Biocontainment of unwanted engineered cells using non-natural amino acids

Abstract

The technology described herein relates to new nuclease and nickase fusions of new CRISPR / Cas systems and their use as a biocontainment kill-switch in modified and / or engineered bacteria.

Description

[0001] Biocontainment of unwanted engineered cells using non-natural amino acids Background

[0002] Microorganisms can be genetically reprogrammed to carry out various functions. Genetically modified (GM) microbes have a wide range of applications across several fields. For example, in the pharmaceutical industry they are used to produce active ingredients in vitro or inside the human or animal body. In the agriculture industry, bacteria are modified to enhance crop growth by improving pest resistance, or by promoting nutrient synthesis or uptake. Some strains are enhanced to combat plant pathogens or promote soil health. In environmental applications, bacteria are engineered to degrade pollutants, including oil spills, heavy metals, and organic waste, aiding in wastewater management and soil recovery. In other industrial applications, bacteria are modified to produce fuels, plastics, and enzymes used in food processing.

[0003] It is difficult to predict how GM microbes may impact different ecosystems. Therefore, there are strict regulations in place to prevent them from being unintentionally released into the environment. In many applications, GM microbes are applied in complex and uncontrolled environments, such as the human gut or the soil, which are not properly isolated from the environment. For such applications, diverse biocontainment strategies have been developed for both prokaryotic and eukaryotic microbes (see for example, Sebesta etal, Front. Plant. Sci., 2(13), 839446, 2022, doi: 10.3389 / fpls.2022.839446; Chang et ai., Nature Comm., 14, 6487, 2023, doi: https: / / doi.org / 10.1038 / s41467-023-42358-4; and George et ai., Nature Comms., 15, 650, 2024, doi:10.1038 / s41467-023-44531-l).

[0004] One of the most common biocontainment strategy is auxotrophy, where a GM microbe is unable to synthesize an essential compound or to utilize an environmentally available compound required for propagation or survival. Examples of such biocontainment practices include natural metabolic auxotrophs created by for example knocking out the thymidylate synthase thyA) or the tetrahydropicolinate synthase (dapA genes in E. coii A more advanced approach is synthetic auxotrophy, where propagation of the GMmicrobe is strictly dependent on the presence of non-natural amino acids. This approach was used to control GM eukaryotic cells as well, see Chang et ai., supra.

[0005] It has further been suggested that human cellular therapies, for example in regenerative medicines and cancer treatments would also benefit from the inclusion of a kill switch, see Di Stasi et ai., N.. Eng. J. Med., 365, 1673-1683, 2011, doi:10.1056 / NE]Moall06152. Such a kill switch could be induced in case of adverse events in such a patient. Such a kill switch would also be applicable in any type of animal cell.

[0006] These synthetic safeguards are more difficult to overcome by horizontal gene transfer or by natural mutations. However, all biocontainment systems that are based on auxotrophy are dependent on a molecule that needs to be provided externally during manufacturing and application of the GM cell. Large scale application of such molecules may also have an undesired environmental impact. Therefore new strategies have been developed which employ 'kill switches' or 'self-destruction mechanisms'. These mechanisms often require the introduction of sophisticated genetic circuits into cells, and are activated under certain conditions. Such conditions can be controlled externally by the addition of certain molecules to the environment, or can be environmental factors outside the site of application. A class of kill switches is based on gene circuits that can switch between two states depending on the presence of the biocontainment signal: (i) maintain essential gene expression (or block toxin gene expression) for survival, and (ii) block essential gene expression (or induce toxin gene expression) to kill the cell. The advantage of these switches is that they can be programmed for different environmental signals and the default state (in the absence of signal) can be freely chosen.

[0007] A second class of kill switches is based on programmable RNA guided nucleases (CRISPR-Cas systems). The advantage of these systems is that they can target the engineered regions in the target cells, and can be easily adapted for use in all types of prokaryotic and eukaryotic cells, thus physically eliminating any DNA sequences that would raise environmental concerns.

[0008] A major difficulty in applying kill switches in complex and unpredictable environments is the choice of the biocontainment signal that triggers cell death. For example, the signal should not occur spontaneously in the environment where the GM cells are applied, should not be toxic to the environment, should be taken up by the target cells, and should be relatively stable to be able to function.

[0009] Summary

[0010] The invention fulfils these needs by proving a new system for biocontainment based on the use of non-natural amino acids to reconstitute expression of an agent capable of performing biocontainment functions.

[0011] There are concerns that genetically engineered cells, especially those which are designed to secrete molecules of interest (MOI, e.g. cells which contain one or more exogenous genes for the conversion of one metabolite into another, or contain one or more exogenous genes which reduce the amounts of a disadvantageous molecule in the local environment), which are not usually secreted, or are secreted in higher amounts than a non-engineered equivalent cell may cause certain issues if and when released into the environment in an uncontrolled manner. Therefore, "kill switches" are designed to be able to be activated when desired to either kill the cell, reduce the amount of the cell or to turn off production of the MOI.

[0012] The present invention relies on the conditional expression of an active peptide product in the presence of a non-natural amino acid (NNAA), where the peptide product can (i) target and introduce a double-stranded or single-stranded DNA break in the chromosome of a cell which is unable to repair said break effectively, resulting in cell death, or (ii) target and modify a gene which is essential for growth of the cell, and the modification prevents expression of the essential gene, resulting in either cell death or a severe reduction in growth or proliferation of the cell, or (iii) target and modify an exogenous nucleic acid which is involved in the production of the MOI, and the modification prevents expression of the exogenous nucleic acid, resulting in reduction or prevention of the production of the MOI. Such a target sequence may be present in either the chromosome of the cell which harbours the peptide product, or in a plasmid or episome comprised by the cell.

[0013] Modified bacteria are proving useful to provide therapeutic effects in patients, such as in humans. For example, a bacteria can be engineered to produce drugs which provide a therapeutic effect in a microbiome (e.g. in any microbiome described herein), or to contain metabolic pathways which convert one detrimental molecule or metabolite into one which either has a neutral effect in the local environment, or indeed even a beneficial effect in said environment. However, there are regulatory concerns with such engineered bacteria, and the use of the peptide products in combination with engineered nonsense or quadruplet codons, tRNA / aaRS pairs and non-natural amino acids (NNAAs) as described herein can provide a biocontainment strategy for such engineered bacteria. Thus, (i) the expression of any exogenous genes can be reduced or prevented by targeted modification of the exogenous nucleic acids, (i) the growth or destruction of the cell in its entirety can be achieved by modification of a gene essential to growth or maintenance of the cell, or (iii) the cell can be destroyed (i.e. killed) by cutting the chromosome of the cell.

[0014] Any part of the disclosure (including the general description and concepts) may be combined with other parts of the disclosure, unless they do not make technical sense. It is not intended that this section or any headed section (which heading are used purely to aid understanding) is self-contained.

[0015] To this end, there is provided:- In a First Configuration

[0016] There is provided one or more nucleic acid(s) comprising:

[0017] a) a first nucleotide sequence comprising a start codon, coding codons and a first stop codon, wherein the coding codons encode a peptide product which kills or inhibits the growth of a target cell, or which modifies a target sequence in a target cell to prevent or reduce expression of one or more exogenous nucleic acids comprised by the target cell, wherein the first nucleotide sequence comprises at least one mutation which,

[0018] i. when the one or more nucleic acid(s) are expressed without the presence of a predefined non-natural amino acid, synthesis of the peptide product is truncated or rendered non-functional; and

[0019] ii. when the one or more nucleic acid(s) are expressed in the presence of the non-natural amino acid produces the peptide product which is functional in the target cell; b) a second nucleotide sequence which expresses a tRNA which is capable of mediating the transfer of the non-natural amino acid into the peptide product of part a)ii. at a position corresponding to a codon which comprises the at least one mutation, and which tRNA is not a substrate for any natural aminoacyl-tRNA synthetase of the target cell; and c) a third nucleotide sequence encoding an aminoacyl-tRNA synthetase which is capable of adding the non-natural amino acid to the tRNA of part b).

[0020] There is alternatively provided one or more nucleic acids comprising:

[0021] a) a first nucleotide sequence comprising a start codon, coding codons and a first stop codon, wherein the coding codons encode an activator peptide product, which activator peptide product modulates a cognate promoter,

[0022] wherein the first nucleotide sequence comprises at least one mutation which,

[0023] i. when the one or more nucleic acid(s) are expressed without the presence of a predefined non-natural amino acid, synthesis of the activator peptide product is truncated or rendered non-functional; and

[0024] ii. when the one or more nucleic acid(s) are expressed in the presence of the non-natural amino acid produces the activator peptide product which is functional to express the peptide product of part e) in the target cell;

[0025] b) a second nucleotide sequence which expresses a tRNA which is capable of mediating the transfer of the non-natural amino acid into the activator peptide product of part a)ii. at a position corresponding to a codon which comprises the at least one mutation, and which tRNA is not a substrate for any natural aminoacyl-tRNA synthetase of the target cell;

[0026] c) a third nucleotide sequence encoding an aminoacyl-tRNA synthetase which is capable of adding the non-natural amino acid to the tRNA of part b); and

[0027] e) a fourth nucleic acid encoding a peptide product which kills or inhibits the growth of a target cell, or which modifies a target sequence in a target cell to prevent or reduce expression of one or more exogenous nucleic acids comprised by the target cell, wherein the peptide product of e) is under control of the cognate promoter sequence, and absence the presence of the activator peptide product of part a), expression of the peptide product of e) is prevented (or reduced).

[0028] In a Second Configuration

[0029] There is provided one or more nucleic acid(s) comprising:

[0030] a) a first nucleotide sequence comprising a start codon, coding codons and a first stop codon, wherein the coding codons would encode a peptide product which kills or inhibits the growth of a target cell, or which modifies a target sequence in a target cell to prevent or reduce expression of one or more exogenous nucleic acids comprised by the target cell, except that at least one (e.g. one) of the coding codons which encodes a natural amino acid residue has been replaced with a second stop codon which is optionally different to the first stop codon, or except that a second stop codon has been inserted between two coding codons, or between the start codon and the first coding codon;

[0031] b) a second nucleotide sequence which expresses a tRNA which is capable of mediating the transfer of a predefined non-natural amino acid into the peptide product of part a) at a position corresponding to the second stop codon, and which tRNA is not a substrate for any natural aminoacyl-tRNA synthetase of the target cell; and

[0032] c) a third nucleotide sequence encoding an aminoacyl-tRNA synthetase which is capable of adding the non-natural amino acid to the tRNA of part b).

[0033] In a Third Configuration

[0034] There is provided one or more nucleic acid(s) encoding:

[0035] a) a first nucleotide sequence comprising a start codon, coding codons, and a first stop codon, wherein the coding codons comprise the nucleotide sequence of SEQ ID No:79, or the coding codons encode the amino acid sequence of SEQ ID No: 80, for example when expressed in the presence of OMTyr;

[0036] b) a second nucleotide sequence encoding a tyrosyl tRNA;

[0037] c) a third nucleotide sequence encoding a tyrosyl tRNA synthetase;

[0038] d) a fourth nucleotide sequence encoding a gRNA or crRNA, comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by a target sequence in the target cell genome;

[0039] e) a fifth nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No:2 (CasS2);

[0040] f) a sixth nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No:4 (CasS4); and

[0041] g) a seventh nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No: 5 (CasS5).

[0042] In a Fourth Configuration

[0043] There is provided one or more nucleic acid(s) encoding:

[0044] a) a first nucleotide sequence comprising the nucleotide sequence of SEQ ID No:79, or a nucleotide sequence encoding the amino acid sequence of SEQ ID No:80;

[0045] b) optionally a second nucleotide sequence encoding a tyrosyl tRNA;

[0046] c) optionally a third nucleotide sequence encoding a tyrosyl tRNA synthetase; and

[0047] d) optionally a fourth nucleotide sequence encoding a gRNA or crRNA, comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by a target sequence in a target cell genome.

[0048] In a Fifth Configuration

[0049] There is provided a vector, or a plurality of vectors comprising the one or more nucleic acid(s) as described elsewhere herein.

[0050] There is also provided a transmissible element (e.g. a plasmid, a virus, a conjugative plasmid, a phage or a phagemid), comprising the one or more nucleic acid(s) as described elsewhere herein. There is also provided a plasmid (e.g. a conjugative plasmid or plasmid comprised by a phage particle) comprising the one or more nucleic acid(s) as described elsewhere herein.

[0051] There is also provided a cell comprising the one or more nucleic acid(s) as described elsewhere herein, or a cell comprising a plasmid as described elsewhere herein.

[0052] In a Sixth Configuration

[0053] There is provided a cell comprising a target sequence which comprises a nuclease cleavage site nucleotide sequence (e.g. a zinc finger or TALEN cleavage site), and further comprising

[0054] the one or more nucleic acid(s) as described elsewhere herein, and

[0055] the one or more exogenous nucleic acids as described elsewhere herein, and

[0056] wherein the peptide product of part a) is a meganuclease, a zinc finger nuclease or a TALEN.

[0057] There is also provided a cell comprising a target sequence which comprises, in 5' to 3' orientation:

[0058] a) a PAM sequence; and

[0059] b) a protospacer sequence,

[0060] and further comprising

[0061] the one or more nucleic acid(s) as described elsewhere herein, and

[0062] the one or more exogenous nucleic acids as described elsewhere herein, and

[0063] wherein the peptide product of part a) is a CRISPR / Cas nuclease or nickase, and

[0064] optionally wherein the target sequence comprises a PAM sequence 5' of the or a protospacer sequence, e.g. wherein the PAM is immediately adjacent to the protospacer sequence.

[0065] There is also provided a cell comprising a target sequence which comprises, in 5' to 3' orientation:

[0066] a) a nuclease cleavage site nucleotide sequence;

[0067] b) optionally a nuclease spacer nucleotide sequence;

[0068] c) a PAM sequence; and

[0069] d) a protospacer sequence,

[0070] and further comprising

[0071] the one or more nucleic acid(s) as described elsewhere herein, and

[0072] optionally the one or more exogenous nucleic acids as described elsewhere herein, and wherein the peptide product of part a) is as described elsewhere herein, and

[0073] optionally wherein the target sequence comprises a PAM sequence 5' of the or a protospacer sequence, e.g. wherein the PAM is immediately adjacent to the protospacer sequence.

[0074] In a Seventh Configuration

[0075] There is provided a pharmaceutical composition (optionally an in vitro composition or wherein the composition is comprised by a medical container) comprising (i) the one or more nucleic acids as described elsewhere herein, or (ii) the cell as described elsewhere herein, or (iii) the vector or transmissible element as described elsewhere herein, and a pharmaceutically acceptable excipient or carrier.

[0076] In an Eighth Configuration

[0077] There is provided a biocontainment method for a target cell, said method comprising

[0078] (I) providing a target cell comprising a target sequence as described elsewhere herein; and (II) exposing the target cell to the predefined non-natural amino acid to produce the peptide product of part a);

[0079] whereby the target cell is killed or the growth of the target cell is inhibited (in particular whereby the target cell is killed).

[0080] There is also provided a biocontainment method for a target cell comprising one or more exogenous nucleotide sequences (e.g. as described elsewhere herein), said method comprising

[0081] (I) providing a target cell comprising a target sequence as described elsewhere herein and the one or more exogenous nucleotide sequences; and

[0082] (II) exposing the target cell to the predefined non-natural amino acid to produce the peptide product of part a);

[0083] whereby the expression of said one or more exogenous nucleotide sequences in the target cell is reduced or prevented.

[0084] There is also provided a method of modifying target cells in a microbiome (e.g. gut of a subject), comprising

[0085] (I) administering to said microbiome (i) a cell as described elsewhere herein, or (ii) a pharmaceutical composition as described elsewhere herein, and

[0086] (II) subsequently administering to the microbiome the predefined non-natural amino acid to produce the peptide product of part a),

[0087] thereby modifying a target sequence in the target cells (in particular whereby the modification of the target cell results in death of the target cell).

[0088] In a Ninth Configuration

[0089] There is provided a method of modifying a target cell to comprise a target sequence wherein the target sequence comprises, in 5' to 3' orientation:

[0090] a) an I-TevI cleavage site nucleotide sequence (optionally as described elsewhere herein); b) an I-TevI spacer nucleotide sequence (optionally as described elsewhere herein); c) a PAM sequence (optionally as described elsewhere herein); and

[0091] d) a protospacer sequence (optionally as described elsewhere herein),

[0092] and optionally wherein sequences a) to d) are each immediately adjacent to each other, said method comprising introducing one or more modifications into the cell to produce the target sequence; and

[0093] further comprising the step of introducing into the cell one or more nucleic acid(s) as described elsewhere herein, one or more vector(s) as described elsewhere herein, a transmissible element as described elsewhere herein, or a plasmid as described elsewhere herein.

[0094] Brief Description of the Drawings

[0095] Figure 1: Schematic representation of the plasmid combinations employed for the TevCasS plasmid targeting assays.

[0096] Figure 2: Graphical representation of the results from the TevCasS plasmid targeting assays. The x-axis corresponds to the drop in the transformation efficiency of a MG1655 strain that expresses TevCasSl and targets the target sequence on a TevCasS target plasmid relative to the transformation efficiency of a MG1655 strain that expresses TevCasSl and does not target the target sequence on a TevCasS target plasmid. The y-axis corresponds to the size of the I-TevI spacer sequence in the target sequence of a TevCasS target plasmid.

[0097] Figure 3: Graphical representation of the results from the TevCasS chromosomal targeting assays. The x-axis corresponds to the drop in the transformation efficiency of a MG1655 strain that expresses TevCasSl and targets a chromosomal TevCasS target sequence relative to the transformation efficiency of a MG1655 strain that expresses TevCasSl and does not target a chromosomal TevCasS target sequence. The y-axis corresponds to the size of the I-TevI spacer sequence in the target sequence of a chromosomal TevCasS target sequence.

[0098] Figure 4: Graphical representation of the TevCasS target cleavage site preferences in a 'Krona plot' form. The sequence of the TevCasS target cleavage site is 5'CN₁N₂N₃G-5'. The inner ring corresponds to Ni, the middle ring corresponds to N2 nucleotide and the outer ring corresponds to N3. In the wheel, every sequence occupies a sector, with an area proportional to the relative enrichment of the sequence.

[0099] Figure 5: E. co / / cells with the expanded genetic code were transformed with a plasmid carrying a toxin gene inactivated by a stop codon (TAG). Transformants were serially diluted and selected in the absence and presence of OMTyr.

[0100] Figure 6: Map of the plasmid carrying all the components of the kill switch, including the elements of the CasS system, the J17 tRNA, and the MjTyrRS gene (p3036).

[0101] Figure 7: Incorporation of the non-natural amino acid OMTyr restores the activity of the stop codon mutant I-TevI-XTEN-CasSl gene. Overnight cultures were grown from colonies of b8315 and b8316 cells. Serial dilutions of cells were spotted on LB Km plates in the presence and absence of 1.5 mM OMTyr. Figure 8: Targeting two chromosomal sites results in improved killing. Overnight cultures were grown from colonies of b8500 and b8501 cells. Serial dilutions of cells were spotted on LB Km plates in the presence and absence of 1.5 mM OMTyr.

[0102] Figure 9: Map of a self-destructing conjugative plasmid carrying the biosynthesis genes for the molecule of interest (MOI) and a non-natural amino acid-inducible CRISPR-Cas system targeting a sequence located in the genes responsible for the production of the MOI. Genes on the plasmid can be expressed from different promoters (P), which can be constitutive or inducible.

[0103] Figure 10: Map of the plasmid carrying the components of the S. mutans CRISPR / Cas system. The system consists of genes encoding the Cas3, Cas5, Cas8, and Cas7 proteins and a CRISPR array targeting the clbl, clbA and clbC genes of the pks operon. The plasmid has the CloDF13 replication origin and the aadA gene providing spectinomycin resistance.

[0104] Figure 11: Incorporation of the non-natural amino acid OMTyr restores the activity of stop codon mutant Cas3 and Cas7 genes. Serial dilutions of cells were spotted on plates in the presence and absence of 1.5 mM OMTyr.

[0105] Detailed Description

[0106] Definitions

[0107] An "aminoacyl-tRNA synthetase", also sometimes referred to in the art as an amino acid-tRNA-ligase, is an enzyme which covalently links a specific amino acid to its corresponding tRNA molecule. This occurs through the transesterification of the amino acid to the CCA tail of the tRNA. Generally, aminoacyl-tRNA synthetases comprise at least a catalytic domain, which carries out the transesterification reaction, and an anticodon binding domain, which recognises the anticodon loop of the tRNA.

[0108] A "beneficial metabolite" refers to a molecule that is beneficial to the local environment of the recipient or donor cell, or to the microbiome as a whole, or indeed causes downstream beneficial effects to the organism which may comprise the cell or microbiome. Beneficial metabolites may not be present, or may be present in too low a concentration in the recipient bacterium which results in a detrimental effect in the recipient (or microbiome or organism), optionally as compared to other bacteria in a microbiome. Beneficial metabolites may be molecules which provide a competitive advantage to the recipient or donor bacterium. In some embodiments, a beneficial metabolite may be a food source, such as a sugar which can be used by the recipient or donor bacterium, thus providing a competitive advantage to the bacteria which are able to use that food source compared to other bacteria in a microbiome which are not able to use that food source.

[0109] As used herein, a "conjugative plasmid" is a particular transmissible element. A conjugative plasmid is a plasmid which, when comprised within a bacterial cell ("host" or "donor" cell, used interchangeably herein) is able to be transferred to another bacterium ("recipient" cell) through the mechanism of bacterial conjugation. Bacterial conjugation is the unidirectional and horizontal transmission of genetic information from one bacterium to another. Conjugative plasmids generally fall into two classes: mobilizable plasmids and self-transmissible plasmids. Mobilizable plasmids comprise at least an origin of transfer (oriT), a relaxase and other genetic information on the plasmid which is transferred to the recipient cell, and require helper functions provided by e.g. a second plasmid or the chromosome of the donor cell to effect the plasmid transfer. On the other hand, self-transmissible plasmids, in addition to the genetic information on the plasmid which is transferred to the recipient cell, also contain all the molecular machinery needed for self-transfer (e.g. for pilus formation and initiation of gene transfer) on the same plasmid. Both mobilizable plasmids and self-transmissible plasmids comprise relaxase genes which recognises the origin of transfer (oriT) and catalyses both the initial cleavage of or / Tin the donor, to produce the DNA strand from the plasmid that will be transferred, as well as the final ligation of the transported DNA in the recipient cell that reconstitutes the conjugated plasmid. Thus, in one embodiment, the conjugative plasmid is a mobilizable plasmid. In another embodiment, the conjugative plasmid is a self-transmissible plasmid. In another embodiment, the conjugative plasmid includes an origin of transfer oriT). Plasmid mobility, mechanisms and structures are described in more detail in Smillie et a!., Microbiol. Mol. Biol. Rev., 74(3): 434-452, 2010 doi: 10.1128 / MMBR.00020-10, which is incorporated herein in its entirety.

[0110] Bacterial cells possessing a conjugative self-transmissible plasmid contain a surface structure (pilus) encoded by the conjugative machinery on the plasmid that is involved in the coupling of donor and recipient cells, and the transfer of the genetic information contained within the plasmid. Conjugation involves contact between cells, and the transfer of genetic traits can be mediated by many plasmids. Among all natural transfer mechanisms, conjugation is the most efficient. For example, F plasmid of E. coli, pCFIO plasmid of Enterococcus faecaiis and pXO16 plasmid of Bacillus thuringiensis employ different mechanisms for the establishment of mating pairs, the sizes of mating aggregates are different, and they have different host ranges within gram-negative (F) as well as gram-positive (pCFIO and pXO16) bacteria. Their plasmid sizes are also different; 54, 100 and 200 kb, respectively. Remarkably, however despite differences in origin and size, those conjugation systems are able to sustain efficient conjugative transfer in liquid medium. The conjugative process permits the protection of plasmid DNA against environmental nucleases, and thus efficient delivery of plasmid DNA into a recipient cell can be obtained. Conjugation functions are naturally plasmid encoded. Numerous conjugative plasmids (and transposons) are known, which can transfer associated genes within one species (narrow host range) or between many species (broad host range). Transmissible plasmids are widespread across the domain of bacteria and similar systems of horizontal gene transfer through pilus structures have recently been described for Archaea. Engineered conjugative plasmids are described in more detail in WO2021 / 037732 (SNIPR Biome ApS), which is incorporated herein in its entirety. The features of such conjugative plasmids and bacterial cells comprising them as described in the claims as filed in WO2021 / 037732 are also incorporated herein by reference. "Constitutive promoter" refers to a promoter that is capable of facilitating continuous transcription of a coding sequence or gene under its control and / or to which it is operably linked in any growth conditions. Constitutive promoters and variants are well known in the art and are described elsewhere herein.

[0111] A "detrimental metabolite" refers to a molecule that is harmful to the local environment of the recipient or donor cell, or to the microbiome as a whole, or indeed causes downstream harmful effects to the organism which may comprise the cell or microbiome. Detrimental metabolites, whilst needed and / or required to a certain degree, may be present in too high a concentration in the recipient bacterium which results in a detrimental effect in the recipient (or microbiome or organism), optionally as compared to other bacteria in a microbiome. Detrimental metabolites may be molecules which cause a fitness cost to the recipient or donor bacterium. In embodiments where the second MOI is a detrimental metabolite, the first MOI is a molecule which either has a neutral effect of the recipient or donor bacterium (or microbiome or organism), or is easily excreted by the cell and / or organism, or is a beneficial metabolite.

