Manipulation of microbial communities

Abstract

The present invention relates to methods and means for temporally controlling the production of a product of interest (eg protein or RNA) in a microbial community of a subject, such as the gut microbial community of a human or animal.

Description

[Technical field]

[0001] The present invention relates to methods and means for temporally controlling the production of a product of interest (e.g., protein or RNA) in a microbial community of interest, such as the intestinal microbial community of a human or animal. For example, microbial communities can be usefully engineered in this way to express products in a controlled and optionally reversible manner. [Background technology]

[0002] Growing evidence undoubtedly links homeostasis in the human microbiome to human health, and in turn, microbiome dysbiosis has been linked to various disease states. For example, microbiome dysbiosis, such as the overgrowth of the bacterial pathogen C. difficile in the gut microbiome, has been linked to severe diarrhea caused by the production of toxins secreted by C. difficile. Another example of a disease associated with microbiome dysbiosis is inflammatory bowel disease (IBD), in which the intestinal inflammatory state is caused, at least in part, by a metabolome associated with the overgrowth of Enterobacteriaceae, under the expression of bacterial species capable of producing short-chain fatty acids.

[0003] Reversal of disease states by restoring healthy microbiomes through fecal microbial transplantation (FMT), in which fecal microbiomes from healthy donors are transplanted into the intestines of patients suffering from dysbiosis, has proven effective, for example, in the treatment of recurrent C. difficile infections. However, FMT is a relatively uncontrolled process and is associated with risks, as demonstrated by the deaths caused by bacterial infections that can be attributed to FMT. A more controlled approach, in which a single strain or a defined collection of strains is added to the dysbiosis gut environment, is currently being explored. Such an approach requires replacement of strains by colonization with new strains added to the collection.

[0004] In some instances, target compounds of interest are identified by the presence of bacterial toxins (against which antibodies have been raised) or the absence of disease-associated IL-22, etc. However, local delivery of relevant protein compounds into the intestinal tract is inefficient and cumbersome due to protein instability in the intestinal environment. Summary of the Invention

[0005] Thus, localized in situ production of relevant molecules (e.g. enzymes, antibodies, nanobodies, small molecules) intracellularly or secreted by live bacteria already established and present in the microbial community allows targeted intervention with minimal impact on the native microbial community. The present invention addresses this need and how to control such production in situ in the microbial community.

[0006] That is, the present invention provides the following configuration.

[0007] In the first configuration At least one nucleic acid vector for the transfer of a microbial population into a host cell, the vector comprising: (a) an expressible nucleotide sequence of interest (NS1) for producing a product of interest (P1) in a host cell, and (b) an expressible nucleotide sequence (NS2) for producing a regulator product (P2) in a host cell, wherein P2 is operable in the host cell to regulate the expression or activity of P1; a nucleic acid comprising (c) NS1 is under the control of a first promoter (e.g., a constitutive or inducible promoter) for expression of P1; (d) A nucleic acid vector, wherein the expression or activity of P2 in a host cell is regulatable by exposure of a host cell containing at least one vector to a regulator agent (R), thereby regulating the expression or activity of P1.

[0008] In one embodiment, At least one nucleic acid vector for the transfer of a microbial population into a host cell, the vector comprising: (a) an expressible nucleotide sequence of interest (NS1) for producing a product of interest (P1) in a host cell, and (b) an expressible nucleotide sequence (NS2) for producing a regulator product (P2) in a host cell, wherein P2 is operable in the host cell to regulate expression of P1; a nucleic acid comprising (c) NS1 is under the control of a first promoter (e.g., a constitutive or inducible promoter) for expression of P1; (d) A nucleic acid vector is provided, wherein expression of P2 in a host cell is regulatable by exposure of a host cell comprising at least one vector to a regulator agent (R), thereby regulating expression of P1.

[0009] At least one nucleic acid vector for the transfer of a microbial population into a host cell, the vector comprising: (a) an expressible nucleotide sequence of interest (NS1) for producing a product of interest (P1) in a host cell, and (b) an expressible nucleotide sequence (NS2) for producing a regulator product (P2) in a host cell, wherein P2 is operable in the host cell to regulate expression of P1; a nucleic acid comprising (c) NS1 is under the control of a first promoter (e.g., a constitutive or inducible promoter) for expression of P1; (d) A nucleic acid vector in which NS2 is under the control of a second promoter that is controllable for expression of P2, and in which binding of a regulator agent (R) to the vector nucleic acid controls the second promoter, thereby controlling expression of P2 and P1.

[0010] Preferably, NS1 and NS2 are contained in the same nucleic acid vector. In another configuration, exposure of a regulator agent (R) to the vector nucleic acid controls the second promoter, thereby controlling P2 and P1.

[0011] Preferably, P2 is operable in the host cell to bind to the vector nucleic acid and control the expression of P1.

[0012] In one embodiment, the invention provides a host cell comprising said at least one vector.

[0013] In one embodiment, P1 is a protein (e.g., an enzyme) of a metabolic pathway in a host cell, and the activity is an activity of P1 in the pathway (e.g., an enzymatic activity). In one example, P1 is a protein inhibitor, and the activity is an inhibitor activity (e.g., an inhibitor of a component of a metabolic pathway). In one example, P1 is a binding agent, such as an antibody or an antibody fragment, such as a single domain antibody (e.g., a nanobody) or an scFv.

[0014] In one embodiment, P2 is an inhibitor of P1 expression. In one embodiment, P2 activity is inhibition of P1 expression. In one embodiment, P2 is a promoter of P1 expression. In one embodiment, P2 activity is promotion of P1 expression.

[0015] In the first aspect of the first configuration A nucleic acid vector for the transfer of a microbial population into a host cell, the vector comprising: (a) an expressible nucleotide sequence of interest (NS1) for producing a product of interest (P1) in a host cell, and (b) an expressible nucleotide sequence (NS2) for producing a regulator product (P2) in a host cell, the nucleotide sequence (NS2) being operable in the host cell such that P2 binds to the vector nucleic acid and controls expression of P1; a nucleic acid comprising (c) NS1 is under the control of a first promoter (e.g., a constitutive or inducible promoter) for expression of P1; (d) A nucleic acid vector in which NS2 is under the control of a second promoter that is controllable for expression of P2, and in which binding of a regulator agent (R) to the vector nucleic acid controls the second promoter, thereby controlling expression of P2 and P1.

[0016] In the second aspect of the first configuration P2 can be expressed in host cells to form a nuclease that can act in the host cell to cleave vector nucleic acid, and the nucleic acid is degraded, thereby downregulating the expression of P1. This is useful for controlling the expression of P1 in a subject, such as a human or animal, microbial population, temporally (i.e., during a given time window). For example, microbial populations can be usefully modified in this way to express P1 in a controlled and optionally reversible manner.

[0017] In the third aspect of the first configuration Each vector (e.g. according to the first embodiment) is a conjugative plasmid. This is useful for allowing the spread of the vector nucleic acid (and thus expression of P1) within the target microbial population. In combination with the first embodiment of the first configuration, this provides a powerful way to controllably alter the degree and timing of expression of P1 in the microbial population.

[0018] In the fourth aspect of the first configuration The host cells are commensal or probiotic bacterial cell species of the human or animal microbial community, preferably cells of a Bacteroides species, which are present and maintained in the natural microbial communities of humans and animals, and thus the present invention provides a means for relatively stable and controllable modification of the microbial community to temporally control P1 expression.

[0019] In the second configuration 1. A method for temporally controlling the production of an expression product in a human or animal subject, comprising: a) administering said at least one vector comprising a nucleic acid to a microbial population of a subject (e.g., the gut microbial population), the microbial population comprising host cells (e.g., bacterial cells), the nucleic acid encoding a product of interest (P1), and optionally the administration being oral or topical; b) transferring nucleic acid into a host cell in the population of microorganisms and expressing P1 in the host cell; and c) after step (b), exposing the microbial population to a regulator agent (R) that controls expression or activity of a regulator product (P2) in the host cell, where P2 is operable in the host cell to control expression or activity of P1, and optionally R upregulates the production of an RNA-guided nuclease / guide RNA complex in the host cell that can target a protospacer contained in a nucleic acid, where the nuclease cleaves the nucleic acid to render expression of P1 non-functional (e.g., by degradation of the cleaved nucleic acid in the cell), and where the nuclease (or a component thereof) and / or the RNA (or a component thereof) are encoded by a nucleic acid. The method includes:

[0020] In the first aspect of the second configuration 1. A method for temporally controlling the production of an expression product in a human or animal subject, comprising: a) administering a vector of the first aspect of said first configuration to a microbial population of a subject (e.g. the gut microbial population), the microbial population comprising host cells (e.g. bacterial cells), the nucleic acid encoding a product of interest (P1), and optionally the administration being oral or topical; b) transferring nucleic acid into a host cell in the population of microorganisms and expressing P1 in the host cell; and c) after step (b), exposing the microbial population to a regulator (R) that controls the expression or activity of P2, where optionally R upregulates the production of an RNA-guided nuclease / guide RNA complex in the host cell that can target a protospacer contained in a nucleic acid, where the nuclease cleaves the nucleic acid to render expression of P1 non-functional (e.g., by degradation of the cleaved nucleic acid in the cell), and where the nuclease (or a component thereof) and / or the RNA (or a component thereof) are encoded by a nucleic acid. The method includes:

[0021] In the third configuration 1. A method for temporally controlling the production of an expression product in a human or animal subject, comprising: a) administering at least one vector of the invention to the microbiome (e.g. the gut microbiome) of a subject, optionally wherein the administration is oral or topical; b) transferring nucleic acid into a host cell in the population of microorganisms and expressing P1 in the host cell; and c) after step (b), exposing the microbial population to R (e.g., by administering R to the subject), where R controls the second promoter, thereby controlling expression of P2 and P1. The method includes:

[0022] In the first aspect of the second or third configuration a) treating or preventing a disease or condition in a human or animal subject by temporally controlling the production of P1 according to the method of the second or third configuration, or b) modifying the microbiome (e.g., the gut microbiome) of a human or animal subject by temporally controlling the production of P1 according to the second or third method of the present invention; method.

[0023] In the fourth configuration A nucleic acid vector for the transfer of a microbial population into a host cell, the vector being contained in a carrier cell (e.g., a bacterial cell); a) a nuclease (optionally an RNA-guided nuclease or a restriction endonuclease) operable in the carrier cell to cleave a chromosome or episome (which is not the vector of the invention) of the carrier cell, optionally resulting in degradation of the chromosome or episome, and / or b) an RNA or a precursor of such an RNA operable in a carrier cell to guide an RNA-guided nuclease, where the RNA guides the nuclease to cleave a chromosome or episome (which is not a vector) of the carrier cell, and optionally the chromosome or episome is degraded; Code the following: A nucleic acid vector, wherein the vector comprises one or more controllable promoters for controlling expression of the (a) nuclease and / or the (b) RNA or component in a carrier cell.

[0024] In the fifth configuration A method of manipulating a microbiome, comprising contacting the microbiome with a plurality of vectors described herein and optionally transferring said vector nucleic acid into target cells of a microbial population.

[0025] A modified microbial population obtained or obtainable by the methods herein, optionally included in a pharmaceutical composition for use as a medicament for treating a disease or condition in a human or animal subject.

[0026] In the sixth configuration (a) an expressible nucleotide sequence of interest (NS1) for producing a product of interest (P1) in a host cell, and (b) an expressible nucleotide sequence (NS2) for producing a regulator product (P2) in a host cell, wherein P2 is operable in the host cell to regulate the expression or activity of P1; A host cell comprising a nucleic acid comprising (c) NS1 is under the control of a first promoter (e.g., a constitutive or inducible promoter) for expression of P1; (d) the expression or activity of P2 in the host cell is regulatable by exposure of the host cell containing at least one vector to a regulator agent (R), thereby regulating the expression or activity of P1; A host cell, wherein the host cell is a bacterial, archaeal, or fungal cell.

[0027] In the seventh configuration In the first aspect A cell comprising a nucleic acid (optionally in any other configuration), wherein the nucleic acid comprises a gene encoding a product of interest (P1), the gene comprises a nucleotide sequence (NS1) encoding P1 and a regulatory region 5' of NS1 comprising a promoter (Px) for regulating expression of NS1, wherein the combination of Px and NS1 is heterologous to the cell, and Px is controllable by xylitol.

[0028] In the second aspect A cell comprising a nucleic acid (optionally in any other configuration), wherein the nucleic acid comprises a gene encoding a product of interest (P1), the gene comprises a nucleotide sequence (NS1) encoding P1 and a regulatory region 5' of NS1 comprising a promoter (Px) for regulating expression of NS1, wherein the combination of Px and NS1 is heterologous to the cell, and Px is controllable by xylose.

[0029] In the third aspect A nucleic acid vector comprising a gene as recited in the first or second embodiment. [Brief description of the drawings]

[0030] [Figure 1]Plasmid map of p1364. The plasmid is annotated by relative gene locations and their functions. The plasmid contains functions for plasmid replication (pBBR ori+rep) and RP4 transfer origin (mob region encompassing oriT and relaxase). araC - Regulatory gene pBAD - Inducible promoter controlled by the presence of arabinose cas3 - Gene encoding E. coli type IE Cas3 protein casA-E - Genes encoding cascade proteins Prham - Inducible promoter controlled by the presence of rhamnose Bba-B1003 terminator - Artificial terminator rmtB - 16S rRNA methylase gene; provides resistance to amikacin and gentamicin sfgfp - Gene encoding superfolder green fluorescent protein kanR - Kanamycin resistance gene mob - Mobilization gene; required for conjugative transfer of DNA pBBRori - Origin of replication pBBR rep - Gene encoding replication initiator protein CRISPR array - Consists of E. coli direct repeats and one spacer [Diagram 2] Conjugative transfer of p1364. Transconjugants formed upon conjugation of p1364 from strain JKE201 encoding the RP4 transgene. A control lacking oriT was included. [Diagram 3] GFP expression from p1364. GFP expression levels from cells harvested at an OD600 of 1. The b52 strain was included as an autofluorescence control. [Figure 4] The self-targeting plasmid is rapidly lost upon CRISPR / cas induction. Percentage of GFP-positive colonies over time for induced and uninduced b52 containing the p1364 self-targeting plasmid. [Diagram 5] PCR screening for p1364 plasmid backbone. Gel electrophoresis shows the absence of p1364 in all GFP-negative colonies picked from the 24 hour time point. [Figure 6]Chromosomal CRISPR / cas targeting allows rapid elimination of the plasmid host. Changes in host cell viability upon induction of the chromosomal (lptA)-targeted CRISPR / cas system in E. coli MG1655. Error bars indicate standard deviation of three replicates. [Figure 7-1] 1A-1D are various schematic diagrams illustrating the pathways in which the present invention may be used. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 7-4] Same as above. [Figure 8-1] (i) Overview of the GFP reporter plasmid with the xylitol inducible control system. (ii) Depicts the genomic region from Morganella morganii strain ZJG812 (genome ID: CP064831.1) with highlighted structural genes for xylitol uptake and metabolism: NAD(P)-dependent alcohol dehydrogenase (IZ184_04875 gene), xylulokinase (xylB), sugar ABC transporter ATP-binding protein (IZ184_04865 gene), ABC transporter permease (IZ184_04860 gene), and substrate-binding domain-containing protein (IZ184_04855). These genes are expressed from a negative inducible promoter (promoter) controlled by a DNA-binding transcriptional regulator of the LacI family (IZ184_04885 gene). A predicted D-lyxose / D-mannose family sugar isomerase (IZ184_04880 gene) is found between the promoter and the repressor. Gene annotation was based on sequence similarity searches against public databases. [Figure 8-2] Same as above. [Figure 9]Activity of xylitol-inducible promoters in response to xylitol. GFP reporter plasmids encoding xylitol-inducible promoter systems with full-length (pSNP1902) or loss-of-function (pSNP103) transcriptional regulators were tested in the bSNP463 strain background in response to increasing amounts of xylitol in the growth medium (LB). Data shown are fluorescence emissions (normalized, see Example 2, section 3.2.2) after 24 hours of incubation. Error bars indicate standard deviation based on three biological replicates. [Figure 10] Activity of the xylitol-inducible promoter in minimal medium containing increasing concentrations of xylitol. A GFP reporter plasmid encoding a xylitol-inducible promoter system with a full-length transcriptional regulator (pSNP1902) was tested in the MG1655 (bSNP230) strain background growing in minimal medium supplemented with glycerol and increasing amounts of xylitol. Data shown are fluorescence emissions (normalized, see Example 2, section 3.2.2) after 24 hours of incubation. Error bars indicate standard deviation based on three biological replicates. [Figure 11] Growth of strains harboring reporter plasmids in minimal medium with and without glycerol and / or xylitol. E. coli MG1655 strain (bSNP230) harboring the xylitol-inducible GFP reporter plasmid pSNP1902 was grown for 24 h in minimal medium supplemented with glycerol (0.4% (v / v)) and with or without xylitol (0.5% (w / v)). Error bars indicate standard deviation based on two biological replicates. The trend from these growth profiles (i.e., increasing amounts of xylitol result in delayed growth) was also observed from the growth profiles of the remaining strains. [Figure 12]Activity of the promoter in the presence of the xylitol ABC transporter system. E. coli bSNP230 strain containing the GFP reporter plasmid (pSNP1902) with or without a coexisting plasmid (pSNP1939) containing the xylitol ABC transporter was grown for 24 h in minimal medium supplemented with 0.4% (v / v) glycerol (A) or 0.4% (w / v) glucose (B) in the presence or absence of xylitol. Cell density-adjusted fluorescence emission after 24 h of incubation is shown. Error bars indicate standard deviation based on two (A) and three (B) biological replicates, respectively. [Figure 13-1] Growth profiles of ABC transporter-harboring strains in minimal medium in the absence or presence of xylitol. The bSNP230 strain containing the GFP reporter plasmid (pSNP1902) with or without a coexisting plasmid (pSNP1939) containing the xylitol ABC transporter was grown for 24 h in minimal medium supplemented with 0.4% (v / v) glycerol (A) or 0.4% (w / v) glucose (B) in the presence or absence of xylitol. Error bars indicate standard deviation based on two (A) and three (B) biological replicates, respectively. [Figure 13-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention relates to methods and means for temporally controlling the production of a product of interest (e.g., protein or RNA, herein referred to as P1) in a microbial community of interest, such as the microbial community of the human or animal gut. For example, the microbial community can be usefully modified in this way to express the product in a controlled and possibly reversible manner. This can be useful for enabling the controlled manipulation of a microbial community of interest in a manner that allows for the production of a desired level of P1, followed by a controlled reduction in expression, e.g., to return to the level of P1 before manipulation. To this end, the present invention provides the following illustrative embodiments.

[0032] In the first configuration, At least one nucleic acid vector for the transfer of a microbial population into a host cell, the vector comprising: (a) an expressible nucleotide sequence of interest (NS1) for producing a product of interest (P1) in a host cell, and (b) an expressible nucleotide sequence (NS2) for producing a regulator product (P2) in a host cell, the nucleotide sequence (NS2) being operable in the host cell such that P2 binds to the vector nucleic acid and controls expression of P1; a nucleic acid comprising (c) NS1 is under the control of a first promoter (e.g., a constitutive or inducible promoter) for expression of P1; (d) A nucleic acid vector is provided, in which NS2 is under the control of a second promoter that is controllable for expression of P2, and binding of a regulator agent (R) to the vector nucleic acid controls the second promoter, thereby controlling expression of P2 and P1 (e.g., R binds to an operator operably linked to the second promoter).

[0033] Said "at least one vector" may relate to one vector or multiple vectors, e.g., a first and a second vector, e.g., two vectors. Where a feature is described herein with respect to one vector or "the vector", the skilled artisan will recognize that the feature can be applied mutatis mutandis to said "at least one vector", e.g., a first and a second vector, or e.g., multiple vectors. A vector may further follow a vector of a fourth configuration. Each of said first and second, two or more vectors may further follow a vector of a fourth configuration. This is advantageous for limiting the presence or spread of NS1 and / or NS2 (i.e., limiting P1 and / or P2, respectively) in a microbial population or a subject containing a microbial population.

[0034] For example, said at least one vector comprises a first vector and a second vector, said first vector comprises NS1 (optionally does not comprise NS2), said second vector comprises NS2 (optionally does not comprise NS1), and said vectors can coexist in a host cell for the expression of P1 and P2. That is, said first vector can comprise NS1 and not comprise NS2, and said second vector can comprise NS2 and not comprise NS1. The first and second vectors can be transferred into the same host cell and coexist in the cell, so that P1 and P2 can be expressed in the cell. For example, said at least one vector is one vector that comprises both NS1 and NS2.

[0035] The microbial community may be in any environment, such as soil or waterways, may be contained in plants, or may be contained in human or animal subjects. As known to those skilled in the art, the microbial community may include bacteria, archaea, fungi, and viruses.

[0036] A first aspect of the first configuration is as follows: A nucleic acid vector for the transfer of a microbial population into a host cell, the vector comprising: (a) an expressible nucleotide sequence of interest (NS1) for producing a product of interest (P1) in a host cell, and (b) an expressible nucleotide sequence (NS2) for producing a regulator product (P2) in a host cell, the nucleotide sequence (NS2) being operable in the host cell such that P2 binds to the vector nucleic acid and controls expression of P1; a nucleic acid comprising (c) NS1 is under the control of a first promoter (e.g., a constitutive or inducible promoter) for expression of P1; (d) providing a nucleic acid vector, wherein NS2 is under the control of a second promoter that is controllable for expression of P2, and wherein binding of a regulator agent (R) to the vector nucleic acid controls the second promoter, thereby controlling expression of P2 and P1.

[0037] As described more fully herein, some advantages of certain aspects of the invention include: Inducible self-targeting of the vector to remove nucleotide sequences of interest already spread in the microbial population to which the vector is exposed, leaving the microbial population free of traces of vector nucleic acid or containing reduced amounts of vector nucleic acid as soon as a desired expression level of, for example, the NS1 product is achieved. Killing of the donor (carrier) bacteria by a CRISPR / cas (or other nuclease) vector-mediated system that upon induction cuts the genome (e.g. chromosome) of the donor cell, killing the donor cell. This is useful to completely eliminate the donor cell population (thus reducing vector transmission in the population) as soon as the desired expression level of the NS1 product is achieved, and also as a safety off-switch system to reduce vector nucleic acid in the population and other environments. Real-time control of NS1 product production. A feedback loop can be employed to remove vector nucleic acid in response to produced compounds or physiological signals. A localized on-off switch that ensures production of the NS1 product at a certain spatial site in the environment, e.g., in the microbiome (e.g., the gut microbiome) in a human or animal subject. It could be.

[0038] Each host cell may be a bacterial, fungal (e.g. yeast) or archaeal cell. Preferably, each host cell is a microbial cell. Preferably, each host cell is a bacterial cell. Preferably, each host cell is an archaeal cell.

[0039] Preferably, each host cell is a cell of a commensal or probiotic bacterial cell species found in the human or animal microbial community. Preferably, each host cell is a cell of a commensal or probiotic bacterial cell species of the human or animal microbial community. Preferably, each host cell is a cell of a microorganism (e.g. a bacterium) of a human or animal gut microbial community species.

[0040] The nucleic acid may be DNA or RNA, for example, the nucleic acid is DNA.

[0041] The host cells associated with the first through third constructs are referred to herein interchangeably as target cells. The host cells associated with the fourth construct are referred to herein interchangeably as carrier or donor cells.

[0042] In one example, P1 is secreted from the host cell. In one example, P1 is expressed from NS1 in the host cell as an amino acid sequence that includes a signal peptide for secretion of P1 from the cell. In one example, P1 is not secreted from the host cell. P1 can be a protein or RNA (e.g., mRNA).

[0043] In one embodiment, P1 is not expressed in the microbial population prior to carrying out the method of the invention.

[0044] P2 can upregulate the expression of P1. In one embodiment, P1 is expressed in a subject before carrying out the method of the present invention, and carrying out the method causes at least 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000% increase in the expression of P1 in the subject. The increase in expression can be determined by determining the relative level of P1 in tissue or liquid samples (e.g., blood samples) obtained from the patient. The increase in expression can be determined by determining the relative level of P1 in fecal samples obtained from the patient.

[0045] P2 can upregulate the expression of P1. In one embodiment, P1 is expressed in a microorganism (e.g., the gut microorganism of a human or animal subject) before carrying out the method of the present invention, and carrying out the method causes an increase in the expression of P1 in the microorganism of at least 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000%. The increase in expression can be determined by determining the relative level of P1 in a sample of the microorganism previously obtained from the patient. The increase in expression can be determined by determining the relative level of P1 in a tissue or liquid sample (e.g., a blood sample) obtained from the patient. The increase in expression can be determined by determining the relative level of P1 in a fecal sample obtained from the patient (e.g., the microorganism is the gut microorganism).

[0046] P2 can downregulate the expression of P1. In one embodiment, the method comprises administering R to a subject in step (c) of the method of the invention, whereby the expression of P1 in the subject is reduced by at least 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000% compared to the expression of P1 immediately before performing step (c). The reduction in expression may be determined by determining the relative level of P1 in a tissue or liquid sample (e.g., a blood sample) obtained from the patient immediately prior to performing step (c) and comparing that level to the level of P1 in a similar sample (i.e., a tissue or liquid sample, respectively) obtained after step (c) is performed (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 3, 4, 5, 6, 7, or 8 weeks after the start of step (c)). The reduction in expression may instead be determined by determining the relative level of P1 in a fecal sample obtained from the patient.

[0047] P2 can downregulate the expression of P1. In one embodiment, the method comprises administering R to the subject in step (c) of the method of the invention, whereby the expression of P1 in the microbial population is reduced by at least 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000% compared to the expression of P1 immediately before performing step (c). The reduction in expression may be determined by determining the relative level of P1 in a tissue or liquid sample (e.g., a blood sample) obtained from the patient immediately prior to performing step (c) and comparing that level to the level of P1 in a similar sample (i.e., a tissue or liquid sample, respectively) obtained after step (c) is performed (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 3, 4, 5, 6, 7, or 8 weeks after the start of step (c)). The reduction in expression may instead be determined by determining the relative level of P1 in a fecal sample obtained from the patient.

[0048] The second promoter may be an inducible promoter (induction of the promoter causes increased expression of P2) or a repressible promoter (repression of the promoter causes decreased expression of P2), i.e., binding of R to the nucleic acid may induce or repress the second promoter.

[0049] For example, promoters herein are selected from Plac and Ptac (containing the lacO operator, lacI repressor), Ptet (containing the tetO operator, tetR repressor), and ParaBad (containing the araO operator, araC repressor). As will be appreciated by those skilled in the art, it is possible to synthetically produce repressible promoters from naturally occurring constitutive promoters by adding the tetO operator (or an analogue thereof) to the sequence and expressing tetR or an analogue to repress them.

[0050] P2 may comprise a nucleic acid (e.g., RNA) or a protein (e.g., a peptide). For example, P2 is a silencing RNA or protein that can bind to a nucleic acid and inhibit the expression of P1, for example, by binding a sequence that overlaps with the first promoter to the first promoter, or by binding to NS1.

[0051] P2 may be operable in the host cell to bind to the vector nucleic acid and downregulate expression of P1, and / or R may upregulate expression of P2. P2 may be operable in the host cell to bind to the vector nucleic acid and upregulate expression of P1.

[0052] P2 may be expressible in a host cell to form a nuclease that can act in the host cell to cleave nucleic acid. Preferably, the cleaved nucleic acid is degraded (optionally the vector containing the nucleic acid is degraded), thereby downregulating the expression of P1 in the host cell. Preferably, the cleaved nucleic acid is degraded, thereby downregulating the expression of P1 in the microbial population. P2 itself may be a nuclease or a component thereof (the component is combined with one or more other components in the host cell to form a nuclease, for example an RNA-guided nuclease). An example of a suitable nuclease is an RNA-guided endonuclease or a restriction endonuclease. For example, nucleases include AatII, AbaSI, Acc65I, AccI, AciI, AclI, AcuI, AfeI, AflII, AflIII, AgeI, AhdI, AleI, Alu I, AlwI, AlwNI, ApaI, ApaLI, ApoI, AscI, AseI, AsiSI, AvaI, AvaII, AvrII, BaeGI, BaeI, BamHI, BanI, BanI I, BbsI, BbvCI, BbvI, BccI, BceAI, BcgI, BciVI, BclI, BfaI, BglI, BglII, BlpI, BmgBI, BmrI, BmtI, BpmI, B puEI, Bpu10I, BsaAI, BsaBI, BsaHI, BsaI, BsaJI, BsaWI, BsaXI, BseRI, BseYI, BsgI, BsiEI, BsiHKAI, BsiW I, BslI, BsmAI, BsmBI, BsmFI, BsmI, BspCNI, BspEI, BspHI, Bsp1286I, BspMI, BsrBI, BsrDI, BsrFI, BsrGI, BsrI, BssHII, BssSI, BstAPI, BstBI, BstEII, BstNI, BstUI, BstXI, BstYI, BstZ17I, Bsu36I, BtgI, BtgZI, BtsCI, BtsIMutI, BtsI, Cac8I, ClaI, CspCI, CviAII, CviKI-1, CviQI, DdeI, DpnI, DraI, DraIII, DrdI, Eae I, EagI, EarI, EciI, Eco53kI, EcoNI, EcoO109I, EcoP15I, EcoRI, EcoRV, Esp3I, FatI, FauI, Fnu4HI, FokI,FseI, FspEI, FspI, HaeII, HaeIII, HgaI, HhaI, HincII, HindIII, HinfI, HinP1I, HpaI, HphI, HpyAV, HpyCH4I II, HpyCH4IV, HpyCH4V, Hpy99I, Hpy188I, Hpy166II, Hpy188III, I-CeuI, I-SceI, KasI, KpnI, LpnPI, MboI, M boII, MfeI, MluCI, MluI, MlyI, MmeI, MnlI, MscI, MseI, MslI, MspA1I, MspI, MspJI, MwoI, NaeI, NarI, Nb.Bbv CI, Nb.BsmI, Nb.BsrDI, Nb.BssSI, Nb.BtsI, NciI, NcoI, NdeI, NgoMIV, NheI, NlaIII, NlaIV, NmeAIII, NotI, N ruI, NsiI, NspI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, Nt.CviPII, PacI, PaqCI, PciI, PflMI , PI-PspI, PI-SceI, PleI, PluTI, PmeI, PmlI, PpuMI, PshAI, PsiI, PspGI, PspOMI, PspXI, PstI, PvuI, PvuII, RsaI, RsrII, SacI, SacII, SalI, SapI, Sau96I, SbfI, ScaI, ScrFI, SexAI, SfaNI, SfcI, SfiI, SfoI, SgrAI, Sm aI, SmlI, SnaBI, SpeI, SphI, SrfI, SspI, StuI, StyD4I, StyI, SwaI, TaqI, TfiI, TseI, Tsp45I, TspRI, Tth111I Restriction nuclease selected from PflFI, XbaI, XcmI, XhoI, XhoI PaeR7I, XmaI, XmaI TspMI, XmnI and ZraI. ,

[0053] In one example, P2 comprises a guided nuclease that is programmable in a host cell to guide the nuclease to a target nucleotide sequence contained in a nucleic acid, and the nuclease can cleave the target sequence, thereby degrading the nucleic acid sequence and inhibiting expression of P1 from the nucleic acid.

