Altering microbial populations and modifying microbiota

Patent metadata

Field	Value
Publication number	US-10463049-B2
Application number	US-201715817135-A
Country	US
Kind code	B2
Filing date	Nov 17, 2017
Priority date	May 6, 2015
Publication date	Nov 5, 2019
Grant date	Nov 5, 2019

Abstract

The invention relates to methods, uses, systems, arrays, engineered nucleotide sequences and vectors for inhibiting bacterial population growth or for altering the relative ratio of sub-populations of first and second bacteria in a mixed population of bacteria. The invention is particularly useful, for example, for treatment of microbes such as for environmental, medical, food and beverage use. The invention relates inter alia to methods of controlling microbiologically influenced corrosion (MIC) or biofouling of a substrate or fluid in an industrial or domestic system.

First claim

The invention claimed is:

1. A method for modifying a mixed population of bacteria, wherein the mixed population of bacteria comprises a first bacterial sub-population and a second bacterial sub-population, wherein the first bacterial sub-population comprises a first bacterial species and the second bacterial sub-population comprises a population of host cells of a second bacterial species, wherein the second bacterial species is a different species than the first bacterial species, the method comprising:
(a) contacting the mixed population of bacteria with an engineered nucleic acid for producing a plurality of different host modifying crRNAs (HM-crRNAs), and
(b) producing the plurality of different HM-crRNAs in the host cells, wherein the plurality of different HM-crRNAs comprises a first nucleotide sequence that hybridizes to a first target sequence in the host cells; and a second nucleotide sequence that hybridizes to a second target sequence in the host cells, wherein the second target sequence is different from the first target sequence; and wherein:
1) the first target sequence is comprised by a first antibiotic resistance gene or RNA thereof and the second target sequence is comprised by a second antibiotic resistance gene or RNA thereof;
2) the first target sequence is comprised by an antibiotic resistance gene or RNA thereof and the second target sequence is comprised by an essential gene or a virulence gene or RNA thereof;
3) the first target sequence is comprised by a first essential gene or RNA thereof and the second target sequence is comprised by a second essential gene or a virulence gene or RNA thereof; or
4) the first target sequence is comprised by a first virulence gene or RNA thereof and the second target sequence is comprised by an essential gene or a second virulence gene or RNA thereof;

wherein:
(i) the plurality of HM-crRNAs is operable with a Type II Cas in the host cells, wherein the engineered nucleic acid and the Type II Cas are comprised by a Type II HM-CRISPR/Cas system in the host cells; and
(ii) the plurality of HM-crRNAs guide the Type II Cas to modify the target sequences in the host cells,
wherein the host cells are modified by the Type II HM-CRISPR/Cas system.

2. The method of claim 1 , wherein the host cells are killed or growth of the host cells is reduced.

3. The method of claim 1 , wherein the Type II Cas is a Cas9.

4. The method of claim 1 , wherein the Type II Cas is encoded by an engineered nucleic acid.

5. The method of claim 1 , wherein the Type II Cas is a functional endogenous Cas of the host cells.

6. The method of claim 1 , wherein the engineered nucleic acid for producing the plurality of HM-crRNAs is present in a phage, phagemid or plasmid.

7. The method of claim 4 , wherein the engineered nucleic acid encoding the Type II Cas is present in a phage, phagemid or plasmid.

8. The method of claim 1 , wherein the mixed population of bacteria is present in a human microbiota.

9. The method of claim 1 , wherein the host cells are gram positive cells.

10. The method of claim 9 , wherein the target sequence is a host target sequence.

11. The method of claim 1 , wherein the Type II Cas is a Streptococcus Cas9.

12. The method of claim 1 , wherein the Type II HM-CRISPR/Cas system comprises an endogenous tracrRNA of the host cells.

13. The method of claim 1 , wherein the Type II HM-CRISPR/Cas system comprises a tracrRNA, and wherein the tracrRNA is encoded by an engineered nucleic acid.

14. The method of claim 13 , wherein the engineered nucleic acid for producing the plurality of HM-crRNAs encodes a single guide RNA comprising the tracrRNA and the plurality of HM-crRNAs.

15. The method of claim 1 , wherein the second bacterial species is C. difficile, E. coli or a Salmonella species.

16. The method of claim 1 , wherein the mixed population of bacteria comprises E. coli and a bacterial species selected from the group consisting of Lactobacillus and Streptococcus.

17. The method of claim 1 , wherein the method inhibits growth of the host cells on a surface.

18. The method of claim 1 , wherein the first bacterial species has a 16s ribosomal RNA-encoding DNA sequence that is at least about 80% identical to a 16s ribosomal RNA-encoding DNA sequence of the second bacterial species, and wherein the growth of the first bacterial species in the mixed population is not inhibited.

19. The method of claim 1 , wherein the first bacterial species is a Firmicutes and the second bacterial species is a Firmicutes.

20. The method of claim 1 , wherein the first bacterial species is a gram positive species and the second bacterial species is a gram positive species.

21. The method of claim 1 , wherein the mixed population of bacteria comprises a third bacterial species.

22. The method of claim 21 , wherein (i) the first bacterial species is a Firmicutes , the second bacterial species is a Firmicutes and the third bacterial species is a human gut commensal species or a human gut probiotic species; or (ii) wherein the first bacterial species is a gram positive species, the second bacterial species is a gram positive species and the third bacterial species is a human gut commensal species or a human gut probiotic species.

23. The method of claim 1 , wherein the host cells are wild-type cells.

24. The method of claim 1 , wherein the method reduces the growth of the host cells by at least 5-fold.

25. The method of claim 1 , wherein the mixed population of bacteria is present in a subject, and wherein the host cells cause a disease in the subject.

26. The method of claim 25 , wherein the mixed population of bacteria is a gut microbiota of a human or an animal.

27. The method of claim 1 , wherein the mixed population of bacteria is present in an industrial or medical fluid, an apparatus, a container, a waterway, water, a beverage, a foodstuff, or a cosmetic, and wherein the host cells cause contamination of the industrial or medical fluid, apparatus, container, waterway, water, beverage, foodstuff or cosmetic.

Assignees

• Snipr Tech Ltd

Inventors

• Clube Jasper
• Sommer Morten
• Grøndahl Christian
• Van Der Helm Eric
• Vazquez-Uribe Ruben

Classifications

• C12N2795/00032

  Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N15/113 Primary

  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 title

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title