Altering microbial populations & modifying microbiota

Patent metadata

Field	Value
Publication number	US-2017196225-A1
Application number	US-201715460962-A
Country	US
Kind code	A1
Filing date	Mar 16, 2017
Priority date	May 6, 2015
Publication date	Jul 13, 2017
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

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, wherein the second species 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 is HM-crRNA is encoded by a respective engineered nucleic acid sequence that is operable with a Cas nuclease in a respective host cell,
wherein the respective engineered nucleic acid sequence and Cas form a HM-CRISPR/Cas system and the engineered nucleic acid sequence comprises
(i) a nucleic acid sequence comprising spacer and repeat sequences encoding said HM-crRNA;
(ii) a nucleic acid sequence encoding a sequence of said HM-crRNA, wherein said HM-crRNA sequence is capable of hybridizing to a host cell target sequence to guide 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 Cas modification 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;
wherein the method reduces host cell population growth by at least 5-fold.

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

3 . 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, wherein the second species 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 is HM-crRNA is encoded by a respective engineered nucleic acid sequence that is operable with a Cas nuclease in a respective host cell,
wherein the respective engineered nucleic acid sequence and Cas form a HM-CRISPR/Cas system and the engineered nucleic acid sequence comprises
(i) a nucleic acid sequence comprising spacer and repeat sequences encoding said HM-crRNA;
(ii) a nucleic acid sequence encoding a sequence of said HM-crRNA, wherein said HM-crRNA sequence is capable of hybridizing to a host cell target sequence to guide 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 Cas modification 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;
wherein the method inhibits host cell population growth on a surface.

4 . 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, wherein the second species 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 is HM-crRNA is encoded by a respective engineered nucleic acid sequence that is operable with a Cas nuclease in a respective host cell,
wherein the respective engineered nucleic acid sequence and Cas form a HM-CRISPR/Cas system and the engineered nucleic acid sequence comprises
(i) a nucleic acid sequence comprising spacer and repeat sequences encoding said HM-crRNA;
(ii) a nucleic acid sequence encoding a sequence of said HM-crRNA, wherein said HM-crRNA sequence is capable of hybridizing to a host cell target sequence to guide 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 Cas modification 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;
wherein the first 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 host cell species, wherein the growth of the first bacteria in the mixed population is not inhibited by said HM-system.

5 . The method of claim 4 , wherein the first species is a Firmicutes and the second species is a Firmicutes.

6 . The method of claim 4 , wherein the first species is a gram positive species and the second species is a gram positive species.

7 . 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, wherein the second species 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 is HM-crRNA is encoded by a respective engineered nucleic acid sequence that is operable with a Cas nuclease in a respective host cell,
wherein the respective engineered nucleic acid sequence and Cas form a HM-CRISPR/Cas system and the engineered nucleic acid sequence comprises
(i) a nucleic acid sequence comprising spacer and repeat sequences encoding said HM-crRNA;
(ii) a nucleic acid sequence encoding a sequence of said HM-crRNA, wherein said HM-crRNA sequence is capable of hybridizing to a host cell target sequence to guide 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 Cas modification 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;
wherein the mixed population of step (a) comprises a third bacterial species.

8 . The method of claim 7 , wherein the third species is E coli.

9 . The method of claim 7 , wherein the first species is a Firmicutes, the second species is a Firmicutes and the third species

Assignees

• Snipr Tech Ltd

Inventors

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

Classifications

• C12N15/70

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

• C12N2795/00032

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

• A61K48/005

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

• C12N15/746

  for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus) · CPC title

• 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