Modification of microbiotic populations and modification of microbiota

Abstract

FIELD: medicine.SUBSTANCE: invention relates to vectors for changing the relative ratio of subpopulations of first and second bacteria in a mixed population of bacteria, as well as to the use of vectors. The vector contains an engineered host-modifying (HM) CRISPR array containing a spacer sequence (HM spacer) and repeats encoding HM-crRNA, wherein the HM-crRNA contains a sequence that is capable of hybridizing to a host target sequence. The second bacteria of the mixed population contain host cells, and the second subpopulation consists of a bacterial species that differs from the species of the first subpopulation. Each host cell has a host target sequence and at least one nucleotide sequence encoding a Cas nuclease endogenous to the host cell. In this case, HM-crRNA directs Cas nuclease to the target to modify the target sequence in the host cell, thus killing the host cell or reducing the growth of the host cell. In this case, the vector is capable of transforming the host cell, resulting in the cell containing the phagemid.EFFECT: treatment or prevention of a condition or disease mediated by bacterial host cells in a human or animal subject.15 cl, 12 dwg, 6 tbl, 8 ex

Claims

1. A nucleic acid vector comprising an engineered host-modifying (HM) CRISPR array for use in the treatment or prevention in a human or animal subject of a condition or disease mediated by bacterial host cells by altering the relative proportion of subpopulations of first and second bacteria in a mixed population of bacteria, wherein the second bacteria comprise host cells, wherein the mixed population is contained in the subject, where the second subpopulation consists of a bacterial species that is different from the species of the first subpopulation, wherein each host cell has a host target sequence and at least one nucleotide sequence encoding a Cas nuclease endogenous to the host cell, wherein the host-modifying (HM) CRISPR array comprises a spacer sequence (HM spacer) and repeats encoding HM-crRNA, wherein the HM-crRNA comprises a sequence that is capable of hybridizing to a host target sequence; wherein the vector is capable of transforming a host cell, resulting in the cell containing the phagemid, where the HM-crRNA directs the Cas nuclease to the target to modify the target sequence in the host cell, thereby killing the host cell or reducing the growth of the host cell.

2. A nucleic acid vector comprising an engineered host-modifying (HM) CRISPR array and a nucleotide sequence encoding a Cas nuclease for use in the treatment or prevention in a human or animal subject of a condition or disease mediated by bacterial host cells by altering the relative ratio of subpopulations of first and second bacteria in a mixed population of bacteria, wherein the second bacteria comprise host cells, wherein the mixed population is contained in the subject, where the second subpopulation consists of a bacterial species that is different from the species of the first subpopulation, where each host cell has a host target sequence, where the vector is capable of transforming a host cell such that the cell contains the phagemid, wherein the engineered host modifying (HM) CRISPR array comprises a spacer sequence (HM spacer) and repeats encoding HM crRNA, wherein the HM crRNA comprises a sequence that is capable of hybridizing to a host target sequence; where the HM-crRNA directs the Cas nuclease to the target to modify the target sequence in the host cell, thereby killing the host cell or reducing the growth of the host cell.

3. Using a nucleic acid-based vector containing an engineered host-modifying (HM) CRISPR / Cas array to alter the relative ratio of subpopulations of primary and secondary bacteria in an ex vivo mixed bacterial population, wherein the secondary bacteria contain host cells, where the second subpopulation consists of a bacterial species that is different from the species of the first subpopulation, wherein each host cell has a host target sequence and at least one nucleotide sequence encoding a Cas nuclease endogenous to the host cell, wherein the engineered host modifying (HM) CRISPR array comprises a spacer sequence (HM spacer) and repeats encoding HM crRNA, wherein the HM crRNA comprises a sequence that is capable of hybridizing to a host target sequence; where the vector is capable of transforming a host cell such that the cell contains the phagemid, where the HM-crRNA directs the Cas nuclease to the target to modify the target sequence in the host cell, thereby killing the host cell or reducing the growth of the host cell.

