IP Library › Granted Patent US 12,264,313
Granted Patent B2
US 12,264,313 · App. 17/280,436 · Granted Apr 1, 2025

Recombinant type I CRISPR-Cas system and uses thereof for genome modification and alteration of expression

Inventors: Rodolphe Barrangou (Raleigh, NC); Claudio Hidalgo Cantabrana (Cary, NC)
Assignee: North Carolina State University
C12N15/102C12N9/22C12N15/111C12N2310/20
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Quick Facts
Patent No.
US 12,264,313
App. No.
17/280,436
Granted
Apr 1, 2025
Kind
B2
Abstract

This invention is directed to recombinant Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays and recombinant nucleic acid constructs encoding Type 1-E CASCADE complexes, plasmids, retroviruses and bacteriophage comprising the same, and methods of use thereof for modifying genomes and expression. Further disclosed are methods of modifying (editing) the genome of target organisms using the constructs.

Claims (56)

1. A method of modifying the genome of a Lactobacillus spp. or Bifidobacterium spp. bacterium, comprising introducing into the Lactobacillus spp. or Bifidobacterium spp. bacterium

(a) a recombinant nucleic acid construct comprising a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array comprising two or more repeat sequences and one or more spacer nucleotide sequence(s), wherein each of the one or more spacer sequences comprises a 3′ end and a 5′ end and is linked at its 5′ end and at its 3′ end to a repeat sequence, and each of the one or more spacer sequences is complementary to a target sequence (protospacer) in the bacterium that is located immediately adjacent (3′) to a protospacer adjacent motif (PAM), wherein the PAM comprises a nucleotide sequence of 5′-NAA-3′, 5′-AAA-3′ or 5′-AA-3′ that is immediately adjacent to and 5′ of the target sequence;

(b) a recombinant nucleic acid construct encoding a Type I-E CRISPR associated complex for antiviral defense complex (Cascade complex) comprising:

a Cse1 polypeptide encoded by the nucleotide sequence of SEQ ID NO:82,

a Cse2 polypeptide encoded by the nucleotide sequence of SEQ ID NO:83,

a Cas7 polypeptide encoded by the nucleotide sequence of SEQ ID NO:84,

a Cas5 polypeptide encoded by the nucleotide sequence of SEQ ID NO:85, and

a Cas6 polypeptide encoded by encoded by the nucleotide sequence of SEQ ID NO:86;

(c) a Cas3 polypeptide or a polynucleotide encoding a Cas3 polypeptide; and

(d) a repair template, thereby modifying the genome of the Lactobacillus spp. or Bifidobacterium spp. bacterium.

2. A method of modifying the genome of a Lactobacillus spp. or Bifidobacterium spp. bacterial cell that comprises an endogenous Type I-E CRISPR-Cas system, comprising introducing into the Lactobacillus spp. or Bifidobacterium spp. bacterial cell

(a) a recombinant nucleic acid construct comprising a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array comprising two or more repeat sequences and one or more spacer nucleotide sequence(s), wherein each of the one or more spacer sequences comprises a 3′ end and a 5′ end and is linked at its 5′ end and at its 3′ end to a repeat sequence, and each of the one or more spacer sequences is complementary to a target sequence (protospacer) in the genome of the bacterial cell, wherein the target sequence is located immediately adjacent (3′) to a protospacer adjacent motif (PAM), wherein the PAM comprises a nucleotide sequence of 5′-NAA-3′, 5′-AAA-3′ or 5′-AA-3′ that is immediately adjacent to and 5′ of the target sequence; and

(b) a repair template, thereby modifying the genome of the bacterial cell wherein the two or more repeat sequences comprise about 20 to about 28 consecutive nucleotides of any one of the nucleotide sequences of SEQ ID NOs: 1 to 68, in any combination, thereby modifying the genome of the Lactobacillus spp. or Bifidobacterium spp. bacterial cell.

3. The method of claim 1 , wherein the recombinant nucleic acid construct comprising a CRISPR array of (a), the recombinant nucleic acid construct encoding a Cascade complex of (b), and the polynucleotide encoding a Cas3 polypeptide of (c) are comprised in a single vector or expression cassette or are comprised in two or three separate vectors and/or expression cassettes.

