Transcriptional control in prokaryotic cells using dna-binding repressors

Abstract

To provide, generally, methods and compositions for transferring a genetic circuit from one prokaryotic cell ("donor cell") to another prokaryotic cell ("recipient cell" or "target cell" used interchangeably herein).SOLUTION: The present disclosure relates to: prokaryotic donor cells comprising (i) a genetic circuit of interest and (ii) one or more expressed transcriptional repressor proteins; and the use of the donor cells in efficient transfer of the genetic circuit into a prokaryotic recipient cell. The genetic circuit includes nucleic acid sequences encoding an RNA molecule or protein of interest.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure generally relates to methods and compositions for transferring genetic circuits from one prokaryotic cell (a "donor cell") to another prokaryotic cell (a "recipient cell" or "target cell", used interchangeably herein). [Background technology]

[0002] Inducible systems that control transcription from a given promoter are useful tools in molecular biology [1]-[3]. Generally, these systems consist of a protein repressor whose binding to its cognate DNA operator depends on the presence of an inducer. In the absence of an inducer, the repressor is able to recognize its operator and prevents transcription from the promoter; when an inducer is present, the repressor can no longer recognize its operator, either by the inducer binding to a domain of the repressor or by changing its 3D conformation, thus activating transcription. Inducers can be chemical (IPTG, sugars, small molecules, proteins, etc.) or physical (heat, light, pressure, pH, etc.). There are many other ways to prevent transcription or reduce the amount of protein made in a cell: conditional degradation of proteins, reducing the copy number of a plasmid in the production strain, etc. These do not repress the promoter itself, but act on other components of the genetic circuit

[22] .

[0003] In some cases, it may be desirable to be able to control the transcription / translation of a particular gene in the host while activating it in the target bacterium in the absence of an inducer. For example, in the case of phagemid transduction of proteins and / or nucleic acids toxic to the target bacterium, e.g. in the case of CRISPR-Cas9-containing circuits

[14] , it is essential that the strain used to produce the particles does not express the toxin protein and / or nucleic acid encoded by the circuit, otherwise the producing strain will die or be very damaged. However, it is necessary that the toxin protein and / or nucleic acid is expressed / transcribed in the target strain to induce either cell death, such as sequence-specific cell death, or any other type of desired function in the target strain. This also applies to any other toxin components injected into the target strain, which may also be toxic to the producing strain. Moreover, it has been shown that constitutive expression of components in a genetic circuit is disadvantageous for the cell, especially if they impose a burden (such as a toxin component, or any component with an undesired function in the producing strain), which makes them prone to deletion or mutation, causing undesired damage of the design and components

[15] . In that sense, having a conditional repressor that acts only in the producing strain is advantageous from an engineering / manufacturing point of view. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2014124226 [Patent Document 2] U.S. Patent Application No. 62 / 771,761 [Patent Document 3] U.S. Patent Application No. 62 / 802,777 [Patent Document 4] U.S. Patent Application No. 62 / 783,258 [Non-patent literature]

[0005] [Non-Patent Document 1] Chemical Reviews 2016, 116 (20) pp. 12655~12687 [Non-Patent Document 2] Kues, U and Stahl, U 1989, Microbiol Rev 53:491-516. [Non-Patent Document 3] Del Solar et al., 1998, Microhio and Molec Biol. Rev 62:434-464. [Non-Patent Document 4] Cotter et al., Nature Reviews Microbiology 11:95, 2013 [Non-Patent Document 5] Jinek et al., Science 2012 [Non-Patent Document 6] Fonfara et al., Nucleic Acids Res 42 (4), 2014 [Non-Patent Document 7] Koonin et al., Nat Rev Microbiol 15(3), 2017 [Non-Patent Document 8] Henkel et al., (Toxins from Bacteria in EXS. 2010; 100: 1-29) [Non-Patent Document 9] Krupovic et al., Arch Virol, 2015 Summary of the Invention [Problem to be solved by the invention]

[0006] A proposed solution has been presented for the specific case of transducing genetic circuits, involving a positive regulator (phage polymerase and its cognate promoter). The target cell may contain either the promoter or the polymerase alone, together with the other components to be transduced by the phagemid

[12]

[13] . However, this is not a practical approach when working with wild-type strains, since it is difficult to transform one of the components, or in environments where it is not possible to preload one of the components of the circuit into the target cell (e.g. the intestinal environment).

[0007] Another possible solution for this would be to express a repressor that controls the expression of the toxin component in trans, i.e. a repressor that is not encoded in the circuitry to be packaged. In this case, the repressor is present only in the producer strain but not in the target strain. However, there is a major problem with this approach: the strain used for the production of the phagemid is of the same species (or at least very closely related) as the strain that needs to be targeted, so the repressor used needs to be carefully selected. For example, by using AraC or LacI, the transcription of the toxin component in the producer strain is effectively repressed in trans, and the phagemids produced in this way inject a "naked" constitutive promoter into the target strain. However, if these are wild-type strains, they will most likely contain their own genes for LacI or AraC, so the injected promoter will be immediately repressed. Even antibiotic inducible systems such as TetR are very commonly found in many wild-type strains. The same thing can happen when using phage master-repressor-promoter pairs: wild-type strains have many prophages in their genome and many of these may encode repressors that recognize the promoter used. [Means for solving the problem]

[0008] Described herein are novel methods and compositions for use in the transfer of a genetic circuit of interest from a prokaryotic cell (herein referred to as a "donor cell") to another prokaryotic cell (herein referred to as a "recipient cell" or "target cell"). In certain embodiments, the prokaryotic cell is a bacterial cell. Additionally, the genetic circuit comprises a nucleic acid of interest under the transcriptional control of a repressor binding sequence (herein also referred to as a promoter / operator). The nucleic acid sequence of interest may encode a protein or RNA of interest.

[0009] The disclosed method and composition are based on the expression of one or more repressor proteins in donor cells and the presence of repressor binding sequences located in a genetic circuit that functions to suppress the transcription of a nucleic acid sequence of interest when present in the donor cell. The genetic circuit does not code for said one or more repressor proteins. Thus, upon transfer of the genetic circuit to a recipient cell, said recipient cell is selected for the lack of expression of said one or more repressor proteins, resulting in the transcription of the nucleic acid sequence of interest.

[0010] Methods are provided for transferring genetic circuits from donor cells to recipient or target cells. The transfer can be accomplished by a variety of different methods. Non-limiting examples of such transfer include bacterial transduction, conjugation, and transformation. In a particular embodiment, a bacterial delivery vehicle, such as a bacteriophage scaffold, is assembled in the donor cell as a means to transfer the genetic circuit to the recipient or target cell with high efficiency. In such cases, the donor cell contains a prophage sequence that provides in trans the bacteriophage components, such as capsid proteins, required for assembly of the genetic circuit into a bacteriophage particle. Additionally, the genetic circuit is engineered to contain a cis-acting packaging signal that mediates encapsidation of the genetic circuit into a capsid.

[0011] Thus, the present invention relates to a method of transferring a genetic circuit from a donor cell to a target cell, comprising contacting the donor cell with the target cell for a time sufficient to allow transfer of the genetic circuit to the target cell, wherein the donor cell expresses a repressor protein that is not encoded by the genetic circuit and is not present in the target cell, and the genetic circuit comprises a nucleic acid sequence of interest under the transcriptional control of a repressor binding sequence. The donor cell may be a bacterial donor cell and the target cell may be a bacterial target cell. The genetic circuit may be packaged in a bacterial delivery vehicle prior to transfer. Preferably, the genetic circuit packaged in the delivery vehicle is a packaged phagemid. The nucleic acid sequence of interest may encode a protein of interest and / or an RNA molecule of interest. In particular, the nucleic acid sequence of interest may code for a protein that is (i) a toxin protein, preferably a protein toxic to bacterial cells, in particular target cells, more particularly a toxin protein selected from the group consisting of holins, endolysins, restriction enzymes, and toxins that affect the survival or proliferation of target cells, (ii) a nuclease, preferably a CRISPR nuclease, and / or a therapeutic protein. Alternatively, or in addition, the nucleic acid sequence of interest may code for an RNA molecule of interest, in particular an RNA molecule of interest selected from the group consisting of mRNA, crRNA, tRNA, iRNA, asRNA, ribozyme RNA, guide RNA, and RNA aptamer. In a particular embodiment, the nucleic acid sequence of interest codes for a CRISPR nuclease and the genetic circuit further comprises a nucleic acid sequence encoding a guide RNA, preferably said nucleic acid sequence encoding a guide RNA under the transcriptional control of a constitutive promoter.In some embodiments, the nucleic acid sequence of interest is a nucleic acid encoding an RNA such as an mRNA, a crRNA, a tRNA, an iRNA (interfering RNA), an asRNA (antisense RNA), a ribozyme RNA, an RNA aptamer or a guide RNA, a CRISPR locus, a toxin gene, a gene encoding an enzyme such as a nuclease or a kinase, a gene encoding a nuclease selected from the group consisting of Cas nuclease, Cas9 nuclease, TALEN, ZFN and meganuclease, a gene encoding a recombinase, a bacterial receptor, a membrane protein, a structural protein or a secreted protein, a gene encoding resistance to an antibiotic or drug in general, a gene encoding a toxin protein or a toxin factor, and a gene encoding a virulence protein or a virulence factor, or any combination thereof. More particularly, the nucleic acid sequence of interest may be selected from the group consisting of nucleic acids encoding one or more of Cas nucleases, Cas9 nucleases, guide RNAs, CRISPR loci, toxins, enzymes, nucleases, kinases, TALENs, ZFNs, meganucleases, recombinases, bacterial receptors, membrane proteins, structural proteins, secreted proteins, proteins conferring resistance to antibiotics or drugs, toxin proteins or toxin factors, virulence proteins, and virulence factors.Preferably, the repressor protein is selected from the group consisting of the repressor proteins listed in Table 1, preferably selected from the group consisting of PhlF, SrpR, LitR, PsrA, AmeR, McbR, QacR, TarA, ButR, Orf2, and ScbR.

[0012] Methods are also provided for the production of packaged phagemids, which are bacterial delivery vehicles for use in, for example, highly efficient transfer of a desired genetic circuit into a recipient or target cell. The present disclosure relates to the use of donor cells described herein that express one or more repressor proteins for controlled expression of a nucleic acid of interest, such as a nucleic acid encoding a toxin or an undesired protein and / or RNA molecule, during the production of a bacterial delivery vehicle of interest. The repressed transcription of a nucleic acid of interest in a donor cell can result from functional positioning of a repressor binding sequence in close proximity to the nucleic acid of interest. Alternatively, the repressed transcription of a nucleic acid of interest in a donor cell can result from functional positioning of a repressor binding sequence away from the nucleic acid of interest. In contrast, when transferred into a recipient or target cell, the nucleic acid of interest is transcribed due to the absence of the one or more repressor proteins in the cell and the absence of a nucleic acid sequence encoding the repressor protein in the genetic circuit.

[0013] In one aspect, a method of producing a delivery vehicle using a donor cell of the invention is provided. In certain embodiments, the delivery vehicle is prepared by introducing a genetic circuit of interest described herein into a donor cell under conditions that allow for the formation of the delivery vehicle. For example, in certain embodiments, the method includes the steps of (i) introducing a genetic circuit of interest into a donor cell; and (ii) allowing sufficient time for replication of the genetic circuit of interest and packaging of the genetic circuit into a delivery vehicle. In one aspect, the method may further include harvesting the delivery vehicle, and optionally purifying the delivery vehicle. In particular, the invention relates to a method of producing a delivery vehicle, comprising the steps of: i) introducing a genetic circuit into a donor cell expressing a repressor protein, the genetic circuit comprising a nucleic acid of interest under the transcriptional control of a repressor binding sequence recognized by the repressor protein, the repressor protein not being encoded by the genetic circuit; and allowing sufficient time for replication of the genetic circuit of interest and packaging of the genetic circuit into a delivery vehicle. The method may further comprise the steps of harvesting the delivery vehicle and optionally purifying the delivery vehicle. Preferably, in said method, the donor cell, preferably a bacterial donor cell, comprises a prophage sequence encoding a protein required in trans for the assembly of the genetic circuit into the delivery vehicle. Preferably, the genetic circuit is a phagemid and / or the delivery vehicle is a bacterial delivery vehicle, more preferably a bacteriophage. In particular, the delivery vehicle may be used in target cells, preferably bacterial target cells, that do not express a repressor protein. The present invention also relates to a delivery vehicle obtained by said method. The present invention also relates to a genetic circuit comprising a transcription promoter controlled by a repressor protein and a nucleic acid sequence of interest placed under the control of said transcription promoter, wherein said genetic circuit does not code for a repressor protein and is packaged in a bacterial delivery vehicle. Preferably, said genetic circuit is a phagemid.The present invention also relates to the use of a delivery vehicle or a genetic circuit of the present invention for transferring a genetic circuit from a donor cell to a target cell, wherein the donor cell expresses a repressor protein and the target cell does not express said repressor protein.

[0014] In yet another aspect, the invention provides a donor cell comprising one or more expressed repressor proteins and a genetic circuit of interest. Preferably, the genetic circuit of interest comprises a nucleic acid sequence of interest placed under the transcriptional control of a repressor binding sequence recognized by said one or more expressed repressor proteins. Furthermore, in certain embodiments for producing a delivery vehicle, the donor cell may also comprise a prophage sequence encoding a protein required in trans for assembly of the genetic circuit of interest into a delivery vehicle. Such proteins include, for example, structural bacteriophage proteins, e.g., capsid proteins. In some specific aspects, the invention relates to a donor cell comprising a genetic circuit comprising a transcriptional promoter controlled by a repressor protein expressed by the donor cell but not encoded by the genetic circuit. Preferably, the genetic circuit comprises a nucleic acid sequence of interest placed under the control of a transcriptional promoter. The donor cell may further comprise a prophage sequence providing in trans bacteriophage components required for assembly of the genetic circuit into a bacteriophage particle. The genetic circuit can further comprise a cis-acting packaging signal that mediates encapsidation of the genetic circuit into a capsid.

[0015] The donor cell of the present disclosure comprises a genetic circuit of interest. In certain embodiments of the present invention, the genetic circuit comprises an expression cassette or transcription cassette with a nucleic acid of interest under the transcriptional control of a repressor binding sequence. Such a nucleic acid of interest may be selected from the group consisting of, for example, a nucleic acid encoding an RNA such as mRNA, crRNA, tRNA, iRNA (interfering RNA), asRNA (antisense RNA), ribozyme RNA, RNA aptamer or guide RNA, a CRISPR locus, a toxin gene, a gene encoding an enzyme such as a nuclease or kinase, a gene encoding a nuclease selected from the group consisting of Cas nuclease, Cas9 nuclease, TALEN, ZFN and meganuclease, a gene encoding a recombinase, a bacterial receptor, a membrane protein, a structural protein or a secreted protein, a gene encoding resistance to antibiotics or drugs in general, a gene encoding a toxin protein or a toxin factor, and a gene encoding a virulence protein or a virulence factor, or any combination thereof. In one embodiment of the present invention, the nucleic acid of interest encodes a therapeutic protein. In another embodiment, the nucleic acid encodes an antisense nucleic acid molecule. In some embodiments, the nucleic acid of interest encodes two or more molecules of interest. In particular, one of these molecules can be a nuclease, such as a Cas nuclease, and another can be a nucleic acid molecule, such as a guide RNA. In one aspect, the nucleic acid of interest encodes a nuclease that performs a cleavage in the genome of the recipient or target cell, or in a plasmid of the recipient or target cell. In some aspects, the cleavage occurs in an antibiotic resistance gene. In another embodiment of the invention, the nucleic acid of interest encodes a nuclease that targets a cleavage in the genome of the recipient or target cell, and the nuclease is designed to stimulate a homologous recombination event for the insertion of the nucleic acid of interest into the genome of the cell.

[0016] Preferably, in the method of the invention or the donor cell of the invention, the genetic circuit is a phagemid and / or the donor cell is a bacterial cell, more preferably a bacterial cell derived from an E. coli species.

[0017] The present disclosure also provides a pharmaceutical or veterinary composition comprising a donor cell, or one or more bacterial delivery vehicles assembled in said donor cell, and a pharma- ceutically acceptable carrier. The present disclosure also provides a pharmaceutical or veterinary composition comprising a delivery vehicle and / or genetic circuit of the present invention, and a pharma- ceutically acceptable excipient. The present disclosure also provides a method for treating a disease or disorder caused by bacteria, preferably a bacterial infection, comprising administering the provided pharmaceutical or veterinary composition to a subject having said disease or disorder in need of treatment. Further provided are (i) a pharmaceutical or veterinary composition disclosed herein for use as a medicament, in particular for use in treating a disease or disorder caused by bacteria, preferably a bacterial infection, and (ii) a use of a pharmaceutical or veterinary composition disclosed herein for the manufacture of a medicament for treating a disease or disorder caused by bacteria, preferably a bacterial infection. A method is provided for reducing the amount of pathogenic and / or antibiotic-resistant bacteria in a bacterial population, in particular a subject having a bacterial infection, comprising contacting the bacterial population with a composition disclosed herein. Further provided is the use of a pharmaceutical or veterinary composition disclosed herein for the manufacture of a medicament for reducing a bacterial population, in particular the amount of pathogenic and / or antibiotic-resistant bacteria, in a subject having a bacterial infection.

[0018] The present disclosure further provides a kit for use in the transfer of a genetic circuit of interest from a donor cell to a recipient or target cell. In one embodiment, the kit comprises (i) a donor cell expressing a repressor protein; and (ii) a genetic circuit of interest. The genetic circuit may comprise an expression cassette into which a nucleic acid of interest may be inserted in functional proximity to a repressor binding sequence recognized by the repressor protein. Optionally, the donor cell of the kit may contain a delivery vehicle, e.g., a prophage sequence for assembly of a bacteriophage protein for packaging the genetic circuit of interest. The kit may further comprise a recipient or target cell, which does not express a repressor protein, thereby allowing expression of the nucleic acid of interest after transfer into the cell.

[0019] For a better understanding of the subject matter disclosed herein and to illustrate how it may be carried out in practice, embodiments will now be described, by way of non-limiting example, with reference to the accompanying drawings in which it is emphasized that particular reference is made to the drawings in which the features shown are by way of example and for purposes of illustration of embodiments of the invention. [Brief description of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram depicting conditional transcriptional control by an interspecies repressor. On the left, a donor strain containing a packaging prophage and a PhlF repressor in trans. The genetic circuit has a packaging signal and a nucleic acid of interest (encoding an actuator protein) under the control of the PphlF promoter. Upon packaging of the genetic circuit, a target or recipient cell can be transduced and the PphlF promoter is active since the recipient cell lacks the PhlF repressor (not present in E. coli). Note that the PhlF repressor can be replaced with a dCas9+gRNA targeting the promoter, RBS or actuator sequence. [Diagram 2]Figure 2 is a diagram depicting transformation of Cas9-containing genetic circuits. Plasmids containing Cas9 under the control of the PphlF promoter and a constitutive sgRNA guide targeting lacZ were transformed into MG1655 (left panel) or MG1656 (right panel). Empty cells (without any other plasmid) are shown at the top, and transformed cells containing an external plasmid encoding the PhlF repressor are shown at the bottom. [Diagram 3] Figure 3 depicts transduction of Cas9-containing genetic circuits. Phagemids containing Cas9 under the control of the PphlF promoter and a constitutive sgRNA guide targeting lacZ were transduced into MG1655 (left panel), MG1655 with the PhlF repressor encoded in the plasmid (middle), or MG1656 (right panel). [Figure 4] Figure 4 is a diagram depicting transformation of Cpf1-containing genetic circuits. A plasmid containing Cpf1 under the control of the PsrpR promoter and a constitutive crRNA guide targeting lacZ (p455) was transformed into MG1655 (left panel) or MG1656 (right panel). Empty cells (without any other plasmid) are shown in the top row, and transformed cells containing an external plasmid encoding the SrpR repressor (pRARE4-SrpR-1.0) are shown below. [Diagram 5] FIG. 5: Packaged phagemids containing Cpf1 under the control of the PsrpR promoter and constitutive crRNA guide targeting lacZ (p455) transduced into MG1655 (left panel), MG1655 with SrpR repressor encoded in the plasmid (middle, pRARE4-SrpR-1.0) or MG1656 (right panel). [Figure 6] Figure 6 depicts a comparison of colony size of strains with and without the SrpR repressor. Both strains were transformed with the PsrpR-Cpf1-LacZ genetic circuit (p841). Top panel, cells containing SrpR in the genome. Bottom panel, cells without the SrpR repressor. Incubation times are indicated at the top. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Disclosed herein is a novel approach for the delivery of genetic circuits to recipient or target cells. In one aspect, a novel method is provided for the production of bacterial delivery vehicles for use in the highly efficient transfer of desired genetic circuits into target cells. The disclosed methods and compositions are based on the use of donor cells that express one or more repressor proteins for the controlled expression of proteins, such as toxin proteins, and / or nucleic acids, such as RNA molecules. This is particularly important, for example, when the genetic circuit is designed to express toxin proteins and / or RNA molecules.

[0022] Provide a donor cell expressing one or more repressor proteins. In a preferred embodiment, the term "donor cell" refers to a donor bacterial cell. As used herein, the term "repressor protein" preferably refers to a protein that binds to a specific site (herein, "repressor binding sequence") on a nucleic acid and prevents transcription of nearby genes. Typically, a repressor protein is a DNA-binding protein that blocks the binding of RNA polymerase to a promoter by its binding to a repressor binding sequence (operator), thus preventing transcription of the gene. The donor cell may be a cell that naturally expresses one or more repressor proteins. Alternatively, the donor cell may be recombinantly engineered to express one or more repressor proteins. Additionally, the donor cell provided comprises a genetic circuit of interest, the genetic circuit containing a nucleic acid of interest under the transcriptional regulation of a repressor binding sequence, and does not contain a nucleic acid encoding a repressor protein that can bind to the repressor binding sequence. At least one repressor protein expressed by the donor cell is capable of binding to said repressor binding sequence, thereby preventing transcription of the nucleic acid of interest, which may encode a protein and / or RNA of interest.

[0023] Repressor proteins that can be used in the donor cell include, for example, those listed in Table 1. In one embodiment, the donor cell expresses a repressor protein selected from the repressor proteins listed in Table 1, and the nucleic acid of interest contained within the genetic circuit is under the transcriptional control of a repressor binding sequence bound by said repressor protein.