[0054] In one example, the nuclease is operable to cleave a target site contained in the vector nucleic acid, the target site comprising: (i) Is it included in NS1? (ii) Not included in NS1; (iii) Included in NS2? (iv) Not included in NS2; (v) is included in a nucleotide sequence of the vector encoding the CRISPR / cas system or a component thereof (e.g., the component is a nucleotide sequence encoding Cas, or the component encodes a crRNA or a guide RNA); (vi) is not contained in the nucleotide sequence of the vector encoding the CRISPR / cas system or a component thereof (e.g., the component is a nucleotide sequence encoding Cas, or the component encodes a crRNA or guide RNA); (vii) contained in a nucleotide sequence encoding a restriction endonuclease; (viii) is not contained in a nucleotide sequence encoding a restriction endonuclease; (ix) contained in the first promoter; or (x) contained in a second promoter.

[0055] The CRISPR / cas system comprises at least one Cas (e.g., Cas3 (optionally also a cascade Cas, e.g., CasA-E), Cas9, Cas12, or Cas13) and a cognate guide RNA that can form a Cas / guide RNA complex to recognize and bind to a protospacer sequence. In the above example, the protospacer is included in the target site. The guide RNA may be a single guide RNA.

[0056] P2 is, a) RNA-guided nucleases; b) an RNA or a precursor of such an RNA that is operable to guide an RNA-guided nuclease; or c) Restriction endonucleases may include:

[0057] Optionally, the precursor is RNA, pre-cRNA, or tracrRNA.

[0058] In one example, P2 can bind to an operator (O) contained in a nucleic acid and operably linked to a first promoter (e.g., O is 5' to the first promoter, e.g., within 200 or 100 kb 5' of the promoter), and when P2 binds to O, expression of P1 is reduced (e.g., eliminated). In one example, P2 can comprise a dead Cas nuclease (e.g., dCas9 or dCas3 or dCas12 or dCas13), and dCas can recognize and bind to a protospacer contained in the nucleic acid to form a dCas / guide RNA complex in the host cell that can disrupt expression of P1 (e.g., by disrupting promoter function of the first promoter).

[0059] Optionally, the guided nuclease is a Cas nuclease, a TALEN, a meganuclease, or a zinc finger nuclease, preferably a Cas nuclease. The nuclease can cleave DNA or RNA, preferably RNA.

[0060] The nuclease may be operable to cleave the nucleic acid at a predetermined sequence motif (target site), optionally at a protospacer sequence or a restriction site. The protospacer may be a CRISPR / Cas protospacer. The restriction site may be cleaved by a restriction endonuclease or any other restriction nuclease disclosed herein. The nucleic acid may contain a plurality (e.g., at least two or three, e.g., 2, 3, 4, 5, 6, 7, 8, or 9) of said motifs. This may be useful for the efficiency of cleavage and destruction of the nucleic acid (or vector containing the nucleic acid).

[0061] P1 may be an amino acid, a protein (e.g., a peptide or polypeptide), or an RNA (e.g., an mRNA or a silencing RNA) for human or animal treatment. For example, P1 is a cytokine, a growth factor, an enzyme, a hormone, or an antibody (or an antibody chain or an antibody fragment). For example, P1 is an antibody chain or an antibody fragment, such as a single domain antibody (also called dAb) or a nanobody. For example, the chain or fragment is a human antibody chain or fragment. For example, P1 is an antibody heavy chain that forms an antibody together with an antibody light chain that is also expressed in a host cell. Preferably, the antibody, chain, or fragment is capable of being secreted from the host cell. Preferably, the antibody, chain, or fragment is secreted from the host cell. For example, P1 is an incretin, such as an incretin peptide or a multimer thereof. Optionally, the incretin is selected from GLP-1, GIP, exendin-4, and insulin.For example, P1 comprises an antigen-binding site of an antibody or a variable domain of an antibody (e.g., a VH and / or a VL domain), such as an antibody selected from the group consisting of ReoPro™, Abciximab, Rituxan™, Rituximab, Zenapax™, Daclizumab, Simulect™, Basiliximab, Synagis™, Palivizumab, Remicade™, Infliximab, Herceptin™, Mylotarg™, Gemtuzumab, , Campath(TM), Alemtuzumab, Zevalin(TM), Ibritumomab, Humira(TM), Adalimumab, Xolair(TM), Omalizumab, Bexxar(TM), Tositumomab, Ra ptiva(TM), Efalizumab, Erbitux(TM), Cetuximab, Avastin(TM), Bevacizumab, Tysabri(TM), Natalizumab, Actemra(TM), Tocilizumab, Vec tibix(TM), Panitumumab, Lucentis(TM), Ranibizumab, Soliris(TM), Eculizumab, Cimzia(TM), Certolizumab, Simponi(TM), Golimumab, Il aris(TM), Canakinumab, Stelara(TM), Ustekinumab, Arzerra(TM), Ofatumumab, Prolia(TM), Denosumab, Numax(TM), Motavizumab, ABThrax (TM), Raxibacumab, Benlysta™, Belimumab, Yervoy™, Ipilimumab, Adcetris™, Brentuximab, Vedotin™, Perjeta™, Pertuzumab, Pembrolizumab, Nivolumab, Atezolizumab, Kadcyla™, Ado-trastuzumab, Keytruda™, Opdivo™, Gazyva™, and Obinutuzumab. For example, P is selected from an insulin peptide, an incretin peptide, or a peptide hormone.For example, the antibody is adalimumab. For example, the antibody is pembrolizumab. For example, the antibody is nivolumab. For example, the antibody is atezolizumab. For example, the antibody is dupilumab. For example, the antibody is tocilizumab. For example, the antibody is sarilumab. For example, the antibody is alirocumab. For example, the antibody is evolocumab. Alternatively, the antibody is an anti-CD38 antibody, an anti-TNFa antibody, an anti-TNFR antibody, an anti-IL-4Ra antibody, an anti-IL-6R antibody, an anti-IL-6 antibody, an anti-VEGF antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-PCSK9 antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-CD138 antibody, an anti-IL-1 antibody. Alternatively, the antibody is selected from the antibodies disclosed on page 40, line 23 to page 43, line 23 of WO2007024715, the disclosures of which are incorporated herein by reference.

[0062] Antigens herein include ABCF1, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, ADORA2A, aggrecan, AGR2, AICDA, AWI, AIG1, AKAP1, AKAP2, AIYIH, AMHR2, ANGPT1, ANGPT2, ANGPTL3, ANGPTL4, ANPEP, APC, APOC1, AR, AZGP1 (zinc-a-glycoprotein), B7.1, B7.2, BAD, BAFF, BAG1, BAI1, BCL2, BCL6, BDNF, BLNK, BLR1 (MDR15), BlyS , BMP1, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, BMPRIA, BMPRIB, BMPR2, BPAG1 (plectin), BRCA1, CI9orflO (IL27w), C3, C4A, C5, C5R1, CANT1, CASP1, C ASP4, CAV1, CCBP2(D6 / JAB61), CCL1(1-309), CCL11(eotaxin), CCL13(MCP-4), CCL15(MIP-id), CCL16(HCC-4), CCL17(TARC), CCL18(PARC), CCL19(M IP-3b), CCL2 (MCP-1), MCAF, CCL20 (MIP-3a), CCL21 (MIP-2), SLC, Exodus-2, CCL22 (MDC / STC-1), CCL23 (M PIF-1), CCL24 (MPIF-2I eotaxin-2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK / ILC), CCL28, CCL3 (MIP-la), CCL4 (M IP-lb), CCL5(RANTES), CCL7(MCP-3), CCL8(mcp-2), CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CCR1(CKR1 / HM145), CCR2(mcp-1RB / RA); CCR3(CKR3 / CMKBR3), CCR4, CCR5(CM KBR5 / ChemR13), CCR6(CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7(CKR7 / EBI1), CCR8(CMKBR8 / TER1 / CKR-L1), CC R9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), CD164, CD19, CD1C, CD20, CD200, CD-22, CD24, CD28, CD3, CD37,CD38、CD3E、CD3G、CD3Z、CD4、CD40、CD40L、CD44、CD45RB、CD52、CD69、CD72、CD74、CD79A、CD79B、CD8、CD80、CD81、CD83、CD86、CDH1(E-カドヘリン)、CDH10、CDH12、CDH13、CDH18、CDH19、CDH20、CDH5、CDH7、CDH8、CDH9、CDK2、CDK3、CDK4、CDK5、CDK6、CDK7、CDK9、CDKN1A(p2IWapl / Cipl)、CDKN1B(p27Kipl)、CDKNIC、CDKN2A(pl6INK4a)、CDKN2B、CDKN2C、CDKN3、CEBPB、CER1、CHGA、CHGB、キチナーゼ、CHST10、CKLFSF2、CKLFSF3、CKLFSF4、CKLFSF5、CKLFSF6、CKLFSF7、CKLFSF8、CLDN3、CLDN7(クラウジン-7)、CLN3、CLU(クラステリン)、CMKLR1、CMKOR1(RDC1)、CNR1、COL18A1、COL1A1、COL4A3、COL6A1、CR2、CRP、CSFl(M-CSF)、CSF2(GM-CSF)、CSF3(GCSF)、CTLA4、CTNNBl(b-カテニン)、CTSB(カテプシンB)、CX3CL1(SCYDi)、CX3CR1(V28)、CXCL1(GROl)、CXCLIO(IP-10)、CXCL11(l-TAC / IP-9)、CXCL12(SDF1)、CXCL13、CXCL14、CXCL16、CXCL2(GR02)、CXCL3(GR03)、CXCL5(ENA-78I LIX)、CXCL6(GCP-2)、CXCL9(MIG)、CXCR3(GPR9 / CKR-L2)、CXCR4、CXCR6(TYMSTR ISTRL33 I Bonzo)、CYB5、CYC1、CYSLTR1、DAB2IP、DES、DKFZp451J0118、DNCL1、DPP4、E2F1、ECGF1、EDG1、EFNAI、EFNA3、EFNB2、EGF、EGFR、ELAC2、ENG、EN01、EN02、EN03、EPHB4、EPO、ERBB2(Her-2)、EREG、ERK8、ESR1、ESR2、F3(TF)、FADD、FasL、FASN、FCER1A、FCER2、FCGR3A、FGF、FGF1(aFGF)、FGF10, FGF11, FGF12, FGF12B, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2(bFGF), FGF20, FGF21, FGF22, FGF23, FGF3(int-2), FGF4( HST), FGF5, FGF6(HST-2), FGF7(KGF), FGF8, FGF9, FGFR3, FIGF(VEGFD), FILL(EPSILON), FILl(ZETA), FU12584, FU25530, FLRTl(Fibronec Chin), FLTl, FOS, FOSLl(FRA-I), FY(DARC), GABRP(GABAa), GAGEB1, GAGEC1, GALNAC4S-65T, GATA3, GDF5, GFI1, GGT1, GM-CSF, GNAS1, GNRH l, GPR2(CCRIO), GPR31, GPR44, GPR81(FKSG80), GRCCIO(CIO), GRP, GSN(gelsolin), GSTPl, HAVCR2, HDAC4, EDAC5, HDAC7A, HDAC9, HGF, HIF1 A, HIP1, histamine and histamine receptor, HLA-A, HLA-DRA, HM74, HMOX1, HUMCYT2A, ICEBERG, ICOSL, 1D2, IFN-a, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNB1, IFN gamma, TFNW1, IGBP1, IGF1, IGF1R, IGF2, IGFBP2, IGFBP3, IGFBP6, IL-1, IL10, IL10RA, IL10RB, IL11, IL11RA, IL-12, IL12 A, IL12B, IL12RB1, IL12RB2, 1L13, IL13RA1, IL13RA2, 1L14, 1L15, IL15RA, IL16, 1L17, IL17B, IL17C, IL17R, 1L18, IL18BP, IL18R1, IL18 RAP, 1L19, ILIA, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL1HY1, IL1R1, IL1R2, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RL1, IL1RL2 IL1RN, 1L2, 1L20, IL20RA, IL21R, 1L22, 1L22R, 1L22RA2, 1L23, 1L24, 1L25, 1L26, 1L27, 1L28A, 1L28B, 1L29, IL2RA,IL2RB, IL2RG, 1L3, 1L30, IL3RA, 1L4, IL4R, 1L5, IL5RA, 1L6, IL6R, IL6ST 130) 1L7 TL7R 1L8 IL8RA IL8RB IL8RB 1L9 IL9R ILK INHA INHBA I NSL3, INSL4, IRAQ, IRAQ2, ITGA1, ITGA2, 1TGA3, ITGA6(a6イテグリン), ITGAV, IT GB3, ITGB4(b4, JAK1, JAK3, JUN, K6HF, KAI1, KDR, MTLG, KLF5(GC). Box BP)、KLF6、KLK10、KLK12、KLK13、KLK14、KLK15、KLK3、KLK4、KLK5、KLK 6. KLK9, KRT1, KRT19 (KRT19), KRT2A, KRTHB6 (Razor Antigen II and KRTHB6), LAMA5 LEP(レプチン), Lingo-p75, Lingo-Troy, LPS, LTA(TNF-b), LTB, LTB4R(G PR16) LTB4R2 LTBR MACMARCKS MAG Omgp MAP2K7(c-Jun) MDK M IB1, M IF, M IP-2, MK167 (Ki-67), MMP2, M MP9, MS4A1, MSMB, MT3 (Manufacturer-ifi), MTSS 1, M UC 1(MYC), MYD88, NCK2, NFKB 1, NFKB2, NGFB(NGF), NGFR, NgR-Lingo, NgR-Nogo66(Nogo), NgR-p75, NgR-Troy, NME1(NM23A), NOX5, NPPB, NROB1 NROB2, NR1D1, NR1D2, NR1H2, NR1H3, NR1H4, NR1I2, NR1I3, NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR2F6, NR3C 1, NR3C2, NR4A1, NR4A2, NR4A3, NR5A1, NR5A2, NR6A1, NRP1, NRP2, NT5E, NTN4, ODZ1, OPRD1, P2RX7, PAP, PARTY, PATE PAWR, PCA3, PCNA, PDGFA, PDGFB, PECAM1, PF4(CXCL4), P.S GF, PGR, PIAS2, PIK3CG, PLAU(uPA), PLG, PLXDC1PPBP (CXCL7), PPID, PR1, PRKCQ, PRKD1, PRL, PROC, PROK2, PSAP, PSCA, PTAFR, PTEN, PTGS2 (COX-2), PTN, RAC2 (p2IRac2), RARB, RGS1, RGS13, RGS3, RNF110 (ZNF144), ROB02, S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2), SCGB2A2 (mammaglobin 1), SCYE1 (endothelial monocyte-activating cytokine), SDF2, SERPINA1, SERPINIA3, SERPINB5 (maspin), SERPINE1 (PAT-i), SERPINF1, SHBG, SLA2, SLC2A2, SL C33A1, SLC43A1, SLIT2, SPPl, SPRRIB(Spri), ST6GAL1, STABl, STAT6, STEAP, STEAP2, TB4R2, TBX21, TCPIO, TDGF1, TEK, TGFA, TGFB1, TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TG FBR3, TH1L, THBS1 (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-i), T]MP3, tissue factor, TLRIO, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TNF, TNF-a, TNFAIP2 (B94), TNFAIP3, TNFRSF1 1A, TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF5, TNFRSF6 (Fas), TNFRSF7, TNFRSF8, TNFRSF9, TNFSFIO (TRAIL), TNFSF1 1 (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF1 5 (VEGI), TNFSF1 8, TNFSF4 (0X40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-lBB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase lia), TP53, TPM 1, TPM2, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TREM 1, TREM2,TRPC6, TSLP, TWEAK, VEGF, VEGFB, VEGFC, versican, VHL C5, VLA-4, XCL1 (lymphotactin), XCL2 (SCM-lb), XCR1 (GPR5 / CCXCR1), YY1, and ZFPM2.

[0063] For example, P1 includes the VEGF binding site of aflibercept, e.g., the anti-VEGF FLT1 and / or KDR domains.

[0064] For example, P1 can be an incretin, an insulin peptide, a GLP-1 (glucagon-like peptide 1 (GLP-1) peptide, a GIP (glucose-dependent insulinotropic polypeptide) peptide, an exendin (e.g., exendin-4) peptide, a peptide hormone, a prolactin or a prolactin peptide, an ACTH or an ACTH peptide, a growth hormone or a growth hormone peptide, a vasopressin or a vasopressin peptide, an oxytocin or an oxytocin peptide, a glucagon or a glucagon peptide, an insulin or an insulin peptide, a somatostatin or a somatostatin peptide, a cholecystokinin or a cholecystokinin peptide, a gastrin or a gastrin peptide, a leptin or a leptin peptide, an antibody binding site (e.g., scFv or Fab) or a variable domain thereof, a TCR binding site (e.g., scTCR) or a domain thereof, a TCR Vα / Vβ binding site or a variable domain thereof, a TCR It comprises (or is) a Vγ / Vδ binding site or variable domain thereof, an antibody single chain variable domain binding site, or an FcAb binding site. In one example, P1 comprises at least one copy of GLP-1 and at least one copy of another incretin (e.g., Exendin-4). For example, there is one copy of GLP-1 and one copy of another incretin. Preferably, in the examples of this paragraph, P1 is a secreted or host cell surface exposed protein. Optionally, any GLP-1 herein is GLP-1(7-37)-Pro9. Optionally, any incretin herein is Exendin-4 or Peptide Y. Optionally, any P1 or incretin herein is DURAGLUTIDE™.

[0065] P1 may comprise an antigen binding site. The binding site herein may be, for example, an antigen (for example, a cytokine or growth factor, such as VEGF or EGFR) binding site of a receptor (for example, KDR or Flt). The binding site herein may be, for example, an Eyelea™, Avastin™, or Lucentis™ binding site for ophthalmology or oncology medical use in humans or animals. If the antigen is VEGF, the vector or method may be for the treatment or prevention of cancer or ophthalmology conditions (for example, wet or dry AMD or diabetic retinopathy), or as an inhibitor of angiogenesis in humans or animals.

[0066] For example, P1 is a component of a metabolic pathway, such as an enzyme or drug in the pathway. For example, P1 is an intracellular enzyme in the target cell (i.e., the host cell). For example, P1 is a secreted enzyme (e.g., secreted from the target cell). Here, the pathway may be in the target cell or outside the target cell. For example, the pathway is an internal pathway of a different cell contained in a microbial community of which the target cell is a component. The different cell may be a carrier cell or may be an endogenous cell of the microbial community (i.e., any cell of the microbial community except the target cell or the carrier cell). The pathway may contain, as a product or intermediate, - Short chain fatty acids (SCFAs), Lipids, indole derivatives or serotonin (e.g., the pathway is for the conversion of tryptophan (Trp) to one or more indole derivatives or serotonin), Hormones or incretins (e.g. GLP-1, oxytocin), One or more antigens (e.g., to stimulate an immune response for vaccination of a subject) may include one or more of:

[0067] Optionally, R upregulates expression of P2, and P2 downregulates expression of P1, and optionally P1 is a component in a metabolic pathway (e.g., in a microbial population or target cell, or in a subject or environment containing a microbial population or target cell), and a product of the pathway downstream of P1 (X) causes regulation of expression of P1 or P2. See, e.g., scenarios 1, 3, and 4 (Figure 7). In one embodiment, X is R or a precursor of R. In one embodiment, X controls a first and / or second promoter. In one embodiment, X upregulates expression of P1 or P2. In one embodiment, X upregulates expression of P1 or P2.

[0068] Optionally, R upregulates expression of P2, and P2 upregulates expression of P1, and optionally P1 is a component in a metabolic pathway (e.g., in a microbial population or target cell, or in a subject or environment containing a microbial population or target cell), and a product of the pathway downstream of P1 (X) causes regulation of expression of P1 or P2. See, e.g., Scenario 2 (Figure 7). In one embodiment, X is R or a precursor of R. In one embodiment, X controls a first and / or second promoter. In one embodiment, X upregulates expression of P1 or P2. In one embodiment, X upregulates expression of P1 or P2.

[0069] In one example, P1 is a secreted or cell surface exposed protein antigen, which is useful for vaccinating a subject, for example, when the antigen is an antigen of a pathogen (e.g., a bacterium or virus, e.g., a coat protein, e.g., a spike protein, e.g., a SARS-Cov or SARS-Cov-2 or influenza antigen).

[0070] In one example, P1 is a secreted antagonist of a target ligand in a subject. For example, binding of P1 to a ligand can be marked to inhibit or neutralize the ligand or to destroy it in a subject (e.g., by immune cells of the subject). For example, the antagonist can include an antibody fragment (e.g., a nanobody or any other antibody single chain variable domain) that contains a binding site for the target ligand. The ligand can be, for example, Curli proteins (e.g. E. coli curli proteins), TMA (trimethylamine), ·gluten, ·Bile acids, Cholesterol or PCSK9, or Bacterial toxins (e.g., toxins encoded by pks (e.g., E. coli pks), C. difficile toxins, V. cholerae toxins, anthrax toxins, or B. fragilis toxins) It may be.

[0071] In one embodiment, P1 is toxic to cells of the same species as the host cells, which may be useful to kill or reduce the growth or proliferation of such cells in a microbial population, such as when the cells are harmful to the health of a subject.

[0072] In one embodiment, P1 is a transcriptional or translational regulator in a cell of the same species as the host cell, i.e., upregulation or downregulation of expression of P1 can favorably affect one or more genes in the genome of the host cell.

[0073] Optionally, R is an amino acid, a protein, a carbohydrate (e.g., a sugar), a lipid, a metal ion, or a nucleic acid (e.g., RNA). R may be a sugar alcohol, such as xylitol, glycerol, arabitol, erythritol, isomalt, HSH, lactitol, maltitol, mannitol, or sorbitol, preferably xylitol. R may be an antibiotic. R may be a metabolite of a metabolic pathway operating in the subject or microbial population. R is optionally a metabolite of P1 or a metabolite produced in a pathway that includes P1. For example, P1 may be a secreted (i.e., secreted from a host cell) enzyme that can act in a pathway in the subject that produces R. For example, P1 may be metabolized in a pathway that produces R (e.g., P1 is metabolized to produce R directly or indirectly). The pathway may be inside the target host cell or outside the target host cell (e.g., in adjacent cells in a microbial population of which the target cell is a component). The effect of R on expression of P1 may (or may not) be dose-dependent. One of skill in the art can readily determine the appropriate amount of R to use to provide the desired effect on a microbial population (e.g., by titrating the dose in an assay).

[0074] As exemplified herein, R can be xylitol. Operons of genes related to sugar conversion / utilization can be induced by the sugar itself through their binding to the relevant transcriptional repressor protein. For this study, we decided to consider negatively repressible promoters predicted to be induced by xylitol. This 5-carbon sugar alcohol has several advantages. First of all, xylitol is generally considered safe for human consumption by the FDA (Xiang et al., 2021). Furthermore, more than half of ingested xylitol is not adsorbed by human cells, but instead reaches the gastrointestinal tract where it is taken up by the microbiome (Livesey, 2003). Finally, xylitol serves as a metabolic product for some bacteria. It is either directly taken up through ABC-type transporter complexes (Madigan et al., 2015) and / or produced through the reduction of the corresponding sugar D-xylose. Xylitol is then typically dehydrogenated and phosphorylated to xylulose-5-phosphate, which is further catabolized via the pentose phosphate pathway.

[0075] For example, R is an amino acid. For example, R is selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In one embodiment, R is tryptophan. For example, R is a protein (e.g., a peptide), for example, R is selected from herring, sakacin A, and sakacin P. For example, R is a bacteriocin. For example, R is a carbohydrate, for example, R is selected from a sugar, for example, 1-arabinose, 1-rhamnose, xylose, and sucrose. For example, R is a metal ion, for example, R is Fe. 2+ , Mn 2+ , Co 2+ , Hg 2+ , and Cu 2+For example, R is a lipid, e.g., proprionate. For example, R is a fatty acid. For example, R is a nucleic acid, e.g., R is RNA. For example, R is a benzene compound, e.g., a substituted benzene compound or benzoic acid.

[0076] Optionally, the vector is an ICE (integral conjugative element), a plasmid (e.g., a conjugative plasmid), a transduction particle (e.g., a phage or a non-self-replicating transduction particle), or a nanoparticle. In one embodiment, the plasmid comprises oriT and oriV. The plasmid may be self-conjugative. The plasmid may be a shuttle plasmid, i.e., a plasmid capable of propagation in at least two different host species. The plasmid may comprise a transposon or ICE (or a mobilizing portion thereof) comprising a nucleic acid comprising NS1 and NS2. For example, the transposon or ICE may be a Bacteroides transposon or ICE. The plasmid may comprise oriT and may be mobilizable in the presence of a conjugation system, e.g., a system found in other plasmids or integrative conjugative elements (ICEs) in the host cell or microbial community. In one example, such a conjugation system is comprised in the genome of the donor cell or carrier cell. In one example, the conjugation system is also or instead comprised in the genome of the recipient cell. The conjugation system may be carried in the chromosome or episome of the carrier cell as described herein (i.e., a system in trans relative to the plasmid). The conjugation system may be contained in the plasmid itself (i.e., a system in cis). The system may be contained in a cell in the microbial population adjacent to the host cell into which the plasmid has been transferred. In this way, the plasmid can spread between adjacent cells, thereby propagating the plasmid vector in the microbial population. This may be useful to amplify expression of P1 in the microbial population. Similarly, autoconjugative plasmids (i.e., the conjugation system is provided along with the oriT on the plasmid) allow the plasmid to spread in the microbial population.

[0077] The vector may be a conjugative plasmid contained in a carrier cell (e.g., a bacterial carrier cell). For example, the carrier cell and the vector are for administration to the microbial community of a human or animal subject. For example, the carrier cell is a commensal or probiotic bacterial cell species of the microbial community of the human or animal. Additionally or alternatively, the carrier cell is a cell of a species of the microbial community of the human or animal gut.

[0078] The conjugative genes of Inc group conjugative plasmids, i.e. conjugative plasmids such as P, N, W, or X, show similarity at the protein level to the VirB system of Agrobacterium, which constitutes itself a prototypic type IV secretion system (T4SS). They are often small and are probably the smallest systems in existence. The genes required for conjugation of proteins homologs in the prototypic VirB plasmid pTI as well as the incN (somewhat related) and incF (distantly related) families of plasmids are shown in Table 4.

[0079] Optionally, the conjugation system is the VirB, IncN, or IncF conjugation system, or a homolog or ortholog thereof.

[0080] Each vector may be a self-conjugative plasmid comprising oriT and a conjugation system for transferring plasmids between cells in a microbial community, the conjugation system being a VirB, IncN, or IncF conjugation system, or a homolog or orthologue thereof. The carrier (or donor) cell and said plasmid vector may comprise a conjugation system between them for transferring plasmids between cells in a microbial community, the conjugation system being a VirB, IncN, or IncF conjugation system, or a homolog or orthologue thereof, and each vector comprising oriT. The host (or recipient) cell and said plasmid vector may comprise a conjugation system between them for transferring plasmids between cells in a microbial community, the conjugation system being a VirB, IncN, or IncF conjugation system, or a homolog or orthologue thereof, and each vector comprising oriT.

[0081] For example, the system is a VirB plasmid pTIm, an IncN plasmid, or an IncF plasmid system, or the system is a homolog or ortholog thereof.

[0082] For example, the system includes (i) the VirB genes virB1-11 and virD4, or homologs or orthologs of said genes, (ii) the IncN genes traA-G, traJ, traL, traM, traN, and traO, or homologs or orthologs of said genes, (iii) the IncF genes traA-E, traG, traH, traK, traL, and ORF196, or homologs or orthologs of said genes, or (iv) the IncF genes traA-I, traK, traL, traM, traW, and traU (and optionally at least one or all of traX, traN, finO, trbI, and trbB), or homologs or orthologs of said genes. For option (iv), see Front Mol Biosci.,2016 Nov 10;3:71.doi:10.3389 / fmolb.2016.00071.eCollection 2016,“Comparative Genomics of the Conjugation Region of F-like Plasmids:Five Shades of F”,Raul Fernandez-Lopez et al.

[0083] According to a fourth configuration, the present invention provides a method for manufacturing a semiconductor device comprising: A nucleic acid vector for the transfer of a microbial population into a host cell, the vector being contained in a carrier cell (e.g., a bacterial cell); c) a nuclease (optionally an RNA-guided nuclease or a restriction endonuclease) operable in the carrier cell to cleave a chromosome or episome (which is not the vector of the invention) of the carrier cell, optionally resulting in degradation of the chromosome or episome; and / or d) an RNA or a precursor of such an RNA operable in a carrier cell to guide an RNA-guided nuclease, where the RNA guides the nuclease to cleave a chromosome or episome (which is not a vector) of the carrier cell, and optionally the chromosome or episome is degraded; Code the following: Also provided is a nucleic acid vector, wherein the vector comprises one or more controllable promoters for controlling expression of the nuclease of (a) and / or the RNA or component of (b) in a carrier cell.

[0084] The vector may have any of the characteristics of a vector disclosed herein. The episome may be a plasmid.

[0085] In one embodiment, the vector comprises an inducible or repressible promoter that controls the expression of the nuclease of (a) and / or the vector comprises an inducible or repressible promoter that controls the expression of the RNA or component of (b), preferably the promoter is an inducible promoter. Alternatively, the promoter is a repressible promoter. In one example, components (a) and (b) are under the control of different promoters. In one example, components (a) and (b) are under the control of a common promoter.

