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Cultures with Improved Phage Resistance

a technology of phages and cultures, applied in the field of cultures with improved phage resistance, can solve the problems of occupying a large space and equipment, phage contamination, and phage contamination, and achieve the effect of reducing the degree of homology to the spacer, reducing or decreasing

Inactive Publication Date: 2011-01-06
DUPONT NUTRITION BIOSCIENCES APS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]The present invention also provides methods for modulating (e.g., 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.
[0050]In addition, the present invention provides methods that facilitate the integration of one or more different bacteriophage sequences into the CRISPR locus of the bacterial strain. In some embodiments, the integration of different bacteriophage sequences in the CRISPR locus of the bacterial strain is a random event. In some alternative embodiments, the integration of different bacteriophage sequences in the CRISPR locus of the bacterial strain is not a random event. Thus, it is not always the same locus of the bacteriophage genome which is integrated into the CRISPR locus of the bacterium. However, once it is integrated it is maintained and thus becomes a robust tag to label and / or track the bacterium. Accordingly, the one or more sequences originating from the bacteriophage genome are not only new to the CRISPR locus of the parent bacterium but are also a label that is unique to each bacterium. There is therefore provided herein methods for labelling (e.g., tagging) and / or identifying bacteria.
[0068]The present invention also provides methods and compositions to utilize CRISPR-cas (i.e., natural mutagenesis, in some preferred embodiments) to construct at least one phage resistant CRISPR variant of at least target microorganism which is then used to generate mutant phage that circumvent CRISPR-cas resistance via mutation within the phage corresponding to a sequence selected from spacer sequences, pseudospacer sequences, proximal sequence, recognition motifs, etc., to enhance the virulence of the phage. In some preferred embodiments, phage with enhanced virulence find use as biocontrol agents.

Problems solved by technology

The preparation of cultures is labor intensive, occupying much space and equipment, and there is a considerable risk of contamination with spoilage bacteria and / or phages during the propagation steps.
The failure of bacterial cultures due to bacteriophage (phage) infection and multiplication is a major problem with the industrial use of bacterial cultures.

Method used

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  • Cultures with Improved Phage Resistance
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  • Cultures with Improved Phage Resistance

Examples

Experimental program
Comparison scheme
Effect test

example 1

Manipulation of Phage-Specific Spacers

[0481]In this Example, experiments conducted to manipulate phage-specific spacers are described. In some experiments, a phage specific spacer was inserted into an existing, functional CRISPR to provide resistance to the corresponding phage are described. The bacterial strain used was Streptococcus thermophilus ST0089 and the phage was phage 2972. S. thermophilus ST0089 is an industrially important strain used in the manufacture of yogurt. It is genetically amenable to manipulation, and susceptible to the well-known virulent phage 2972.

[0482]The CRISPR loci were determined in strain ST0089. This was determined preferentially by sequencing the entire genome of ST0089. Alternatively, the CRISPR loci are identified via PCR using primer sets with sequences identical to S. thermophilus CRISPR elements previously identified.

[0483]Once identified, the CRISPR loci sequences were determined as well as the proximal regions containing the relevant cas genes...

example 2

Integration of CRISPR Spacer(s)

[0500]In these experiments, integration of a CRISPR spacer into the CRISPR locus was shown to provide resistance against a bacteriophage to which the CRISPR spacer shows identity. In these experiments, S. thermophilus strain DGCC7710RH1 was produced.

[0501]Streptococcus thermophilus strain DGCC7710 (deposited at the French “Collection Nationale de Cultures de Microorganismes” under number CNCM I-2423) possesses at least 3 CRISPR loci: CRISPR1, CRISPR2, and CRISPR3. In S. thermophilus strains CNRZ1066 and LMGI8311 for which the complete genome sequence is known (Bolotin et al., Microbiol., 151:2551-1561 [2005]. CRISPR1 is located at the same chromosomal locus: between str0660 (or stu0660) and str0661 (or stu0661).

[0502]In strain DGCC7710, CRISPR1 is also located at the same chromosomal locus, between highly similar genes. CRISPR1 of strain DGCC7710 contains 33 repeats (including the terminal repeat), and thus 32 spacers.

[0503]All these spacers are differ...

example 3

Construct Integration and Knockout

[0524]In this Example, methods used for construct integration and knockout are described.

[0525]The strains used in these experiments were:

S. thermophilus DGCC7710 parent strain, sensitive to phages 858 and 2972

S. thermophilus DGCC7778 CRISPR mutant resistant to 858

S. thermophilus DGCC7778cas1KO

S. thermophilus DGCC7778cas4KO

S. thermophilus DGCC7778RT

S. thermophilus DGCC7778RT′

S. thermophilus DGCC7710R2CRISPR mutant resistant to 2972

S. thermophilus DGCC7710R2S1S2

E. coli EC1,000 provided pOR128 (See, Russell and Klaenhammer, Appl. Environ. Microbiol., 67:43691-4364 [2001])

Escherichia coli pCR2.1TOPO provided pTOPO (See, Invitrogen catalog #K4500-01)

[0526]The following plasmids were used in these experiments:

pTOPO, a plasmid used for sub-cloning of the various constructs

pTOPOcas1ko contains an integral fragment of cas1

pTOPOcas4ko contains an integral fragment of cas4

pTOPOS1S2 contains the S1S2 spacer construct

pTOPO RT contains the RT terminal repeat con...

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Abstract

The present invention provides methods and compositions related to modulating the resistance of a cell against a target nucleic acid or a transcription product thereof. In some preferred embodiments, the present invention provides compositions and methods for the use of one or more cas genes or proteins for modulating the resistance of a cell against a target nucleic acid or a transcription product thereof. In some embodiments, the present invention provides methods and compositions that find use in the development and use of strain combinations and starter culture rotations. In additional embodiments, the present invention provides methods for labelling and / or identifying bacteria. In some preferred embodiments, the present invention provides methods for the use of CRISPR loci to determine the potential virulence of a phage against a cell and the use of CRISPR-cas to modulate the genetic sequence of a phage for increased virulence level. In still further embodiments, the present invention provides means and compositions for the development and use of phages as biocontrol agents.

Description

FIELD OF THE INVENTION[0001]The present invention provides methods and compositions related to modulating the resistance of a cell against a target nucleic acid or a transcription product thereof. In some preferred embodiments, the present invention provides compositions and methods for the use of one or more cas genes or proteins for modulating the resistance of a cell against a target nucleic acid or a transcription product thereof. In some embodiments, the present invention provides methods and compositions that find use in the development and use of strain combinations and starter culture rotations. In additional embodiments, the present invention provides methods for labelling and / or identifying bacteria. In some preferred embodiments, the present invention provides methods for the use of CRISPR loci to determine the potential virulence of a phage against a cell and the use of CRISPR-cas to modulate the genetic sequence of a phage for increased virulence level. In still further...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/74C12N15/74C12N1/21A23L1/00A23C9/12A23K20/195
CPCA23C9/123A23C9/1238A23C2220/202C12N1/20C12N15/746C12Q1/70C12N2795/00011A23Y2240/75C12Q2525/151A23V2400/249
Inventor BARRANGOU, RODOLPHEFREMAUX, CHRISTOPHEHORVATH, PHILIPPEROMERO, DENNISBOYAVAL, PATRICK
Owner DUPONT NUTRITION BIOSCIENCES APS
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