[0024] [Table 1A]

[0025] [Table 1B]

[0026] Repressor binding sequences corresponding to such repressor proteins are well known in the art, and the skilled person can easily select a functional pair of repressor protein / repressor binding sequence. In some embodiments, the donor cell expresses one or several repressor proteins corresponding to one or several repressor binding sequences contained in the genetic circuit. The repressor protein and / or the repressor binding sequence contained in the genetic circuit expressed by the donor cell can be heterologous to the donor cell, i.e., not naturally occurring in said donor cell. In particular, the repressor protein and / or the repressor binding sequence contained in the genetic circuit expressed by the donor cell can be from a different bacterial species, preferably a different bacterial genus, than the donor cell. Preferably, the repressor protein and / or the repressor binding sequence contained in the genetic circuit expressed by the donor cell is endogenous to the donor cell, i.e., naturally occurring in said donor cell.

[0027] In another embodiment, the repressor protein is a CRISPR nuclease that lacks nuclease activity. In this embodiment, the repressor protein is used in combination with a guide RNA that targets a sequence required for transcription of the nucleic acid of interest. In this embodiment, the donor cell thus expresses said guide RNA in addition to the CRISPR nuclease that acts as a repressor protein. In particular, the guide RNA may target a control sequence, such as a promoter, an RBS or a repressor binding sequence, that is operably linked to the nucleic acid of interest. Alternatively, the guide RNA may target a non-control sequence, such as the coding region of the nucleic acid of interest. In this case, said non-control sequence should be considered as a repressor binding sequence. Thanks to this guide RNA, the CRISPR nuclease that lacks nuclease activity can bind to the target sequence without inducing any destruction, thereby preventing the transcription of the nucleic acid of interest without altering the integrity of the genetic circuit. CRISPR nucleases that lack nuclease activity, such as death Cas9 (dCas9), are well known to those skilled in the art.

[0028] In addition to expressing the repressor protein, the donor cell may further comprise a packaging prophage that provides the necessary components in trans for the assembly of a bacterial delivery vehicle, e.g., a bacteriophage scaffold. Once produced, the delivery vehicle may be advantageously used to transfer a genetic circuit of interest into a recipient or target cell. The absence of the repressor protein in the recipient or target cell results in the expression of the transferred genetic circuit of interest.

[0029] As used herein, the term "genetic circuit" preferably refers to a nucleic acid construct, preferably a linear or circular double-stranded DNA molecule, comprising one or more nucleic acids of interest operably linked to a control sequence comprising a transcription promoter ("promoter") and a repressor binding sequence (recognized by at least one repressor protein expressed by the donor cell). The genetic circuit may further comprise one or more additional nucleic acid sequences operably linked to one or several control sequences comprising a transcription promoter. In particular, the genetic circuit may comprise one or more nucleic acids of interest operably linked to a control sequence comprising a promoter and a repressor binding sequence (recognized by at least one repressor protein expressed by the donor cell), as well as one or more nucleic acid sequences operably linked to a constitutive promoter. For example, in certain embodiments, the genetic circuit comprises a nucleic acid of interest encoding a CRISPR nuclease operably linked to a control sequence comprising a promoter and a repressor binding sequence (recognized by at least one repressor protein expressed by the donor cell), and a nucleic acid sequence encoding a guide RNA operably linked to a constitutive promoter. The term "operably linked" refers to an arrangement in which the control sequence is placed in an appropriate position relative to the nucleic acid of interest in such a way that the control sequence directs the expression of said nucleic acid. Optionally, the genetic circuit may comprise other control sequences such as leader sequences, polyadenylation sequences, propeptide sequences, ribozymes, hairpin-forming sequences, ribosome binding sites, signal peptide sequences and / or transcription terminators. In some preferred embodiments, the genetic circuit is a plasmid or a phagemid, i.e., comprises a genetic sequence that signals for packaging. More preferably, the genetic circuit is a phagemid.

[0030] In one aspect, a method for producing a delivery vehicle using the donor cell line of the present invention is provided. As used herein, the term "delivery vehicle" preferably refers to any means that allows the transfer of a genetic circuit into a cell. Preferably, the delivery vehicle allows the transfer of a genetic circuit into a bacterial cell ("bacterial delivery vehicle"). In certain embodiments, the delivery vehicle is prepared by introducing a genetic circuit described herein into a suitable donor cell under conditions that allow the formation of the delivery vehicle. For example, in certain embodiments, the method includes the steps of (i) introducing a genetic circuit of interest into a donor cell; and (ii) allowing sufficient time for the replication of the genetic circuit and packaging of the genetic circuit into the delivery vehicle. More specifically, the method of producing a delivery vehicle may comprise the steps of: (i) introducing a genetic circuit of interest as defined above and a prophage sequence encoding proteins required in trans for assembly of the genetic circuit into a delivery vehicle (e.g., a bacteriophage scaffold protein) into a donor cell, preferably a bacterial donor cell, as defined above; and (ii) allowing sufficient time for replication of the genetic circuit and packaging of the genetic circuit into the bacterial delivery vehicle. In one aspect, the method may further comprise the steps of harvesting the delivery vehicle, and optionally purifying the delivery vehicle.

[0031] In yet another aspect, the invention provides a donor cell comprising a repressor protein expression cassette comprising a sequence encoding a repressor protein. The donor cell may comprise a repressor protein expression cassette comprising a sequence encoding a repressor protein, or several sequences encoding several repressor proteins. Furthermore, in any of these embodiments, the donor cell may comprise a prophage sequence encoding a protein required in trans for packaging of a nucleic acid payload of interest contained in the genetic circuit (or the entire genetic circuit) into a delivery vehicle. Such proteins include, for example, structural bacteriophage proteins, such as capsid proteins.

[0032] A method is provided that allows the transfer of a genetic circuit comprising a nucleic acid of interest encoding a protein or RNA molecule of interest from a donor cell to a desired recipient or target cell. The genetic circuit and donor cell may be as defined above. The target cell is preferably a bacterial cell and does not express a repressor protein found to negatively regulate the transcription of the nucleic acid of interest contained in the genetic circuit in the donor cell. The method may include contacting the donor cell with the recipient or target cell for a sufficient time for the transfer of the genetic circuit. Alternatively, if a bacterial delivery vehicle is produced (e.g., using a donor cell comprising a bacteriophage scaffold), the method may include contacting the recipient or target cell with the bacterial delivery vehicle produced in the donor cell. The transferred genetic circuit comprises a nucleic acid of interest under the control of a repressor binding sequence that is negatively regulated by the donor cell expressing the repressor protein. In such a case, the transcription of the nucleic acid of interest encoding the protein or RNA molecule of interest is repressed by the expression of the repressor protein in the donor cell. The methods provided herein allow a genetic circuit comprising nucleic acids encoding one or more proteins or RNA molecules of interest to be transferred into a desired target host cell that does not express, or preferably naturally lacks, a repressor protein, thereby allowing transcription of a payload of interest.

[0033] In certain embodiments of the invention, the nucleic acid of interest is selected from the group consisting of a nucleic acid encoding an RNA such as mRNA, crRNA, tRNA, iRNA (interfering RNA), asRNA (antisense RNA), ribozyme RNA, RNA aptamer or guide RNA, a CRISPR locus, a toxin gene, a gene encoding an enzyme such as a nuclease or kinase, a gene encoding a nuclease selected from the group consisting of Cas nuclease, Cas9 nuclease, TALEN, ZFN and meganuclease, a gene encoding a recombinase, a bacterial receptor, a membrane protein, a structural protein or a secreted protein, a gene encoding resistance to antibiotics or drugs in general, a gene encoding a toxin protein or a toxin factor, and a gene encoding a virulence protein or a virulence factor, or any combination thereof. The nucleic acid of interest may also encode a bacterial transporter or pore, or a secretion system. The protein encoded by the nucleic acid of interest may be modified or engineered to include an external feature such as adding or removing a function (e.g., dCas9), adding a secretion signal to a protein that is not normally secreted, or adding an exogenous peptide in a loop.

[0034] In one embodiment of the present invention, the nucleic acid of interest encodes a therapeutic protein. In another embodiment, the nucleic acid of interest encodes an antisense nucleic acid molecule. In some embodiments, the nucleic acid of interest encodes two or more molecules of interest. In particular, one of these molecules can be a nuclease, such as a Cas nuclease, and another molecule can be a nucleic acid molecule, such as a guide RNA. In one aspect, the methods and compositions provided herein allow for the transfer of a genetic circuit comprising a nucleic acid of interest encoding a nuclease that targets cleavage of a host bacterial cell genome or a host bacterial cell plasmid. In some embodiments, the nuclease that mediates cleavage occurs in an antibiotic resistance gene. In some other embodiments, the nuclease that mediates cleavage of the host bacterial cell genome is designed to stimulate a homologous recombination event for insertion of the nucleic acid of interest into the genome of the bacterial cell.

[0035] Methods and compositions are provided that allow for the transfer of a genetic circuit comprising a nucleic acid of interest that encodes a protein or RNA molecule of interest into a desired target or recipient cell. As used herein, the term "transfer" refers to any means that allows for the transfer of a genetic circuit into a recipient or target cell. Such means include, for example, transduction, conjugation, and transformation. In some embodiments, a delivery vehicle can be used to transfer the genetic circuit from a donor cell to a target cell. Delivery vehicles encompassed by the present invention include, but are not limited to, bacteriophage scaffolds, viral scaffolds, chemical-based delivery vehicles (e.g., cyclodextrin, calcium phosphate, cationic polymers, cationic liposomes), protein- or peptide-based delivery vehicles, lipid-based delivery vehicles, nanoparticle-based delivery vehicles, non-chemical-based delivery vehicles (e.g., transformation, electroporation, sonoporation, optical transfection), particle-based delivery vehicles (e.g., gene guns, magnetofection, impalefection, biolistics, cell-penetrating peptides) or donor bacteria (conjugation). In a preferred embodiment, the delivery vehicle is a bacteriophage scaffold, i.e., derived from a natural, evolved or engineered capsid.

[0036] Any combination of delivery vehicles is also encompassed by the present invention. A delivery vehicle can also refer to a bacteriophage-derived scaffold and can be derived from a natural, evolved or engineered capsid. In some embodiments, the delivery vehicle is a payload, since bacteria are naturally capable of absorbing payloads (e.g., genetic circuits) from the environment by themselves.

[0037] As used herein, the term "payload" refers to any one or more nucleic acid sequences, such as the genetic circuits disclosed herein, and / or amino acid sequences, or a combination of both (such as, but not limited to, peptide nucleic acid or peptide oligonucleotide conjugates), that are transferred to a recipient or target cell by a delivery vehicle. The term "payload" may also refer to a plasmid, vector, or cargo. A payload may be a phagemid or phasmid derived from a natural, evolved, or engineered bacteriophage genome. A payload may consist of only a portion of a phagemid or phasmid derived from a natural, evolved, or engineered bacteriophage genome.

[0038] As used herein, the term "nucleic acid" refers to a sequence of at least two nucleotides covalently linked together that may be single-stranded or double-stranded, or contain portions of both single-stranded and double-stranded sequences. The nucleic acids of the present invention may be naturally occurring, recombinant, or synthetic. The nucleic acids may be in the form of circular or linear sequences, or a combination of both forms. The nucleic acids may be both DNA, genomic or cDNA, or RNA or a combination of both. The nucleic acids may contain any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine, hypoxanthine, isocytosine, 5-hydroxymethylcytosine, and isoguanine. Other examples of modified bases that may be used in the present invention are detailed in Chemical Reviews 2016, 116 (20) 12655-12687. The term "nucleic acid" also encompasses any nucleic acid analogs that may contain other backbones, including, but not limited to, phosphoramide, phosphorothioate, phosphorodithioate, O-methyl phosphoramidite linkages and / or deoxyribonucleotide and ribonucleotide nucleic acids. Any combination of the above features of nucleic acids is also encompassed by the present invention.

[0039] The genetic circuit used in the present invention may also include an origin of replication. Origins of replication for use in the disclosed methods and compositions are known in the art and have been identified from species-specific plasmid DNA (e.g., CoIE1, R1, pT181, pSC101, pMB1, R6K, RK2, p15a, etc.), from bacterial viruses (e.g., φX174, M13, F1 and P4), and from bacterial chromosomal origins of replication (e.g., oriC). Such sequences allow for replication of the genetic circuit in, for example, bacterial cells, such as donor cells and / or target cells. In one embodiment, the phagemid (genetic circuit) according to the present disclosure includes a bacterial origin of replication that is functional in donor cells, target cells or recipient cells.

[0040] Alternatively, the genetic circuits of the present disclosure do not include any functional bacterial origin of replication or contain an origin of replication that is inactive in the target bacterium, and therefore cannot replicate by themselves once introduced into a target or recipient cell.

[0041] In one embodiment, the origin of replication on the genetic circuit or plasmid packaged in the delivery vehicle is inactive in the target bacterium, meaning that this origin of replication is not functional in the target cell, thus preventing unwanted plasmid replication.

[0042] In one embodiment, the genetic circuit or plasmid comprises a bacterial origin of replication that is functional in a donor bacterial cell, e.g., a donor bacterial cell used for production of bacterial viral particles.

[0043] Genetic circuits or plasmids replication depends on host enzymes and genetic circuit or plasmid regulatory cis and trans determinants. For example, some genetic circuits or plasmids may have determinants recognized in almost all Gram-negative bacteria and may act correctly in each host during replication initiation and regulation. Other genetic circuits or plasmids may retain this ability only in some bacteria (Kues, U and Stahl, U 1989, Microbiol Rev 53:491-516).

[0044] Genetic circuits or plasmids can replicate by three general mechanisms: theta-type, strand displacement, and rolling circle initiated at replication origins (reviewed by Del Solar et al., 1998, Microhio and Molec Biol. Rev 62:434-464). These replication origins contain sites necessary for the interaction of the genetic circuit or plasmid and / or host-encoded proteins.

[0045] As used herein, the origin of replication may be of medium copy number, such as the colE1 ori from pBR322 (15-20 copies per cell) or the R6K plasmid (15-20 copies per cell), or may be of high copy number, such as the pUC ori (500-700 copies per cell), pGEM ori (300-400 copies per cell), pTZ ori (>1000 copies per cell) or pBluescript ori (300-500 copies per cell).

[0046] In one embodiment, the bacterial origins of replication included in the genetic circuit are ColE1, pMB1 and variants (such as pBR322, pET, pUC), p15a, ColA, ColE2, pOSAK, pSC101, R6K, IncW (such as pSa), IncFII, pT181, P1, F IncP, IncC, IncJ, IncN, IncP1, IncP4, IncQ, IncH11, RSF1010, CloDF13, NTP16, R1, f5, pPS10, pC194, pE194, BBR1, pBC1, pEP2, pWVO1, pLF1311, pAP1, pWKS1, pLS1, pLS11, pUB6060, pJD4, pIJ101, pSN22, pAMbeta1, pIP5 01, pIP407, ZM6100(Sa), pCU1, RA3, pMOL98, RK2 / RP4 / RP1 / R68, pB10, R300B, pRO1614, pRO1600, pECB2, pCM1, pFA3, RepFIA, RepFIB, RepFIC, pYVE439-80, R387, phasyl, RA1, TF-FC2, pMV158 and pUB113.

[0047] More preferably, the bacterial origin of replication is an E. coli origin of replication selected from the group consisting of ColE1, pMB1 and variants (such as pBR322, pET, pUC), p15a, ColA, ColE2, pOSAK, pSC101, R6K, IncW (such as pSa), IncFII, pT181, P1, F IncP, IncC, IncJ, IncN, IncP1, IncP4, IncQ, IncH11, RSF1010, CloDF13, NTP16, R1, f5 and pPS10.

[0048] More preferably, the bacterial origin of replication is selected in the group consisting of pC194, pE194, BBR1, pBC1, pEP2, pWVO1, pLF1311, pAP1, pWKS1, pLS1, pLS11, pUB6060, pJD4, pIJ101, pSN22, pAMbeta1, pIP501, pIP407, ZM6100(Sa), pCU1, RA3, pMOL98, RK2 / RP4 / RP1 / R68, pB10, R300B, pRO1614, pRO1600, pECB2, pCM1, pFA3, RepFIA, RepFIB, RepFIC, pYVE439-80, R387, phasyl, RA1, TF-FC2, pMV158 and pUB113.

[0049] Even more preferably, the bacterial origins of replication are ColE1 and p15A.

[0050] The genetic circuit may include a phage origin of replication. In particular, the delivered nucleic acid sequence according to the present disclosure may also include a phage origin of replication that, by virtue of the complementation of the complete phage genome, can initiate replication of the delivered nucleic acid sequence for subsequent encapsulation into a different capsid. The phage origin of replication may be engineered to act as a bacterial origin of replication without the need to package any phage particles.

[0051] The phage origin of replication contained in the genetic circuit or delivered nucleic acid sequence of the present disclosure can be any origin of replication found in a phage.

[0052] Preferably, the phage origin of replication may be the wild-type or non-wild-type sequence of M13, f1, φX174, P4, lambda, P2, 186, lambda-like, HK022, mEP237, HK97, HK629, HK630, mEP043, mEP213, mEP234, mEP390, mEP460, mEPx1, mEPx2, phi80, mEP234, T2, T4, T5, T7, RB49, phiX174, R17, PRD1 P1-like, P2-like, P22, P22-like, N15 and N15-like bacteriophages.

[0053] More preferably, the phage origin of replication is selected in the group consisting of M13, f1, φX174, P4, and lambda phage origins of replication.

[0054] In certain embodiments, the phage origin of replication is a lambda or P4 origin of replication.

[0055] The genetic circuit disclosed herein comprises a nucleic acid sequence of interest under the transcriptional control of a repressor binding sequence. In the disclosed method, the transcription of the nucleic acid of interest is suppressed in donor cells while active in target cells. In one embodiment, the nucleic acid of interest is a programmable nuclease circuit that is delivered to the target cell, i.e., the nucleic acid of interest encodes a programmable nuclease system that is delivered to the target cell. The programmable nuclease system comprises a programmable nuclease, i.e., a nuclease that can mediate sequence-specific removal of a target gene of interest in a target cell. In particular, this programmable nuclease system may be capable of mediating sequence-specific removal in vivo of bacteria that contain a target gene of interest (e.g., a gene harmful to humans). Programmable nucleases that can be used in the methods of the present invention include, for example, CRISPR nucleases (also referred to as "CRISPR-associated proteins" or "Cas nucleases"), preferably type I, type II CRISPR nucleases, TALEN nucleases, ZFN nucleases, meganucleases and recombinases, and any variants (evolved or engineered variants) thereof. These nucleases can be used separately or in combination, i.e., the programmable nuclease system can include one or several programmable nucleases. In certain embodiments, the programmable nuclease is selected from the engineered variants of the type II CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) system of Streptococcus pyogenes. Other programmable nucleases that may be used include other CRISPR-Cas systems, engineered TALEN (Transcription Activator-Like Effector Nuclease) variants, engineered zinc finger nuclease (ZFN) variants, natural, evolved or engineered meganuclease or recombinase variants, and any combination or hybrid of programmable nucleases.Depending on the type of nuclease used in the method, the programmable nuclease system may further comprise one or several additional components.In particular, for the programmable nuclease in CRISPR nuclease, the programmable nuclease system preferably further comprises guide RNA to find and selectively cut target sequence.Therefore, the engineered distributed nuclease circuit provided herein can be used to selectively cut the DNA that codes for the gene of interest in target cell, such as toxin gene, virulence factor gene, antibiotic resistance gene, remodeling gene or regulatory gene (see WO2014124226).

[0056] Other sequences of interest, preferably programmable, may be included in the nucleic acid sequence delivered for delivery to the target cell. In one embodiment, the nucleic acid of interest encodes a molecule that affects the survival or growth of the target cell, preferably a target bacterium. In embodiments in which the target cell is a bacterium, such a molecule may be selected to bring about cell death (bactericidal effect) or to prevent the growth of said bacterium (bacteriostatic effect). For example, the nucleic acid sequence of interest may encode a holin, an endolysin, a restriction enzyme, or a toxin that affects the target cell, preferably affects the survival or growth of the target cell.

[0057] In certain embodiments, the nucleic acid of interest encodes a bacteriocin. Bacteriocins can be proteinaceous toxins produced by bacteria that kill or inhibit the growth of other bacteria. Bacteriocins are categorized in several ways, including by the producing strain, common resistance mechanisms, and mechanism of death. Such bacteriocins have been described from Gram-negative bacteria (e.g., microcins, colicin-like bacteriocins, and tylosins) and from Gram-positive bacteria (e.g., class I, class II, class III, or class IV bacteriocins). The nucleic acid of interest may also encode a transporter required for secretion of the toxin into the extracellular space.

[0058] In more particular embodiments, the nucleic acid sequence of interest comprises a sequence encoding a toxin selected from the group consisting of a microcin, a colicin-like bacteriocin, a tylosin, a class I, a class II, a class III and a class IV bacteriocin.

[0059] In certain embodiments, the corresponding immunity polypeptides (i.e., antitoxins) may be used to protect bacterial cells for production and encapsidation purposes of the delivered nucleic acid sequence (see review by Cotter et al., Nature Reviews Microbiology 11:95, 2013, incorporated herein by reference in its entirety), but are not present in the pharmaceutical composition and within the target bacteria to which the nucleic acid of interest is delivered.

[0060] In some other embodiments, expression of the transferred nucleic acid of interest in the target cell does not result in cell death, for example, the nucleic acid of interest may encode a reporter gene that provides, for example, a luminescent or fluorescent signal.

[0061] In some other embodiments, the nucleic acid of interest may encode a protein, particularly an enzyme, that performs a useful function in the target cell, such as modifying the composition of the target cell's metabolism, its environment, or the host that contains the target cell.

[0062] In certain embodiments, the nucleic acid sequence of interest is selected from the group consisting of a nucleic acid encoding an RNA such as an mRNA, a crRNA, a tRNA, an iRNA (interfering RNA), an asRNA (antisense RNA), a ribozyme RNA, an RNA aptamer or a guide RNA (gRNA), a CRISPR locus, a gene encoding an enzyme such as a nuclease or a kinase, a gene encoding a nuclease selected from the group consisting of a Cas nuclease, a Cas9 nuclease, a TALEN, a ZFN or a meganuclease, a recombinase, a bacterial receptor, a membrane protein, a structural protein, a secreted protein, or a gene encoding resistance to antibiotics or drugs in general, a gene encoding a toxin protein or toxin factor, and a gene encoding a virulence protein, a virulence factor, a bacterial transporter or a bacterial pore, and any combination thereof. The protein encoded by the nucleic acid of interest can also be modified or engineered to include external features such as adding or removing a function (e.g., dCas9), adding a secretion signal to a protein that is not normally secreted, adding an exogenous peptide in a loop, etc.