[0086] In one embodiment, the vector comprises an inducible promoter that controls expression of the nuclease of (a). In one embodiment, the vector comprises an inducible promoter that controls expression of the RNA or component of (b). Alternatively, the vector comprises a repressible promoter that controls expression of the RNA or component of (b).

[0087] In one embodiment, the vector comprises a repressible promoter that controls expression of the nuclease of (a). In one embodiment, the vector comprises an inducible promoter that controls expression of the RNA or component of (b). Alternatively, the vector comprises a repressible promoter that controls expression of the RNA or component of (b).

[0088] Optionally, the guided nuclease is Cas nuclease, TALEN, meganuclease, or zinc finger nuclease, preferably Cas nuclease.For example, the guided nuclease is Cas3.For example, the guided nuclease is Cas9.For example, the guided nuclease is Cas12 (for example, Cas12a).For example, the guided nuclease is Cas13 (for example, Cas13a).

[0089] The nuclease in the fourth configuration is the same guided nuclease as in the first configuration.

[0090] The nuclease in the fourth configuration is operable to cleave the chromosome or episome in the carrier cell at a predetermined sequence motif, optionally a protospacer sequence or a restriction site. Preferably, the cleavage of the chromosome or episome of the carrier cell kills the carrier cell or reduces the growth or proliferation of the carrier cell, most preferably the cell is killed. This is useful for reducing the transmission of the vector nucleic acid, for example when a microbial population or environment is exposed to the vector. This provides a useful method of controlling the expression of a product from the vector, for example in a recipient cell into which the vector nucleic acid has been transferred.

[0091] In one example, the vector contains an oriT for transfer into a host cell, and optionally the vector is a conjugative plasmid.

[0092] In a preferred embodiment, a) the vector is a conjugative plasmid; b) the vector comprises an inducible promoter that controls expression of the nuclease of (a), and / or the vector comprises an inducible promoter that controls expression of the RNA or component; c) optionally, the guided nuclease is a Cas nuclease; Cleavage of the chromosome or episome of the carrier cell kills the carrier cell or reduces the growth or proliferation of the carrier cell.

[0093] According to the fourth configuration, A nucleic acid vector for the transfer of a microbial population into a host cell, the vector being contained in a carrier cell (e.g., a bacterial cell); a) a nuclease (optionally an RNA-guided nuclease or a restriction endonuclease) operable in the carrier cell to cleave a chromosome or an episome (which is not the vector of the invention) of the carrier cell, optionally resulting in degradation of the chromosome or episome, and / or b) an RNA or a precursor of such an RNA operable in a carrier cell to guide an RNA-guided nuclease, where the RNA guides the nuclease to cleave a chromosome or episome (which is not a vector) of the carrier cell, and optionally the chromosome or episome is degraded; Code the following: the vector comprises one or more controllable promoters for controlling expression of the nuclease of (a) and / or the RNA or component of (b) in the carrier cell; Nucleic acid vectors are provided in which chromosomal or episomal cleavage of the carrier cell kills the carrier cell or reduces the growth or proliferation of the carrier cell, preferably resulting in cell death.

[0094] A plurality of carrier cells containing the vector is also provided. By cutting, the number of carrier cells in the plurality is increased to at least 10 5 1 / 10 6 1 / 10th or 1 / 10th 7 one part, e.g. 10 5 1 / 10 7 1 / 10th or 1 / 10th 5 1 / 10 8 1 / 10th or 1 / 10th 5 1 / 10 9The number of the carrier cells may be reduced to at least 10 times. Those skilled in the art are familiar with determining the fold death or reduction in cells, for example, using a cell sample representative of a microbial or cell population. For example, the degree of death or reduction is determined using a cell sample, for example, a sample obtained from a subject to which the carrier cells of the present invention have been administered, or an environmental sample (e.g., an aqueous, water, or soil sample) obtained from an environment (e.g., a water source, a waterway, or a field) that has come into contact with the carrier cells of the present invention. For example, by cutting, the number of the carrier cells in the plurality may be reduced to at least 10 times. 5 1 / 10 6 1 / 10th or 1 / 10th 7 1 / 2 reduction, optionally each of the plurality of carrier cells comprises at least 100,000, 1,000,000, or 10,000,000 carrier cells. Optionally, the plurality of carrier cells is included in a cell population of a microorganism population, and at least 5, 6, or 7 log10 cells of the population are killed by severing, optionally each of the plurality of carrier cells comprises at least 100,000, 1,000,000, or 10,000,000 carrier cells. Optionally, at least 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or 99.99999% of the cells of the plurality of carrier cells are killed by severing.

[0095] A method is provided for killing a plurality of carrier cells in a population of microorganisms, the carrier cells comprising the vector of a fourth configuration, the method comprising upregulating (e.g., by inducing a controllable promoter) expression of (a) nuclease and / or (b) RNA or component in the carrier cells, thereby cleaving the genome of the carrier cells and killing the cells. Preferably, the chromosome of the cells is cleaved. The method optionally kills at least 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or 99.99999% of the cells of said plurality of carrier cells. In one example, the method kills all (or essentially all) cells of said plurality of carrier cells. In one example, the method kills 100% (or about 100%) of the plurality of carrier cells. Preferably, all carrier cells in the population of microorganisms are cells of said plurality.

[0096] Preferably, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the carrier cells are killed.

[0097] The conjugative plasmids herein may be autoconjugative plasmids (i.e., the plasmid contains oriT and encodes all the proteins necessary to mobilize the plasmid for conjugative transfer between cells).

[0098] For any of the configurations herein, the host cell (e.g., a donor, carrier, or recipient cell) is a cell of a species found in the human or animal microbiome (e.g., the gut microbiome). In a preferred example, the species is a Bacteroides species. In a preferred example, the species is E coli. The host cell may be a cell of a commensal or probiotic bacterial cell species of the human or animal microbiome. For example, the species is selected from any species in Table 1, preferably a Bacteroides species (e.g., Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, or Bacteroides ovatus) or a Clostridioides species (e.g., Clostridioides difficle or Clostridium disporicum).

[0099] In one embodiment, a) P1 is a protein or RNA (e.g., a silencing RNA) for human or animal therapy; b) P2 comprises (i) a crRNA (e.g., comprised in a single guide RNA) operable in a host cell to guide a Cas nuclease to bind to a protospacer sequence comprised in a nucleic acid for cleavage of the protospacer, optionally resulting in degradation of the nucleic acid, thereby downregulating expression of P1, or (ii) a precursor of such a crRNA (e.g., a pre-cRNA).

[0100] Human or animal therapy herein may be the treatment or prophylaxis of a disease or condition in a human or animal.

[0101] For any of the configurations herein, the crRNA may be included in a guide RNA, e.g., a single guide RNA. For example, the single guide RNA includes a crRNA and a tracrRNA operable in a recipient and / or carrier (donor) cell with Cas9 to cleave a cognate target nucleic acid sequence.

[0102] The present invention provides a vector according to any of the configurations herein for use as a medicament.

[0103] The medicament may be for treating or preventing a disease in a human or animal subject, for example when included in a formulation for oral administration to the subject.

[0104] A pharmaceutical composition comprising a vector according to any of the configurations herein and a pharma- ceutically acceptable carrier, diluent or excipient, and optionally an antacid.

[0105] A tablet, suppository, pill, capsule, or liquid formulation for administration to the gastrointestinal tract of a human or animal subject comprising a vector according to any of the configurations herein.

[0106] Optionally, the tablet, pill, or capsule comprises an enteric coating.Optionally, the tablet, pill, capsule, or liquid formulation is for oral administration pharmaceutical use.

[0107] In one example, the composition, tablet, suppository, pill, capsule, or formulation herein comprises a pharmaceutical agent selected from those listed in Table 3.

[0108] The present invention relates to A method is provided for temporally controlling the production of an expression product in a human or animal subject, the method comprising: a) administering a vector comprising a nucleic acid to a microbial population of a subject (e.g., the gut microbial population), the microbial population comprising host cells (e.g., bacterial cells), the nucleic acid encoding a product of interest (P1), and optionally the administration being oral or topical; b) transferring nucleic acid into a host cell in the population of microorganisms and expressing P1 in the host cell; c) after step (b), exposing the microbial population to a regulator (R) that upregulates the production in the host cell of an RNA-guided nuclease / guide RNA complex that can target a protospacer contained in the nucleic acid, where the nuclease cleaves the nucleic acid and expression of P1 becomes non-functional (e.g., by degradation of the cleaved nucleic acid in the cell), and the nuclease (or a component thereof) and / or the RNA (or a component thereof) are encoded by the nucleic acid. Includes.

[0109] The present invention relates to A method is provided for temporally controlling the production of an expression product in a human or animal subject, the method comprising: a) administering a vector, composition, tablet, suppository, pill, capsule or liquid formulation according to any preceding claim to the microbiome of a subject (e.g. the gut microbiome), optionally wherein the administration is oral or topical; b) transferring nucleic acid into a host cell in the population of microorganisms and expressing P1 in the host cell; and c) after step (b), exposing the microbial population to R (e.g., by administering R to the subject), where R controls the second promoter, thereby controlling expression of P2 and P1. Includes.

[0110] Step (b) may be for any desired time between time points T1 and T2. For example, for any of the methods, step (a) of the method is initiated at a first time (T1) and step (c) is initiated at a second time (T2). Optionally, T2 is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or 3, 4, 5, 6, 7, or 8 weeks after T1. Preferably, T2 is at least 0.5 hours after T1. Preferably, T2 is at least 1 day after T1. Preferably, T2 is at least 1 week after T1. Preferably, T2 is at least 1 month after T1. Preferably, T2 is at least 2 months after T1. Preferably, T2 is at least 3 months after T1. Preferably, T2 is at least 3 months after T1. Preferably, T2 is at least 4 months after T1. Preferably, T2 is at least 5 months after T1. Preferably, T2 is at least 6 months after T1. Preferably, T2 is at least 12 months after T1. Preferably, T2 is at least 18 months after T1. For example, T2 is 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) months after T1. For example, T2 is 1 to 8 weeks after T1. That is, in this way, the method is a method of temporally controlling expression.

[0111] i. a method for treating or preventing a disease or condition in a human or animal subject by temporally controlling the production of P1 according to the methods of the present invention; or ii. A method for modifying the microbial community (e.g., the gut microbial community) of a human or animal subject by temporally controlling the production of P1 according to the methods of the invention. is provided.

[0112] For example, a method is provided for modifying the metabolome of a human or animal subject by carrying out the method of the present invention. For example, expression of P1 causes the secretion or sequestration of one or more metabolites in the subject (e.g., in the target or cell containing the vector nucleic acid). For example, expression of P1 causes the metabolism of a compound by changing a pathway in the subject's microbial population (e.g., in the target cell) to produce a tryptophan sink, e.g., tryptophan is used in the microbial population (e.g., in the target cell) to produce AhR (aryl hydrocarbon receptor) ligands). For example, expression of P1 causes the metabolism or modification of a chemical substance, such as a therapeutic drug, in the microbial population. Such "elicitation" can be performed because P1 is a component of a metabolic pathway in the microbial population (e.g., in the target cell), e.g., P1 is a protein, such as an enzyme.

[0113] The disease or condition may be any disease or condition described herein.The human or animal microbiome may be any microbiome described herein.

[0114] The administration in step (a) may be oral, topical (e.g., applied to the skin), buccal, rectal, vaginal, parenteral, intravenous, intramuscular, inhalation, subcutaneous, ocular, or intranasal. Preferably, oral administration is used. Preferably, topical administration is used.

[0115] In one example, the vector is included in a fecal microbial transplant (FMT). Administration in step (a) may be by rectal administration of an enema or FMT containing the vector.

[0116] In step (b), a vector (eg, a plasmid) containing the nucleic acid can be transferred.

[0117] P1 can be an expression product that is therapeutically or prophylactically useful in a subject.

[0118] In one embodiment, in step (c), P1 expressed from the nucleic acid is a regulator (R) or a component of a pathway that produces R, whereby the expression feedback loop of P1 negatively controls further expression of P1. For example, P1 is an enzyme of a metabolic pathway. For example, P1 is an intracellular enzyme in a target cell. For example, P1 is an enzyme that is secreted (e.g., secreted from a target cell). In one example, expression of P1 causes the production of R (e.g., in a microbial population, e.g., in a target cell).

[0119] The pathways described herein may be in the target cell or may be outside the target cell. For example, the pathways are pathways in different cells contained in the microbial community of which the target cell is a component. The different cells may be carrier cells (i.e., donor cells) or may be endogenous cells of the microbial community (i.e., any cell of the microbiome other than the target cell or carrier cell).

[0120] In one example, R upregulates a second promoter in step (c), and P2 downregulates expression of P1, and optionally upregulation of the second promoter causes production of a guided nuclease or restriction endonuclease that cleaves the nucleic acid in the host cell, resulting in degradation of the nucleic acid, thereby downregulating expression of P1.

[0121] In one example, P2 can upregulate the expression of P1 in a host cell, and R downregulates a second promoter in step (c), thereby downregulating the expression of P1.

[0122] The downregulation of P1 in step (c) may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% less than the expression of P1 in step (b). For example, the downregulation of P1 in step (c) may be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% less expression of P1 in the presence of R during a period (PD1) compared to the expression of P1 in step (b) during a period (PD2), where PD1 and PD2 are the same length of time. For example, PD1 and PD2 are 1 minute, 1 hour, 1 day, 1 week, 1 month, 6 months, or 12 months, respectively. The expression of P1 may be determined in steps (b) and (c) by evaluating the expression of P1 in the samples of the microorganisms collected during steps (b) and (c), respectively. For example, where the microbial population is the intestinal microbial population of a human or animal subject, the respective sample may be a faecal sample of the subject.

[0123] Any upregulation of P1 expression may be, for example, an increase in P1 of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% compared to the expression of P1 in the immediately preceding step (b). Any downregulation of P1 expression may be, for example, a decrease in P1 of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% compared to the expression of P1 in the immediately preceding step (b).

[0124] Any upregulation of P2 expression may be, for example, an increase in P2 of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% compared to the expression of P2 in the immediately preceding step (c). Any downregulation of P2 expression may be, for example, a decrease in P2 of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% compared to the expression of P2 in the immediately preceding step (c). The vectors, compositions, tablets, suppositories, pills, capsules, or liquid formulations described herein may be for use in the methods described herein, for example, the vectors, compositions, suppositories, pills, capsules, or formulations are administered to a subject by oral or topical administration.

[0125] The host, carrier and / or target cells may be bacterial cells. Alternatively, instead of bacterial cells, the host, carrier and / or target cells may be archaeal cells.

[0126] In one example, the method modifies the genome of a cell, for example, modifies the chromosome or episome (e.g., plasmid) of a host cell.For example, following the introduction of vector nucleic acid into a host cell, a copy of NS1 is inserted into the chromosome or episome of the host cell.In one example, expression of a nuclease (e.g., P2 or a nuclease operable with P2, for example, when P2 comprises a guide RNA) results in the cleavage of NS1 in the chromosome or episome and, optionally, the death of the host cell.

[0127] The carrier cell and the target cell may be cells of the same order, family or genus, for example as shown in the examples.

[0128] Preferably, the agent comprises a CRISPR / Cas system or a component thereof. The agent may be a crRNA or guide RNA that guides a Cas nuclease in a target cell to a target protospacer sequence, where the Cas cleaves the target sequence resulting in the death of the target cell. For example, the plasmid may encode a plurality of different crRNAs or guide RNAs, such as a first cRNA or gRNA that includes a spacer sequence capable of guiding a Cas in a target cell to a first protospacer sequence and a second cRNA or gRNA that includes a spacer sequence capable of guiding a Cas in a target cell to a second protospacer sequence, where the protospacer sequences are different (e.g., different chromosomal sequences of the target cell). Each protospacer may be comprised in an essential gene, a virulence gene, or an antibiotic resistance gene in the genome of the target cell. Each protospacer sequence may be 10-60 nucleotides in length, such as 15-50, 15-40, 15-30, or 15-20 nucleotides in length. The target sequence may be a chromosomal sequence of the target cell. The target sequence may be an episomal sequence in the target cell. The plasmid may encode one or more of the Cas nucleases, optionally Cas9, Cas3, or Cpf1.

[0129] For example, the target sequence is contained in a plant microorganism. The carrier cell can be a Pseudomonas cell, optionally a P fluorescens cell. Optionally, the carrier and target cells are cells of the same genus or species, and optionally both are Pseudomonas cells. For example, the target cell is a P syringae or aeruginosa cell, and the carrier is a Pseudomonas (e.g., P fluorescens) cell.

[0130] Preferably, the carrier cells are cells of a strain or species that is not pathogenic to the organism (e.g., a plant, animal, or human) that contains the target cells. The carrier cells may be cells of a strain or species that is commensal or probiotic to the organism (e.g., a plant, animal, or human) that contains the target cells, e.g., probiotic or commensal in the intestine of the organism.

[0131] For example, the target sequence is contained in a plant microorganism. In one example, the carrier cell comprises a chitinase class I exoenzyme and / or the genome of the carrier cell encodes a chitinase class I exoenzyme. Optionally, the carrier cell in this example is a Pseudomonas, e.g., a P fluorescens cell. In one example, the carrier cell comprises a pep1 gene. Optionally, the carrier cell in this example is a Pseudomonas, e.g., a P fluorescens cell.

[0132] In one example, the carrier cells are motile bacterial cells. Optionally, the target cells are included in the plant microbiome, in this example the carrier cells are Pseudomonas, e.g., P fluorescens cells.

[0133] For example, each target cell is a lag phase cell, a log phase cell, or a stationary phase cell. For example, each carrier cell is a lag phase cell, a log phase cell, or a stationary phase cell.

[0134] For example, the target cell is included in a plant microorganism. Optionally, the target cell is a Pseudomonas (optionally P fluorescens or P aeruginosa) cell, Erwinia (optionally Ecorotovora), Xanthomonas, Agrobacterium, Burkholdi, Clavibacterium, Enterobacteria, Pantoae, Pectobacterium (e.g. P atrosepticum), Rhizobium, Streptomyces (e.g. S scabies), Xylella (e.g. X fastidiosa), Candidatus (e.g. C liberibacter), Phytoplasma, Ralstonia (e.g. R solanacearum), or Dickeya (e.g. D dadantii) cell.

[0135] Each target cell (e.g., a plurality of target cells) may be a cell of a genus or species disclosed in Tables 1 or 2. Each target cell (e.g., a plurality of target cells) may be contained in a plant or the plant's environment (e.g., soil) and may be selected from a genus or species disclosed in Table 1. Each carrier cell (e.g., a plurality of carrier cells) may be a cell of a genus or species disclosed in Tables 1 or 2.

[0136] The method may be carried out in vitro or ex vivo.

[0137] The target cells are (a) a plant microbiome (e.g., the microbiome of any plant part disclosed herein); (b) an animal or human microbial community (e.g., the microbial community of any human or animal organ or tissue or part disclosed herein); or (c) The microbial community of soil, fertilizer, food, or drink may be included in

[0138] For example, target cells may be contained within the microbial population of plant leaves, stems, roots, seeds, bulbs, flowers, or fruits.

[0139] Optionally, the microbial community herein is the microbial community of the gut, lungs, kidneys, ureters, bladder, blood, vagina, eyes, ears, nose, penis, bowel, liver, heart, tongue, hair, or skin.

[0140] For example, the target cells are cells of a species found in soil.

[0141] The method can be performed with a first cell population comprising a plurality of carrier cells in contact with a second cell population comprising a plurality of target cells, where copies of said plasmid are conjugatively transferred from the carrier cells to the target cells, thereby killing some or all of the cells of the second population or inhibiting growth or proliferation of the cells of the second population (e.g., at least 50, 60, 70, 80, 90, or 95% at 5 or 10 hours or 1 day after initiation of the method). Preferably, the cells of the second population are killed.

[0142] Optionally, the second and third method configurations are performed on a plurality of target cells in a population of microorganisms by exposing the plurality of target cells to a plurality of vectors (e.g., a plurality of copies of a vector comprising NS1 and NS2, or a plurality of copies of a first vector comprising NS1 and a plurality of copies of a second vector comprising NS2). The method optionally modifies at least 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or 99.99999% of the cells of the plurality of target cells, such that they are capable of expressing P1. In one example, the method is performed on a population of the target cells (or the plurality of target cells), and the method modifies all (or essentially all) of the cells (or the plurality of cells) of the population. In one example, the method is performed on a population of the target cells (or the plurality of cells), and the method modifies 100% (or about 100%) of the cells (or the plurality of cells) of the population.

[0143] Preferably, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the target cells are modified.

[0144] Optionally, the target cell is a Pseudomonas (e.g., P syringae) cell, e.g., the cell is in a crop, e.g., a tomato plant. For example, the biomass of leaves, fruits, ears, seeds, grains, heads, pods, stems, trunks, tubers, and / or roots is increased by the method. For example, the dry biomass of leaves or fruits, the wet biomass of leaves or fruits, or the number of flowers is increased by the method, e.g., expression of P1 is beneficial to plant fertility or growth. For example, the average biomass or number is increased in a plurality of plants subjected to the method of the present invention.

[0145] The increase in biomass (e.g., average biomass or number) may be at least 5, 10, 15, 20, 25, 30, 40, or 50% increase compared to the biomass of plants containing the target bacteria but not exposed to the carrier bacteria. The increase in plant biomass may be determined by measuring the weight of harvested material (e.g., fruit, grain, cane, leaf, tuber, nut, or seed) per harvested area and comparing the measurement of harvested material from plants treated according to the present invention with the same area of ​​harvested material from plants of the same species and strain that were not treated according to the present invention, where all plants are grown under the same conditions, e.g., in the same field. In some systems, a unit of volume, e.g., bushel, is used instead of a unit of weight.

[0146] In one aspect, the method is a method of promoting plant growth or germination of a seed of a plant, the method being carried out with a first cell population comprising a plurality of carrier cells in contact with a second cell population comprising a plurality of target cells, and copies of said nucleic acid are transferred from the carrier cells to the target cells, whereby some or all of the cells of the second population are modified to express P1, and wherein a seed comprises said target cells, and said growth or germination is promoted.

[0147] Promoting germination may be shortening the time to onset of germination and / or shortening the duration of germination. Promoting germination may be increasing the percentage of seeds that germinate (e.g., by at least 5, 10, 15, or 20%) among the plurality of seeds exposed to the carrier cells of the method.

[0148] Each seed may include a target cell on the surface of the seed.

[0149] The method provides for an increase in germination (e.g., average germination) in a plurality of seeds exposed to carrier cells, which is an increase of at least 5, 10, 15, 20, 25, 30, 40, or 50% compared to the germination of seeds containing the target bacteria but not exposed to carrier cells.

[0150] The method may be useful for treating pre-emerged seedlings with pathogens present that halt successful germination. Each seedling may contain target cells on the leaves and / or stems of the seedling.

[0151] The method provides for an increase in growth (e.g., average growth) in a plurality of seedlings exposed to carrier cells, which is at least 5, 10, 15, 20, 25, 30, 40, or 50% increased compared to the growth of seedlings containing the target bacteria but not exposed to carrier cells.

[0152] In one aspect, the method is a method of increasing leaf chlorophyll (e.g., chlorophyll a and / or b) production in a plant, the method being carried out with a first cell population comprising a plurality of carrier cells in contact with a second cell population comprising a plurality of target cells, where copies of said nucleic acid are transferred from the carrier cells to the target cells, thereby modifying some or all of the cells of the second population to express P1, where the plant comprises said target cells (optionally in their leaves and / or stems, or in the apoplast of the plant), thereby modifying the target cells to increase chlorophyll in the plant. Measurement of chlorophyll may be measured, for example, by spectroscopy, high performance liquid chromatography (HPLC), or fluorimetry.

[0153] In one aspect, the method is a method of modifying target cells contained in a biofilm, wherein the biofilm is contained in a subject or on a surface, and wherein the biofilm comprises the target cells, and the method is performed with a first cell population comprising a plurality of carrier cells in contact with a second cell population comprising a plurality of target cells, and copies of the nucleic acid are transferred from the carrier cells to the target cells, thereby modifying the target cells in the biofilm to express P1, and optionally the method is performed ex vivo or in vitro.

[0154] The subject may be a human or an animal, and optionally the surface is a pulmonary surface.

[0155] The subject may be a plant, and optionally the biofilm is contained on the leaves, stems, roots, or stems of the plant.

[0156] The surface may be comprised in a domestic or industrial apparatus or vessel, for example a fermentation vessel.

[0157] Further provided is a carrier bacterial cell for administration to a microbial population comprising a target cell (e.g. for use in a method according to the invention), wherein the carrier cell comprises a conjugative plasmid, the plasmid being a vector of the invention, and the carrier cell is capable of conjugating to a target cell, and the plasmid is transferred to the target cell to modify the target cell to express P1.

[0158] The carrier cell may be any carrier cell or carrier cell disclosed herein. The target cell may be any carrier cell or target cell disclosed herein.

[0159] A carrier cell (e.g., a bacterial cell) is provided that contains the vector of the present invention. The present invention also provides a plurality of such carrier cells (e.g., the cells are genetically identical or all of the cells encode the same P1). Alternatively, the plurality of cells encodes different P1 proteins.

[0160] A pharmaceutical composition comprising a plurality of carrier cells of the invention for administration to a human or animal subject to modify a plurality of bacterial target cells contained in the subject to express P1, wherein a vector of the invention (e.g., a conjugative plasmid) encoding P1 can be introduced (e.g., by conjugation) from the carrier cells to the target cells, and P1 is produced in the target cells.

[0161] Preferably, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the target cells are so modified.

[0162] The multiple target cells are at least 10 7 , 10 8 , 10 9 , 1010 , 10 11 , or 10 12 For example, the plurality of target cells may be contained in the microbial community of the gut, blood, lung, oral cavity, liver, kidney, bladder, ureter, or skin of a subject.

[0163] Methods are provided for treating or preventing a disease or condition in a subject, comprising contacting the subject (e.g., the gut microbial community when the subject is a human or animal) with a composition comprising a plurality of carrier cells of the invention, and a vector of the invention (e.g., a conjugative plasmid) encoding P1 is transferred (e.g., by conjugation) from the carrier cells to a target cell, and P1 is produced in the target cell, thereby treating or preventing the disease or condition.

[0164] Use of a plurality of carrier cells of the invention in the manufacture of a composition for administration to a subject or environment (e.g. soil) for modifying bacterial target cells contained in the subject or environment to express P1, wherein the target cells are contacted with the carrier cells, a vector of the invention (e.g. a conjugative plasmid) encoding P1 is introduced from the carrier cells (e.g. by conjugation) into the target cells, and P1 is produced in the target cells.

[0165] For example, the subject is a human or an animal. For example, the subject is a mammal. For example, the subject is a bird, a fish, a protozoan, or an insect. For example, the animal is a livestock animal. For example, the animal is a dog, a cat, a horse, a cow, a sheep, or a pig.

[0166] For example, the subject is a plant, and optionally the method includes contacting the plant (e.g., one or more stems and / or one or more leaves of the plant, or the apoplast of the plant) with a composition comprising a plurality of carrier cells.

[0167] There is provided the use of a carrier cell of the invention in the manufacture of a composition for modifying bacterial target cells ex vivo or when the target cells are not in a human or animal (e.g. when the target cells are in a plant or soil, or in an ex vivo human microbiome sample), wherein the target cells are contacted with the carrier cell and the carrier cell conjugates with the target cells, whereby the vector nucleic acid is introduced into the target cells and P1 is expressed in the target cells.

[0168] For example, each vector is a conjugative plasmid, and the carrier cell conjugates (or can conjugate) with the target cell and transfers the plasmid to the target cell by conjugation. Optionally, the plasmid contains an origin of transfer (oriT) and a gene for self-conjugation, so that the plasmid can be conjugatively transferred from the target cell to additional cells (where the target cell and the additional cells are contained in a microbial population).

[0169] Optionally, the use comprises use of said plurality of carrier cells for modifying said plurality of target cells, the target cells being comprised in a plant or a plant environment (e.g. soil), and said modification comprising: a) increasing the biomass of the plant or parts thereof (e.g. increasing the biomass of leaves, fruits, ears, seeds, grains, heads, pods, stems, trunks, tubers, and / or roots), (or due to an increase in the biomass of the plant or parts thereof); b) promoting (or intended to promote) the germination of one or more seeds of a plant; c) increasing (or being due to) the amount of chlorophyll in the leaves of the plant; and / or d) reducing (or being for the reduction of) biofilms in plants; The biofilm comprises target cells (eg, Pseudomonas cells).

[0170] Optionally, the target cell or cells are in an environment, such as in soil, or in an environment for a growing plant. For example, P1 is a promoter of plant growth (e.g., a fertilizer).

[0171] Examples of target cells For example, each target cell is a Bacteriodes cell, for example, contained in a human or animal subject. For example, each target cell is a Clostridiales cell, for example, contained in a human or animal subject.

[0172] For example, each target cell is a Gram-positive bacterial cell (e.g., a Staphylococcus (e.g., S aureus, e.g., methicillin-resistant Staphylococcus aureus (MRSA)), Streptococcus pneumoniae, Clostridium difficile, Enterococcus spp., or Listeria monocytogenes cell). For example, each target cell is a Gram-negative bacterial cell (e.g., an Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Stenotrophomonas maltophilia, Campylobacter jejuni, Pseudomonas aeruginosa, Neisseria gonorrhoeae, Vibrio cholerae, or Salmonella spp. cell). For example, each target cell is a cell of a genus or species disclosed in Table 1 herein or Table 2 herein.

[0173] See Journal of Plant Pathology (2010), 92(3), 551-592 Edizioni ETS Pisa, 2010 551, LETTER TO THE EDITOR, “COMPREHENSIVE LIST OF NAMES OF PLANT PATHOGENIC BACTERIA, 1980-2007”, CT Bull et al, the disclosure of which is incorporated herein by reference, providing examples of bacterial genera, species and strains that are important for plants and may be target genera, species and strains of the present invention. Examples are disclosed in Table 1 herein.

[0174] For example, each target cell is resistant to a fluoroquinolone, a β-lactam (e.g., methicillin), a tetracycline, or a linezolid antibiotic. For example, each target cell is resistant to vancomycin, e.g., the cell is a vancomycin-resistant Enterococcus cell.

[0175] For example, the respective target cell may be an Azotobacter, Burkholderia, Cupriavidus, Enterococcus, Lysobacter, Paucimonas, Paraburkholderia, Ralstonia, Stenotrophomonas, Variovorax, Xanthomonas, or Pseudomonas cell, e.g., the target cell is contained in a plant.