[0112] "Exogenous" or "heterologous" are used interchangeably and refer to a nucleotide sequence that is not normally found in a given cell in nature. The given cell is usually the donor or recipient bacterium, in particular the recipient bacterium. Thus, an exogenous or heterologous nucleic acid may be one which is not naturally found in the recipient bacterium, i.e. the nucleic acid is exogenous to the recipient bacterium. As used herein, a heterologous sequence encompasses a nucleic acid sequence (or amino acid sequence) that is exogenously introduced into a given cell. A heterologous gene includes a native gene, or fragment thereof, that has been introduced into the cell. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include a native coding sequence that is a portion of a chimeric gene to include non-native regulatory regions that is reintroduced into the cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non-native cell. Thus, a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and / or the polypeptide which it encodes; and / or two or more nucleic acid sequences that are not found in the same relationship to each other in nature. For plasmids, such as conjugative plasmid, a heterologous gene is one which has been engineered to be included on such plasmid (e.g. conjugative plasmid). However, in alternative embodiments relating to plasmids, such as conjugative plasmids, a heterologous or exogenous gene is heterologous to the recipient bacterium.

[0113] As used herein an "exporter" refers to a membrane-integrated protein which is capable of exporting a molecule (such as a naturally occurring cellular substrate or metabolite, an MOI, or any other peptide, protein or small molecule) out of the bacterial cell. As discussed elsewhere herein, in some bacteria there are no endogenous exporters of MOIs, and thus the cell must rely on diffusion of the MOIs to remove the MOIs from the cell. Without being bound by theory, the export of MOIs in gram-negative bacteria may be particularly problematic, due to the presence of both an inner and outer membrane. Thus, the addition of a heterologous exporter capable of exporting the MOI in the transmissible element may provide further benefits in this type of bacterial cell. In other bacteria, there may be endogenous exporters which export substrates which are used in the production of the second MOI. When these substrates are exported from the cell, they are no longer available to be used in the production of the second MOI, thus potentially decreasing the amount of second MOI available in the cell to be converted into the first MOI. It may be desirable to deactivate or reduce the ability of these exporters to export the endogenous substrates from the cell. The ability of any given protein to act as an exporter can be easily measured by those skilled in the art. Identification of putative exporters can be achieved through literature searches as well as by using protein and genetic databases, such as GenBank, which are well-known to those skilled in the art.

[0114] As used herein an "importer" refers to a membrane-integrated protein which is capable of importing a molecule (such as a naturally occurring cellular substrate or metabolite, a second MOI or any other peptide, protein or small molecule) into the bacterial cell. As discussed elsewhere herein, in some bacteria there are no endogenous importers of second MOIs and / or of substrates which are used in the production of the second MOI, and thus the cell must rely on diffusion of the second and / or such substrates MOIs to import them into the cell. Thus, the addition of a heterologous importer capable of importing the second MOI and / or substrates which are used in the production of the second MOI in the transmissible element may provide further benefits in this type of bacterial cell. The ability of any given protein to act as an importer can be easily measured by those skilled in the art. Identification of putative importers can be achieved through literature searches as well as by using protein and genetic databases, such as GenBank, which are well-known to those skilled in the art.

[0115] An "inducible promoter" refers to a promoter which transcribes a coding sequence or gene under its control and / or to which it is operably linked in the presence of an inducer of said promoter. The inducer may be one or more environmental condition(s) and / or one or more inducing molecule(s).

[0116] A "microbiome," as used herein, refers to the totality of microbes in a particular environment (e.g. in / on an organism, in a marine environment (e.g. ocean), and / or in a terrestrial environment (e.g. soil)). In some embodiments, a microbiome may refer to the totality of microbes that reside, or are stably maintained, for example, on the surface and in deep layers of the skin, in the saliva and oral mucosa, in the conjunctiva, and in the gastrointestinal tracts of an organism. The microbiome may exist within any of the organs described elsewhere herein.

[0117] "Peptide", "polypeptide" and "protein" are used interchangeably herein.

[0118] "Phage" or "bacteriophage" are used interchangeably and refer to obligate intracellular parasites that multiply inside bacteria by co-opting some or all of the host biosynthetic machinery. Phage genomes come in a variety of sizes and shapes. Most phages range in size from 24-200 nm in diameter. Phages contain nucleic acid (i.e., genome) and proteins, and may be enveloped by a lipid membrane. Depending upon the phage, the genome can be either DNA or RNA, and can exist in either circular or linear forms. The size of the phage genome varies depending upon the phage. The simplest phages have genomes that are only a few thousand nucleotides in size, while the more complex phages may contain more than 100,000 nucleotides in their genome, and in rare instances more than 1,000,000. The number and amount of individual types of protein in phage particles will vary depending upon the phage.

[0119] A "phagemid" refers to a bacteriophage-derived vector containing the replication origin of a plasmid and the packaging site of a bacteriophage. Examples of phagemids that may be used in accordance with the present disclosure include, without limitation, M13-derived phagemids containing the fl origin for filamentous bacteriophage packaging such as, for example, pBluescript II SK (+ / -) and KS (+ / -) phagemids, pBC SK and KS phagemids, pADL and Pl-based phagemids (see, e.g. Westwater CA et al., Microbiology 148, 943-50 (2002); Kittleson JT et al., ACS Synthetic Biology 1, 583-89 (2012); Mead DA et al., Biotechnology 10, 85-102 (1988)). Other phagemids may be used and, for example, can be made to work with packaging systems from natural, engineered or evolved bacteriophage.

[0120] "Preventing" as used herein in relation to transcription or expression of a gene or protein refers to a complete ablation of transcription or expression.

[0121] "Reducing" as used herein in relation to transcription or expression of a gene or protein refers to a reduction in gene transcription or expression, such as at least a 50% reduction of transcription or expression. In one embodiment, the reduction is at least a 60%, at least a 70%, at least an 80% reduction. In one embodiment, the reduction is at least an 85% reduction. In one embodiment, the reduction is at least a 90% reduction. In one embodiment, the reduction is at least a 95% reduction. In one embodiment, the reduction is at least a 97% reduction. In one embodiment, the reduction is at least a 99% reduction. In one embodiment, the reduction is a 100% reduction.

[0122] As used herein, with respect to treatment methods, "prevention" includes a reducing of the risk of contracting the disease. The "treatment or prevention" may be complete or partial treatment or prevention, i.e. a reduction, but not complete reduction of the disease / condition or symptoms thereof; or a reducing of the risk but not total prevention of the disease / condition or a symptom thereof. Similarly, the methods treat or prevent (i.e. reduces the risk of) an undesirable symptom of the disease or condition or the therapy.

[0123] A "therapeutic metabolite" refers to a molecule that is able to act in the local environment of the donor or recipient cell to provide a therapeutic effect. This may be by, for example, counteracting the presence of another detrimental molecule or causing a downstream reaction which provides the therapeutic effect.

[0124] A "transmissible element" as used herein refers to any means which can be transferred to a recipient bacterium, including, but not limited to a plasmid, a conjugative plasmid, a phage or a phagemid. In some embodiments, a transmissible element may include other mobile genetic elements, such as transposons and the like. In a particular embodiment, the transmissible element is selected from a conjugative plasmid, and phage and a phagemid. In another particular embodiment, the transmissible element is a conjugative plasmid.

[0125] A "tRNA" refers to transfer RNA which is a small (typically ~75-90 nucleotides in length) RNA molecule which plays a key role in protein synthesis. Each tRNA comprises an acceptor stem, a T loop, a D loop and an important anticodon loop. It is this anticodon loop which recognises a codon (naturally, a 3-nucleotide codon) in the messenger RNA (mRNA). At the acceptor stem of the tRNA molecule, attached to a CCA tail, is the amino acid which will be added to the emerging peptide or polypeptide by the ribosome. The amino acid is added to the acceptor stem of the tRNA by an aminoacyl-tRNA synthetase which is specific for both the tRNA and the amino acid.

[0126] Any percentage identity herein may be at least (about) 70%. For example, any percentage identity herein may be at least (about) 80%. For example, and in particular, any percentage identity herein may be at least (about) 90%. For example, and in particular, any percentage identity herein may be at least (about) 95%. For example, any percentage identity herein may be at least (about) 96%. For example, any percentage identity herein may be at least (about) 97%. For example, any percentage identity may be at least (about) 98%. For example, any percentage identity herein may be at least (about) 99%.

[0127] Any percent identity may be at least (about) 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to any given sequence.

[0128] Percent identity for amino acid sequences are determined using the blastP algorithm with the following parameters: - The default parameters are adjusted for short input sequences, the expect threshold is set at 0.05 and the length of the seed sequence that initiates an alignment is set at 6. Regions of low compositional complexity are masked. The employed scoring matrix is 'BLOSUM62', with scoring costs to create and extend a gap being 11 and 1 respectively. Conditional compositional score matrix adjustment is employed to compensate for amino acid composition of compared sequences.

[0129] Percent identity for nucleotide sequences are determined using the blastn algorithm with the following parameters: - The default parameters are automatically adjusted for short input sequences, the expect threshold is set at 0.05 and the length of the seed sequence that initiates an alignment is set at 28. Regions of low compositional complexity are masked. The query sequence is masked while producing seed sequences used to scan databases, but not masked for extensions. Matches are scored as +1 and mismatches are scored as -2.

[0130] For example, an amino acid sequence described herein is identical to the reference SEQ ID No, except for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes, in particular 1 to 5, for example 1 to 3, such as 1 or 2, e.g. 1 amino acid change. For example, a nucleic acid sequence described herein is identical to the reference SEQ ID No, except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotide changes. For example, an amino acid sequence described herein is identical to the reference SEQ ID No except for a total number of amino acid changes wherein the total number is no more than (about) 5, 10, 15, 20, 25 or 30% of the number of amino acids in the reference sequence. For example, a nucleotide sequence described herein is identical to the reference SEQ ID No except for a total number of nucleotide changes wherein the total number is no more than (about) 5, 10, 15, 20, 25 or 30% of the number of nucleotides in the reference sequence.

[0131] Non-natural amino acids and their corresponding tRNAs and RNA synthetases to recognise specific codons

[0132] Many different non-natural amino acids (NNAAs) exist, and have been exploited for a number of different functions in peptide and protein molecules, such as providing detectable labels (e.g. fluorescent side chains), preventing or reducing proteolysis, changing the lipophilic profile, changing the secondary structure. As expected with such a range of functions, NNAAs may be considered as "analogues" or as "surrogates", depending on whether they resemble natural amino acids, or differ significantly in their structure. For the purposes herein, it is desirable that the NNAA is structurally similar to the natural amino acid occurring in the protein product at the given location where the NNAA will be inserted in the final product. Thus, the NNAA may be a structural analogue of a naturally occurring amino acid which would have been included in the peptide product, except for the presence of the at least one mutation in the first nucleotide sequence. For a review of NNAAs, see for example, Castro et al., Biomolecules, 13(6), 981, 2023, doi:10.3390 / biom13060981; Li et al., Biosci. Rep., 42(8), BSR20220168, 2022, doi:10.1042 / BSR20220168; and Adhikari et al., RSC Adv., 11, 38126-38145, 2021, doi:10.1039 / DlRA07028B, each of which is incorporated herein by reference in its entirety.

[0133] In the present application, the NNAA is used to activate the kill-switch properties of the peptide product in the target cell. Without the addition of the NNAA, and because the nucleic acid sequence has been mutated (e.g. to replace a coding codon with a non-sense codon, or a quadruplet codon) any peptide product which is expressed is non-functional (e.g. by truncation, or by a frameshift mutation which results in expression of a different amino acid sequence after the frameshift, which cannot function, for example because this sequence is unable to fold correctly, or does not form a functional active site). When the NNAA is added to the cell, the expression of a tRNA and amino-acyl-tRNA synthetase (aaRS) results in the addition of the NNAA to the nascent polypeptide chain of the peptide product, restoring its functionality and resulting in either killing or inhibiting the growth of the target cell, or in modification of a target sequence in the target cell which prevents or reduces expression of one or more exogenous nucleic acids in the target cell.

[0134] Thus, there is provided one or more nucleic acid(s) comprising:

[0135] a) a first nucleotide sequence comprising a start codon, coding codons and a first stop codon, wherein the coding codons encode a peptide product which kills or inhibits the growth of a target cell, or which modifies a target sequence in a target cell to prevent or reduce expression of one or more exogenous nucleic acids comprised by the target cell, wherein the first nucleotide sequence comprises at least one mutation which, iii. when the one or more nucleic acid(s) are expressed without the presence of a predefined non-natural amino acid, synthesis of the peptide product is truncated or rendered nonfunctional; and

[0136] iv. when the one or more nucleic acid(s) are expressed in the presence of the non-natural amino acid produces the peptide product which is functional in the target cell; b) a second nucleotide sequence which expresses a tRNA which is capable of mediating the transfer of the non-natural amino acid into the peptide product of part a)ii. at a position corresponding to a codon which comprises the at least one mutation, and which tRNA is not a substrate for any natural aminoacyl-tRNA synthetase of the target cell; and

[0137] c) a third nucleotide sequence encoding an aminoacyl-tRNA synthetase which is capable of adding the non-natural amino acid to the tRNA of part b).

[0138] There is also provided one or more nucleic acid(s) comprising:

[0139] a) a first nucleotide sequence comprising a start codon, coding codons and a first stop codon, wherein the coding codons would encode a peptide product which kills or inhibits the growth of a target cell, or which modifies a target sequence in a target cell to prevent or reduce expression of one or more exogenous nucleic acids comprised by the target cell, except that at least one (e.g. one) of the coding codons which encodes a natural amino acid residue has been replaced with a second stop codon which is optionally different to the first stop codon, or except that a second stop codon has been inserted between two coding codons, or between the start codon and the first coding codon;

[0140] b) a second nucleotide sequence which expresses a tRNA which is capable of mediating the transfer of a predefined non-natural amino acid into the peptide product of part a) at a position corresponding to the second stop codon, and which tRNA is not a substrate for any natural aminoacyl-tRNA synthetase of the target cell; and

[0141] c) a third nucleotide sequence encoding an aminoacyl-tRNA synthetase which is capable of adding the non-natural amino acid to the tRNA of part b).

[0142] The NNAA tRNA / aaRS pair (encoded by the second and third nucleotide sequences respectively) may be orthogonal to the endogenous tRNA / aaRS pairs occurring in the target cell. Orthogonal tRNA / aaRS pairs are unable to (significantly) charge the tRNAs of the target cell with the NNAA, and the tRNA / aaRS pairs of the target cell will be unable to (significantly) charge the NNAA tRNA / aaRS pair. Thus, the tRNA encoded by the second nucleic acid sequence is not a substrate for any natural aminoacyl-tRNA synthetase of the target cell. This ensures that the peptide product is not expressed (or is only minimally expressed) in the absence of the NNAA. By "not a substrate", it is not intended that the tRNA encoded by the second nucleic acid is never able to be charged by a naturally occurring amino acid, but that the tRNA has a much stronger ability to be charged with the NNAA than to be charged with any naturally occurring amino acid. Thus, the tRNA encoded by the second nucleic acid may be capable of introducing the predefined NNAA (at least) 3-fold more frequently than any naturally occurring amino acid. In some examples, the NNAA may be introduced at least 4-fold (e.g. 5-fold, 6-fold, 7-fold, and in particular at least 10-fold) more frequently than any naturally occurring amino acid. Particularly, the NNAA may be introduced at least 20-fold (e.g. 50-fold, and in particular at least 100-fold) more frequently than any naturally occurring amino acid. Additionally or alternatively, the tRNA encoded by the second nucleic acid may be capable of binding (i.e. being charged by) the predefined NNAA (at least) 3-fold more than any naturally occurring amino acid. In some examples, the NNAA may be bound (i.e. charged) at least 4-fold (e.g. 5-fold, 6-fold, 7-fold, and in particular at least 10-fold) more than any naturally occurring amino acid. Particularly, the NNAA may be bound (i.e. charged) at least 20-fold (e.g. 50-fold, and in particular at least 100-fold) more frequently than any naturally occurring amino acid.

[0143] The tRNA / aaRS pair used to incorporate the NNAA (NNAA tRNA / aaRS pair, encoded by the second and third nucleotide sequence respectively) is usually a mutated tRNA / aaRS pair. Mutations to tRNAs to permit their charging with a predetermined NNAA may be carried out for example using compartmentalised partnered replication (CPR), as described in Figure 1 and the corresponding methods in Maranhao et al., ACS Synth. Biol, 2016, doi:10.1021 / acssynbio.6b00145, which is incorporated herein by reference in its entirety. Generally, any given tRNA can be permissively mutated at the CCA tail end to allow charging with a NNAA without interfering with the ability of the tRNA to recognise any given codon, because codon binding is mediated by the anti-codon loop of the tRNA (see Figure 1 of Anderson et al., infra). Thus, starting with any given naturally occurring tRNA / aaRS pair, the aaRS can be mutated (e.g. by directed evolution) to provide specificity for the predetermined NNAA over any naturally occurring amino acid, and to add that NNAA to the tRNA, which may also need to be mutated to receive the NNAA. Some methods (as described in Maranhao et al., supra) for high throughput screening of mutated tRNA / aaRS pairs involve the use of a GFP variant where the nucleotide sequence has an introduced stop codon (or quadruplet codon) in place of a surface-exposed tyrosine. The functional fluorescent protein can be obtained (i) by mis-charging of the 'artificial' stop codon with a naturally occurring amino acid because a native tRNA recognises the stop codon and introduces a natural amino acid (base check that GFP not reconstituted by the native charging enzymes of the cell), or (ii) by mis-charging of the 'artificial' stop codon with a predetermined NNAA, but not the engineered tRNA / aaRS pair, because a native tRNA / aaRS is capable of charging and adding the NNAA (by the addition of the NNAA to the native charging system in the cell); or (iii) mis-charging of the 'artificial' stop codon with a naturally occurring amino acid by the engineered tRNA / aaRS pair (by the addition of the engineered tRNA / aaRS pair to the native charging system in the cell); or (iv) charging by a NNAA using the heterologous / engineered charging enzyme (by the addition of both the engineered tRNA / aaRS pair and the NNAA to the native charging system in the cell). If only experiment (iv) provides the fluorescent GFP, then the NNAA is not recognised by the native charging system and the engineered tRNA / aaRS pair does not charge naturally-occurring amino acids. Methods for mutating the anticodon loops of tRNAs to recognise quadruplet codons or nonsense codons are described elsewhere herein.

[0144] The aaRS encoded by the third nucleotide sequence is capable of adding the predetermined NNAA to the tRNA encoded by the second nucleotide sequence. This aaRS should not be capable of (significantly) adding any naturally occurring amino acid to the tRNA.

[0145] Determining whether any given aaRS or mutated aaRS (expressed from the third nucleotide sequence) is capable of adding the NNAA to the tRNA (expressed by the second nucleotide sequence) may be carried out, for example, by the methods described in Tamura et al., J. Biol. Chem., 269(35), 22173-22177, 1994, PMID:8071341 (see Figure 2 and corresponding methods); Wang eta / ., infra and Maranhao et al., supra each of which is incorporated herein by reference in its entirety.

[0146] A skilled person is also able to test the orthogonality of any given NNAA tRNA / aaRS pair, for example using the methods described in Anderson et al., PNAS, 101(20), 7566-7571, 2004, doi:10.1073 / pnas.0401517101, which is incorporated herein by reference in its entirety.

[0147] Use of tRNA / aaRS pairs for NNAAs in yeast and mammalian cells are summarised in Liu and Schultz, infra.

[0148] In some applications of the technology, the expression of only one copy of each of the second and third nucleotide sequences may not provide enough of the tRNA and / or the aaRS. Thus, the one or more nucleic acids may comprise two or more (e.g. between 2 and 5) copies of the second nucleotide sequence. Additionally or alternatively, the one or more nucleic acids may comprise two or more (e.g. between 2 and 5) copies of the third nucleotide sequence.

[0149] In the absence of the NNAA, the peptide product may not be expressed or may be expressed only in truncated form. In particular embodiments, the mutation in the first nucleotide sequence results in the introduction (by replacement or addition) of a second (premature) stop codon (for example, introduction of TAG, TAA, or TGA before the first stop codon, or a frameshift mutation which introduces a stop codon after the frameshift mutation but before the first stop codon). In any embodiment herein, in the absence of the NNAA, the second stop codon truncates the synthesis of the peptide product, rendering it non-functional.

[0150] Thus, in any embodiment, the at least one mutation in the first nucleotide sequence is the replacement of a coding codon with a second stop codon. Alternatively, in any embodiment, the at least one mutation in the first nucleotide sequence is the insertion of a second stop codon into the first nucleotide sequence after the start codon and before the stop codon. In these examples, the second stop codon is selected from an amber nonsense codon (TAG), an ochre nonsense codon (TAA) and an opal nonsense codon (TGA), and is different from the first stop codon. Further, in these examples, when the one or more nucleic acid(s) are expressed without the presence of the predefined non-natural amino acid, synthesis of the peptide product is truncated at the amino acid residue encoded by the coding codon immediately preceding the second stop codon. Expression of the one or more nucleic acid(s) without the presence of the predefined non-natural amino acid (e.g. in part a)i.) may be in vitro in media which does not contain the predefined non-natural amino acid. Expression of the one or more nucleic acid(s) without the presence of the predefined non-natural amino acid (e.g. in part a)i.) may be in vitro in media which contains the non-natural amino acid. In an example, the in vitro expression of the peptide product of part a) is measured in an assay measuring the number of colony forming units (CFU) of the target cell in the presence and absence of the non-natural amino acid. A statistically significant decrease in the CFU of the target cell in the presence of the non-natural amino acid indicates that the peptide product is functional for killing or inhibiting the growth of the target cell, for example the assay as described in Example 2.1.3.

[0151] For measuring whether a peptide product prevents or reduce expression of one or more exogenous nucleic acids comprised by the target cell, the amount of expression of the one or more exogenous nucleic acids (or the amount of the first MOI produced by the one or more exogenous nucleic acids) can be measured in the presence and absence of the non-natural amino acid. A statistically significant decrease in the expression of the one or more exogenous nucleic acids, or a statistically significant reduction in the production of the first MOI in the presence of the non-natural amino acid indicates that the peptide product is functional for preventing or reducing expression of one or more exogenous nucleic acids comprised by the target cell.

[0152] The Examples, in particular Example 2.2.1 and 2.2.2 hereinbelow, show methods for testing reconstitution of functionality of a toxin-based peptide product (Example 2.2.1) and an RNA-guided nuclease-based peptide product (Example 2.2.2).

[0153] Truncations are particularly interesting, because the truncation can be designed such that little or no peptide product is formed, thus ensuring that no residual killing activity is observed when the NNAA is not present. For example, the second (premature) stop codon (whether introduced as a non-sense codon or frameshift mutation resulting in such downstream non-sense codon) is introduced within the first 5 (such as the first 4, 3, or 2) coding codons of the first nucleotide sequence encoding the peptide product. For example, the second (premature) stop codon (whether introduced as a nonsense codon or frameshift mutation resulting in such downstream non-sense codon) is introduced within the first 10 (such as the first 9, 8, 7, or 6, in particular 7) coding codons of the first nucleotide sequence encoding the peptide product. In a particular example, the second stop codon is introduced immediately after the start codon and before the first coding codon. In another example, the second (premature) stop codon (whether introduced as a non-sense codon or frameshift mutation resulting in such downstream non-sense codon) is introduced within the first 20% (for example, the first 15%, 10% or 5%, in particular the first 10%) of coding codons of the first nucleotide sequence encoding the peptide product.

[0154] A skilled person has access to numerous protein databases (Genbank, wwPDB, RSCB PDB, RefSeq, Alphafold and the like) which contain annotated sequences of various protein products, identifying individual sub-domains and their functions. Many of the peptide products described herein comprise catalytic domains, or other domains or series of residues which confer upon said peptide product the ability to kill or inhibit the growth of a target cell, or to modify a target sequence in a target cell to prevent or reduce expression of one or more exogenous nucleic acids comprised by the target cell. Examples include, but are not limited to nuclease domains, DNA cutting domains, or another active site / cata lytic domain (e.g. in the RNases, kinases and acetyltransferases of toxin-antitoxin systems). Thus, in another example, the peptide product comprises a catalytic domain and the second (premature) stop codon (whether introduced as a non-sense codon or frameshift mutation resulting in such downstream non-sense codon) is introduced (e.g. inserted) before the coding codon which encodes the first amino acid of the catalytic domain. In another example, the peptide product comprises a catalytic domain and the second (premature) stop codon replaces a coding codon for a residue before the catalytic domain. This ensures that, while some peptide product is translated in the absence of the NNAA, it will not be able to perform its usual function, as the catalytic domain is not translated in the truncated product.

[0155] Whilst truncations are useful to ensure that, in the absence of the NNAA, no functionality of the kill switch is observed, the corollary to this is to ensure that, when the NNAA is added to the peptide product in the final functional form, the NNAA does not interfere with the functionality of the peptide product. Thus, it may be important for some peptide products to consider the positioning of the mutation which allows the introduction of the NNAA (e.g. by tRNA recognition of a quadruplet codon or of a nonsense codon) with respect to the three-dimensional structure of the peptide product.

[0156] Thus, in some examples, the NNAA replaces a (naturally occurring) amino acid residue which is (known or predicted to be) a surface amino acid residue of the peptide product (for example, by replacement of the coding codon for the (naturally occurring) amino acid residue with a nonsense codon or with a quadruplet codon), e.g. when the one or more nucleic acid(s) are expressed in the presence of the NNAA. In other examples, the NNAA is inserted between two coding codons which encode two amino acid residues on the surface of the peptide product (for example, by insertion of a nonsense codon or with a quadruplet codon between two coding codons for (naturally occurring) amino acid residues which are (known or predicted to be) surface amino acid residues of the peptide product), e.g. when the one or more nucleic acid(s) are expressed in the presence of the NNAA. In particular examples, the second stop codon replaces a coding codon which encodes an amino residue which is (known or predicted to be) a surface amino acid residue of the peptide product. Alternatively, the second stop codon is inserted between two coding codons which encode two amino acid residues (which are known or predicted to be) on the surface of the peptide product.

