulating (eg. conferring or increasing) the resistance of a bacterial cell against a target nucleic acid or transcription product in a bacteriophage thereof comprising the steps of:

(i) identifying one or more pseudo CRISPR spacers in a bacteriophage genome that is capable of providing resistance to the target nucleic acid or transcription product thereof;

(ii) preparing a recombinant nucleic acid comprising at least one cas gene and at least two CRISPR repeats together with said identified one or more pseudo CRISPR spacers; and

(iii) introducing said recombinant nucleic acid into said bacterial cell thus to render the bacterial cell resistant to said target nucleic acid or transcription product thereof.

63. The method according to claim 61 or claim 62, wherein the target nucleic acid in the bacteriophage is a highly conserved nucleic acid sequence.

64. The method according to any of claims 61-63, wherein the target nucleic acid in the bacteriophage encodes a host specificity protein.

65. The method according to any of claims 61-64, wherein the target nucleic acid in the bacteriophage encodes a protein that is essential for survival, replication or growth of the bacteriophage.

66. The method according to claim 65, wherein the target nucleic acid in the to bacteriophage encodes a helicase, a primase, a head or tail structural protein, a protein with a conserved domain (eg. holin, lysin, and others) or a conserved sequences amongst important phage genes.

62. A method for modulating the resistance of a bacterial cell comprising one or more cas genes or proteins and two or more CRISPR repeats against a target nucleic acid or transcription product thereof in a bacteriophage comprising the steps of:

(i) identifying one or more pseudo CRISPR spacers in a bacteriophage that is capable of providing resistance to a target nucleic acid or transcription product thereof;

(ii) identifying one or more CRISPR spacers in a bacterial cell in which resistance is to be modulated; and

(iii) modifying the sequence of the CRISPR spacer(s) in the bacterial cell in which resistance is to be modulated such that the CRISPR spacer(s) has a higher degree of homology to the pseudo CRISPR spacer(s) of the bacteriophage against which resistance is to be modulated.

67. A method for determining the resistance of a cell against a target nucleic acid or a transcription product thereof comprising identifying one or more functional CRISPR repeat-cas combinations and one or more CRISPR spacers in the cell.

68. The method according to claim 67, comprising the additional step of comparing the sequence of the one or more CRISPR spacers in the cell with the sequence of the target nucleic acid.

69. The method according to claim 67 or claim 68, comprising the additional step of determining the resistance profile of the cell.

83. A nucleotide sequence comprising or consisting of the sequence set forth in any of SEQ ID Nos. 7-10 and SEQ ID Nos. 359 to 405 or a variant, fragment, homologue or derivative thereof.

84. An amino acid sequence encoded by the nucleotide sequence according to claim 83.

85. A construct or vector comprising one or more of the nucleotide sequences according to claim 83.

86. A host cell into which has been incorporated one or more of the nucleotide sequences according to claim 83 or the construct or vector according to claim 85.