[0176] For example, each target cell is an E. coli cell.

[0177] For example, each target cell is a Klebsiella cell, for example, the target cells are contained in a plant.

[0178] For example, the respective target cell may be an Azotobacter, Burkholderia, Cupriavidus, Lysobacter, Paraburkholderia, Ralstonia, Variovorax, Xanthomonas, or Pseudomonas cell, e.g., the target cell may be comprised in a plant.

[0179] For example, each target cell is a cell of a Pseudomonas species, optionally wherein the species is selected from Pseudomonas aeruginosa, Pseudomonas amygdali, Pseudomonas asturiensis, Pseudomonas avellanae, Pseudomonas cerasi, Pseudomonas chlororaphis, Pseudomonas cichorii, Pseudomonas coronafaciens, Pseudomonas otitidis, Pseudomonas putida, Pseudomonas salegens, Pseudomonas savastanoi, Pseudomonas syringae, and Pseudomonas viridiflava, e.g., the target cell is contained in a plant.

[0180] For example, each target cell is a cell of a species selected from Azotobacter chroococcum, Azotobacter salinestris, Burkholderia ambifaria, Burkholderia cenocepacia, Burkholderia lata, Burkholderia pyrrocinia, Cupriavidus basilensis, Cupriavidus necator, Cupriavidus taiwanensis, Lysobacter gummosus, Paraburkholderia sprentiae, Paraburkholderia terricola, Ralstonia pseudosolanacearum, Ralstonia solanacearum, Variovorax paradoxus, Xanthomonas arboricola, Xanthomonas axonopodis, Xanthomonas campestris, Xanthomonas citri, Xanthomonas euvesicatoria, and Xanthomonas perforans, e.g., the target cell is contained in a plant.

[0181] For example, the respective target cell may be a Stenotrophomonas, Enterococcus, Paucimonas, or Pseudomonas cell, for example, the target cell may be comprised in a plant.

[0182] For example, each target cell is a cell of a Pseudomonas species, optionally wherein the species is selected from Pseudomonas amygdali, Pseudomonas asturiensis, Pseudomonas avellanae, Pseudomonas cerasi, Pseudomonas chlororaphis, Pseudomonas cichorii, Pseudomonas coronafaciens, Pseudomonas putida, Pseudomonas savastanoi, Pseudomonas syringae, and Pseudomonas viridiflava, e.g., the target cell is contained in a plant.

[0183] For example, each target cell is a cell of a species selected from Stenotrophomonas rhizophila, Enterococcus faecalis, Paucimonas lemoignei, Pseudomonas amygdali, Pseudomonas asturiensis, Pseudomonas avellanae, Pseudomonas cerasi, Pseudomonas chlororaphis, Pseudomonas cichorii, Pseudomonas coronafaciens, Pseudomonas putida, Pseudomonas savastanoi, Pseudomonas syringae, and Pseudomonas viridiflava, e.g., the target cell is contained in a plant.

[0184] Examples of carrier cells For example, the carrier is an E. coli cell (e.g., an E. coli, K12, Nissle, or S17 cell), e.g., the cell is for administration to a human or animal subject, e.g., to treat or prevent a disease or condition. For example, each carrier cell is a Bacteroides cell, e.g., the cell is for administration to a human or animal subject, e.g., to treat or prevent a disease or condition. For example, each carrier cell is a Clostridiales cell, e.g., the cell is for administration to a human or animal subject, e.g., to treat or prevent a disease or condition.

[0185] For example, each carrier cell is a gram-positive bacterial cell.For example, each carrier cell is a gram-negative bacterial cell.For example, the carrier cell is a cell of the genus or species disclosed in Table 1 of WO2017211753 (this table and the disclosure of each genus and species are individually incorporated herein for the disclosure of the genus or species of the cell used in the present invention).

[0186] For example, the carrier cell is a cell of the phylum Proteobacteria, the class Gammaproteobacteria, the order Pseudomonadales, or the family Pseudomonadaceae. In a preferred embodiment, the carrier is a Pseudomonas (e.g., P fluorscens) cell and the target cell is, for example, contained in a plant.

[0187] For example, each carrier cell may be a Klebsiella cell and the target cell may be, for example, contained in a plant.

[0188] For example, the carrier is a gram positive cell, such as a Bacillus (eg, Bacillus subtilis) or Clostridiales (eg, Clostridium butyricum) cell.

[0189] In one example, the subject is a crustacean, which may be selected from shrimp, crayfish, crabs, lobsters, clams, scallops, oysters, prawns, and mussels.

[0190] The subject can be any subject disclosed herein. The subject can be an animal, such as a livestock animal, such as a bird (e.g., a poultry, or a chicken or turkey) or a pig.

[0191] Alternatively, the subject is a plant, for example, the target bacterium is a plant pathogenic bacterium. In one example, the target bacterium is a Pseudomonas, for example P syringae or P aeruginosa.

[0192] Alternatively, the carrier and target cells are archaeal cells. For example, the target cells are methanobacterium cells. For example, the target cells are methanogen cells. For example, the target cells are Methanobacterium bryantii Methanobacterium formicum · Methanobrevibacter arboriphilicus · Methanobrevibacter gottschalkii · Methanobrevibacter ruminantium Methanobrevibacter smithii · Methanococcus chunghsingensis Methanococcus burtonii Methanococcus aeolicus Methanococcus deltae Methanococcus jannaschii Methanococcus maripaludis Methanococcus vannielii · Methanocorpusculum labreanum · Methanoculleus bourgensis (Methanogenium olentangyi and Methanogenium bourgense) · Methanoculleus marisnigri · Methanoflorens stordalenmirensis · Methanofollis liminatans · Methanogenium cariaci · Cold methanogen · Organophilic methanogenium · Methanogenium wolfei · Methanomicrobium mobile · Methanopyrus kandleri · Methanoregula boonei · Methanosaeta concilii · Methanosaeta thermophila · Methanosarcina acetivorans · Methanosarcina barkeri · Methanosarcina mazei · Methanosphaera stadtmanae · Methanospirillium hungatei · Methanothermobacter defluvii (Methanobacterium defluvii) · Methanothermobacter thermautotrophicus (Methanobacterium thermoautotrophicum) · Methanothermobacter thermoflexus (Methanobacterium thermoflexum) · Methanothermobacter wolfei(Methanobacterium wolfei) Methanothrix sochngenii The present invention includes one or more species of cells selected from the group consisting of:

[0193] Optionally, the target cells are not pathogenic to the subject, e.g., if the method is a non-medical method, hi one example, the method is a cosmetic method.

[0194] In examples, optionally, the subject or animal is a livestock animal, such as a cow, sheep, goat, or chicken (preferably a cow). Optionally, for example when the subject is an animal (e.g. a domestic or wild animal), the target cell is a zoonotic bacterial cell, for example a cell of a species selected from Bacillus anthracis, Mycobacterium bovis (e.g. when the animal is a bovine), Campylobacter spp (e.g. when the animal is a poultry animal), Mycobacterium marinum (e.g. when the animal is a fish), Shiga toxin producing E. coli (e.g. when the animal is a ruminant animal), Listeria spp (e.g. when the animal is a bovine or ovine), Chlamydia abortus (e.g. when the animal is a ovine), Coxiella burnetii (e.g. when the animal is a bovine, ovine or caprine), Salmonella spp (e.g. when the animal is a poultry animal), Streptococcus suis (e.g. when the animal is a porcine), and Corynebacterium (e.g. C ulcerans) (e.g. when the animal is a bovine).

[0195] In one example, a plurality of carrier cells described herein (e.g., carrier cells of any configuration, aspect, example, or embodiment described herein) are administered to a subject, and the carrier cells comprise a nucleic acid encoding P1.

[0196] In one example, each animal is a chicken. In one example, each animal is a cow (e.g., a beef or dairy cow).

[0197] Optionally, the method modifies target cells in the gastrointestinal tract of a human or animal subject. Optionally, the method modifies target cells in the jejunum, ileum, colon, liver, spleen, or cecum of a subject. Optionally, the animal is avian and the method modifies target cells in the cecum of the avian. In one example, the method is performed on a group of animals (optionally a flock or herd), some or all of the animals comprising the target cells.

[0198] Optionally, the plasmid contains an RP4 origin of transfer (oriT). The plasmid may be any type of plasmid disclosed herein.

[0199] P2 P2 may be any antibacterial agent or component thereof disclosed herein, preferably a guided nuclease programmed to cleave one or more target sequences in a target cell. Suitable nucleases may be TALENs, meganucleases, zinc finger nucleases, or Cas nucleases. For example, the agent comprises one or more components (e.g., Cas nucleases and / or guide RNAs or crRNAs) of a CRISPR / Cas system operable in a target cell to cleave a protospacer sequence contained in the target cell (e.g., contained in a vector, whereby the cleaved vector is degraded in the target cell). For example, the system is operable to cleave at least two or three different protospacer sequences contained in a vector of the present invention. Optionally, P2 is operable to cleave multiple different protospacer sequences contained in the vector (and optionally further operable to cleave protospacer sequences in the genome of the carrier cell, e.g., in the chromosome or episome of the carrier cell, which cleavage is lethal to the carrier cell. As explained elsewhere, this is useful, if desired, to reduce or remove carrier cells from a subject or microbial population, e.g., after a desired level of expression of P1 in the target cells has been obtained). Optionally, the agent comprises one or more components of a CRISPR / Cas system operable to cleave at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different protospacer sequences contained in the vector and / or the genome of the carrier cell (e.g., contained in the chromosome of the carrier cell).

[0200] In one embodiment, P2 is (a) comprising a guide nuclease capable of recognizing and cleaving a protospacer sequence contained in the vector in a target cell; and / or (b) encoding a guide RNA or crRNA of the CRISPR / Cas system that operates with a Cas nuclease to cleave a protospacer sequence contained in the vector in a target cell; The nuclease can cleave the vector in the target cell, and the vector is degraded in the target cell.

[0201] This is useful as an "off switch" to remove the vector from the target cell, for example, to down-regulate the ability of P1 expression in a plurality of target cells that have already been contacted with carrier cells and into which the vector of the present invention has been transferred for expression of P1. For example, this can be usefully used to completely remove the target microbial population, so that it can produce less or no P1 after cleavage of the vector nucleic acid has occurred. In this way, it is possible to modify the target microbial population (e.g., the human gut microbial population) in a first step, for example, to produce P1 (e.g., when expression of P1 is useful for treating or preventing a disease or condition, or for non-medical use), and then reduce or remove the expression of P1 to restore the microbial population to a partially modified or unmodified state.

[0202] Any administration of the cells herein to a subject may be oral administration. Any administration of the cells herein to a subject may be preferably to the GI tract. Any administration of the cells herein to a subject may be systemic, intranasal, or inhalation administration.

[0203] The following definitions are provided: Homolog: A gene, nucleotide, or protein sequence related to a second gene, nucleotide, or protein sequence by descent from a common ancestral DNA or protein sequence. The term "homolog" applies to the relationship between genes separated by the event of gene duplication. Orthologs: Orthologs are gene, nucleotide, or protein sequences in different species that have evolved by speciation from a common ancestral gene, nucleotide, or protein sequence. Orthologs usually retain the same function during evolution.

[0204] Optionally, each P2 is a guide RNA. Optionally, each vector (e.g., a plasmid) encodes multiple guide RNAs or crRNAs of the CRISPR / Cas system, and the guide RNAs or crRNAs are operable with Cas nuclease in the target cell to recognize multiple protospacer sequences contained in the vector and / or the genome of the carrier cell, and optionally the protospacer sequences include one or more nucleotide sequences of a selected gene. In one example, the vector further or instead encodes a Cas, such as Cas9, Cas3, Cpf1, Cas12, Cas13, CasX, or CasY.

[0205] In one example, the Cas herein is type I, type II, type III, type IV, type V, or type VI Cas, preferably type I or type II Cas.

[0206] In one example, the vector also encodes Cas3 and cognate Cascade proteins (e.g., CasA, B, C, D, and E). Optionally, the Cas (and the Cascade) is an E. coli Cas (and the Cascade).

[0207] The plasmid may comprise one or more CRISPR spacers, each spacer consisting of 20-40, 25-35, or 30-35 consecutive nucleotides, for example selected from:

[0208] Optionally, the plasmid contains the RP4 origin of transfer (oriT) and / or the p15A origin of replication.

[0209] In one example, the plasmid is a conjugative phagemid.

[0210] In one example, the vector encodes Cas3 and optionally one or more cascade proteins (e.g., one or more of CasA, B, C, D, and E). In one embodiment, the vector encodes Cas3 and CasA, B, C, D, and E. In one embodiment, the vector encodes E. coli Cas3 and CasA, B, C, D, and E. Optionally, the guided nuclease (e.g., Cas3) is a type IA, -B, -C, -D, -E, -F, or -U Cas.

[0211] In one example, P2 in any configuration, aspect, example, option, or embodiment herein comprises one or more components of a CRISPR / Cas system operable in a target cell to cleave a protospacer sequence contained in a vector.Furthermore, in one example, P2 in any configuration, aspect, example, option, or embodiment herein comprises one or more components of a CRISPR / Cas system (e.g., the same system as in the first sentence of this paragraph) operable in a carrier cell to cleave a protospacer sequence contained in the genome of the carrier cell (e.g., a chromosomal or episomal sequence whose cleavage is lethal to the carrier cell).This is useful for completely removing carrier cells from a subject (e.g., from the gut microbiome) when expression of P1 is no longer required.

[0212] In one example, the system is operable to cleave at least three different protospacer sequences contained in the genome of the vector or carrier cell.

[0213] In one example, the vector is (a) encodes a guided nuclease capable of recognizing and modifying a nucleic acid sequence of a carrier cell, the sequence being contained in an endogenous chromosome or episome of the carrier cell, such that the nuclease cleaves the chromosome or episome, killing the carrier cell or inhibiting the growth or proliferation of the target cell; and / or (b) It encodes the guide RNA or crRNA of the CRISPR / Cas system that works with the Cas nuclease in the carrier cell to cleave the protospacer sequence contained in the cell.

[0214] The expression of (a) and / or (b) may be inducible by exposure of carrier cells to a regulator, such as R. For example, exposure of carrier cells and target cells to R induces the production of P2, where P2 comprises components (a) and / or (b), thereby cleaving the vector nucleic acid in the target cells and also cleaving the genome of the carrier cells. This results in the death of the carrier cells and degradation of the vector in the target cells. This is useful, as the vector and carrier cells can be completely removed from the subject after a desired amount of P1 is expressed in the subject.

[0215] Optionally, the Cas, Cascade protein, gRNA, and crRNA are Cas, Cascade protein, gRNA, and crRNA of E. coli K12 (MG1655), respectively. Optionally, the vector lacks nucleotide sequences encoding Cas1 and Cas2 proteins.

[0216] In an embodiment, the action of P2 (e.g., components (a) and / or (b)) in the carrier cells reduces the growth or proliferation of the carrier cells (e.g., by at least 40, 50, 60, 70, 80, or 90% compared to growth in the absence of P2 therein).

[0217] For example, the carrier cells may be included in a pharmaceutical for treating or preventing a disease or condition in humans or animals, in a growth promoter for administration to animals to promote their growth, to kill zoonotic bacteria in animals, for administration to livestock as a pesticide, as a pesticide to be applied to plants, or as a fertilizer for plants.

[0218] An advantage may be that the carrier cells are used as producer cells in which the vectors of the invention can replicate (eg prior to (eg in vitro) and / or after administration to a subject).

[0219] Plasmid examples The method of delivery of the vector can be by bacterial conjugation, a natural process in which a donor bacterium (carrier bacterium) transfers plasmid DNA from itself to a recipient bacterium (target bacterium). The donor bacterium transforms its surface structure into pili that can be thought of as a syringe or drinking straw through which the DNA is delivered. The donor pili bind to the receptive recipient surface, and this event triggers the DNA transfer process. A plasmid is suitable for this conjugation process, where the plasmid contains the DNA encoding the agent of the present invention.

[0220] Transfer of DNA by conjugation can only occur in "susceptible recipients" but generally not in recipients carrying similar types of plasmids. Because conjugation is via a pilus bridge, it is possible that the bridge will attach to itself, i.e., to the donor bacterium rather than to the recipient. This can result in a futile cycle of transferring plasmid DNA to itself. That is, plasmids naturally encode incompatibility factors. One is a protein arrayed on the surface that prevents the pili from binding to bacteria that display surface proteins, such as itself or any other bacterium carrying the same plasmid. In addition, plasmids naturally encode another incompatibility system that tightly controls the copy number of the plasmid inside the bacterium. That is, if the conjugation event somehow evades surface exclusion and initiates DNA transfer by conjugation, the recipient will prevent the establishment of the plasmid, since the plasmid already maintains its current copy number and will not accept and maintain additional undesired copies.

[0221] In one embodiment of the present invention, the plasmid is a member of a plasmid incompatibility group and the target cell does not contain a plasmid of that group. Optionally, the plasmid of the present invention is a member of incompatibility group P (i.e., the plasmid is an incP plasmid). For example, in Enterobacteriaceae, the following is a non-exclusive list of potential plasmids that can be used for delivery: IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, InclO, Incl, IncA, IncB, IncC, IncH, IncIa, InclIc, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, IncS, IncT, and / or IncW. That is, optionally, the target cell is an Enterobacteriaceae cell and the vector of the invention is a plasmid, and the plasmid is selected from IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, InclO, Incl, IncA, IncB, IncC, IncH, IncIa, InclIc, IncI1, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, IncS, IncT, and IncW plasmids.

[0222] Preferably, the subject is a human or animal and the plasmid is an IncI plasmid, such as an IncI1 or IncI2 plasmid.

[0223] In one example, the carrier cell of the present invention comprises two or more plasmids, each plasmid comprising DNA encoding P1 and P2, respectively (P1 / P2 may be identical in the cell, or the cell may comprise different P1 and / or P2). Optionally, a first of said plasmids is a member of a first incompatibility group, and the target cell does not comprise a plasmid of said first group, and a second of said plasmids is a member of a second incompatibility group, and the target cell does not comprise a plasmid of said second group. For example, the carrier cell may comprise an incP plasmid (e.g., a CRISPR-Cas system or a component thereof (e.g., encoding a first crRNA or guide RNA that targets a first protospacer sequence of the vector)) encoding P1 and P2, and the carrier cell further comprises an incF1 plasmid (e.g., an anti-carrier cell CRISPR-Cas system or a component thereof (e.g., encoding a second crRNA or guide RNA that targets a protospacer sequence of the genome of the carrier cell)) encoding P1 and P2. The protospacers may comprise different nucleotide sequences. Optionally, the carrier cell comprises a group of plasmids comprising 2, 3, 4, 5, 6 or more different types of plasmids, each of which can be conjugatively transferred to the target cell, and the plasmids encode different P1 and / or P2 products. For example, the plasmids encode different cRNAs or gRNAs that target different protospacers contained in the genome of the vector, the carrier cell, and / or the target cell. For example, the group of plasmids comprises up to n different types of plasmids, and the plasmids are members of up to n different incompatibility groups, such as a group selected from IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, IncO, Incl, IncA, IncB, IncC, IncH, IncIa, IncIc, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, IncS, IncT, and IncW. For example, n=2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0224] For example, the carrier cell comprises (i) a first vector (e.g., a plasmid) that encodes a first type of CRISPR / Cas system P2 or encodes a component of said system that targets a first protospacer contained in the vector, and (ii) a second vector (e.g., a plasmid) that encodes a second type of CRISPR / Cas system P2 or encodes a component of said system that targets a second protospacer contained in the vector or the genome of the carrier cell (e.g., the chromosomal sequence of the carrier cell), and the first and second types are different. For example, the first type is a type I system and the second type is a type II system (e.g., the first vector encodes Cas3, Cascade, and crRNA or a guide RNA that can operate with Cas3 and Cascade to modify the first protospacer in the target cell, and the second vector encodes Cas9 and crRNA or a guide RNA that can operate with Cas9 to modify the second protospacer in the target cell). Alternatively, Cas3 and Cascade are encoded by endogenous target cell genes, and the first vector encodes a crRNA or guide RNA that can operate with endogenous Cas3 and Cascade to modify the first protospacer in the target cell. Alternatively, Cas9 is encoded by endogenous target cell genes, and the second vector encodes a crRNA or guide RNA that can operate with endogenous Cas9 to modify the second protospacer in the target cell. Optionally, Cas3 and Cascade are encoded by endogenous target cell genes, and Cas9 is encoded by the second vector.

[0225] In place of type I and type II systems, the present invention in one embodiment provides a first vector (e.g., a plasmid) encoding a type I CRISPR / Cas system (or a component thereof, e.g., Cas3 or crRNA or gRNA) and a second vector (e.g., a plasmid) encoding a type III CRISPR / Cas system (or a component thereof). In place of type I and type II systems, the present invention in one embodiment provides a first vector encoding a type I CRISPR / Cas system (or a component thereof) and a second vector encoding a type IV CRISPR / Cas system (or a component thereof). In place of type I and type II systems, the present invention in one embodiment provides a first vector encoding a type I CRISPR / Cas system (or a component thereof) and a second vector encoding a type V CRISPR / Cas system (or a component thereof). In place of type I and type II systems, the present invention in one embodiment provides a first vector encoding a type I CRISPR / Cas system (or a component thereof) and a second vector encoding a type VI CRISPR / Cas system (or a component thereof).

[0226] In place of type I and type II systems, the present invention in one embodiment provides a first vector encoding a type II CRISPR / Cas system (or a component thereof, e.g. Cas9 or crRNA or gRNA) and a second vector encoding a type III CRISPR / Cas system (or a component thereof). In place of type I and type II systems, the present invention in one embodiment provides a first vector encoding a type II CRISPR / Cas system (or a component thereof) and a second vector encoding a type IV CRISPR / Cas system (or a component thereof). In place of type I and type II systems, the present invention in one embodiment provides a first vector encoding a type II CRISPR / Cas system (or a component thereof) and a second vector encoding a type V CRISPR / Cas system (or a component thereof). In place of type I and type II systems, the present invention in one embodiment provides a first vector encoding a type II CRISPR / Cas system (or a component thereof) and a second vector encoding a type VI CRISPR / Cas system (or a component thereof).

[0227] In place of type I and type II systems, the present invention in one embodiment provides a first vector encoding a type V CRISPR / Cas system (or a component thereof, e.g. Cas12a or crRNA) and a second vector encoding a type III CRISPR / Cas system (or a component thereof). In place of type I and type II systems, the present invention in one embodiment provides a first vector encoding a type V CRISPR / Cas system (or a component thereof) and a second vector encoding a type IV CRISPR / Cas system (or a component thereof). In place of type I and type II systems, the present invention in one embodiment provides a first vector encoding a type V CRISPR / Cas system (or a component thereof) and a second vector encoding a type V CRISPR / Cas system (or a component thereof). In place of type I and type II systems, the present invention in one embodiment provides a first vector encoding a type V CRISPR / Cas system (or a component thereof) and a second vector encoding a type VI CRISPR / Cas system (or a component thereof).

[0228] In place of the type I and type II systems, the present invention in one embodiment provides a first and second vector (e.g., a plasmid) encoding a type I CRISPR / Cas system (or a component thereof), respectively. In place of the type I and type II systems, the present invention in one embodiment provides a first and second vector encoding a type II CRISPR / Cas system (or a component thereof), respectively. In place of the type I and type II systems, the present invention in one embodiment provides a first and second vector encoding a type III CRISPR / Cas system (or a component thereof), respectively. In place of the type I and type II systems, the present invention in one embodiment provides a first and second vector encoding a type IV CRISPR / Cas system (or a component thereof), respectively. In place of the type I and type II systems, the present invention in one embodiment provides a first and second vector encoding a type V CRISPR / Cas system (or a component thereof), respectively. In place of the type I and type II systems, the present invention in one embodiment provides a first and second vector encoding a type VI CRISPR / Cas system (or a component thereof), respectively.

[0229] Optionally, the plasmids are members of different incompatibility groups, e.g., a group selected from IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, InclO, Incl, IncA, IncB, IncC, IncH, IncIa, InclIc, IncI1, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, IncS, IncT, and IncW. In one example herein, the target cell is an Enterobacteriaceae cell.

[0230] Advantageously, the carrier cells are for treating or preventing infection of target cells in a human or animal subject (eg, dog, cat, horse, chicken, cow, sheep, goat, pig, fish, or crustacean).

[0231] Advantageously, the carrier cells are of a species that is probiotic to said subject or a species that is probiotic to humans or animals (e.g. chickens). For example, the carrier cells are probiotic Bacteriodetes (e.g. Bacteriodes) cells, for example, the subject is a human. For example, the carrier cells are probiotic Clostridiales cells, for example, the subject is a human. For example, the carrier cells are probiotic E coli cells. For example, the carrier cells are probiotic Bacillus cells, for example, the subject is a plant.

[0232] Advantageously, each vector (e.g., virus or plasmid) encodes one or more guide RNAs or one or more crRNAs that can hybridize to the respective vector target nucleic acid sequences in the target cell. For example, each vector encodes 2, 3, 4, 5, 6, 7, 7, 9, or 10 (or more than 10) different gRNAs or different crRNAs that hybridize to the respective target sequences, where the target sequences are different from each other. For example, three different gRNAs or crRNAs are encoded by each vector. For example, two different gRNAs or crRNAs are encoded by each vector. For example, three different gRNAs or crRNAs are encoded by each vector. For example, four different gRNAs or crRNAs are encoded by each vector. For example, three different gRNAs or crRNAs are encoded by each vector. For example, five different gRNAs or crRNAs are encoded by each vector. For example, six different gRNAs or crRNAs are encoded by each vector. For example, seven different gRNAs or crRNAs are encoded by each vector. For example, 8 different gRNAs or crRNAs are encoded by each vector. For example, 9 different gRNAs or crRNAs are encoded by each vector. For example, 10 different gRNAs or crRNAs are encoded by each vector. For example, 11 different gRNAs or crRNAs are encoded by each vector. For example, 12 different gRNAs or crRNAs are encoded by each vector. For example, 13 different gRNAs or crRNAs are encoded by each vector.

[0233] In one example, the target cell is a Salmonella cell (e.g., where the subject is a chicken). In one example, the target cell is a Campylobacter cell (e.g., where the subject is a chicken). In one example, the target cell is an Edwardsiella cell (e.g., where the subject is a fish or crustacean, such as a catfish, or a shrimp, or a prawn). In one example, the target cell is a Bacteriodetes (e.g., a Bacteriodes) cell. In one example, the target cell is a Clostridiales cell. In one example, the target cell is an E coli cell.

[0234] Optionally, each plasmid contains expressible tra1 and / or tra2 modules or homologs thereof for conjugative transfer of the plasmid between cells. Any episome herein may be a plasmid.

[0235] Optionally, each plasmid contains an expressible operon of the tra1 and / or tra2 module or its homologue for conjugal transfer of the plasmid between cells.

[0236] Optionally, each plasmid is a modified RK2 or R6K plasmid, the modification comprising the insertion of nucleotide sequences encoding P1 and P2.

[0237] Optionally, each plasmid contains an oriV, such as an oriV of IncI (e.g., IncI1 or IncI2), an RK2, or an R6K plasmid, or a homolog thereof. Optionally, each plasmid contains an oriV of IncI (e.g., IncI1 or IncI2), an RK2, or an R6K plasmid, or a homolog thereof.

[0238] Optionally, each plasmid includes an oriT, such as the oriT of an IncI1 or IncI2 plasmid. Optionally, each plasmid is a modified IncI (e.g., IncI1 or IncI2) plasmid. The modification (for the first through third configurations, and optionally for the fourth configuration) includes the insertion of nucleotide sequences encoding P1 and P2.

[0239] Optionally, P2 comprises one or more components of a CRISPR / Cas system operable in the target cell to cleave a protospacer sequence contained in the vector or the genome of the carrier cell, e.g., the protospacer sequence is contained in the chromosome of the carrier cell.

[0240] In one embodiment, cleavage herein kills the carrier cell or causes degradation of the vector in the target cell, or alternatively, cleavage inhibits the growth or proliferation of the target cell.

[0241] Optionally, P2 encodes a guide RNA or crRNA of a CRISPR / Cas system that can operate with a Cas nuclease to cleave a protospacer sequence contained in the vector in a target cell.

[0242] In one example of the first to third configurations, the protospacer is contained in a gene necessary for the survival or maintenance of the vector in a cell. In one example of the fourth configuration, the protospacer is contained in a gene necessary for the survival of the carrier cell.

[0243] Optionally, each vector (e.g., a virus (e.g., a phage) or a plasmid) comprises a gene encoding a product, which is essential for survival or growth of the carrier cell when in an environment lacking that product, and the carrier cell chromosome does not comprise an expressible gene encoding that product, and optionally the vector nucleic acid is the only episomal nucleic acid contained in the carrier cell that encodes that product. For example, the gene is selected from aroA, argH, hisD, leuB, lysA, metB, proC, thrC, pheA, tyrA, trpC, and pflA genes, or the gene is an antitoxin gene, and optionally the vector encodes the cognate toxin.

[0244] For example, the carrier cell is an E. coli (e.g., a Nissle, F18, or S17 E. coli strain) cell. For example, the carrier cell is a Bacillus (e.g., B. subtilis), Enterococcus, or Lactobacillus cell, and the subject is, for example, a plant.

[0245] Optionally, each carrier cell is for administration to a microbial population of a human or animal subject for medical use.

[0246] For example, medical applications are for treating or preventing a disease as disclosed herein. For example, medical applications are for treating or preventing a condition as disclosed herein.

[0247] Optionally, the medical use is for the treatment or prevention of a disease or condition mediated by said target cells. Optionally, the medical use is for the treatment or prevention of a disease or condition mediated by cells of a microbial community that also includes said target cells. For example, P1 is secreted from a target cell and acts to kill or modify the growth or metabolism of adjacent cells in the microbial community.

[0248] Optionally, the carrier cells are for administration to a human or animal to promote the growth or weight of the human or animal. Optionally, the carrier cells are for administration to a human or animal to reduce the growth or weight of the human or animal. Optionally, the carrier cells are for administration to a human or animal to reduce obesity in the human or animal.

[0249] In one embodiment, the administration is to a human to promote growth or weight in the human.

[0250] Optionally, the facilitation is not a medical treatment. Optionally, the facilitation is a medical treatment.