[0063] In some embodiments, the nucleic acid of interest encodes a CRISPR system. Typically, a CRISPR system contains two different elements: i) an endonuclease, in this case a CRISPR-associated nuclease (Cas or "CRISPR-associated protein") and ii) a guide RNA. The structure of the guide RNA may depend on the nature of the Cas nuclease. In particular, the guide RNA (gRNA or sgRNA) may be in the form of a chimeric RNA consisting of a combination of a CRISPR (RNAcr) bacterial RNA and an RNAtracr (trans-activating RNA CRISPR) (Jinek et al., Science 2012). The gRNA combines the targeting specificity of the cRNA, which corresponds to the "spacing sequence" that serves as a guide to the Cas protein, with the structural properties of the Rtracr in a single transcript. Such a guide RNA is necessary, for example, when the Cas nuclease is Cas9. Alternatively, the guide RNA may only comprise RNAcr. Such a guide RNA is necessary, for example, when the Cas nuclease is Cpf1. When gRNA and Cas proteins are expressed simultaneously in a cell, the target genomic sequence can be permanently disrupted (and, depending on the location, cause loss of the targeted and surrounding sequences and / or cell death) or modified. The modification can be guided by a repair matrix. In general, CRISPR systems include two major classes that depend on the nuclease mechanism of action. Class 1 consists of multi-subunit effector complexes and includes types I, III and IV. Class 2 consists of single-unit effector modules, like Cas9 nuclease, and includes types II (II-A, II-B, II-C, II-C variants), V (VA, VB, VC, VD, VE, V-U1, V-U2, V-U3, V-U4, V-U5) and VI (VI-A, VI-B1, VI-B2, VI-C, VI-D).

[0064] The nucleic acid of interest according to the present disclosure may include a nucleic acid sequence encoding a Cas protein. A variety of CRISPR enzymes are available for use as the sequence of interest of the plasmid. In some embodiments, the CRISPR enzyme is a type II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some other embodiments, the CRISPR enzyme catalyzes RNA cleavage. In one embodiment, the CRISPR enzyme may be bound to an sgRNA. In certain embodiments, the sgRNA targets a gene selected from the group consisting of antibiotic resistance genes, virulence proteins or virulence factor genes, toxin proteins or virulence factor genes, bacterial receptor genes, membrane protein genes, structural protein genes, secreted protein genes, genes that generally express resistance to drugs, or genes that cause harmful effects to the host (host including the target cell).

[0065] Non-limiting examples of Cas proteins, either as part of a multi-subunit effector or as single unit effectors, include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas11 (SS), Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), C2c4, C2c8, C2c5, C2c10, C2c9, Cas13a (C2c2), Cas13b (C2c6), Cas13c (C2c7), Cas13d, Csa5, Csc 1, Csc2, Cse1, Cse2, Csy1, Csy2, Csy3, Csf1, Csf2, Csf3, Csf4, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csn2, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx1 6, CsaX, Csx13, Csx1, Csx15, SdCpf1, CmtCpf1, TsCpf1, CmaCpf1, PcCpf1, ErCpf1, FbCpf1, UbcCpf1, AsCpf1, LbCpf1, homologs thereof, orthologues thereof, variants thereof, or modified versions thereof. In some embodiments, the CRISPR enzyme cleaves both strands of the target nucleic acid at a protospacer adjacent motif (PAM) site.

[0066] In certain embodiments, the CRISPR enzyme is any Cas9 protein, such as any naturally occurring bacterial Cas9, as well as any variants, homologs, or orthologues thereof.

[0067] By "Cas9" is meant the protein Cas9 (also called Csn1 or Csx12) or a functional protein, peptide or polypeptide fragment thereof, i.e. capable of interacting with a guide RNA and exerting an enzymatic activity (nuclease) that allows it to perform a double-stranded break in the DNA of the target genome. Thus, "Cas9" refers, for example, to a modified protein in which domains of the protein that are not essential for the predefined function of the protein have been truncated to remove them, in particular domains that are not required for interaction with the gRNA.

[0068] As used in the context of this disclosure, sequences encoding Cas9 (whole protein or fragments thereof) can be obtained from any known Cas9 protein (Fonfara et al., Nucleic Acids Res 42 (4), 2014; Koonin et al., Nat Rev Microbiol 15(3), 2017). Examples of Cas9 proteins useful in the present disclosure include, but are not limited to, the Cas9 proteins of Streptococcus pyogenes (SpCas9), Streptococcus thermophiles (St1Cas9, St3Cas9), Streptococcus mutans, Staphylococcus aureus (SaCas9), Campylobacter jejuni (CjCas9), Francisella novicida (FnCas9), and Neisseria meningitides (NmCas9).

[0069] In another particular embodiment, the CRISPR enzyme is any Cas12a, Cas13a, or Cas13d protein, such as any naturally occurring bacterial Cas12a, Cas13a, or Cas13d, as well as any variants, homologs, or orthologues thereof.

[0070] As used in the context of the present disclosure, sequences encoding Cpf1 (Cas12a) (whole protein or fragments thereof) can be obtained from any known Cpf1 (Cas12a) protein (Koonin et al., 2017). Examples of Cpf1 (Cas12a) proteins useful in the present disclosure include, but are not limited to, the Cpf1 (Cas12a) proteins of Acidaminococcus sp., Lachnospiraceae bacteriu, and Francisella novicida.

[0071] The sequence encoding Cas13a (whole protein or a fragment thereof) can be obtained from any known Cas13a (C2c2) protein (Abudayyeh et al., 2017). Examples of Cas13a (C2c2) proteins useful in the present disclosure include, but are not limited to, the Cas13a (C2c2) protein of Leptotrichia wadei (LwaCas13a).

[0072] The sequence encoding Cas13d (whole protein or fragments thereof) can be obtained from any known Cas13d protein (Yan et al., 2018). Examples of Cas13d proteins useful in the present disclosure include, but are not limited to, Cas13d proteins from Eubacterium siraeum and Ruminococcus sp.

[0073] In certain embodiments, the nucleic acid of interest encodes a CRISPR / Cas system, preferably a CRISPR / Cas9 system, for the reduction of gene expression or inactivation of a gene selected from the group consisting of antibiotic resistance genes, virulence factor or protein genes, toxin factor or protein genes, bacterial receptors, membrane proteins, structural proteins, genes expressing secreted proteins, genes expressing resistance to drugs in general, or genes causing a deleterious effect in the host.

[0074] In one embodiment, the CRISPR system is used to target and inactivate virulence factors. Virulence factors can be any substance produced by a pathogen that alters host-pathogen interactions by increasing the degree of damage inflicted on the host. Virulence factors are used by pathogens in many ways, including, for example, in cell adhesion or colonization of a niche in the host, to avoid the host's immune response, to facilitate invasion and release from the host cell, to obtain nutrients from the host, or to inhibit other physiological processes in the host. Virulence factors can include enzymes, endotoxins, adhesion factors, motility factors, factors involved in complement evasion, and factors that promote biofilm formation. For example, such targeted virulence factor genes include, but are not limited to, E. coli virulence factor genes, such as, but not limited to, EHEC-HlyA, Stx1 (VT1), Stx2 (VT2), Stx2a (VT2a), Stx2b (VT2b), Stx2c (VT2c), Stx2d (VT2d), Stx2e (VT2e), and Stx2f (VT2f), Stx2h (VT2h), stx2k, fimA, fimF, fimH, neuC, kpsE, sfa, foc, iroN, aer, iha, papC, papGI, papGII, papGIII, hlyC, cnf1, hra, sat, ireA, usp The targeted virulence factor gene may be ompT, ibeA, malX, fyuA, irp2, traT, afaD, ipaH, eltB, estA, bfpA, eaeA, espA, aaiC, aatA, TEM, CTX, SHV, csgA, csgB, csgC, ​​csgD, csgE, csgF, csgG, csgH, T1SS, T2SS, T3SS, T4SS, T5SS, T6SS (secretion system). For example, such targeted virulence factor gene may be Shigella dysenteriae virulence factor gene, such as, but not limited to, stx1 and stx2. For example, such targeted virulence factor gene may be Yersinia pestis virulence factor gene, such as, but not limited to, yscF (plasmid-borne (pCD1) T3SS outer needle subunit).For example, such a targeted virulence factor gene can be a Francisella tularensis virulence factor gene, such as, but not limited to, fslA. For example, such a targeted virulence factor gene can be a Bacillus anthracis virulence factor gene, such as, but not limited to, pag (anthrax toxin, cell-associated protective antigen). For example, such a targeted virulence factor gene can be a Vibrio cholera virulence factor gene, such as, but not limited to, ctxA and ctxB (cholera toxin), tcpA (toxin coregulator pilus), and toxT (master virulence regulator). For example, such targeted virulence factor genes can be Pseudomonas aeruginosa virulence factor genes, such as, but not limited to, pyoverdine (e.g., sigma factor pvdS, biosynthetic genes pvdL, pvdl, pvdJ, pvdH, pvdA, pvdF, pvdQ, pvdN, pvdM, pvdO, pvdP, transporter genes pvdE, pvdR, pvdT, opmQ), siderophore pyochelin (e.g., pchD, pchC, pchB, pchA, pchE, pchF and pchG), and toxins (e.g., exoU, exoS and exoT). For example, such targeted virulence factor genes can be Klebsiella pneumoniae virulence factor genes, such as, but not limited to, fimA (adherence, type 1 pilus major subunit), and cps (capsular polysaccharide). For example, such targeted virulence factor genes can be Acinetobacter baumannii virulence factor genes, such as, but not limited to, ptk (capsular polymerization), and epsA (assembly). For example, such targeted virulence factor genes can be Salmonella enterica Typhi virulence factor genes, such as, but not limited to, MIA (invasion, SPI-1 regulator), ssrB (SPI-2 regulator), and those associated with bile resistance, including, but not limited to, the efflux pump genes acrA, acrB, and tolC.For example, such targeted virulence factor genes can be Fusobacterium nucleatum virulence factor genes, such as, but not limited to, FadA and TIGIT. For example, such targeted virulence factor genes can be Bacteroides fragilis virulence factor genes, such as, but not limited to, bft.

[0075] In another embodiment, the CRISPR system, preferably the CRISPR / Cas9 system, is capable of expressing an antibiotic resistance gene, such as, but not limited to, GyrB, ParE, ParY, AAC(1), AAC(2'), AAC(3), AAC(6'), ANT(2"), ANT(3"), ANT(4'), ANT(6), ANT(9), APH(2"), APH(3"), APH(3'), APH(4), APH(6), APH(7"), APH(9), ArmA, RmtA, RmtB, RmtC, Sgm, AER, BLA1, CTX-M, KPC, SHV, TEM, BlaB, CcrA, IMP, NDM, VIM, ACT, AmpC, CMY, LAT, PDC, OXA β-lactamase, mecA, Omp36, OmpF, PIB, bla (blaI, blaR1) and mec (mecI, mecR1) operons, chloramphenicol acetyltransferase (CAT), chloramphenicol phosphotransferase, ethambutol-resistant arabinosyltransferase (EmbB), MupA, MupB, integral membrane protein MprF, Cfr 23S rRNA methyltransferase, rifampin ADP-ribosyltransferase (Arr), rifampin glycosyltransferase, rifampin monooxygenase, rifampin phosphotransferase, DnaA, RbpA, rifampin-resistant beta-subunit of RNA polymerase (RpoB), Erm 23SrRNA methyltransferase, Lsa, MsrA, Vga, VgaB, streptogramin Vgb lyase, Vat acetyltransferase, fluoroquinolone acetyltransferase, fluoroquinolone resistance DNA topoisomerase, fluoroquinolone resistance GyrA, GyrB, ParC, quinolone resistance protein (Qnr), FomA, FomB, FosC, FosA, FosB, FosX, VanA, VanB, VanD, VanR, VanS, lincosamide nucleotidyltransferase (Lin), EreA, EreB, GimA, Mgt, Ole, macrolide phosphotransferase (MPH) , MefA, MefE, Mel, streptothricin acetyltransferase (sat), Sul1, Sul2, Sul3, sulfonamide resistance FolP, tetracycline inactivation enzyme TetX, TetA, TetB, TetC, Tet30, Tet31, TetM, TetO, TetQ, Tet32, Tet36, MacAB-TolC, MsbA, MsrA, VgaB, EmrD, EmrAB-TolC, NorB, GepA, MepA, AdeABC, AcrD, MexAB-OprM, mtrCDE, EmrE, adeR, acrR, baeSR, mexR, phoPQ, mtrR, or any antibiotic resistance gene listed in the Comprehensive Antibiotic Resistance Database (CARD https: / / card.mcmaster.ca / ).

[0076] In another embodiment, the CRISPR system, preferably the CRISPR / Cas9 system, is used to target and inactivate bacterial toxin genes. Bacterial toxins can be classified as either exotoxins or endotoxins. Exotoxins are produced and actively secreted; endotoxins remain part of the bacterium. The response to bacterial toxins can include severe inflammation and can result in suppuration. Such toxins can be, for example, botulinum neurotoxins, tetanus toxins, staphylococcal toxins, diphtheria toxins, anthrax toxins, alpha toxins, pertussis toxins, shiga toxins, heat-stable enterotoxins (E. coli ST), colibactins, BFT (Bacteroides fragilis toxins) or any toxins described in Henkel et al. (Toxins from Bacteria in EXS. 2010; 100: 1-29).

[0077] The terms "donor cell", "target cell" and "recipient cell" as used herein preferably refer to a prokaryotic cell, more preferably a bacterial cell. In particular, a "donor" cell, a "target" cell or a "recipient" cell disclosed herein may be any bacterium that is or may be present in a mammalian organism. It may be any commensal, symbiotic or pathogenic bacterium of the microflora or microbiome.

[0078] The microbiome contains a variety of endogenous bacterial species, any of which may be targeted according to the present disclosure. In some embodiments, when a bacterial delivery vehicle is used, the genus and / or species of the targeted endogenous bacterial cells may depend on the type of bacteriophage used to prepare the bacterial delivery vehicle. For example, some bacteriophages exhibit tropism or preferentially target specific host species of bacteria. Other bacteriophages do not exhibit such tropism and may be used to target many different genera and / or species of endogenous bacterial cells.

[0079] Examples of bacterial cells that can be used as donor or target cells include, but are not limited to, Yersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp., Bordetella spp., Neisseria spp., Aeromonas spp., Francisella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Haemophilus spp., Brucella spp., and the like. spp.), Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp., Erysipelothrix spp., Salmonella spp., Streptomyces spp., Streptococcus spp., Staphylococcus spp., Bacteroides spp. spp., Prevotella spp., Clostridium spp., Bifidobacterium spp., Clostridium spp., Brevibacterium spp., Lactococcus spp., Leuconostoc spp., Actinobacillus spp.), Selnomonas spp., Shigella spp., Zymonas spp., Mycoplasma spp., Treponema spp., Leuconostoc spp., Corynebacterium spp., Enterococcus spp., Enterobacter spp., Pyrococcus spp., Serratia spp., Morganella spp., Parvimonas spp., Fusobacterium spp. spp., Actinomyces spp., Porphyromonas spp., Micrococcus spp., Bartonella spp., Borrelia spp., Brucelia spp., Campylobacter spp., Chlamydophilia spp., Cutibacterium spp., Propionibacterium spp., Gardnerella spp., Ehrlichia spp., Haemophilus spp. spp., Leptospira spp., Listeria spp., Mycoplasma spp., Nocardia spp., Rickettsia spp., Ureaplasma spp., and Lactobacillus spp., and mixtures thereof.

[0080] Preferably, the bacteria may be selected from the group consisting of Yersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Salmonella spp., Streptococcus spp., Staphylococcus spp., Bacteroides spp., Clostridium spp., Shigella spp., Enterococcus spp., Enterobacter spp., Listeria spp., Cutibacterium spp., Propionibacterium spp., Fusobacterium spp., Porphyromonas spp., and Gardnerella spp.

[0081] In some embodiments, the bacterial cells of the present disclosure that may be used as donor or target cells are anaerobic bacterial cells (e.g., cells that do not require oxygen for growth). Anaerobic bacterial cells include facultative anaerobic cells, such as, but not limited to, Escherichia coli, Shewanella oneidensis, Gardnerella vaginalis, and Listeria. Anaerobic bacterial cells also include obligate anaerobic cells, such as Bacteroides, Clostridium, Cutibacterium, Propionibacterium, Fusobacterium, and Porphyromona species. In humans, anaerobic bacteria are most commonly found in the digestive tract. In some specific embodiments, the target bacteria (target cells) are therefore bacteria that are most commonly found in the digestive tract. The bacterial delivery vehicle, preferably a bacteriophage, used to prepare the bacterial viral particles, which in turn can target (e.g., specifically target) anaerobic bacterial cells and specifically deliver plasmids according to their specific spectrum known to those skilled in the art.

[0082] In some embodiments, bacterial cells that may be used as donor or target cells include, but are not limited to, Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides distasonis, Bacteroides vulgatus, Clostridium leptum, Clostridium coccoides, Staphylococcus aureus, Bacillus subtilis, Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis, and the like. lactis, Leuconostoc lactis, Actinobacillus actinobycetemcomitans, cyanobacteria, Escherichia coli, Helicobacter pylori, Selnomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, Treponema denticola, Bacillus thuringiensis, Staphylococcus lugdunensis lugdunensis, Leuconostoc oenos, Corynebacterium xerosis, Lactobacillus plantarum, Lactobacillus rhamnosusrhamnosus, Lactobacillus casei, Lactobacillus acidophilus, Enterococcus faecalis, Bacillus coagulans, Bacillus cereus, Bacillus popillae, Synechocystis strain PCC6803, Bacillus liquefaciens, Pyrococcus abyssi, Selenomonas nominantium, Lactobacillus hilgardii, Streptococcus ferus, Lactobacillus pentosus, Bacteroides fragilis, Staphylococcus epidermidis, Streptomyces phaechromogenes, Streptomyces ghanaenis, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, Morganella morganii, Citrobacter freundii freundii, Propionibacterium freudenreichii, Pseudomonas aerigunosa, Parvimonas micramicra, Prevotella intermedia, Fusobacterium nucleatum, Prevotella nigrescens, Actinomyces israelii, Porphyromonas endodontalis, Porphyromonas gingivalis, Micrococcus luteus, Bacillus megaterium, Aeromonas hydrophila, Aeromonas caviae, Bacillus anthracis, Bartonella henselae henselae, Bartonella Quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Chlamydia pneumoniae pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinumbotulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria, Cutibacterium acnes (formerly Propionibacterium acnes), Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecium, Francisella tularensis, Haemophilus influenza, Legionella pneumophila pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitides, Nocardia asteroids, Rickettsia rickettsia, Salmonella enteritidis, Salmonella typhityphi, Salmonella paratyphi, Salmonella typhimurium, Shigella flexnerii, Shigella dysenteriae, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Gardnerella vaginalis, Streptococcus viridans, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia pestis pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Actinobacter baumanii, Pseudomonas aerignosa, and mixtures thereof, and preferably, the bacterial cells that may be used as donor or target cells are selected from the group consisting of Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, and Enterobacter aerogenes, and mixtures thereof.

[0083] In one embodiment, the bacterium that may be used as a donor or target cell is E. coli.

[0084] In one embodiment, the bacteria that may be used as donor or target cells are pathogenic bacteria. The bacteria may be pathogenic bacteria.

[0085] The bacteria that may be used as donor or target cells may be antibacterial resistant bacteria, preferably selected from the group consisting of extended spectrum beta-lactamase producing (ESBL) Escherichia coli, ESBL Klebsiella pneumoniae, vancomycin resistant Enterococcus (VRE), methicillin resistant Staphylococcus aureus (MRSA), multidrug resistant (MDR) Acinetobacter baumannii, MDR Enterobacter species, and combinations thereof. Preferably, the bacteria may be selected from the group consisting of extended spectrum beta-lactamase producing (ESBL) Escherichia coli strains.

[0086] In some particular embodiments, the donor cells and / or target cells are probiotics. As used herein, the term "probiotics" preferably includes, but is not limited to, lactobacilli, bifidobacteria, streptococci, enterococci, propionibacteria, yeast, lactobacilli, bifidobacteria, or proteobacteria.

[0087] Alternatively, bacteria that may be used as donor or target cells may be bacteria of the microbiome of a given species, preferably bacteria of the human microbiota.

[0088] In some embodiments, the donor cells and target cells are phylogenetically close, preferably from the same phylum, class, order, family, genus or species, more preferably from the same genus or species.

[0089] In certain embodiments, the present disclosure is directed to a method for transferring a genetic circuit from a donor cell to a target or recipient cell by producing a bacterial delivery vehicle containing the genetic circuit described herein. The bacterial delivery vehicle may be prepared from a bacterial virus. The bacterial delivery vehicle is selected to be capable of introducing the genetic circuit into the target bacterial cell. The bacterial delivery vehicle may be engineered to target a specific bacterium (see, e.g., U.S. Patent Application Nos. 62 / 771,761; 62 / 802,777; and 62 / 783,258, each of which is incorporated herein in its entirety).

[0090] The bacterial virus from which the bacterial delivery vehicle can be derived is preferably a bacteriophage. Optionally, the bacteriophage is selected from the Order Caudovirales, based on the taxonomy of Krupovic et al., Arch Virol, 2015.

[0091] Bacteriophages include those from the Myoviridae family (for example, but not limited to, the genus Cp220virus, genus Cp8virus, genus Ea214virus, genus Felixolvirus, genus Mooglevirus, genus Suspvirus, genus Hp1virus, genus P2virus, genus Kayvirus, genus P100virus, genus Silviavirus, genus Spo1virus, genus Tsarbombavirus, genus Twortvirus, genus Cc31virus, genus Jd18virus, genus Js98virus, genus Kp15virus, genus Moonvirus, genus Rb49virus, genus Rb69virus, genus S16virus, genus Schizot4virus, genus Sp18virus, genus T4virus, genus Cr3virus, genus Se1virus, genus V5virus, genus Abouovirus, genus Agatevirus, genus Agrican357virus, genus Ap22virus, genus Arv1virus, genus B4virus, genus B5virus, genus B69virus, genus B7virus, genus B8virus, genus B9virus, genus B10virus, genus B11virus, genus B12virus, genus B13virus, genus B14virus, genus B15virus, genus B16virus, genus B17virus, genus B19virus, genus B20virus, genus B21virus, genus B22virus, genus B23virus, genus B24virus, genus B25virus, genus B26virus, genus B27virus, genus B28virus, genus B29virus, genus B29virus, genus B29virus, genus Viruses, Bastille virus, Bc431 virus, Bcep78 virus, Bcepmu virus, Biquarta virus, Bxz1 virus, Cd119 virus, Cp51 virus, Cvm10 virus, Eah2 virus, El virus, Hapuna virus, Jimmer virus, Kpp10 virus, M12 virus, Machina virus, Martha virus, Msw3 virus, Mu virus, Myohalo virus, Nit1 virus, P1 virus, Pakpuna virus, Pbuna virus, Phikz virus, Rheph4 virus, Rsl2 virus, Rsluna virus, Secunda5 virus, Sep1 virus, Spn3 virus, Svuna virus, Tg1 virus, Vhml virus and Wph virus).