[0157] Because of the requirement for non-functionality of the peptide product in the absence of the NNAA, and the requirement for functionality of the peptide product in the presence of the NNAA, in particular embodiments, the positioning of the codon for the NNAA (e.g. nonsense codon or quadruplet codon) is at a position which is (known or predicted to be) on the surface of the peptide product (when correctly folded), and the mutation is (e.g. when the NNAA is encoded by a nonsense codon) or results in (when the NNAA is encoded by a quadruplet codon resulting in a frameshift mutation which introduces a second stop codon) a truncation of the peptide product before the catalytic domain of the peptide product.

[0158] Any vector, nucleic acid or nucleotide sequence described herein may be codon optimized for use in a human, animal (e.g. mammalian, rodent, mouse or rat), plant or fungus cell (or any of the other cells described herein). The vectors, nucleic acids and / or nucleotide sequences described herein may be codon optimized for use in a eukaryotic cell. Vectors and nucleic acid sequences for use in eukaryotic cells may comprise nuclear localisation sequences (NLSs). NLSs facilitate entry of the proteins and polypeptides described herein into eukaryotic cells. NLSs are known to those skilled in the art, and include, but are not limited to monopartite or bipartite NLSs. The NLS sequence may be from an SV40 Large T-antigen (see Kalderon et al., Cell, 39(3), 499-509, 1984, doi: https: / / doi.org / 10.1016 / 0092-8674(84)90457-4, which is incorporated herein by reference in its entirety), from nucleoplamin, importin a, c-myc, EGL-13, TUS-protein, hnRNP Al or yeast transcription repressor Mata2.

[0159] Optionally, any vector, nucleic acid or nucleotide sequence herein described is codon optimized for use in a prokaryotic cell, e.g. a bacterial or archaeal cell, in particular an E. coli cell.

[0160] Amber nonsense codons (TAG)

[0161] In most eukaryotes and prokaryotes, the opal nonsense codon (TGA) is used at a significantly higher frequency than the amber nonsense codon (TAG) as termination codon of protein-coding genes, see Trexler et al., Sci. Rep., 13, 14294, 2023, doi:10.1038 / s41598-023-41410-z, which is incorporated herein by reference in its entirety. As it is relatively rare as a stop codon in many species, this makes it attractive as a second stop codon for introduction into the first nucleotide sequence, because there is less chance of off-target effects for other native proteins in the target cell being transcribed through a TAG stop codon and introducing unwanted phenotypes. That said, in this particular use of the inclusion of an NNAA in the peptide product with a view to killing or inhibiting the growth of a target cell, or to modifying a target sequence in a target cell to prevent or reduce expression of one or more exogenous nucleic acids comprised by the target cell, should another off-target gene be transcribed through a stop codon (any of TAG, TAA and TGA) resulting in death of the target cell, then this is not necessarily as much of an issue as for other applications of this technology.

[0162] In E. coli for example, the TAG stop codon is used 0.0003% compared to the other 63 codons. In humans, mice and rats that number is 0.0005%, see https: / / www.genscript.com / tools / codon-frequency-table. In all these species, the TAG stop codon has a lower frequency than TAA and TGA. The inventors chose this amber nonsense codon for use in the proof of concept Examples below.

[0163] Thus, in a particular example, in any embodiment herein, the second stop codon is an amber nonsense codon (TAG). Several groups have developed mutant tRNA / aaRS pairs with the ability to recognise the TAG stop codon. One of the most studied and well-characterised tRNA / aaRS pair with the ability to be charged with a predetermined NNAA and to recognise a TAG stop codon is the M. jannaschii tyrosyl tRNA / TyrRS in combination with O-Methyl tyrosine as the predetermined NNAA, see Wang eta / ., infra, Zhang etai., infra, Maranhao et al., supra, and Chen etai., infra. Other tyrosine derivatives have been added (e.g. 3-halo-tyrosines), and could be used in the present application, but these are likely to be more disruptive to the function of the peptide product due to the inclusion of the halo groups which are not usually seen in naturally occurring amino acids.

[0164] Thus in a particularly useful example, the second stop codon is an amber nonsense codon (TAG); and the non-natural amino acid is O-methyl tyrosine (OMTyr); and the second nucleic acid of part b) encodes a tyrosyl tRNA; and the third nucleic acid of part c) encodes a tyrosyl tRNA synthetase.

[0165] The tyrosyl tRNA may be a J17 tyrosyl tRNA, for example a mutated or engineered J17 tyrosyl tRNA. The J17 tyrosyl tRNA may be from Methanococcus jannaschii. In a particular example, the second nucleotide sequence comprises the nucleotide sequence of residues 97 to 173 of SEQ ID No:71.

[0166] The tyrosyl tRNA synthetase may be a Methanococcus jannaschii tyrosyl tRNA synthetase (MjTyrRS), for example a mutated or engineered Methanococcus jannaschii tyrosyl tRNA synthetase. In a particular example, the third nucleotide sequence comprises the nucleotide sequence of SEQ ID No:72. Alternatively, the third nucleotide sequence encodes a tyrosyl tRNA synthetase comprising the amino acid sequence of SEQ ID No:73.

[0167] Because OMTyr is structurally similar to the natural amino acid tyrosine, it is expected that the substitution of OMTyr for tyrosine in the peptide product is less likely to cause issues with the folding and functionality of the peptide product, if it is included outside of any catalytic domain, and in particular in a surface-exposed position. Thus, in any embodiment, the TAG codon replaces a tyrosine coding codon (TAC or TAT). In a particular example, the TAG codon replaces a surface-exposed tyrosine coding codon, which is optionally not comprised by the catalytic domain of the peptide product.

[0168] Similarly, OMTyr is structurally similar to the natural amino acid phenylalanine, due to the presence of the benzyl ring, and is expected to be a good candidate for replacement of phenylalanine residues in the peptide product, if they are present in the correct locations. Thus, in any embodiment, the TAG codon replaces a phenylalanine coding codon (TTT or TTC). In a particular example, the TAG codon replaces a surface-exposed phenylalanine coding codon, which is optionally not comprised by the catalytic domain of the peptide product.

[0169] Other tRNA / aaRS pairs recognising the amber nonsense codon (TAG) in mammalian cells have been described, see for example, Koher et al., PNAS, 98(25), 14310-14315, 2001, doi: www.pnas.org / cgi / doi / 10.1073 / pnas.251438898, which is incorporated herein by reference in its entirety. Ochre nonsense codons (TAA)

[0170] Although the ochre nonsense codon (TAA) is used more frequently in many species than the amber nonsense codon (TAG), the ochre stop codon is still an attractive option for the second stop codon in the present utility. As mentioned above, even if another off-target gene be transcribed through another ochre stop codon, which results in the death of the cell, then this off-target effect would result in killing or inhibiting the growth of the target cell, thus turning off the expression of any exogenous nucleic acids comprised by said target cell.

[0171] Several groups have developed mutant tRNA / aaRS pairs with the ability to recognise the TAA stop codon, see for example Liu and Schultz, Annu. Rev. Biochem., 79, 413-444, 2010, doi:10.1146 / annurev.biochem.052308.105824, and Chatterjee et al., Biochemistry, 52(10), 2013, doi:10.1021 / bi4000244. One of the most studied and well-characterised tRNA / aaRS pair with the ability to be charged with a predetermined NNAA and to recognise a TAA stop codon is the Methanosarcina barkeri or the Methanosarcina mazei-derived pyrrolysyl tRNA / aaRS pair. Whilst these tRNA / aaRS pairs have been successfully charged with a number of predetermined NNAAs, such as allyloxycarbonyl lysine, boc-lysine and a number of other lysine derivatives, pyrrolysine is a particularly useful NNAA for the present utility, as it is relatively unreactive and has similar properties to naturally occurring amino acids, lysine and arginine.

[0172] Thus, in one example, the second stop codon is an ochre nonsense codon (TAA); and the non-natural amino acid is pyrrolysine; and the second nucleic acid of part b) encodes a pyrrolysyl tRNA; and the third nucleic acid of part c) encodes a pyrrolysyl tRNA synthetase.

[0173] The pyrrolysyl tRNA may be a Methanosarcina barkeri pyrrolysyl tRNA, for example a mutated or engineered Methanosarcina barkeri pyrrolysyl tRNA. In a particular example, the second nucleotide sequence comprises the nucleotide sequence of SEQ ID No:36.

[0174] The pyrrolysyl tRNA synthetase may be a Methanosarcina barkeri pyrrolysyl tRNA synthetase, for example a mutated or engineered Methanosarcina barkeri pyrrolysyl tRNA synthetase. In a particular example, the third nucleotide sequence comprises the nucleotide sequence of SEQ ID No:37. Alternatively, the third nucleotide sequence encodes a pyrrolysyl tRNA synthetase comprising the amino acid sequence of SEQ ID No:38.

[0175] In a particular embodiment, the pyrrolysyl tRNA and pyrrolysyl tRNA synthetase pair are both from Methanosarcina barkeri, e.g. any of the Methanosarcina barkeri tRNA / aaRS described above.

[0176] The pyrrolysyl tRNA may be a Methanosarcina mazei pyrrolysyl tRNA, for example a mutated or engineered Methanosarcina mazei pyrrolysyl tRNA. In a particular example, the second nucleotide sequence comprises the nucleotide sequence of SEQ ID No:30.

[0177] The pyrrolysyl tRNA synthetase may be a Methanosarcina mazei pyrrolysyl tRNA synthetase, for example a mutated or engineered Methanosarcina mazei pyrrolysyl tRNA synthetase. In a particular example, the third nucleotide sequence comprises the nucleotide sequence of SEQ ID No:31. Alternatively, the third nucleotide sequence encodes a pyrrolysyl tRNA synthetase comprising the amino acid sequence of SEQ ID No:32.

[0178] In a particular embodiment, the pyrrolysyl tRNA and pyrrolysyl tRNA synthetase pair are both from Methanosarcina mazei, e.g. any of the Methanosarcina mazei tRNA / aaRS described above.

[0179] Because pyrrolysine is structurally related to the nature amino acid lysine, but comprises a more bulky pyrrole ring, it is expected that the substitution of pyrrolysine for a lysine which is surface-exposed in the peptide product is less likely to cause issues with the folding and functionality of the peptide product. The surface-exposed lysine should not be part of the catalytic domain of the peptide product. Thus, in any embodiment, the TAA codon replaces a surface-exposed lysine coding codon (AAA or AAG), which is optionally not comprised by the catalytic domain of the peptide product.

[0180] Similarly, pyrrolysine is structurally similar to the natural amino acid arginine, due to the length of the cardon side change and it's basic nature, and is expected to be a good candidate for replacement of arginine residues in the peptide product, if they are present in the correct locations. Thus, in any embodiment, the TAA codon replaces a surface-exposed arginine coding codon (AGA or AGG), which is optionally not comprised by the catalytic domain of the peptide product.

[0181] Other tRNA / aaRS pairs recognising the ochre nonsense codon (TAA) in mammalian cells have been described, see for example, Koher et al., supra.

[0182] Opal nonsense codons (TGA)

[0183] Although the opal nonsense codon (TGA) is used more frequently in many species that then amber nonsense codon (TAG), the opal stop codon is still an attractive option for the second stop codon in the present applications. As mentioned above, even if another off-target gene be transcribed through another opal stop codon, which results in the death of the cell, then this off-target effect would result in killing or inhibiting the growth of the target cell, thus turning off the expression of any exogenous nucleic acids comprised by said target cell.

[0184] Several groups have developed mutant tRNA / aaRS pairs with the ability to recognise the TGA stop codon, see for example Albers et al., Nature Comms., 12, 3850, 2021, doi:https: / / doi.org / 10.1038 / s41467-021-24076-xand Zhang et al., PNAS, 101(24), 8882-8887, 2004, doi: www.pnas.org / cgi / doi / 10.1073 / pnas.0307029101. In the present application, 5-hydroxytryptophan is a useful NNAA, as it is relatively unreactive and has similar properties to naturally occurring amino acid, tryptophan.

[0185] Thus, in one example, the second stop codon is an opal nonsense codon (TGA); and the nonnatural amino acid is 5-hydroxy-L-tryptophan; and the second nucleic acid of part b) encodes a 5-hydroxytryptophan tRNA; and the third nucleic acid of part c) encodes a 5-hydroxytryptophan tRNA tRNA synthetase.

[0186] The hydroxytryptophan tRNA may be a Bacillus subtih's tryptophan tRNA, for example a mutated or engineered Bacillus subtih's tryptophan tRNA. In another example, the hydroxytryptophan tRNA is a variant of a Bacillus so / stryptophan tRNA. In a particular example, the second nucleotide sequence comprises the nucleotide sequence of SEQ ID No:33.

[0187] The 5-hydroxytryptophan tRNA synthetase may be a Bacillus subtilis tryptophan tRNA synthetase, for example a mutated or engineered Bacillus subtilis tryptophan tRNA synthetase. In another example, the 5-hydroxytryptophan tRNA synthetase is a variant of a Bacillus subtilis tryptophan tRNA synthetase. In a particular example, the third nucleotide sequence comprises the nucleotide sequence of SEQ ID No:34. Alternatively, the third nucleotide sequence encodes a 5-hydroxytryptophan tRNA synthetase comprising the amino acid sequence of SEQ ID No: 35.

[0188] Because 5-hydroxytryptophan is structurally similar to the natural amino acid tryptophan, it is expected that the substitution of 5-hydroxytryptophan for tryptophan in the peptide product is less likely to cause issues with the folding and functionality of the peptide product, if it is included outside of any catalytic domain, and in particular in a surface-exposed position. Thus, in any embodiment, the TGA codon replaces a tryptophan coding codon (TGG). In a particular example, the TGA codon replaces a surface-exposed tryptophan coding codon, which is optionally not comprised by the catalytic domain of the peptide product.

[0189] Quadruplet codons resulting in frameshift mutations

[0190] In addition to the repurposing of non-sense codons to be recognised by a tRNA which is charged with a NNAA by a specific aminoacyl-tRNA synthetase (aaRS), tRNAs have been engineered to recognise four-base codons ("quadruplet codon"). Engineering of any given tRNA to enable it to recognise a quadruplet codon, rather than a natural 3-base codon is possible by engineering an expanded anticodon loop into the tRNA, usually to 8 nucleotides instead of the usual 7. As for other tRNAs for NNAAs, specificity for the NNAA (e.g. the engineered quadruplet tRNA (qtRNA) should not be a substrate of any naturally occurring aaRS) and the quadruplet codon (and not any triplet codon) is important. In parallel, the aaRS may need engineering to recognise the new qtRNA and / or the NNAA to be added and not any other naturally occurring tRNA.

[0191] Engineering of the anticodon loop may be achieved by directed evolution of tRNAs, for example by designing libraries of mutations in the tRNA anticodon loop and screening, see e.g. Anderson et al., supra. In addition, the catalytic domain of the aaRS may need to be mutated such that the aaRS is only capable of adding the NNAA to the qtRNA and not any naturally occurring amino acid. Methods for achieving this are also described in Anderson et al., supra.

[0192] Any of the tRNAs which recognise the non-sense codons above, and are already paired with a cognate aaRS which is capable of charging a NNAA are particularly interesting starting points for simply engineering the anticodon loop of the tRNA to generate a new qtRNA, without (necessarily) needing to also mutate the aaRS.

[0193] Thus, in one example, the mutation (in the first nucleic acid sequence) is the replacement of a coding codon with a non-natural quadruplet codon which results in a frameshift mutation. Such a replacement may involve removing all three nucleotides of a coding codon and replacing them with four new nucleotides of a quadruplet codon. Alternatively, such a replacement may involve the insertion of a single nucleotide (expanding a triplet codon to a quadruplet codon). Alternatively, such an replacement may be a combination of insertion(s) and replacement(s) of nucleotides to achieve a quadruplet codon in the first nucleic acid. As an example, a coding GGC codon may be replaced with GCAC, where the C replaces the G of the original coding codon and the A is inserted between the second G and final C of the original coding codon. Any combination of insertions and replacements are contemplated herein.

[0194] In another example, the mutation (in the first nucleic acid sequence) is the insertion of a nonnatural quadruplet codon between two coding codons which results in a frameshift mutation. In this example, four new nucleotides are inserted between two original coding codons.

[0195] In another example, the first nucleic acid is not mutated, but a sequence of four naturally occurring bases is recognised by the qtRNA / aaRS pair, resulting in a frameshift mutation.

[0196] In these examples, the frameshift mutation results in synthesis of the peptide product being truncated or rendered non-functional by the frameshift mutation, when (e.g. in part a)i.) the one or more nucleic acid(s) are expressed without the presence of the predefined non-natural amino acid, The introduction of the quadruplet codon results in the synthesis of the peptide product being truncated or rendered non-functional when the one or more nucleic acid(s) are expressed without the presence of the predefined non-natural amino acid. For example, the frameshift mutation introduces a second (premature) stop codon downstream of the quadruplet codon. By analysing the nucleotide sequence of the peptide product, a skilled person is able to determine appropriate locations in the first nucleotide sequence for the introduction of the quadruplet codon, which will result in the desired premature stop codon. Thus, in an example, the frameshift mutation results in a second stop codon being introduced into the first nucleotide sequence, such that (for example in part a)i.), when the one or more nucleic acid(s) are expressed without the presence of the predefined non-natural amino acid, synthesis of the peptide product is truncated at the amino acid encoded by the coding codon immediately preceding the second stop codon.

[0197] In the case of a quadruplet codon, the first nucleotide sequence may continue to be expressed without having engineered in a premature stop codon into the frameshifted sequence. In these scenarios, a longer peptide product may be expressed, but is in fact rendered non-functional by the change in amino acid sequence which is as a result of said frameshift mutation. Again, a skilled person is able to determine from the nucleotide sequence encoding the peptide product how any given quadruplet codon (i.e. inserted, mutated or rendered recognisable by the qtRNA / aaRS pair) will affect the resulting amino acid sequence of the peptide product. The various databases and programmes mentioned herein may be able to predict whether the new amino acid sequence will be able to fold correctly, and, even in the absence of such a prediction, the skilled person is able to experimentally test (e.g. in high throughput methods, as described elsewhere herein) whether such a translated peptide product is functional to kill or inhibit the growth of a target cell, or to modify a target sequence in a target cell to prevent or reduce expression of one or more exogenous nucleic acids comprised by the target cell, when it expressed without the addition of the predetermined NNAA. Thus, in any example relating to quadruplet codons, (e.g. in part a)i.), when the one or more nucleic acid(s) are expressed without the presence of the predefined non-natural amino acid, the peptide product is rendered non-functional by the frameshift mutation.

[0198] Several groups have developed mutant tRNA / aaRS pairs with the ability to recognise non-naturally occurring quadruplet codons, see for example Neumann H, et al., Nature, 464, 441-444, 2010, doi:10.1038 / nature08817 and Anderson et al., supra, P useful qtRNA / aaRS pair with the ability to charge a NNAA and to recognise an AGGA quadruplet codon is described in Anderson et al., the identity and mutations of the orthogonal tRNA / aaRS pair derived from the type I lysyl-tRNA synthetase of Pyrococcus horikoshii are hereby incorporated herein by reference in their entirety. In this case the NNAA is homoglutamine. Thus, in one example, the non-natural quadruplet codon is AGGA, and the non-natural amino acid is homoglutamine, the second nucleic acid of part b) encodes an AK514 homoglutamine tRNA; and the third nucleic acid of part c) encodes a homoglutamine tRNA-synthetase.

[0199] Alternatively, the tRNA may be a mutated or engineered lysyl tRNA, such as a mutated or engineered lysyl tRNA from P. horikoshii, for example a mutated or engineered Type I lysyl tRNA from P. horikoshii, as described in Anderson et al., supra. In an example, the anticodon loop of the variant tRNA comprises the sequence CUUCCUAA. In a particular example, the tRNA is an AK514 homoglutamine tRNA, for example the tRNA described in Figure 1 of Anderson et al., supra, which is incorporated herein by reference.

[0200] Alternatively, the third nucleic acid sequence encodes a mutated or engineered lysyl tRNA synthetase, such as a mutated or engineered lysyl tRNA synthetase from P. horikoshii, as described in Anderson et al., supra. In an example, the mutated lysyl tRNA synthetase comprises a mutation at Glu-41 and / or Tyr-268 of the lysyl tRNA synthetase from P. horikoshii, as described in Anderson et al., supra. In particular, the lysyl tRNA synthetase from P. horikoshii comprises an Ile-41 and / or Ser-268 mutation, such as both an Ile-41 mutation and a Ser-268 mutation.

[0201] Because homoglutamine is structurally and functionally similar to the natural amino acids asparagine and glutamine it is expected that the substitution of homoglutamine for either asparagine and glutamine in the peptide product is less likely to cause issues with the folding and functionality of the peptide product, if it is included outside of any catalytic domain, and in particular in a surface-exposed position. Thus, in any embodiment, the AGGA replaces a coding codon which encodes an asparagine residue (AAT or AAC). In a particular example, the AGGA codon replaces a surface-exposed asparagine coding codon, which is optionally not comprised by the catalytic domain of the peptide product.

[0202] Glutamine is similar to homoglutamine because they both have hydrogen bond-donating and -accepting properties. Thus, alternatively, in any embodiment, the AGGA replaces a coding codon which encodes a glutamine residue (CAA or CAG). In a particular example, the AGGA codon replaces a surface-exposed glutamine coding codon, which is optionally not comprised by the catalytic domain of the peptide product.

[0203] Nuclease-based peptide products

[0204] In a particular embodiment, the peptide product of part a) (e.g. as part of the one or more nucleic acid(s), transmissible elements, cells or compositions described elsewhere herein) is a nuclease or nickase which kills or inhibits the growth of the target cell by modifying a target sequence comprised by the genome of the target cell. Alternatively, the nuclease or nickase modifies the target sequence in the target cell to prevent or reduce expression of the one or more exogenous nucleic acids comprised by the genome (e.g. chromosome or episome) of the target cell.

[0205] In a particular example, the peptide product in a nuclease.

[0206] Nuclease systems that may be used as a peptide product are well known in the art. Thus, the peptide product may be a nuclease. The peptide product may be a nuclease and may further comprise a gRNA or crRNA. Examples of such nucleases are described in Piller et al., RNA, 3(3), 279-290, 1997, PMID:9056765, which is incorporated herein by reference in its entirety.

[0207] In one embodiment, the nuclease is selected from a meganuclease, a zinc finger, a TALEN. There is also provided a target cell comprising a target sequence which comprises a nuclease cleavage site nucleotide sequence (e.g. a zinc finger or TALEN cleavage site) and the one or more exogenous nucleic acids as described elsewhere herein. In one embodiment, the target sequence is a non-native (i.e. exogenous) sequence to the cell.

[0208] There is also provided a target cell comprising a target sequence which comprises a nuclease cleavage site nucleotide sequence (e.g. a zinc finger or TALEN cleavage site), the one or more exogenous nucleic acids as described elsewhere herein, and the one or more nucleic acid(s) as described elsewhere herein, and wherein the peptide product of part a) is a meganuclease, a zinc finger nuclease or a TALEN.

[0209] The target sequence may be an RNA. The target sequence may be a single stranded DNA. The target sequence may be comprised by the chromosome of the target cell. Alternatively, the target sequence in the target cell may be comprised by a plasmid or episome of the cell.

[0210] The target sequence may be a sequence within the one or more exogenous nucleic acids. In this example, the nuclease reduces or prevents the expression of the one or more exogenous nucleic acids. The target sequence may be located within an essential gene of the target cell. In this example, the nuclease prevents the expression of the essential gene, and the target cell is thereby killed, or the growth of the cell is reduced. CRISPR / Cas-based peptide products

[0211] CRISPR / Cas based guided nucleases are particularly attractive as peptide products for use in the present applications because of their ease of design and specificity to their target sequences. In a particular example, the peptide product is a CRISPR / Cas nuclease. In an example, the nuclease is selected from a Type I, Type II, Type III or Type V nuclease. In a particular example, the peptide product is comprised by a Type I or Type III CRISPR / Cas system in combination with at least one Cascade protein.

[0212] In an example, the peptide product is a CRISPR / Cas nickase, such as a Type I, Type II, Type III or Type V nickase.

[0213] Such CRISPR / Cas nucleases are used in the present applications in combination with a fourth nucleotide sequence which expresses a gRNA or crRNA. gRNAs are generally a single RNA sequence, whereas a crRNA is processed after transcription of a pre-crRNA, which optionally complexes with the tracrRNA. Both gRNA and crRNA comprises one or more repeat sequences and a spacer sequence. The spacer sequence is complementary to a protospacer sequence in the target sequence, and provides the nuclease with the precise location to bind and cut (or nick) the target sequence. Sequences of specific CRISPR / Cas proteins from various source strains can be found in various databases, such as https: / / crisprcas.i2bc.paris-saclay.fr / .

[0214] In any of the descriptions of gRNA or crRNAs (e.g. expressed from the fourth nucleotide sequence) relating to CRISPR / Cas systems as peptide products, in an example, the nuclease (or nickase) forms a complex with the gRNA or crRNA which comprises a first spacer that is cognate to a first protospacer in the target sequence. In one embodiment, the nuclease (or nickase) forms a ribonucleoprotein complex with a gRNA or crRNA which comprises a first spacer that is cognate to a first protospacer in the target sequence. In one embodiment, the gRNA or crRNA comprises two repeat sequences (e.g. two repeat sequences).

[0215] The gRNA or crRNA may comprise a first spacer that is cognate to a first protospacer in the target sequence, wherein the protospacer is immediately adjacent to a Protospacer Adjacent Motif (PAM) sequence in the target sequence.

[0216] To improve the effectiveness of the ability of the CRISPR / Cas nuclease or nickase systems as peptide products to perform the function of either killing or inhibiting the growth of the target cell by modifying a target sequence comprised by the genome of the target cell, or preventing or reducing expression of the one or more exogenous nucleic acids comprised by the genome of the target cell, the crRNA or gRNA (e.g. expressed from the or a fourth nucleotide sequence) may comprise a second which hybridises to a second protospacer comprised by the target cell. Alternatively, the one or more nucleic acid(s) comprises a further nucleotide sequence encoding a gRNA or crRNA comprising one or more repeat sequences and a second spacer sequence which hybridises to a second protospacer comprised by the target cell. The second protospacer may be immediately adjacent to a Protospacer Adjacent Motif (PAM). The second protospacer may be in the chromosome of the target cell. The second protospacer may be comprised by the one or more exogenous nucleic acids described elsewhere herein.

