LACTOCOCCUS CRISPR-CAS SEQUENCES

• US 20130288251A1
• Filed: 10/20/2011
• Published: 10/31/2013
• Est. Priority Date: 10/20/2010
• 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: Horvath, Philippe, Romero, Dennis, Millen, Anne M.
• Granted Patent: US 9,951,341 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C07K 14/195 from bacteria
• 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)

Abstract

The present invention relates to a nucleic acid comprising a Lactococcus CRISPR repeat region and/or a Lactococcus CRISPR spacer region.

Claims

1. A nucleic acid comprising a Lactococcus CRISPR repeat region and/or a Lactococcus CRISPR spacer region and/or a Lactococcus cas gene.

2. The nucleic acid according to claim 1, wherein the Lactococcus CRISPR spacer region is selected from the group consisting of SEQ ID Nos:

25 to 60 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to any one of SEQ ID Nos;

25 to 60, or a sequence capable of hybridizing under stringent conditions to any one of SEQ ID Nos;

25 to 60.

3. The nucleic acid according to claim 1, wherein the Lactococcus CRISPR repeat region is corresponding to SEQ ID No:

24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to SEQ ID No;

24, or a sequence capable of hybridizing under stringent conditions to SEQ ID No;

24.

4. The nucleic acid according to claim 1 wherein the Lactococcus cas gene is selected from the group consisting of SEQ ID Nos:

2, 3, 5, 7, 9, 11, 13, 15, 17, and/or 21, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to any one of SEQ ID No;

2, 3, 5, 7, 9, 11, 13, 15, 17, and/or 21, or a sequence capable of hybridizing under stringent conditions to any one of SEQ ID No;

2, 3, 5, 7, 9, 11, 13, 15, 17, and/or 21.

6. The nucleic acid of claim 1 comprising SEQ ID NO:

22 or a portion thereof comprising at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or 200 nucleotides.

7. The nucleic acid of claim 1 further comprising at least one nucleic acid sequence selected from the group consisting of SEQ ID NOs:

2, 3, 5, 7, 9, 11, 13, 15, 17, and/or 21, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to any one of SEQ ID No;

2, 3, 5, 7, 9, 11, 13, 15, 17, and/or 21, or a sequence capable of hybridizing under stringent conditions to any one of SEQ ID No;

2, 3, 5, 7, 9, 11, 13, 15, 17, and/or 21.

8. A vector (e.g. a plasmid) comprising a nucleic acid sequence according to claim 1.

9. A host cell having altered phage resistance comprising a nucleic acid according to claim 1.

10. The host cell of claim 9, wherein the host cell is recombinant.

11. The host cell of claim 9 wherein said host cell is a bacterial cell.

12. The host cell of claim 11 wherein said bacterial cell is a lactic acid bacterial cell.

13. The host cell of claim 12, wherein the lactic acid bacterial cell is a bacterial cell selected from Bifidobacterium species, a Brevibacterium species, a Propionibacterium species, a Lactococcus species, a Streptococcus species, a Lactobacillus species including the Enterococcus species, Pediococcus species, a Leuconostoc species and Oenococcus species.

14. The host cell of claim 12 wherein the lactic acid bacterial cells is a Lactococcus cell, preferably a Lactococcus chungangensis, Lactococcus fujiensis, Lactococcus garvieae, Lactococcus lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. tructae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis cell, more preferably a Lactococcus lactis cell.

15. A method for preparing a variant bacterial strain with altered phage resistance comprising:

(a) transforming bacteria with a nucleic acid comprising a Lactococcus CRISPR spacer sequence or a vector according to claim 8;

(b) contacting the transformed bacteria with a phage; and

(c) isolating transformed bacteria that exhibit altered resistance to the phage.

16. The method according to claim 15, wherein the Lactococcus CRISPR spacer sequence is selected from at least one of SEQ ID NOs:

25-60 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to any one of SEQ ID Nos;

25 to 60 or a sequence capable of hybridizing under stringent conditions to any one of SEQ ID Nos;

25 to 60.