[0251] Optionally, the use includes administration of a plurality of carrier cells to a microbial population (e.g., the intestinal microbial population) of a human or animal subject, the microbial population including target cells, and the vector nucleic acid is transferred into the target cells for expression of P1 in the target cells, thereby killing or reducing the growth or proliferation of microbial population cells in the subject.

[0252] Optionally, the use includes administration of a plurality of carrier cells to a microbial population (e.g., the intestinal microbial population) of a human or animal subject, the microbial population including target cells, and the vector nucleic acid is transferred into the target cells for expression of P1 in the target cells, thereby promoting the growth or metabolism of the microbial population cells in the subject.

[0253] Optionally, the use includes administering a plurality of carrier cells to a microbial community (e.g., gut microbial community) of a human or animal subject, the microbial community including target cells, the vector nucleic acid being transferred into the target cells for expression of P1 in the target cells, P1 being a protein (e.g., an enzyme) in a metabolic pathway in the cells of the microbial community, for example, P1 being secreted by the target cells and taken up by further cells in the microbial community for use of P1 in a metabolic pathway in the further cells.

[0254] Optionally, the use includes administration of a plurality of carrier cells to a microbial community (e.g., gut microbial community) of a human or animal subject, the microbial community includes target cells, and the vector nucleic acid is transferred into the target cells for expression of P1 in the target cells, where P1 is a protein capable of sequestering a substance (e.g., a protein, a peptide, a nucleic acid (e.g., RNA), a carbohydrate (e.g., sugar or a precursor thereof), an amino acid, a lipid, a fatty acid, an ion, or a chemical) in the subject. For example, P1 is secreted from the target cells and taken up by further cells in the microbial community for use of P1 as a sequestering agent in the further cells. For example, P1 is secreted by the target cells for use of P1 as a sequestering agent in the subject, for example, in the microbial community, organs, tissues, cells, or bloodstream of the subject.

[0255] For example, the plant herein in any configuration or embodiment of the present invention is selected from a tomato plant, a potato plant, a wheat plant, a corn plant, a maize plant, an apple plant, a bean-producing plant, a pea plant, a beetroot plant, a stone fruit plant, a barley plant, a hop plant, and a grass. For example, the plant is a tree, such as a palm, a horse chestnut, a pine, an oak, or a hardwood. For example, the plant is a fruit-producing plant selected from strawberry, raspberry, blackberry, red currant, kiwi, banana, apple, apricot, avocado, cherry, orange, clementine, tangerine, grapefruit, prune, date, fig, lime, lemon, melon, mango, pear, olive, or grape. Optionally, the plant is a dicotyledonous plant. Optionally, the plant is a flowering plant. Optionally, the plant is a monocotyledonous plant.

[0256] In one example, the weight (i.e., biomass) of the plant is dry weight. For example, the method is for increasing the dry weight of a plant (e.g., within 1 or 2 weeks of said administration). Optionally, the increase is at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% increase compared to a control plant of the same species or strain that is not administered with carrier cells, where all plants are kept under the same environmental conditions. For example, such increase is within 1, 2, 3, 4, 5, 6, or 8 weeks after the first administration of carrier cells. In one example, the method is for increasing the dry weight of leaves and / or fruits of a plant, such as a tomato plant.

[0257] In one example, the weight is wet weight. For example, the method is for increasing the wet weight of a plant (e.g., within 1 or 2 weeks of said administration). Optionally, the increase is achieved by the addition of carrier cells. of The increase is at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% compared to a control plant of the same species or strain that is not administered, where all plants are kept under the same environmental conditions. For example, such increase is within 1, 2, 3, 4, 5, 6, or 8 weeks after the first administration of carrier cells. In one example, the method is for increasing the dry weight of leaves and / or fruits of plants, such as tomato plants.

[0258] For example, the microbial community may be contained in the leaves, stems, roots, or stems of a plant.

[0259] The target bacteria (or target cells) may be included in the microbiome of a plant. In one example, the microbiome is included in a leaf. In one example, the microbiome is included in a xylem. In one example, the microbiome is included in a phloem. In one example, the microbiome is included in a root. In one example, the microbiome is included in a tuber. In one example, the microbiome is included in a bulb. In one example, the microbiome is included in a seed. In one example, the microbiome is included in an exocarp, epicarp, mesocarp, or endocarp. In one example, the microbiome is included in a fruit, such as a simple, compound, or multi-flowered fruit. In one example, the microbiome is included in a seed or embryo, such as a seed coat, cotyledons, cotyledons, or root. In one example, the microbiome is included in a flower, such as a pedicel, sepals, petals, stamens, filaments, anthers, or pistils. In one example, the microbiome is included in a root, such as a taproot system, or a fibrous root system. In one example, the microbial community is contained in a leaf, e.g., in the leaf blade, petiole, or stipule. In one example, the microbial community is contained in a stem, e.g., in the bark, epidermis, phloem, cambium, xylem, or pith.

[0260] For example, the biofilm is contained in the lung of a subject, for example, the target cell is a Pseudomonas (e.g., P aeruginosa) cell. This may be useful when the subject is a human suffering from a pulmonary disease or condition, for example, pneumonia or cystic fibrosis, where P1 is a therapeutic protein expressed by the modified target cell of the biofilm. For example, the biofilm is contained in an animal or human organ disclosed herein. For example, the biofilm is contained in a human or animal microbial community disclosed herein.

[0261] Optionally, the surface is an ex vivo surface, such as a surface contained in a household or industrial device or container.

[0262] Optionally, the target cell is comprised in a biofilm, such as a biofilm disclosed herein.

[0263] Pharmaceutical compositions, livestock growth promotion compositions, soil amendments, herbicides, plant fertilizers, compositions for sterilization of food or food ingredients, dental compositions, personal hygiene compositions or disinfectant compositions (e.g., for household or industrial use) comprising a plurality of carrier cells are provided.

[0264] As used herein, a carrier cell is, for example, a commensal or probiotic cell for administration to a human or animal subject. For example, the carrier cell is commensal in the microbiome of a human or animal subject (e.g., the gut or blood microbiome), and the carrier is for administration to the subject. In one example, the carrier cell is a prokaryotic cell. In one example, the carrier cell is a bacterial cell (and optionally the target cell is a bacterial cell). In one example, the carrier cell is an archaeal cell (and optionally the target cell is an archaeal cell).

[0265] Optionally, the carrier cell is a gram-positive bacterial cell and the target cell is a gram-positive bacterial cell.

[0266] Optionally, the carrier cell is a gram-positive bacterial cell and the target cell is a gram-negative bacterial cell.

[0267] Optionally, the carrier cell is a gram-negative bacterial cell and the target cell is a gram-positive bacterial cell.

[0268] Optionally, the carrier cell is a gram-negative bacterial cell and the target cell is a gram-negative bacterial cell.

[0269] Optionally, the carrier cell is a Bacteriodes bacterial cell and the target cell is a Gram-positive bacterial cell. Optionally, the carrier cell is a Bacteriodes bacterial cell and the target cell is a Gram-negative bacterial cell. Optionally, the carrier cell is a Bacteriodes bacterial cell and the target cell is a Bacteriodes bacterial cell. Optionally, the carrier cell is a Bacteriodes bacterial cell and the target cell is an E coli bacterial cell. Optionally, the carrier cell is an E coli bacterial cell and the target cell is a Bacteriodes bacterial cell. For example, among these options, the subject is a human or an animal, preferably a human.

[0270] Optionally, the carrier cell is a Clostridiales bacterial cell and the target cell is a Gram-positive bacterial cell. Optionally, the carrier cell is a Clostridiales bacterial cell and the target cell is a Gram-negative bacterial cell. Optionally, the carrier cell is a Bacteriodes bacterial cell and the target cell is a Clostridiales bacterial cell. Optionally, the carrier cell is a Clostridiales bacterial cell and the target cell is an E. coli bacterial cell. Optionally, the carrier cell is an E. coli bacterial cell and the target cell is a Clostridiales bacterial cell. For example, in these options, the subject is a human or an animal, preferably a human.

[0271] Optionally, the carrier cell is an Escherichia (e.g., E. coli) bacterial cell and the target cell is a Gram-positive bacterial cell. Optionally, the carrier cell is an Escherichia (e.g., E. coli) bacterial cell and the target cell is a Gram-negative bacterial cell. Optionally, the carrier cell is a Bacteriodes bacterial cell and the target cell is an Escherichia (e.g., E. coli) bacterial cell. Optionally, the carrier cell is an Escherichia (e.g., E. coli) bacterial cell and the target cell is an E. coli bacterial cell. Optionally, the carrier cell is an E. coli bacterial cell and the target cell is an Escherichia (e.g., E. coli) bacterial cell. For example, in these options, the subject is a human or an animal, preferably a human.

[0272] Optionally, the carrier cell is a Bacillus bacterial cell and the target cell is a Gram-positive bacterial cell. Optionally, the carrier cell is a Bacillus bacterial cell and the target cell is a Gram-negative bacterial cell. Optionally, the carrier cell is a Bacillus bacterial cell and the target cell is a Salmonella bacterial cell. Optionally, the carrier cell is a Bacillus bacterial cell and the target cell is an E coli bacterial cell. Optionally, the carrier cell is an E coli bacterial cell and the target cell is a Pseudomonas bacterial cell. For example, in these options, the subject is a plant.

[0273] Optionally, the carrier cell is an E. coli bacterial cell and the target cell is a gram-positive bacterial cell.

[0274] Optionally, the carrier cell is an E coli bacterial cell and the target cell is a gram-negative bacterial cell.

[0275] Optionally, the carrier cell is an E coli bacterial cell and the target cell is a Salmonella bacterial cell.

[0276] Optionally, the carrier cells are E coli bacterial cells and the target cells are E coli bacterial cells.

[0277] Optionally, the carrier cell is an E coli bacterial cell and the target cell is a Pseudomonas bacterial cell.

[0278] The Bacillus cell herein is optionally a B subtilis cell.

[0279] Optionally, the carrier cell is a probiotic or commensal Bacteriodes bacterial cell for administration to a human or animal subject. Optionally, the carrier cell is a probiotic or commensal Clostridiales bacterial cell for administration to a human or animal subject. Optionally, the carrier cell is a probiotic or commensal E coli bacterial cell for administration to a human or animal subject. Optionally, the carrier cell is a probiotic or commensal Bacillus bacterial cell for administration to a human or animal subject.

[0280] Optionally, the plasmid herein is a closed circular DNA.

[0281] In one embodiment, the vector (e.g., plasmid) nucleic acid is DNA. Optionally, the DNA is dsDNA. In one embodiment, the vector DNA is ssDNA. In one embodiment, the vector (e.g., plasmid) nucleic acid is RNA.

[0282] In one example, the target cells are of a species that does not cause nosocomial infections in humans.

[0283] Optionally, the target cell is from a microbial community of an animal, such as a poultry animal (e.g., a chicken), a pig, a cow, a fish (e.g., a catfish or salmon), or a crustacean (e.g., a shrimp or lobster). Optionally, the microbial community is a gut microbial community. For example, the target cell is a cell from a gut biofilm of a human or an animal (e.g., a chicken). For example, the target cell is a cell from an ex vivo gut biofilm sample. For example, the target cell is a cell from a lung biofilm of a human or an animal (e.g., a chicken). For example, the target cell is a cell from an ex vivo lung biofilm sample. For example, the target cell is a cell from a skin biofilm of a human or an animal (e.g., a chicken). For example, the target cell is a cell from an ex vivo skin biofilm sample.

[0284] In one embodiment, each plasmid comprises oriV and / or oriT. In one embodiment, each plasmid comprises bacterial oriV and / or oriT.

[0285] In one embodiment, the plasmid contains oriV and does not encode any replication proteins (eg, pir or trfA) that can operate with oriV to initiate replication of the plasmid.

[0286] In one example, the present invention relates to a composition comprising a plurality of carrier cells of the present invention. Optionally, all of the carrier cells comprise the same said vector (e.g., a plasmid). Optionally, the plurality comprises a first subpopulation of carrier cells (first cells) and a second subpopulation of carrier cells (second cells), the first cells comprising the same first said vector and the second cells comprising the same second said vector (which is different from the first vector of the first cells). For example, the first vector encodes a first guide RNA or crRNA and the second vector encodes a second guide RNA or crRNA, the first guide RNA / crRNA can hybridize to a first protospacer sequence contained in the vector in the first target cell and the second guide RNA / crRNA can hybridize to a second protospacer sequence in the carrier cell or the target cell, where the protospacers are different.

[0287] Optionally, the composition is contained in a liquid (e.g., an aqueous liquid or in water), and the composition comprises carrier cells at 1×10 3 ~1×10 10 (For example, 1×10 4 ~1×10 10 , 1x10 4 ~1x10 9 , 1x10 4 ~1x10 8 , 1x10 4 ~1x10 7 , 1x10 3 ~1x10 10 , 1x10 3 ~1x10 9 , 1x10 3 ~1x10 8 , 1x10 3 ~1x10 7 , 1x10 5 ~1x10 10 , 1x10 5 ~1x10 9 , 1x10 5 ~1x10 8 , 1x10 5 ~1x10 7 , 1x10 6 ~1x10 10 , 1x10 6 ~1x109 , 1×10 6 ~1×10 8 , or 1 × 10 6 ~1×10 7 ) cfu / ml. For example, the liquid is, for example, a beverage for human or animal consumption. For example, the beverage is a livestock beverage, for example a poultry beverage (i.e. a beverage for consumption by poultry, for example chickens).

[0288] In one example, the composition is a dietary (e.g., dietary supplement) composition for human or animal consumption. In one example, the composition is a slimming composition for human or animal consumption. In one example, the composition is a growth promoting composition for human or animal consumption. In one example, the composition is a bodybuilding composition for human consumption. In one example, the composition is a probiotic composition for human or animal consumption. In one example, the composition is a bactericidal composition for human or animal consumption. In one example, the composition is an insecticidal composition for human or animal consumption. In one example, the composition is a zoonotic disease control composition for animal consumption.

[0289] In one example, the composition includes vitamins in addition to the carrier cells. In one example, the composition includes vitamins A, B (e.g., B12), C, D, E, and / or K in addition to the carrier cells. In one example, the composition includes lipids in addition to the carrier cells. In one example, the composition includes carbohydrates in addition to the carrier cells. In one example, the composition includes proteins and / or amino acids in addition to the carrier cells. In one example, the composition includes minerals in addition to the carrier cells. In one example, the composition includes metal ions (e.g., Mg) in addition to the carrier cells. 2+ , Cu 2+ , and / or Zn 2+ In one example, the composition includes sodium ions, potassium ions, magnesium ions, calcium ions, manganese ions, iron ions, cobalt ions, copper ions, zinc ions, and / or molybdenum ions.

[0290] In one example, the composition is a plant fertilizer composition. In one example, the composition is a herbicide. In one example, the composition is a pesticide composition for application to plants.

[0291] In any embodiment or example, where appropriate, the plant is, for example, a crop. The plant is, for example, wheat. The plant is, for example, corn. The plant is, for example, maize. The plant is, for example, a fruit tree. The plant is, for example, a vegetable plant. The plant is, for example, a tomato plant. The plant is, for example, a potato plant. The plant is, for example, a grass plant. The plant is, for example, a flowering plant. The plant is, for example, a tree. The plant is, for example, a shrub.

[0292] In one example, the composition is for application to the environment, and the environment is an outdoor environment (eg, application to a field or a waterway or reservoir).

[0293] In one example, the composition is included in a food product or food ingredient (e.g., for human or animal consumption). In one example, the composition is included in a beverage or beverage ingredient (e.g., for human or animal consumption).

[0294] In one example, the target cells are biofilm cells found in humans, for example, the biofilm is a biofilm of the intestine, skin, lungs, eyes, nose, ears, gastrointestinal tract (GI tract), stomach, hair, kidneys, urethra, bronchioles, oral cavity, mouth, liver, heart, anus, rectum, bladder, bowel, intestine, penis, vagina, or scrotum. In one example, the target cells are biofilm cells of an animal, for example, the biofilm is a biofilm of the intestine, skin, lungs, eyes, nose, ears, gastrointestinal tract (GI tract), cecum, jejunum, ileum, colon, stomach, hair, feathers, scales, kidneys, urethra, bronchioles, oral cavity, mouth, liver, spleen, heart, anus, rectum, bladder, bowel, intestine, penis, vagina, or scrotum. For example, the biofilm is a biofilm of the cecum of an avian (e.g., chicken). For example, the biofilm is a biofilm of the gastrointestinal tract (GI tract), cecum, jejunum, ileum, colon, or stomach of an avian (eg, chicken).

[0295] In one example, any method herein is ex vivo. In one example, any method herein is in vivo. In one example, any method herein is in vitro. In one example, any method herein is carried out in an environment, such as domestic (e.g., in a house), industrial (e.g., in a factory), or agricultural environment (e.g., in a field). In one example, any method herein is carried out in or on a container, or on a surface.

[0296] In one example, each vector (e.g., a plasmid) encodes one or more components of a CRISPR / Cas system operable to effect cleavage of a protospacer by the vector in a target cell (e.g., a protospacer comprises 10-20, 10-30, 10-40, 10-100, 12-15, or 12-20 contiguous nucleotides that can hybridize to a crRNA or gRNA encoded by the vector in a target cell). For example, the system is a Type I, Type II, Type III, Type IV, or Type V CRISPR / Cas system.

[0297] In one example, each vector encodes Cas9 (and optionally a second different Cas, e.g., Cas3, Cas9, Cpf1, Cas13a, Cas13b, or Cas10), and / or Cas3 (and optionally a second different Cas, e.g., Cas3, Cas9, Cpf1, Cas13a, Cas13b, or Cas10). In one example, each vector encodes a Cas selected from Cas3, Cas9, Cpf1, Cas13a, Cas13b, and Cas10. Additionally or alternatively, the vector encodes a guide RNA or crRNA or tracrRNA. For example, the guide RNA or crRNA or tracrRNA is cognate to (i.e., operable with) the first Cas in the target cell.

[0298] In one example, the Cas herein is Cas9. In one example, the Cas herein is Cas3. The Cas may be identical to the Cas encoded by the target bacterium.

[0299] In one embodiment, each plasmid is a shuttle vector.

[0300] Optionally, the target cell lacks a functional endogenous CRISPR / Cas system prior to transfer of the vector therein, e.g., the vector encodes components of an exogenous CRISPR / Cas system that are functional in the target cell. One embodiment provides a medicament comprising a plurality of carrier cells of the invention, each target cell optionally according to this paragraph, for administration to a human or animal subject for medical use.

[0301] In one example, the composition of the present invention is a herbicide, a pesticide, an insecticide, a plant fertilizer, or a cleaning agent.

[0302] Optionally, the target bacteria herein are included in the microbiome of a subject, such as the gut microbiome, or the microbiome of the skin, scalp, hair, eye, ear, mouth, throat, lung, blood, rectum, anus, vagina, scrotum, penis, nose, or tongue.

[0303] In one example, the subject (e.g., a human or animal) is further administered a pharmaceutical agent, either simultaneously or sequentially with administration of the carrier cells. In one example, the pharmaceutical agent is an antibiotic, an antibody or antibody fragment (e.g., an scFv, nanobody, or Fab), an immune checkpoint inhibitor (e.g., an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody), an adoptive cell therapy (e.g., CAR-T therapy), or a vaccine.

[0304] In one embodiment, the vector encodes a guided nuclease, such as a Cas nuclease, TALEN, zinc finger nuclease, or meganuclease. That is, P2 can comprise a guided nuclease, such as a Cas nuclease, TALEN, zinc finger nuclease, or meganuclease. Optionally, the vector encodes a restriction nuclease that can cut the genome of the vector and / or carrier cell (e.g., the chromosome of the carrier cell).

[0305] Optionally, the composition is a pharmaceutical composition for use in the practice of medicine on human or animal subjects. Optionally, the composition is not a pharmaceutical.

[0306] In one example, the animal is a livestock or companion pet animal (e.g., a cow, pig, goat, sheep, horse, dog, cat, or rabbit). In one example, the animal is an insect (an insect at any stage of its life cycle, e.g., egg, larva, or pupa). In one example, the animal is a protozoan. In one example, the animal is a cephalopod.

[0307] Optionally, the composition is a herbicide, a pesticide, a food or beverage processing agent, a food or beverage additive, a petrochemical or fuel treatment agent, a water purification agent, a cosmetic additive, a detergent additive, or an environmental (e.g., soil) additive or cleaning agent.

[0308] For example, the carrier bacterium is a Lactobacillus (e.g., L reuteri or L lactis), E coli, Bacillus, or Streptococcus (e.g., S thermophilus) bacterium. Usefully, the carrier can provide the plasmid with protection from the surrounding environment. The use of the carrier can be useful for oral administration or other routes where the carrier can provide the vector with protection from the acidic stomach or other harsh environment in the subject. Furthermore, the carrier can be formulated into a beverage, such as a probiotic drink, such as adapted Yakult™, Actimel™, Kevita™, Activia™, Jarrow™, or similar drink for human consumption.

[0309] Optionally, the carrier cell or composition is for administration to human or animal subjects for medical use, comprising killing target bacteria with P1 or its metabolites produced in the target cell, where the target bacteria is mediated as a disease or condition in the subject.In one example, when the subject is a human, the subject is not an embryo.In one example, the carrier cell is probiotic in the subject.

[0310] Optionally, the environment is a soil biota, i.e., a plant, a plant part (e.g., a leaf, fruit, vegetable, or flower) or a plant product (e.g., pulp), water, a waterway, a fluid, a foodstuff or a component thereof, a beverage or a component thereof, a medical device, a cosmetic, a detergent, blood, a bodily fluid, a medical apparatus, an industrial apparatus, an oil rig, a petrochemical process, a storage or transport apparatus, a vehicle, or a container.

[0311] Optionally, the environment is an ex vivo bodily fluid (e.g., urine, blood, a blood product, sweat, tears, sputum, or saliva), bodily solids (e.g., feces), or tissue of a human or animal subject to which the composition has been administered.

[0312] Optionally, the environment is an in vivo bodily fluid (e.g., urine, blood, a blood product, sweat, tears, sputum, or saliva), bodily solid (e.g., feces), or tissue of a human or animal subject to which the composition is administered.

[0313] In one embodiment, the plasmid is a phagemid or a cloning vector (e.g., a shuttle vector, e.g., a pUC vector). In one embodiment, the plasmid is a conjugative plasmid.

[0314] Optionally, P2 comprises a DNA sequence encoding one or more components of a CRISPR / Cas system, such as one or more components of a type I cascade (e.g., CasA).

[0315] Optionally, P2 comprises a DNA sequence encoding a guided nuclease, such as a Cas nuclease, a TALEN, a zinc finger nuclease, or a meganuclease.

[0316] In one example, the carrier cell or composition is contained in a medical container, such as a syringe, vial, IV bag, inhaler, eye dropper, or nebulizer. In one example, the carrier cell or composition is contained in a sterile container. In one example, the carrier cell or composition is contained in a medically compatible container. In one example, the carrier cell or composition is contained in a fermentation vessel, such as a metal, glass, or plastic vessel. In one example, the carrier cell or composition is contained in an agricultural device. In one example, the carrier cell or composition is contained in a food production or processing device. In one example, the carrier cell or composition is contained in a horticultural device. In one example, the carrier cell or composition is contained in a farm device. In one example, the carrier cell or composition is contained in a petrochemical recovery or processing device. In one example, the carrier cell or composition is contained in a distillation device. In one example, the carrier cell or composition is contained in a cell culture vessel (e.g., having a volume of at least 50, 100, 1000, 10000, or 100000 liters). Additionally or alternatively, the target cells are contained in any of these devices or others.

[0317] In one example, the carrier cells or composition are included in a pharmaceutical in combination with instructions or a packaging label that includes instructions for administering the pharmaceutical to a human or animal subject, for example, by oral, IV, subcutaneous, intranasal, intraocular, vaginal, topical, rectal, or inhalation administration. In one example, the carrier cells or composition are included in an oral pharmaceutical formulation. In one example, the carrier cells or composition are included in an intranasal or intraocular pharmaceutical formulation. In one example, the carrier cells or composition are included in a personal hygiene composition (e.g., shampoo, soap, or deodorant) or a cosmetic formulation. In one example, the carrier cells or composition are included in a cleaning formulation. In one example, the carrier cells or composition are included in a cleaning formulation, for example, for cleaning a medical or industrial device or equipment. In one example, the carrier cells or composition are included in a foodstuff, foodstuff ingredient, or foodstuff processing agent. In one example, the carrier cells or composition are included in a beverage, beverage ingredient, or beverage processing agent. In one example, the carrier cells or composition are included in a medical bandage, textile, plaster, or swab. In one example, the carrier cells or composition are included in a herbicide or insecticide. In one example, the carrier cell or composition is included in an insecticide.

[0318] In one example, the CRISPR / Cas component is a component of a type I CRISPR / Cas system. In one example, the CRISPR / Cas component is a component of a type II CRISPR / Cas system. In one example, the CRISPR / Cas component is a component of a type III CRISPR / Cas system. In one example, the CRISPR / Cas component is a component of a type IV CRISPR / Cas system. In one example, the CRISPR / Cas component is a component of a type V CRISPR / Cas system. In one example, the CRISPR / Cas component comprises a nucleotide sequence encoding Cas9 (e.g., S pyogenes Cas9, S aureus Cas9, or S thermophilus Cas9). In one example, the CRISPR / Cas component comprises a nucleotide sequence encoding Cas3 (e.g., E coli Cas3, C dificile Cas3, or Salmonella Cas3). In one example, the CRISPR / Cas component comprises a nucleotide sequence encoding Cpf. In one example, the CRISPR / Cas component comprises a nucleotide sequence encoding CasX. In one example, the CRISPR / Cas components include a nucleotide sequence encoding CasY.

[0319] Optionally, the target bacterium is a Gram-negative bacterium (e.g., a Spirobacterium or a Vibrio bacterium). Optionally, the target bacterium is a Gram-positive bacterium. Optionally, the target bacterium is a Mycoplasma, a Chlamydia, a Spirochete, or a Mycobacterium. Optionally, the target bacterium is a Streptococcus (e.g., a pyogenes or a thermophilus). Optionally, the target bacterium is a Staphylococcus (e.g., aureus, e.g., MRSA). Optionally, the target bacterium is an E. coli (e.g., O157:H7), for example, where the Cas is vector-encoded or where the nuclease (e.g., Cas3) activity of an endogenous target cell Cas is derepressed. Optionally, the target bacterium is a Pseudomonas (e.g., a syringae or aeruginosa). Optionally, the target bacterium is a Vibro (e.g., a cholerae (e.g., O139) or a vulnificus). Optionally, the target bacterium is Neisseria (e.g., gonorrhoeae or meningitidis). Optionally, the target bacterium is Bordetella (e.g., pertussis). Optionally, the target bacterium is Haemophilus (e.g., influenzae). Optionally, the target bacterium is Shigella (e.g., dysenteriae). Optionally, the target bacterium is Brucella (e.g., abortus). Optionally, the target bacterium is a Francisella host. Optionally, the target bacterium is Xanthomonas. Optionally, the target bacterium is Agrobacterium. Optionally, the target bacterium is Erwinia. Optionally, the target bacterium is Legionella (e.g., pneumophila). Optionally, the target bacterium is Listeria (e.g., monocytogenes). Optionally, the target bacterium is Campylobacter (e.g., jejuni). Optionally, the target bacterium is Yersinia (e.g., pestis). Optionally, the target bacterium is Borelia (e.g., burgdorferi). Optionally, the target bacterium is Helicobacter (e.g., pylori).Optionally, the target bacterium is Clostridium (e.g., dificile or botulinum). Optionally, the target bacterium is Erlichia (e.g., chaffeensis). Optionally, the target bacterium is Salmonella (e.g., typhi or enterica, e.g., serovar typhimurium, e.g., DT104). Optionally, the target bacterium is Chlamydia (e.g., pneumoniae). Optionally, the target bacterium is a Parachlamydia host. Optionally, the target bacterium is Corynebacterium (e.g., amycolatum). Optionally, the target bacterium is Klebsiella (e.g., pneumoniae). Optionally, the target bacterium is Enterococcus (e.g., faecalis or faecim, e.g., linezolid resistant). Optionally, the target bacterium is Acinetobacter (e.g., baumannii, e.g., multidrug resistant).

[0320] Further examples of target cells are as follows: (a) Optionally, the target bacteria is a Staphylococcus aureus cell that is resistant to an antibiotic selected from, for example, methicillin, vancomycin, linezolid, daptomycin, quinupristin, dalfopristin, and teicoplanin. (b) Optionally, the target bacteria is a Pseudomonas aeruginosa cell that is resistant to an antibiotic selected from, for example, cephalosporins (e.g., ceftazidime), carbapenems (e.g., imipenem or meropenem), fluoroquinolones, aminoglycosides (e.g., gentamicin or tobramycin), and colistin. (c) Optionally, the target bacterium is, for example, a Klebsiella (e.g., pneumoniae) cell that is resistant to a carbapenem. (d) Optionally, the target bacteria is a Streptococcus (e.g., thermophilus, pneumoniae, or pyogenes) cell that is resistant to an antibiotic selected from, e.g., erythromycin, clindamycin, beta-lactams, macrolides, amoxicillin, azithromycin, and penicillin. (e) Optionally, the target bacterium is a Salmonella (e.g., serovar Typhi) cell that is resistant to an antibiotic selected from, e.g., ceftriaxone, azithromycin, and ciprofloxacin. (f) Optionally, the target bacterium is a Shigella cell that is resistant to an antibiotic selected from, for example, ciprofloxacin and azithromycin. (g) Optionally, the target bacterium is a mycobacterium tuberculosis cell that is resistant to an antibiotic selected from, for example, resistance to isoniazid (INH), rifampicin (RMP), fluoroquinolones, amikacin, kanamycin, and capreomycin, and azithromycin. (h) Optionally, the target bacterium is, for example, an Enterococcus cell that is resistant to vancomycin. (i) Optionally, the target bacterium is an Enterobacteriaceae cell that is resistant to an antibiotic selected from, for example, cephalosporins and carbapenems. (j) Optionally, the target bacteria is an E. coli cell that is resistant to an antibiotic selected from, for example, trimethoprim, itoroflantoin, cephalexin, and amoxicillin. (k) Optionally, the target bacterium is a Clostridium (e.g., a Clostridium dificile cell) that is resistant to an antibiotic selected from, e.g., a fluoroquinolone antibiotic and a carbapenem. (l) Optionally, the target bacteria is a Neisseria gonnorrhoea cell that is resistant to an antibiotic selected from, for example, cefixime (e.g., an oral cephalosporin), ceftriaxone (an injectable cephalosporin), azithromycin, and tetracycline. (m) Optionally, the target bacterium is an Acinetobacter baumannii cell that is resistant to an antibiotic selected from, for example, beta-lactams, meropenems, and carbapenems. (n) Optionally, the target bacterium is a Campylobacter (e.g., jejuni) cell that is resistant to an antibiotic selected from, e.g., ciprofloxacin and azithromycin. (o) Optionally, the target cell produces beta (β)-lactamase (e.g., ESBL-producing E. coli or ESBL-producing Klebsiella). (p) Optionally, the target cell is a bacterial cell that is resistant to an antibiotic recited in any one of examples (a)-(n).

