Altering microbial populations and modifying microbiota

Patent metadata

Field	Value
Publication number	US-11844760-B2
Application number	US-202016813615-A
Country	US
Kind code	B2
Filing date	Mar 9, 2020
Priority date	May 6, 2015
Publication date	Dec 19, 2023
Grant date	Dec 19, 2023

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 modified bacterial population produced by a method of modifying a mixed population of bacteria, wherein the mixed population 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 target host cells of a second bacterial species, wherein the second bacterial species is a different species than the first bacterial species, wherein cells of the first bacterial subpopulation comprise a conjugative plasmid comprising: (i) an origin of transfer (oriT) and (ii) an engineered nucleic acid sequence for producing a target host modifying crRNA (HM-crRNA), the method comprising
a. transferring the conjugative plasmid from cells of the first bacterial subpopulation to the target host cells, and
b. producing the HM-crRNA in the target host cells,

wherein the HM-crRNA is operable with a Cas nuclease in the target host cells,
wherein the engineered nucleic acid sequence and the Cas nuclease are comprised by a HM-CRISPR/Cas system, and
wherein
the HM-crRNA comprises a nucleic acid sequence that is capable of hybridizing to a target sequence in the target host cells to guide the Cas nuclease to modify the target sequence in the target host cells;

whereby the target host cells are killed or growth of the target host cells is reduced, thereby reducing the proportion of the target host cells and altering the relative ratio of the first and second bacterial sub-populations in the modified population of bacteria.

2. The modified bacterial population of any claim 1 , wherein the population is in a container for medical use, a container for nutritional use or a sterilised container.

3. The modified bacterial population of claim 2 , wherein the sterilized container is an inhaler, a syringe, or a container connected to an IV needle.

4. The modified bacterial population of claim 1 , wherein the mixed population of bacteria comprises E. coli.

5. The modified bacterial population of claim 1 , wherein the method reduces the target host cells by at least 5-fold.

6. The modified bacterial population 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, wherein the growth of the first bacterial species in the mixed population of bacteria is not inhibited by the HM-CRISPR/Cas system.

7. The modified bacterial population of claim 6 , wherein: (i) the first bacterial species is a Firmicutes and the second bacterial species is a Firmicutes; or (ii) the first bacterial species is a gram positive species and the second bacterial species is a gram positive species.

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

9. The modified bacterial population of claim 8 , 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) 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.

10. The modified bacterial population of claim 1 , wherein the Cas nuclease is an endogenous Cas nuclease of the target host cells.

11. The modified bacterial population of claim 1 , wherein the method comprises contacting the mixed population of bacteria with a plurality of engineered nucleic acid sequences for producing different HM-crRNAs.

12. The modified bacterial population of claim 11 , wherein each engineered nucleic acid sequence comprises a HM-CRISPR array for producing (i) a first HM-crRNA comprising a nucleic acid sequence that is capable of hybridizing to a first target sequence of the target host cells to guide Cas nuclease modification to the first target sequence; and (ii) a second HM-crRNA comprising a nucleic acid sequence that is capable of hybridizing to a second target sequence of the target host cells to guide Cas nuclease modification to the second target sequence, wherein the first target sequence and the second target sequence are different, wherein the Cas nuclease modification of the first target sequence or the second target sequence kills the target host cells or reduces growth of the target host cells, thereby reducing the proportion of the target host cells and altering the relative ratio of the first and second bacterial sub-populations in the mixed population of bacteria.

13. The modified bacterial population of claim 12 , wherein:
(i) 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;
(ii) 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;
(iii) 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
(iv) 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.

14. The modified bacterial population of claim 1 , wherein the first bacterial species and the second bacterial species are human, human gut, non-human animal or environmental microbiota species.

15. The modified bacterial population of claim 1 , wherein the target host cells are wild-type cells.

16. The modified bacterial population of claim 1 , wherein the mixed population of bacteria comprises E. coli and Lactobacillus bacteria.

17. The modified bacterial population of claim 1 , wherein the mixed population of bacteria comprises E. coli, Streptococcus and Lactobacillus bacteria.

18. The modified bacterial population of claim 1 , wherein the target host cells are E. coli cells.

19. The modified bacterial population of claim 1 , wherein the target host cells are Streptococcus thermophilus cells.

20. The modified bacterial population of claim 1 , wherein the HM-CRISPR/Cas system comprises a tracrRNA, and wherein the tracrRNA is encoded by an engineered nucleic acid sequence.

21. The modified bacterial population of claim 1 , wherein the engineered nucleic acid sequence for producing the HM-crRNA encodes a single guide RNA (HM-gRNA) comprising a sequence of the HM-crRNA.

22. The modified bacterial population of claim 1 , wherein the Cas nuclease is a Cas nuclease of Type I CRISPR system.

23. The modified bacterial population of claim 1 , wherein the Cas nuclease is a Cas9.

24. The modified bacterial population of claim 1 , wherein the HM-CRISPR/Cas system comprises an endogenous tracrRNA of the target host cells.

25. A bacterial transplant medicament comprising the modified bacterial population of claim 1 .

Assignees

• Snipr Tech Ltd

Inventors

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

Classifications

• A61K31/7105 Primary

  Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links · CPC title

• A01N63/00

  Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates (containing compounds of determined constitution A01N27/00 - A01N59/00; unicellular algae A01N65/03) · CPC title

• A01N63/50

  Isolated enzymes; Isolated proteins (peptides A01N37/46) · CPC title

• A01N63/60

  Isolated nucleic acids · CPC title

• A61K31/711

  Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links · CPC title