[0217] The crRNA or gRNA (e.g. expressed from the or a fourth nucleotide sequence) may comprise a third spacer which hybridises to a third protospacer comprised by the target cell. Alternatively, the one or more nucleic acid(s) comprises a further nucleotide sequence encoding a gRNA or crRNA comprising one or more repeat sequences and a third spacer sequence which hybridises to a third protospacer comprised by the target cell. The third protospacer may be immediately adjacent to a Protospacer Adjacent Motif (PAM). The third protospacer may be in the chromosome of the target cell. The third protospacer may be comprised by the one or more exogenous nucleic acids described elsewhere herein.

[0218] PAMs for each type of CRISPR / Cas system are well-known and are characterised in various databases and scientific publications.

[0219] The protospacer may be an RNA. The protospacer may be a single stranded DNA.

[0220] The protospacer in the target cell may be comprised by the chromosome of the cell. In target cells which are bacterial cells, the cutting of chromosomal DNA results in cell death, as bacteria are not able to effectively replicate DNA with double strand breaks. Alternatively, the protospacer in the target cell may be comprised by a plasmid or episome of the cell. The plasmid may be destroyed by the cutting action of the nuclease (i.e. the plasmid is cured). If the plasmid comprises the one or more exogenous nucleic acids, then the curing of the plasmid results in the prevention of the expression of the one or more exogenous nucleic acids.

[0221] The protospacer may be a sequence within the one or more exogenous nucleic acids. In this example, the nuclease or nickase reduces or prevents the expression of the one or more exogenous nucleic acids.

[0222] The protospacer may be located within an essential gene of the cell. In this example, the nuclease or nickase prevents the expression of the essential gene, and the target cell is thereby killed, or the growth of the cell is reduced.

[0223] In some examples, the target cell may comprise more than one copy of the same protospacer sequence (and optionally the PAM sequence and / or any nuclease cleavage site nucleotide sequence and / or any nuclease spacer nucleotide sequence). In one example, the target cell comprises two copies of the same protospacer sequence. In another example, the target cell comprises three, four or five copies of the same protospacer sequence. If the protospacer is comprised by a transposon or by a ribsosomal gene, then the number of copies of the protospacer sequence can be higher, for example up to 15 copies. The protospacer may be chosen as a naturally occurring sequence within the target cell, or may be engineered into the target cell. When there is more than one protospacer in the target cell, the protospacers may be a combination of naturally occurring and engineered sequences. Where the peptide product comprises a nickase, it may be beneficial to include two protospacer sequences in the target cell, in particular when the protospacers are separated by (about) 20 nucleotides or fewer. In a particular example, each protospacer is on a separate strand of DNA. In such cases, the nickase may form a staggered end cut, which is less likely to be successfully repaired by cellular machinery, particularly in eukaryotic cells.

[0224] Furthermore, where the cells are eukaryotic cells, and the peptide product comprises a nuclease it may be beneficial to include two protospacers, which are reasonably close to each other, e.g. separated by (about) 20 nucleotides or fewer. In such cases, when both protospacers are cut, it is more likely that a genetic deletion will result and the gene will be inactivated, than a repair via INDEL formation (which may still result in a viable gene product, and must be present on both chromosomes). For eukaryotic cells where the peptide product comprises a nickase, four protospacers may be included, such that two staggered end cuts are produced and the DNA between the two staggered cut ends is deleted from the genome to inactivate the gene.

[0225] In these cases, all the protospacers are located within the same target sequence (e.g. the same essential gene, or the same nucleic acid of the one or more exogenous nucleic acids).

[0226] The gRNA or crRNA may comprise a (first, second, third...) spacer that is cognate to a (first, second, third...) protospacer in a target sequence, wherein the spacer sequence is from (about) 25 to 65 nucleotides in length, see for example Shmakov et al., mBio, 8(5), 1128, 1997, doi:https: / / doi.org / 10.1128 / mbio.01397-17. The spacer sequence may be from (about) 25 to 39 nucleotides in length. The spacer sequence may be from (about) 28 to 32 nucleotides in length. The spacer sequence may be about 32 nucleotides in length. The spacer sequence may be 32 nucleotides in length. The spacer may be (about) 70% (such as (about) 80%, or (about) 90%, or (about) 95%) complementary to the protospacer sequence in the target sequence. The spacer may be 100% complementary to the protospacer sequence in the target sequence.

[0227] Nucleotides 1 to 28 of the (first, second, third...) spacer may be identical to the complement of nucleotides 1 to 28 of the (first, second, third...) protospacer sequence which is immediately 5' of the PAM sequence in the target sequence.

[0228] The spacer may be (about) 90% complementary to the protospacer. The spacer may be (about) 70% complementary to the protospacer. The spacer may be (about) 80% complementary to the protospacer. The spacer may be (about) 95% complementary to the protospacer.

[0229] The spacer may be identical to the complement of the protospacer in the target sequence. The spacer may be identical to the complement of the protospacer in the target sequence across its entire length. The spacer may be (about) 80% (for example (about) 90%) identical to the complement of the protospacer in the target sequence across its entire length. The spacer may 100% complementary to the protospacer across its entire length.

[0230] The spacer may be identical to the complement of the protospacer in the target sequence across the first 1 to 28 nucleotides which are immediately 5' of the PAM sequence in the target sequence, and (about) 80% (e.g. (about) 90%) identical across the rest of the nucleotides in the protospacer.

[0231] In an example, the spacer sequence comprises the nucleotide sequence of SEQ ID No:69. Optionally, the gRNA or crRNA is 15-100 nucleotides long. For example, the gRNA or crRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long.

[0232] The spacer sequence may comprise at least 10 contiguous nucleotides that is complementary to the protospacer sequence. For example, the gRNA or crRNA comprises a spacer sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to the protospacer sequence.

[0233] The gRNA or crRNA may comprise a spacer sequence having at least 10 consecutive nucleotides which are complementary to the protospacer sequence. The gRNA or crRNA may comprise a spacer sequence having at least 15 consecutive nucleotides which are complementary to the protospacer sequence. The gRNA or crRNA may comprise a spacer sequence having at least 20 consecutive nucleotides which are complementary to the protospacer sequence. The gRNA or crRNA may comprise a spacer sequence having at least 25 consecutive nucleotides which are complementary to the protospacer sequence. The gRNA or crRNA may comprise a spacer sequence having at least 28 consecutive nucleotides which are complementary to the protospacer sequence.

[0234] The spacer and protospacer may be from about 10 to 40 nucleotides in length. For example, the spacer and protospacer may be between about 25 and 40 nucleotides, such as between about 25 and 38 nucleotides, such as between about 28 and 35 nucleotides. The spacer and protospacer may be from about 25 to 39 nucleotides in length. The spacer and protospacer may be from about 29 to 35 (e.g. from about 30 to 34, or from about 31 to 33) nucleotides in length. For example, the spacer and protospacer may be about 32 nucleotides in length. For example, the spacer and protospacer may be about 28 nucleotides in length. For example, the spacer and protospacer may be 32 nucleotides in length. For example, the spacer and protospacer may be 28 nucleotides in length.

[0235] The gRNA or crRNA comprises at least one repeat sequence. In an example, the gRNA or crRNA comprises two repeat sequences. The repeat sequence may be any sequence which is capable of forming a hairpin loop and being recognised by the specific CRISPR / Cas system. The repeat sequence may be about 20 to 25 nucleotides (e.g. 22 or 23 nucleotides) in length.

[0236] The gRNA or crRNA may comprise at least one repeat sequence comprising the nucleotide sequence of SEQ ID No:6. The gRNA or crRNA may comprise two repeat sequences comprising the nucleotide sequence of SEQ ID No:6. For example, the repeat sequence is SEQ ID NO:6 or SEQ ID No: 70 or a sequence that is identical except for 1, 2, 3, 4, or 5 changes, in particular 1 or 2, e.g. 1 change. The repeat sequence may be a nucleotide sequence which is at least about 80%, e.g. about 90% (such as at least about 95, 96, 97 or 98%) identical to the nucleotide sequence of SEQ ID No:6. The gRNA or crRNA may comprise at least one repeat sequence comprising the nucleotide sequence of SEQ ID No:70. The gRNA or crRNA may comprise two repeat sequences comprising the nucleotide sequence of SEQ ID No: 70. The repeat sequence may be a nucleotide sequence which is at least about 80%, e.g. about 90% (such as at least about 95, 96, 97 or 98%) identical to the nucleotide sequence of SEQ ID No:70.

[0237] In examples where a second, third... spacer is contemplated, the crRNA or gRNA may comprise further repeat sequences as required.

[0238] The crRNA may be encoded by a CRISPR array.

[0239] Type I nucleases are useful, and have been previously shown to be effective in killing target cells, see for example WO2016 / 177682A1 (SNIPR Technologies, Limited) and W02024 / 003301A1 (SNIPR Biome, ApS). Thus, in a particular example, the peptide product is a Type I nuclease which is a Cas3 nuclease, and wherein the one or more nucleic acid(s) further comprises d) a fourth nucleotide sequence which expresses a gRNA or crRNA comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence.

[0240] In another example, the peptide product is a Type I nuclease which is selected from a Cas3' and Cas3, or a Cas3 (in particular a Cas3) nuclease, and wherein the one or more nucleic acid(s) further comprises d) a fourth nucleotide sequence which expresses a gRNA or crRNA comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence. In these examples, the Cas3 nuclease, or the Cas3' and Cas3 polypeptides may be in combination with a further one or more nucleotide sequence(s) encoding one or more of a Cast, a Cas2, a Cas4, a Cas5, a Cas6 and a Cas7 protein. The Cas3 nuclease, or the Cas3' and Cas3 polypeptides may be in combination with a further one or more nucleotide sequence(s) encoding each of a Cast, a Cas2, a Cas4, a Cas5, a Cas6 and a Cas7 protein. The Cas3 nuclease, or the Cas3' and Cas3 polypeptides may be in combination with a further one or more nucleotide sequence(s) encoding one or more of a Cast, a Cas2, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8 and a Casll protein. The Cas3 nuclease, or the Cas3' and Cas3 polypeptides may be in combination with a further one or more nucleotide sequence(s) encoding each of a Cast, a Cas2, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8 and a Casll protein. In some nomenclature of Type I systems, a Cas8 protein is also referred to as a CasA protein, and a Casll protein is referred to as a CasB protein. It has further been shown that in Type I systems, Casl, Cas2 and Cas4 are not required for genome editing, as they are involved in spacer acquisition in the native system. Thus, in these engineered systems, the Cas3 nuclease, or the Cas3' and Cas3 polypeptides may be in combination with a further one or more nucleotide sequence(s) encoding one or more of a Cas5, a Cas6, a Cas7, a Cas8 and a Casll protein. The Cas3 nuclease, or the Cas3' and Cas3 polypeptides may be in combination with a further one or more nucleotide sequence(s) encoding each of a Cas5, a Cas6, a Cas7, a Cas8 and a Casll protein.

[0241] In particular applications a Type I-C Cas system may be used, as shown in Example 4. Thus, in a particular example, the peptide product is a Type I-C nuclease Cas3 nuclease in combination with at least one (in particular all of) a Cascade protein selected from a Cas5, a Cas7 and a Cas8 protein, and at least one of the Cas3 nuclease and Cascade protein(s) are expressed from the first nucleotide sequence, and wherein the one or more nucleic acid(s) further comprises d) a fourth nucleotide sequence which expresses a gRNA or crRNA comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence. The peptide product comprising the at least one mutation may be the Cas3 nuclease. The peptide product comprising the at least one mutation may be the Cas5 protein. The peptide product comprising the at least one mutation may be the Cas7 protein. The peptide product comprising the at least one mutation may be the Cas8 protein.

[0242] In particular, the Type I-C Cas nuclease and the at least one (in particular all of) Cascade protein(s) selected from Cas5, a Cas7 and a Cas8 proteins are from Streptococcus mutans, see for Example Serbanescu eta, J. Bacteriol., 197(4), 749-761, 2015, doi: 10.1128 / JB.02333-14.

[0243] A particular amino acid sequence for Cas3 (from Streptococcus mutans) is provided in SEQ ID No: 106. In this Cas3 sequence (see https: / / www.uniprot.org / uniprotkb / Q8DSL6 / entry), the first nuclease domain can be found between amino acid residues 10 to 213 (HD nuclease domain). In order to effectively truncate the functionality of the Cas3, a stop codon can be positioned early in this first nuclease domain to prevent any functionality in the absence of the NNAA. Thus, in one embodiment, the first nucleotide sequence encodes the Cas3 nuclease, and the sequence would encode an amino acid sequence of SEQ ID No: 106 or a homologue or orthologues thereof, except that at least one (in particular, one) of the coding codons which encodes an amino acid has been replaced with a second stop codon.

[0244] In an example, at least one (e.g. one) of the coding codons which encodes a residue selected from F22, F34, F42 and F47 (in particular F22 and F34, most particularly F22) has been replaced with a second stop codon. Optionally the first stop codon is not the same as the second stop codon. In particular the second stop codon is TAG, and optionally the first stop codon is not TAG.

[0245] In this example, the Cascade proteins may comprise each of a Cas5, a Cas8 and a Cas7 protein. The one or more nucleic acids may comprise a further nucleotide sequence encoding a Cas5 protein comprising the nucleotide sequence of SEQ ID No: 107 (or a nucleotide sequence having at least 90% [or 95%] identity thereto). The one or more nucleic acids may comprise a further nucleotide sequence encoding a Cas8 protein comprising the nucleotide sequence of SEQ ID No: 108 (or a nucleotide sequence having at least 90% [or 95%] identity thereto). The one or more nucleic acids may comprise a further nucleotide sequence encoding a Cas7 protein comprising the nucleotide sequence of SEQ ID No: 109 (or a nucleotide sequence having at least 90% [or 95%] identity thereto).

[0246] However, as mentioned above, the peptide product could be any of the Cascade proteins. A particular amino acid sequence for Cas7 (from Streptococcus mutant) is provided in SEQ ID No: 109. The Cas7 protein forms the backbone of the Cascade complex. In order to effectively truncate the functionality of the Cas7, a stop codon can be positioned early in this sequence to prevent any functionality in the absence of the NNAA, see for example, Example 4 herein. Thus, in another embodiment, the first nucleotide sequence encodes the Cas7 protein, and the sequence would encode an amino acid sequence of SEQ ID No: 109 or a homologue or orthologues thereof, except that at least one (in particular, one) of the coding codons which encodes an amino acid has been replaced with a second stop codon.

[0247] In an example, at least one (e.g. one) of the coding codons which encodes F8 has been replaced with a second stop codon. Optionally the first stop codon is not the same as the second stop codon. In particular the second stop codon is TAG, and optionally the first stop codon is not TAG.

[0248] In this example, the Type I-C system may further comprise each of a Cas3 nuclease, a Cas5 protein and a Cas8 protein. The one or more nucleic acids may comprise a further nucleotide sequence encoding a Cas3 nuclease comprising the nucleotide sequence of SEQ ID No: 106 (or a nucleotide sequence having at least 90% [or 95%] identity thereto). The one or more nucleic acids may comprise a further nucleotide sequence encoding a Cas5 protein comprising the nucleotide sequence of SEQ ID No: 107 (or a nucleotide sequence having at least 90% [or 95%] identity thereto). The one or more nucleic acids may comprise a further nucleotide sequence encoding a Cas8 protein comprising the nucleotide sequence of SEQ ID No: 108 (or a nucleotide sequence having at least 90% [or 95%] identity thereto).

[0249] In particular applications a Type I-E Cas system may be used. Thus, in an example, the peptide product is a Type I-E nuclease Cas3 nuclease in combination with at least one (in particular all of) a Cascade protein selected from a CasA, a CasB, Cas5, a Cas6, and a Cas7 protein, and at least one of the Cas3 nuclease and Cascade protein(s) are expressed from the first nucleotide sequence, and wherein the one or more nucleic acid(s) further comprises d) a fourth nucleotide sequence which expresses a gRNA or crRNA comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence. The peptide product comprising the at least one mutation may be the Cas3 nuclease. The peptide product comprising the at least one mutation may be the CasA protein. The peptide product comprising the at least one mutation may be the CasB protein. The peptide product comprising the at least one mutation may be the Cas5 protein. The peptide product comprising the at least one mutation may be the Cas6 protein. The peptide product comprising the at least one mutation may be the Cas7 protein.

[0250] In particular, the Type I-E Cas nuclease and the at least one (in particular all of) Cascade protein(s) selected from CasA, a CasB, Cas5, a Cas6, and a Cas7 proteins are from Escherichia coii, see for Example Gencay etai., Nature Biotech., 42, 265-274, 2024.

[0251] A particular amino acid sequence for Cas3 (from E. coii) is provided in SEQ ID No: 110. In this Cas3 sequence (which structure the inventors have predicted by comparison to https: / / www.uniprot.org / uniprotkb / E0J256 / entry), the first nuclease domain is predicted to be found between amino acid residues 31 to 230 (HD nuclease domain). In order to effectively truncate the functionality of the Cas3, a stop codon can be positioned early in this first nuclease domain to prevent any functionality in the absence of the NNAA. Thus, in one embodiment, the first nucleotide sequence encodes the Cas3 nuclease, and the sequence would encode an amino acid sequence of SEQ ID No: 110 or a homologue or orthologues thereof, except that at least one (in particular, one) of the coding codons which encodes an amino acid has been replaced with a second stop codon.

[0252] In an example, at least one (e.g. one) of the coding codons which encodes a residue selected from Y4, F17, F18, F20, Y35, Y49, Y63 and F64 has been replaced with a second stop codon. Optionally the first stop codon is not the same as the second stop codon. In particular the second stop codon is TAG, and optionally the first stop codon is not TAG.

[0253] In this example, the Cascade proteins may comprise each of a CasA, a CasB, Cas5, a Cas6, and a Cas7 protein. The one or more nucleic acids may comprise a further nucleotide sequence encoding a CasA protein comprising the nucleotide sequence of SEQ ID No: 111 (or a nucleotide sequence having at least 90% [or 95%] identity thereto). The one or more nucleic acids may comprise a further nucleotide sequence encoding a CasB protein comprising the nucleotide sequence of SEQ ID No: 112 (or a nucleotide sequence having at least 90% [or 95%] identity thereto). The one or more nucleic acids may comprise a further nucleotide sequence encoding a Cas7 protein comprising the nucleotide sequence of SEQ ID No: 113 (or a nucleotide sequence having at least 90% [or 95%] identity thereto). The one or more nucleic acids may comprise a further nucleotide sequence encoding a Cas5 protein comprising the nucleotide sequence of SEQ ID No: 114 (or a nucleotide sequence having at least 90% [or 95%] identity thereto). The one or more nucleic acids may comprise a further nucleotide sequence encoding a Cas6 protein comprising the nucleotide sequence of SEQ ID No: 115 (or a nucleotide sequence having at least 90% [or 95%] identity thereto).

[0254] However, as mentioned above, the peptide product could be any of the Cascade proteins. A particular amino acid sequence for Cas7 (from E. co!i) is provided in SEQ ID No: 113. The Cas7 protein forms the backbone of the Cascade complex. In order to effectively truncate the functionality of the Cas7, a stop codon can be positioned early in this sequence to prevent any functionality in the absence of the NNAA. Thus, in another embodiment, the first nucleotide sequence encodes the Cas7 protein, and the sequence would encode an amino acid sequence of SEQ ID No: 113 or a homologue or orthologues thereof, except that at least one (in particular, one) of the coding codons which encodes an amino acid has been replaced with a second stop codon.

[0255] In an example, at least one (e.g. one) of the coding codons which encodes a residue selected from F4 and Y13 has been replaced with a second stop codon. Optionally the first stop codon is not the same as the second stop codon. In particular the second stop codon is TAG, and optionally the first stop codon is not TAG.

[0256] In this example, the Type I-E system may further comprise each of a Cas3 nuclease, CasA, a CasB, Cas5 and a Cas6 protein. The one or more nucleic acids may comprise a further nucleotide sequence encoding a Cas3 nuclease comprising the nucleotide sequence of SEQ ID No: 110 (or a nucleotide sequence having at least 90% [or 95%] identity thereto). The one or more nucleic acids may comprise a further nucleotide sequence encoding a CasA protein comprising the nucleotide sequence of SEQ ID No: 111 (or a nucleotide sequence having at least 90% [or 95%] identity thereto). The one or more nucleic acids may comprise a further nucleotide sequence encoding a CasB protein comprising the nucleotide sequence of SEQ ID No: 112 (or a nucleotide sequence having at least 90% [or 95%] identity thereto). The one or more nucleic acids may comprise a further nucleotide sequence encoding a Cas5 protein comprising the nucleotide sequence of SEQ ID No: 114 (or a nucleotide sequence having at least 90% [or 95%] identity thereto). The one or more nucleic acids may comprise a further nucleotide sequence encoding a Cas6 protein comprising the nucleotide sequence of SEQ ID No: 115 (or a nucleotide sequence having at least 90% [or 95%] identity thereto).

[0257] Alternatively, the peptide product is a Type II Cas9 nuclease, and wherein the one or more nucleic acid(s) further comprises d) a fourth nucleotide sequence which expresses a gRNA or crRNA (e.g. a gRNA), comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence. Type II CRISPR / Cas systems are attractive because, firstly, they comprise only a single nuclease peptide, thus reducing the complexity of expression and assembly in the target cell. Secondly, they are compatible with a single guide RNA, which is easier and more convenient to design and encode than a more complicated crRNA systems (which requires processing from the pre-crRNA by other components of the CRISRP / Cas system). Thirdly, Cas9 molecules have been extensively studied and mutated, including for their use in eukaryotic cells (e.g. by the inclusion of a nuclear localisation sequence, as described elsewhere herein), thus making them particularly suited for applications where the target cell is a eukaryotic cell. See, for example, Schindele et al., Nature Comms., 13, 1502, 2022, doi:https: / / doi.org / 10.1038 / s41467-022-29130-w, which is incorporated herein by reference in its entirety.

[0258] A particular amino acid sequence for Cas9 is provided in SEQ ID No:40. In this Cas9 sequence, the first catalytic domain can be found between amino acid residues 1 to 62 (RuvC-I domain). In order to effectively truncate the functionality of the Cas9, a stop codon can be positioned early in this first catalytic domain to prevent any functionality in the absence of the NNAA. Thus, in one embodiment, the first nucleotide sequence encoding the Cas9 nuclease would encode an amino acid sequence of SEQ ID No:40 or a homologue or orthologues thereof, except that at least one (in particular, one) of the coding codons which encodes an amino acid has been replaced with a second stop codon.

[0259] In an example, at least one (e.g. one) of the coding codons which encodes a residue selected from Y5, Y25 and F32 has been replaced with a second stop codon. Optionally the first stop codon is not the same as the second stop codon. In particular the second stop codon is TAG, and optionally the first stop codon is not TAG.

[0260] In the Cas9 sequence of SEQ ID No:40, other domains which perform the nuclease activity can be found between amino acid residues 718 and 1102 (RuvC-II, the HNH and RuvC-III domains). In order to effectively truncate the functionality of the Cas9, a stop codon can alternatively be positioned in this region to prevent functionality of the nuclease in the absence of the NNAA. Thus, in an alternative embodiment, the first nucleotide sequence encoding the Cas9 nuclease would encode an amino acid sequence of SEQ ID No:40 or a homologue or orthologue thereof, except that at least one (in particular, one) of the coding codons which encodes an amino acid residue selected from Y812, Y814, Y815, Y823, Y836, F846, Y882 and F897 has been replaced with a second stop codon, and optionally wherein the first stop codon is not the same as the second stop codon. In an alternative embodiment, the first nucleotide sequence encoding the Cas9 nuclease would encode an amino acid sequence of SEQ ID No:40 or a homologue or orthologue thereof, except that at least one (in particular, one) of the coding codons which encodes an amino acid residue selected from F916, Y943, F966, F972, Y973, Y981 and Y988 has been replaced with a second stop codon, and optionally wherein the first stop codon is not the same as the second stop codon.

[0261] In one example, the second stop codon is TAG. In particular, the second stop codon is TAG and the first stop codon is not TAG. In a particular embodiment, the non-natural amino acid is 0-methyl tyrosine (OMTyr); and the second nucleic acid of part b) encodes a tyrosyl tRNA (as described elsewhere herein); and the third nucleic acid of part c) encodes a tyrosyl tRNA synthetase (as described elsewhere herein).

[0262] For other CRISPR / Cas sequences, the methodology described in Spencer & Zhang et al., Nature Sci. Rep., 7,16836, 2017, doi:https: / / doi.org / 10.1038 / s41598-017-17081-y can be used to determine domains and activity to guide the choice of positioning of the second stop codon.

[0263] Alternatively, the peptide product is a Type III CaslO nuclease and wherein the one or more nucleic acid(s) further comprises d) a fourth nucleotide sequence which expresses a gRNA or crRNA, comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence. The CaslO nuclease may be in combination with a further one or more nucleotide sequence(s) encoding one or more of a Casl, a Cas2, a Cas5, a Cas6, a Cas7 and a Casll protein. The CaslO nuclease may be in combination with a further one or more nucleotide sequence(s) encoding each of a Casl, a Cas2, a Cas5, a Cas6, a Cas7 and a Casll protein. It has further been shown that in Type III systems, Casl and Cas2 are not required for genome editing, as they are involved in spacer acquisition in the native system. Thus, in these engineered systems, the CaslO nuclease may be in combination with a further one or more nucleotide sequence(s) encoding one or more of a Cas5, a Cas6, a Cas7 and a Casll protein. The CaslO nuclease, may be in combination with a further one or more nucleotide sequence(s) encoding each of a Cas5, a Cas6, a Cas7 and a Casll protein

[0264] Alternatively, the peptide product is a Type V Casl2 nuclease and wherein the one or more nucleic acid(s) further comprises d) a fourth nucleotide sequence which expresses a gRNA or crRNA, comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence. In particular, the peptide product is a Type V Casl2 nuclease which is Cpfl, and wherein the one or more nucleic acid(s) further comprises d) a fourth nucleotide sequence which expresses a gRNA or crRNA, comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence. The Casl2 nuclease may optionally be in combination with a further one or more nucleotide (e.g. a further one) sequence(s) encoding a Cas4 polypeptide. However, Casl2 is known to function without the presence of the Cas4 in systems designed for engineering, as Cas4 is involved in spacer acquisition in the native system, and this function is not required for these purposes.