[0321] In one example, the target cell is a cell of a species selected from Shigella, E coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas, and Enterobacter, e.g., the subject is a plant. Optionally, the composition includes a carrier cell capable of conjugatively transferring the vector nucleic acid to a target cell of a species selected from two or more of Shigella, E coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas, and Enterobacter, e.g., the subject is a plant, either individually or in combination.

[0322] In one example, the reduction in target cell growth or proliferation is at least 50, 60, 70, 80, 90, or 95%.

[0323] In embodiments, the plasmid comprises a screenable or selectable marker gene, for example, a selectable marker gene is an antibiotic resistance gene.

[0324] The carrier bacteria may be of the following species or genera: for example, species found in warm-blooded animals (e.g., domestic vertebrates); for example, species found in humans; for example, species found in plants. Preferably, non-pathogenic bacteria that colonize non-sterile parts of the human or animal body (e.g., skin, gastrointestinal tract, urogenital area, mouth, nasal passages, throat and upper respiratory tract, ears, and eyes) are utilized as carrier cells, and in one example, the method of the present invention is used to combat infection of such parts of the human or animal body by target cell bacteria. In another embodiment, the infection is a systemic infection. Examples of carrier bacterial species include, but are not limited to, non-pathogenic strains of Escherichia coli (E. coli F18, S17, and Nissle strain of E. coli), various species of Lactobacillus (e.g., L. casei, L. plantarum, L. paracasei, L. acidophilus, L. fermentum, L. zeae, and L. gasseri), or other non-pathogenic or probiotic skin or GI colonizing bacteria, such as Lactococcus, Bifidobacteria, Eubacteria, and bacterial minicells, which are cells that lack nucleoids that are destined to die but still have the ability to transfer plasmids (e.g., Adler et al., Proc. Natl. Acad. Sci. USA 57;321-326, 1970; Frazer and Curtiss III, Current Topics in Microbiology and Immunology 69:1-84, 1975; Curtiss (See U.S. Patent No. 4,968,619 to Ill.). In some embodiments, the target recipient cells are pathogenic bacteria found in humans, animals, or plants, for example on the skin or in the digestive tract, urogenital area, mouth, nasal passages, throat and upper respiratory tract, eyes, and ears.Of particular interest for targeting and eradication are pathogenic strains of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus pneumoniae, and other species, Enterobacter spp., Enterococcus spp., and Mycobacterium tuberculosis.

[0325] The present invention has application in a wide variety of settings or environments, such as therapeutic, agricultural or other settings, including but not limited to those described in U.S. Patent Nos. 6,271,359, 6,261,842, 6,221,582, 6,153,381, 6,106,854, and 5,627,275, others are discussed herein and still others will be readily apparent to one of ordinary skill in the art.

[0326] A single carrier cell line may have two or more types of such vectors (e.g., they encode different P1s), and in another example, two or more different carrier bacterial strains, each containing one or more such vectors, may be combined to produce multiple different P1 products in a subject.

[0327] The present invention has applications in human treatment as well as various veterinary, agricultural, horticultural, and food processing applications. For human and veterinary applications, the following modes of administration of the carrier bacteria of the present invention are contemplated depending on the cell population or tissue targeted for protection: topical, oral, nasal, ocular, otic, pulmonary (e.g., by inhaler), ophthalmic, rectal, genitourinary, subcutaneous, intraperitoneal, and intravenous. The bacteria may be provided as a pharmaceutical composition in a delivery vehicle suitable for the mode of administration selected for the patient to be treated. The term "patient" or "subject" as used herein may mean a human or an animal (an animal may be particularly useful, for example, as a model for the clinical efficacy of a particular donor strain, or may be a domesticated animal or livestock). Commercially relevant animals are chickens, turkeys, ducks, catfish, salmon, cod, herring, lobsters, shrimp, prawns, cattle, sheep, goats, pigs, goats, geese, or rabbits.

[0328] For example, to deliver the carrier bacteria to the gastrointestinal tract or nasal passages, the preferred mode of administration may be oral ingestion or nasal aerosol, or feeding (either alone or incorporated into the subject's feed or food and / or beverage, such as drinking water). In this regard, the carrier cells may be included in the feed of livestock (or farmed or companion animals), e.g., the carrier bacteria are included in a feed additive for livestock. Alternatively, the additive is an additive to beverages (e.g., water) for livestock. It is noted that probiotic bacteria, such as Lactobacillus acidophilus, are sold as gel capsules that contain a freeze-dried mixture of bacterial cells and a solid support, such as mannitol. When the gel capsule is ingested with liquid, the freeze-dried cells are rehydrated and become viable and colonizing bacteria. That is, in a similar manner, the carrier bacterial cells of the present invention may be provided as gel capsules or bulk freeze-dried powder formulations, e.g., for sprinkling on food or beverages. The rehydrated viable bacterial cells then inhabit and / or colonize sites throughout the upper and / or lower digestive system and thereafter come into contact with the target bacteria.

[0329] For topical application, the carrier bacteria may be formulated as an ointment or cream for application to the affected skin surface.Ointment or cream formulations are also suitable for rectal or vaginal delivery, along with other standard formulations, such as suppositories.Appropriate formulations for topical, vaginal or rectal administration are well known to medicinal chemists.

[0330] The present invention may be useful for topical or mucosal administration to treat a variety of bacterial infections or unwanted conditions associated with bacteria. Some representative examples of these uses include: (1) conjunctivitis caused by Haemophilus sp. and corneal ulcers caused by Pseudomonas aeruginosa; (2) otitis externa caused by Pseudomonas aeruginosa; (3) chronic sinusitis caused by many gram-positive cocci and gram-negative bacilli, or general decontamination of the bronchial tubes; (4) cystic fibrosis associated with Pseudomonas aeruginosa; (5) enteritis caused by Helicobacter pylori (e.g., to treat or prevent gastric ulcers), Escherichia coli, Salmonella typhimurium, Campylobacter, or Shigella sp.; (6) open wounds, e.g., surgical or non-surgical, e.g., as a prophylactic measure; (7) burns to remove Pseudomonas aeruginosa or other gram-negative pathogens; (8) bacterial infections, e.g., bacterial infections caused by Propionobacter sp. These include acne caused by P. acnes, (9) nasal or skin infections caused, for example, by Methicillin-resistant Staphylococcus aureus (MSRA), (10) body odor caused, for example, by gram-positive anaerobic bacteria (i.e., the use of carrier cells in deodorants), (11) bacterial vaginosis associated, for example, with Gardnerella vaginalis or other anaerobic bacteria, and (12) treatment of gingivitis and / or dental caries caused by various organisms.

[0331] In one example, the target cells are E. coli cells and the disease or condition to be treated or prevented in humans is an infection of the uterine tract or a respiratory related infection, such as pneumonia, sepsis, septicaemia, or HUS.

[0332] In other embodiments, the carrier cells of the present invention have application in treating surfaces, such as in a method of treating such surfaces or environments containing target bacteria, for removing or attenuating undesirable target bacteria or modifying bacteria on surfaces, which method includes contacting the surface or environment with the carrier bacteria of the present invention, transferring the vector nucleic acid of the present invention from the carrier to the target bacteria, and expressing P1 in the target cells. For example, surfaces used in surgery, catheter insertion, and other invasive procedures may be treated to prevent infection of subjects by bacterial contamination on the surface. It is contemplated that the methods and compositions of the present invention can be used to treat and control bacterial contamination on many surfaces, objects, materials, and the like, such as medical or first aid equipment, nursing and kitchen equipment, and surfaces.

[0333] Pharmaceutical preparations or other compositions containing carrier bacteria may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to a physically discrete unit of pharmaceutical preparation appropriate for the patient or plant or environment or surface to be treated. Each dosage should contain an amount of carrier bacteria calculated to produce the desired antimicrobial effect associated with the selected carrier. Procedures for determining appropriate dosage units are well known to those skilled in the art. Dosage units may be proportionally increased or decreased based on the weight of the patient, plant, surface, or environment. The appropriate concentration to achieve eradication of pathogenic target cells (e.g., contained in the patient's tissue) may be determined by calculation of dosage concentration curves known in the art.

[0334] Other uses of the carrier bacteria of the present invention are also contemplated. These include various agricultural, horticultural, environmental, and food processing applications. For example, in agriculture and horticulture, various plant pathogenic bacteria may be targeted to minimize plant disease. One example of a suitable plant pathogen for targeting is Erwinia (e.g., E amylovora, the causative agent of fire blight). A similar strategy may be utilized to reduce or prevent the death of cut flowers. For veterinary or animal husbandry, the carrier cells of the present invention may be incorporated into animal feed (chicken, pig, poultry, goat, sheep, fish, shellfish, or cattle feed) to reduce bioburden or eliminate certain pathogenic organisms (e.g., Salmonella in chickens, turkeys, or other poultry). In other embodiments, the present invention may be applied to meat or other foods to eliminate undesirable or pathogenic bacteria (e.g., E. coli O157:H7 in meat, or Proteus spp., which contribute to the "fishy" odor of seafood).

[0335] Environmental uses include, for example, engineering a carrier bacterium, such as Bacillus thurengiensis and one of its conjugative plasmids, to deliver and conditionally express an insecticide in addition to or instead of an antimicrobial agent (e.g., to control mosquitoes that transmit malaria or West Nile virus). In such applications, formulation of the carrier bacterium as a solution, aerosol, or gel capsule is contemplated, as in agriculture and horticulture, or in the other applications mentioned above.

[0336] In one example, the plasmid is an engineered RK2 plasmid (e.g., when the microbial community is a human, animal, or plant microbial community) (i.e., an RK2 plasmid modified by recombinant DNA techniques, or a descendant of such a modified plasmid). Plasmid RK2 is a broad-host-range plasmid capable of replicating in 29 (and probably many more) Gram-negative species (Guiney and Lanka, 1989, p 27-54. In CMThomas (ed) Promiscous plasmids in gram-negative bacteria. London, Ltd London United Kingdom.). Plasmid RK2 is a self-transmissible plasmid of 60 kb whose complete nucleotide sequence is known (Pansegrau et al., 1994, J. Mol. Biol. 239, 623-663). Derived from this large plasmid, a minimal replicon was obtained that lacks all genes except the trfA gene, which encodes the Rep protein of the plasmid, designated TrfA, and the origin of vegetative replication, oriV. For a review of RK2 replication and its regulation by the TrfA protein, see Helinski et al., 1996 (In Escherichia coli and Salmonella Cellular and Molecular Biology, Vol. 2 (ed. F. Neidhardt, et al., 2295-2324, ASM Press, Washington DC).

[0337] In one example, the plasmid (e.g., when the microbial community is a human, animal, or plant microbial community) is an engineered R6K plasmid (i.e., an R6K plasmid that has been modified by recombinant DNA techniques, or a progeny of such a modified plasmid).

[0338] The present invention is optionally for or used in a method for industrial or domestic use, for example for or in agriculture, oil or petroleum industry, food or beverage industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aerospace industry, automotive industry, biotechnology industry, medical industry, health care industry, dental industry, energy industry, consumer products industry, pharmaceutical industry, mining industry, cleaning industry, forestry industry, fishery industry, leisure industry, recycling industry, cosmetics industry, plastics industry, pulp or paper industry, textile industry, clothing industry, leather or suede or animal leather industry, tobacco industry, or steel industry.

[0339] The invention is optionally for use in industry or the environment is an industrial environment and the industries are medical and healthcare, pharmaceutical, human food, feed, fertiliser for plants, beverages, dairy, meat processing, agriculture, livestock, poultry, fish and seafood, veterinary medicine, oil, gas, petrochemical, water treatment, sewage treatment, packaging, electronics and computers, personal healthcare and toiletries, cosmetics, dentistry, non-medical dentistry, ophthalmology, non-medical ophthalmology, mining and mineral processing, metal mining and metal processing, quarrying, aviation, automotive, railways. , transportation, space, environment, soil treatment, pulp and paper, clothing manufacturing, dyes, printing, adhesives, air conditioning, solvents, biodefense, vitamin supplementation, cryopreservation, textile steeping and production, biotechnology, chemicals, industrial cleaning products, household cleaning products, soaps and detergents, consumer products, forestry, fisheries, leisure, recycling, plastics, leather, leather and suede, waste disposal, funerals and contracting, fuel, construction, energy, steel, and tobacco industries.

[0340] In one example, the plasmid comprises a CRISPR array, where the array comprises one or two or more different spacers (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more spacers) for targeting the vector and / or genome of the carrier bacterium.

[0341] In one example, the target bacteria is included in an environment, as follows: In one example, the environment is a human organism, such as the oral or intestinal organism, or the bloodstream. In one example, the environment is not an environment in or on a human. In one example, the environment is not an environment in or on a non-human animal. In one embodiment, the environment is an air environment. In one embodiment, the environment is an agricultural environment. In one embodiment, the environment is an oil or petroleum recovery environment, such as an oil or petroleum field or well. In one example, the environment is an environment in or on food or beverages for consumption by humans or non-human animals. In one example, the environment is a marine environment, such as seawater or on a ship (e.g., ballast water of a ship or boat).

[0342] In one example, the environment is a human or animal organism (e.g., the gut, vagina, scalp, axilla, skin, or oral cavity organism). In one example, the target bacteria is contained in a human or animal organism (e.g., the gut, vagina, scalp, axilla, skin, or oral cavity organism).

[0343] In one example, the carrier bacteria or composition of the present invention is administered or intended for administration intranasally, topically, or orally to humans or non-human animals. Those skilled in the art who wish to treat a human or animal organism can determine the best route of administration depending on the organism of interest. For example, if the organism is an intestinal organism, administration may be nasal or oral. If the organism is a scalp or axillary organism, administration may be topical. If the organism is an oral or pharyngeal organism, administration may be oral.

[0344] In one example, the environment includes drinking water (e.g., waterways or drinking water for human consumption) or soil. The water is optionally in a heating, cooling, or industrial system, or in a drinking water storage vessel.

[0345] In one example, the carrier and / or target bacteria are Firmicutes selected from Anaerotruncus, Acetanaerobacterium, Acetitomaculum, Acetivibrio, Anaerococcus, Anaerofilum, Anaerosinus, Anaerostipes, Anaerovorax, Butyrivibrio, Clostridium, Capracoccus, Dehalobacter, Dialister, Dorea, Enterococcus, Ethanoligenens, Faecalibacterium, Fusobacterium, Gracilibacter, Guggenheimella, Hespellia, Lachnobacterium, Lachnospira, Lactobacillus, Leuconostoc, Megamonas, Moryella, Mitsuokella, Oribacterium, Oxobacter, Papillibacter, Proprionispira, Pseudobutyrivibrio, Pseudoramibacter, Roseburia, Ruminococcus, Sarcina, Seinonella, Shuttleworthia, Sporobacter, Sporobacterium, Streptococcus, Subdoligranulum, Syntrophococcus, Thermobacillus, Turibacter, and Weisella.

[0346] In one example, the carrier bacteria, composition, use, or method is for reducing pathogenic infection or rebalancing intestinal or oral biofilm, for example for treating or preventing obesity or disease in humans or animals, or for treating or preventing GI conditions (e.g., Crohn's disease, IBD, or enteritis).For example, the vector, carrier bacteria, composition, use, or method is for knocking down Salmomnella, Campylobacter, Erwinia, Xanthomonous, Edwardsiella, Pseudomonas, Klebsiella, Pectobacterium, Clostridium dificile, or E coli bacteria in intestinal biofilm in humans or animals or plants, preferably humans or animals.

[0347] In one example, the animal is a chicken, e.g., the target bacterium is Salmomnella or Campylobacter. In one example, the animal is a fish (e.g., catfish or salmon) or a crustacean (e.g., prawn or lobster), e.g., the target bacterium is Edwardsiella. In one example, the plant is a potato plant, e.g., the target bacterium is Pectobacterium. In one example, the plant is a cabbage plant, e.g., the target bacterium is Xanthomonous (e.g., X campestris). In one example, the plant is a marijuana plant, e.g., the target bacterium is Pseudomonas (e.g., P cannabina or P amygdali), Agrobacterium (e.g., Atumefaciens), or Xanthomonas (e.g., X campestris). In one example, the plant is a hemp plant and the target bacterium is, for example, a Pseudomonas (eg, P cannabina or P amygdali), Agrobacterium (eg, Atumefaciens) or Xanthomonas (eg, X campestris).

[0348] Optionally, the environment or the target bacteria is comprised in an intestinal microbiofilm, a skin microbiofilm, an oral microbiofilm, a throat microbiofilm, a hair microbiofilm, an axillary microbiofilm, a vaginal microbiofilm, a rectal microbiofilm, anal microbiofilm, an eye microbiofilm, a nasal microbiofilm, a tongue microbiofilm, a lung microbiofilm, a liver microbiofilm, a kidney microbiofilm, a genital microbiofilm, a penile microbiofilm, a scrotal microbiofilm, a mammary microbiofilm, an ear microbiofilm, a urethral microbiofilm, a labial microbiofilm, an organ microbiofilm, or a dental microbiofilm. Optionally, the environment or the target bacteria is comprised in a plant (e.g., tobacco, a crop, a fruit tree, a vegetable, or tobacco, e.g., on or comprised in a plant) or in an environment (e.g., soil or water or a waterway or an aqueous liquid).

[0349] Diseases and Conditions In one example, the carrier cell or composition is for treating a disease or condition in an animal or human. In one example, the disease or condition is caused or mediated by the presence of a protein or metabolite in a human or animal subject, and expression of P1 by a target cell comprising a vector nucleic acid causes a reduction in the protein or metabolite in the subject. In one example, the disease or condition is caused or mediated by the absence of a protein or metabolite in a human or animal subject, and expression of P1 by a target cell comprising a vector nucleic acid causes an increase in the protein or metabolite in the subject. In one example, the disease or condition is caused by an undesirably high level of a protein or metabolite in a human or animal subject, and expression of P1 by a target cell comprising a vector nucleic acid causes a decrease in the protein or metabolite in the subject. In one example, the disease or condition is caused by an undesirably low level of a protein or metabolite in a human or animal subject, and expression of P1 by a target cell comprising a vector nucleic acid causes an increase in the protein or metabolite in the subject. The metabolite may be a protein, a peptide, an amino acid, a carbohydrate, a sugar, a lipid, a fatty acid, or an ion (e.g., a metal ion). The metabolite may be toxic to human cells of the human subject. The metabolite may be a hormone, a growth factor, or an antibiotic. The metabolite may be a mineral. The metabolite may be a salt. The metabolite may be a nucleic acid, such as RNA (e.g., mRNA) or DNA.

[0350] In one example, the disease or condition is cancer, an inflammatory or autoimmune disease or condition, such as obesity, diabetes, IBD, a GI tract condition, or an oral condition.

[0351] Optionally, the disease or condition of the human or animal subject is: (a) a neurodegenerative disease or condition, (b) a brain disease or condition; (c) a disease or condition of the CNS; (d) memory loss or damage; (e) a cardiac or cardiovascular disease or condition, such as a heart attack, stroke, or atrial fibrillation; (f) liver disease or condition; (g) kidney disease or condition, e.g., chronic kidney disease (CKD); (h) a disease or condition of the pancreas; (i) a pulmonary disease or condition, such as cystic fibrosis or COPD; (j) gastrointestinal disease or condition; (k) any disease or condition of the throat or mouth; (l) eye disease or condition; (m) a disease or condition of the reproductive organs, such as a disease or condition of the vagina, labia, penis, or scrotum; (n) A sexually transmitted disease or condition, such as gonorrhea, HIV infection, syphilis, or chlamydia infection; (o) ear disease or condition; (p) skin diseases or conditions; (q) a heart disease or condition; (r) nasal diseases or conditions; (s) a blood disease or condition, such as anemia, e.g. anemia of chronic disease or cancer; (t) viral infection, (u) Pathogenic bacterial infection; (v) cancer, (w) an autoimmune disease or condition, e.g., SLE; (x) an inflammatory disease or condition, such as rheumatoid arthritis, psoriasis, dermatitis, asthma, ulcerative colitis, colitis, Crohn's disease, or IBD; (y) autism; (z) ADHD, (aa) bipolar disorder, (bb) ALS (amyotrophic lateral sclerosis), (cc) osteoarthritis, (dd) congenital or developmental defects or conditions; (ee) miscarriage, (ff) blood coagulation status; (gg) bronchitis, (hh) dry or wet AMD, (ii) neovascularization (e.g., in tumors or the eye); (jj) the common cold, (kk) epilepsy, (ll) fibrosis, e.g. liver or lung fibrosis, (mm) fungal diseases or conditions, such as thrush; (nn) a metabolic disease or condition, such as obesity, anorexia, diabetes, type I or type II diabetes, (oo) ulcers, e.g. gastric or skin ulcers, (pp) dry skin, (qq) Sjögren's syndrome, (rr) Cytokine storm, (ss) deafness, loss of or damage to hearing; (tt) metabolic slowing or acceleration (i.e., slowing or acceleration compared to the average for the subject's weight, sex, and age). (uu) Fertility disorders, e.g. infertility or subfertility; (vv) Jaundice; (lol) Skin rash, (xx) Kawasaki disease, (yy) Lyme disease, (zz) allergies, e.g., allergies to nuts, grass, pollen, house dust mites, cat or dog fur or dander; (aaa) malaria, typhoid fever, tuberculosis, or cholera; (bbb) Depression, (ccc) Mental retardation, (ddd)microcephaly, (eee) Malnutrition, (fff) conjunctivitis, (ggg) pneumonia, (hhh) Pulmonary embolism, (iii) pulmonary hypertension, (jjj) Bone disorders, (kkk) Sepsis or septic shock, (lll) sinusitis, (mmm) stress (e.g. occupational stress), (nnn) thalassemia, anemia, von Willebrand disease, or hemophilia, (ooo) Shingles or herpes, (ppp) Menstruation, (qqq) Decrease in sperm count is selected from.

[0352] Neurodegenerative or CNS Diseases or Conditions for Treatment or Prevention According to the Invention In one example, the neurodegenerative or CNS disease or condition is selected from the group consisting of Alzheimer's disease, geriatric psychosis, Down's syndrome, Parkinson's disease, Creutzfeldt-Jakob disease, diabetic neuropathy, Parkinsonism, Huntington's disease, Machado-Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy, and Creutzfeldt-Creutzfeldt-Jakob disease. For example, the disease is Alzheimer's disease. For example, the disease is Parkinsonism.

[0353] In an example where the method of the present invention is carried out on a human or animal subject to treat a CNS or neurodegenerative disease or condition, the method induces the downregulation of Treg cells in the subject, thereby promoting the entry of systemic monocyte-derived macrophages and / or Treg cells across the choroid plexus into the subject's brain, thereby treating, preventing, or reducing the progression of the disease or condition (e.g., Alzheimer's disease). In one embodiment, the method induces an increase in IFN-gamma in the subject's CNS system (e.g., in the brain and / or CSF). In one example, the method restores nerve fibers and / or reduces the progression of nerve fiber damage. In one example, the method restores nerve myelin and / or reduces the progression of nerve myelin damage. In one example, the methods of the present invention treat or prevent a disease or condition disclosed in WO2015136541 and / or the methods can be used in conjunction with any of the methods disclosed in WO2015136541 (the disclosures of this document are incorporated by reference in their entirety herein to provide a disclosure of such methods, diseases, conditions, and potential therapeutic agents, e.g., agents such as immune checkpoint inhibitors, e.g., anti-PD-1, anti-PD-L1, anti-TIM3, or other antibodies disclosed herein, that can be administered to a subject to effect treatment and / or prevention of CNS and neurodegenerative diseases and conditions).

[0354] Cancer for treatment The cancers that can be treated include non-vascularized or substantially non-vascularized tumors, as well as vascularized tumors.Cancers can include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) or can include solid tumors.The types of cancers that can be treated by the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemia or lymphatic malignancies, benign and malignant tumors, and malignant diseases, e.g., sarcomas, carcinomas, and melanomas.Adult tumors / cancers and pediatric tumors / cancers are also included.

[0355] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematopoietic) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.

[0356] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or areas of fluid. Solid tumors can be benign or malignant. The names of the various types of solid tumors are derived from the type of cells that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovial tumors, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytic lipocarcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, , testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors (e.g., gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germinomas, medulloblastomas, schwannomas, craniopharyogiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0357] Autoimmune Diseases for Treatment or Prevention 1. Acute disseminated encephalomyelitis (ADEM) 2. Acute necrotizing hemorrhagic leukoencephalitis 3. Addison's disease 4. Agammaglobulinemia 5. Alopecia areata 6. Amyloidosis 7. Ankylosing spinal cord disease 8. Anti-GBM / anti-TBM nephritis 9. Antiphospholipid syndrome (APS) 10. Autoimmune angioedema 11. Autoimmune aplastic anemia 12. Autoimmune autonomic neuropathy 13. Autoimmune hepatitis 14. Autoimmune hyperlipidemia 15. Autoimmune immunodeficiency 16.Autoimmune inner ear disease (AIED) 17. Autoimmune myocarditis 18. Autoimmune oophoritis 19. Autoimmune pancreatitis 20. Autoimmune retinitis 21. Autoimmune thrombocytopenic purpura (ATP) 22. Autoimmune thyroid disease 23. Autoimmune urticaria 24. Axonal and neuronal neuropathy 25. Barrow's disease 26. Behcet's disease 27. Bullous pemphigoid 28. Cardiac myopathy 29. Castleman's disease 30. Celiac disease 31. Chagas Disease 32. Chronic Fatigue Syndrome 33. Chronic inflammatory demyelinating polyneuropathy (CIDP) 34. Chronic recurrent multifocal ostomyelitis (CRMO) 35. Churg-Strauss syndrome 36. Cicatricial pemphigoid / benign mucous membrane pemphigoid 37. Crohn's disease 38. Kogans Syndrome 39.Cold agglutinin disease 40. Congenital heart block 41. Coxsackie myocarditis 42.CREST disease 43. Essential mixed cryoglobulinemia 44. Demyelinating neuropathy 45. Dermatitis herpetiformis 46. ​​Dermatomyositis 47. Devic's disease (neuromyelitis optica) 48. Discoid Loops 49. Dressler syndrome 50. Endometriosis 51. Eosinophilic esophagitis 52. Eosinophilic fasciitis 53. Erythema nodosum 54. Experimental allergic encephalomyelitis 55. Evans syndrome 56.Fibromyalgia 57. Fibrinating alveolitis 58.Giant cell arteritis (temporal arteritis) 59. Giant cell myocarditis 60. Glomerulonephritis 61. Goodpasture syndrome 62. Polyangiitis endoblastomatosis (GPA) (previously called Wegener's endoblastomatosis) 63. Graves' disease 64. Guillain-Barre syndrome 65. Hashimoto's encephalitis 66. Hashimoto's thyroiditis 67.Hemolytic anemia 68. Henoch-Schonlein purpura 69. Herpes gestationis 70. Hypogammaglobulinemia 71. Idiopathic thrombocytopenic purpura (ITP) 72. IgA nephropathy 73. IgG4-related sclerosing disease 74. Immunomodulatory Lipoproteins 75. Inclusion body myositis 76.Interstitial cystitis 77. Juvenile arthritis 78. Juvenile diabetes (type 1 diabetes) 79. Juvenile myositis 80. Kawasaki syndrome 81. Lambert-Eaton Syndrome 82.Leukocytoclastic vasculitis 83. Lichen planus 84. Lichen sclerosus 85.Ligny conjunctivitis 86. Linear IgA disease (LAD) 87. Lupus (SLE) 88. Lyme disease, chronic 89. Meniere's disease 90. Microscopic polyangiitis 91.Mixed connective tissue disease (MCTD) 92. Mooren's ulcer 93. Mucca-Habermann Disease 94. Multiple sclerosis 95.Myasthenia gravis 96. Myositis 97. Narcolepsy 98. Neuromyelitis Optica (Debicchi's) 99. Neutropenia 100. Ocular cicatricial pemphigoid 101.Optic neuritis 102. Relapsing rheumatism 103. PANDAS (Pediatric autoimmune neuropsychiatric disorders associated with streptococcus) 104. Paraneoplastic cerebellar degeneration 105. Paroxysmal nocturnal hemoglobinuria (PNH) 106. Parry-Romberg syndrome 107. Parsonage-Turner syndrome 108. Pars planitis (peripheral uveitis) 109. Pemphigus 110. Peripheral neuropathy 111. Perivenous encephalomyelitis 112. Pernicious anemia 113. POEMS syndrome 114. Polyarteritis nodosa 115. Autoimmune polyglandular syndromes types I, II, and III 116. Rheumatic polymyalgia 117. Polymyositis 118. Post-myocardial infarction syndrome 119. Postpericardiotomy syndrome 120. Progesterone dermatitis 121. Primary biliary cirrhosis 122. Primary sclerosing cholangitis 123. Psoriasis 124. Psoriatic arthritis 125. Idiopathic pulmonary fibrosis 126. Pyoderma gangrenosum 127. Erythroblastic aplasia 128. Raynaud's phenomenon 129. Reactive arthritis 130. Reflex sympathetic dystrophy 131. Reiter's syndrome 132. Relapsing polychondritis 133.Restless legs syndrome 134. Retroperitoneal fibrosis 135. Rheumatic fever 136. Rheumatoid arthritis 137. Sarcoidosis 138. Schmidt syndrome 139.Scleritis 140. Scleroderma 141. Sjögren's syndrome 142. Sperm and testicular autoimmunity 143. Stiff person syndrome 144. Subacute bacterial endocarditis (SBE) 145. Susac syndrome 146.Sympathetic ophthalmia 147.Takayasu arteritis 148. Temporal arteritis / giant cell arteritis 149. Thrombocytopenic purpura (TTP) 150. Tolosa-Hunt Syndrome 151. Transverse myelitis 152.1 diabetes 153.Ulcerative colitis 154.Undifferentiated Connective Tissue Disease (UCTD) 155. Uveitis 156. Vasculitis 157. Vesicular bullous skin disease 158. Vitiligo 159. Wegener's granulomatosis (now called granulomatosis with polyangiitis (GPA))

[0358] Inflammatory Diseases for Treatment or Prevention 1. Alzheimer's Disease 2. Ankylosing spondylitis 3. Arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis) 4. Asthma 5. Atherosclerosis 6. Crohn's disease 7. Colitis 8. Dermatitis 9.Diverticulitis 10. Fibromyalgia 11.Hepatitis 12.Irritable Bowel Syndrome (IBS) 13. Systemic lupus erythematosus (SLE) 14. Nephritis 15. Parkinson's disease 16. Ulcerative colitis

[0359] For example, the composition comprising the carrier cells is a human or animal food and / or drink (e.g., mixed into drinking water for livestock consumption). When provided in drink, the vector may be contained in carrier bacteria, which may be present in a concentration of 1×10 3 ~1×10 10 (For example, 1×10 4 ~1×10 10 , 1x10 4 ~1x10 9 , 1x10 4 ~1x10 8 , 1x10 4 ~1x10 7 , 1x10 3 ~1x10 10 , 1x10 3 ~1x10 9 , 1x10 3 ~1x10 8 , 1x10 3 ~1x10 7 , 1x10 5 ~1x10 10 , 1x10 5 ~1x10 9 , 1x10 5 ~1x10 8 , 1x10 5 ~1x10 7 , 1x10 6 ~1x10 10 , 1x10 6 ~1x10 9 , 1×10 6 ~1×10 8 , or 1 × 10 6 ~1×10 7 When provided in a beverage, the vector may be contained in a carrier bacterium, the carrier bacterium being at least 1×10 8 The amount of cfu / ml present in the beverage may be, for example, a human or an animal (eg, poultry, e.g., chickens).