4. Use of a nucleic acid-based vector containing an engineered host-modifying (HM) CRISPR / Cas array and a nucleotide sequence encoding a Cas nuclease to alter the relative ratio of subpopulations of first and second bacteria in an ex vivo mixed population of bacteria, wherein the second bacteria contain host cells, where the second subpopulation consists of a bacterial species that is different from the species of the first subpopulation, where each host cell has a host target sequence, where the vector is capable of transforming a host cell such that the cell contains the phagemid, wherein the engineered host modifying (HM) CRISPR array comprises a spacer sequence (HM spacer) and repeats encoding HM crRNA, wherein the HM crRNA comprises a sequence that is capable of hybridizing to a host target sequence; where the HM-crRNA directs the Cas nuclease to the target to modify the target sequence in the host cell, thereby killing the host cell or reducing the growth of the host cell.

5. Use according to paragraph 3 or 4 for the treatment of industrial or ex vivo medical fluids, surfaces, devices or containers; or for the treatment of waterways, water, beverages, food or cosmetic product, where the host cell(s) are contained in or on the liquid, surface, device, container, waterways, water, beverage, food or cosmetic product.

6. The use or vector for use according to any of the preceding paragraphs, wherein alternatively the HM-crRNA and tracrRNA are contained in a single guide RNA (gRNA).

7. The use or vector for use according to any of the preceding paragraphs, wherein the growth of the population of host cells is reduced by at least 5 times compared to the growth of the population of said host cells not transformed with said HM array or nucleotide sequence encoding said gRNA.

8. Use according to paragraphs 3, 4 or any of paragraphs 5-7 in case of dependence on paragraph 3 or 4, wherein the growth of the host cell population on the surface is inhibited.

9. The use or vector for use according to any one of claims 1 or 3 or any one of claims 5 to 8 in case of dependence on any of claims 1 or 3, wherein each vector lacks a sequence encoding a Cas nuclease.

10. The use or vector for use according to any of the preceding paragraphs, wherein the vector comprises a DNA sequence for expressing a tracrRNA sequence.

11. The use or vector for use according to any of the preceding paragraphs, wherein the mixed population comprises S. thermophilus, L. lactis and E. coli.

12. The use or vector for use according to any of the preceding paragraphs, wherein the Cas nuclease is a Cas nuclease type I, a Cas nuclease type II or a Cas nuclease type III.

13. A vector for use or use according to any of the preceding claims, wherein the vector comprises nucleotide sequences for expressing a plurality of different crRNAs, optionally contained in a gRNA, wherein a first of said crRNAs is capable of hybridizing to a first nucleotide sequence in said host cell; and a second of said crRNAs is capable of hybridizing to a second nucleotide sequence in said host cell, wherein said second sequence differs from said first sequence; and (a) the first sequence is contained in an antibiotic resistance gene or RNA thereof, and the second sequence is contained in an antibiotic resistance gene or RNA thereof; optionally, where the genes are different; (b) the first sequence is contained in an antibiotic resistance gene or its RNA, and the second sequence is contained in an essential or virulence gene or its RNA; (c) the first sequence is contained in an essential gene or its RNA, and the second sequence is contained in an essential or virulence gene or its RNA; or (d) the first sequence is contained in a virulence gene or its RNA, and the second sequence is contained in a vital gene or a virulence gene or its RNA.

14. A vector for use or a use according to any of claims 1, 3 and 6-12 in case of dependence on any of claims 1 or 3, wherein the vector comprises more than 1.4 kb of exogenous DNA sequence, wherein the exogenous DNA encodes one or more components of the CRISPR / Cas system and comprises a designed array for expression of HM-crRNA or gRNA in host cells; wherein at least two different cRNAs or gRNAs are encoded by the exogenous DNA, optionally by at least two HM-CRISPR arrays.

15. The vector of any one of claims 1, 3 and 6-14 in the case of dependence on claim 1 or 3, wherein the vector lacks a nucleotide sequence encoding a Cas nuclease that is related to cRNA or gRNA.

Images