[0265] A particular amino acid sequence for Cpfl is provided in SEQ ID No:41. In this Cpfl sequence, a catalytic domain can be found between amino acid residues 893-1300. In order to effectively truncate the functionality of the Cpfl, a stop codon can be positioned early (for example, from residues 893 to 1066) in this catalytic domain to prevent any functionality in the absence of the NNAA. Thus, in one embodiment, the first nucleotide sequence encoding the Cpfl nuclease which would encode an amino acid sequence of SEQ ID No:41 or a homologue or orthologue thereof, except that at least one (e.g. one) of the coding codons which encodes an amino acid has been replaced with a second stop codon.

[0266] In an example, at least one (e.g. one) of the coding codons which encodes a residue selected from F896, Y925, Y926, F941, Y953, Y984, Y999, F1005, F1010, F1012, F1017, Y1024, Y1037, F1040, F1045, Y1055, F1061, and F1064 (and particularly selected from F896, Y925, Y926, F941, Y953, Y984, Y999, F1005) has been replaced with a second stop codon, and optionally wherein the first stop codon is not the same as the second stop codon.

[0267] In particular, the second stop codon is TAG and the first stop codon is not TAG. In a particular embodiment, the non-natural amino acid is O-methyl tyrosine (OMTyr); and the second nucleic acid of part b) encodes a tyrosyl tRNA (as described elsewhere herein); and the third nucleic acid of part c) encodes a tyrosyl tRNA synthetase (as described elsewhere herein).

[0268] The Casl2 may be a compact Casl2f nuclease (e.g. 422-603 amino acids), for example, any of those described in Karvelis et a!., NAR, 48(9), 5016-5023, 2020, doi: https: / / doi.org / 10.1093 / nar / gkaa208, which is incorporated herein by reference in its entirety.

[0269] The peptide product may be a nickase. The nickase may be a DNA nickase. The nickase may be an RNA nickase. The nickase may be selected from Nt. BstNBI, Nb. BsrDI, Nb. BtsI, Nt. AIwI, Nb. BbvCI, Nt. BbvCI, and Nb. BsmI (available from New England BioLabs). The nickase may be a synthetic Cas nickase (e.g. nCas9 or Cas9D10A).

[0270] There is provided a target cell comprising a target sequence which comprises, in 5' to 3' orientation:

[0271] a) a PAM sequence (as described elsewhere herein); and

[0272] b) a protospacer sequence (as described elsewhere herein), and further comprising the one or more exogenous nucleic acids as described elsewhere herein.

[0273] There is also provided a target cell comprising a target sequence which comprises, in 5' to 3' orientation:

[0274] a) a PAM sequence (as described elsewhere herein); and

[0275] b) a protospacer sequence (as described elsewhere herein),

[0276] and further comprising

[0277] the one or more exogenous nucleic acids as described elsewhere herein, and

[0278] the one or more nucleic acid(s) as described elsewhere herein, and

[0279] wherein the peptide product of part a) is a CRISPR / Cas nuclease or nickase.

[0280] The target sequence may comprises a PAM sequence 5' of the protospacer sequence, e.g. wherein the PAM is immediately adjacent to the protospacer sequence

[0281] In one embodiment, sequences a) and b) are each immediately adjacent to each other. In one embodiment, a) is a non-native (i.e. exogenous) sequence to the cell. In another embodiment, b) is a non-native (i.e. exogenous) sequence to cell. In another embodiment, a) and b) are non-native (i.e. exogenous) sequences to cell.

[0282] I-TevI-CasS nuclease-based

[0283]

[0284] The present inventors previously discovered a new CRISPR / Cas system, termed CasS, which was characterised and described in WO2024 / 047151A1 (SNIPR Biome ApS), which is incorporated herein by reference. In that application, they also developed fusion proteins with an I-TevI nuclease, which provided the new CasS system with nuclease functionality. In the examples below, they have harnessed this system, in combination with mutations to introduce stop codons in the nucleotide sequence and second and third nucleotide sequences to provide a precise kill switch system. This kill switch system has numerous applications, which are contemplated herein. In any of the embodiments relating to this I-TevI-CasS skill switch system, there is provided a first nucleotide sequence encoding at least one I-TevI-CasS fusion protein comprising at least one mutation to introduce a non-natural quadruplet codon or a nonsense codon.

[0285] There is thus provided a peptide product which is a fusion protein complex comprising a fusion protein in a complex with further proteins, and wherein the fusion protein comprises a protein of the formula A-B-C (in 5' to 3' orientation), wherein:

[0286] A is an I-Tev nuclease (for example any of the I-TevI proteins described herein, for example having the amino acid sequence of SEQ ID No: 5);

[0287] B is optionally present, and when present comprises a linker (for example any of the linkers as described herein, in particular an XTEN linker, for example having the amino acid sequence of SEQ ID No: 17);

[0288] C is a CasS protein selected from Cas-Sl and Cas-S4 (in particular a CasSl protein); and the further proteins in complex with the fusion protein comprise a Cas-S2 and Cas-S5 protein and a Cas-Sl or Cas-S4 protein (in particular a CasS4 protein) as required such that the fusion protein complex comprises at least a Cas-Sl, Cas-S2, Cas-S4 and a Cas-S5 protein,

[0289] and the peptide product further comprises a gRNA or a crRNA as described elsewhere herein, e.g. expressed from a fourth nucleotide sequence.

[0290] In an embodiment, the fusion protein complex may further comprise a Cas-S3 protein. There is also provided a) a first nucleotide sequence which expresses the fusion protein of the formula A-B-C (in the presence of the NNAA) and additional nucleotide sequences (e.g. a fifth, sixth, seventh... nucleotide sequence) which expresses the further proteins of the fusion protein complex. There is also provided a first vector which expresses a) a first nucleotide sequence which expresses the fusion protein of the formula A-B-C and a second vector which expresses the further proteins of the fusion protein complex.

[0291] In an embodiment, the fusion protein complex comprising an I-Tev nuclease introduces a double stranded break within the target sequence. For example, when the target sequence comprises the sequence of SEQ ID No:42, the fusion protein complex introduces a break after the second C on the forward strand of the target sequence and after the first T on the reverse strand of the target sequence (as the cleavage site nucleotide sequence on the reverse strand comprises the sequence 5'-CGTTG-3')- The I-TevI nuclease nicks the forward strand immediately after the second C, and immediately after the first T on the reverse strand. The double nicking of the cleavage site results in a staggered double stranded DNA break and each site of the break harbours single stranded, dinucleotide overhangs; 5'-AC-3' for the forward strand and 5'-GT-3' for the reverse strand.

[0292] However, the inventors contemplate that other nucleases or nickases could be substituted for the I-TevI described in the specific examples.

[0293] Thus, the peptide product (e.g. when expressed in the presence of the NNAA) may comprise: I. a polypeptide (Px) comprising an amino acid sequence that is at least 80% (in particular at least 90%) identical to a sequence selected from SEQ ID Nos: 1-5; and

[0294] II. a heterologous polypeptide (Py) which is a nuclease or a nickase (e.g. nuclease), and (e.g. wherein the one or more nucleic acids) further comprises:

[0295] d) (for example a fourth nucleotide sequence encoding) a gRNA or crRNA, comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by a target sequence in the target cell genome.

[0296] The nucleases and nickases which can be Py are any of the nucleases or nickases described elsewhere herein.

[0297] The peptide product may be in the form of a protein complex, further complexed with the gRNA or crRNA to form a ribonucleoprotein complex. The peptide product may comprise a fusion of different protein subunits. The peptide product may comprise one of a CasSl, a CasS2, a CasS3, a CasS4 or a CasS5 protein as described elsewhere herein (including % homologies). The peptide product may comprise two proteins selected from a CasSl, a CasS2, a CasS3, a CasS4 and a CasS5 protein as described elsewhere herein (including % homologies). The peptide product may comprise three proteins selected from a CasSl, a CasS2, a CasS3, a CasS4 and a CasS5 protein as described elsewhere herein (including % homologies). The peptide product may comprise four proteins selected from a CasSl, a CasS2, a CasS3, a CasS4 and a CasS5 protein as described elsewhere herein (including % homologies). The peptide product may comprise all of a CasSl, a CasS2, a CasS3, a CasS4 and a CasS5 protein as described elsewhere herein (including % homologies).

[0298] The peptide product comprising Px and Py may be in the form of a peptide product complex which comprises at least two further polypeptides (e.g. encoded by at least e) a fifth and f) a sixth nucleotide sequence),

[0299] wherein the two further polypeptides each have an amino acid sequence that is at least 80% (in particular at least 90%) identical to a sequence selected from the sequences of SEQ ID No:l, SEQ ID No:4 and SEQ ID No:5, and

[0300] wherein the peptide product comprises amino acid sequences that are at least 80% (in particular at least 90%) identical to each of the sequences of SEQ ID No:l, SEQ ID No:4 and SEQ ID No:5.

[0301] The peptide product comprising Px and Py may be in the form of a peptide product complex which comprises at least three further polypeptides (e.g. encoded by at least e) a fifth, f) a sixth, and g) a seventh nucleotide sequence), and wherein the peptide product comprises amino acid sequences that are at least 80% (in particular at least 90%) identical to each of the sequences of SEQ ID No:l, SEQ ID No:2, SEQ ID No:4 and SEQ ID No:5.

[0302] The peptide product comprising Px and Py may be in the form of a peptide product complex which comprises at least four further polypeptides (e.g. encoded by at least e) a fifth, f) a sixth, g) a seventh, and h) an eighth nucleotide sequence), and wherein the peptide product complex comprises amino acid sequences that are at least 80% (in particular at least 90%) identical to each of the sequences of SEQ ID No:l, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4 and SEQ ID No:5.

[0303] In a particular example, Px comprises an amino acid sequence that is selected from an amino acid sequence that is at least 80% (in particular at least 90%) identical to the sequence selected from the sequences of SEQ ID Nos:l and 4, and Px is fused to Py to form a fusion protein;

[0304] wherein the peptide product is in the form of a fusion protein complex which comprises at least two further polypeptides (e.g. encoded by at least e) a fifth and f) a sixth nucleotide sequence), wherein the two further polypeptides each have an amino acid sequence that is at least 80% (in particular at least 90%) identical to a sequence selected from the sequences of SEQ ID No:2 and SEQ ID No:5, and

[0305] g) optionally (e.g. a seventh nucleotide sequence which encodes) a polypeptide which is at least 80% (in particular at least 90%) identical to a sequence selected from SEQ ID Nos:l and 4 and is not based on the amino acid sequence of the polypeptide of Px; and h) optionally (e.g. an eighth nucleotide sequence which encodes) a polypeptide which is at least 80% (in particular at least 90%) identical to SEQ ID No:3.

[0306] In a particular example, Px is fused to Py to form a fusion protein.

[0307] The peptide product (when expressed in the presence of the NNAA) may comprise a fusion protein comprising a Cas-Sl fused to a heterologous polypeptide (Py) which is a nuclease or a nickase.

[0308] The peptide product (when expressed in the presence of the NNAA) may comprise a fusion protein comprising a Cas-S2 fused to a heterologous polypeptide (Py) which is a nuclease or a nickase.

[0309] The peptide product (when expressed in the presence of the NNAA) may comprise a fusion protein comprising a Cas-S3 fused to a heterologous polypeptide (Py) which is a nuclease or a nickase.

[0310] The peptide product (when expressed in the presence of the NNAA) may comprise a fusion protein comprising a Cas-S4 fused to a heterologous polypeptide (Py) which is a nuclease or a nickase.

[0311] The peptide product (when expressed in the presence of the NNAA) may comprise a fusion protein comprising a Cas-S5 fused to a heterologous polypeptide (Py) which is a nuclease or a nickase.

[0312] In one embodiment Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:l and the peptide product is in the form of a fusion protein complex which comprises two further polypeptides which each comprise an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:2 and SEQ ID No: 5.

[0313] In one embodiment Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:3 and the peptide product is in the form of a fusion protein complex which comprises two further polypeptides which each comprise an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:2 and SEQ ID No: 5.

[0314] In one embodiment Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:4 and the peptide product is in the form of a fusion protein complex which comprises two further polypeptides which each comprise an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:2 and SEQ ID No:5.

[0315] In one embodiment, Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:l, and the peptide product is in the form of a fusion protein complex which comprises three further polypeptides, wherein the three further polypeptides each comprise an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:3, SEQ ID No:2 and SEQ ID No:5.

[0316] In one embodiment, Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:l, and the peptide product is in the form of a fusion protein complex which comprises three further polypeptides, wherein the three further polypeptides each comprise an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:4, SEQ ID No:2 and SEQ ID No:5. In one embodiment, Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:3, and the peptide product is in the form of a fusion protein complex which comprises three further polypeptides, wherein the three further polypeptides each comprise an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:l, SEQ ID No:2 and SEQ ID No:5.

[0317] In one embodiment, Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:3, and the peptide product is in the form of a fusion protein complex which comprises three further polypeptides, wherein the three further polypeptides each comprise an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:4, SEQ ID No:2 and SEQ ID No:5.

[0318] In one embodiment, Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:4, and the peptide product is in the form of a fusion protein complex which comprises three further polypeptides, wherein the three further polypeptides each comprise an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:l, SEQ ID No:2 and SEQ ID No:5.

[0319] In one embodiment, Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:4, and the peptide product is in the form of a fusion protein complex which comprises three further polypeptides, wherein the three further polypeptides each comprise an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:3, SEQ ID No:2 and SEQ ID No:5.

[0320] In one embodiment, Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:l, and the peptide product is in the form of a fusion protein complex which comprises four further polypeptides, wherein the four further polypeptides each comprise an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:2, SEQ ID No:5, SEQ ID No:3 and SEQ ID No:4.

[0321] In one embodiment, Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:3, and the peptide product is in the form of a fusion protein complex which comprises four further polypeptides, wherein the four further polypeptides each comprise an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:l, SEQ ID No:2, SEQ ID No:4 and SEQ ID No:5.

[0322] In one embodiment, Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:4, and the peptide product is in the form of a fusion protein complex which comprises four further polypeptides, wherein the four further polypeptides each comprise an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:l, SEQ ID No:2, SEQ ID No:3 and SEQ ID No:5.

[0323] The peptide product (when expressed in the presence of the NNAA) may comprise a fusion protein complex comprising: a) (for example expressed from a first nucleotide sequence) a fusion protein polypeptide (Px), wherein Px

[0324] I. comprises an amino acid sequence that is at least (about) 80% identical (e.g. (about) 90% identical) to a sequence selected from SEQ ID Nos: 1 and 4; and II. is fused to a heterologous polypeptide (Py) which is a nuclease or a nickase; b) (for example expressed from a fifth nucleotide sequence) a polypeptide which comprises an amino acid sequence that is at least (about) 80% identical (e.g. (about) 90% identical) to SEQ ID No: 2;

[0325] c) (for example, expressed from a sixth nucleotide sequence) a polypeptide which comprises an amino acid sequence that is at least (about) 80% identical (e.g. (about) 90% identical) to SEQ ID No: 5;

[0326] d) (for example, expressed from a fourth nucleotide sequence) a crRNA comprising a spacer sequence that is cognate to a first protospacer in a target sequence; and

[0327] e) optionally (for example expressed from a seventh nucleotide sequence) a polypeptide which is at least (about) 80% identical (e.g. (about) 90% identical) to a sequence selected from SEQ ID Nos:l and 4 and is not based on the amino acid sequence of the polypeptide recited in part a) I.; and f) optionally (for example expressed from an eighth nucleotide sequence) a polypeptide which is at least (about) 80% identical (e.g. (about) 90% identical) to SEQ ID No:3.

[0328] The peptide product (when expressed in the presence of the NNAA) may comprise a fusion protein complex comprising:

[0329] a) (for example expressed from a first nucleotide sequence) a fusion protein polypeptide (Px), wherein Px

[0330] I. comprises a Cas-Sl or Cas-S4 protein; and

[0331] II. is fused to a heterologous polypeptide (Py);

[0332] b) (for example expressed from a fifth nucleotide sequence) a Cas-S2 protein;

[0333] c) (for example expressed from a sixth nucleotide sequence) a Cas-S5 protein;

[0334] d) (for example expressed from a fourth nucleotide sequence) a crRNA comprising a spacer sequence that is cognate to a first protospacer in a target sequence; and

[0335] e) optionally (for example expressed from a seventh nucleotide sequence) a protein which is selected from a Cas-Sl and a Cas-S4 protein, and is of a different CasS protein class to the protein recited in part a) I.; and

[0336] f) optionally (for example expressed from an eighth nucleotide sequence) a Cas-S3 protein. In an example, the fusion protein comprises 2 or more of Cas-Sl to S5, e.g. comprises Cas-Sl and S2, or S4 and S5. The peptide product (when expressed in the presence of the NNAA) may comprise an ITevI-CasSl 5'-3' fusion protein (as described elsewhere herein), and optionally further comprises one or more of:

[0337] e) (for example expressed from an fifth nucleotide sequence) a polypeptide which comprises an amino acid sequence that is at least (about) 80% identical (e.g. (about) 90% identical) to SEQ ID No:2 (CasS2);

[0338] f) (for example expressed from an sixth nucleotide sequence) a polypeptide which comprises an amino acid sequence that is at least (about) 80% identical (e.g. (about) 90% identical) to SEQ ID No:3 (CasS3);

[0339] g) (for example expressed from an seventh nucleotide sequence) a polypeptide which comprises an amino acid sequence that is at least (about) 80% identical (e.g. (about) 90% identical) to SEQ ID No:4 (CasS4); and / or

[0340] h) (for example expressed from an eighth nucleotide sequence) a polypeptide which comprises an amino acid sequence that is at least (about) 80% identical (e.g. (about) 90% identical) to SEQ ID No: 5 (CasS5).

[0341] In a particular example, the peptide product comprising an I-TevI-CasSl 5'-3' fusion protein (Px and Py) further comprises each of e), g) and h), for example as a fusion protein complex.

[0342] In any of the peptide products comprising Px and Py described herein, the peptide product may comprise an I-TevI-XTEN-CasSl 5'-3' fusion protein (Px and Py). The I-TevI-XTEN-CasSl 5'-3' fusion protein (Px and Py) may further comprise each of e), g) and h), for example as a fusion protein complex.

[0343] In any of the peptide products comprising Px and Py described herein, Py may be fused (directly or indirectly) to the N-terminus of the CasS protein (Px). In any of the peptide products comprising Px and Py described herein, in particular, Py may be fused (directly or indirectly) to the C-terminus of the CasS protein (Px).

[0344] Fusion may be direct or indirect (e.g. via a peptide linker, such as a (G4S)nlinker, wherein n=l, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Px may be fused to Py via a linker. The linker may be a polypeptide linker. A peptide linker may be from about 10 to 20 amino acids (e.g. about 16 amino acids) in length. A peptide linker may be from about 8 to 12 amino acids (e.g. about 10 amino acids) in length. The linker may be an XTEN linker. A peptide linker may comprise the amino acid sequence of SEQ ID No: 17. A peptide linker (e.g. an XTEN linker) may be encoded by a nucleotide sequence of SEQ ID No: 16. Linkers may be as described elsewhere herein.

[0345] As it relates to peptide products comprising Px and Py described herein, "heterologous" refers to a polypeptide that is not naturally found fused to or associated with Px. In an example, Py comprises an I-Tev nuclease or mutH protein.

[0346] In any of the peptide products comprising Px and Py described herein, Py is a nuclease. Py may comprise an I-Tev nuclease, such as an I-TevI nuclease, for example as described elsewhere herein. In a particular example, the I-TevI nuclease is fused to the N-terminus of Px which comprises an amino acid sequence that is at least 80% (in particular at least 90%) identical to the sequence of SEQ ID No:l.

[0347] In an example, the I-TevI nuclease is a protein comprising the amino acid sequence of SEQ ID No:45. In one embodiment, the I-TevI nuclease is encoded by a nucleic acid sequence which encodes an amino acid sequence comprising the amino acids of SEQ ID: No:45. In one embodiment, the I-TevI nuclease is encoded by a nucleic acid sequence of SEQ ID: No:44. In an example, the I-TevI nuclease is a protein which has an amino acid sequence which is at least about 80% identical (e.g. is about 85%, 90%, 95%, 96%, 97% or 98% identical) to the amino acid of SEQ ID No:45 and is capable of cleaving an I-TevI cleavage site nucleotide sequence having the nucleotide sequence of SEQ ID No:42 and is capable of recognising an I-TevI spacer nucleotide sequence having the nucleotide sequence of SEQ ID No:43.

[0348] In any of the peptide products comprising Px and Py described herein, Py is a nuclease, such as any of the nucleases disclosed herein, in particular an I-TevI nuclease. Py may be a DNA nuclease. Py may be an RNA nuclease. Py may be a nickase or a dead nuclease.

[0349] In any of the peptide products comprising Px and Py described herein, the I-TevI nuclease (Py) is fused to the N-terminus of an amino acid sequence (Px, e.g. anyCasS protein described herein). In one embodiment, the I-TevI nuclease is fused to the N-terminus of an amino acid sequence that is at least (about) 80% (e.g. (about) 90%) identical to the sequence of SEQ ID No: 1. The I-TevI nuclease may be as described elsewhere herein.

[0350] In any of the peptide products comprising Px and Py described herein, in one example, the I-TevI nuclease does not comprise a complete DNA binding domain. The I-TevI nuclease may be devoid of a complete DNA binding domain.

[0351] I-TevI comprises 245 amino acids, and consists of an N-terminal catalytic domain and a C-terminal DNA-binding domain that are connected by a long, flexible linker. The crystal structure of the DNA-binding domain of I-TevI (comprising residues 130 to 245) complexed with the 20-bp primary binding region of its DNA target, reveals the presence of a zinc finger (comprising residues 151 to 167) that makes backbone contacts with the DNA from the minor groove, an elongated segment containing a minor groove-binding a-helix (comprising residues 183 to 194) and a helix-turn-helix (comprising residues 204 to 245). The N-terminal catalytic domain was shown to comprise residues 1-92. Biochemical data have shown that the zinc finger does not contribute to the DNA-binding affinity or to the specificity of the enzyme, but rather that it has a novel function and acts as a distance determinant that controls the relative positions of the catalytic and DNA-binding domains, see for example, Roey eta / ., 2005, DOI:10.1007 / 0-387-27421-9_7, which is incorporated herein by reference in its entirety.

[0352] Kleinstiver et al., G3 Genes|Genomes|Genetics, 4(6), 1155-1165, 2014, doi: https: / / doi.org / 10.1534 / g3.114.011445 showed that various truncations of the N-terminal catalytic domain retain activity when fused in a TALEN construct. The largest construct was residues 1 to 206 of I-Tevl. The smallest that was functional when fused to a TALEN was resides 1 to 162.

[0353] Thus, in one embodiment, the I-Tevl does not comprise a complete helix-turn-helix domain. The I-Tevl may comprise the amino acid sequence of SEQ ID No:45. The I-Tevl may not comprise a helix-turn-helix domain. The I-Tevl may comprise amino acids 1 to 195-203 of SEQ ID No:45. The I-TevI may not comprise the minor groove-binding a-helix. The I-Tevl may comprise amino acids 1 to 167 of SEQ ID No:45. The I-Tevl may not comprise a complete zinc finger domain. The I-Tevl may comprise amino acids 1 to 162 of SEQ ID No:45.

[0354] In any of the peptide products comprising Px and Py described herein, in one example, the I-TevI nuclease (Py) comprises an N-terminal catalytic domain.

[0355] Thus, the I-Tevl may comprise amino acids 1 to 92 of SEQ ID No:45. The I-Tevl may comprise the amino acid sequence of SEQ ID No:45.

[0356] In any of the peptide products comprising Px and Py described herein, the I-Tevl nuclease (Py) may be a bacteriophage I-Tevl nuclease.

[0357] In any of the peptide products comprising Px and Py described herein, the I-Tevl nuclease (Py) may be a T4 bacteriophage I-Tevl. The I-Tevl nuclease may be from a T4 bacteriophage. The I-TevI nuclease may be encoded by the intron of bacteriophage T4. The I-Tevl nuclease may comprise amino acids 1 to 206 of a naturally occurring I-Tevl nuclease. The I-Tevl nuclease may comprise an amino acid sequence of SEQ ID No:45. The sequence may differ from SEQ ID No:45 by the incorporation of the NNAA. The I-Tevl nuclease may be encoded by a nucleotide sequence of SEQ ID No:44. The sequence may differ from SEQ ID No:44 by the incorporation of at least one mutation to incorporate a second stop codon or a non-natural quadruplet codon.

[0358] In any of the peptide products comprising Px and Py described herein, wherein Py is an I-Tev nuclease for example as described by a given sequence or portion thereof, the sequence may differ in the peptide product by the incorporation (e.g. addition or substitution) of the predetermined NNAA.