[0092] Bacteriophages include those from the family Podoviridae (e.g., but not limited to, the genus Fri1virus, Kp32virus, Kp34virus, Phikmvvirus, Pradovirus, Sp6virus, T7virus, Cp1virus, P68virus, Phi29virus, Nona33virus, Pocjvirus, Tl2011virus, Bcep22virus, Bpp1virus, Cba41virus, Dfl12virus, and the like). The virus may be selected from the genera: Bacteroides, Ea92, Epsilon15, F116, G7c, Jwalpha, Kf1, Kpp25, Lit1, Luz24, Luz7, N4, Nonana, P22, Page, Phieco32, Prtb, Sp58, Una961 and Vp5.

[0093] Bacteriophages include those from the family Siphoviridae (e.g., but not limited to, those of the genera Cam virus, Lika virus, R4 virus, Acadian virus, Cooper virus, Pg1 virus, Pipefish virus, Rosebush virus, Brujita virus, Che9c virus, Hawkeye virus, Plot virus, Jersey virus, K1g virus, Sp31 virus, Lmd1 virus, Una4 virus, Bongo virus, Re virus, and the like). y virus genus, Butters virus genus, Charlie virus genus, Redi virus genus, Baxter virus genus, Nymphadora virus genus, Bignus virus genus, Fishburne virus genus, Phayonce virus genus, Kp36 virus genus, Rogue1 virus genus, Rtp virus genus, T1 virus genus, Tls virus genus, Ab18 virus genus, Amigo virus genus, Anatole virus genus, Andromeda virus genus, Attis virus genus, Barnyard virus genus, Bernal13 virus genus, Biseptimavirus, Bronvirus, C2virus, C5virus, Cba181virus, Cbastvirus, Cecivirus, Che8virus, Chivirus, Cjw1virus, Corndogvirus, Cronusvirus, D3112virus, D3virus, Decurrovirus, Demosthenesvirus, Doucettevirus, E125virus, Eiauvirus, Ff47virus, Gaiavirus, Gilesvirus, Gordonvirus Virus genera, Gordtnk virus genus, Harrison virus genus, Hk578 virus genus, Hk97 virus genus, Jenst virus genus, Jwx virus genus, Kellezio virus genus, Korra virus genus, L5 virus genus, lambda virus genus, Laroye virus genus, Liefie virus genus, Marvin virus genus, Mudcat virus genus, N15 virus genus, Nonag virus genus, Np1 virus genus, Omega virus genus, P12002 virus genus, P12024 virus genus, P23 virus genus, P70 virus genus,Pa6virus genus, Pamx74virus genus, Patiencevirus genus, Pbi1virus genus, Pepy6virus genus, Pfr1virus genus, Phic31virus genus, Phicbkvirus genus, Phietavirus genus, Phifelvirus genus, Phijl1virus genus, Pis4avirus genus, Psavirus genus, Psimunavirus genus, Rdjlvirus genus, Rer2virus genus, Sap6virus genus, Send513virus genus, Septima3virus genus, Seuratvirus genus, Sextaecvirus genus, Sfi11virus genus, Sfi21dt1virus genus, Sitaravirus The virus may be selected from the genera: Sarcovirus, Sk1virus, Slashvirus, Smoothievirus, Soupsvirus, Spbetavirus, Ssp2virus, T5virus, Tankvirus, Tin2virus, Titanvirus, Tm4virus, Tp21virus, Tp84virus, Triavirus, Trigintaduovirus, Vegasvirus, Vendettavirus, Wbetavirus, Wildcatvirus, Wizardvirus, Woesvirus, Xp10virus, Ydn12virus, and Yuavirus.

[0094] The bacteriophage may be selected from the family Ackermannviridae (such as, but not limited to, the genera Ag3, Limestone, Cba120 and Vi1).

[0095] In some cases, the bacteriophage is not part of the Caudovirales but belongs to a family with an unclassified order, such as, but not limited to, the family Tectiviridae (e.g., genera Alphatectiviridae, Betatectiviridae), the family Corticoviridae (e.g., the genus Corticovirus), the family Inoviridae (e.g., the genus Fibrovirus, Habenivirus, Inovirus, Lineavirus, Plectrovirus, Saevirus, and the like). tivirus, Vespertiliovirus), Cystoviridae (e.g. Cystovirus), Leviviridae (e.g. Allolevivirus, Levivirus), Microviridae (e.g. Alpha3microvirus, G4microvirus, Phix174microvirus, Bdellomicrovirus, Chlamydiamicrovirus, Spiromicrovirus) and Plasmaviridae (e.g. Plasmavirus).

[0096] In some cases, the bacteriophage is a targeted archaea that is not part of the Caudovirales but is from a family with an unclassified order, such as, but not limited to, Ampullaviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Liposthrixviridae, Pleolipoviridae, Rudiviridae, Salterprovirus, and Bicaudaviridae.

[0097] A non-exhaustive list of bacterial genera and their known host-specific bacterial viruses is presented in the following paragraphs. The bacterial delivery vehicles disclosed herein may be engineered from the following phages, as non-limiting examples: Synonyms and orthographic variations are indicated in brackets. Homophones are repeated the various times they occur (e.g., D, D, d). Unnamed phages are indicated by "NN" next to their genus and their number in brackets.

[0098] Bacteria of the genus Actinomyces can be infected by the following phages: Av-I, Av-2, Av-3, BF307, CT1, CT2, CT3, CT4, CT6, CT7, CT8 and 1281.

[0099] Bacteria of the genus Aeromonas can be infected by the following phages: AA-I, Aeh2, N, PMl, TP446, 3, 4, 11, 13, 29, 31, 32, 37, 43, 43-10T, 51, 54, 55R.1, 56, 56RR2, 57, 58, 59.1, 60, 63, Aehl, F, PM2, 1, 25, 31, 40RR2.8t, (syn= 44R), (syn= 44RR2.8t), 65, PM3, PM4, PM5 and PM6.

[0100] <h2 style=";text-align:left;direction:ltr">A、aizl、Al-KI、B 、BCJAl、BCl、BC2、BLLl、BLl、BP142、BSLl、BSL2、BS l、BS3、BS8、BS15、BS18、BS22、BS26、BS28、BS31、BS104、BS105、BS106、BTB、B1715V1、C、CK-I、Coll、Co rl, CP-53, CS-I, Csi, D, D, D, D5, entl, FP8, FP9, Fsi, FS2, FS3, FS5, FS8, FS9, G, GH8, GT8, GV-I, GV-2, GT-4, g3, gl2, gl3, gl4, gl6, gl7, g21, g23, g24, g29, H2, kenl, KK-88, Kuml, Kyul, J7W-1, LP52 (syn= LP-52)、L7、Mexl、MJ-I、mor2、MP-7、MplO、MP12、MP14、MP15、Neol、N° 2、N5、N6P、PBCl、PBLA、PBPl、P2、Sa、SF2、SF6、Shal、Sill、SP02、(syn= ΦSPP1)、SPβ、STI、Sti、SU-Il、t、TbI、Tb2、Tb5、TbIO、Tb26、Tb51、Tb53、Tb55、 Tb77、Tb97、Tb99、Tb560、Tb595、Td8、Td6、Tdl5、TgI、Tg4、Tg6、Tg7、Tg9、TgIO、 TgIl、Tgl3、Tgl5、Tg21、Tinl、Tin7、Tin8、Tinl3、Tm3、Tocl、Togl、toll、TP-I 、TP-10vir、TP-15c、TP-16c、TP-17c、TP-19、TP35、TP51、TP-84、Tt4、Tt6、type A、type B、type C、type D、type E、Tφ3、VA-9、W、wx23、wx26、Yunl、α、γ、pl l、φmed-2、φT、φμ-4、φ3T、φ75、φlO5、(syn= φlO5)、IA、IB、1-97A、1-97B、2、2、3、3、3、5、12、14、20、30、35、36、37、38、41C、51、6 3、64、138D、I、II、IV、NN-バチルス(13)、alel、Arl、AR2、AR3、AR7、AR9、Bace-11、(syn=11) Bastille BLl BL2 BL3 BL4 BL5 BL6 BL8 BL9 BP1 BS28, BS80, Ch, CP-51, CP-54, D-5, darl, denl, DP-7, entl. FoSi, FoS2, FS4, FS6, FS7, G, gall, gamma, Gel, GF-2, Gsi, GT -I、GT-2、GT-3、GT-4、GT-5、GT-6、GT-7、GV-6、gl5、19、110、I si, K, MP9, MP13, MP21, MP23, MP24, MP28, MP29, MP30, MP32, M P34, MP36, MP37, MP39, MP40, MP41, MP43, MP44, MP45, MP47, M P50, NLP-I, No.l, N17, N19, PBSl, PKl, PMBl, PMB12, PMJl, S. SPOl, SP3, SP5, SP6, SP7, SP8, SP9, SplO, SP-15, SP50 (syn= SP-50) SP82 SST subl SW Tg8 Tgl2 Tgl3 Tgl4 thul thuΛ thuS Tin4 Tin23 TP-13 TP33 TP50 TSP-I type V type VI, V, Vx, β22, φe, φNR2, φ25, φ63, 1, 1, 2, 2C, 3NT, 4, 5, 6 7, 8, 9, 10, 12, 12, 17, 18, 19, 21, 138, III, 4(B. megateriwn))、4(B.セァリス(B. sphaericus)) AR13 BPP-IO BS32 BS107 Bl B2 GA-I GP-IO GV-3 GV-5 g8 MP20 MP27 MP49N f PP5, PP6, SF5, Tgl8, TP-I, Versailles, φl5, φ29, 1-97, 837 / IV, mι-バチルス(1), BatlO, BSLlO, BSLI l, BS6, BSI l, BS16, BS23, BslOl, BS102, gl8, morl, PBLl, SN45, thu2, thu3, TmI, Tm2, ​​TP-20, TP21, TP52, type F, type G, type IV, HN-BacMus(3), BLE (syn=θc), BS2, BS4, BS5, BS7, BlO, B12, BS20, BS21, F, MJ-4, PBA12, AP50, AP50-04, AP50-11, AP50-23, AP50-26, AP50-27 and Bam35. The following Bacillus specific phages were deleted: DLP10716, DLP-11946, DPB5, DPB12, DPB21, DPB22, DPB23, GA-2, M, No. IM, PBLB, PBSH, PBSV, PBSW, PBSX, PBSY, PBSZ, phi, Spa, type 1 and μ.

[0101] Bacteria of the genus Bacteriodes can be infected by the following phages: crAss-phage, ad I2, Baf-44, Baf-48B, Baf-64, Bf-I, Bf-52, B40-8, Fl, βl, φAl, φBrOl, φBrO2, 11, 67.1, 67.3, 68.1, mt-Bacteroides (3), Bf42, Bf71, HN-Bdellovibrio (1), and BF-41.

[0102] Bacteria of the genus Bordetella can be infected by the following phages: 134 and NN-Bordetella (3).

[0103] Bacteria of the genus Borrellia can be infected by the following phages: NN-Borrellia (1) and NN-Borrellia (2).

[0104] Bacteria of the Brucella genus can be infected by the following phages: A422, Bk, (syn= Berkeley), BM29, Foi, (syn= Fol), (syn= FQl), D, FP2, (syn= FP2), (syn= FD2), Fz, (syn= Fz75 / 13), (syn= Firenze 75 / 13), (syn= Fi), Fi, (syn= Fl), Fim, (syn= Fim), (syn= Fim), FiU, (syn= FlU), (syn= FiU), F2, (syn= F2), F3, (syn= F3), F4, (syn= F4), F5, (syn= F5), F6, F7, (syn= F7), F25, (syn= F25), (syn= £25), F25U, (syn= F25u), (syn= F25U), (syn= F25V), F44, (syn- F44), F45, (syn= F45), F48, (syn= F48), I, Im, M, MC / 75, M51, (syn= M85), P, (syn= D),S708,R,Tb,(syn= TB),(syn= Tbilisi),W,(syn= Wb),(syn= Weybridge),X,3,6,7,10 / 1,(syn= 10),(syn= F8),(syn= F8),12m,24 / 11,(syn= 24),(syn= F9), (syn= F9), 45 / 111, (syn= 45), 75, 84, 212 / XV, (syn= 212), (syn= Fi0), (syn= FlO), 371 / XXIX, (syn= 371), (syn= Fn), (syn= Fl l) and 513.

[0105] Bacteria of the genus Burkholderia can be infected by the following phages: CP75, NN-Burkholderia (1) and 42.

[0106] Bacteria of the Campylobacter genus can be infected by the following phages: C type, NTCC12669, NTCC12670, NTCC12671, NTCC12672, NTCC12673, NTCC12674, NTCC12675, NTCC12676, NTCC12677, NTCC12678, NTCC12679, NTCC12680, NTCC12681, NTCC12682, NTCC12683, NTCC12684, 32f, 111c, 191, NN-Campylobacter (2), Vfi-6, (syn= V19), VfV-3, V2, V3, V8, V16, (syn= Vfi-1), V19, V20 (V45), V46, (syn= Vfi-2), V5, (syn= V5-1), V6, (syn= V6-2), V7, (syn= V7-3), V8, (syn= V7-4), V9, (syn= V9-5), V10, (syn= V9-6), V11, (syn= V9-7), V12, (syn= V9-8), V13, (syn= V9-9), V14, (syn= V9-1), V15, (syn= V9-1), V16, (syn= V9-1), V17, (syn= V9-2), V18, (syn= V19-3), V19, (syn= V19-4), V20 (syn= V19-5), V21, (syn= V19-5), V22, (syn= V19-5), V23, (syn= V13, V13, V14, (syn= V13, V1 V-45) and NN-Campylobacter (1).

[0107] Bacteria of the genus Chlamydia can be infected by the following phages: Chpl.

[0108] Bacteria of the genus Clostridium can be infected by the following phages: CAKl, CA5, Ca7, Ceβ, (syn= 1C), Ceγ, Cldl, c-n71, c-203 Tox-, Deβ, (syn= ID), (syn= lDt0X+), HM3, KMl, KT, Ms, Nal, (syn= Naltox+), PA135Oe, Pfo, PL73, PL78, PL81, Pl, P50, P5771, P19402, lCt0X+, 2Ct0X\ 2D3 (syn= 2Dt0X+), 3C, (syn= 3Ctox+), 4C, (syn= 4Ct0X+), 56, III-l, NN-Clostridium (61), NBlt0X+, αl, Cal, HMT, HM2, PFl5 P-23, P-46, Q-05, Q-oe, Q-16, Q-21, Q-26, Q-40, Q-46, S111, SA02, WA01, WA03, Wm, W523, 80, C, CA 2, CA3, CPTl, CPT4, cl, c4, c5, HM7, H11 / A1, H18 / Ax, FWS23, Hi58ZA1, K2ZA1, K21ZS23, ML, NA2t0X; Pf2, Pf3, Pf4, S9ZS3, S41ZA1, S44ZS23, α2, 41, 112ZS23, 214 / S23, 233 / Ai, 234 / S23, 235 / S23, II-l, II-2, II-3, NN-Clostridium (12), Cal, Fl, K, S2, 1, 5 and NN-Clostridium (8).

[0109] Bacteria of the genus Corynebacterium can be infected by the following phages: CGKl (deletion), A, A2, A3, AlOl, A128, A133, A137, A139, A155, A182, B, BF, B17, B18, B51, B271, B275, B276, B277, B279, B282, C, capi, CCl, CGl, CG2, CG33, CL31, Cog, (syn= CG5), D, E, F, H, HI, hqi, hq2, 11ZH33, Ii / 31, J, K, K, (syn= Ktox"), L, L, (syn= L), IL, L ... Ltox+), M, MC-I, MC-2, MC-3, MC-4, MLMa, N, O, ovi, ov2, ov3, P, P, R, RP6, RS29, S, T, U, UB1, ub2, UH1, UH3, uh3, uh5, uh6, β, (syn= βtox+), βhv64, βvir, γ, (syn= γtoχ-), γl9, δ, (syn= δ'ox+), p, (syn= ptoχ-), Φ9, φ984, ω, IA, 1 / 1180, 2, 2 / 1180, 5 / 1180, 5ad / 9717, 7 / 4465, 8 / 4465, 8ad / 10269, 10 / 9253, 13Z9253, 15 / 3148, 21 / 9253, 28, 29, 55, 2747, 2893, 4498 and 5848.

[0110] Bacteria of the genus Enterococcus are infected by the following phages: DF78, Fl, F2, 1, 2, 4, 14, 41, 867, Dl, SB24, 2BV, 182, 225, C2, C2F, E3, E62, DS96, H24, M35, P3, P9, SblOl, S2, 2BII, 5, 182a, 705, 873, 881, 940, 1051, 1057, 21096C, NN-Enterococcus (1), Pel, Fl, F3, F4, VD13, 1, 200, 235 and 341.

[0111] Bacteria of the genus Eysipelothrix can be infected by the following phages: NN-Eiysipelothrix (1).

[0112] Bacteria of the genus Escherichia can be infected by the following phages: BW73, B278, D6, D108, E, El, E24, E41, FI-2, FI-4, FI-5, HI8A, Ffl8B, i, MM, Mu, (syn= mu), (syn= MuI), (syn= Mu-I), (syn= MU-I), (syn= MuI), (syn= μ), 025, PhI-5, Pk, PSP3, Pl, PlD, P2, P4(deletion), Sl, Wφ, φK13, φR73(deletion), φl, φ2, φ7, φ92, ψ(deletion), 7 A, 8φ, 9φ, 15 (deletion), 18, 28-1, 186, 299, HH-Escherichia (2), AB48, CM, C4, C16, DD-VI, (syn= Dd-Vi), (syn= DDVI), (syn= DDVi), E4, E7, E28, fIl, FI3, H, Hl, H3, H8, K3, M, N, ND-2, ND-3, ND4, ND-5, ND6, ND-7, Ox-I (syn= oXl), (syn= HF), Ox-2 (syn= 0x2), (syn= 0X2), Ox-3, Ox-4, Ox-5, (syn= 0X5), Ox-6, (syn= 66F), (syn= φ66t), (syn= φ66t-)5 0111, PhI-I, RB42, RB43, RB49, RB69, S, SaI-I, Sal-2, Sal-3, Sal-4, Sal-5, Sal-6, TC23, TC45, TuII * -6, (syn= TuII * ), TuIP-24, TuII *46、TuIP-60、T2、(syn= ganuTia)、(syn= γ)、(syn= PC)、(syn= P.C.)、(syn= T-2)、(syn= T2)、(syn= P4)、T4、(syn= T-4)、(syn= T4)、T6、T35、αl、1、IA、3、(syn= Ac3)、3A、3T+、(syn= 3)、(syn= Ml)、5φ、(syn= φ5)、9266Q、CFO103、HK620、J、K、KlF、m59、no. A、no. E、no. 3、no. 9、N4、sd、(syn= Sd)、(syn= SD)、(syn= Sa)3 (syn= sd)、(syn= SD)、(syn= CD)、T3、(syn= T-3)、(syn= T3)、T7、(syn= T-7)、(syn= T7)、WPK、W31、ΔH、φC3888、φK3、φK7、φK12、φV-1、Φ04-CF、Φ05、Φ06、Φ07、φl、φl.2、φ20、φ95、φ263、φlO92、φl、φll、(syn= φW)、Ω8、1、3、7、8、26、27、28-2、29、30、31、32、38、39、42、933W、NN-エシェリキア(1)、Esc-7-11、AC30、CVX-5、Cl、DDUP、eCl、EC2、E21、E29、Fl、F26S、F27S、Hi、HK022、HK97、(syn= ΦHK97)、HK139、HK253、HK256、K7、ND-I、no.D, PA-2, q, S2, Tl, (syn= α), (syn= P28), (syn= TI), (syn= Tx), T3C, T5, (syn= T-5), (syn= T5), UC-I, w, β4, γ2, λ(syn= lambda), (syn= Φλ), ΦD326, φγ, Φ06, Φ7, Φ10, φ80, χ, (syn= χi), (syn= φχ), (syn= φχi), 2, 4, 4A, 6, 8A, 102, 150, 168, 174, 3000, AC6, AC7, AC28, AC43, AC50, AC57, AC81, AC95, HK243, KlO, ZG / 3A, 5, 5A, 21EL, H19-J, 933H, O157 typing phages 1-16, JES-2013, 121Q, 172-1, 1720a-02, ADB-2, AKVF33, av-05, bV_EcoS_AHP42, bV_EcoS_AHP24, bC_EcoS_AHS24, bV_EcoS_AKS96 and CBA120.

[0113] Bacteria of the genus Fusobacterium are infected by the following phages: NN-Fusobacterium (2), fv83-554 / 3, fv88-531 / 2, 227, fv2377, fv2527 and fv8501.

[0114] Bacteria of the genus Haemophilus are infected by the following phages: HP1, S2 and N3.

[0115] Bacteria of the genus Helicobacter are infected by the following phages: HPl and ^^-Helicobacter (1).

