Altering microbial populations & modifying microbiota

Patent metadata

Field	Value
Publication number	US-2016333348-A1
Application number	US-201615160405-A
Country	US
Kind code	A1
Filing date	May 20, 2016
Priority date	May 6, 2015
Publication date	Nov 17, 2016
Grant date	—

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

We claim:

1 . A method of modifying a mixed population of microbiota bacteria, the mixed population comprising a first and a second bacterial sub-population wherein the first sub-population comprises a first microbiota species and the second sub-population comprises a host cell population of a second microbiota species which is a different species than the first microbiota species the method comprising
a. combining the mixed population of microbiota bacteria with multiple copies of engineered nucleic acid sequences encoding host modifying (HM) crRNAs, and b. expressing HM-crRNAs in host cells, wherein each HM-crRNA is encoded by a respective engineered nucleic acid sequence and is operable with an endogenous Cas nuclease expressed in a respective host cell, wherein said respective engineered nucleic acid sequence and Cas form a HM-CRISPR/Cas system and the engineered sequence comprises (i) spacer and repeat sequences encoding a HM-crRNA; (ii) the HM-crRNA comprising a sequence that is capable of hybridizing to a host cell target sequence to guide endogenous Cas nuclease to the target sequence in the host cell; and optionally the HM-system comprises a tracrRNA sequence or a DNA sequence expressing a tracrRNA sequence; whereby HM-crRNAs guide endogenous Cas activity to modify of host target sequences in host cells, whereby host cells are killed or the host cell population growth is reduced, thereby reducing the proportion of said host cell population and altering the relative ratio of said sub-populations of bacteria in the mixed bacterial population.

2 . (canceled)

3 . The method of claim 1 , comprising reducing host cell population growth by at least 5-fold compared to the growth of a control population of host cells that have not received said Cas modification.

4 . The method of claim 1 , wherein the host cell population is on a surface, whereby host cell population growth is inhibited on said surface.

5 . The method of claim 1 , wherein the first microbiota species comprises a 16s ribosomal RNA-encoding DNA sequence that is at least 80% identical to an 16s ribosomal RNA-encoding DNA sequence of the host cell species, wherein the growth of the first bacteria in the mixed population is not inhibited by said HM-system.

6 . The method of claim 1 , wherein the first microbiota species is a human gut commensal species and/or a human gut probiotic species.

7 . The method of claim 1 , wherein the first microbiota species is a Bacteroidetes (eg, Bacteroides ) and optionally the host cells are gram positive bacterial cells.

8 . The method of claim 1 , wherein the host cell population consists of Firmicutes cells.

9 . The method of claim 6 , wherein the host cell population consists of Firmicutes cells.

10 . The method of claim 7 , wherein the host cell population consists of are Firmicutes cells.

11 . The method of claim 1 , wherein for each host cell the system comprises components according to (i) to (iv):—
(i) at least one nucleic acid sequence encoding an endogenous Cas nuclease having endogenous Cas nuclease activity;
(ii) an engineered HM-CRISPR array comprising a spacer sequence and repeats encoding a HM-crRNA, the HM-crRNA comprising a sequence that hybridises to a host cell target sequence to guide said Cas to the target in the host cell to modify the target sequence;
(iii) an optional tracrRNA sequence or a DNA sequence expressing a tracrRNA sequence;
(iv) wherein said components of the system are split between the host cell and at least one nucleic acid vector that transforms the host cell, whereby the HM-crRNA guides endogenous Cas to the target to modify the host target sequence in the host cell; and
wherein the target sequence is modified by the endogenous Cas whereby the host cell is killed or host cell growth is reduced;
the method comprising introducing the vectors of (iv) into host cells and expressing said HM-crRNA in the host cells, allowing HM-cRNA to hybridise to host cell target sequences to guide endogenous Cas to the targets in the host cells to modify target sequences, whereby host cells are killed or host cell growth is reduced, thereby altering the relative ratio of said sub-populations in the mixed population of bacteria.

12 . (canceled)

13 . The method of claim 11 , wherein each vector is a virus or phage.

14 . The method of claim 11 , wherein each target sequence is adjacent a NNAGAAW or NGGNG protospacer adjacent motif (PAM).

15 . The method of claim 1 , wherein alternatively HM-crRNA and tracrRNA are comprised by a single guide RNA (gRNA), the method comprising introducing said gRNA into host cells or expressing the gRNA in host cells.

16 . The method of claim 1 wherein each of the first and second species is a respective Firmicutes species and the growth of the first bacteria is not inhibited by the HM-system.

17 . The method of claim 1 wherein each of the first and second species is a respective gram-positive species and the growth of the first bacteria is not inhibited by the HM-system.

18 . The method of claim 1 for treating a host cell infection of a human or animal subject, the method comprising exposing the host cells to a first antibiotic, wherein target sequences are each comprised by an antibiotic resistance gene for resistance to said first antibiotic, wherein the host cell infection is treated in the subject.

19 . The method of claim 1 for treating or reducing the risk of a disease or condition in a human or animal subject, wherein the disease or condition is mediated by said second bacterial species, wherein the first bacterial species is probiotic, commensal or symbiotic with humans and wherein the first bacterial species cells do not comprise said target sequence, wherein target sequence modification by said Cas is carried out and growth of the host cells is inhibited in said subject but growth of first bacterial species cells is not inhibited, wherein the disease or condition is treated or risk of the disease or condition in said subject is reduced.

20 . The method of claim 1 for treating an industrial or medical fluid, surface, apparatus or container; or for treating a waterway, water, a beverage, a foodstuff or a cosmetic, wherein said host cells are comprised by or on the fluid, surface, apparatus, container, waterway, water, beverage, foodstuff or cosmetic, wherein host cells growth is inhibited, thereby carrying out said treatment.

21 . The method of claim 1 , wherein each host cell is a Staphylococcus, Streptococcus, Pseudomonas, Salmonella, Listeria, E coli, Desulfovibrio, Vibrio or Clostridium cell.

22 . The method of claim 1 , wherein each target sequence is comprised by an antibiotic resistance gene, virulence gene or essential gene of the host cell.

23 . The method of claim 7 for increasing the proportion of Bacteroides in the mixed population, wherein said increase is carried out.

24 . The method of claim 23 wherein the proportion of B thetaiotomicron and/or B. fragilis is increased.

25 . The method of claim 7 wherein the relative ratio of Bacteroidetes versus Firmicutes or gram-positive host cells comprised by the mixed population is increased.

26 . The method of claim 25 wherein the proportion of B. thetaiotomicron and/or B. fragilis is increased.

27 . The method of claim 1 for favouring commensal or symbiotic Bacteroidetes in a hum

Assignees

• Snipr Tech Ltd

Inventors

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

Classifications

• 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

• A61K45/06

  Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca · CPC title

• C12N1/20

  Bacteria; Culture media therefor · 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

• C12N9/16

  acting on ester bonds (3.1) · CPC title