TUNING MICROBIAL POPULATIONS WITH PROGRAMMABLE NUCLEASES

• US 20150064138A1
• Filed: 09/03/2014
• Published: 03/05/2015
• Est. Priority Date: 09/05/2013
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: LU, TIMOTHY KUAN-TA, CITORIK, ROBERT JAMES, MIMEE, MARK KYLE
• Granted Patent: US 10,760,065 B2
• Time in Patent Office: Days
• US Class Current: 424/93.2
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• 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 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Various aspects and embodiments of the invention are directed to methods and compositions for reversing antibiotic resistance or virulence in and/or destroying pathogenic microbial cells such as, for example, pathogenic bacterial cells. The methods include exposing microbial cells to a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease targeted to one or multiple genes of interest.

Claims

1. A method, comprising delivering to microbial cells in vivo a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease that targets a gene or nucleotide sequence of interest.

2. The method of claim 1, wherein the programmable nuclease is an RNA-guided nuclease.

3. The method of claim 2, wherein the engineered autonomously distributed circuit contains a Cas9 nuclease, a guide RNA (gRNA), and/or a transactivating small RNA (tracrRNA) derived from the Type II CRISPR-Cas system of Streptococcus pyogenes.

4. The method of claim 3, wherein the gRNA and the tracrRNA form a chimeric single-guide RNA molecule.

5. The method of claim 1, wherein the programmable nuclease is a transcriptional activator like (TAL)-effector nuclease (TALEN) or a zinc finger nuclease (ZFN).

7. The method of claim 1, wherein the microbial cells are Gram-negative bacterial cells or Gram-positive bacterial cells.

9. The method of claim 1, wherein the microbial cells are pathogenic microbial cells or non-pathogenic bacterial cells.

10. The method of claim 9, wherein the microbial cells are pathogenic microbial cells that are selected from extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli, ESBL Klebsiella pneumonia, Pseudomonas aeruginosa, vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant (MDR) Acinetobacter baumannii, and MDR Enterobacter spp.

12. The method of claim 1, wherein the gene or nucleotide sequence of interest is located episomally or chromosomally.

14. The method of claim 1, wherein the gene or nucleotide sequence of interest is a virulence factor gene, a toxin gene, an antibiotic resistance gene, a remodeling gene or a modulatory gene.

18. The method of claim 1, wherein the nucleotide sequence of interest comprises 16S ribosomal DNA (rDNA).

19. The method of claim 1, wherein the delivery vehicle is a bacteriophage, engineered particle derived from a bacteriophage, or a donor bacterial cell comprising a conjugative plasmid.

20. The method of claim 19, wherein the delivery vehicle is a phagemid.

22. The method of claim 19, wherein the delivery vehicle is a probiotic bacterial cell and/or a commensal bacterial cell.

23. The method of claim 1, wherein the delivery vehicle is a nanoparticle-based delivery vehicle, chemical-based delivery vehicle, a non-chemical-based delivery vehicle, a particle-based delivery vehicle, or a viral-based delivery vehicle.

24. The method of claim 1, wherein the programmable nuclease is operably linked to an inducible promoter.

6. (canceled)

8. (canceled)

11. (canceled)

13. (canceled)

15-17. -17. (canceled)

21. (canceled)

25. A phagemid, a bacteriophage or a donor bacterial cell comprising an engineered autonomously distributed circuit that contains at least one nucleic acid encoding a programmable nuclease that targets a gene or nucleotide sequence of interest.

26-67. -67. (canceled)

68. A nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease that targets a virulence factor or an antibiotic resistance gene.

79. A library comprising a plurality of nucleic acids of claim 68.

69-78. -78. (canceled)

80-86. -86. (canceled)

87. A method, comprising delivering to microbial cells in vivo

(a) a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease that targets a gene or nucleotide sequence of interest;

and

(b) a nucleic acid encoding an reporter construct that is responsive to double-stranded DNA breaks.

88. (canceled)