Altering microbial populations and modifying microbiota

Patent metadata

Field	Value
Publication number	US-12514867-B2
Application number	US-202117392827-A
Country	US
Kind code	B2
Filing date	Aug 3, 2021
Priority date	May 6, 2015
Publication date	Jan 6, 2026
Grant date	Jan 6, 2026

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 of modifying a mixed population of bacteria, wherein the mixed population of bacteria comprises cells of a first bacterial species of Bacillus subtilis , host cells of a second bacterial species, and a third bacterial species Lactobacillus lactis , and 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 a plurality of vectors comprising a first nucleic acid sequence encoding a Cas nuclease and a second nucleic acid sequence for producing a host modifying crRNA (HM-crRNA), and (b) expressing the Cas nuclease and the HM-crRNA in the host cells, wherein the HM-crRNA is operable with the Cas nuclease in the host cells, wherein the second 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 host cells to guide the Cas nuclease to cut the target sequence in the host cells of the second bacterial species; whereby the host cells of the second bacterial species are killed or growth of the host cells is reduced, thereby reducing the proportion of the host cells of the second bacterial species and altering the relative ratio of the first and second bacterial species in the mixed population of bacteria; and wherein the host cells of the second bacterial species are killed by at least 1000-fold or growth of the host cells of the second bacterial species is reduced by at least 1000-fold, and wherein the first bacterial species is not killed or growth of first bacterial species is not reduced.

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

3 . The method of claim 1 , wherein the first bacterial species has a 16s ribosomal RNA-encoding DNA sequence that is at least 80% identical to an 16s ribosomal RNA-encoding DNA sequence of the second bacterial species.

4 . The method of claim 1 , wherein expression of the Cas nuclease is induced in the host cells, whereby the Cas nuclease and the HM-crRNA are combined in the host cells.

5 . The method of claim 1 , wherein step (b) comprises inducing production of the HM-crRNA in the host cells.

6 . The method of claim 1 , wherein the target sequence is comprised by an essential gene, an antibiotic resistance gene, or a virulence gene of the host cells.

7 . The method of claim 1 , wherein the second bacterial species is an Enterobacteriaceae species.

8 . The method of claim 1 , wherein the second bacterial species is Escherichia coli.

9 . The method of claim 1 , wherein the mixed population is in a human gut microbiota.

10 . The method of claim 1 , wherein Cas expression is inducible in the host cells.

11 . The method of claim 1 , wherein the second nucleic acid sequence for producing the HM-crRNA is operably linked to an inducible promoter.

12 . The method of claim 1 , wherein the host cells are Firmicutes cells.

13 . The method of claim 1 , wherein each vector is a virus, a phage, a phagemid, or a plasmid.

14 . The method of claim 1 , wherein the host cells are Staphylococcus, Streptococcus, Pseudomonas, Salmonella, Listeria, E coli, Desulfovibrio, Vibrio or Clostridium cells.

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

16 . The method of claim 1 , wherein the target sequence is comprised by an essential gene that is required for protein production.

17 . The method of claim 1 , wherein the Cas nuclease is a Cas nuclease of Type I or Type II CRISPR system.

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

19 . A method of modifying a mixed population of bacteria, wherein the mixed population of bacteria comprises cells of a first bacterial species and host cells of a second bacterial species, and 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 a plurality of vectors comprising a first nucleic acid sequence encoding a Cas nuclease and a second nucleic acid sequence for producing a host modifying crRNA (HM-crRNA), and (b) expressing the Cas nuclease and the HM-crRNA in the host cells, wherein the HM-crRNA is operable with the Cas nuclease in the host cells, wherein the second 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 host cells to guide the Cas nuclease to cut the target sequence in the host cells of the second bacterial species; wherein the target sequence is present in multiple copies in the host cells of the second bacterial species; whereby the host cells of the second bacterial species are killed or growth of the host cells is reduced, thereby reducing the proportion of the host cells of the second bacterial species and altering the relative ratio of the first and second bacterial species in the mixed population of bacteria; and wherein the host cells of the second bacterial species are killed by at least 1000-fold or growth of the host cells of the second bacterial species is reduced by at least 1000-fold, and wherein the first bacterial species is not killed or growth of first bacterial species is not reduced.

Assignees

• Snipr Tech Ltd

Inventors

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

Classifications

• A61K2300/00

  Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00 · CPC title

• C12N2795/00032

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

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N1/20

  Bacteria; Culture media therefor · CPC title

• A61K48/005

  characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered · CPC title