[0360] Optionally, the guided nuclease is any guided nuclease disclosed herein, such as Cas, TALEN, meganuclease, or zinc finger nuclease.In one example, the component is a crRNA or guide RNA that can operate in target cells with its cognate Cas nuclease.The Cas nuclease can be any Cas nuclease disclosed herein.The Cas nuclease can be the endogenous Cas of the target cell, or can be encoded by an exogenous nucleic acid administered to the animal.

[0361] According to the fifth configuration, Provided are methods of manipulating a microbiome (e.g., any microbiome disclosed herein) comprising contacting the microbiome with a plurality of vectors described herein (e.g., by combining the microbiome with a carrier cell described) and optionally transferring said vector nucleic acid to target cells of the microbial population.

[0362] A modified microbial population obtained or obtainable by the methods herein, optionally included in a pharmaceutical composition for use as a medicament for treating a disease or condition in a human or animal subject.

[0363] Any of the features disclosed in the context of the first to fourth arrangements may be applied to the fifth arrangement, mutatis mutandis.

[0364] concept The present invention also provides the following concept.

[0365] 1. (a) an expressible nucleotide sequence of interest (NS1) for producing a product of interest (P1) in a host cell, and (b) an expressible nucleotide sequence (NS2) for producing a regulator product (P2) in a host cell, wherein P2 is operable in the host cell to regulate the expression or activity of P1; A host cell comprising a nucleic acid comprising (c) NS1 is under the control of a first promoter (e.g., a constitutive or inducible promoter) for expression of P1; (d) the expression or activity of P2 in the host cell is regulatable by exposure of the host cell containing at least one vector to a regulator agent (R), thereby regulating the expression or activity of P1; A host cell, wherein the host cell is a bacterial, archaeal, or fungal cell.

[0366] Optionally, the nucleic acid is contained in at least one nucleic acid vector for transfer from a host cell to a cell of the microbial community, for example, the host cell and the cell of the microbial community are bacterial cells.

[0367] For example, the fungal cell is a yeast cell. The bacterial cell may be a cell of any bacterial species or genus disclosed herein.

[0368] 2. A cell according to Concept 1, (a) the nucleic acid is contained in at least one nucleic acid vector in the cell; (b) NS1 is contained in a nucleic acid vector of the cell and NS2 is contained in a chromosome of the cell; (c) NS1 is contained in a chromosome of the cell and NS2 is contained in a nucleic acid vector of the cell; or (d) A cell, in which NS1 is contained in a chromosome of the cell and NS2 is contained in a chromosome of the cell.

[0369] 3. The cell of any one of Concept 2(a)-(c), wherein the or each vector is a conjugative plasmid for the transfer of a microbial population contained in a human or animal subject into a cell.

[0370] 4. A cell according to any of the preceding concepts, which is a cell of a commensal or probiotic bacterial species of the human or animal microbiome, optionally an E coli cell or a Bacteroides cell.

[0371] 5. A cell of any of the preceding concepts, wherein NS2 is under the control of a second promoter that is controllable for expression of P2, and binding of a regulator agent (R) to the vector nucleic acid controls the second promoter, thereby controlling expression of P2 and P1.

[0372] 6. The cell of any of the preceding concepts, wherein P2 comprises an RNA-guided nuclease (optionally a Cas nuclease), and the nuclease is operable to cleave a nucleic acid at a predetermined sequence motif.

[0373] 7. A cell according to Concept 6, (a) the sequence motif is contained in the cell's chromosome and cleavage causes cell death, or the sequence motif is contained (on the cell's chromosome or on a vector) in a gene that contains NS1 for production of P1 and cleavage downregulates production of P1, or (b) A cell, wherein the sequence motif is contained in said vector comprising NS1.

[0374] This is useful to facilitate degradation of the vector, thereby reducing or inhibiting production of P1, for example a cleaved vector is degraded in the cell.

[0375] 8. A cell of any of the preceding concepts for treating or preventing a disease or condition in a human or animal subject, wherein the cell is administered to the subject's microbiome (optionally the gut microbiome) to produce P1 in the subject, thereby treating or preventing the disease or condition in the subject.

[0376] Control with xylitol or xylose More than half of ingested xylitol in humans is not adsorbed by human cells, but instead reaches the gastrointestinal tract where it is taken up by the microbiome (Livesey, 2003), making it useful for controlling our switch in the GI tract. As exemplified herein, we have advantageously found that xylitol induces expression by releasing the repression that transcriptional regulators exert on the promoter. Furthermore, E. coli strains generally lack xylose reductase and xylitol dehydrogenase, which are typically required for the metabolism of xylitol (Ge et al., 2018). It was recognized that this may be useful for more precise or sustained control by xylitol if the strain cannot metabolize xylitol. If the strain cannot metabolize xylitol, the use of lower doses of xylitol is possible. That is, in one example, the cell of the present invention is an E. coli cell and the promoter is controllable by xylitol.

[0377] 1. A cell (optionally according to any of the preceding concepts) comprising a nucleic acid, wherein the nucleic acid comprises a gene encoding a product of interest (P1), wherein the gene comprises a nucleotide sequence (NS1) encoding P1 and a regulatory region 5' of NS1 comprising a promoter (Px) for regulating expression of NS1, wherein the combination of Px and NS1 is heterologous to the cell, and wherein Px is controllable by xylitol or xylose.

[0378] For example, NS1 is not found in wild-type cells of the same species as the cell of the present invention. For example, NS1 is a non-bacterial (e.g., animal, human, mammalian, or plant) sequence. Preferably, NS1 is a human sequence.

[0379] For example, NS1 is an endogenous sequence of the cell and Px is heterologous to the cell.

[0380] 2. The cell of concept 1, wherein the promoter is a xylitol or xylose-regulatable promoter of Morganella species, optionally M morganii.

[0381] For example, M morganii is Morganella morganii strain ZJG812.

[0382] 3. The cell of concept 1 or 2, wherein the promoter comprises SEQ ID NO:3 or a nucleotide sequence that is at least 70% identical to SEQ ID NO:3.

[0383] For example, any percent identity herein is at least 70, 80, 90, 95, 96, 97, 98, or 99%.

[0384] Optionally, the cell comprises a nucleotide sequence encoding SEQ ID NO: 4 or a promoter that can be controlled by xylitol. For example, the sequence further encodes a repressor that is cognate to the promoter. For example, the sequence further encodes a xylitol isomerase.

[0385] 4. The cell of any one of concepts 1 to 3, wherein Px is homologous to a xylitol or xylose-controllable promoter of Morganella morganii.

[0386] For example, the promoter is a xylitol-regulatable promoter, e.g., the promoter is repressible by a repressor and xylitol can derepress the repressor.

[0387] 5. The cell of any one of concepts 1 to 4, wherein the genome of the cell encodes a repressor capable of repressing Px, and wherein xylitol and / or xylose are capable of derepressing the repressor.

[0388] 6. The cell of concept 5, wherein the repressor is encoded by SEQ ID NO:1 or a nucleotide sequence that is at least 70% identical to SEQ ID NO:1.

[0389] For example, the identity is at least 70, 80, 90, 95, 96, 97, 98, or 99%.

[0390] For example, the repressor comprises SEQ ID NO:2 or an amino acid sequence which is at least 70% identical to SEQ ID NO:2.

[0391] 7. The cell of any one of concepts 1 to 6, comprising a xylitol transporter, optionally a xylitol ABC transporter.

[0392] Our data surprisingly suggest that the presence of a xylitol transporter increases the sensitivity of the donor strain to xylitol in the growth medium.

[0393] 8. The cell of any one of concepts 1 to 7, wherein the cell lacks a xylitol isomerase gene.

[0394] For example, the genome of the cell contains a xylitol-inducible promoter of a Morganii species and excludes a sugar isomerase gene. For example, the cell contains a xylitol-inducible promoter of a Morganii species and excludes a nucleotide sequence encoding a xylitol isomerase.

[0395] In one example, the genome of the cell contains the xylABC operon from Morganella morganii. Optionally, the operon lacks a nucleotide sequence encoding xylitol isomerase.

[0396] 9. A nucleic acid vector comprising a gene as recited in any one of concepts 1 to 8.

[0397] 10. The vector of concept 9, which is a plasmid (optionally a conjugative plasmid), a transposon, a phagemid, or a phage.

[0398] In one example, a cell of the invention comprises any of the vectors of the invention described herein.

[0399] It will be understood that the specific embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of the invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize or be able to ascertain, without more than routine study, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the invention and are included in the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the invention pertains. All patent publications and patent applications, and all U.S. equivalent patent applications and patents, are incorporated herein by reference to the same extent as if each individual patent publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification can mean "one," which is also consistent with the meanings of "one or more," "at least one," and "one or more than one." Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to mean alternatives only or the alternatives are not mutually exclusive, however, the present disclosure supports the definition to mean alternatives only and "and / or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method employed to determine the value, or the variation that exists among study subjects.

[0400] As used in this specification and the claims, the words "comprising" (and any of its forms, e.g., comprise and comprise), "having" (and any of its forms, e.g., have and has), "including" (and any of its forms, e.g., includes and include), or "containing" (and any of its forms, e.g., contains and contain) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0401] As used herein, the term "or combinations thereof" or the like refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB, if order is important in the particular context. Continuing with this example, combinations including repeats of one or more items or terms, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, etc., are expressly included. Those skilled in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0402] Unless otherwise clear from the context, any part of this disclosure may be read in combination with any other part of this disclosure.

[0403] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that changes can be made to the compositions and / or methods, and to the steps or sequence of steps of the methods described herein, without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. EXAMPLES

[0404] [Example 1] Construction of conjugative and self-targeting producing CGV Background of Example 1 The pBBR1-based plasmids used contain oriT that can be mobilized by the RP4 plasmid transfer machinery carried in trans by the E. coli JKE201 strain that will carry the plasmid (see Harms, A., Liesch, M., Korner, J., Quebatte, M., Engel, P. and Dehio, C., 2017. A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effectors of Bartonella. PLOS Genetics, 13(10), p. e1007077). A gene encoding E. coli type IE Cas3 was cloned under the control of the pBad promoter (which is induced by exposure to arabinose). A nucleotide sequence encoding a superfolder green fluorescent protein (sfGFP) and an sfGFP-targeting CRISPR array controlled by a rhamnose-inducible promoter is inserted into the replicon. The array encodes one or more crRNAs for the formation of a guide RNA that is cognate to Cas3 and is operable to target the sfGFP sequence and guide Cas3 to that sequence and cleave it.

[0405] Plasmid components: pBBR1-msc2, a mobilizable shuttle and expression vector, · oriT from the RP4 plasmid; Kanamycin antibiotic resistance marker; · rmtB (16S rRNA methylase gene), conferring resistance to amikacin and gentamicin; · an E. coli CRISPR / cas system with a pBAD promoter (wherein the system comprises nucleotide sequences encoding IE-type Cas3, cascade proteins A-E under the control of the pBAD promoter, and the system also comprises a CRISPR array); · S.pyogenes terminator after cascade; A rhamnose promoter in front of the CRISPR array, which is programmed to target the sfGFP gene (containing one spacer that can hybridize to the sfGFP sequence); and · Constitutively expressed sfGFP gene.

[0406] Ingredient order: an array containing a kanamycin resistance gene, pBBR1-msc2, oriT from the RP4 plasmid, araC, the pBad promoter, the E. coli Cas3 IE-type sequence, the cascade sequence, the S. pyogenes terminator, the rhamnose promoter, a spacer capable of hybridizing to the sfGFP sequence, rmtB (16S rRNA methylase gene) conferring resistance to amikacin and gentamicin, and the sfGFP gene.

[0407] Methods of cloning: Skeleton p1075 containing the pBBR1 replicon and CRISPR cas system was used as a template for cloning the sfGFP gene, the E. coli IE-type Cas3 and S. pyogenes terminator after the cascade, the CRISPR array with the sfGFP target, and the rhamnose promoter to control the CRISPR array with the sfGFP target (see plasmid map in Figure 1). Transformation, selection, and sequence validation Transformation procedure: The final construct (p1364) is electroporated into the strain of interest (NEB 10-beta cells for cloning or JKE201 strain for further mating). Correct colonies are selected for three antibiotic markers (kanamycin, gentamicin, amikacin). Colonies have a distinctive green color (due to expression of GFP) and are confirmed using colony PCR with primers specific for the construct.

[0408] Sequence verification is performed by subjecting purified plasmids to sequencing with construct-specific primers covering the entire construct.

[0409] See Figure 1 for a plasmid map highlighting the major components of the final plasmid, p1364.

[0410] [Example 2] Conjugative transfer method from E. coli donor strain JKE201 to recipient E. coli b52 strain: JKE201 strain was transformed with p1364, and positive clones were selected by kanamycin LB plates (supplemented with DAP) and further verified by positive GFP signal. One colony of the transformed JKE201 / p1364 donor strain as well as the b52 (C-1a-CGSC strain obtained from Coli Genetic Stock Center) recipient strain were grown to log phase in liquid LB (JKE201 supplemented with DAP). The two cultures were then mixed 5:1 (JKE201 / p1364:b52), spotted on solid LB agar and incubated overnight.

[0411] After overnight conjugation, the mixture was scraped off, dissolved in liquid LB, and plated onto LB plates containing kanamycin but no DAP to select for only b52 exconjugants, which were counted relative to the total number of recipients on the LB plates.

[0412] The results are shown in Figure 2.

[0413] p1364 is transferred to the b52 model strain at high frequency. Figure 2 shows the number of exconjugants formed as a result of RP4 oriT upon transfer of p1364 and the resulting absence of exconjugants in the absence of oriT.

[0414] [Example 3] Production of a protein of interest from a mobilizable plasmid in the "producing bacterial" strain (recipient strain) b52 Transconjugant recipient cells (b52 cells containing plasmid p1364) were generated as described in Example 2. Transconjugant conjugative plasmid p1364 containing sfGFP expression construct with self-targeting CRISPR array was grown in LB medium to OD600 of 1. Plasmid-free b52 strain was grown to the same level for use as negative / autofluorescence control. Each culture was transferred to a microtiter plate and measured in a Synergy™ H1 plate reader. sfGFP production was quantified using an excitation wavelength of 485 nm and an emission wavelength of 510 nm.

[0415] The results are shown in Figure 3. GFP fluorescence was determined at OD = 0.7 in b52 cells with p1364 plasmid and in b52 control cells without plasmid. GFP fluorescence was almost 5-fold higher than the background fluorescence of the b52 control strain, indicating reliable expression of sfGFP.

[0416] [Example 4] Curing of conjugative plasmids from recipient strain b52 Transconjugant b52 cells (from Example 2) carrying the p1364 plasmid were grown for 2 hours with selection for p1364 (kanamycin) and then washed with LB to remove residual antibiotic.

[0417] The resuspended culture was then split into two 1 ml portions and inducer (1% arabinose, 10 mM rhamnose) was added to one of the tubes to induce the self-targeting Cas machinery. Immediately after induction, cells from both cultures were plated onto LB plates (no inducer added was time 0). Samples from each tube (with and without induction) were then plated onto LB plates at 1 hour and 24 hours after induction. All plates were incubated overnight, after which the percentage of sfGFP-expressing cells was determined by colony counting in a blue light transilluminator (365 nm, UV benchtop transilluminator, VWR®).

[0418] The results are shown in Figure 4, which shows the percentage of GFP positive cells over time for induced and uninduced cultures. Induction of the CRIPSR system and corresponding arrays in P1364 allows rapid curing of self-targeting plasmids. The system allows for curing of more than 70% of the plasmids within 1 hour and elimination of more than 95% of the plasmids within 24 hours.

[0419] To verify that the loss of sfGFP was indeed due to loss of the plasmid, a plasmid backbone specific PCR was performed on 14 randomly selected colonies from the 24 hour plate, see the resulting gel image in Figure 5.

[0420] [Example 5] CRISPR-mediated eradication of donor strains Conjugative plasmid constructs were made that can be introduced into host cells to produce donor cells. After the plasmid is transferred from donor cells to recipient cells by conjugation, it becomes the vector for the self-targeting CRISPR / array that targets the gene of interest (e.g. GFP in the above example) and the plasmid in recipient cells.

[0421] The plasmid was engineered so that it could target the genome of the host cell upon induction of the CRISPR / cas system by arabinose. The plasmid was generated using the RP4 plasmid as a starting point. The E. coli IE-type Cas3 cascade and subsequent CRISPR array, which can be induced by arabinose to produce guide RNA targeting the E. coli chromosomal gene lptA, were integrated into the RP4 plasmid using recombinant techniques.

[0422] The Cas system and array containing the arabinose promoter were amplified from a cloning vector containing them along with a spectinomycin resistance gene and araC.

[0423] The PCR product was flanked by homology arms allowing insertion of the CRISPR / cas-araC-spectinomycin cassette into the tetracycline gene of RP4.

[0424] Recombination procedures were performed. Briefly, JKE201 cells containing the apSIM5 recombinant plasmid and containing the lptA mutant allele not targeted by the array were grown in LB supplemented with DAP and chloramphenicol to an OD of 0.5. The lambda RED system was then induced for 15 min at 42°C, the culture was placed on ice and the cells were prepared for transformation by washing with cold MiliQ water. Cells were transformed with the PCR product at 18 kv, allowed to recover for 12 h and plated on selection plates containing DAP and spectinomycin. The resulting recombinant clones were confirmed by PCR.

[0425] Positive clones and JKE201 containing wild-type RP4 plasmid (negative control without CRISPR / Cas) were grown for 4 hours in liquid LB with spectinomycin. Wild-type MG1655 cells were grown to the same OD and mixed with JKE201 / RP4::Cas and JKE201 / RP4 to transfer the plasmid and amplify it in this strain. The mixture was resuspended in LB without DAP to remove the initial JKE201 donor strain. The resulting population of MG1655 containing the plasmid (the newly formed plasmid donor cells) was now plated on LB plates with or without arabinose (arabinose for CRISPR / cas induction) to activate self-targeting and killing of donor cells by cleaving the lptA target contained in the donor chromosome. The population containing wild-type RP4 plasmid was plated on the same medium as a control. After overnight incubation, the surviving cells were counted and presented in Figure 6.

[0426] The results shown in Figure 6 demonstrate that upon induction of CRISPR / cas, robust counterselection of strains carrying one or more targets of interest occurs. The engineered conjugative plasmid presented here can discriminate between two closely related strains in mixed cultures, suggesting its usefulness for precise removal of donor strains associated with the microbiome, such as in vivo in human or animal subjects. If additional targets are added to the array, multiple targets can be hit simultaneously. The multiple target approach is an ideal implementation of a stringent biological containment strategy that essentially combines the two approaches presented here of donor cell genome and plasmid targeting, thereby allowing complete removal of a product (pathway) of interest from the microbiome in a controllable manner.

[0427] [Example 6] Illustrative Routes Employing the Vectors, Cells, and Methods of the Invention Various metabolic pathway schemes can be envisioned in which the present invention operates. For example, the pathway can be in any environment, including a microbial community, for example in a human or animal subject. An example of the scheme is provided in FIG. 7. The pathway scheme operates in a subject, for example limited to the microbial community (or even limited to the target cells that received the vector nucleic acid encoding P1 and P2). Alternatively, part of the scheme (at least the part that includes the target cells) occurs in the microbial community, and another part occurs outside the microbial community of the subject, for example in the environment where metabolites are secreted from the target cells and metabolized in downstream parts of the pathway scheme. In one embodiment, R is produced as a metabolite (in the target cells or in different cells, for example in different cells of the microbial community), which provides R to control the expression of P2 (and therefore P1) in a feedback loop.

[0428] R upregulates expression of P2, P2 downregulates expression of P1, and optionally P1 is a component of a metabolic pathway (e.g., in a microbial population or target cell, or in a subject or environment containing a microbial population or target cell), and a product (X) in the pathway downstream of P1 causes regulation of expression of P1 or P2. See, e.g., scenarios 1, 3, and 4 (Figure 7). In one embodiment, X is R or a precursor of R. In one embodiment, X controls a first and / or second promoter. In one embodiment, X upregulates expression of P1 or P2. In one embodiment, X upregulates expression of P1 or P2.

[0429] R upregulates expression of P2, P2 upregulates expression of P1, and optionally P1 is a component of a metabolic pathway (e.g., in a microbial population or target cell, or in a subject or environment that includes a microbial population or target cell), and a product (X) in the pathway downstream of P1 causes regulation of expression of P1 or P2. See, e.g., Scenario 2 (Figure 7). In one embodiment, X is R or a precursor of R. In one embodiment, X controls a first and / or second promoter. In one embodiment, X upregulates expression of P1 or P2. In one embodiment, X upregulates expression of P1 or P2.

[0430] [Example 7] Vector elimination by CRISPR self-targeting in Bacteroides 1. Abstract In this study, we developed and tested a plasmid containing a CRISPR / Cas system, which we term CRISPR-guided vector (CGV), that can be removed from the population of Bacteroides thetaiotaomicron using a vector-mediated spacer that targets the CGV itself (self-targeting CGV). The CGV contained an inducible type IB CRISPR / Cas operon from Clostridioides difficile alongside five spacers that target the CGV. We demonstrated that upon induction, the CRISPR / Cas system removed the CGV from over 99.9% of the Bacteroides population without killing the host cells in the process.

[0431] the purpose Purpose 1 We deliver the self-targeting CGV to Bacteroides thetaiotaomicron VPI-5482 (bSNP2978) via conjugation from an Escherichia coli donor and show that the CGV can be efficiently removed upon CRISPR / Cas induction. Purpose 2 We demonstrate that self-targeting CGV is cleared from populations of Bacteroides thetaiotaomicron VPI-5482 (bSNP2978) without off-target effects.

[0432] 2. Materials and Methods 2.1 Bacterial strains and growth conditions Escherichia coli strains were grown at 37°C in lysogeny broth (LB) at 250 RPM or on solid LB agar plates (made with 1.5% (w / v) agar). For plasmid selection, the medium was supplemented with ampicillin (Amp, 100 μg / ml) or spectinomycin (Spc, 400 μg / ml). For growth of auxotrophic strains, the medium was additionally supplemented with diaminopimelic acid (DAP, 40 μg / ml).

[0433] Bacteroides thetaiotaomicron VPI-5482 (bSNP2978) grows only anaerobically, but does not die under aerobic conditions. For this reason, an "M45 Variable Atmosphere Workstation" (Don Whitley, Yorkshire, UK) was used and set up to contain a mixture of the gases N2, H2, and CO2 in a ratio of 92:6:2. All solutions and media intended for Bacteroides were reduced under these conditions for approximately 24 hours before use.

[0434] All Bacteroides strains were routinely grown at 37°C in brain heart infusion (BHI) broth supplemented with cysteine ​​(1 g / l), hemin (5 mg / l), and NaHCO3 (0.2% (w / v)). This medium, designated BHIpp, was based on a recent Bacteroides protocol (Bacic & Smith, 2008). When growth on solid medium was required, BHIpp was additionally supplemented with agar (1.5% (w / v)). For plasmid selection, the medium was supplemented with erythromycin (Erm, 10 μg / l). For counterselection of E. coli donor strains, BHIpp medium was supplemented with gentamicin (Gm, 30 μg / l).

[0435] 2.2 CGV Design and Assembly CGV pSNP1599 is based on a shuttle plasmid, which can be delivered via conjugation from an E. coli (bSNP3235) based donor strain to a Bacteroides thetaiotaomicron VPI-5482 recipient strain (bSNP2978).

[0436] To construct pSNP1599, five spacers complementary to the protospacers found in the plasmid itself (details can be found in Appendix C) were first assembled into a CRISPR array and then cloned into the shuttle plasmid. In the same cloning reaction, a type IB CRISPR / Cas operon from Clostridioides difficile 630Δerm (designated CdCas) was also cloned into the plasmid.

[0437] Expression of the vector-mediated CRISPR / Cas system was driven by a rhamnose-inducible promoter (P rha ), which allowed for controlled excision of the plasmid. Detailed information on the construction of pSNP1599 can be found in Appendix B.

[0438] 2.3 Conjugal transfer of CGV from E. coli to B. thetaiotaomicron First, the bSNP3235-based donor strain carrying the plasmid of interest was grown aerobically in 30 ml of LB medium supplemented with DAP, whereas the bSNP2978 recipient was grown anaerobically in 5 ml of BHIpp broth. The donor and recipient cultures were grown to optical densities (OD ) of 0.4 ± 0.2 and 0.15 ± 0.05, respectively. 600 Once the total volume of the donor and recipient cells reached 10 ml, they were mixed using 25 ml of donor and 2.5 ml of recipient. The mixture was centrifuged (9000×g for 10 min), the supernatant was removed, and the pellet was resuspended in 100 μl of PBS. The resuspended pellet was spotted onto a BHIpp agar plate and incubated aerobically at 37° C. for 18 hours to promote conjugation.

[0439] Mating spots were collected and resuspended in 500 μl of pre-reduced BHIpp broth (from this point on, the remaining experiments were carried out in the anaerobic workstation). Ten-fold serial dilutions were prepared in PBS (dilution range 10 0 ~10 -6), then spotted onto selective medium (BHIpp+Gm+Erm agar) with and without inducer. Plates were packaged in plastic bags and incubated anaerobically at 37°C for 2-3 days. Any colony forming units (CFUs) of exconjugants arising from the plates were counted. Two examples of plates after 3 days of incubation are shown in Figure 10.

[0440] 2.4 Plasmid curing assay A colony of bSNP2978 (verified by sequencing) containing intact self-targeting CGV pSNP1599 was inoculated into 5 ml of BHIpp broth supplemented with gentamicin and erythromycin and grown anaerobically overnight. The culture was then washed three times (each wash consisting of centrifugation at 4500 × g for 2 min, removal of the supernatant, and resuspension in 5 ml of PBS) to remove all traces of erythromycin.

[0441] Two 5 μl aliquots of the washed cultures were transferred to separate tubes containing 5 ml of fresh, pre-reduced BHIpp broth. One tube received rhamnose (10 mM) as the inducer, and the other received an equal volume of milliQ HO. Induced and uninduced cultures were grown anaerobically over the course of a day, and 200 μl aliquots were taken from each culture at 0, 1, 3, 6, and 24 hours post-induction.

[0442] Each collected aliquot was serially diluted 10-fold in PBS (dilution range 10 0 ~10 -9 ) were prepared and spotted (2.5 μl per dilution) onto BHIpp agar plates supplemented with gentamicin (non-selective plates) or gentamicin and erythromycin (selective plates). Plates were packaged in plastic bags and incubated anaerobically for 2-3 days. CFU were counted. Experiments were performed in three biological replicates. Two examples of plates after 3 days of incubation are shown in Figure 11.

[0443] 3.Results 3.1 Estimation of the efficacy of CRISPR-mediated CGV removal We generated a self-targeting CGV, named pSNP1599, that contains a type I CRISPR / Cas system (CdCas) from C. difficile and five spacers that target the CGV itself. Upon induction with rhamnose, the CdCas system is activated, which leads to CRISPR targeting, which results in the elimination of the CGV.

[0444] CGV was conjugated to the model strain Bacteroides thetaiotaomicron VPI-5482 (bSNP2978) by mating with an E. coli (bSNP3235)-based donor strain, and the curing efficiency was tested by plating the mating mixture with and without rhamnose on solid medium to select for CGV. As a control, the empty plasmid pSNP1380 was conjugated via separate matings.

[0445] Conjugation experiments showed that induction produced 1000-fold less transconjugants on selective media compared to uninduced plates, effectively eliminating CGV (approximately 10 4 CFU / ml and approx. 7 CFU / ml) (Figure 8). Conjugation of the empty control plasmid shows no difference in exconjugants regardless of induction.

[0446] 3.2 Testing off-target effects of self-targeting CGV To test whether self-targeting CGVs could be eliminated without killing the Bacteroides host, bSNP2978(pSNP1599) was grown with and without rhamnose (Fig. ​(Fig.9A,top). ​Aliquots were taken at various time points, serial dilutions were made, and spread onto solid media plates with and without erythromycin (an antibiotic marker for GCVs) (Fig.9A,bottom), allowing us to estimate the size of the plasmid-containing and total populations, respectively.

[0447] One hour after induction, approximately 99% of the cells in the population had lost their CGV (Figure 9B). This difference gradually increased throughout the experiment. As expected, for uninduced cultures, the number of cells was similar regardless of selection, suggesting that our CGV was stable in Bacteroides, despite the active removal of the CGV by the addition of inducer.

[0448] 4. Conclusion A novel CGV harboring a self-targeting CRISPR / Cas system (CdCas) can be efficiently removed from >99.9% of cells in a population of B. thetaiotaomicron upon induction with rhamnose.