17. The method according to claim 15, wherein the nucleic acid comprises a repeat-spacer unit sequence, wherein the repeat sequence corresponds to SEQ ID NO:

24 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to SEQ ID No;

24 or a sequence capable of hybridizing under stringent conditions to SEQ ID No;

24, and the spacer sequence is selected from at least one of SEQ ID NOs;

25 to 60 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to any one of SEQ ID Nos;

25 to 60 or a sequence capable of hybridizing under stringent conditions to any one of SEQ ID Nos;

25 to 60.

18. The method according to claim 16, wherein the nucleic acid comprises SEQ ID NO:

22 or a portion thereof comprising at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or 200 nucleotides.

19. The method according to claim 15, wherein the nucleic acid further comprises at least one of SEQ ID Nos:

2, 3, 5, 7, 9, 11, 13, 15, 17, and/or 21.

20. The method according to claim 15, wherein the bacterial strain is a lactic acid bacterial strain.

21. The method according to claim 20 wherein the bacterial strain is lactic acid bacterial strain selected from Bifidobacterium species, a Brevibacterium species, a Propionibacterium species, a Lactococcus species, a Streptococcus species, a Lactobacillus species including the Enterococcus species, Pediococcus species, a Leuconostoc species and Oenococcus species.

22. The method according to claim 20 wherein the lactic acid bacterial cells is a Lactococcus species strain, more preferably a Lactococcus chungangensis, Lactococcus fujiensis, Lactococcus garvieae, Lactococcus lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. tructae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis strain, most preferably a Lactococcus lactis strain.

23. A variant bacterial strain obtained using the method according to claim 15.

36. A method for tagging a bacterial strain comprising the steps of:

(a) transforming bacteria with a nucleic acid comprising a Lactococcus CRISPR spacer sequence or a vector according to claim 8; and

(b) isolating transformed bacteria that exhibit altered resistance to the phage or

(c) isolating transformed bacteria containing the Lactococcus CRISPR spacer sequence.

37. A variant bacterial cell comprising a nucleic acid sequence according to claim 1.

38. Use of a nucleic acid according claim 1 for modulating the resistance of a cell against a target nucleic acid or a transcription product thereof.

39. A cell culture comprising a host cell according to claim 9.

40. A cell culture according to claim 39, wherein the cell culture is a bacterial culture selected from the group consisting of a starter culture, a probiotic culture, a dietary supplement culture.

41. A product comprising the cell culture of claim 39.

42. A product according to claim 41 wherein said product is selected from the group consisting of:

a food product, a feed product, a personal care product, a health care product, a veterinary product and a dietary supplement.

43. A process for preparing a product according to claim 41.

5. A nucleic acid comprising:

—

a) at least one nucleic acid sequence corresponding to SEQ ID No;

24, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to SEQ ID No;

24, or a sequence capable of hybridizing under stringent conditions to SEQ ID No;

24; and

/or

b) at least one nucleic acid sequence selected from the group consisting of SEQ ID Nos;

25 to 60 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to any one of SEQ ID Nos;

25 to 60, or a sequence capable of hybridizing under stringent conditions to any one of SEQ ID Nos;

25 to 60; and

/or

c) at least one nucleic acid sequence selected from the group consisting of SEQ ID Nos;

2, 3, 5, 7, 9, 11, 13, 15, 17, and/or 21, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% identity to any one of SEQ ID No;

2, 3, 5, 7, 9, 11, 13, 15, 17, and/or 21, or a sequence capable of hybridizing under stringent conditions to any one of SEQ ID No;

2, 3, 5, 7, 9, 11, 13, 15, 17, and/or 21.

24. A method for strain typing Lactococcus comprising:

(a) amplifying genomic DNA from a strain of interest using at least one primer pair, wherein the genomic DNA comprises at least a portion of a sequence of a CRISPR array;

(b) detecting an amplicon generated in step (a), whereby said detected amplicon indicates the strain type,

wherein each primer of a pair is complementary to at least a portion of a repeat sequence of the CRISPR array, whereby the amplicon generated comprises at least one spacer sequence located in the CRISPR array and wherein the repeat sequence of the CRISPR array corresponds to SEQ ID NOs;

24.