[0116] The bacteria of the genus Klebsiella are infected by the following phages: AIO-2, KI4B, Kl6B, Kl9, (syn= K19), Kl14, Kl15, Kl21, Kl28, Kl29, KI32, Kl33, Kl35, Kl106B, Kl171B, Kl181B, Kl832B, AIO-I, AO-I, AO-2, AO-3, FC3-10, K, Kl1, (syn= kIl), Kl2, (syn= K12), Kl3, (syn= K13), (syn= Kl 70 / 11), Kl4, (syn= K14), Kl5, (syn= K15), Kl6, (syn= K16), Kl7, (syn= K17), Kl8, (syn= K18), Kl19, (syn= K19), Kl27, (syn= K127), Kl31, (syn= K131), Kl35, Kl171B, II, VI, IX, CI-I, Kl4B, Kl8, Kl11, Kl12, Kl13, Kl16, Kl17, Kl18, Kl20, Kl22, Kl23, Kl24, Kl26, Kl30, Kl34, Kl106B, kIi65B, Kl328B, KLXI, K328, P5046, 11, 380, III, IV, VII, VIII, FC3-11, Kl2B, (syn= K12B), Kl25, (syn= K125), Kl42B, (syn= K142), (syn= K142B), Kl181B, (syn= kIl 81), (syn= K1181B), Kl765 / !, (syn= K1765 / 1), Kl842B, (syn= K1832B), Kl937B, (syn= K1937B), Ll, φ28, 7, 231, 483, 490, 632 and 864 / 100.

[0117] Bacteria of the genus Lepitospira are infected by the following phages: lEl, LE3, LE4 and NN-leptospira (1).

[0118] Listeria bacteria are infected by the following phages: A511, 01761, 4211, 4286, (syn=BO54), A005, A006, A020, A500, A502, A511, Al 18, A620, A640, B012, B021, B024, B025, B035, B051, B053, B054, B055, B056, BlOl, BI lO, B545, B604, B653, C707, D441, HSO47, HlOG, H8 / 73, H19, H21, H43, H46, H107, H108, HI lO, H163 / 84, H312, H340, H387, H391 / 73, H684 / 74, H924A, PSA, U153, φMLUP5, (syn= P35), 00241, 00611, 02971A, 02971C, 5 / 476, 5 / 911, 5 / 939, 5 / 11302, 5 / 11605, 5 / 11704, 184, 575, 633, 699 / 694, 744, 9 00, 1090, 1317, 1444, 1652, 1806, 1807, 1921 / 959, 1921 / 11367, 1921 / 11500, 1921 / 11566, 1921 / 12460, 1921 / 12582, 1 967, 2389, 2425, 2671, 2685, 3274, 3550, 3551, 3552, 4276, 4277, 4292, 4477, 5337, 5348 / 11363, 5348 / 11646, 5348 / 12430, 5348 / 12434, 10072, 11355C, 11711A, 12029, 12981, 13441, 90666, 90816, 93253, 907515, 910716, and NN-Listeria (15).

[0119] Bacteria of the genus Morganella are infected by the following phages:47.

[0120] Bacteria of the Mycobacterium genus are infected by the following phages: 13, aGl, aLi, ATCC 11759, A2, B.C3, BG2, BKl, BK5, butyricum, BI, B5, B7, B30, B35, Clark, Cl, C2, DNAIII, DSP1, D4, D29, GS4E, (syn= GS4E), GS7, (syn= GS-7), (syn= GS7), iPa, lacticola, Legendre, Leo, L5, (syn= ΦL-5), MC-I, MC-3, MC-4, minetti, MTPHI l, Mx4, MyF3P / 59a, phlei, (syn= phlei 1), phlei 4, Polonus II, rabinovitschi, smegmatis, TM4, TM9, tMlO, TM20, Y7, YlO, φ630, IB, IF, IH, 1 / 1, 67, 106, 1430, Bl, (syn= Bol), B24, D, D29, FK, FS, HP, Polonus I, Roy, Rl, (syn= Rl-Myb), (syn= Ri), 11, 31, 40, 50, 103a, 103b, 128, 3111-D, 3215-D and NN-Mycobacterium (1).

[0121] Bacteria of the genus Neisseria are infected by the following phages: Group I, group II and NP1.

[0122] Bacteria of the genus Nocardia are infected by the following phages: MNP8, NJ-L, NS-8, N5 and TtiN-Nocardia.

[0123] Bacteria of the genus Proteus are infected by the following phages: Pm5, 13vir, 2 / 44, 4 / 545, 6 / 1004, 13 / 807, 20 / 826, 57, 67b, 78, 107 / 69, 121, 9 / 0, 22 / 608, 30 / 680, PmI, Pm3, Pm4, Pm6, Pm7, Pm9, PmIO, PmI l, Pv2, πl, φm, 7 / 549, 9B / 2, 10A / 31, 12 / 55, 14, 15, 16 / 789, 17 / 971, 19A / 653, 23 / 532, 25 / 909, 26 / 219, 27 / 953, 32A / 909, 33 / 971, 34 / 13, 65, 5006M, 7480b, VI, 13 / 3a, Clichy 12, π2600, φχ7, 1 / 1004, 5 / 742, 9, 12, 14, 22, 24 / 860, 2600 / D52, Pm8 and 24 / 2514.

[0124] Bacteria of the genus Providencia are infected by the following phages: PL25, PL26, PL37, 9211 / 9295, 9213 / 921 Ib, 9248, 7 / R49, 7476 / 322, 7478 / 325, 7479, 7480, 9000 / 9402 and 9213 / 921 Ia.

[0125] Bacteria of the genus Pseudomonas are infected by the following phages: PfI, (syn = Pf-I), Pf2, Pf3, PP7, PRRl, 7s, im-Pseudomonas(1), AI-I, AI-2, B17, B89, CB3, Col2, Col11, Col18, Col21, C154, C163, C167, C2121, E79, F8, ga, gb, H22, K1, M4, N2, Nu, PB-I, (syn = PBl), pfl6, PMN17, PPl, PP8, Psal, PsPl, PsP2, PsP3, PsP4, PsP5, PS3, PS17, PTB80, PX4, PX7, PYO1, PYO2, PYO5, PYO6, PYO9, PYO10, PYO13, PYO14, PYO16, PYO18, PYO19, PYO20, PYO29, PYO32, PYO33, PYO35, PYO36, PYO37, PYO38, PYO39, PYO41, PYO42, PYO45, PYO47, PYO48, PYO64, PYO69, PYO103, PlK, SLPl, SL2, S2, UNL-I, wy, Yai, Ya4, Yan, φBE, φCTX, φC17, φKZ, (syn = ΦKZ), φ-LT, Φmu78, φNZ, φPLS-1, φST-1, φW-14, φ-2, 1 / 72, 2 / 79, 3, 3 / DO, 4 / 237, 5 / 406, 6C, 6 / 6660, 7, 7v, 7 / 184, 8 / 280, 9 / 95, 10 / 502, 11 / DE, 12 / 100, 12S, 16, 21, 24, 25F, 27, 31, 44, 68, 71, 95, 109, 188, 337, 352, 1214, HN-Pseudomonas(23), A856, B26, CI-I, CI-2, C5, D, gh-1, Fl16, HF, H90, K5, K6, Kl04, K109, K166, K267, N4, N5, O6N-25P, PE69, Pf, PPN25, PPN35, PPN89, PPN91, PP2, PP3, PP4, PP6, PP7, PP8, PP56, PP87, PPl14. PP206, PP207, PP306, PP651, Psp231a, Pssy401, Pssy 9220 psi PTB2 PTB20 PTB42 PXl PX3 pXlO PX12 PX14 PYO70, PYO71, R, SH6, SH133, tf, Ya5, Ya7, φBS, ΦKf77, φ- MC, ΦmnF82, φPLS27, φPLS743, φS-1, 1, 2, 2, 3, 4, 5, 6, 7, 7 8, 9, 10, 11, 12, 12B, 13, 14, 15, 14, 15, 16, 17, 18, 19, 20 20, 21, 21, 22, 23, 23, 24, 25, 31, 53, 73, 119x, 145, 0, 267, 284, 308, 525, NN-Ch BI-I, C22, D3, D37, D40, D62, D3112, F7, FlO, gd, ge, gξ Hwl2, Jb 19. KFl, L°, OXN-32P, O6N-52P, PCH-I, PC13-1, PC35-1, PH2, PH51 PH93, PH132, PMW, PM13, PM57, PM61, PM62, PM63, PM69, PM105, PMl 13 PM681, PM682, PO4, PPl, PP4, PP5, PP64, PP65, PP66, PP71, PP86, PP88, PP92, PP401, PP711, PP891 Pssy41, Pssy42, Pssy403, Pssy404, Pssy420, Pssy923, PS4, PS-IO, Pz, SDl, SLl, SL3, SL5, SM, φC5, φCl l, φCl l-1, φC13, φC15, φMO, φX, φO4, φl l φ240, 2, 2F, 5, 7m, 11, 13, 13 / 441, 14, 20, 24, 40, 45, 49, 61, 73, 148, 160, 198, 218, 222, 236, 242, 246, 249, 258, 269, 295, 297, 309, 318, 342, 350, 351, 357-1, 400-1, HN- GlOl, M6, M6a, Ll, PB2, Pssyl5, Pssy4210, Pssy4220, PYO12, PYO34, PYO4 9. PYO50, PYO51, PYO52, PYO53, PYO57, PYO59, PYO200, PX2, PX5, SL4, φO3, φO6, and 31214.

[0126] Bacteria of the genus Rickettsia are infected by the following phages: NN-Rickettsia.

[0127] Bacteria of the genus Salmonella are infected by the following phages: b, Beccles, CT, d, Dundee, f, FeIs 2, GI, GUI, GVI, GVIII, k, K, i, j, L, 01, (syn= 0-1), (syn= O1), (syn= OI), (syn= 7), 02, 03, P3, P9a, PlO, Sab3, Sab5, SanlS, Sanl7, SI, Taunton, ViI, (syn= ViI), 9, im Salmonella (1), NI, N-5, N-IO, N-17, N-22, 11, 12, 16-19, 20.2, 36, 449C / C17 8, 966A / C259, a, BAOR, e, G4, GUI, L, LP7, M, MG40, N-18, PSA68, P4, P9c, P22, (syn= (syn= φl

[40] ), 1,422, 2, 2.5, 3b, 4, 5, 6,14(18), 8, 14(6,7), 10, 27, 28B, 30, 31, 32, 33 , 34, 36, 37, 39, 1412, SNT-3, 7-11, 40.3, c, C236, C557, C625, C966N, g, GV, G5, Gl 73, h, IRA, Jersey, MB78, P22-1, P22-3, P22-12, Sabl, Sab2, Sab2, Sab4, Sanl, San2, San3, San4, San6, San7, San8, San9, Sanl3, Sanl4, Sanl6, Sanl8, Sanl9, San20, San21, San22, San23, San24, San25, San26, SasLl, SasL2, SasL3, SasL4, SasL5, SlBL, SII, ViII, φl, 1, 2, 3a, 3al, 1010, Ym-Salmonella (1), N-4, SasL6 and 27.

[0128] Serratia bacteria are infected by the following phages: A2P, PS20, SMB3, SMP, SMP5, SM2, V40, V56, ic, ΦCP-3, ΦCP-6, 3M, 10 / la, 20A, 34CC, 34H, 38T, 345G, 345P, 501B, SMB2, SMP2, BC, BT, CW2, CW3, CW4, CW5, Lt232, L2232, L34, L.228, SLP, SMPA, V. .43, σ, φCWl, ΦCP6-1, ΦCP6-2, ΦCP6-5, 3T, 5, 8, 9F, 10 / 1, 2OE, 32 / 6, 34B, 34CT, 34P, 37, 41, 56, 56D, 56P, 6OP, 6 1 / 6, 74 / 6, 76 / 4, 101 / 8900, 226, 227, 228, 229F, 286, 289, 290F, 512, 764a, 2847 / 10, 2847 / 1Oa, L.359 and SMBl.

[0129] You can also use the following expression: Fsa(syn= a)(syn=d=(FSD=2d)2d). W2d). HVIII)、SKγ66、(syn= gamma 66)、(syn= yββ)、(syn= γ66b)、SKm、(syn= SIIIb)5 (syn= UI)、SKw、(syn=SIII)、(S=(syn=IC)、、(Syn=IC IV). SIVA.) (syn= IVA)、sKvi、(syn= KVI)、(syn= Svi)、(syn= Svm)、(syn= VIII)、SKVΠIA、(syn= SvmA=)、(syn= SvmA=)、 VIIIA). K29)、FlO、(syn= fSlO)、(syn= K31)、(syn= alpha)、(syn= fSa)、(syn= Kl 8)、(syn= α)、I2、(syn= a)、、、= K19)、、= K19 G35). K2)、(syn= KII)、(syn= Sn)、(syn= II)、(syn= Sm)、(syn= SsIV)、(syn= IV)、SK1Va、(syn= Swab)(sIVa=syn=) iV T35)、(syn= 35-50-R)、STvm、(syn=T8345)、(syn= 8345-SO-SR)、W1、(syn= D8)、(syn= FSD8)、W2a、(syn= D2A)、(syn= FS2a)、DD-2、Sf6、fSi、、=F F6). 1881-SO-R)、γ66、(syn= gamma 66a)、(syn= Ssγ66)、φ2、bIl、DDVII、(syn= DD7)、FSD2b、(syn= W2B)、=FS2、(syn=Fsyn=2、). F2)、FS4、(syn= F4)、(syn= F4)、FS5、(syn= F5)、(syn= F5)、FS9、(syn= F9)、(syn= F9)、FI l、P2-S0-S、(syn=G6)-SO、(syn=G36)、 G36). HXn)、SKI、KI、(syn= S1)、(syn= SsI)、(syn= Svπ)、(syn= SsVII)、(syn= KIX)、(syn= S1x) SsIX)、SKXII、(syn= KXII)、(syn= Sxn)、(syn= SsXII)、sTi、STffl、STrv、STVi、STvπ、S70、S206、U2-S0-S、3 210-SO-S、3859-SO-S、4020-SO-S、φ3、φ5、φ7、φ8、ϕ9、φlO、ϕl l、φl3、ϕl4、φl8、SHm、(syn= Hπi)、sHχi、(syn= HXt)、(syn= Sχi)、(syn= Sχi)、(syn=X=I ) Ss

[0130] <h2 style=";text-align:left;direction:ltr">A、EW、K、Ph5、Ph9:A、EW、K、Ph5、Ph9 (syn= Sb-I)、S3K、Twort、ΦSK311、φ812、06、40、58、119、130、131、200、1623、STCl、(syn= stcl)、STC2、(syn= stc2)、44AHJD、68、aCl、AC2、A6"C"、A9"C"、b581、CA-I、CA-2、CA-3、CA-4、CA-5、DI l, L39x35, L54a, M42, Nl, N2, N3, N4, N5, N7, N8, NlO, Ni l, N12, N13, N14, N16, Ph6, Phl2, Phl4, UC-18, U4, U15, Sl, S2, S3, S4, S5, X2, Z1, φB5-2, φD, ω, 11, (syn= φl l), (syn= P11-M15)、15、28、28A、29、31、31B、37、42D、(syn= P42D)、44A、48、51、52、52A、(syn= P52A)、52B、53、55、69、71、(syn= P71)、71A、72、75、76、77、79、80、80α、82、82A、83 A、84、85、86、88、88A、89、90、92、95、96、102、107、1 08、111、129-26、130、130A、155、157、157A、165、187 、275、275A、275B、356、456、459、471、471A、489、58 1、676、898、1139、1154A、1259、1314、1380、1405、15 63、2148、2638A、2638B、2638C、2731、2792A、2792B、2818、2835、2848A、3619、5841、12100、AC3、A8、AlO A13, b594n, D, HK2, N9, N15, P52, P87, Sl, S6, Z4, φRE, 3A, 3B, 3C, 6, 7, 16, 21, 42B, 42C, 42E, 44, 47, 47A5 47C、51、54、54x1、70、73、75、78、81、82、88、93、94、1 01、105、110、115、129 / 16、174、594n、1363 / 14、2460 and mS-スタフィロコッカス(1)。<h2 style=";text-align:left;direction:ltr">

[0131] Bacteria of the genus Streptococcus are infected by the following phages: EJ-I, NN-Streptococcus (1), a, Cl, FL0Ths, H39, Cp-I, Cρ-5, Cp-7, Cp-9, Cp-IO, AT298, A5, alO / Jl, alO / J2, alO / J5, alO / J9, A25, BTI l, b6, cAl, c20-l, c20-2, DP-I, Dp-4, DTl, ET42, elO, FA101, FEThs, Fκ, FKKIOI, FKLIO, FKP74, FKH, FLOThs, FyIOl, fl, F10, F20140 / 76, g, GT-234, HB3, (syn= HB-3), HB-623, HB-746, M102, O1205, φO1205, PST, PO, Pl, P2, P3, P5, P6, P8, P9, P9, P12, P13, P14, P49, P50, P5 1, P52, P53, P54, P55, P56, P57, P58, P59, P64, P67, P69, P71, P73, P75, P76, P77, P82, P83, P88, sc, sch, sf, SfIl 1, (syn= sFiI l), (syn= φSFill), (syn= φSfil l), (syn= φSfil l), sfil9, (syn= sFil9), (syn= φSFil9), (syn= φSfil9), Sfi21, (syn= sFi21), (syn= φSFi21), (syn= φSfi21), ST0, STX, st2, ST2, ST4, S3, (syn=φS3), s265, φ17, φ42, φ57, φ80, φ81, φ82, φ83, φ84, φ85, φ86, φ87, φ88, φ89, φ90, φ91, φ92, φ93, φ94, φ95, φ96, φ97 , φ98, φ99, φlOO, φlOl, φlO2, φ227, φ7201, ωl, ω2, ω3, ω4, ω5, ω6, ω8, ωlO, 1, 6, 9, 1OF, 12 / 12, 14, 17SR, 19S, 24, 50 / 33, 50 / 34, 55 / 14, 55 / 15, 70 / 35, 70 / 36, 71 / ST15, 71 / 45, 71 / 46, 74F, 79 / 37, 79 / 38, 80 / J4, 80 / J9, 80 / ST16, 80 / 15, 80 / 47, 80 / 48, 101, 103 / 39, 103 / 40, 121 / 41, 121 / 42, 123 / 43, 123 / 44, 124 / 44, 337 / ST17, and mStreptococcus (34).

[0132] Bacteria of the genus Treponema are infected by the following phages: NN-Treponema (1).

[0133] The structure of the solvent is as follows: CTXΦ and fs (syn= si) fs2, Ivpf5, Vfl2, Vf33, VPIΦ, VSK, v6, 493, CP-Tl, ET25, kappa, K13 9. Labol) XN-69P, OXN-86, O6N-21P, PB-I, P147, rp-1, SE3, VA-I (syn= VcA-I), VcA-2, VPl, VP2, VP4, VP7, VP8, VP9, ​​vPlO, VP17, VP18, VP19, X29 (syn= 29). d'Herelle)、t、ΦHAWI-1、ΦHAWI-2、ΦHAWI-3、ΦHAWI-4、ΦHAWI-5、ΦHAWI-6、ΦHAWI-7、 SHOW-8, ΦSHOW-9, ΦSHOW-10, ΦHCl-1, ΦHC1-2, ΦHC1-3, ΦHC1-4, ΦHC2-1,>HC2-2, ΦH C2-3, ΦHC2-4, ΦHC3-1, ΦHC3-2, ΦHC3-3, ΦHD1S-1, ΦHD1S-2, ΦHD2S-1, ΦHD2S-2, ΦHD2 S-3, ΦHD2S-4, ΦHD2S-5, ΦHDO-1, ΦHDO-2, ΦHDO-3, ΦHDO-4, ΦHDO-5, ΦHDO-6, ΦKL-33, Φ KL-34, ΦKL-35, ΦKL-36, ΦKWH-2, ΦKWH-3, ΦKWH-4, ΦMARQ-1, ΦMARQ-2, ΦMARQ-3, ΦMOA T-1, ΦO139, ΦPEL1A-1, ΦPEL1A-2, ΦPEL8A-1, ΦPEL8A-2, ΦPEL8A-3, ΦPEL8C-1, ΦPEL8C -2, ΦPEL13A-1, ΦPEL13B-1, ΦPEL13B-2, ΦPEL13B-3, ΦPEL13B-4, ΦPEL13B-5, ΦPEL1 B-6, ΦPEL13B-7, ΦPEL13B-8, ΦPEL13B-9, ΦPEL13B-10, φVP143, φVP253, Φ16, φl38, 1- II, 5, 13, 14, 16, 24, 32, 493, 6214, 7050, 7227 II (syn= group II) (syn=φ2), V, VIII, ~m-Vibrio (13), KVP20, KVP40, nt-1, O6N-22P, P68, el, e2, e3, e4, e5, FK, G, I, K, nt-6, Nl, N2, N3, N4, N5, O6N-34 P, OXN-72P, OXN-85P, OXN-100P, P, Ph-I, PL163 / 10, Q, S, T, φ92, 1-9, 37, 51, 57, 70A-8, 72A-4, 72A-10, 110A-4, 333, 4996, I (syn= group I), III (syn= group III), VI, (syn= A-Saratov), ​​VII, IX, lOA-1, 110A-5, 110A-7, hv-1, OXN-52P, P13, P38, P53, P65, P108, Pill, TPl3 VP3, VP6, VP12, VP13, 70A-3, 70A-4, 70A-10, 72A-1, 108A-3, 109-B1, 110A-2, 149, (syn= φl49), IV, (syn= group IV), NN-Vibrio (22), VP5, VP11, VP15, VP16, α1, α2, α3a, α3b, 353B and HN-Vibrio (7).

[0134] Bacteria of the genus Yersinia are infected by the following phages: H, HI, H-2, H-3, H-4, Lucas 110, Lucas 303, Lucas 404, YerA3, YerA7, YerA20, YerA41, 3 / M64-76, 5 / G394-76, 6 / C753-76, 8 / C239-76, 9 / F18167, 1701, 1710, PST, 1 / F2852-76, D'Herelle, EV, H, Kotljarova, PTB, R, Y, YerA41, φYerO3-12, 3, 4 / C1324-76, 7 / F783-76, 903, 1 / M6176 and Yer2AT.

