Bifidobacteria crispr sequences
AssigneeDanisco A/S
InventorsRodolph Barrangou, Philippe Horvath, Dennis A. Romero, Lindsay L. Traeger
Life of this patent
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Abstract
The invention relates to CRISPR sequences found in bifidobacteria and their uses.
Claims54
-45. (canceled)
An isolated nucleic acid comprising a Bala1 spacer sequence selected from SEQ ID NOs:
- 3-24, a Bala1 repeat sequence selected from SEQ ID NO:
- 2 and sequence variants thereof, a CRISPRo spacer sequence selected from SEQ ID NOs:
- 43-52, 61-67, 76-82, 93-101, 110-116, 126-133, and 144-152 or a CRISPRo repeat sequence selected from SEQ ID NOs:
- 32-42, 53-60, 68-75, 83-92, 102-109, 117-125, and 134-143.
An isolated nucleic acid comprising a sequence capable of hybridizing under stringent conditions to a portion of a bifidobacteria CRISPR locus selected from the group consisting of Bala1 (SEQ ID NO:
- 1), CRISPRo9a (SEQ ID NO:
- 25), CRISPRo9b (SEQ ID NO:
- 26), CRISPRo91 (SEQ ID NO:
- 27), CRISPRoI 64 (SEQ ID NO:
- 28), CRISPRO228 (SEQ ID NO:
- 29), CRISPRo245 (SEQ ID NO:
- 30), and CRISPRo327 (SEQ ID NO:
- 31).
A vector comprising a Bala1 spacer sequence selected from SEQ ID NOs:
- 3-24, a Bala1 repeat sequence selected from SEQ ID NO:
- 2 and sequence variants thereof, a CRISPRo repeat sequence selected from SEQ ID NOs:
- 32-42, 53-60, 68-75, 83-92, 102-109, 117-125, and 134-143 or a CRISPRo spacer sequence selected from SEQ ID NOs:
- 43-52, 61-67, 76-82, 93-101, 110-116, 126-133, and 144-152.
A recombinant strain of bacteria with altered phage resistance comprising a Bala1 spacer sequence selected from SEQ ID NOs:
- 3-24, a Bala1 repeat sequence selected from SEQ ID NO:
- 2 and variants thereof, a CRISPRo spacer sequence selected from SEQ ID NOs:
- 43-52, 61-67, 76-82, 93-101, 110-116, 126-133, and 144-152 or a CRISPRo repeat sequence selected from SEQ ID NOs:
- 32-42, 53-60, 68-75, 83-92, 102-109, 117-125, and 134-143.
A method for preparing a recombinant bacteria strain with altered phage resistance comprising:
- (a) transforming bacteria with a nucleic acid comprising a Bala1 spacer sequence selected from
- SEQ ID NOs:
- 3-24 or a CRISPRo spacer sequence selected from SEQ ID NOs:
- 43-52, 61-67, 76-82, 93-101, 110-116, 126-133, and 144-152;
- (b) contacting the transformed bacteria with a phage;
- and
- (c) isolating the transformed bacteria that exhibit altered resistance to the phage.
A method for strain typing comprising:
- (a) amplifying a genomic DNA sequence from a strain of interest using an at least one primer pair, thereby creating an amplicon, wherein the genomic DNA sequence comprises at least a portion of a sequence of a CRISPR locus;
- (b) detecting the amplicon generated in step (a), whereby said detected amplicon indicates a strain type.
A strain typing kit comprising:
- (a) a container including 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 a genomic DNA sequence including a CRISPR locus such that the primer pair amplifies at least a portion of a repeat or spacer sequence of a CRISPR locus;
- and (b) a container including a detection mixture comprising a probe capable of hybridizing under stringent conditions to at least a portion of the CRISPR locus amplified by the primer pair.
A method for tagging a bifidobacterial strain comprising:
- (a) exposing a parent bifidobacterial strain to a phage;
- (b) selecting a phage insensitive mutant;
- (c) comparing a CRISPR locus 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 locus sequence of the phage insensitive mutant indicates that the strain is tagged.
Dependencies are inferred from the claim text.