[0449] It was also demonstrated that the survival and proliferation of B. thetaiotaomicron cells was not affected by the induced self-targeting CGV, suggesting that CRISPR / Cas-mediated removal of CGV has no off-target effects.

[0450] Appendix A: Bacterial strains used in this study

[0451] [Table 1]

[0452] Appendix B: Construction and cloning of self-targeting CGV pSNP1599 CRISPR array design and assembly A CRISPR array was designed by identifying five 37 bp protospacers in the backbone of the shuttle plasmid, each flanked by a consensus type IB PAM sequence (5'-CCW-3') (details in Appendix C). A preliminary sequence similarity search (BLASTn, not shown) did not predict any off-targets on the bSNP2978 chromosome for any of the five selected spacers. A repeat sequence (5'-GTTTTATATTAACTAAGTGGTATGTAAAT-3') compatible with the type IB CRISPR / Cas system was selected.

[0453] Appendix C:

[0454] [Table 2]

[0455] [Example 8] Evaluation of xylitol-inducible promoters from Morganella morganii. Abstract A predicted xylitol-inducible promoter from the bacterium Morganella morganii was cloned and examined in various Escherichia coli strain backgrounds. It was demonstrated that the promoter was repressed by the transcriptional regulator protein IZ184_04885, and repression could be gradually relieved by addition of xylitol to the growth medium or completely by mutating the transcriptional regulator. Inclusion of the xylitol ABC transporter system also increased the sensitivity of the promoter to xylitol.

[0456] the purpose Purpose 1 We demonstrate that repression of a xylitol-inducible promoter can be relieved by adding increasing amounts of xylitol to the growth medium. Purpose 2 We demonstrate that repression of a xylitol-inducible promoter is circumvented by mutating the transcriptional regulator protein IZ184_04885. Purpose 3 We will examine whether expression of the xylitol ABC transporter increases the sensitivity of the xylitol-inducible promoter to xylitol in the medium.

[0457] material and method Bacterial strains and growth conditions for plasmid cloning All strains of Escherichia coli used in this study are listed in Table 5, Appendix A. For cloning, E. coli strains were grown at 250 RPM in lysogeny broth (LB) or on solid LB agar plates (made with 1.5% (w / v) agar) at 37°C. For plasmid selection, the medium was supplemented with tetracycline (10 μg / ml) and / or spectinomycin (Spc, 100 μg / ml).

[0458] Plasmid construction GFP reporter plasmid Based on the annotated genome sequence of Morganella morganii strain ZJG812 (genome ID: CP064831.1), a 1213 bp DNA sequence (excluding the sugar isomerase gene IZ184_04880) encoding a xylitol-inducible regulatory system was defined and ordered from Integrated DNA technologies™ (Coralville, IA, USA) as a gBlock fragment. The gBlock DNA fragment was further amplified by PCR using CloneAmp HiFi PCR Premix from Takara Bio (Mountain View, CA, USA) with primers SEM1770 and SEM1771. In parallel, the backbone of plasmid pSNP1248 was PCR amplified using CloneAMP HiFi PCR Premix with primers SEM1282 and SEM1769 to encode a fluorescent reporter gene (gfp), a low copy number origin of replication (cloDF13), and a spectinomycin resistance marker (Spc R A complete list of primers used in this study can be found in Table 6, Appendix B.

[0459] The amplified DNA fragment was treated with DpnI in the usual way (1 hour at 37°C, followed by 20 minutes at 80°C) and purified by gel extraction using the MinElute kit from Qiagen (Hilden, Germany) according to the manufacturer's instructions. The purified control system DNA was inserted into the linearized pSNP1248 backbone with the In-Fusion® HD cloning kit from Takara Bio according to the manufacturer's instructions. Chemically competent bSNP2480 cells were transformed with DNA from the In-Fusion® reaction mixture. The transformed cells were cultured in LB+Spc 100 The plasmids were spread on agar plates and incubated overnight. Plasmids were purified from individual colonies using Qiagen's QIAprep Spin Miniprep Kit according to the manufacturer's instructions and subsequently verified by Sanger sequencing. A positive clone was registered as plasmid pSNP1902. Furthermore, an unexpected clone (IZ184_04885) containing a 595 bp deletion located within the open reading frame (ORF) of a transcription regulator was identified and registered as plasmid pSNP1903.

[0460] Xylitol ABC transporter plasmid The 3728 bp xylABC operon (Genome ID: CP064831.1) from Morganella morganii strain ZJG812 was ordered as two gBlock DNA fragments from Integrated DNA Technologies™. The plasmid backbone was prepared by PCR amplification of plasmid pSNP639 using CloneAmp HiFi PCR Premix and primers SEM1814 and SEM1815. The amplified fragment was DpnI treated and purified by gel extraction following the same method as described in section 3.2.1.

[0461] The two gBlock DNA fragments were fused with the linearized backbone DNA using Takara Bio's In-Fusion® HD Cloning kit according to the manufacturer's instructions. Chemically competent bSNP2522 cells were transformed with DNA from the In-Fusion® reaction mixture. Transformed cells were cultured in LB+Tet 10 The plasmids were spread on agar plates and incubated overnight. Plasmids were purified from individual colonies using Qiagen's QIAprep Spin Miniprep Kit according to the manufacturer's instructions and subsequently verified by Sanger sequencing. The positive clone was deposited as plasmid pSNP1939. A complete list of plasmids used in this study can be found in Table 7, Appendix C.

[0462] GFP expression assay for characterization of xylitol-inducible promoters Preparation of model strains The constructed GFP reporter plasmids (pSNP1902 and pSNP1903), xylitol ABC transporter-containing plasmid (pSNP1939), and control plasmids (Table 7, see Appendix C) were transformed individually or in combination into the model strain E. coli MG1655 (bSNP230) and the probiotic E. coli isolate (bSNP463) by electroporation. The transformed cells were cultured in LB+Spc medium depending on whether the cells were transformed with the GFP reporter plasmid, the xylitol ABC transporter plasmid, or both, respectively. 100 -, LB+Tet 10 - or LB+Spc 100 +Tet 10 The clones were spread onto agar plates. The plates were incubated overnight. Three colonies were picked from each strain and used as three biological replicates in the subsequent 24-hour gfp expression assay.

[0463] 24-h gfp expression assay Triplicate colonies were inoculated into 5 ml of LB medium or 5 ml of M9 minimal medium broth (recipe found in Appendix D) supplemented with Tet (10 μg / ml) and / or Spc (100 μg / ml) and incubated overnight (approximately 16 hours) at 37°C and 250 RPM. Cultures reached an optical density (OD ) of 0.8 ± 0.3 in M9 minimal medium and 1.4 ± 0.2 in LB medium. 600 ) will be reached.

[0464] Each overnight culture was then diluted 100-fold in fresh medium supplemented with the appropriate antibiotic (as above) and distributed into three to six new cultures supplemented with different concentrations of xylitol ranging from 0% to 5% (the exact concentrations of xylitol are listed in the relevant figure in section 4). The cultures were then transferred in 200 μl aliquots into black 96-well microtiter plates with clear bottoms. To normalize the fluorescence emission, one row of wells in the plate was dedicated to cells containing the control plasmids pSNP958 and pSNP1617 that do not code for gfp (to estimate the autofluorescence) and another row was dedicated to medium without any cells (to estimate the background fluorescence). The microtiter plate was sealed with a Breath-Easy® sealing membrane from Merck (Whitehouse station, NJ, USA) and placed in a Synergy H1 microplate reader from Agilent Biotek (Winooski, VT, USA) programmed to incubate for 24 hours at 37° C. with constant stirring. OD was measured every 10 minutes throughout the incubation. 600 Both GFP fluorescence (excitation 485 nm, emission 516 nm, gain 90) were measured. Recorded fluorescence emissions were normalized according to the corresponding cell density and corrected for background and autofluorescence levels.

[0465] result Inducible promoter criteria and selection We developed a biocontainment strategy that uses the CRISPR / Cas system as an off-switch for a production circuit or strain, which requires that the CRISPR / Cas system is under tight transcriptional control by a promoter that is activated upon addition of an exogenous inducer molecule.

[0466] Within the field of molecular microbiology, operons of genes related to sugar transformation / utilization can be induced by the sugar itself, as sugars bind and affect transcriptional repressor proteins. For this study, we decided to look at negatively inducible promoters predicted to be induced by xylitol. This five-carbon sugar alcohol has several advantages. First of all, xylitol is generally regarded as safe for human consumption by the FDA (Xiang et al., 2021). Furthermore, more than half of ingested xylitol does not adsorb to human cells, but instead reaches the gastrointestinal tract where it is taken up by the microbiome (Livesey, 2003), making it useful for controlling switches in the GI tract. Finally, xylitol is a known metabolic product for certain bacteria. It is either directly taken up through an ABC-type transporter complex (Madigan et al., 2015) and / or produced through the reduction of the corresponding sugar D-xylose. Xylitol is subsequently dehydrogenated and phosphorylated to xylulose-5-phosphate, which is further catabolized via the pentose phosphate pathway.

[0467] From an old PhD thesis (Gallo, 1991), we found an operon in the genome of the bacterium Morganella morganii strain ZJG812 that contains genes related to xylitol uptake and metabolism (details are summarized in Figure 8(ii)). This operon is located downstream of a ∼1800 bp regulatory region that consists, among other elements, of a negatively inducible promoter and the cognate transcriptional regulator protein IZ184_04885 (details in Appendix E) to which xylitol has been suggested to bind.

[0468] Estimation of promoter activity in response to xylitol in the growth medium For this study, we constructed two reporter plasmids consisting of a green fluorescent protein (GFP) reporter gene located downstream of a xylitol-inducible regulatory system without (pSNP1902, Figure 8(i)A) or with (pSNP1903, Figure 8(ii)B) a loss-of-function deletion located in the ORF of a transcriptional regulator.

[0469] The reporter plasmid consists of a green fluorescent protein (GFP) reporter gene located downstream of a predicted xylitol-inducible promoter controlled by a DNA-binding transcriptional regulator of the LacI family (gene IZ184_04885).Two reporter plasmids were generated carrying either (A) the full-length transcriptional regulator (plasmid pSNP1902) or (B) a 595-bp loss-of-function (LOF) deletion in the ORF of the transcriptional regulator (plasmid pSNP1903).

[0470] The probiotic isolate bSNP463 was transformed with GFP reporter plasmids, i.e., pSNP1902 and pSNP1903, and incubated for 24 h in the presence of increasing amounts of xylitol, after which the optical density (OD 600 ) and by measuring the green fluorescence emitted from the cultures. The results of these tests are summarized in Figure 9. It is observed how the fluorescence intensity per cell increases up to 6-fold by adding xylitol to a strain containing a GFP reporter plasmid together with the full-length transcriptional regulator (Figure 9A). When the transcriptional regulator is mutated by a 595 bp deletion (Figure 9B), the basal fluorescence intensity is about 30-fold higher than that of the strain with the full-length transcriptional regulator, and the fluorescence intensity does not change upon addition of xylitol. This suggests that xylitol induces expression by releasing the repression that the transcriptional regulator exerts on the promoter.

[0471] Testing of the GFP reporter plasmid (pSNP1902) carrying the full-length transcriptional regulator was repeated in minimal medium broth with E. coli MG1655 strain (bSNP230) with similar results (shown in Figure 10). It should be noted that for these tests, the concentration of xylitol applied was low, as increasing concentrations of xylitol showed a slower growth rate of the bacteria when grown in minimal medium supplemented with glycerol. Furthermore, when all carbon sources except xylitol were omitted from the minimal medium, the E. coli strain did not grow at all (as explained by the growth profile shown in Figure 11). This is likely because E. coli strains generally lack xylose reductase and xylitol dehydrogenase, which are typically required for the metabolism of xylitol (Ge et al., 2018). This would be useful for more precise and sustained control with xylitol if the strain is unable to metabolize xylitol. If the strain is unable to metabolize xylitol, the use of lower doses of xylitol would be possible.

[0472] Testing whether the presence of a xylitol ABC transporter increases the sensitivity of xylitol-inducible promoters The activity of a negative inducible promoter will depend on the import of the inducer molecule into the cell. In this context, we hypothesized that expression of the predicted xylitol ABC transporter operon (described in section 4.1) would allow more xylitol to enter the cell, thereby increasing the sensitivity of the promoter. To test this, the bSNP230 construct, which already contains the GFP reporter plasmid pSNP1902, was transformed with plasmid pSNP1939 and tested by the same 24-h assay used previously. For these experiments, we used a defined M9 minimal medium containing glycerol or glucose as carbon source for growth. Glucose was chosen because it is the preferred sugar for bacterial growth, but it may also have an inhibitory effect on promoters controlling alternative metabolic pathways, such as those related to xylitol. Glycerol was chosen because it was previously shown by us (data not shown) not to affect expression from xylitol-inducible promoters. The results are summarized in Figure 12.

[0473] The bSNP230 strain was used, containing a GFP reporter plasmid (pSNP1902) with or without a coexisting plasmid (pSNP1939) containing the xylitol ABC transporter. The strain was grown for 24 h in minimal medium supplemented with 0.4% (v / v) glycerol (A) or 0.4% (w / v) glucose (B) in the presence or absence of xylitol. The cell density-adjusted fluorescence emission after 24 h of incubation is shown at the top. Error bars indicate standard deviation based on two (A) and three (B) biological replicates, respectively. The corresponding growth curves are shown in Figure 13.

[0474] At these low concentrations of xylitol, strain bSNP230 / pSNP1902 did not exhibit any change in fluorescence intensity (although higher concentrations of xylitol in different experiments could increase fluorescence). However, for strain bSNP230 / pSNP1902p+pSNP1939, an increase in fluorescence intensity was observed when xylitol was added. This effect was more pronounced when minimal medium was supplemented with glycerol (Figure 13A) than with glucose (Figure 13B). These data suggest that the presence of the xylitol ABC transporter increases the sensitivity of reporter plasmid-carrying strains to xylitol in the growth medium.

[0475] conclusion The xylitol-inducible promoter from Morganella morganii is repressed by the transcriptional regulator protein IZ184_04885 when tested in various E. coli strain backgrounds. This repression was gradually relieved by adding xylitol to the growth medium or completely by mutating IZ184_04885. The sensitivity of the promoter to xylitol in the growth medium was further enhanced by including the xylitol ABC transporter system.

[0476] References Gallo, MA (1991). Molecular characterization of xylitol catabolic pathways in the Enterobacteriaceae. Conrell University. Ge, X., Chang, C., Zhang, L., Cui, S., Luo, X., Hu, S., Qin, Y., & Li, Y. (2018). Conversion of Lignocellulosic Biomass Into Platform Chemicals for Biobased Polyurethane Application. In Advances in Bioenergy (Vol. 3, pp. 161-213). Elsevier. https: / / doi.org / 10.1016 / BS.AIBE.2018.03.002 Livesey, G. (2003). Health potential of polyols as sugar replacers, with emphasis on low glycaemic properties. Nutrition Research Reviews, 16(2), 163-191. https: / / doi.org / 10.1079 / nrr200371 Madigan, M. T., Martinko, J. M., Bender, K. S., Buckley, D. H., Stahl, D. A., & Brock, T. (2015). Microbial Metabolism. In Brock Biology of Microorganisms (14th ed.). Pearson. Xylitol enhancements synthesis of propionate in the colon via cross-feeding of gut microbiota. Microbiome, 9(1), 1-21. https: / / doi.org / 10.1186 / s40168-021-01029-6

[0477] appendix Appendix A: Bacterial strain background

[0478] [Table 3]

[0479] Appendix B: Primer

[0480] [Table 4]

[0481] Appendix C: Plasmids

[0482] [Table 5]

[0483] Appendix D: M9 Minimal Medium Recipe The M9 minimal medium used in this study consists of a medium salt solution supplemented with a carbon source (glucose or glycerol), B1 vitamins, magnesium, calcium, and an iron source.

[0484] Media Salt Solution is a composition of four important salts. It is recommended to prepare it as a 5x stock solution according to Table 8 below.

[0485] [Table 6]

[0486] All remaining stock solutions needed to make M9 media salts solution are routinely prepared by weighing out the appropriate amount of components, dissolving it in distilled HO with constant stirring, and filter sterilizing (0.2 μm pore size). Once prepared, mix the stock solutions together as described in Table 9 below.

[0487] [Table 7]

[0488] Appendix E: DNA and amino acid sequences of the LacI family DNA-binding transcriptional regulator IZ184_04885 IZ184_04885 DNA sequence (5'-→3') (SEQ ID NO: 1) IZ184_04885 amino acid sequence (SEQ ID NO:2) MAEPRTQKVTLENVAIAIANVSKITASRAFSQPDKVHPETLRRILDAADKIGYVVNAAARSLRAKSSRTIGIVSPDMSNPFFGGLAKRITLEAYNAGYDTLMFDSYESRENEARIIDKLIGYNVDAIILSVVSAERVYRPAYMKQLELLNIPVILVDRELDAKACSG VYIDNLDCGLQAGRYLLSQKADNVVIVSGPEDSNVAQDRVTGMVAGLHGQVSSVNVLHADFLMDEAFKVTDHYLKYHPAPDYFVGCNNQISLGIIKACIRHNLIPQKDVSLFSIDEVSHADIYGFNFPCISHDLQEIAWQAINMAVRRATDRSAPASKVVVRGLLKS

[0489] Appendix F: Further Sequences Promoter sequence (5'-→3') (SEQ ID NO:3) cctgaatgatatcgttatcattatagccttgtcacagatagcaaagatgtgacccggaagaccctttgccgaatactacatgagtagatcagactttatttaataaaagtttaaccatcatcacataattataataaatattgctctattatccgcatcatagtaatgatatcgatatcattttaatgaaaggaaatgatc A sequence (SEQ ID NO: 4) containing (5' to 3') the sequences of the repressor, isomerase, and promoter of Morganella morganii strain ZJG812.

[0490] This sequence can be used in the present invention to provide a repressor and a promoter.

[0491]

Table 8

[0492]

Table 9

[0493]

Table 10

[0494]

Table 11

[0495]

Table 12

[0496]

Table 13

Claims

1. At least one nucleic acid vector for use in a method of treating a human or animal subject comprising transferring the vector into host cells of a microbial population of the subject, The vector is (a) an expressible nucleotide sequence of interest (NS1) for producing a product of interest (P1) in said host cell, wherein P1 is said therapeutic amino acid, protein, or RNA, or P1 is an enzyme of a metabolic pathway in the host cell; and (b) an expressible nucleotide sequence (NS2) for producing a regulator product (P2) in the host cell, wherein P2 is operable in the host cell to regulate expression or activity of P1, and wherein P2 is expressible in the host cell to form a nuclease operable in the host cell to cleave the nucleic acid, thereby down-regulating expression of P1. a nucleic acid comprising (c) NS1 is under the control of a first promoter (optionally a constitutive or inducible promoter) for expression of P1; (d) the expression or activity of P2 in the host cell is regulatable by exposing the host cell containing the at least one vector to a regulator agent (R), thereby regulating the expression or activity of P1; and The method of treatment comprises the controlled reduction in the production and subsequent expression of P1 in the subject's microbial population. Nucleic acid vectors.

2. At least one nucleic acid vector for use according to claim 1, said vector comprising: P2 is operable in the host cell to bind to vector nucleic acid and regulate expression of P1; NS2 is under the control of a second promoter that is controllable for expression of P2, and binding or exposure of a regulator agent (R) to said vector nucleic acid controls said second promoter, thereby controlling expression of P2 and P1, and optionally said at least one vector is one vector containing both NS1 and NS2; and / or The nucleic acid vector, wherein P2 is operable in the host cell to bind to the vector nucleic acid and downregulate expression of P1.

3. Nucleic acids are degraded, and / or 3. The vector for use according to claim 1 or 2, wherein the nuclease is operable to cleave the nucleic acid at a predetermined sequence motif, optionally a protospacer sequence or a restriction site, and wherein the nucleic acid comprises a plurality of said motifs.

4. P2, (a) an RNA-guided nuclease; (b) an RNA or a precursor of such an RNA operable to guide an RNA-guided nuclease; or (c) a restriction endonuclease, such as a Cas nuclease, a TALEN, a meganuclease, or a zinc finger nuclease, preferably a Cas nuclease; 3. A vector for use according to claim 1 or 2, comprising: (i) P1 is toxic to cells of the same species as the host cell, and / or (ii) P1 is a regulator of transcription or translation in a cell of the same species as the host cell, and / or (iii) R is an amino acid, a protein, a carbohydrate (optionally a sugar), a lipid, a metal ion, or a nucleic acid, or R is a sugar alcohol (optionally xylitol), and / or (iv) A vector for use according to claim 1 or 2, which is an ICE (integrative conjugative element), a plasmid (optionally a conjugative plasmid), a transduction particle (optionally a phage or a non-self-replicating transduction particle), or a nanoparticle. (iv) An ICE (integral conjugative element), a plasmid (optionally a conjugative plasmid), a transduction particle (optionally a phage or a non-self-replicating transduction particle), or a nanoparticle, wherein the vector is contained in a carrier cell, optionally a conjugative plasmid contained in a carrier cell (optionally a bacterial carrier cell) for administration to a microbial population of a human or animal subject; and further optionally, 3. A vector for use according to claim 1 or 2, wherein the carrier cells are cells of a commensal or probiotic bacterial cell species of the human or animal microbiome and / or the carrier cells are cells of a human or animal intestinal microbiome species.

7. A vector for use according to claim 1 or 2, (iv) an ICE (integral conjugative element), a plasmid (optionally a conjugative plasmid), a transduction particle (optionally a phage or a non-self-replicating transduction particle), or a nanoparticle, said vector being contained in a carrier cell, optionally a nucleic acid vector for transfer of a microorganism into a host cell, said vector being contained in a carrier cell, optionally a conjugative plasmid, said vector being contained in a carrier cell (optionally a bacterial carrier cell) for administration to a microorganism of a human or animal subject; Further optionally, the carrier cells are commensal or probiotic bacterial cell species of the human or animal microbiome and / or the carrier cells are species of the human or animal intestinal microbiome, And the vector, (a) a nuclease (optionally an RNA-guided nuclease or a restriction endonuclease) operable in said carrier cell to cleave a chromosome or episome (which is not said vector) of said carrier cell, optionally resulting in degradation of said chromosome or episome; and / or (b) an RNA or a precursor of such an RNA operable in said carrier cell to guide an RNA-guided nuclease, said RNA guiding said nuclease to cleave a chromosome or episome (which is not said vector) of said carrier cell, optionally resulting in degradation of said chromosome or episome; Code the vector comprises one or more controllable promoters for controlling expression of the nuclease of (a) and / or the RNA or component of (b) in the carrier cell, and optionally the vector comprises an inducible or repressible promoter that controls the expression of the nuclease of (a), and / or an inducible or repressible promoter that controls the expression of the RNA or component of (b), preferably the promoter is an inducible promoter, and further optionally The guided nuclease is a Cas nuclease, a TALEN, a meganuclease, or a zinc finger nuclease, preferably a Cas nuclease, and further optionally The vector, wherein the nuclease is operable to cleave a chromosome or episome in the carrier cell at a predetermined sequence motif, optionally a protospacer sequence or a restriction site.

8. A vector for use according to claim 7, wherein cleavage of the chromosome or episome of the carrier cell kills the carrier cell or reduces the growth or proliferation of the carrier cell, preferably resulting in the death of the cell, and / or containing oriT for transfer into said host cell, optionally a conjugative plasmid; and / or (a) the vector is a conjugative plasmid; (b) the vector comprises an inducible promoter that controls expression of the nuclease of (a), and / or the vector comprises an inducible promoter that controls expression of the RNA or component; (c) optionally, the guided nuclease is a Cas nuclease; (d) chromosomal or episomal cleavage of the carrier cell kills the carrier cell or reduces the growth or proliferation of the carrier cell; and Optionally, the species is selected from any species in Table 1, preferably a Bacteroides species or a Clostridioides species.

9. the host cell is a cell of a species found in the human or animal microbiome (optionally the gut microbiome), and / or the host cells are cells of a commensal or probiotic bacterial cell species of the human or animal microbiome, and optionally 3. The vector for use according to claim 1 or 2, wherein said species is selected from any species of Table 1, preferably a Bacteroides species or a Clostridioides species.

10. (a) P1 is a protein or RNA for human or animal therapy; 3. The vector for use of claim 1 or 2, wherein (b) P2 comprises (i) a crRNA operable in the host cell to guide a Cas nuclease to bind to a protospacer sequence contained in the nucleic acid for cleavage of the protospacer, optionally resulting in degradation of the nucleic acid, thereby downregulating expression of P1, or (ii) a precursor of such a crRNA.

11. A vector for use according to any one of claims 1 to 10, comprising: The method is (a)(i) administering at least one vector comprising a nucleic acid to the subject's microbial population; (ii) transferring the nucleic acid into the host cells contained in the microbial population and expressing P1 in the host cells; and (iii) after step (ii), exposing the microbial population to a regulator agent (R) that controls the expression or activity of a regulator product P2 in the host cells, wherein P2 is operable in the host cells to control the expression or activity of P1.

12. A vector for use according to claim 1 or 2, wherein the method comprises: (a) administering at least one vector to the subject's microbiome (optionally the gut microbiome), wherein the microbiome comprises host cells, and optionally the administration is oral or topical; (b) transferring the nucleic acid into the host cells contained in the microorganisms and expressing P1 in the host cells; (c) after step (b), exposing the microbial population to a regulator agent (R) that upregulates the production in the host cells of an RNA-guided nuclease / guide RNA complex that can target a protospacer comprised in the nucleic acid, wherein the nuclease cleaves the nucleic acid and expression of P1 becomes non-functional (optionally by degradation of the cleaved nucleic acid in the cell), and the nuclease (or a component thereof) and / or RNA (or a component thereof) encoded by the nucleic acid. The vector comprising:

13. A bacterial, archaeal or fungal host cell for use in a method of treatment of a human or animal subject, comprising: (a) the cells an expressible nucleotide sequence (NS1) of interest for producing a product of interest (P1) in said host cell, wherein P1 is an amino acid, protein, or RNA for said therapeutic, or P1 is an enzyme of a metabolic pathway in the host cell; and an expressible nucleotide sequence (NS2) for producing a regulator product (P2) in said host cell, wherein P2 is operable in said host cell to regulate expression or activity of P1, and wherein P2 is expressible in said host cell to form a nuclease operable in said host cell to cleave said nucleic acid, thereby down-regulating expression of P1; a nucleic acid comprising (b) NS1 is under the control of a first promoter (optionally a constitutive or inducible promoter) for expression of P1; (c) the expression or activity of P2 in the host cell is regulatable by exposure of the host cell to a regulator agent (R), thereby regulating the expression or activity of P1; and The method comprises administering the cells to a microbial population of a subject to produce P1 and subsequently to a controlled reduction in expression of P1 in the subject.

14. A cell for use according to claim 13. a. said nucleic acid is contained in at least one nucleic acid vector in said cell (optionally the or each vector is a conjugative plasmid for transfer into cells of a microbial population contained in a human or animal subject); b. NS1 is comprised in a nucleic acid vector of said cell and NS2 is comprised in a chromosome of said cell (optionally the or each vector is a conjugative plasmid for transfer into cells of a microorganism contained in a human or animal subject); c. NS1 is comprised in the chromosome of said cell and NS2 is comprised in a nucleic acid vector of said cell (optionally the or each vector is a conjugative plasmid for transfer into cells of a microorganism contained in a human or animal subject); or d. The cell, wherein NS1 is contained in a chromosome of the cell and NS2 is contained in a chromosome of the cell.

15. A cell for use according to claim 13 or 14, comprising: (i) cells of commensal or probiotic bacterial species of the human or animal microbiome, optionally E. coli cells or Bacteroides cells, and / or (ii) NS2 is under the control of a second promoter that is controllable for expression of P2, and binding of a regulator agent (R) to the vector nucleic acid controls the second promoter, thereby controlling expression of P2 and P1; and / or (iii) P2 comprises an RNA-guided nuclease (optionally a Cas nuclease), wherein said nuclease is operable to cleave said nucleic acid at a predetermined sequence motif; and optionally (a) the sequence motif is contained in a chromosome of the cell and the cleavage causes death of the cell, or the sequence motif is contained (on the chromosome of the cell or on a vector) in a gene that contains NS1 for the production of P1 and the cleavage downregulates the production of P1; or (b) the sequence motif is contained in the vector containing NS1; The cells.

16. A vector for use as claimed in claim 1 or 2, or a cell for use as claimed in claim 13 or 14, wherein the vector or cell is provided in combination with a pharmaceutically acceptable carrier, diluent or excipient, optionally an antacid, and / or the vector or cell is comprised in a tablet, suppository, pill, capsule, or liquid formulation for administration to the gastrointestinal tract of a human or animal subject.

17. A cell comprising a nucleic acid for use according to claim 13 or 14, said nucleic acid comprising a gene encoding a product of interest (P1), said gene comprising a nucleotide sequence (NS1) encoding P1 and a regulatory region 5' of NS1 comprising a promoter (Px) for regulating expression of NS1, the combination of Px and NS1 being heterologous to said cell, and Px being controllable by xylitol or xylose, and optionally the promoter is a xylitol- or xylose-regulatable promoter of Morganella species, optionally M morganii; (i) the promoter comprises SEQ ID NO:3 or a nucleotide sequence that is at least 70% identical to SEQ ID NO:3; and / or (ii) Px is homologous to a xylitol- or xylose-regulatable promoter of Morganella morganii; (iii) the genome of the cell encodes a repressor capable of repressing Px, and xylitol and / or xylose are capable of derepressing the repressor; and optionally the repressor is encoded by SEQ ID NO: 1 or a nucleotide sequence which is at least 70% identical to SEQ ID NO: 1, and / or (iv) a xylitol transporter, optionally including a xylitol ABC transporter; and / or (v) The cell lacking a xylitol isomerase gene.

18. A vector for use according to claim 1 or 2, or a cell for use according to claim 13 or 14, comprising: (i) P1 is an enzyme in a metabolic pathway that includes one or more short-chain fatty acids (SCFAs) as products or intermediates; or (ii) P1 is an enzyme in a metabolic pathway that includes an indole derivative as a product or intermediate (where, for example, the pathway is for the conversion of tryptophan (Trp) to an indole derivative); or (iii) P1 is an enzyme in a metabolic pathway that includes one or more hormones or incretins as products or intermediates; or (iv) P1 is an amino acid or protein for the treatment; The vector or the cell.

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