Method for modulating resistance

• US 9,879,269 B2
• Filed: 06/06/2012
• Issued: 01/30/2018
• 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: Barrangou, Rodolphe, Boyaval, Patrick, Horvath, Philippe, Fremaux, Christophe, Romero, Dennis A.
• Primary Examiner(s): Marvich, Maria
• Application Number:
  US13/490,050
• Publication Number:
  US 20130011828A1
• Time in Patent Office: 2,064 Days
• Field of Search: None
• 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. A method for conferring resistance in a bacterial cell to a bacteriophage, the method comprising introducing into the bacterial cell a nucleic acid sequence comprising at least one Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (cas) gene operably linked to a regulatory sequence and at least two CRISPRs and at least one CRISPR spacer, wherein the at least one CRISPR spacer is flanked by CRISPRs of the at least two CRISPRs wherein the at least two CRISPRs, the at least one CRISPR spacer and the at least one cas gene form a functional combination that confers the resistance;

the at least one CRISPR spacer is a nucleic acid sequence 100% identical to a sequence of the bacteriophage genome thus to confer the resistance to said bacteriophage.

2. The method of claim 1 wherein the CRISPR spacer is heterologous to the at least one cas gene or the at least two CRISPRs.

3. A method for conferring resistance in a bacterial cell against a bacteriophage, the method comprising introducing into the bacterial cell a nucleic acid sequence comprising at least one Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (cas) gene operably linked to a regulatory sequence and at least two CRISPRs and at least one CRISPR spacer, wherein the at least one CRISPR spacer is flanked by CRISPRs of the at least two CRISPRs wherein the at least two CRISPRs, the at least one CRISPR spacer and the at least one cas gene form a functional combination and the at least one CRISPR spacer is a nucleic acid sequence 100% identical to a sequence of the bacteriophage genome thus conferring the resistance to said bacteriophage.

4. The method according to claim 3, wherein the spacers are from a different organism than the bacterial cell.

5. The method according to claim 3, wherein the at least one cas gene and the at least two CRISPRs naturally co-occur in the same bacterial cell.

6. A method for modulating the resistance of a bacterial cell to a bacteriophage wherein the bacterial cell comprises at least one or more cas genes wherein each of the at least one or more cas genes is operably linked to a regulatory sequence, and at least two CRISPRs, the method comprising the steps of (i) identifying one or more CRISPR spacers that confer resistance to the bacteriophage in an organism different than the bacterial cell (ii) preparing a nucleotide sequence comprising the at least two CRISPRs together with the one or more CRISPR spacers, wherein each of the identified one or more CRISPR spacers is flanked by CRISPRs of the at least two of the identified CRISPRs, and wherein the at least two identified CRISPRs form a functional combination with the at least one or more cas genes of the bacterial cell and the identified one or more CRISPR spacers that confers the resistance;

and (iv) introducing said nucleotide sequence into the bacterial cell thus to modulate the resistance to said bacteriophage.

7. A method for suppressing the resistance of a bacterial cell to a bacteriophage wherein the bacterial cell comprises at least one or more cas genes and at least two CRISPRs, the method comprising the step of modifying the bacterial cell by deleting the at least one or more cas genes or the at least two CRISPRs of the at least two CRISPRs in the bacterial cell wherein the cas/CRISPR functional combination mediates the resistance and the deletion results in suppressing the resistance of the bacterial cell to the bacteriophage.

8. The method according to claim 7, wherein the at least one or more cas genes or Cas proteins and the at least two CRISPRs naturally co-occur in the same bacterial cell.

9. A method for modulating the resistance of a bacterial cell against a bacteriophage wherein the bacterial cell comprises at least one or more cas genes or Cas proteins and at least two CRISPRs and one or more CRISPR spacers wherein each of the one or more CRISPR spacers is flanked by CRISPRs of the at least two of the CRISPRs, the method comprising:

(i) identifying one or more CRISPR spacers in an organism that is not the bacterial cell wherein the organism is resistant to the bacteriophage, wherein the one or more identified CRISPR spacers is a nucleic acid sequence 100% identical to a sequence of the bacteriophage genome; and

(ii) modifying the one or more identified CRISPR spacers of the bacterial cell to have a higher or lesser degree of homology to the CRISPR spacer of the organism than the bacterial cell to render the modulated resistance to the bacteriophage.