[0135] More preferably, the bacteriophage used as the bacterial delivery vehicle is selected from the group consisting of Salmonella virus SKML39, Shigella virus AG3, Dickeja virus Limestone, Dickeja virus RC2014, Escherichia virus CBA120, Escherichia virus PhaxI, Salmonella virus 38, Salmonella virus Det7, Salmonella virus GG32, Salmonella virus PM10, Salmonella virus SFP10, Salmonella virus SH19, Salmonella virus SJ3, Escherichia virus ECML4, Salmonella virus ECML5, Salmonella virus ECML6, Salmonella virus ECML7, Salmonella virus ECML8, Salmonella virus ECML9, Salmonella virus ECML10, Salmonella virus ECML11, Salmonella virus ECML12, Salmonella virus ECML13, Salmonella virus ECML14, Salmonella virus ECML15, Salmonella virus ECML16, Salmonella virus ECML17, Salmonella virus ECML18, Salmonella virus ECML19, Salmonella virus ECML20, Salmonella virus ECML21, Salmonella virus ECML22, Salmonella virus ECML23, Salmonella virus ECML24, Salmonella virus ECML25, Salmonella virus ECML26, Salmonella virus ECML27, Salmonella virus ECML28, Salmonella virus ECML29, Salmonella virus ECML30, Salmonella virus ECML31, Salmonella virus ECML32, Salmonella virus ECML33, Salmonella virus ECML34, Salmonella virus ECML35, Salmonella virus ECML36, Salmonella virus ECML37, Salmonella virus ECML38, Salmonella virus ECML40, Salmonella virus ECML41, Salmonella virus ECML42 Salmonella Marshall, Salmonella virus Maynard, Salmonella virus SJ2, Salmonella virus STML131, Salmonella virus ViI, Erwinia virus Ea2809, Klebsiella virus 0507KN21, Serratia virus IME250, Serratia virus MAM1, Campylobacter virus CP21, Campylobacter virus CP220, Campylobacter virus CPt10, Campylobacter virus IBB35, Campylobacter virus CP81, Campylobacter virus CP30A, Campylobacter virus CP40A, Campylobacter virus CP50A, Campylobacter virus CP60A, Campylobacter virus CP70A, Campylobacter virus CP81B, Campylobacter virus CP90B, Campylobacter virus CP10C, Campylobacter virus CP11C, Campylobacter virus CP120C, Campylobacter virus CP130C, Campylobacter virus CP140C, Campylobacter virus CP150C, Campylobacter virus CP160C, Campylobacter virus CP170C, Campylobacter virus CP180C, Campylobacter virus CP190C, Campylobacter virus CP20C, Campylobacter virus CP21C, Campylobacter virus CP2 ... Rux CPX, Campylobacter virus NCTC12673, Erwinia virus Ea214, Erwinia virus M7, Escherichia virus AYO145A, Escherichia virus EC6, Escherichia virus HY02, Escherichia virus JH2, Escherichia virus TP1, Escherichia virus VpaE1, Escherichia virus wV8, Salmonella virus FelixO1, Salmonella virus HB2014, Salmonella virus Mushroom, Salmonella virus UAB87, Citrobacter virus Moogle, Citro Bactervirus Mordin, Escherichiavirus SUSP1, Escherichiavirus SUSP2, Aeromonasvirus phiO18P, Haemophilusvirus HP1, Haemophilusvirus HP2, Pasteurellavirus F108, Vibriovirus K139, Vibriovirus Kappa, Burkholderiavirus phi52237, Burkholderiavirus phiE122, Burkholderiavirus phiE202, Escherichiavirus 186, Escherichiavirus P4, Escherichiavirus P2, Escherichiavirus Wphi,Mannheimia virus PHL101, Pseudomonas virus phiCTX, Ralstonia virus RSA1, Salmonella virus Fels2, Salmonella virus PsP3, Salmonella virus SopEphi, Yersinia virus L413C, Staphylococcus virus G1, Staphylococcus virus G15, Staphylococcus virus JD7, Staphylococcus virus K, Staphylococcus virus MCE2014, Staphylococcus virus P108, Staphylococcus virus Rodi, Staphylococcus virus S2 53, Staphylococcal virus S25-4, Staphylococcal virus SA12, Listeria virus A511, Listeria virus P100, Staphylococcal virus Remus, Staphylococcal virus SA11, Staphylococcal virus Stau2, Bacillus virus Camphawk, Bacillus virus SPO1, Bacillus virus BCP78, Bacillus virus TsarBomba, Staphylococcal virus Twort, Enterococcal virus phiEC24C, Lactobacillus virus Lb338-1, Lactobacillus virus Bacillus virus LP65, Enterobacter virus PG7, Escherichia virus CC31, Klebsiella virus JD18, Klebsiella virus PKO111, Escherichia virus Bp7, Escherichia virus IME08, Escherichia virus JS10, Escherichia virus JS98, Escherichia virus QL01, Escherichia virus VR5, Enterobacter virus Eap3, Klebsiella virus KP15, Klebsiella virus KP27, Klebsiella virus Matisse, Klebsiella virus Miro, Cyto Citrobactervirus Merlin, Citrobactervirus Moon, Escherichiavirus JSE, Escherichiavirus phi1, Escherichiavirus RB49, Escherichiavirus HX01, Escherichiavirus JS09, Escherichiavirus RB69, Shigellavirus UTAM, Salmonellavirus S16, Salmonellavirus STML198, Vibriovirus KVP40, Vibriovirus nt1, Vibriovirus ValKK3, Escherichiavirus VR7, Escherichiavirus VR20, Escherichiavirus VR25,Escherichia virus VR26, Shigella virus SP18, Escherichia virus AR1, Escherichia virus C40, Escherichia virus E112, Escherichia virus ECML134, Escherichia virus HY01, Escherichia virus Ime09, Escherichia virus RB3, Escherichia virus RB14, Escherichia virus T4, Shigella virus Pss1, Shigella virus Shfl2, Yersinia virus D1, Yersinia virus PST, Acinetobacter virus 133, Aeromonas virus 65, Aeromonas virus Virus Aeh1, Escherichia virus RB16, Escherichia virus RB32, Escherichia virus RB43, Pseudomonas virus 42, Cronobacter virus CR3, Cronobacter virus CR8, Cronobacter virus CR9, Cronobacter virus PBES02, Pectobacterium virus phiTE, Cronobacter virus GAP31, Escherichia virus 4MG, Salmonella virus SE1, Salmonella virus SSE121, Escherichia virus FFH2, Escherichia virus FV3, Escherichia virus JES2013, Escherichia virus V5, Brevibacillus virus Abouo, Brevibacillus virus Davies, Bacillus virus Agate, Bacillus virus Bobb, Bacillus virus Bp8pC, Erwinia virus Deimos, Erwinia virus Ea35-70, Erwinia virus RAY, Erwinia virus Simmy50, Erwinia virus Special G, Acinetobacter virus AB1, Acinetobacter virus AB2, Acinetobacter virus AbC62, Acinetobacter virus A P22, Arthrobactervirus ArV1, Arthrobactervirus Trina, Bacillusvirus AvesoBmore, Bacillusvirus B4, Bacillusvirus Bigbertha, Bacillusvirus Riley, Bacillusvirus Spock, Bacillusvirus Troll, Bacillusvirus Bastille, Bacillusvirus CAM003, Bacillusvirus Bc431, Bacillusvirus Bcp1, Bacillusvirus BCP82, Bacillusvirus BM15, Bacillusvirus Deepblue, Bacillusvirus JBP901,Burkholderia virus Bcep1, Burkholderia virus Bcep43, Burkholderia virus Bcep781, Burkholderia virus BcepNY3, Xanthomonas virus OP2, Burkholderia virus BcepMu, Burkholderia virus phiE255, Aeromonas virus 44RR2, Mycobacterium virus Alice, Mycobacterium virus Bxz1, Mycobacterium virus Dandelion, Mycobacterium virus HyRo, Mycobacterium virus I3, Mycobacterium Mycobacterium virus Nappy, Mycobacterium virus Sebata, Clostridium virus phiC2, Clostridium virus phiCD27, Clostridium virus phiCD119, Bacillus virus CP51, Bacillus virus JL, Bacillus virus Shanette, Escherichia virus CVM10, Escherichia virus ep3, Erwinia virus Asesino, Erwinia virus EaH2, Pseudomonas virus EL, Halomonas virus HAP1, Vibrio virus VP882, Brevibacillus virus Jim mer, Brevibacillus virus Osiris, Pseudomonas virus Ab03, Pseudomonas virus KPP10, Pseudomonas virus PAKP3, Sinorhizobium virus M7, Sinorhizobium virus M12, Sinorhizobium virus N3, Erwinia virus Machina, Arthrobacter virus Brent, Arthrobacter virus Jawnski, Arthrobacter virus Martha, Arthrobacter virus Sonny, Edwardsiella virus MSW3, Edwardsiella virus PEi21, Escherichia virus Mu, Shigella virus SfMu, Halobacterium virus phiH, Bacillus virus Grass, Bacillus virus NIT1, Bacillus virus SPG24, Aeromonas virus 43, Escherichia virus P1, Pseudomonas virus CAb1, Pseudomonas virus CAb02, Pseudomonas virus JG004, Pseudomonas virus PAKP1, Pseudomonas virus PAKP4, Pseudomonas virus PaP1, Burkholderia virus BcepF1, Pseudomonas virus 141, Pseudomonas virus Ab28,Pseudomonas virus DL60, Pseudomonas virus DL68, Pseudomonas virus F8, Pseudomonas virus JG024, Pseudomonas virus KPP12, Pseudomonas virus LBL3, Pseudomonas virus LMA2, Pseudomonas virus PB1, Pseudomonas virus SN, Pseudomonas virus PA7, Pseudomonas virus phiKZ, Rhizobium virus RHEph4, Ralstonia virus RSF1, Ralstonia virus RSL2, Ralstonia virus RSL1, Aeromonas virus 25, A Aeromonas virus 31, Aeromonas virus Aes12, Aeromonas virus Aes508, Aeromonas virus AS4, Stenotrophomonas virus IME13, Staphylococcus virus IPLAC1C, Staphylococcus virus SEP1, Salmonella virus SPN3US, Bacillus virus 1, Geobacillus virus GBSV1, Yersinia virus R1RT, Yersinia virus TG1, Bacillus virus G, Bacillus virus PBS1, Microcystis virus Ma-LMM01, Vibrio virus MAR, Vibriovirus Acinetobacter virus VHML, Vibriovirus VP585, Bacillus virus BPS13, Bacillus virus Hakuna, Bacillus virus Megatron, Bacillus virus WPh, Acinetobacter virus AB3, Acinetobacter virus Abp1, Acinetobacter virus Fri1, Acinetobacter virus IME200, Acinetobacter virus PD6A3, Acinetobacter virus PDAB9, Acinetobacter virus phiAB1, Escherichia virus K30, Klebsiella virus K5, Klebsiella virus K11, Klebsiella Virus Kp1, Klebsiella virus KP32, Klebsiella virus KpV289, Klebsiella virus F19, Klebsiella virus K244, Klebsiella virus Kp2, Klebsiella virus KP34, Klebsiella virus KpV41, Klebsiella virus KpV71, Klebsiella virus KpV475, Klebsiella virus SU503, Klebsiella virus SU552A, Pantoea virus Limelight, Pantoea virus Limezero, Pseudomonas virus LKA1, Pseudomonas virus phiKMV,Xanthomonas virus f20, Xanthomonas virus f30, Xylella virus Prado, Erwinia virus Era103, Escherichia virus K5, Escherichia virus K1-5, Escherichia virus K1E, Salmonella virus SP6, Escherichia virus T7, Kluyvera virus Kvp1, Pseudomonas virus gh1, Prochlorococcus virus PSSP7, Synechococcus virus P60, Synechococcus virus Syn5, Streptococcus Cassvirus Cp1, Streptococcusvirus Cp7, Staphylococcusvirus 44AHJD, Streptococcusvirus C1, Bacillusvirus B103, Bacillusvirus GA1, Bacillusvirus phi29, Kurthiavirus 6, Actinomycesvirus Av1, Mycoplasmavirus P1, Escherichiavirus 24B, Escherichiavirus 933W, Escherichiavirus Min27, Escherichiavirus PA28, Escherichiavirus Stx2 II, Shigella virus 7502Stx, Shigella virus POCJ13, Escherichia virus 191, Escherichia virus PA2, Escherichia virus TL2011, Shigella virus VASD, Burkholderia virus Bcep22, Burkholderia virus Bcepil02, Burkholderia virus Bcepmigl, Burkholderia virus DC1, Bordetella virus BPP1, Burkholderia virus BcepC6B, Cernophaga virus Cba41, Cernophaga virus Cba172, Dinortheobacter virus DFL12, Erwinia virus Ea9-2, Erwinia virus Rus Frozen, Escherichia virus phiV10, Salmonella virus Epsilon15, Salmonella virus SPN1S, Pseudomonas virus F116, Pseudomonas virus H66, Escherichia virus APEC5, Escherichia virus APEC7, Escherichia virus Bp4, Escherichia virus EC1UPM, Escherichia virus ECBP1, Escherichia virus G7C, Escherichia virus IME11, Shigella virus Sb1, Achromobacter virus Axp3, Achromobacter virus JWAlpha, Edwardsiella virus KF1, Pseudomonas virus KPP25,Pseudomonas virus R18, Pseudomonas, Virus Ab09, Pseudomonas virus LIT1, Pseudomonas virus PA26, Pseudomonas virus Ab22, Pseudomonas virus CHU, Pseudomonas virus LUZ24, Pseudomonas virus PAA2, Pseudomonas virus PaP3, Pseudomonas virus PaP4, Pseudomonas virus TL, Pseudomonas virus KPP21, Pseudomonas virus LUZ7, Escherichia virus N4, Salmonella virus 9NA, Salmonella virus SP069, Salmonella virus BTP1, Salmonella virus HK620, Salmonella Bacillus virus P22, Salmonella virus ST64T, Shigella virus Sf6, Bacillus virus Page, Bacillus virus Palmer, Bacillus virus Pascal, Bacillus virus Pony, Bacillus virus Pookie, Escherichia virus 172-1, Escherichia virus ECB2, Escherichia virus NJ01, Escherichia virus phiEco32, Escherichia virus Septima11, Escherichia virus SU10, Brucella virus Pr, Brucella virus Tb, Escherichia virus Pollock, Salmonella virus FSL SP-058, Salmonella virus FSL SP-076, Helicobacter virus 1961P, Helicobacter virus KHP30, Helicobacter virus KHP40, Hamiltonella virus APSE1, Lactococcus virus KSY1, Phormidium virus WMP3, Phormidium virus WMP4, Pseudomonas virus 119X, Roseobacter virus SIO1, Vibrio virus VpV262, Vibrio virus VC8, Vibrio virus VP2, Vibrio virus VP5, Streptomyces virus Amela, Streptomyces virus phiCAM, Streptomyces virus Aaronocolus, Streptomyces virus Caliburn, Streptomyces virus Danzina, Streptomyces virus Hydra, Streptomyces virus Izzy, Streptomyces virus Lannister, Streptomyces virus Lika, Streptomyces virus Sujidade, Streptomyces virus Zemlya, Streptomyces virus ELB20,Streptomyces virus R4, Streptomyces virus phiHau3, Mycobacterium virus Acadian, Mycobacterium virus Baee, Mycobacterium virus Reprobate, Mycobacterium virus Adawi, Mycobacterium virus Bane1, Mycobacterium virus BrownCNA, Mycobacterium virus Chrisnmich, Mycobacterium virus Cooper, Mycobacterium virus JAMaL, Mycobacterium virus Nigel, Mycobacterium Mycobacterium Virus Stinger, Mycobacterium Virus Vincenzo, Mycobacterium Virus Zemanar, Mycobacterium Virus Apizium, Mycobacterium Virus Manad, Mycobacterium Virus Oline, Mycobacterium Virus Osmaximus, Mycobacterium Virus Pg1, Mycobacterium Virus Soto, Mycobacterium Virus Suffolk, Mycobacterium Virus Athena, Mycobacterium Virus Bernardo, Mycobacterium Virus Rus Gadjet, Mycobacterium Virus Pipefish, Mycobacterium Virus Godines, Mycobacterium Virus Rosebush, Mycobacterium Virus Babsiella, Mycobacterium Virus Brujita, Mycobacterium Virus Che9c, Mycobacterium Virus Sbash, Mycobacterium Virus Hawkeye, Mycobacterium Virus Plot, Salmonella Virus AG11, Salmonella Virus Ent1, Salmonella Virus f18SE, Salmonella Virus J ersey, Salmonella virus L13, Salmonella virus LSPA1, Salmonella virus SE2, Salmonella virus SETP3, Salmonella virus SETP7, Salmonella virus SETP13, Salmonella virus SP101, Salmonella virus SS3e, Salmonella virus wksl3, Escherichia virus K1G, Escherichia virus K1H, Escherichia virus K1ind1, Escherichia virus K1ind2, Salmonella virus SP31, Leuconostoc virus Lmd1, Leuconostoc virus LN03,Leuconostoc virus LN04, Leuconostoc virus LN12, Leuconostoc virus LN6B, Leuconostoc virus P793, Leuconostoc virus 1A4, Leuconostoc virus Ln8, Leuconostoc virus Ln9, Leuconostoc virus LN25, Leuconostoc virus LN34, Leuconostoc virus LNTR3, Mycobacterium virus Bongo, Mycobacterium virus Rey, Mycobacterium virus Butters, Mycobacterium virus M ichelle, Mycobacterium virus Charlie, Mycobacterium virus Pipsqueaks, Mycobacterium virus Xeno, Mycobacterium virus Panchino, Mycobacterium virus Phrann, Mycobacterium virus Redi, Mycobacterium virus Skinnyp, Gordonia virus BaxterFox, Gordonia virus Yeezy, Gordonia virus Kita, Gordonia virus Zirinka, Gordonia virus Nymphadora, My Mycobacterium virus Bignuz, Mycobacterium virus Brusacoram, Mycobacterium virus Donovan, Mycobacterium virus Fishburne, Mycobacterium virus Jebeks, Mycobacterium virus Malithi, Mycobacterium virus Phayonce, Enterobacter virus F20, Klebsiella virus 1513, Klebsiella virus KLPN1, Klebsiella virus KP36, Klebsiella virus PKP126, Klebsiella virus Sushi, Escherichia Virus AHP42, Escherichia virus AHS24, Escherichia virus AKS96, Escherichia virus C119, Escherichia virus E41c, Escherichia virus Eb49, Escherichia virus Jk06, Escherichia virus KP26, Escherichia virus Rogue1, Escherichia virus ACGM12, Escherichia virus Rtp, Escherichia virus ADB2, Escherichia virus JMPW1, Escherichia virus JMPW2, Escherichia virus T1, Shigella virus PSf2, Shigella virus Shfl1,Citrobactervirus Stevie, Escherichiavirus TLS, Salmonellavirus SP126, Cronobactervirus Esp2949-1, Pseudomonasvirus Ab18, Pseudomonasvirus Ab19, Pseudomonasvirus PaMx11, Arthrobactervirus Amigo, Propionibacteriumvirus Anatole, Propionibacteriumvirus B3, Bacillusvirus Andromeda, Bacillusvirus Blastoid, Bacillusvirus Curly, Bacillusvirus Eoghan, Bacillusvirus Russ Finn, Bacillus virus Glittering, Bacillus virus Riggi, Bacillus virus Taylor, Gordonia virus Attis, Mycobacterium virus Barnyard, Mycobacterium virus Konstantine, Mycobacterium virus Predator, Mycobacterium virus Bernal 13, Staphylococcus virus 13, Staphylococcus virus 77, Staphylococcus virus 108 PVL, Mycobacterium virus Bron, Mycobacterium virus Fait h1, Mycobacterium virus Joedirt, Mycobacterium virus Rumpelstiltskin, Lactococcus virus bIL67, Lactococcus virus c2, Lactobacillus virus c5, Lactobacillus virus Ld3, Lactobacillus virus Ld17, Lactobacillus virus Ld25A, Lactobacillus virus LLKu, Lactobacillus virus phiLdb, Cellulophaga virus Cba121, Cellulophaga virus Cba171, Cellulophaga virus Cba181, Cellulophaga virus ST , Bacillus Virus 250, Bacillus Virus IEBH, Mycobacterium Virus Ardmore, Mycobacterium Virus Avani, Mycobacterium Virus Boomer, Mycobacterium Virus Che8, Mycobacterium Virus Che9d, Mycobacterium Virus Deadp, Mycobacterium Virus Drane, Mycobacterium Virus Dorothy, Mycobacterium Virus Dotproduct, Mycobacterium Virus Drago, Mycobacterium Virus Fruitloop,Mycobacterium Virus Gumbie, Mycobacterium Virus Ibhubesi, Mycobacterium Virus Llij, Mycobacterium Virus Mozy, Mycobacterium Virus Mutaforma13, Mycobacterium Virus Pacc40, Mycobacterium Virus PMC, Mycobacterium Virus Ramsey, Mycobacterium Virus Rockyhorror, Mycobacterium Virus SG4, Mycobacterium Virus Shauna1, Mycobacterium Virus Shilan, Mycobacterium Virus Mycobacterium virus Spartacus, Mycobacterium virus Taj, Mycobacterium virus Tweety, Mycobacterium virus Wee, Mycobacterium virus Yoshi, Salmonella virus Chi, Salmonella virus FSLSP030, Salmonella virus FSLSP088, Salmonella virus iEPS5, Salmonella virus SPN19, Mycobacterium virus 244, Mycobacterium virus Bask21, Mycobacterium virus CJW1, Mycobacterium virus Eureka, Mycobacterium virus Mycobacterium virus Kostya, Mycobacterium virus Porky, Mycobacterium virus Pumpkin, Mycobacterium virus Sirduracell, Mycobacterium virus Toto, Mycobacterium virus Corndog, Mycobacterium virus Firecracker, Rhodobacter virus RcCronus, Pseudomonas virus D3112, Pseudomonas virus DMS3, Pseudomonas virus FHA0480, Pseudomonas virus LPB1, Pseudomonas virus MP22, Pseudomonas virus Pseudomonas virus MP29, Pseudomonas virus MP38, Pseudomonas virus PA1KOR, Pseudomonas virus D3, Pseudomonas virus PMG1, Arthrobacter virus Decurro, Gordonia virus Demosthenes, Gordonia virus Katyusha, Gordonia virus Kvothe, Propionibacterium virus B22, Propionibacterium virus Doucette, Propionibacterium virus E6, Propionibacterium virus G4, Burkholderia virus phi6442,Burkholderia virus phi1026b, Burkholderia virus phiE125, Edwardsiella virus eiAU, Mycobacterium virus Ff47, Mycobacterium virus Muddy, Mycobacterium virus Gaia, Mycobacterium virus Giles, Arthrobacter virus Captnmurica, Arthrobacter virus Gordon, Gordonia virus GordTnk2, Paenibacillus virus Harrison, Escherichia virus EK99P1, Escherichia virus HK578, Escherichia virus JL1, Escherichia virus SSL2009a, Escherichia virus YD2008s, Shigella virus EP23, Sodalis virusSO1, Escherichiavirus HK022, Escherichiavirus HK75, Escherichiavirus HK97, Escherichiavirus HK106, Escherichiavirus HK446, Escherichiavirus HK542, Escherichiavirus HK544, Escherichiavirus HK633, Escherichiavirus mEp234, Escherichiavirus mEp235, Escherichiavirus mEpX1, Escherichiavirus mEpX2, Escherichiavirus mEp043, Escherichiavirus mEp213, Escherichiavirus mEp237, Escherichiavirus mEp390, Escherichiavirus mEp460, Escherichiavirus mEp505, Escherichiavirus mEp506, Brevibacillus virus Jenst, Achromobactervirus 83-24, Achromobactervirus JWX, Arthrobactervirus Kellezzio, Arthrobactervirus Kitkat, Arthrobactervirus Bennie, Arthrobactervirus DrRobert, Arthrobactervirus Glenn, Arthrobactervirus HunterDalle, Arthrobactervirus Joann, Arthrobactervirus Korra, Arthrobactervirus Preamble, Arthrobactervirus Pumancara, Arthrobactervirus Wayne, Mycobacteriumvirus Alma, Mycobacteriumvirus Arturo, Mycobacteriumvirus Astro, Mycobacteriumvirus Backyardigan,Mycobacterium virus BBPiebs31, Mycobacterium virus Benedict, Mycobacterium virus Bethlehem, Mycobacterium virus Billknuckles, Mycobacterium virus Bruns, Myco, Mycobacterium virus Bxb1, Mycobacterium virus Bxz2, Mycobacterium virus Che12, Mycobacterium virus Cuco, Mycobacterium virus D29, Mycobacterium virus Doom, Mycobacterium virus Ericb, Mycobacterium virus Euphoria, Mycobacterium virus George, Mycobacterium virus Gladiator, Mycobacterium virus Goose, Mycobacterium virus Hammer, Mycobacterium virus Heldan, Mycobacterium virus Jasper, Mycobacterium virus JC27, Mycobacterium virus Jeffabunny, Mycobacterium virus JHC117, Mycobacterium virus KBG, Mycobacterium virus Kssjeb, Mycobacterium virus Kugel, Mycobacterium virus L5, Mycobacterium virus Lesedi, Mycobacterium virus LHTSCC, Mycobacterium virus lockley, Mycobacterium virus Marcell, Mycobacterium virus Microwolf, Mycobacterium virus Mrgordo, Mycobacterium virus Museum, Mycobacterium virus Nepal, Mycobacterium virus Packman, Mycobacterium virus Peaches, Mycobacterium virus Perseus, Mycobacterium virus Pukovnik, Mycobacterium virus Rebeuca, Mycobacterium virus Redrock, Mycobacterium virus Ridgecb, Mycobacterium virus Rockstar, Mycobacterium Mycobacterium virus Saintus, Mycobacterium virus Skipole, Mycobacterium virus Solon, Mycobacterium virus Switzer, Mycobacterium virus SWU1, Mycobacterium virus Ta17a, Mycobacterium virus Tiger, Mycobacterium virus Timshel, Mycobacterium virus Trixie, Mycobacterium virus Turbido, Mycobacterium virus Twister, Mycobacterium virus U2, Mycobacterium virus Violet,Mycobacterium virus Wonder, Escherichia virus DE3, Escherichia virus HK629, Escherichia virus HK630, Escherichia virus lambda, Arthrobacter virus Laroye, Mycobacterium virus Halo, Mycobacterium virus Liefie, Mycobacterium virus Marvin, Mycobacterium virus Mosmoris, Arthrobacter virus Circum, Arthrobacter virus Mudcat, Escherichia virus N15, Escherichia virus 9g, Escherichia virus Escherichia virus JenK1, Escherichia virus JenP1, Escherichia virus JenP2, Pseudomonas virus NP1, Pseudomonas virus PaMx25, Mycobacterium virus Baka, Mycobacterium virus Courthouse, Mycobacterium virus Littlee, Mycobacterium virus Omega, Mycobacterium virus Optimus, Mycobacterium virus Thibault, Polaribacter virus P12002L, Polaribacter virus P12002S, Non-Ravens virus P12024L, Non-Lavens virus P12024S, Thermus virus P23-45, Thermus virus P74-26, Listeria virus LP26, Listeria virus LP37, Listeria virus LP110, Listeria virus LP114, Listeria virus P70, Propionibacterium virus ATCC29399BC, Propionibacterium virus ATCC29399BT, Propionibacterium virus Attacne, Propionibacterium virus Keiki, Propionibacterium virus Kubed ... Onibacterium virus Lauchelly, Propionibacterium virus MrAK, Propionibacterium virus Ouroboros, Propionibacterium virus P91, Propionibacterium virus P105, Propionibacterium virus P144, Propionibacterium virus P1001, Propionibacterium virus P1.1, Propionibacterium virus P100A, Propionibacterium virus P100D, Propionibacterium virus P101A, Propionibacterium virus P104A,Propionibacterium virus PA6, Propionibacterium virus Pacnes201215, Propionibacterium virus PAD20, Propionibacterium virus PAS50, Propionibacterium virus PHL009M11, Propionibacterium virus PHL025M00, Propionibacterium virus PHL037M02, Propionibacterium virus PHL041M10, Propionibacterium virus PHL060L00, Propionibacterium virus PHL067M0 ... Propionibacterium virus PHL070N00, Propionibacterium virus PHL071N05, Propionibacterium virus PHL082M03, Propionibacterium virus PHL092M00, Propionibacterium virus PHL095N00, Propionibacterium virus PHL111M01, Propionibacterium virus PHL112N00, Propionibacterium virus PHL113M01, Propionibacterium virus PHL114L00, Propionibacterium virus PHL116M00, Propionibacterium virus PHL117M01, Propionibacterium virus PHL118M01, Propionibacterium virus PHL119M02, Propionibacterium virus PHL120M03, Propionibacterium virus PHL121M04, Propionibacterium virus PHL122M04, Propionibacterium virus PHL123M04, Propionibacterium virus PHL124M05, Propionibacterium virus PHL125M06, Propionibacterium virus PHL126M07, Propionibacterium virus PHL127M08, Propionibacterium virus PHL128M09, Propionibacterium virus PHL130M01, Propionibacterium virus PHL131M01, Propionibacterium virus PHL132M00, Propionibacterium virus PHL133M01, Propionibacterium virus PHL134L00, Propionibacterium virus PHL135M01, Propionibacterium virus PHL136M02, Propionibacterium virus PHL137M03, Pro Propionibacterium virus PHL117M00, Propionibacterium virus PHL117M01, Propionibacterium virus PHL132N00, Propionibacterium virus PHL141N00, Propionibacterium virus PHL151M00, Propionibacterium virus PHL151N00, Propionibacterium virus PHL152M00, Propionibacterium virus PHL163M00, Propionibacterium virus PHL171M01, Propionibacterium virus PHL179M00, Propionibacterium virus PHL179M00 Propionibacterium virus PHL194M00, Propionibacterium virus PHL199M00, Propionibacterium virus PHL301M00, Propionibacterium virus PHL308M00, Propionibacterium virus Pirate, Propionibacterium virus Procrass1, Propionibacterium virus SKKY, Propionibacterium virus Solid, Propionibacterium virus Stormborn, Propionibacterium virus Wizzo, Pseudomonas virus PaMx28,Pseudomonas virus PaMx74, Mycobacterium virus Patience, Mycobacterium virus PBI1, Rhodococcus virus Pepy6, Rhodococcus virus Poco6, Propionibacterium virus PFR1, Streptomyces virus phiBT1, Streptomyces virus phiC31, Streptomyces virus TG1, Caulobacter virus Karma, Caulobacter virus Magneto, Caulobacter virus phiCbK, Caulobacter virus Rogue, Caulobacter virus Swift, Staphylococcal virus 11, Staphylococcal virus 29, Staphylococcal virus 37, Staphylococcal virus 53, Staphylococcal virus 55, Staphylococcal virus 69, Staphylococcal virus 71, Staphylococcal virus 80, Staphylococcal virus 85, Staphylococcal virus 88, Staphylococcal virus 92, Staphylococcal virus 96, Staphylococcal virus 187, Staphylococcal virus 52a, Staphylococcal virus 80al pha, Staphylococcal virus CNPH82, Staphylococcal virus EW, Staphylococcal virus IPLA5, Staphylococcal virus IPLA7, Staphylococcal virus IPLA88, Staphylococcal virus PH15, Staphylococcal virus phiETA, Staphylococcal virus phiETA2, Staphylococcal virus phiETA3, Staphylococcal virus phiMR11, Staphylococcal virus phiMR25, Staphylococcal virus phiNM1, Staphylococcus Virus phiNM2, Staphylococcus virus phiNM4, Staphylococcus virus SAP26, Staphylococcus virus X2, Enterococcus virus FL1, Enterococcus virus FL2, Enterococcus virus FL3, Lactobacillus virus ATCC8014, Lactobacillus virus phiJL1, Pediococcus virus cIP1, Aeromonas virus pIS4A, Listeria virus LP302, Listeria virus PSA, Methanobacterium virus psiM1, Roseobacter virus RDJL1,Roseobactervirus RDJL2, Rhodococcusvirus RER2, Enterococcusvirus BC611, Enterococcusvirus IMEEF1, Enterococcusvirus SAP6, Enterococcusvirus VD13, Streptococcusvirus SPQS1, Mycobacteriumvirus Papyrus, Mycobacteriumvirus Send513, Burkholderiavirus KL1, Pseudomonasvirus 73, Pseudomonasvirus Ab26, Pseudomonasvirus Kakheti25, Escherichiavirus Cajan , Escherichia virus Seurat, Staphylococcus virus SEP9, Staphylococcus virus Sextaec, Streptococcus virus 858, Streptococcus virus 2972, Streptococcus virus ALQ132, Streptococcus virus O1205, Streptococcus virus Sfi11, Streptococcus virus 7201, Streptococcus virus DT1, Streptococcus virus phiAbc2, Streptococcus virus Sfi19, Streptococcus virus Sfi21, Pae Paenibacillus virus Diva, Paenibacillus virus Hb10c2, Paenibacillus virus Rani, Paenibacillus virus Shelly, Paenibacillus virus Sitara, Paenibacillus virus Willow, Lactococcus virus 712, Lactococcus virus ASCC191, Lactococcus virus ASCC273, Lactococcus virus ASCC281, Lactococcus virus ASCC465, Lactococcus virus ASCC532, Lactococcus virus Bibb29, Lactococcus virus bIL17 0, Lactococcal virus CB13, Lactococcal virus CB14, Lactococcal virus CB19, Lactococcal virus CB20, Lactococcal virus jj50, Lactococcal virus P2, Lactococcal virus P008, Lactococcal virus sk1, Lactococcal virus Sl4, Bacillus virus Slash, Bacillus virus Stahl, Bacillus virus Staley, Bacillus virus Stills, Gordonia virus Bachita, Gordonia virus ClubL, Gordonia virus OneUp,Gordoniavirus Smoothie, Gordoniavirus Soups, Bacillusvirus SPbeta, Vibriovirus MAR10, Vibriovirus SSP002, Escherichiavirus AKFV33, Escherichiavirus BF23, Escherichiavirus DT57C, Escherichiavirus EPS7, Escherichiavirus FFH1, Escherichiavirus H8, Escherichiavirus slur09, Escherichiavirus T5, Salmonellavirus 118970sal2, Salmonellavirus Shivani, Salmonellavirus SPC35 , Salmonella virus Stitch, Arthrobacter virus Tank, Tsukamurella virus TIN2, Tsukamurella virus TIN3, Tsukamurella virus TIN4, Rhodobacter virus RcSpartan, Rhodobacter virus RcTitan, Mycobacterium virus Anaya, Mycobacterium virus Angelica, Mycobacterium virus Crimd, Mycobacterium virus Fionnbarth, Mycobacterium virus Jaws, Mycobacterium virus Larva, Mycobacterium virus Ma cncheese, Mycobacterium virus Pixie, Mycobacterium virus TM4, Bacillus virus BMBtp2, Bacillus virus TP21, Geobacillus virus Tp84, Staphylococcus virus 47, Staphylococcus virus 3a, Staphylococcus virus 42e, Staphylococcus virus IPLA35, Staphylococcus virus phi12, Staphylococcus virus phiSLT, Mycobacterium virus 32HC, Rhodococcus virus RGL3, Paenibacillus virus Vegas, Gordonii Avirus Vendetta, Bacillus virus Wbeta, Mycobacterium virus Wildcat, Gordonia virus Twister6, Gordonia virus Wizard, Gordonia virus Hotorobo, Gordonia virus Monty, Gordonia virus Woes, Xanthomonas virus CP1, Xanthomonas virus OP1, Xanthomonas virus phil7, Xanthomonas virus Xop411, Xanthomonas virus Xp10, Streptomyces virus TP1604, Streptomyces virus YDN12,Alphaproteobacterial virus phiJl001, Pseudomonas virus LKO4, Pseudomonas virus, Pseudomonas virus M6, Pseudomonas virus MP1412, Pseudomonas virus PAE1, Pseudomonas virus Yua, Pseudoalteromonas virus PM2, Pseudomonas virus phi6, Pseudomonas virus phi8, Pseudomonas virus phi12, Pseudomonas virus phi13, Pseudomonas virus phi2954, Pseudomonas virus phiNN, Pseudomonas virus phiYY, Vibrio virus fs1, Vibrio virus VGJ, Ralstonia virus RS603, Ralstonia virus RSM1, Ral stoniavirus RSM3, Escherichiavirus M13, Escherichiavirus I22, Salmonellavirus IKe, Acholeplasmavirus L51, Vibriovirus fs2, Vibriovirus VFJ, Escherichiavirus If1, Propionibacterium virus B5, Pseudomonasvirus Pf1, Pseudomonasvirus Pf3, Ralstoniavirus PE226, Ralstoniavirus RSS1, Spiroplasmavirus SVTS2, Stenotrophomonasvirus PSH1, Stenotrophomonasvirus SMA6, Stenotrophomonasvirus SB1, Stenotrophomonasvirus SB2, Stenotrophomonasvirus SB3, Stenotrophomonasvirus SB4, Stenotrophomonasvirus SB5, Stenotrophomonasvirus SB6, Stenotrophomonasvirus SB7, Stenotrophomonasvirus SB8, Stenotrophomonasvirus SB9, Stenotrophomonasvirus SB10, Stenotrophomonasvirus SB11, Stenotrophomonasvirus SB12, Stenotrophomonasvirus SB13, Stenotrophomonasvirus SB14, Stenotrophomonasvirus SB15, Stenotrophomonasvirus SB16, Stenotrophomonasvirus SB17, Stenotrophomonasvirus SB19, Stenotrophomonasvirus SB20, Stenotrophomonasvirus SB21, Stenotrophomonasvirus SB22, Stenotrophomonasvirus SB23, Stenotrophomonasvirus SB24, Stenotrophomonasvirus SB25, Stenotrophomonasvirus SB26, Stenotrophomonasvirus SB27, Stenotrophomonasvirus SB28, Stenotrophomonasvirus SB29, Stenotrophomonasvirus SB30, Stenotrophomonasvirus SB31, Stenotrophomonasvirus SB3 Virus SMA7, Stenotrophomonas virus SMA9, Vibriovirus CTXphi, Vibriovirus KSF1, Vibriovirus VCY, Vibriovirus Vf33, Vibriovirus VfO3K6, Xanthomonas virus Cf1c, Spiroplasma virus C74, Spiroplasma virus R8A2B, Spiroplasma virus SkV1CR23x, Escherichia virus FI, Escherichia virus Qbeta, Escherichia virus BZ13, Escherichia virus MS2, Escherichia virus alpha3, Escherichia virus IL Escherichiavirus ID21, Escherichiavirus ID32, Escherichiavirus ID62, Escherichiavirus NC28, Escherichiavirus NC29, Escherichiavirus NC35, Escherichiavirus phiK, Escherichiavirus St1, Escherichiavirus WA45, Escherichiavirus G4, Escherichiavirus ID52, Escherichiavirus Talmos, Escherichiavirus phiX174, Bdellovibriovirus MAC1, Bdellovibriovirus MH2K, Chlamydiavirus Chp1, Chlamydiavirus Chp2,Chlamydia virus CPAR39, Chlamydia virus CPG1, Spiroplasma virus SpV4, Acholeplasma virus L2, Pseudomonas virus PR4, Pseudomonas virus PRD1, Bacillus virus AP50, Bacillus virus Bam35, Bacillus virus GIL16, Bacillus virus Wip1, Escherichia virus phi80, Escherichia virus RB42, Escherichia virus T2, Escherichia virus T3, Escherichia virus T6, Escherichia virus VT2-Sa, Escherichia virus VT1-Sakai, Escherichia virus VT2-Sakai, Escherichia virus CP-933V, Escherichia virus P27, Escherichia virus Stx2phi-I, Escherichia virus Stx1phi, Escherichiavirus Stx2phi-II, Escherichiavirus CP-1639, based on Escherichiavirus BP-4795, Escherichiavirus 86, Escherichiavirus Min27, Escherichiavirus 2851, Escherichiavirus 1717, Escherichiavirus YYZ-2008, Escherichiavirus EC026_P06, Escherichiavirus ECO103_P15, Escherichiavirus ECO103_P12, Escherichiavirus ECO111_P16, Escherichiavirus ECO111_P11, Escherichiavirus VT2phi_272, Escherichiavirus TL-2011c, Escherichiavirus P13374, Escherichiavirus Sp5,