4. The method of claim 2 , wherein the recombinant nucleic acid construct comprising a CRISPR array is comprised in an expression cassette or a vector.

5. The method of claim 2 , wherein the two or more repeat sequences comprise any one of the nucleotide sequences of SEQ ID NOs: 1 to 68, in any combination.

6. The method of claim 1 , wherein the recombinant nucleic acid construct comprising a CRISPR array of (a), the recombinant nucleic acid construct encoding the Cascade complex of (b), and the polynucleotide encoding a Cas3 polypeptide of (c) are operably linked to a single promoter or are operably linked to two or three separate promoters.

7. The method of claim 1 , wherein the target sequence is located in:

a coding (sense) strand of a gene;

a non-coding (antisense) strand of a gene;

a coding (sense) strand of an intragenic region of a gene; or

a non-coding (antisense) strand of an intragenic region of a gene.

8. The method of claim 7 , wherein the target sequence is located on a chromosome, an extrachromosomal nucleic acid, a plasmid, or a mobile genetic element.

9. The method of claim 1 , wherein modifying the genome comprises making a deletion, an insertion, a single base pair addition, a single base pair substitution, a single base pair removal, a stop codon insertion, a conversion of one base pair to another base pair, or any combination thereof.

10. The method of claim 1 , wherein the Cas3 polypeptide comprises the amino acid sequence of SEQ ID NO: 119, wherein the Cas3 polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 87, or wherein the Cas3 polypeptide comprises the amino acid sequence of SEQ ID NO: 119 and the Cas3 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:87.

11. A method of altering the expression of a target gene in a Lactobacillus spp. or Bifidobacterium spp. bacterium, comprising introducing into the Lactobacillus spp. or Bifidobacterium spp. bacterium

(a) a recombinant nucleic acid construct comprising a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array comprising two or more repeat sequences and one or more spacer nucleotide sequence(s), wherein each spacer sequence is linked at its 5′ end and at its 3′ end to a repeat sequence, and the spacer sequence is complementary to a target sequence (protospacer) from a target genome that is located immediately adjacent (3′) to a protospacer adjacent motif (PAM), wherein the PAM comprises a nucleotide sequence of 5′-NAA-3′, 5′-AAA-3′ or 5′-AA-3′ that is immediately adjacent to and 5′ of the target sequence; and

(b) a recombinant nucleic acid construct encoding a Type I-E CRISPR associated complex for antiviral defense complex (Cascade complex) comprising:

a Cse1 polypeptide encoded by the nucleotide sequence of SEQ ID NO:82,

a Cse2 polypeptide encoded by the nucleotide sequence of SEQ ID NO:83,

a Cas7 polypeptide encoded by the nucleotide sequence of SEQ ID NO:84,

a Cas5 polypeptide encoded by the nucleotide sequence of SEQ ID NO:85, and

a Cas6 polypeptide encoded by encoded by the nucleotide sequence of SEQ ID NO:86, thereby altering expression of the target gene in the Lactobacillus spp. or Bifidobacterium spp. bacterium.

12. The method of claim 11 , wherein the recombinant nucleic acid construct comprising a CRISPR array of (a) and the recombinant nucleic acid construct encoding a Cascade complex of (b) are comprised in a single vector or are comprised in separate vectors.

13. The method of claim 11 , wherein the two or more repeat sequences comprise any one of the nucleotide sequences of SEQ ID NOs: 1 to 68, in any combination.

14. The method of claim 11 , wherein the recombinant nucleic acid construct comprising a CRISPR array of (a) and the recombinant nucleic acid construct encoding the Cascade complex of (b) are operably linked to a single promoter or are operably linked to separate promoters.

15. The method of claim 11 , wherein the target sequence is located in:

a coding (sense) strand of a gene;

a non-coding (antisense) strand of a gene;

a coding (sense) strand of an intragenic region of a gene; or

a non-coding (antisense) strand of an intragenic region of a gene.