10. The method according to claim 9, wherein the modification of the one or more CRISPR spacer(s) of the bacterial cell is modification to become identical to the sequence of the one or more CRISPR spacers of the organism and the modulating the resistance is conferring the resistance to the bacteriophage.

11. The method according to claim 9, wherein the modification is modifying the sequence of the one or more CRISPR spacer(s) of the bacterial cell to reduce its(their) homology to the one or more CRISPR spacers of the organism and the modulating the resistance is decreasing the resistance to the bacteriophage.

12. A method for suppressing the resistance of a bacterial cell to a bacteriophage, wherein the bacterial cell comprises at least one or more cas genes or Cas proteins and at least two CRISPRs and one or more CRISPR spacers having a nucleic acid sequence 100% identical to a sequence of the bacteriophage genome wherein each of the one or more CRISPR spacers is flanked by CRISPRs of the at least two of the CRISPRs, the method comprising (i) identifying the one or more CRISPR spacers having a nucleic acid sequence 100% identical to a sequence of the bacteriophage genome, and (ii) deleting the one or more spacers identified in (i).

13. A method for modulating the lysotype of a bacterial cell comprising one or more cas genes or Cas proteins and at least two CRISPRs and at least one or more CRISPR spacers wherein the one or more cas genes are operably linked to a regulatory sequence, the method comprising the steps of:

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

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

14. A method for modulating the resistance of a bacterial cell against a bacteriophage, the method comprising the steps of:

(i) identifying one or more pseudo CRISPR spacers in a bacteriophage genome that provides resistance to the bacteriophage, wherein the one or more pseudo spacers are present in the genome of the bacteriophage (ii) preparing a nucleotide sequence comprising at least one cas gene operably linked to a regulatory sequence and at least two CRISPRs together with the identified one or more pseudo CRISPR spacers, wherein each of the at least one CRISPR spacer is flanked by at least two of the CRISPRs and the at least two CRISPRs, the at least one CRISPR spacer and the cas gene form a functional combination that confers resistance; and

(iii) introducing said nucleotide sequence into said bacterial cell thus to render the bacterial cell resistant to said bacteriophage.

15. A method for modulating the resistance of a bacterial cell against a bacteriophage wherein the bacterial cell comprises one or more cas genes or Cas proteins and at least two CRISPRs, wherein the one or more cas genes are operably linked to a regulatory sequence, the method comprising the steps of:

(i) identifying one or more pseudo CRISPR spacers in a bacteriophage that can provide the resistance of the bacteria cell against the bacteriophage;

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

(iii) modifying the sequence of the one or more CRISPR spacer(s) in the bacterial cell in which resistance is to be modulated such that the one or more CRISPR spacer(s) has a higher degree of homology to the one or more pseudo CRISPR spacer(s) of the bacteriophage than the unmodified CRISPR spacer in the bacterial cell against which resistance is to be modulated thus to render the bacterial cell with the increased resistance.

16. A method for rendering a bacterial cell resistant to a bacteriophage wherein the bacterial cell comprises at least one or more cas genes, at least two CRISPRs and one or more CRISPR spacers wherein each of the one or more CRISPR spacers is flanked by CRISPRs of the at least two of the CRISPRs, the method comprising:

(i) identifying one or more CRISPR spacers in an organism that is not the bacterial cell wherein the organism is resistant to the bacteriophage, wherein the one or more identified CRISPR spacers is a nucleic acid sequence 100% identical to a sequence of the bacteriophage genome; and

(ii) inserting the one or more identified CRISPR spacers of the organism into the bacterial cell, such that each of the one or more spacers of the organism is flanked by two CRISPRs wherein the bacterial cell is rendered resistant to the bacteriophage.