[0136] One of the most popular is BW73 and B278. D6, D108, E, El, E24, E41, FI-2, FI-4, FI-5, H I8A, Ffl8B, i, MM, Mu, 025, PhI-5, Pk, PSP3, P l, PlD, P2, P4, Sl, Wφ, φK13, φl, φ2, φ7, φ92, 7 A, 8φ, 9φ, 18, 28-1, 186, 299, HH-Ch E4, E7, E28, FIl, FI3, H, Hl, H3, H8, K3, M, N, ND-2, ND-3, ND4, ND-5, ND 6, ND-7, Ox-I, Ox-2, Ox-3, Ox-4, Ox-5, Ox-6, PhI-I, RB42, RB43, RB49 RB69, S, SaI-I, Sal-2, Sal-3, Sal-4, Sal-5, Sal-6, TC23, TC45, TuII * -6, TuIP-24, TuII*46, TuIP-60, T2, T4, T6, T35, αl, 1, IA 3A, 3T+, 5φ, 9266Q, CFO103, HK620, J, K, KlF, m59, no. A、no. Yes, no. 3、no. 9, N4, sd, T3, T7, WPK, W31, ΔH, φC3888, φK3, φK7, φK12, φV-1, Φ04-CF, Φ 05, Φ06, Φ07, φl, φl.2, φ20, φ95, φ263, φlO92, φl, φll, Ω8, 1, 3, 7, 8, 27. 28-2. 29. 30. 31. 32. 38. 39. 42. 933W C30, CVX-5, Cl, DDUP, ECl, EC2, E21, E29, Fl, F26S, F27S, Hi, HK022, HK 97, HK139, HK253, HK256, K7, ND-I, PA-2, q, S2, Tl,) T3C, T5, UC-I, w. β4, γ2, λ, ΦD326, φγ, Φ06, Φ7, Φ10, φ80, χ, 2, 4, 4A, 6, 8A, 102, 150, 168; 174, 3000, AC6, AC7, AC28, AC43, AC50, AC57, AC81, AC95, HK243, KlO, Z G / 3A, 5, 5A, 21EL, H19-J and 933H It's a great way to get your hands on.

[0137] In embodiments in which the genetic circuit is packaged into a bacterial delivery vehicle, the genetic circuit may include a nucleic acid sequence that signals for packaging and the donor cell may express a bacteriophage scaffold protein. The sequence that signals for packaging and the bacteriophage scaffold protein will be selected by one of skill in the art depending on the nature of the bacteriophage to be used as a bacterial delivery vehicle.

[0138] In some embodiments, the bacterial donor cells and / or bacterial recipient cells of the present invention may be used in the presence of prebiotics that enhance their growth or any other desired function of the bacterial donor cells and / or bacterial recipient cells. Prebiotics include, but are not limited to, amino acids, biotin, fructo-oligosaccharides, galacto-oligosaccharides, hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan), inulin, chitin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, gums (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galacto-oligosaccharides, pectins (e.g., xylogalacturonan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fibers (e.g., soy fiber, sugar beet fiber, pea fiber, corn bran, and oat fiber), and xylo-oligosaccharides.

[0139] In a further embodiment, the nucleic acid sequence of interest contained in the genetic circuit and under the transcriptional control of the repressor binding sequence encodes a protein that confers resistance to an antibiotic. As used herein, the term "antibiotic" preferably refers to penicillins, such as penicillin G, penicillin K, penicillin N, penicillin O, penicillin V, methicillin, benzylpenicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, epicillin, carbenicillin, ticarcillin, temocillin, mezlocillin, and piperacillin; cephalosporins, such as cephalosporins, ... phosphates, such as cephacetrile, cefadroxil, cephalexin, cephaloglycin, cephalonium, cephaloridine, cephalothin, cephapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cephradine, cefroxadine, ceftezole, cefaclor, cefonicid, cefprozil, cefuroxime, cefuzonam, cefmetazole, cefotetan, cefoxitin, loracarbef, cefbuperazone, cefminox, cefotetan, cefoxitin, cefotiam, Cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefovecin, cefpimizole, cefpodoxime, cefteram, ceftamele, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, latamoxef, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, flomoxef, ceftobiprole , ceftaroline, ceftolozane, cephaloram, cefaparole, cefcanel, cefedrolol, cephempidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, cefoxazole, cefrotil, cefsumide, ceftioxide, cefraacetim, and nitrocefin; polymyxins, such as Polysporin, Neosporin, Polymyxin B, and Polymyxin E, rifampicins, such as Rifampicin, Rifapentine, and Rifaximin; fidaxomicin;Quinolones, e.g., cinoxacin, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, losoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, grepafloxacin, levofloxacin, pazufloxacin, temafloxacin, tosufloxacin, clinafloxacin, gatifloxacin, gemifloxacin, moxifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, de Lafloxacin, Nemonoxacin, and Zavofloxacin; Sulfonamides such as Sulfafurazole, Sulfacetamide, Sulfadiazine, Sulfadimidine, Sulfafurazole, Sulfisomidine, Sulfadoxine, Sulfamethoxazole, Sulfamoxole, Sulfanitran, Sulfadimethoxine, Sulfamethoxypyridazine, Sulfamethoxydiazine, Sulfadoxine, Sulfamethpyrazine, and Terephthyl; Macrolides such as Azithromycin, Clarithromycin, Erythromycin, Fidaxomicin, Telithromycin, carbomycin A, josamycin, kitasamycin, midecamycin, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, and roxithromycin; ketolides such as telithromycin and cethromycin; luoloketolides such as solithromycin; lincosamides such as lincomycin, clindamycin, and pirlimycin; tetracyclines such as demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline; Aminoglycosides, such as amikacin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, sisomicin, tobramycin, paromomycin, and streptomycin; ansamycins, such as geldanamycin, herbimycin, and rifaximin; carbacephems, such as loracarbef; carbapenems, such as ertapenem, doripenem, imipenem (or cilastatin), and meropenem; glycopeptides, such as teicoplanin, vancomycin, telavancin, dalbavancin, and oritavancin;nitrofurans, such as furazolidone, and nitrofurantoin; oxazolidinones, such as linezolid, pozizolid, radezolid, and torezolid; teixobactin, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifabutin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin (or dalfopristin), thiamphenicol, tigecycline, tinidazole, trimethoprim, alatrofloxacin, fidaxomicin, nalidixic acid, rifampin, derivatives and combinations thereof. In particular, the term "protein that confers resistance to antibiotics" may refer to a protein that confers resistance to any of these antibiotics.

