COMPOSITIONS AND METHODS FOR DOWNREGULATING PROKARYOTIC GENES

• US 20140113376A1
• Filed: 05/31/2012
• Published: 04/24/2014
• Est. Priority Date: 06/01/2011
• Status: Abandoned Application

• Current Assignee: Yeda Research and Development Co. Ltd. (Weizmann Institute of Science)
• Original Assignee: Yeda Research and Development Co. Ltd. (Weizmann Institute of Science)
• Inventors: Sorek, Rotem, Amitai, Gil
• Application Number:
  US14/123,267
• Publication Number:
  US 20140113376A1
• Time in Patent Office: Days
• US Class Current: 435/471
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 2320/30 Special therapeutic applications

Abstract

An isolated polynucleotide is disclosed. The polynucleotide comprises a clustered, regularly interspaced short palindromic repeat (CRISPR) array nucleic acid sequence wherein at least one spacer of the CRISPR is sufficiently complementary to a portion of at least one prokaryotic gene so as to down-regulate expression of the prokaryotic gene and further comprises a nucleic acid sequence encoding at least one CRISPR associated (CAS) polypeptide of a repeat associated mysterious protein (RAMP) family. Uses of the polynucleotides and pharmaceutical compositions comprising the polynucleotides are also disclosed.

Claims

1. An isolated polynucleotide, comprising

(i) a clustered, regularly interspaced short palindromic repeat (CRISPR) array nucleic acid sequence wherein at least one spacer of said CRISPR is sufficiently complementary to a portion of at least one prokaryotic gene so as to down-regulate expression of said prokaryotic gene;

and

(ii) a nucleic acid sequence encoding at least one CRISPR associated (CAS) polypeptide of a repeat associated mysterious protein (RAMP) family.

2. The isolated polynucleotide of claim 1, wherein said at least one spacer comprises at least two spacers, each being sufficiently complementary to a portion of different prokaryotic genes so as to down-regulate expression of said different prokaryotic genes.

3. The isolated polynucleotide of claim 1, wherein said at least one spacer comprises 26-72 base pairs.

4. The isolated polynucleotide of claim 1, wherein said prokaryotic gene is a bacterial gene.

14. The isolated polynucleotide of claim 1, further comprising a nucleic acid sequence encoding a CRISPR leader sequence.

15. The isolated polynucleotide of claim 1, wherein said at least one CRISPR associated (CAS) polypeptide of a RAMP family is of a mesophilic organism.

18. The isolated polynucleotide of claim 1, wherein said nucleic acid sequence encoding at least one CRISPR associated (CAS) polypeptide of a RAMP family comprises a sequence encoding a RAMP module.

20. A nucleic acid construct comprising the isolated polynucleotide of claim 1.

21. The nucleic acid construct of claim 20, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:

1, 6 and 11.

22. The nucleic acid construct of claim 20, comprising a nucleic acid sequence encoding at least one polypeptide having at sequence selected from the group consisting of SEQ ID NOs:

1354-1360.

5-13. -13. (canceled)

16-17. -17. (canceled)

19. (canceled)

23-26. -26. (canceled)

27. A nucleic acid construct system comprising:

(i) a first nucleic acid construct comprising an isolated polynucleotide having a clustered, regularly interspaced short palindromic repeat (CRISPR) array nucleic acid sequence wherein at least one spacer of said CRISPR is sufficiently complementary to a portion of at least one prokaryotic gene so as to down-regulate expression of said prokaryotic gene; and

(ii) a second nucleic acid construct comprising an isolated polynucleotide having a nucleic acid sequence encoding at least one CRISPR associated (CAS) polypeptide of a repeat associated mysterious protein (RAMP) family.

28. The nucleic acid construct system of claim 27, wherein said at least one CAS polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:

1354-1360.

29. The nucleic acid construct system of claim 28, wherein said first nucleic acid construct comprises a leader sequence upstream to said at least one spacer as set forth in SEQ ID NO:

1374.

30. The nucleic acid construct system of claim 27, wherein a repeat sequence of said CRISPR array is as set forth in SEQ ID NO:

1369, 1373, 1374 or 1375.

31-34. -34. (canceled)

35. A method of down-regulating expression of a gene of a prokaryotic cell, the method comprising introducing into the cell a CRISPR system polynucleotide encoding a CRISPR array and at least one CRISPR associated (CAS) polypeptide of a repeat associated mysterious protein (RAMP) family, wherein a spacer of said CRISPR array is sufficiently complementary with a portion of the gene to down-regulate expression of the gene, thereby down-regulating expression of gene of a prokaryotic cell.

36. The method of claim 35, wherein the gene is not introduced into the cell by a bacteriophage.

37. The method of claim 35, wherein the gene is integrated into a chromosome of the cell.

38. The method of claim 35, wherein the gene is endogenous to the prokaryotic cell.

39. The method of claim 35, wherein the gene is epichromosomal.

40. The method of claim 35, further comprising introducing into the cell a naï

ve CRISPR array system.

41-44. -44. (canceled)