25. The method of claim 24, wherein the strain is a Lactococcus chungangensis, Lactococcus fujiensis, Lactococcus garvieae, Lactococcus lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. tructae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis strain, most preferably a Lactococcus lactis strain.

26. The method of claim 24, wherein each primer of a pair is complementary to at least a portion of a spacer sequence of the CRISPR array.

27. The method of claim 26, wherein the CRISPR array comprises at least two spacer sequences, and each primer of a pair is complementary to at least a portion of a different spacer sequence, whereby the amplicon generated comprises at least one repeat sequence located between the two spacer sequences.

28. The method of claim 26, wherein one primer of the pair is complementary to the spacer sequence adjacent to the first repeat of the CRISPR array.

29. The method according to claim 24, wherein the CRISPR array comprises SEQ ID NO:

22 or a portion thereof comprising at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or 200 nucleotides.

30. The method according to claim 24, wherein the amplicon is detected by hybridization to an immobilized probe.

31. A strain typing kit comprising:

(a) a container of an amplification mixture comprising a DNA polymerase, an amplification buffer, and at least one primer pair, wherein each primer of the pair is complementary to a portion of genomic DNA such that the primer pair amplifies at least a portion of a repeat or spacer sequence of a CRISPR array selected from at least one of SEQ ID Nos;

24 to 60; and

(b) a container of a detection mixture comprising a probe capable of hybridizing under stringent conditions to at least a portion of the CRISPR array amplified by the primer pair.

32. A method for tagging a lactococcal strain comprising:

(a) exposing a parent lactococcal strain to a phage;

(b) selecting a phage insensitive mutant;

(c) comparing a CRISPR array sequence or a portion thereof from the parent strain and the phage insensitive mutant strain;

whereby the presence of an additional repeat-spacer unit in the CRISPR array sequence of the phage insensitive mutant indicates that the strain is tagged.

34. The method of claim 32 wherein the strain is a Lactococcus chungangensis, Lactococcus fujiensis, Lactococcus garvieae, Lactococcus lactis, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. tructae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis strain, most preferably a Lactococcus lactis strain.

35. The method according to claim 32, wherein the CRISPR array comprises SEQ ID NO:

22 or a portion thereof comprising at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180 or 200 nucleotides.

33. A method for tagging a lactococcal strain comprising the steps of:

(a) exposing a parent lactococcal strain comprising at least a portion of a CRISPR array to at least one nucleic acid sequence to produce a mixture of Lactococcus comprising at least one bacteriophage resistant strain comprising a modified CRISPR array;

(b) selecting said bacteriophage resistant strain from said mixture of bacteria;

(c) selecting said bacteriophage resistant strains comprising an additional nucleic acid fragment in said modified CRISPR array from said bacteriophage resistant strains selected in step (b) whereby the presence of a modified CRISPR array sequence of the phage insensitive mutant indicates that the strain is tagged; and

(d) isolating said tagged strain, wherein said strain comprises an additional nucleic acid fragment in said modified CRISPR array.

44. A method for generating CRISPR-escape phage mutants comprising:

(a) obtaining;

at least one parent phage and a phage-resistant lactococcal bacterial strain comprising at least one CRISPR locus, wherein the CRISPR locus comprises a nucleic acid sequence that is at least about 95% identical to at least one protospacer sequence in the genome of the at least one parent phage;

(b) exposing the at least one parent phage to the phage-resistant lactococcal bacterial strain, under conditions such that at least one phage variant is produced; and

(c) selecting the at least one phage variant, wherein the at least one phage variant exhibits the ability to infect the phage-resistant lactococcal bacterial strain and is a CRISPR-escape phage mutant.

45. A CRISPR-escape phage mutants obtainable (preferably obtained) using the methods of claim 44.

46. A method for controlling bacterial (preferably lactococcal) populations in a product comprising exposing compositions comprising at least one CRISPR-escape phage mutant according to claim 45 to a fermentation medium, wherein the fermentation medium contains at least one population of undesirable bacteria, under conditions such that the population of the undesirable bacteria is reduced, and the fermentation medium is used to generate the product.

47-52. -52. (canceled)