[0140] The present invention provides pharmaceutical or veterinary compositions comprising one or more bacterial delivery vehicles produced using donor cells as disclosed herein, preferably using the methods of the present invention for producing bacterial delivery vehicles, and a pharma- tically acceptable carrier. The present invention also provides pharmaceutical or veterinary compositions comprising recipient or target cells, e.g., probiotics, into which a genetic circuit is transferred as disclosed herein, and a pharma- tically acceptable carrier. The present invention also provides pharmaceutical or veterinary compositions comprising donor cells, e.g., probiotics, as disclosed herein, i.e., comprising a genetic circuit and expressing a repressor protein, and a pharma-tically acceptable carrier. In general, for pharmaceutical use, the bacterial delivery vehicle may be formulated as a pharmaceutical product or a composition comprising at least one bacterial delivery vehicle and at least one pharma- tically acceptable carrier, diluent or excipient, and optionally one or more additional pharma- tically active compounds. Such formulations may be in a form suitable for administration orally, parenterally (e.g., by intravenous, intramuscular or subcutaneous injection, or intravenous infusion), topically, by inhalation, by skin patch, by implant, by suppository, etc. Such dosage forms may be solid, semi-solid, or liquid, depending on the method and route of administration. For example, a formulation for oral administration may be provided with an enteric coating to allow the synthetic bacterial delivery vehicle in the formulation to survive the stomach environment and pass into the intestine. More generally, a synthetic bacterial delivery vehicle formulation for oral administration may be formulated suitable for delivery to any desired part of the gastrointestinal tract. Additionally, suitable suppositories may be used for delivery to the gastrointestinal tract. A variety of pharmaceutically acceptable carriers, diluents, and excipients useful in bacterial delivery vehicle compositions are known to those skilled in the art.

[0141] The pharmaceutical or veterinary compositions according to the present disclosure may further comprise a pharma- ceutically acceptable vehicle. Solid pharma- ceutically acceptable vehicles may include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coating agents, or tablet disintegrating agents. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes, and ion exchange resins.

[0142] Pharmaceutical or veterinary compositions may be prepared as sterile solid compositions that can be suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. Pharmaceutical or veterinary compositions of the present disclosure may be orally administered in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleic acid esters of sorbitol and its anhydrides copolymerized with ethylene oxide), and the like. Particles according to the present disclosure may also be orally administered in either liquid or solid composition form. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, as well as liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for enteral administration include sterile solutions, emulsions, and suspensions.

[0143] The bacterial delivery vehicle produced by the production method of the present disclosure can be dissolved or suspended in a pharma- ceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture of both, or a pharma- ceutically acceptable oil or lipid. The liquid vehicle can contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity adjusters, stabilizers, or osmolality adjusters. Suitable examples of liquid vehicles for oral and enteral administration include water (partially containing additives as described above, such as cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, such as glycols) and their derivatives, and oils (e.g., coconut oil and peanut oil). For parenteral administration, the vehicle can also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for enteral administration. The liquid vehicle for pressurized compositions can be halogenated hydrocarbon or other pharma- ceutically acceptable propellant.

[0144] For transdermal administration, the pharmaceutical or veterinary compositions can be formulated in the form of an ointment, cream or gel, and penetration can be enhanced using suitable penetrants or surfactants, such as, for example, dimethylsulfoxide, dimethylacetamide, and dimethylformamide.

[0145] For transmucosal administration, nasal spray, rectal or vaginal suppository can be used.Active compound can be incorporated into any of the known suppository bases by methods known in the art.Examples of such bases include cocoa butter, polyethylene glycol (carbowax), polyethylene monostearate sorbitan, and mixtures thereof with other compatible materials that modify their melting point or dissolution rate.

[0146] Also provided is a method for treating a disease or disorder caused by bacteria, preferably a bacterial infection, using the compositions disclosed herein. In this embodiment, a genetic circuit containing a nucleic acid of interest is transferred into one or more bacteria causing the disease or disorder, i.e., the target cells. The method comprises administering a pharmaceutical or veterinary composition disclosed herein, preferably a therapeutically effective amount of a pharmaceutical or veterinary composition disclosed herein, to a subject having a disease or disorder caused by bacteria, preferably a bacterial infection, in need of treatment. Further provided is a pharmaceutical or veterinary composition disclosed herein for use as a medicament, in particular in the treatment of a disease or disorder caused by bacteria, preferably in the treatment of a bacterial infection. Also provided is the use of a pharmaceutical or veterinary composition disclosed herein for the manufacture of a medicament for treating a disease or disorder caused by bacteria, preferably a bacterial infection.

[0147] In some embodiments, the subject is a mammal, hi some particular embodiments, the subject is a human.

[0148] Diseases or disorders caused by bacteria include abdominal cramps, acne vulgaris, acute epiglottitis, arthritis, bacteremia, bloody diarrhea, botulism, brucellosis, brain abscess, chancroid, chlamydia, Crohn's disease, conjunctivitis, cholecystitis, colorectal cancer, polyposis, intestinal dysbiosis, Lyme disease, diarrhea, diphtheria, duodenal ulcer, endocarditis, erysipelothricosis, enteric fever, fever, glomerulonephritis, gastroenteritis, gastric ulcer, Guillain-Barre syndrome, tetanus, gonorrhea, gingivitis, inflammatory bowel disease, and irritable bowel syndrome. , leptospirosis, leprosy, listeriosis, tuberculosis, Lady Windermere syndrome, legionnaires' disease, meningitis, mucopurulent conjunctivitis, multidrug resistant bacterial infection, multidrug resistant bacterial carriage, myonecrotizing gas gangrene, Mycobacterium avium complex, neonatal necrotizing enterocolitis, nocardiosis, hospital-acquired infection, otitis, periodontitis, pharyngitis, pneumonia, peritonitis, purpuric fever, Rocky Mountain spotted fever, dysentery, syphilis, sinusitis, sigmoiditis, septicemia, subcutaneous abscess, tularemia, tracheobronchitis, tonsillitis, typhoid fever, ulcerative colitis, urinary infection, and whooping cough.

[0149] The bacterial infection may be selected from the group consisting of skin infections, such as acne, intestinal infections, such as esophagitis, gastritis, enteritis, colitis, sigmoiditis, proctitis, and peritonitis, urinary tract infections, vaginal infections, female upper genital tract infections, such as salpingitis, endometritis, oophoritis, metritis, parametritis, and pelvic peritoneal infections, respiratory tract infections, such as pneumonia, intra-amniotic infections, odontogenic infections, endodontic infections, fibrosis, meningitis, bloodstream infections, hospital-acquired infections, such as catheter-associated infections, hospital-acquired pneumonia, postpartum infections, hospital-acquired gastroenteritis, hospital-acquired urinary tract infections, or combinations thereof. Preferably, the bacterial infection according to the present disclosure is caused by bacteria that exhibit antibiotic resistance. In certain embodiments, the infection is caused by bacteria that can be used as the bacteria listed above, donor cells, or target cells.

[0150] Also provided are methods for treating a bacterial infection comprising administering to a subject having a bacterial infection in need of treatment a provided pharmaceutical or veterinary composition, particularly a therapeutically effective amount of a provided pharmaceutical or veterinary composition. A "therapeutically effective amount" is the amount necessary to treat a targeted disease or disorder or to produce a desired effect, such as the amount necessary to treat a disease or disorder caused by a bacterium, particularly a bacterial infection, when administered to a subject.

[0151] Provided is a method for reducing a bacterial population, particularly the amount of pathogenic and / or antibiotic resistant bacteria, in a subject having a bacterial infection, comprising contacting the bacterial population with a pharmaceutical or veterinary composition disclosed herein, or a bacterial delivery vehicle disclosed herein. Further provided is the use of a pharmaceutical or veterinary composition disclosed herein, or a bacterial delivery vehicle disclosed herein, for the manufacture of a medicament for reducing a bacterial population, particularly the amount of pathogenic and / or antibiotic resistant bacteria, in a subject having a bacterial infection.

[0152] The present disclosure also relates to the pharmaceutical or veterinary composition of the present invention for use in the treatment of metabolic disorders, including, for example, obesity, type 2 diabetes, and non-alcoholic fatty liver disease. Indeed, emerging evidence indicates that these disorders are characterized by changes in the composition of the gut microbiota and its metabolic products

[31] . Thus, the pharmaceutical or veterinary composition may be used to deliver a nucleic acid of interest in some gut bacteria that can change the composition of the gut microbiota (e.g., by inducing the death of some bacteria) and its metabolic products (e.g., by inducing the expression, overexpression, or secretion by said bacteria of some molecules, e.g., molecules that have a beneficial role in metabolic inflammation). The present disclosure also relates to the use of the pharmaceutical or veterinary composition of the present invention for the manufacture of a medicament for the treatment of metabolic disorders, including, for example, obesity, type 2 diabetes, and non-alcoholic fatty liver disease. The present disclosure also relates to methods for treating metabolic disorders, including, for example, obesity, type 2 diabetes, and non-alcoholic fatty liver disease, comprising administering to a subject having a metabolic disorder in need of treatment, a provided pharmaceutical or veterinary composition, particularly a therapeutically effective amount of a provided pharmaceutical or veterinary composition.

[0153] In certain embodiments, the present disclosure relates to a pharmaceutical or veterinary composition of the present invention for use in the treatment of pathologies involving bacteria of the human microbiome, such as inflammatory and autoimmune diseases, cancer, infectious diseases, or brain disorders. The present disclosure also relates to a method for treating said pathologies involving bacteria of the human microbiome, comprising administering to a subject having a pathology and in need of treatment, a provided pharmaceutical or veterinary composition, in particular a therapeutically effective amount of a provided pharmaceutical or veterinary composition, and also to the use of the pharmaceutical or veterinary composition disclosed herein for the manufacture of a medicament for treating a pathology involving bacteria of the human microbiome. Indeed, some bacteria of the microbiome may secrete molecules that induce and / or enhance the development of inflammatory or autoimmune diseases, or cancer, without causing any infectious disease. More specifically, the present disclosure also relates to modifying the composition of the microbiome to improve the efficacy of immunotherapies, for example, based on CAR-T (chimeric antigen receptor T) cells, TIL (tumor infiltrating lymphocytes), and Treg (regulatory T cells), also known as suppressor T cells. Modifying the composition of the microbiome to improve the efficacy of immunotherapies may also include the use of immune checkpoint inhibitors known in the art, such as, but not limited to, PD-1 (programmed cell death protein 1) inhibitors, PD-L1 (programmed death ligand 1) inhibitors, and CTLA-4 (cytotoxic T lymphocyte-associated protein 4).

[0154] Some bacteria in the microbiome can also secrete molecules that affect the brain.

[0155] Therefore, a further object of the present disclosure is a method for controlling the microbiome of a subject, comprising administering in said subject an effective amount of a pharmaceutical composition disclosed herein.

[0156] In certain embodiments, the present disclosure also relates to a personalized treatment method for an individual in need of treatment for a bacterial infection, comprising: i) obtaining a biological sample from the individual and determining a group of bacterial DNA sequences from the sample; ii) identifying one or more pathogenic strains or species present in the sample based on the sequence determination; and iii) administering to the individual a pharmaceutical composition according to the present disclosure capable of recognizing or delivering a packaged genetic circuit for each pathogenic strain or species identified in the sample.

[0157] Preferably, the biological sample contains pathogenic and non-pathogenic species, and following administration of a pharmaceutical or veterinary composition according to the present disclosure to an individual, the amount of pathogenic bacteria on or in the individual is reduced, but the amount of non-pathogenic bacteria is not reduced.

[0158] In another particular embodiment, the present disclosure relates to a pharmaceutical or veterinary composition according to the present disclosure for use in improving the efficacy of a drug. Indeed, it is known that some bacteria of the microbiome, while not pathogenic in themselves, are capable of metabolizing drugs and modifying them into ineffective or harmful molecules.

[0159] In another particular embodiment, the present disclosure relates to in situ bacterial production of any compound of interest, including therapeutic compounds, e.g., mammalian prophylactic and therapeutic vaccines. The compound of interest encoded by the nucleic acid of interest contained in the genetic circuit can be produced within the target bacterium, secreted from the target bacterium, or expressed on the surface of the target bacterium. In a more particular embodiment, the compound of interest is an antigen expressed on the surface of the target bacterium for prophylactic and / or therapeutic vaccination.

[0160] The present disclosure also relates to non-therapeutic uses of the compositions disclosed herein. For example, non-therapeutic uses can be cosmetic uses or uses for improving the health of a subject, particularly a subject that does not suffer from a disease. Thus, the present disclosure also relates to cosmetic or non-therapeutic compositions comprising the compositions of the present disclosure.

[0161] The present disclosure further provides a kit for use in the transfer of a genetic circuit of interest from a donor cell to a recipient or target cell. In one embodiment, the kit comprises (i) a donor cell expressing a repressor protein; and (ii) a genetic circuit of interest. The genetic circuit may be as defined above, and may in particular comprise an expression cassette into which a nucleic acid of interest may be inserted (operably linked) in functional proximity to a repressor binding sequence recognized by the repressor protein. In another embodiment of the invention, or in addition, the donor cell of the kit may contain a prophage sequence for the generation of a delivery vehicle, e.g., packaging of the genetic circuit of interest. The kit may further comprise a recipient or target cell, which does not express a repressor protein, thereby allowing expression of the nucleic acid of interest after transfer into the cell.

[0162] For a better understanding of the subject matter disclosed herein, and to illustrate how it may be carried out in practice, embodiments will now be described, by way of non-limiting example, with reference to the accompanying drawings in which: EXAMPLES

[0163] With particular reference to the examples, it is emphasized that the features shown are by way of example and for purposes of illustration of embodiments of the invention.

[0164] The following example demonstrates the use of interspecies promoter-repressor pairs for use in a novel system for the production of delivery vehicles. In this case, the repressor expressed in trans in the production strain is from a different bacterial species, ideally a phylogenetically different bacterial species. The use of interspecies repressors is a very advantageous tool to control the expression of a given protein or the transcription of a genetic circuit component only in strains that contain the heterologous repressor in trans. Nevertheless, care should be taken when following this approach, and not to use repressors that may be found ubiquitous, such as tryptophan synthesis repressors, since it has been shown that they are not orthogonal when transferring them to other species

[16] . However, as bacteria exist in environments with different characteristics, they have evolved specific repressors that recognize certain signals that may not be present in all other organisms. Two recent publications show that the use of interspecies repressors in E. coli is possible

[10]

[17] , providing a source of potential interspecies repressor-operator pairs even when the inducer is not known. Finally, another solution involves the expression of an inactive Cas9 molecule (dCas9) and a gRNA / tracrRNA that targets the promoter, RBS or coding sequence of the toxin moiety. dCas9 has no nuclease activity but is able to bind and block transcription from the region targeted by the gRNA

[18]

[19] .

[0165] Figure 1 depicts conditional transcriptional regulation by an interspecies repressor. On the left, a production strain containing a packaging prophage and the PhlF (interspecies) repressor in trans. The payload contains the packaging signal and the P phlF The desired sequence (actuator) is under the control of a promoter. Upon packaging of the phagemid particle, the target strain can be transduced, P phlF The promoters are active because they lack the PhlF repressor (not present in E. coli). Note that the PhlF repressor can be replaced with a dCas9+gRNA targeting the promoter, RBS, or actuator sequence.

[0166] As a proof of concept, P phlF A plasmid was constructed containing Cas9 under the control of a promoter (SEQ ID NO: 3), an sgRNA targeting LacZ, and a lambda phage cos signal. Cas9 targeting LacZ has been used previously in a different setting

[20] . We first attempted to transform this plasmid into two strains of E. coli: MG1655 (wt strain) and MG1656 (containing a deletion in lacZ). As expected, transformation of MG1655 did not yield colonies because the Cas9 circuitry targets its genome, which is toxic. In contrast, transformation into MG1656 gave colonies (Figure 2). Furthermore, transformation into cells containing the PhlF repressor (SEQ ID NO: 1, coding sequence: SEQ ID NO: 2) expressed in trans in a separate plasmid gave colonies in both cases because the repressor confers protection against Cas9 activity. The difference in size could be due to the fact that constitutive expression of Cas9 has been shown to be toxic

[21] .

[0167] FIG. 2 depicts transformation of Cas9-containing circuits. phlF A plasmid containing Cas9 under the control of a promoter and a constitutive sgRNA guide targeting lacZ (SEQ ID NO: 4) was transformed into MG1655 (left panel) or MG1656 (right panel). Empty cells (without any other plasmid) are shown at the top, and transformed cells containing an external plasmid encoding the PhlF repressor (SEQ ID NO: 5) are shown at the bottom.

[0168] To test whether such a system could also be transduced, the PhlF repressor was integrated into the genome of a production strain that also lacked the lacZ gene and thus was not targeted by Cas9. The production strain carrying this plasmid grew normally (data not shown). Phagemids were produced according to a standard heat induction protocol

[23] and titrated against MG1655 and MG1656 (Figure 3). Transduction of the Cas9-LacZ circuit into MG1655 did not give colonies, whereas colonies were recovered in the case of MG1656, as in the case of transduction.

[0169] Figure 3 demonstrates the transduction of Cas9-containing circuits. phlF A phagemid containing Cas9 under the control of a promoter and a constitutive sgRNA guide targeting lacZ (SEQ ID NO: 4) was transduced into MG1655 (left panel), MG1655 (middle) with the PhlF repressor encoded in the plasmid (SEQ ID NO: 5) or MG1656 (right panel).

[0170] Since the PhlF repressor does not naturally occur in E. coli, this system can be used to repress expression of a toxin protein (in this case, Cas9) in a production strain while allowing expression in another E. coli strain. In this particular case, an sgRNA guide was used that does not target the production strain, but since Cas9 is repressed, the system also allows for the production of phagemid particles that encode sgRNAs that target its genome. EXAMPLES

[0171] A plasmid was constructed containing the Cpf1 nuclease under the control of the PsrpR promoter (SEQ ID NO: 8), the crRNA targeting LacZ, and the lambda phage cos signal (p455, SEQ ID NO: 9). These plasmids were first transformed into two strains of E. coli: MG1655 (wild type strain) and MG1656 (containing a deletion in the lacZ gene). As expected, transformation of MG1655 did not yield colonies because the Cpf1 circuit is targeted to its genome, which is toxic. In contrast, transformation into MG1656 gave colonies (Figure 4). Furthermore, transformation into cells containing the SrpR repressor expressed in trans in another plasmid (pRARE4-SrpR-1.0, SEQ ID NO: 10) gave colonies in both cases because the repressor confers protection against Cpf1 activity (Figure 4).

[0172] To test whether such a system could also be transduced, the SrpR repressor (SEQ ID NO: 6, coding sequence: SEQ ID NO: 7) was integrated into the genome of a production strain that also lacks the lacZ gene and is therefore not targeted by Cpf1. Packaged phagemids were produced following the standard heat induction protocol as shown for the PhlF repressor data and quantified in MG1655 with or without the SrpR repressor supplied in trans, and in MG1656 (Figure 5). Transduction of the Cpf1-LacZ circuit into MG1655 gave an almost 4 log reduction in the number of colonies recovered, reflecting the high toxicity of the circuit, while in the case of MG1655 supplemented with the SrpR repressor, colonies were recovered to a similar number as in cells not containing the lacZ target (MG1656).

[0173] Finally, the addition of repressors to the genome of the production strain that can repress the payload in trans can help reduce the burden of circuits that would otherwise be constitutively expressed. Because doubling times are reduced due to the plasmid burden, cells become smaller, which can be detrimental to upscaling due to the longer incubation times required to reach a certain OD. Furthermore, and perhaps more importantly, circuits that contain constitutively expressed components are unstable and prone to rapid disruption as cells find ways to remove the metabolic burden

[24] -

[30] . This is especially true when their expression levels are high, as in the case of the Cpf1 circuit shown above, which can result in large economic losses if the circuit is disrupted during large-scale fermentation.

[0174] FIG. 6 shows the addition of the SrpR repressor, which confers an advantage to production strains encoding the Cpf1-LacZ circuit in that expression of Cpf1 is higher than in FIG. 4 and FIG. 5 (p841, SEQ ID NO: 11). srpR- Colony size was monitored following transformation of the Cpf1-LacZ circuit into production strains that either contained or did not contain the SrpR repressor. Cells were transformed and incubated overnight at 30° C. on chloramphenicol LB agar and colony size was followed after 15, 17, 19 and 22 hours. As can be seen in FIG. 6, colonies are clearly visible at 17 hours in the production strain containing the genomic SrpR repressor but not without. Even after 22 hours of incubation, colonies of the production strain without the repressor are visibly smaller than those containing SrpR, indicating that introduction of the repressor in trans reduces the metabolic burden in the production strain.

[0175] (References) TIFF2025023091000003.tif157168TIFF2025023091000004.tif219170TIFF2025023091000005.tif48170

Claims

[Claim 1] 1. A method for transferring a genetic circuit from a donor cell to a target cell, comprising contacting a donor cell with a target cell for a time sufficient to allow transfer of the genetic circuit to the target cell, wherein the donor cell expresses a repressor protein that is not encoded by the genetic circuit and is not present in the target cell, and the genetic circuit comprises a nucleic acid sequence of interest under the transcriptional control of a repressor binding sequence.