16. The method of claim 15 , wherein the gene or the intergenic region of the gene is located on a chromosome, an extrachromosomal nucleic acid, a plasmid, or a mobile genetic element.

17. The method of claim 1 , wherein the Lactobacillus spp. or Bifidobacterium spp. bacterium comprises Lactobacillus acidophilus ( L. acidophilus ), L. brevis, L. bulgaricus, L. plantarum, L. rhamnosus, L. fermentum, L helveticus, L. salivarius, L. gasseri, L. reuteri L. crispatus, L. casei, Bifidobacterium animalis lactis, Bifidobacterium longum, Bifidobacterium bifidum or Bifidobacterium breve.

18. The method of claim 2 , wherein the Lactobacillus spp. or Bifidobacterium spp. bacterial cell comprises a cell from Lactobacillus acidophilus ( L. acidophilus ), L. brevis, L. bulgaricus, L. plantarum, L. rhamnosus, L. fermentum, L helveticus, L. salivarius, L. gasseri, L. reuteri L. crispatus, L. casei, Bifidobacterium animalis lactis, Bifidobacterium longum, Bifidobacterium bifidum or Bifidobacterium breve.

19. The method of claim 11 , wherein the Lactobacillus spp. or Bifidobacterium spp. bacterium comprises Lactobacillus acidophilus ( L. acidophilus ), L. brevis, L. bulgaricus, L. plantarum, L. rhamnosus, L. fermentum, L helveticus, L. salivarius, L. gasseri, L. reuteri L. crispatus, L. casei, Bifidobacterium animalis lactis, Bifidobacterium longum, Bifidobacterium bifidum or Bifidobacterium breve.

20. The method of claim 3 , wherein the vector is a recombinant plasmid, bacteriophage, or retrovirus.

21. The method of claim 4 , wherein the vector is a recombinant plasmid, bacteriophage, or retrovirus.

22. The method of claim 5 , wherein the two or more repeat sequences comprise about 20 to about 28 consecutive nucleotides of any one of the nucleotide sequences of SEQ ID NOs:1, 10, 19, 28, 37, 42, 51, or 60, in any combination.

23. The method of claim 6 , wherein the single promoter or the two or three separate promoters comprise the nucleotide sequence of any of SEQ ID NOs:69 to 72.

24. The method of claim 1 , wherein the recombinant nucleic acid construct comprising a CRISPR array of (a), the recombinant nucleic acid construct encoding the Cascade complex of (b), and the polynucleotide encoding a Cas3 polypeptide of (c) are operably linked to a single terminator sequence or are operably linked to two or three separate terminator sequences.

25. The method of claim 24 , wherein the terminator sequence or the two or three separate terminator sequences comprise the nucleotide sequence of any one of SEQ ID NOs:77 to 81.

26. The method of claim 12 , wherein the vector is a recombinant plasmid, bacteriophage, or retrovirus.

27. The method of claim 13 , wherein the two or more repeat sequences comprise about 20 to about 28 consecutive nucleotides of any one of the nucleotide sequences of SEQ ID NOs:1, 10, 19, 28, 37, 42, 51, or 60, in any combination.

28. The method of claim 14 , wherein the promoter or the separate promoters comprise the nucleotide sequence of any of SEQ ID NOs:69 to 72.

29. The method of claim 11 , wherein the recombinant nucleic acid construct comprising a CRISPR array of (a) and the recombinant nucleic acid construct encoding the Cascade complex of (b) are operably linked to a single terminator sequence or are operably linked to separate terminator sequences.

30. The method of claim 29 , wherein the terminator or the separate terminator sequences comprise the nucleotide sequence of any of SEQ ID NOs:77 to 81.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 8, 2021
From: BARRANGOU, RODOLPHE; CANTABRANA, CLAUDIO HIDALGO
To: NORTH CAROLINA STATE UNIVERSITY
Reel/Frame 056467/0885 →
Continuity (2)
Provisional Application 62739669 · Oct 1, 2018
Related Publication 20220056433A1 · Feb 24, 2022
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