[0359] In particular embodiments, the first nucleotide sequence encodes a peptide product comprising Px and Py (as described herein), in particular comprising an I-Tevl nuclease (as described herein). The first nucleotide sequence may, in particular examples, comprise a replacement of a tyrosine coding codon (TAC or TAT) for a TAG nonsense codon. The first nucleotide sequence may, in particular examples comprise a replacement of a phenylalanine coding codon (TTT or TTC) for a TAG nonsense codon. In these examples, the TAG nonsense codons may be recognised by the tRNA / aaRS pair encoded by the second and third nucleotide sequences (as described elsewhere herein), in particular by a tRNA / aaRS which is capable of adding OMTyr as the NNAA. Thus, the amino acid sequence of any peptide product comprising Px and Py herein may differ in any position from the stated sequence (in particular at a tyrosine or phenylalanine position) by the replacement of OMTyr for the original amino acid. Similarly, the first nucleotide sequence encoding any peptide product comprising Px and Py herein may differ in any codon from the stated sequence (in particular at a tyrosine or phenylalanine position) by the replacement of a nonsense codon (in particular TAG) for the original coding codon (in particular a codon for tyrosine or phenylalanine).

[0360] Thus, the first nucleotide sequence encoding the ITevI-XTEN-CasSl 5'-3' fusion protein comprises, in 5'-3' direction, the nucleotide sequence of SEQ ID No:44, the nucleotide sequence of SEQ ID No: 16, and the nucleotide sequence of SEQ ID No:l, except for at least one mutation to introduce a or the non-natural quadruplet codon. In a particular embodiment, the first nucleotide sequence encoding the ITevI-XTEN-CasSl 5'-3' fusion protein comprises, in 5'-3' direction, the nucleotide sequence of SEQ ID No:44, the nucleotide sequence of SEQ ID No: 16, and the nucleotide sequence of SEQ ID No:l, except for at least one mutation to introduce a or the second stop codon. In some examples, the second stop codon does not replace codons in SEQ ID No:44 encoding F77 or F96 of SEQ ID No:45.

[0361] Alternatively, the first nucleotide sequence encoding the ITevI-XTEN-CasSl 5'-3' fusion protein encodes, in 5'-3' direction, an amino acid sequence of SEQ ID No:45, an amino acid sequence of SEQ ID No: 17, and an amino acid sequence of SEQ ID No:7, except for at least one mutation in said first nucleotide sequence to introduce a or the non-natural quadruplet codon. In particular, the first nucleotide sequence encoding the ITevI-XTEN-CasSl 5'-3' fusion protein encodes, in 5'-3' direction, an amino acid sequence of SEQ ID No:45, an amino acid sequence of SEQ ID No: 17, and an amino acid sequence of SEQ ID No:7, except for at least one mutation in said first nucleotide sequence to introduce a or the second stop codon. In some examples, the second stop codon does not replace codons encoding F77 or F96 of SEQ ID No:45. In this case the amino acid sequences of SEQ ID Nos:45, 17 and 7 will differ by the incorporation of a NNAA when expressed in the presence of said NNAA.

[0362] In another example, the ITevI-XTEN-CasSl 5'-3' fusion protein is encoded by a nucleotide sequence of SEQ ID No:79. This sequence comprises a mutation to introduce a TAG stop codon at codon 206. Alternatively, the first nucleotide sequence encoding the ITevI-XTEN-CasSl 5'-3' fusion protein encodes an amino acid sequence of SEQ ID No: 80, for example when expressed in the presence of OMTyr.

[0363] There is provided one or more nucleic acid(s) encoding:

[0364] a) a first nucleotide sequence comprising a start codon, coding codons, and a first stop codon, wherein the coding codons comprise the nucleotide sequence of SEQ ID No:79;

[0365] b) a second nucleotide sequence encoding a tyrosyl tRNA;

[0366] d) a third nucleotide sequence encoding a tyrosyl tRNA synthetase;

[0367] e) a fourth nucleotide sequence encoding a gRNA or crRNA, comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by a target sequence in the target cell genome;

[0368] f) a fifth nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No:2 (CasS2); g) a sixth nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No:4 (CasS4); and

[0369] h) a seventh nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No: 5 (CasS5).

[0370] There is also provided one or more nucleic acid(s) encoding:

[0371] a) a first nucleotide sequence comprising a start codon, coding codons, and a first stop codon, wherein the coding codons encode the amino acid sequence of SEQ ID No:80, for example when expressed in the presence of OMTyr;

[0372] b) a second nucleotide sequence encoding a tyrosyl tRNA;

[0373] d) a third nucleotide sequence encoding a tyrosyl tRNA synthetase;

[0374] e) a fourth nucleotide sequence encoding a gRNA or crRNA, comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by a target sequence in the target cell genome;

[0375] f) a fifth nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No: 2 (CasS2);

[0376] g) a sixth nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No:4 (CasS4); and

[0377] h) a seventh nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No: 5 (CasS5).

[0378] There is provided one or more nucleic acid(s) encoding a) a first nucleotide sequence comprising the nucleotide sequence of SEQ ID No: 79. There is also provided one or more nucleic acid(s) encoding a) a nucleotide sequence encoding the amino acid sequence of SEQ ID No:80. The amino acid sequence of SEQ ID No:80 may be expressed when the one or more nucleic acids are expressed in the presence of OMTyr and the second and third nucleotide sequences of part b) and c) respectively. The one or more nucleic acids in this example may further comprise b) a second nucleotide sequence encoding a tyrosyl tRNA (e.g. as described elsewhere herein). The one or more nucleic acids in this example may further comprise c) a third nucleotide sequence encoding a tyrosyl tRNA synthetase (e.g. as described elsewhere herein). The one or more nucleic acids in this example may further comprise d) a fourth nucleotide sequence encoding a gRNA or crRNA, comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by a target sequence in a target cell genome (e.g. as described elsewhere herein).

[0379] The inventors have devised a particular configuration of the elements of the ITevI-CasS fusion protein complex, tRNA / aaRS pair and crRNA, driven by different promoters, in the examples below. Extrapolating from this example, in a particular embodiment, there is provided four nucleic acid(s), wherein: the first nucleic acid comprises the first nucleotide sequence of part a) and, if present, each of the fifth nucleotide sequence of part e), the sixth nucleotide sequence of part f), the seventh nucleotide sequence of part g), and the eighth nucleotide sequence of part h);

[0380] the second nucleic acid comprises the second nucleotide sequence of part b);

[0381] the third nucleic acid comprises the third nucleotide sequence of part c); and

[0382] the fourth nucleic acid sequence comprises the fourth nucleotide sequence of part d); and wherein the first, second, third and fourth nucleic acids are each under the control of a separate promoter. The four promoters may be the same or different. The promoters may be any of the promoters described herein.

[0383] In particular, the first nucleic acid of the four nucleic acids in under the control of a promoter of SEQ ID No: 101. In particular, the second nucleic acid of the four nucleic acids in under the control of a promoter of SEQ ID No: 103. In particular, the third nucleic acid of the four nucleic acids in under the control of a promoter comprised by the nucleic acid sequence of SEQ ID No: 104. In particular, the fourth nucleic acid of the four nucleic acids in under the control of a promoter of SEQ ID No: 102.

[0384] The crRNA or gRNA to be used with the I-Tev-CasS nucleases described herein may comprise any of the features described herein, regarding spacer length, numbers of spacers, complementarity of the spacer to the protospacer sequence, and sequences and numbers of repeat sequences.

[0385] For embodiments relating to peptide products comprising a fusion protein in which Py comprises a CasS protein, the spacer sequence may comprise the nucleotide sequence of SEQ ID No:69.

[0386] There is provided a target cell comprising a target sequence which comprises, in 5' to 3' orientation:

[0387] a) a PAM sequence (as described elsewhere herein); and

[0388] b) a protospacer sequence (as described elsewhere herein),

[0389] and further comprising the one or more exogenous nucleic acids as described elsewhere herein. Optionally, sequences a) and b) are each immediately adjacent to each other.

[0390] There is provided a target cell comprising a target sequence which comprises, in 5' to 3' orientation:

[0391] a) a nuclease cleavage site nucleotide sequence;

[0392] b) optionally a nuclease spacer nucleotide sequence;

[0393] c) a PAM sequence; and

[0394] d) a protospacer sequence,

[0395] and further comprising the one or more exogenous nucleic acids as described elsewhere herein. Optionally, sequences a) to d) are each immediately adjacent to each other.

[0396] There is also provided a target cell comprising a target sequence which comprises, in 5' to 3' orientation: a) a nuclease cleavage site nucleotide sequence; (as described elsewhere herein); b) optionally a nuclease spacer nucleotide sequence; (as described elsewhere herein); c) a PAM sequence; (as described elsewhere herein); and

[0397] d) a protospacer sequence (as described elsewhere herein),

[0398] and further comprising

[0399] the one or more exogenous nucleic acids as described elsewhere herein, and

[0400] optionally the one or more nucleic acid(s) as described elsewhere herein, and

[0401] wherein the peptide product of part a) is a CasS-based nuclease or nickase (as described elsewhere herein). Optionally, sequences a) to d) are each immediately adjacent to each other. In one particular embodiment, the nuclease spacer nucleotide sequence is present. In another embodiment, the nuclease spacer nucleotide sequence is absent. The target sequence may comprise a PAM sequence 5' of the or a protospacer sequence, e.g. wherein the PAM is immediately adjacent to the protospacer sequence.

[0402] There is provided a target cell comprising a target sequence which comprises, in 5' to 3' orientation:

[0403] a) an I-TevI cleavage site nucleotide sequence (as described elsewhere herein);

[0404] b) an I-TevI spacer nucleotide sequence (as described elsewhere herein);

[0405] c) a PAM sequence (as described elsewhere herein); and

[0406] d) a protospacer sequence (as described elsewhere herein),

[0407] and further comprising the one or more exogenous nucleic acids as described elsewhere herein. Optionally, sequences a) to d) are each immediately adjacent to each other. In one embodiment, one or more of a) to d) are non-native (i.e. exogenous) sequence(s) to the cell. In another embodiment, all of a) to d) are non-native (i.e. exogenous) sequences to cell. In another embodiment, a) and / or b) are non-native (i.e. exogenous) sequences to cell. The target sequence may comprise a PAM sequence 5' of the or a protospacer sequence, e.g. wherein the PAM is immediately adjacent to the protospacer sequence.

[0408] There is also provided a target cell comprising a target sequence which comprises, in 5' to 3' orientation:

[0409] a) an I-TevI cleavage site nucleotide sequence (as described elsewhere herein);

[0410] b) an I-TevI spacer nucleotide sequence (as described elsewhere herein);

[0411] c) a PAM sequence (as described elsewhere herein); and

[0412] d) a protospacer sequence (as described elsewhere herein),

[0413] and further comprising

[0414] the one or more exogenous nucleic acids as described elsewhere herein, and the one or more nucleic acid(s) as described elsewhere herein, and

[0415] wherein the peptide product of part a) comprises an I-Tev-CasS nuclease (as described elsewhere herein).

[0416] Optionally, sequences a) to d) are each immediately adjacent to each other. In one embodiment, one or more of a) to d) are non-native (i.e. exogenous) sequence(s) to the cell. In another embodiment, all of a) to d) are non-native (i.e. exogenous) sequences to cell. In another embodiment, a) and / or b) are non-native (i.e. exogenous) sequences to cell. The target sequence may comprise a PAM sequence 5' of the or a protospacer sequence, e.g. wherein the PAM is immediately adjacent to the protospacer sequence.

[0417] In examples where the peptide product comprises a fusion protein in which Py comprises a nuclease which is an I-TevI nuclease, the I-TevI nuclease recognises an I-TevI cleavage site nucleotide sequence which is 5' or 3' (e.g. 5') of the protospacer in the target sequence comprised by the target cell.

[0418] In one example, the I-TevI cleavage site nucleotide sequence is 5' of the protospacer in the target sequence. The I-TevI cleavage site nucleotide sequence may be between (about) 3 and 7 nucleotides in length, for example between (about) 4 and 6 nucleotides in length. The I-TevI cleavage site nucleotide sequence may be about 5 nucleotides in length. The I-TevI cleavage site nucleotide sequence may comprise the motif 5'-CNNNG-3'. The I-TevI cleavage site nucleotide sequence may be selected from 5'-CCACG-3', 5'-CACCG-3', 5'-CATAG-3', 5'-CTAAG-3', 5'-CTGAG-3', 5'-CTTAG-3', 5'-CCTCG-3', 5'-CAGCG-3', 5'-CACTG-3', 5'-CGACG-3', 5'-CGATG-3', 5'-CTCCG-3', 5'-CTACG-3', 5'-CTGTG-3', 5'-CTGCG-3', 5'-CCGTG-3', 5'-CCTAG-3', 5'-CCATG-3', 5'-CAAAG-3', 5'-CAGAG-3', 5'-CATTG-3' and 5'-CATCG-3'. The I-TevI cleavage site nucleotide sequence may be selected from 5'-CCACG-3', 5'-CACCG-3', 5'-CATAG-3', 5'-CTAAG-3', 5'-CTGAG-3', 5'-CTTAG-3', 5'-CCTCG-3' and 5'-CAGCG-3', or is selected from 5'-CCACG-3', 5'-CACCG-3', 5'-CATAG-3', 5'-CTAAG-3' and 5'-CTGAG-3'. The I-TevI cleavage site nucleotide sequence may be selected from 5'-CCACG-3', 5'-CACCG-3' and 5'-CATAG-3'. The I-TevI cleavage site nucleotide sequence may be selected from 5'-CCACG-3' and 5'-CACCG-3'. The I-TevI cleavage site nucleotide sequence may have, in particular the nucleotide sequence of SEQ ID No:42.

[0419] In examples where the peptide product comprises a fusion protein in which Py comprises a nuclease which is an I-TevI nuclease, the I-TevI nuclease recognises an I-TevI spacer nucleotide sequence which is 5' or 3' (e.g. 5') of the protospacer in the target sequence.

[0420] In one example, the I-TevI spacer nucleotide sequence is 5' of the protospacer in the target sequence. The I-TevI spacer nucleotide sequence may be between (about) 10 and 35 nucleotides in length, such as from 15 to 35, or from 20 to 35 in length. The I-TevI spacer nucleotide sequence may be between (about) 29 and 33 nucleotides in length. The I-TevI spacer nucleotide sequence may be between (about) 30 and 32 nucleotides in length. The I-TevI spacer nucleotide sequence may be (about) 31 nucleotides in length. The I-TevI spacer nucleotide sequence may at least (about) 80% (e.g. (about) 90%) identical to the nucleotide sequence of SEQ ID No:43. The I-TevI spacer nucleotide sequence may be 100% identical to the nucleotide sequence of SEQ ID No:43.

[0421] In examples where the peptide product comprises a fusion protein in which Py comprises a nuclease which is an I-TevI nuclease, the target sequence comprises, in 5' to 3' orientation:

[0422] a) an I-TevI cleavage site nucleotide sequence;

[0423] b) an I-TevI spacer nucleotide sequence;

[0424] c) a PAM sequence; and

[0425] d) a protospacer sequence.

[0426] In one embodiment, sequences a) to d) are each immediately adjacent to each other. In one embodiment, one or more of a) to d) are non-native (i.e. exogenous) sequence(s) to the cell. In another embodiment, all of a) to d) are non-native (i.e. exogenous) sequences to cell. In another embodiment, a) and / or b) are non-native (i.e. exogenous) sequences to cell.

[0427] In examples where the peptide product comprises a fusion protein in which Py comprises a CasS protein for targeting the nuclease, the Protospacer Adjacent Motif (PAM) sequence in the target sequence may be selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'-ACG-3', 5'-AAT-3', 5'-ACA-3', 5'-ACT-3', 5'-ATC-3', 5'-ATA-3', 5'-GAG-3', 5'-TAG-3', 5'-ACC-3', 5'-AGG-3', 5'-ATT-3', 5'-GAC-3' and 5'-GTG-3'.

[0428] The protospacer may be immediately adjacent to a Protospacer Adjacent Motif (PAM) sequence in the target sequence selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'-ACG-3', 5'-AAT-3', 5'-ACA-3', 5'-ACT-3', 5'-ATC-3', 5'-ATA-3', 5'-GAG-3' and 5'-TAG-3'. The protospacer may be immediately adjacent to a PAM sequence in the target sequence selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'-ACG-3', 5'-AAT-3' and 5'-ACA-3'. The protospacer may be immediately adjacent to a PAM sequence in the target sequence selected from 5'-AAC-3' and 5'-ATG-3'. The protospacer may be immediately adjacent to a PAM sequence in the target sequence which is 5'-AAC-3'.

[0429] Base Editor peptide products

[0430] In a particular embodiment, the peptide product of a) encodes a nucleic acid modifier which is a base editor.

[0431] Base editors that may be used as a peptide product of part a) are well known in the art. In this example, the peptide product comprises a base editor, optionally wherein the base editor further comprises a modified nuclease that is modified to be unable to perform DNA double strand breaks, while retaining its DNA binding capacity and is fused to a domain to perform base editing. Many base editors known in the art comprise a fusion of a modified nuclease (e.g. a CRISPR-based nuclease, see elsewhere herein) fused to a base editing domain. Base editors are described in general, for example, in Rees eta / ., Nature Genetics Reviews, 19, 2018, 770-788, which is incorporated herein by reference in its entirety. Thus, base editors include systems where CRISPR / Cas protein(s) (such as dead-Cas9 (dCas9) or nickase Cas9 (nCas9)) is / are fused to a cytosine or adenosine deaminase domain and directed to the target sequence to make the desired modification. Thus, in one embodiment, the peptide product encodes a dCas9 or nCas9 fused to a cytosine or adenosine deaminase domain and a guide RNA (or crRNA).

[0432] In examples using a CRISPR / Cas-based base editor, the one or more nucleic acid(s) further comprises d) a fourth nucleotide sequence which expresses a gRNA or crRNA comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence.

[0433] In one embodiment, the base editor is selected from:

[0434] A. Cytosine Base Editors (CBEs) that convert C: G into T: A (Komor eta / ., Nature, 533:420-4, 2016, incorporated herein by reference in its entirety)

[0435] CBEs rely on ssDNA cytidine deaminase among which: APOBEC1, rAPOBECl, APOBEC1 mutant or evolved version (evoAPOBECl), and APOBEC homologs (APOBEC3A (eA3A), Anc689), Cytidine deaminase 1 (CDA 1), evoCDA 1, FERNY, evoFERNY.

[0436] B. Adenine Base Editors (ABEs) that convert A: T into G: C (Gaudelli eta / ., Nature, 551 (7681), 464-471, 2017, incorporated herein by reference in its entirety)

[0437] ABEs rely on deoxyadenosine deaminase activity of a tandem fusion TadA-TadA* where TadA* is an evolved version of TadA, an E. co / itRNA adenosine deaminase enzyme, able to convert adenosine into inosine on ssDNA. TadA* include TadA-8a-e and TadA-7.10.

[0438] C. Cytosine Guanine Base Editors (CGBEs) that convert C: G into G: C (Chen et a!., Biorxiv, 2020; Kurt eta / ., Nature Biotechnology, 2020, both incorporated herein by reference in its entirety) CGBEs generally consist of a nickase CRISPR / Cas protein fused to a cytosine deaminase (rAPOBEC) and base excision repair proteins, such as rXRCCl, or of a nickase CRISPR / Cas protein fused to a rat APOBEC1 variant (R33A) protein and an E. co / Aderived uracil DNA N-glycosylase (ellNG).

[0439] D. Cytosine Adenine Base Editors (CABEs) that convert C: G into A: T (Zhao et a!., Nature Biotechnology, 2020, incorporated herein by reference in its entirety)

[0440] CABEs generally consist of a nickase CRISPR / Cas protein (e.g. a Cas9 nickase), a cytidine deaminase (e.g. AID), and a uracil-DNA glycosylase (Ung).

[0441] E. Adenine Cytosine Base Editors (ACBEs) that convert A: T into C: G (W02020 / 181180, incorporated herein by reference in its entirety)

[0442] ACBEs generally include a nucleic acid programmable DNA-binding protein and an adenine oxidase.

[0443] F. Adenine Thymine Base Editors (ATBEs) that convert A: T into T: A (W02020 / 181202, incorporated herein by reference in its entirety) ATBEs generally consist of a CRISPR / Cas nickase protein (e.g. a Cas9 nickase) and one or more adenosine deaminase or an oxidase domain.

[0444] G. Thymine Adenine Base Editor (TABE) that convert T: A into A: T (W02020 / 181193; W02020 / 181178; W02020 / 181195, each of which is incorporated herein by reference in its entirety).

[0445] TABEs generally consist of a CRISPR / Cas nickase protein (e.g. a Cas9 nickase) and an adenosine methyltransferase, a thymine alkyltransferase, or an adenosine deaminase domain.

[0446] Further additional modules can be added to base editors to increase precision, modularity and editing efficiency. These include the addition of one or two uracil DNA glycosylase inhibitor domain(s) (UGI) to prevent base excision repair mechanism to revert base edition. Another module that can be added is Mu-GAM that decreases the insertion-deletion rate by inhibiting DNA repair by the non-homologous end joining mechanism (NHEJ) in the cell. For CRISPR / Cas based base editing systems, the CRISPR / Cas system may rely on nickase activity (for example by mutating Cas9 to form the well-known nCas9 D10A). The use of a nickase cuts the non-edited strand, and favours its repair and thus the fixing of the edited base.

[0447] In an example, the base editor is selected from the group consisting of: POBEC1, rAPOBECl, APOBEC1 mutant or evolved version (evoAPOBECl), APOBEC homologs (APOBEC3A (eA3A), Anc689), Cytidine deaminase 1 (CDA1), evoCDA 1, FERNY, evoFERNY, BE1, BE2, BE3, BE4, BE4-GAM, HF-BE3, Sniper-BE3, Target-AID, Target-AID-NG, ABE, EE-BE3, YE1-BE3, YE2-BE3, YEE-BE3, BE-PLUS, SaBE3, SaBE4, SaBE4-GAM, Sa(KKH)-BE3, VQR-BE3, VRER-BE3, EQR-BE3, xBE3, Casl2a-BE, Ea3A-BE3, A3A-BE3, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, ABE8e, SpRY-ABE, SpRYCBE, SpG-CBE4, SpG-ABE, SpRY-CBE4, SpCas9-NG-ABE, SpCas9-NG-CBE4, enAsBEl.l, enAsBE1.2, enAsBE1.3, enAsBE1.4, AsBEl.l, AsBE1.4, CRISPR-Abest, CRISPR-Cbest, eA3A-BE3 and AncBE4. Examples of DNA-based editor proteins include, but are not limited to BE1, BE2, BE3, BE4, BE4-GAM, HF-BE3, Sniper-BE3, Target-AID, Target-AID-NG, ABE, EE-BE3, YE1-BE3, YE2-BE3, YEE-BE3, BE-PLUS, SaBE3, SaBE4, SaBE4-GAM, Sa(KKH)-BE3, VQR-BE3, VRER-BE3, EQR-BE3, xBE3, Casl2a-BE, Ea3A-BE3, A3A-BE3, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, ABE8e, SpRY-ABE, SpRYCBE, SpG-CBE4, SpG-ABE, SpRY-CBE4, SpCas9-NG-ABE, SpCas9-NG-CBE4, enAsBEl.l, enAsBE1.2, enAsBE1.3, enAsBE1.4, AsBEl.l, AsBEl.4, CRISPR-Abest, CRISPR-Cbest, eA3A-BE3 and AncBE4.

[0448] The base editor may be a sea lamprey base editor. In one embodiment, the base editor is a PmCDAl cytidine deaminase having the amino acid sequence encoded by SEQ ID No: 19. In one embodiment, the base editor is from sea lamprey.

[0449] When the peptide product of a) is a CRISPR / Cas-based base editor, there is provided a cell comprising a target sequence which comprises, in 5' to 3' orientation:

[0450] a) a PAM sequence; and

[0451] b) a protospacer sequence,

[0452] and further comprising the one or more nucleic acid(s) as described elsewhere herein (in particular as described for parts b) and c), and

[0453] the one or more exogenous nucleic acids as described elsewhere herein (for the production of a first MOI or the conversion of a second MOI to the first MOI), and

[0454] wherein the peptide product of part a) is a base editor (e.g. any of the base editors described herein), and wherein the base editor is configured to target the PAM of a) and protospacer of b) and optionally wherein the target sequence comprises the PAM sequence 5' of the protospacer sequence, e.g. wherein the PAM is immediately adjacent to the protospacer sequence.

[0455] Other base editors are known which are not based on a CRISPR / Cas system, such as the interbacteria I DddA, which catalyses the deamination of cytidines within dsDNA, which was engineered to split the DddA into two halves, then fused to transcription activator-like effector array proteins, and a uracil glycosylase inhibitor (UGI), see Mok et al., Nature, 583, 2020, 631-637, doi: doi.org / 10.1038 / s41586-020-2477-4. Thus, in one example, the base editor comprises two fusion proteins as described in Figure 3a of Mok eta / ., supra, which is incorporated herein by reference for the structure and composition of the TALE-split DddAtox fusions.

[0456] Another non-CRISPR / Cas based base editor is described in Lim eta / ., Nature Communications, 13, 366, 2022, doi: https: / / doi.org / 10.1038 / s41467-022-27962-0. The authors describe zinc-finger DNA-binding proteins, the split interbacteria I toxin deaminase DddAtox, and a uracil glycosylase inhibitor (UGI), together termed a zinc finger deaminase (ZFD) which catalyze targeted C-to-T base conversions which achieve base editing at frequencies of up to 60% in nuclear DNA and 30% in mtDNA. Thus, in another example, the base editor comprises a zinc-finger DNA-binding protein, a split interbacteria I toxin deaminase DddAtox, and a UGI, in particular as described in Figure la of Lim eta / ., supra, which is incorporated herein by reference for the structure and composition of the zinc finger deaminases.

[0457] In examples wherein the peptide product of a) is a base editor, in an example, the base editor kills or inhibits the growth of the target cell by modifying a target sequence comprised by the genome of the target cell. Alternatively, the base editor modifies the target sequence in the target cell to prevent or reduce expression of the one or more exogenous nucleic acids comprised by genome of the target cell. In an example, base editors may target essential genes, in which case the protospacer may be located within an essential gene of the cell. In this example, the base editor prevents the expression of the essential gene, and the target cell is thereby killed, or the growth of the cell is reduced.

