Altering microbial populations and modifying microbiota
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.
1. A method for reducing growth of a bacterial population comprising bacterial host cells, the method comprising:
(a) infecting the host cells of the bacterial population with a phage comprising an engineered nucleic acid for producing a host modifying crRNA (HM-crRNA); and
(b) producing the HM-crRNA in an infected host cell of the bacterial population;
wherein
(i) the HM-crRNA is operable with a Cas nuclease in the host cell, wherein the engineered nucleic acid and the Cas nuclease are comprised by a HM-CRISPR/Cas system in the host cell; and
(ii) the HM-crRNA comprises a nucleotide sequence that is capable of hybridizing to a target sequence in the host cell to guide the Cas nuclease to cut the target sequence in the host cell, and
wherein the target sequence is cut by the HM-CRISPR/Cas system, and
wherein the phage expresses a holin and/or an endolysin for host cell lysis;
wherein growth of the bacterial population is reduced.
2. The method of claim 1 wherein the Cas nuclease is a Cas9.
3. The method of claim 1 , wherein the Cas nuclease is a Type I Cas nuclease.
4. The method of claim 1 , wherein the Cas nuclease is encoded by the engineered nucleic acid for producing the HM-crRNA.
5. The method of claim 1 , wherein the Cas nuclease is an endogenous Cas nuclease of the host cell.
6. The method of claim 1 , wherein the engineered nucleic acid for producing the HM-crRNA comprises an origin of replication (ori) and a packaging site.
7. The method of claim 1 , wherein in step (a) the bacterial population comprising host cells is comprised by a mixed population of microbiota bacteria, wherein the mixed population further comprises E. coli and L. lactis bacteria that do not comprise the target sequence, and wherein growth of the E. coli or L. lactis is not reduced.
8. The method of claim 1 , wherein the target sequence is a chromosomal sequence of the host cell.
9. The method of claim 1 , wherein the HM-CRISPR/Cas system is a type II HM-CRISPR/Cas system that comprises an endogenous tracrRNA of the host cell.
10. The method of claim 1 , wherein the HM-CRISPR/Cas system is a Type II HM-CRISPR/Cas system that comprises a tracrRNA, and wherein the tracrRNA is encoded by the engineered nucleic acid for producing the HM-crRNA.
11. The method of claim 1 , wherein the engineered nucleic acid for producing the HM-crRNA encodes a single guide RNA comprising a tracrRNA and the HM-crRNA.
12. The method of claim 1 , wherein the host cells are C. difficile, E. coli, Klebsiella, Pseudomonas aeruginosa, Helicobacter pylori or Salmonella cells.
13. The method of claim 1 , wherein the engineered nucleic acid comprises a first HM-CRISPR array for producing the HM-crRNA of step (a) and one or more further HM-CRISPR arrays.
14. The method of claim 4 , wherein the engineered nucleic acid comprises a first HM-CRISPR array for producing the HM-crRNA of step (a) and one or more further HM-CRISPR arrays.
15. The method of claim 4 , wherein the engineered nucleic acid comprises more than one Cas-encoding nucleic acids.
16. The method of claim 14 , wherein the engineered nucleic acid comprises more than one Cas-encoding nucleic acids.
17. The method of claim 1 , wherein the phage is Caudovirales phage.
18. The method of claim 17 , wherein the host cells are Staphylococcus aureus host cells.
19. The method of claim 1 , wherein the host cells are E. coli host cells.
20. The method of claim 1 , wherein the phage expresses an endolysin, and wherein the endolysin is a phage phi11, phage Twort, phage P68, phage phiWMY or phage K endolysin.
21. The method of claim 1 , wherein the bacterial population comprising the bacterial host cells is comprised by a waterway microbiota, a water microbiota, a human or animal gut microbiota, a human or animal oral cavity microbiota, a human or animal lung microbiota, a human or animal ocular microbiota, a human or animal ear microbiota, a human or animal nose microbiota, a human or animal anal microbiota, a human or animal throat microbiota, a human or animal vaginal microbiota, a human or animal skin or hair microbiota, a human or animal bloodstream microbiota, a human or animal scalp microbiota, or a human or animal armpit microbiota.
22. The method of claim 19 , wherein the phage is a lambda or T4 phage.
23. The method of claim 1 , wherein the engineered nucleic acid comprises a T7 promoter for expression of the HM-crRNA in the host cell.
24. The method of claim 1 , wherein the growth of the bacterial population is reduced by at least 5-fold.
25. The method of claim 1 , wherein the method further comprises exposing the bacterial population to an antibiotic.