[0458] Toxic peptide products

[0459] In any example herein, the peptide product of part a) (e.g. as part of the one or more nucleic acid(s), transmissible elements, cells or compositions described elsewhere herein) is a peptide toxin. The peptide toxin may be a bacteriocin. The peptide toxin may be a lysin. The peptide toxin may be a toxic peptide of a toxin -antitoxin system. The toxin is toxic to at least the target cell. The toxin may additionally be toxic to other cells in the local environment, such as in a microbiome of a subject. The peptide toxin may be a molecule that causes cell death by lysing cell membranes, degrading cellular DNA, or other mechanisms.

[0460] Examples of toxin -antitoxins include but are not limited to peml-pemK genes of plasmid R100, the phd-doc genes of phage Pl, the ccdA-ccdB genes of plasmid F, mazE-mazF (also known as chpAI-chpAK) of E. co / ZK-12, sof-gefc E. coli K-12, kicA-kicBc E. co / / K-12, re / B-relEc E. co / i\\-12, and chpBI-chpBK of E. coli.-12 (toxin in bold). See Westwater eta / ., Antimicrobial agents and chemotherapy, 47, 1301-1307, 2003; doi:10.1128 / AAC.47.4.1301-1307.2003.

[0461] Other examples of toxins and lysis proteins are described in in Knudsen, eta / ., Appl. Environ. Microbiol., 57, 85-92, 1991; and Callura, et al., Proc. Natl Acad. Sci. USA, 107, 15898-15903, 2010, each of which is incorporated herein by reference in its entirety. Molecules which are toxic because they are capable of the degradation of essential proteins are discussed in Chan, et a / ., Nat. Chem. Biol., 12, 82-86, 2016 which is incorporated herein by reference in its entirety.

[0462] Antimicrobial peptides include natural antimicrobial peptides, for example from frog's skin, human sweat or ant's venom. Classes of antimicrobial peptides include: anionic, linear cationic a-helical, cationic peptides enriched for specific amino acids, anionic and cationic peptides that contain cysteine and form disulphide bonds, and anionic and cationic peptide fragments of larger proteins. For a discussion of these classes, see the review paper Brogden, Nat Rev Microbiol, 3, 238-250, 2005, doi:10.1038 / nrmicrol098, which is incorporated herein by reference in its entirety. In particular, see Table 1 of Brogen for specific examples of antimicrobial peptides, which peptides are intended to form part of the subject-matter of this disclosure.

[0463] In one example, the peptide antimicrobial is selected from a-defensin, p-defensin, plectasin, Protegrin-1, LL-37, nisin, mersacidin, polymyxin B. Other peptide antimicrobials are described in Figure 2 of Hancock & Sahl, Nat. Biotechnol., 24(12), 1551, 2006, doi:10.1038 / nbtl267, which is incorporated herein by reference in its entirety. In particular, see Table 1 of Hancock & Sahl for specific examples of antimicrobial peptides, which peptides are intended to form part of the subject-matter of this disclosure.

[0464] W02010 / 141135A2 (Trustees of Boston University & Massachusetts Institute of Technology), which is incorporated herein by reference in its entirety, describes a number of toxic peptide molecules. Thus, in an example, the peptide product is selected from Indolicidin (SEQ ID No:6 of W02010 / 141135A2), Cecropin PI (SEQ ID No: 11 of W02010 / 141135A2), Dermaseptin (SEQ ID No: 14 of W02010 / 141135A2), Ponericin WI (SEQ ID No:44 of W02010 / 141135A2), Ponericin W3 (SEQ ID No:40 of W02010 / 141135A2), Ponericin W4 (SEQ ID No: 18 of W02010 / 141135A2), Ponericin W5 (SEQ ID No:42 of W02010 / 141135A2) and Ponericin W6 (SEQ ID No:22 of W02010 / 141135A2) or variants thereof. In another example, the peptide product comprises any of the amino acid sequences of SEQ ID No: 10 or SEQ ID Nos:36-45 of W02010 / 141135A2. Each of the sequences of W02010 / 141135A2 which are referred to herein are incorporated by reference and intended to form part of the subject-matter of this disclosure.

[0465] Other examples of antimicrobial peptides include, but are not limited to, mefloquine, venturicidin A, antimycin, myxothiazol, stigmatellin, diuron, iodoacetamide, potassium tellurite hydrate, aDL-vinylglycine, N-ethylmaleimide, L-a I lyg lycine, diaryquinoline, betaine aldehyde chloride, acivcin, psicofuraine, buthionine sulfoximine, diaminopemelic acid, 4-phospho-D-erythronhydroxamic acid, motexafin gadolinium and / or xycitrin or modified versions or analogues thereof.

[0466] W02010 / 141135A2 also described a lytic enzyme, LysK. Thus, in one example, the peptide product is LysK or a functional fragment thereof, e.g. the functional fragment of LysK is CHAP165 (SEQ ID NO: 71 of W02010 / 141135A2 which sequence is incorporated by reference and intended to form part of the subject-matter of this disclosure).

[0467] In the examples below, the inventors provided proof of concept using both a nuclease peptide product and using a peptide toxin, RalR. Thus, in one example, the peptide product (e.g. encoded by the first nucleotide sequence) is a RalR peptide toxin. The peptide product (e.g. encoded by the first nucleotide sequence) may be encoded by a nucleotide sequence comprising SEQ ID No:76. The first nucleotide sequence encoding the peptide product may comprise a nucleotide sequence comprising SEQ ID No:76. The peptide product (e.g. encoded by the first nucleotide sequence) may the comprise the amino acid sequence of SEQ ID No:77, for example when expressed in the presence of OMTyr.

[0468] In some examples, the toxic peptide product targets a target site comprising a target sequence. The target sequence may be comprised by the chromosome of the target cell. Alternatively, the target sequence in the target cell may be comprised by a plasmid or episome of the cell.

[0469] The target sequence may be an RNA. The target sequence may be a single stranded DNA. The target sequence may be located within an essential gene of the target cell. In this example, the toxic peptide product prevents the expression of the essential gene, and the target cell is thereby killed, or the growth of the cell is reduced. Alternatively, the toxic peptide product may have non-specific toxicity, whereby the toxic peptide product kills or reduces the growth of the target cell by modification of the target sequence, and wherein the target sequence is the genome of the target cell. The modification may be, for example, non-specific cutting of the DNA by an endonuclease (as is the case for RalR)

[0470] Peptide products which block transcription of the one or more exogenous nucleic acids

[0471] Peptide products which perform gene silencing functions may also be useful as peptide products. The gene to be silenced and / or down-regulated may be an essential gene or may be involved in the production of the first MOI in the target cell.

[0472] In some applications, nucleases which are mutated such that they are no longer able to cleave DNA can be used to silence genes, which may be particularly useful to prevent or reduce expression of the one or more exogenous nucleic acids comprised by the target cell. Suitable nuclease systems for gene silencing include, but are not limited to CRISPR / Cas systems, such as dCas9, type IV CRIPSR / Cas systems or type I CRISPR / Cas systems in which the nuclease (Cas3) has been removed, a mutated TALEN or a mutated zinc finger protein.

[0473] Thus, in one embodiment, the peptide product of part a) (e.g. as part of the one or more nucleic acid(s), transmissible elements, cells or compositions described elsewhere herein) is a nuclease which cannot introduce double stranded breaks in the target sequence in the target cell, and the nuclease is directed to, or, if present, the gRNA (or crRNA) directs the nuclease to, the target sequence to modify said target sequence and prevent or reduce expression of the one or more exogenous nucleic acids comprised by the target cell. In another embodiment, the peptide product is a nuclease which cannot introduce double stranded breaks in the target sequence in the target cell, and the nuclease is directed to, or, if present, the gRNA (or crRNA) directs the nuclease to, modify a target sequence which kills or inhibits the growth of the target cell.

[0474] The peptide product may be a restriction enzyme which has been modified to prevent cutting of DNA or RNA.

[0475] The peptide product may be a transcription activator that activates transcription of the target sequence in the target cell. In one example, the transcription activator may re-activate a normally-inactive repressor of an essential gene within the target cell. Thus, when the peptide product comprising such a transcription activator is expressed in the presence of the NNAA, it is capable of causing transcription of the previously-inactive repressor, which represses transcription of the essential gene within the target cell, thereby killing or inhibiting the growth of the target cell.

[0476] In a particular embodiment, the peptide product comprises a CRISPRi system. Such a peptide product may comprise a CRISPRi system encoding a Cas9 (dead-Cas9) fused to one or more transcriptional repressor(s), and d) a gRNA or crRNA which directs the CRISPRi system to the target sequence to modify said target sequence and prevent or reduce expression of the one or more exogenous nucleic acids comprised by the target cell. In another embodiment, the peptide product comprises a CRISPRi system encoding a Cas9 (dead-Cas9) fused to one or more transcriptional repressor(s), and d) a gRNA or crRNA which directs the CRISPRi system to the target sequence modify a target sequence which kills or inhibits the growth of the target cell.

[0477] Any CRISPRi system herein may encode a system selected from: dCas9-KRAB, dCas9-KRAB-MeCP2 and dCas9-SALLl-SDS3. Escobar et al., ACS Synth. Biol., 11(10), 3239-3250, 2022, doi: https: / / doi.org / 10.1021 / acssynbio.2c00156, which is incorporated herein by reference in its entirety, describes that a number of diverse Cas proteins (various nuclease-deactivated Cas9, Casl2a, Casl2e, or Casl2j proteins) are compatible with a Kruppel associated box (KRAB) domain to provide CRISPRi in eukaryotic human cells. See in particular Figure 3.

[0478] In a particular embodiment, the CRISPRi system is a CasS-based CRISPRi system, for example as described in the Examples hereinbelow. Example 4 of WO2024 / 047151A1 shows that CasS2, CasS4 and CasS5 together are able to work together to reduce transcription of target genes. Thus, any CRISRPi system described herein may comprise:

[0479] I. a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No:2;

[0480] a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No:4; and

[0481] III. a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No: 5; and

[0482] d) a guide RNA or crRNA (e.g. expressed from the fourth nucleotide sequence) comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence.

[0483] Any of sequences L, IL, or III. may be comprised by the first nucleotide sequence. In an example, the nucleotide sequences of I. and II. are comprised by the first nucleotide sequence of part a), and the nucleotide sequence of III. is comprised by e) a fifth nucleotide sequence. In another example, the nucleotide sequences of II. and III. are comprised by the first nucleotide sequence of part a), and the nucleotide sequence of I. is comprised by e) a fifth nucleotide sequence. In another example, the nucleotide sequences of I. and III. are comprised by the first nucleotide sequence of part a), and the nucleotide sequence of II. is comprised by e) a fifth nucleotide sequence. In another example, the nucleotide sequences of L, II. and III. are all comprised by the first nucleotide sequence of part a).

[0484] The CRISPRi system may further comprise IV. a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No:l, and / or V. a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 90% identical to SEQ ID No:3.

[0485] In one example, the CRISPRi system includes sequences of I., IL, III., and IV. In another example, the CRISPRi system includes sequences of I., IL, III., and V. In another example, the CRISPRi system includes sequences of L, IL, III., IV. and V. Any of sequences L, IL, III., IV., or V. may be comprised by the first nucleotide sequence of part a). In an example, all of sequences L, IL, III., IV., or V. are comprised by the first nucleotide sequence of part a).

[0486] For use in eukaryotic cells, any CasS-based CRISPRi system described herein may further comprise a KRAB domain (see for example, Pfam: PF01352, https: / / www.ebi.ac.uk / interpro / entry / pfam / PF01352 / , or UnitProt: Q96PE6). The KRAB domain may be fused to the N- or C-terminus of any of the CasS2, CasS4 or CasS5 proteins, optionally via a linker peptide. The linker peptide may be any linker described herein. The KRAb domain may comprise a sequence of SEQ ID No: 105.

[0487] The target sequence may be a sequence within the one or more exogenous nucleic acids. In this example, the peptide product reduces or prevents the expression of the one or more exogenous nucleic acids. The target sequence may be located within an essential gene of the target cell. In this example, the peptide product prevents the expression of the essential gene, and the target cell is thereby killed, or the growth of the cell is reduced.

[0488] Peptide products which modify DNA or RNA to create toxicity

[0489] Also disclosed herein are peptide products (of part a) which are toxic to the target cells through non-specific DNA and / or RNA modifications. These peptide products can be expressed in the presence of the NNAA to kill or reduce the growth of the target cell.

[0490] In one example, the peptide product of part a) is a deaminase.

[0491] The deaminase may be a cytosine deaminase. Cytosine deaminases modify random cytosine bases to uracil bases, which result in a cytosine to thymine change in the genetic code, because the new uracil base pairs with adenine, rather than guanine (which the original cytosine pairs with). When sufficient random C-to-U mutations have occurred in the target cell (hypermutation), the cell will die, because the DNA code has been sufficiently scrambled. In an example, the cytosine deaminase is APOBEC. In an example, the cytosine deaminase is AID, see Zhang etai., "Activation-induced cytidine deaminase-based in vivo continuous evolution system enables rapid protein engineering", BioRxiv, 2023, doi: https: / / doi.org / 10.1101 / 2023.01.17.524385. In an example, the cytosine deaminase is a bacterial cytosine deaminase. See Conticello, Genome Biology, 9, 229, 2008, doi: https: / / doi.org / 10.1186 / gb-2008-9-6-229. In this example, the cytosine deaminase peptide product of part a) kills or inhibits the growth of the target cell by modifying (e.g. hypermutating) a nucleotide sequence comprised by the genome (e.g. chromosome) of the target cell.

[0492] The deaminase may be a adenine deaminase. Adenine deaminases modify random adenine bases to inosine bases, which result in an adenine to guanine change in the genetic code, because the new inosine base pairs with cytosine, rather than thymine (which the original adenine pairs with). When sufficient random A-to-G mutations have occurred in the target cell (hypermutation), the cell will die, because the DNA code has been sufficiently scrambled. In an example, the cytosine deaminase is TadA, see for example, Sun et ai., Advanced Science, 12(34), e06644, 2025, doi: https: / / doi.org / 10.1002 / advs.202506644. In this example, the adenine deaminase peptide product of part a) kills or inhibits the growth of the target cell by modifying (e.g. hypermutating) a nucleotide sequence comprised by the genome (e.g. chromosome) of the target cell.

[0493] In one example, the peptide product of part a) is Mom (phage p). Mom originates from bacteriophage Mu (p), where it adds a methylcarbomyl chemical group onto DNA, which protects it from degradation by a number of different restriction enzymes, see for example, Karambelkar etai., Nucleic Acids Research, 48(10), 5294-5305, 2020, doi:https: / / doi.org / 10.1093 / nar / gkaa319. In this example, the Mom adds the methylcarbomyl group to the DNA which will inhibit DNA binding by various transcription factors, RNA polymerases, or otherwise interfere with DNA replication, causing cellular toxicity. In this example, the Mom peptide product of part a) kills or inhibits the growth of the target cell by modifying (e.g. by random addition of a methylcarbomyl moiety) a nucleotide sequence comprised by the genome (e.g. chromosome) of the target cell. Other bacteria have DNA sequence specific modification systems which add other chemical moieties to various nucleotide bases, which could also be used as a peptide product of a). One such example is the bacterial Dam methylase which methylates adenosine in DNA comprising a "GATC" motif. Thus Dam methylase could be the peptide product of a) which kills or inhibits the growth of the target cell by methylating target sequences which comprise GATC in the genome (e.g. chromosome) of the target cell.

[0494] Transcription regulatory proteins as peptide products

[0495] It is already mentioned herein that the peptide product of a) could be a transcriptional activator or de-repressor of an essential gene, which would directly kill or reduce growth of the target cell. However, this system could also be used to cause expression of an exogenously-introduced toxic compound. In this example, such transcription regulatory proteins are peptide products (rather than DNA or RNA constructs), and are referred to herein as "activator peptide products". For example, if a CRISPR / Cas based nuclease system (designed either to target a protospacer to kill the target cell or to reduce or prevent expression of the exogenous nucleic acids for the production of the first MOI or the conversion of the second MOI to the first MOI) were operably linked to a promoter which, in the absence of a transcriptional activator or de-repressor did not transcribe the CRISPR / Cas based nuclease system, then the transcription regulatory protein could be engineered such that it is only expressed in the presence of the NNAA, such expression causing the promoter associated with the CRISPR / Cas system to express the CRISPR / Cas system and thereby target and kill the cell or reduce expression of the one or more exogenous nucleic acids.

[0496] There is therefore alternatively provided one or more nucleic acids comprising:

[0497] a) a first nucleotide sequence comprising a start codon, coding codons and a first stop codon, wherein the coding codons encode an activator peptide product, which activator peptide product modulates a cognate promoter,

[0498] wherein the first nucleotide sequence comprises at least one mutation which,

[0499] i. when the one or more nucleic acid(s) are expressed without the presence of a predefined non-natural amino acid, synthesis of the activator peptide product is truncated or rendered non-functional; and

[0500] ii. when the one or more nucleic acid(s) are expressed in the presence of the non-natural amino acid produces the activator peptide product which is functional to express the peptide product of part e) in the target cell;

[0501] b) a second nucleotide sequence which expresses a tRNA which is capable of mediating the transfer of the non-natural amino acid into the activator peptide product of part a)ii. at a position corresponding to a codon which comprises the at least one mutation, and which tRNA is not a substrate for any natural aminoacyl-tRNA synthetase of the target cell;

[0502] c) a third nucleotide sequence encoding an aminoacyl-tRNA synthetase which is capable of adding the non-natural amino acid to the tRNA of part b); and e) a fourth nucleic acid encoding a peptide product which kills or inhibits the growth of a target cell, or which modifies a target sequence in a target cell to prevent or reduce expression of one or more exogenous nucleic acids comprised by the target cell, wherein the peptide product of e) is under control of the cognate promoter sequence, and absence the presence of the activator peptide product, expression of the peptide product of e) is prevented (or reduced). Any peptide products or features of peptide products described elsewhere for part a) may be encoded by the fourth nucleic acid of e) in this example. It will be appreciated by the skilled person that, when used for this purpose, no engineering of the nucleic acid encoding the peptide product (to be expressed only in the presence of the NNAA) would be required, as it is the nucleic acid encoding the activator peptide product which would be engineered for expression only in the presence of the NNAA.

[0503] Transcription regulatory proteins which induce (activate) transcription are known in the art. Some are known to bind to their cognate promoter sequence to cause activation of transcription, whereas others are known to bind in regions nearby the cognate promoter. Some directly cause transcription of their cognate promoter (e.g. by directly interacting with RNA polymerase and recruiting it to the promoter or facilitate a post-binding step in transcription initiation), whereas others indirectly cause such transcription (e.g. by inducing a curvature in DNA to promote RNA polymerase binding to the promoter)

[0504] In this example, the peptide product of a) may be a transcription regulatory protein activator that activates transcription of a fourth nucleic acid. In one example, the transcription regulatory protein activator may re-activate a normally-inactive repressor. Thus, when the peptide product comprising such a transcription regulatory protein activator is expressed in the presence of the NNAA, it is capable of causing transcription of the previously-inactive fourth nucleic acid (encoding a peptide product which kills or inhibits the growth of a target cell, or which modifies a target sequence in a target cell to prevent or reduce expression of one or more exogenous nucleic acids comprised by the target cell). Examples of such transcription regulatory protein activators and cognate promoters include the CII activator of the Lambda phage (SEQ ID No: 116) and the pRE promoter (SEQ ID No: 117), which causes an increase of ~700-fold expression of the downstream nucleotide sequence when in the presence of the CII activator. When the NNAA is OMTyr, the nucleic acid encoding the CII activator of lambda phase is modified to introduce a premature stop codon, because the amino acid sequence of SEQ ID No: 116 does not comprise F or Y early in it's sequence.

[0505] In this example, the peptide product of a) may be a transcription regulatory protein that derepresses transcription of a fourth nucleic acid that is normally not transcribed. In one example, the transcription regulatory protein is an antiterminator that allows transcription through a transcriptional terminator sequence that would otherwise stop transcription. Thus, when the peptide product comprising such a transcription regulatory antiterminator protein is expressed in the presence of the NNAA, it is capable of causing transcription of the previously-inactive fourth nucleic acid (encoding a peptide product which kills or inhibits the growth of a target cell). Examples of such transcription regulatory antiterminator proteins and cognate promoters include the antiterminator N protein of lambda phage (SEQ ID No: 118), its cognate pL promoter (SEQ ID No: 119) and tL terminator sequence (SEQ ID No: 120). The tL transcriptional terminator sequence prevents the normal expression of the downstream nucleotide sequence from the pL promoter. However, in the presence of an active antiterminator N protein, the N protein binds the NutL site on the RNA transcript from the pL promoter, and consequently RNA polymerase does not stop at the termination signal and transcribes the downstream nucleotide sequence.

[0506] Exogenous nucleic acids comprised bv the target cell

[0507] The cells which are targeted by the peptide products described herein are engineered or non-naturally occurring cells and comprise one or more exogenous nucleic acids. Various arrangements in the target cell of the one or more nucleic acid(s) and the one or more exogenous nucleic acids are contemplated.

[0508] In one embodiment, a particular target cell may be engineered to include, for example, one or more exogenous nucleic acids in its genome (e.g. chromosome or episome), and the one or more nucleic acid(s) is comprised by a transmissible element which is subsequently or separately delivered to the target cell. In such embodiments, the engineered target cell may be administered or contacted with an environment, organism or microbiome, and the one or more exogenous nucleic acids produce the desired effect in that environment, organism or microbiome. When it is desired to stop expression of the one or more exogenous nucleic acids, the transmissible element comprising the one or more nucleic acid(s) is administered or contacted with an environment, where the transmissible element is capable of delivering the one or more nucleic acid(s) to the target cell. Simultaneously or subsequently to this, the non-natural amino acid is administered or contacted with the target cells, such that the peptide product is expressed and the target sequence is modified to prevent or reduce expression of the one or more exogenous nucleic acids.

[0509] In addition to this basic configuration of the one or more nucleic acid(s) being comprised by a transmissible element, and the one or more exogenous nucleic acids being comprised by the genome of the target cell, it is contemplated herein that one or more components of the one or more nucleic acids may be comprised by the genome of the target cell, and at least one component of the one or more nucleic acid(s) is comprised by the transmissible element. For example, the transmissible element may comprise only the nucleic acid(s) required for production of the peptide product, and the genome of the target cell may additionally comprise the nucleic acids for expression of the tRNA / aaRS pair. Alternatively, the transmissible element may comprise only the nucleic acid(s) required for expression of the tRNA / aaRS pair, and the genome of the target cell may additionally comprise the nucleic acids for production of the peptide product, as well as the one or more exogenous nucleic acids. Of course, the nucleic acids for expression of the tRNA / aaRS pair need not be comprised by the same element (i.e. target cell genome or transmissible element), and may also be split. In another embodiment, the one or more nucleic acid(s), vectors or transmissible elements may comprise both the one or more nucleic acid(s) and the one or more exogenous nucleic acids, which are delivered to the target cell at the same time. In such embodiments, the transmissible element may be for example a conjugative plasmid, phage or packaged phagemid, which delivers both the one or more nucleic acid(s) and the one or more exogenous nucleic acids to a cell which is already established or present in an environment, organism or microbiome. The target cell is thus modified such that it is able to express the one or more exogenous nucleic acids and produce the desired effect in that environment, organism or microbiome. When it is desired to stop expression of the one or more exogenous nucleic acids, the non-natural amino acid is administered or contacted with the target cells in the environment, organism or microbiome, such that the peptide product is expressed and the target sequence is modified to prevent or reduce expression of the one or more exogenous nucleic acids, or the target cell is killed or its growth is reduced.

[0510] In other particular embodiments, the cell may be a engineered cell which comprises both the one or more nucleic acid(s) and the one or more exogenous nucleic acids, which may be for example engineered into the genome (e.g. chromosome or episome) of the cell. In such embodiments, the cell may be delivered directly to an environment, organism or microbiome, and the one or more exogenous nucleic acids are expressed to produce the desired effect in that environment. When it is desired to stop expression of the one or more exogenous nucleic acids, the non-natural amino acid is administered or contacted with the target cells in the environment, organism or microbiome, such that the peptide product is expressed and the target sequence is modified to prevent or reduce expression of the one or more exogenous nucleic acids.

[0511] In an example, the one or more exogenous nucleic acids comprised by the target cell are for the production of a first molecule of interest (MOI). In all of these arrangements, the one or more exogenous nucleic acids may, for example, directly produce a first molecule of interest (MOI), or may convert a molecule which is found in the local cellular environment (termed a second MOI) into a different first MOI.

[0512] The one or more exogenous nucleic acids for production of the first MOI may be comprised by one or more operons. Where production of the first MOI is direct, the one or more exogenous nucleic acids for production of the first MOI may be comprised by one or more operon (which may include any signal peptide or exporter as described herein. Where the one or more exogenous nucleic acids encodes one or more protein(s) for the conversion of a second MOI to the first MOI, the one or more proteins may be comprised by one or more operons, in particular by a single operon.

[0513] Metabolic pathways, exogenous genes, exporters and importers may be those described in W02016 / 141108, W02016 / 201380, WO2017 / 074566, WO2017 / 123418, WO2017 / 136792, WO2019 / 014391, WO2019 / 232415, W02020 / 097424, W02020 / 232063, W02020 / 257610, W02020 / 257707, WO2021 / 146394, WO2021 / 146397, WO2021 / 163421, WO2021 / 173808, WO2021 / 188618, WO2021 / 188819, WO2021 / 242897, WO2022 / 072636, WO2022 / 146718, WO2022 / 150779, W02022 / 204406, WO2022 / 221273 and WO2023 / 225667 (all owned by Synlogic Operating Company, Inc); US10,993,930B2, US10,300,043B2, US11,878,00262, WO2020 / 123483, WO2022 / 169909 (all owned by Novome Biotechnologies, Inc), each of which is incorporated herein by reference in its entirety.

[0514] Direct production of a first MOI

[0515] In any embodiment herein, the one or more exogenous nucleic acids may be comprised by a transmissible element comprising the one or more nucleic acid(s). Alternatively, the one or more exogenous nucleic acids may be comprised by the target cell.

[0516] The one or more exogenous nucleic acids may be for the production of a first molecule of interest (MOI) in the target cell. The one or more exogenous nucleic acids may encode the first MOI. The one or more exogenous nucleic acids may comprise or consist of an exogenous gene for the production of the first MOI.

[0517] The first MOI may be a therapeutic molecule, such as a peptide molecule (e.g. an antibody fragment, a hormone such as GLP-1, an interleukin, a cytokine, a chemokine, an eukaryotic growth factor, or an enzyme) or a small molecule (e.g. a metabolite such as L-DOPA, indole-3-acetic acid, butyrate etc).

[0518] The one or more exogenous nucleic acids for the production of a first MOI may encode an MOI which is a therapeutic molecule.

[0519] In one embodiment, the first MOI is a therapeutic peptide molecule, e.g. a therapeutic protein. Therapeutic proteins may be used to, for example, replace a protein that is deficient or abnormal, augment an existing biological pathway, provide a novel function or activity, interfere with a molecule or organism, and / or deliver other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins.

[0520] Therapeutic proteins include antibody fragments, hormones, interleukins, cytokines, chemokines, eukaryotic growth factors and / or enzymes.

[0521] The first molecule of interest may be a therapeutic protein, e.g. an antibody, antibody fragment (such as an antibody single variable domain), TCR (T-cell receptor) binding site, TCR variable domain, hormone, incretin, growth factor, anti-cancer agent, neurotransmitter or enzyme.

[0522] When the first MOI is a protein molecule (e.g. a therapeutic protein molecule), a transport signal peptide (SP) may be needed so that the cell can secrete the active substances (first MOI) out of the cell. SPs can be used with all types of cell. Examples of SPs in bacteria are described in Kaushik et al., Front. Physiol., 13, 2022, doi: 10.3389 / fphys.2022.933153. Examples of SPs used in yeast are described in Xue eta / ., Advanced Science, 10(2), 2203433, 2023, doi: 10.1002 / advs.202203433, and Durmusoglu, eta / ., Microb. Cell Fact., 22(109), 2023, doi:10.1186 / sl2934-023-02117-y. For a review of SPs across species, including eukaryotes, see Owji et a / ., Eur. J. Cell Biol., 97(6), 422-441, 2018, doi:10.1016 / j.ejcb.2018.06.003. Each of these reference is incorporated herein by reference in its entirety.

[0523] Thus, the one or more exogenous nucleic acids may further encode a sequence encoding a signal peptide for the secretion of the first MOI to the periplasm of the target cell, to the cell surface of the target cell, or to the extracellular space outside of the target cell.

[0524] In particular, when the cell or target cell is a bacterial cell, a bacterial transport signal peptide (SP) may be needed so that the bacteria can secrete the active substances (first MOI) out of the bacteria. SPs are generally short peptides with 16-30 amino acids and are N-terminal extensions of secreted proteins. They act as a target and recognition signal for signal peptidases which remove SPs from the translocated protein across the cytoplasmic membrane and cell wall. This process will result in the extracellular release of the mature protein or peptide.

[0525] Thus, when the target cell is a bacterial cell, a signal peptide may also be required to aid secretion of the first MOI (e.g. therapeutic protein molecule) to the periplasm of the target bacterial cell, to the cell surface of the target bacterial cell, or to the extracellular space outside of the target bacterial cell. In any embodiment herein, when the target cell is a bacterial cell, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) may further comprise a (exogenous) nucleic acid encoding a signal peptide for secretion of the first MOI (e.g. therapeutic protein molecule) to the periplasm of the target bacterial cell. In any embodiment herein, when the target cell is a bacterial cell, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) may further comprise a (exogenous) nucleic acid encoding a signal peptide for secretion of the first MOI (e.g. therapeutic protein molecule) to the cell surface of the target) bacterial cell. In any embodiment herein, when the target cell is a bacterial cell, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) may further comprise a (exogenous) nucleic acid encoding a signal peptide for secretion of the first MOI (e.g. therapeutic protein molecule) to the extracellular space outside of the target bacterial cell.

[0526] Further, as is known in the art, diffusion of molecules through cell membrane(s) (for example in bacteria, both in gram-negative and gram-positive bacteria) can limit the amount of a particular molecule which diffuses to the local environment. Without being bound by theory, in general, cellular (e.g. bacterial) membrane(s) are hydrophobic, so the more hydrophilic the first MOI is, the less likely it is to be able to cross the membrane. However, certain molecules which are detrimental to the cell (for example toxins, bacteriocins, etc) are exported more often, along with certain molecules which provide specific extra-cellular functions (such as molecules in bacteria associated with quorum sensing, iron acquisition, etc). In addition, some molecules are actively transported through the membrane(s) by dedicated exporters. For example, excess amino acids may be exported from the cell to maintain homeostasis, such as the alaE exporter of alanine, and the leuE exporter of leucine both found in E. coH, or exporters which export a certain class of molecule, for example, the setA transporter found in E. coH exports various sugar molecules. As another example, in yeast, S. cerevisiae, two monocarboxylate permeases have been identified: Jenl which mediates the transport of lactate, pyruvate, acetate and propionate; and Ady2 which mediates the transport of acetate, propionate, formate and lactate. Poor function of transporters in human cells have been implicated in hyperuricemia / gout (see Stiburkova et a!., Arthritis Res. Ther., 21, 77, 2019, doi: https: / / doi.org / 10.1186 / sl3075-019-1860-8). The addition of heterologous, functioning transporters in human cells may prove to have therapeutic benefits.

[0527] Thus, in many cells (e.g. bacteria), secretion of MOIs may be limited by the rate of diffusion. Thus, the cells may be engineered to express heterologous exporters which are capable of exporting an first MOI across the membrane(s) of the cell (e.g. bacteria). Heterologous exporters are able to form and function within the cell, e.g. bacterial membrane. This leads to an increased secretion of the first MOI into the local environment, and contributes to the reduced fitness disadvantage of the expression of the first MOI within the cell, e.g. bacteria, because (without being bound by theory) the first MOI is removed from the cytoplasm and / or periplasm into the local environment.

[0528] Thus, in any embodiment herein, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) may further comprise a nucleic acid encoding one or more exporter(s) of the first MOI (e.g. the therapeutic protein molecule or metabolite) from the cell (e.g. bacterium). In any embodiment herein, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) may further comprise an exogenous nucleic acid encoding one or more exporter(s) of the first MOI (e.g. the therapeutic protein molecule or metabolite) from the cell (e.g. bacterium). In a particular embodiment, the nucleic acid encodes an exporter of the first MOI.

[0529] In one embodiment, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) encodes, in 5' to 3' direction, a promoter, a sequence encoding a signal peptide, at least one nucleic acid for the expression of the first MOI (e.g. therapeutic protein) and optionally a further nucleic acid sequence encoding one or more exporter(s) of the first MOI (e.g. therapeutic protein) from the cell (e.g. bacterium). In a particular embodiment, the nucleic acid encodes an exporter of the first MOI.

[0530] The therapeutic molecule may be an antibody therapy. In particular, the therapeutic molecule is an antibody fragment. The therapeutic molecule may comprise an antibody (or in particular an antibody fragment) comprising the binding domains of any of the specific antibody molecules described herein. The therapeutic molecule may comprise an antibody fragment which binds to any of the targets of any of the antibody molecules described herein. Fc fusion proteins are also contemplated.

[0531] The antibody therapy may be an immune checkpoint inhibitor antibody or fragment thereof. The antibody or fragment thereof may be selected from an anti-PD-Ll, anti-PD-1, anti-CTLA4, anti-TIM3, anti-TNFa superfamily member (such as an anti-TNFa, TNFR1 or BAFF), anti-IL6R, anti-IL-4Ra, or anti-PCSK9. Examples of antibodies, antibody fragments, and / or Fc fusion proteins include, without limitation, Abagovomab, Abciximab, Actoxumab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, Anifrolumab, Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab (or tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bivatuzumab mertansine, Blinatumomab, Blosozumab, Brentuximab vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, Cedelizumab, Certolizumab, pegol, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Conatumumab, Concizumab, Crenezumab, Dacetuzumab, Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elotuzumab, Elsilimomab, Enavatuzumab, Enlimomab pegol, Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab, Ibalizu mab, Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lambrolizumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab, Lirilumab, Lodelcizumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Margetuximab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Panobacumab, Parsatuzumab, Pascolizumab, Pateclizumab, Patritumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, TGN, Ticilimumab (or tremelimumab), Tildrakizumab, Tigatuzumab, TNX-, Tocilizumab (or atlizumab), Toralizumab, Tositumomab, Tovetumab, Tralokinumab, Trastuzumab, TRBS, Tregalizumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vantictumab, Vapaliximab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab, Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab and Zolimomab aritox.

[0532] Therapeutic hormones include glucagon-like peptide-1 (GLP-1) or GLP-t and insulin. Other examples of peptide hormones include, without limitation, Amylin (or Islet Amyloid Polypeptide), Antimullerian hormone (or Mullerian inhibiting factor or hormone), Adiponectin, Adrenocorticotropic hormone (or corticotropin), Angiotensi nogen and angiotensin, Antidiuretic hormone (or vasopressin, arginine vasopressin), Atrial-natriuretic peptide (or atriopeptin), Brain natriuretic peptide, Calcitonin, Cholecystokinin, Corticotropin-releasing hormone, Enkephalin, Endothelin, Erythropoietin, Follicle-stimulating hormone, Galanin, Gastrin, Ghrelin, Glucagon, Gonadotropin-releasing hormone, Growth hormone-releasing hormone, Human chorionic gonadotropin, Human placental lactogen, Growth hormone, Inhibin, Insulin, Insulin-like growth factor (or somatomedin), Leptin, Lipotropin, Luteinizing hormone, Melanocyte stimulating hormone, Motilin, Orexin, Oxytocin, Pancreatic polypeptide, Parathyroid hormone, Prolactin, Prolactin releasing hormone, Relaxin, Renin, Secretin, Somatosta tin, Thrombopoietin, Thyroid-stimulating hormone (or thyrotropin), and Thyrotropin-releasing hormone.

[0533] Examples of interleukins include, without limitation, interleukin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17. The interleukin may be interleukin-2, interleukin-4, interleukin-6, interleukin-7, interleukin-10, interleukin-11 or nterleukin-13. The interleukin may in particular be interleukin 1-17.

[0534] Examples of cytokines include, without limitation pegfilgratim, anakinra, emfilermin, denileukin diftitox, interferon-a, erythropoietin, granulocyte-macrophage colony stimulating factor, interleukin-1 receptor antagonist, granulocyte colony stimulating factor, Cintredekin besudotox, Romiplostim, Regramostim, Albinterferon Alfa-2B, Maxy-G34, Thrombopoietin, stem cell factors, erythropoiesis stimulating agents, Avotermin, Nagrestipen, Binetrakin, Dulanermin, Muplestim, Molgramostim, Balugrastim, Lipegfilgrastim, CD40-ligand, Leridistim, Bempegaldesleukin, Pegilodecakin, ALT-801, Teceleukin, Lorukafusp-a, edodekin-a, viral macrophage inflammatory protein-II (vMIP), interferon-K, Efineptakin-a and Tengonermin. Examples of interferons (IFNs) include, without limitation, IFN-a, IFN-β, IFN-ω and IFN-y. Examples of chemokines include, without limitation, CC chemokines (e.g. CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 and CCR10), CXC chemokines (e.g. CXCR1, XCR2, CXCR3, CXCR4, CXCR5, CXC6 and CXCR7), C chemokines or CX3C chemokines (e.g. CX3CR1).

[0535] Examples of growth factors include, without limitation, Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell line-derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nerve growth factor (NGF) and other neurotrophins, Platelet-derived growth factor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha (TGF-a), Transforming growth factor beta (TGF-p), Tumor necrosis factor-alpha (TNF-a), Vascular endothelial growth factor (VEGF), placental growth factor (PIGF), Foetal Bovine Somatotrophin (FBS) and IL-1-IL7.

[0536] Examples of enzymes include, without limitation, any of the enzymes assigned an Enzyme Commission Number (EC) number (e.g. EC1-EC6) by the International Union of Biochemistry and Molecular Biology (IUBMB) (Webb, Edwin C. Enzyme nomenclature 1992: recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes. San Diego: Published for the International Union of Biochemistry and Molecular Biology by Academic Press. ISBN 0-12-227164-5 (1992), incorporated herein by reference). Other examples include: styrene monooxygenase (StyAB), toluene dioxygenase (TODC1C2AB), luciferase and lactase. In some embodiments, the enzyme is toluene dioxygenase. In some embodiments, the enzyme is styrene monoxygenase.

[0537] In any embodiment, the first MOI is a therapeutic molecule described in Table 1 below. Other therapeutic molecules include, without limitation, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, and thrombolytics. Other examples include those that bind non-covalently to target (e.g. monoclonal antibodies or fragments thereof), those that affect covalent bonds (e.g. enzymes), and those that exert activity without specific interactions (e.g. serum albumin).

[0538] Therapeutic molecules include recombinant therapeutic proteins, used to treat, for example, cancers, immune disorders, infections and / or other diseases.

[0539] In one embodiment, the therapeutic protein is selected from Etanercept, Bevacizumab, Rituximab, Adalimumab, Infliximab, Trastuzumab, Insulin glargine, Epoetin alfa, Pegfilgrastim, Ranibizumab, Darbepoetin alfa, Interferon beta-la, Insulin aspart, Rhu insulin, Octocog alfa, Insulin lispro, Cetuximab, Peginterferon alfa-2a, Interferon beta-lb, Eptacog alfa, Insulin aspart, OnabotulinumtoxinA, Epoetin beta, Rec antihemophilic factor, Filgrastin, Insulin detemir, Natalizumab, Insulin (humulin) and Palivizumab. Examples of Fc fusion proteins include, without limitation, Etanercept, Alefacept, Abatacept, Rilonacept, Romiplostim, Belatacept and Aflibercept.

[0540] Examples of anticoagulants and / or blood factors include, without limitation, Protein C, Protein S, and antithrombin, Factors I-VIII, prothrombinase, prothrombin, thrombin von Willebrand Factor (vWF), fibrinogen, fibrin and fibrinopeptides.

[0541] Examples of bone morphogenetic proteins (BMPs) include, without limitation, BMP1-BMP7, BMP8a, BMP8b, BMP10, and BMP15.

[0542] Other examples of therapeutic proteins include, without limitation, Insulin (blood glucose regulator), Pramlintide acetate (glucose control), Growth hormone GH (growth failure), Pegvisoman (growth hormone receptor antagonist), Mecasermin (IGF1, growth failure), Factor VIII (coagulation factor), Factor IX (coagulation factor, Protein C concentrate (anti-coagulation), al-proteinase inhibitor (anti-trypsin inhibitor), Erythropoietin (stimulates erythropoiesis), Filgrastim (granulocyte colonystimulating factor, G-CSF; stimulates neutrophil proliferation), Sargramostim 36, 37 (granulocytemacrophage colony-stimulating factor, GM-CSF), Oprelvekin (interleukin-11, IL-11), Human follicle-stimulating hormone (FSH), Human chorionic gonadotropin (HCG), Lutropin-a (human luteinizing hormone), Interleukin 2 (IL-2), Interleukin-1 Receptor Agonist, Denileukin diftitox (fusion of IL-2 and Diphtheria toxin), Interferon alfacon 1 (consensus interferon), Interferon-a2a (IFN-a2a), Interferon-a2b (IFN-a2b), Interferon-an3 (IFN-an3), Interferon-pia (rIFN-p), Interferon-plb (rIFN-p), Interferon-ylb (IFNy), Salmon calcitonin (32-amino acid linear polypeptide hormone), Teriparatide (part of human parathyroid hormone 1-34 residues), Exenatide (Incretin mimetic with actions similar to glucagon-like peptide 1), Octreotide (octapeptide that mimics natural somatostatin), Dibotermin-a (recombinant human bone morphogenic protein 2), Recombinant human bone morphogenic protein 7, Histrelin acetate (gonadotropin-releasing hormone; GnRH), Palifermin (Keratinocyte growth factor, KGF), Becaplermin (platelet-derived growth factor, PDGF), Nesiritide (recombinant human B-type natriuretic peptide), Lepirudin (recombinant variant of hirudin, another variant is Bivalirudin), Anakinra (interleukin 1 (IL-1) receptor antagonist), Enfuviritide (an HIV-1 gp41-derived peptide), £-Glucocerebrosidase (hydrolyzes to glucose and ceramide), Alglucosidase-a (degrades glycogen), Laronidase (digests glycosaminoglycans within lysosomes), Idursulfase (cleaves O-sulfate preventing GAGs accumulation), Galsulfase (cleave terminal sulphage from GAGs), Agalsidase-0 (human a-galactosidase A, hydrolyzes glycosphingolipids), Lactase (digest lactose), Pancreatic enzymes (lipase, amylase, protease; digest food), Adenosine deaminase (metabolizes adenosine), Tissue plasminogen activator (tPA, serine protease involved in the breakdown of blood clots), Factor Vila (serine protease, causes blood to clot), Drotrecogin-a (serine protease, human activated protein C), Trypsin (serine protease, hydrolyzes proteins), Botulinum toxin type A (protease, inactivates SNAP-25 which is involved in synaptic vesicle fusion), Botulinum toxin type B (protease that inactivates SNAP-25 which is involved in synaptic vesicle fusion), Collagenase (endopeptidase, digest native collagen), Human Table 1: MOIs produced bv genetically modified cells

[0543]

[0544]

[0545] Table produced from Omer etal., supra and Zhou eta!., Engineering Microbiol., 2(3), 100034, 2022, doi:https: / / doi.org / 10.1016 / j.engmic.2022 100034

[0546] Zeng etal., Appl. Microbiol. Biotechnol., 101, 7177-7186, 2017, doi:10.1007 / s00253-017-8410-6

[0547] Carvalho etai., Microb. Cel. Fact., 16, 27-0624, 2017, doi:10.1186 / sl2934-017-0624-x

[0548] An etal J. Microbiol., 59, 202-21, 2021, doi:10.1007 / sl2275-021-0562-5

[0549] Ciacma etal., Microb. Cel. Fact., 17, 018-1028, 2018, doi:10.1186 / sl2934-018-1028-2

[0550] Yue etal., Microb. Cel. Fact., 19, 213, 2020, doi:10.1186 / sl2934-020-01466-2

[0551] Gusmao-Silva etal., Front. Immunol., 11, 562905, 2020, doi: 10.3389 / fimmu.2020.562905

[0552] Zhang etal., PLoS One, 12, e0188960, 2017, doi:10.1371 / journal. pone.0188960

[0553] Peng etal., Sci. Rep., 8, 6435, 2018, doi:10.1038 / s41598-018-24879-x

[0554] Mao etal., Sci. Transl. Med., 10, eaao2586, 2018, doi:10.1126 / scitranslmed.aao2586 Kikuchi eta!., Biochem. Biophysical Res. Commun., 493(1), 306-312, 2017, doi:10.1016 / j.bbrc.2017.09.026 Guo et al., J. Pharm. Biomed. Sci., 7(6), 2017, doi:10.20936 / JPBMS / 170602

[0555] Liu eta / ., Int. Immunopharmacology, 57, 25-32, 2018, doi:10.1016 / j.intimp.2018.02.004

[0556] Geldart eta / ., Bioeng. Translational Med., 3, 197-208, 2018, doi:10.1002 / btm2.10107 Praveschotinunt eta / ., Nat. Commun., 10, 5580, 2019, doi:10.1038 / s41467-019-13336-6

[0557] Yoon eta / ., Eur. J. Cancer, 70, 48-61, 2017, doi: 10.1016 / j.ejca.2016.10.010

[0558] Puurunen eta!., Nat. Metab., 3, 1125-1132, 2021, https: / / doi.org / 10.1038 / s42255-021-00430-7 Chowdhury eta / ., Nat. Med., 25, 1057-1063, 2019, https: / / doi.org / 10.1038 / s41591-019-0498-z

[0559] Zeng eta / ., Appl. Microbiol. Biotechnol., 101, 7177-7186, 2017, https: / / doi.org / 10.1007 / s00253-017-8410-6 Chiabai eta / ., BMC Biotechnol., 19, 38, 2019, https: / / doi.org / 10.1186 / sl2896-019-0518-6 deoxyribonuclease I (endonuclease, DNase I, cleaves DNA), Hyaluronidase (hydrolyzes hyaluronan), Papain (cysteine protease, hydrolyzes proteins), L-Asparaginase (catalyzes the conversion of L-asparagine to aspartic acid and ammonia), Rasburicase (urate oxidase, catalyzes the conversion of uric acid to allantoin), Streptokinase (Anistreplase is anisoylated plasminogen streptokinase activator complex (APSAC)), and Antithrombin III (serine protease inhibitor).

[0560] Examples of small molecule therapeutics include beneficial metabolites. Therapeutics include GLP1, GLP2, IL10, IL22, TNF-a, a STING agonist such as ci-di-AMP and human epidermal growth factor. Beneficial metabolites include, without limitation, L-DOPA, butyrate, propionate, acetate, GLP1, GLP2, IL10, IL22, indole, tryptophan, indole-3-acetic acid (IAA), arginine, tryptophan, kynurenine and kynurenic acid.

[0561] Production of TNF-a (first MOI) is described in WO2022 / 072636A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety.

[0562] Production of the STING agonist, ci-di-AMP (first MOI), using the dacA gene from Listeria monocytogenes is disclosed in WO2022 / 150779A1 and W02020 / 097424A1 (both Synlogic Operating Company, Inc.), each of which is incorporated herein by reference in its entirety. This MOI is useful in treating cancers.

[0563] Production of human epidermal growth factor (hEGF, first MOI), which uses the human EGF cDNA sequence (NCBI accession number:gq214314, codon-optimised for expression in E. coli), with N-terminal sec secretion signal sequences of either PhoA (21 amino acids), PcIB (22 amuio acids), or OmpA (21 amino acids) is disclosed in WO2022 / 221273A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety. Other expression constructs which could be used in the present disclosure for expression of a first MOI are disclosed in Example 1 therein.

[0564] In one embodiment, the one or more exogenous nucleic acids does not comprise any reporter genes. In one embodiment, the one or more exogenous nucleic acids does not comprise any antibiotic resistance genes.

[0565] First MOIs which are toxic to cells

[0566] A particular application of the current technology is as a kill-switch for cells which contain a conjugative plasmid which deliver MOIs to neighbouring cells (see for example, Neil et al, Communications Biology, 3, 2020, 523, doi: https: / / doi.org / 10.1038 / s42003-020-01253-0). One such example is wherein the donor cell comprises a conjugative plasmid which contains an MOI which is capable of removing plasmids containing anti-microbial resistance (AMR) genes of a second target bacterium (see for example Neil etal, Mol. Syst. Biol., 17(10), 2021, el0335, doi: 10.15252 / msb.202110335) or removing AMR genes located on the chromosome of a second target bacterium, or killing or reducing the growth of a second target bacterium. The second target bacterium may be a pathogenic bacterium, or a bacterium which is pathogenic in certain subjects. In such cases, it may be desired, or even required by the regulatory authorities to have the donor bacterium comprise a kill-switch such that the donor bacterium may be destroyed, along with any conjugative plasmid it contains, or such that the conjugative plasmid within the donor bacterium is destroyed, rendering the donor bacterium no longer a donor, but a bacterium which is no longer able to conjugate MOIs to neighbouring cells. Conjugative plasmids comprising such MOIs are described for example in WO2019 / 18551A1 (SNIPR Biome ApS), see Example 4 in particular, and in WO2020 / 229372 (Folium Food Science Limited), infra, see Examples 1 to 7, in particular.

[0567] Thus, in a specific example, the first MOI may be a nuclease or nickase. The nuclease or nickase may be as described for the peptide products of part a), in particular a CRISPR / Cas nuclease or nickase elsewhere herein (including any Cascade proteins and gRNAs and / or crRNA). In this example, the protospacer is comprised by an antimicrobial resistance gene of a second bacterium (e.g. a pathogenic bacterium) or is comprised by the genome (e.g. chromosome) of a second bacterium (e.g. a pathogenic bacterium).

[0568] In another example, the first MOI may be selected from a meganuclease, a zinc finger nuclease or a TALEN. The meganuclease, zinc finger nuclease or TALEN may be as described for the peptide products of part a) elsewhere herein. In this example, the target of the meganuclease, zinc finger nuclease or TALEN would be the genome (e.g. chromosome or conjugative plasmid) of a second bacterium (e.g. a pathogenic bacterium).

[0569] In another example, the first MOI may be a base editor. The base editor (in particular a CRISPR / Cas based base editor) may be as described for the peptide products of part a) elsewhere herein. In this example, the target of the base editor would be to downregulate or disrupt expression of an essential gene comprised by a second bacterium (e.g. a pathogenic bacterium).

[0570] In another example, the first MOI may be a peptide toxin. The peptide toxin may be as described for the peptide products of part a) elsewhere herein. In this example, the peptide toxin is toxic to a second bacterium (e.g. a pathogenic bacterium).

[0571] It will be appreciated by the skilled person that, when the first MOI is the same as a peptide product of a) as described elsewhere herein, no nucleic acid encoding the MOI would be required for the MOI (i.e. the nucleotide sequence encoding the first MOI does not need to be able to incorporate any NNAA when the first MOI is the same as a peptide product of a)). This is because it is the nucleic acid encoding the peptide product of part a) (which is a peptide product encoded by a different nucleic acid) which would be engineered for expression only in the presence of the NNAA.

[0572] In all of these examples, the first MOI may target an antimicrobial resistance gene of a second bacterium (e.g. a pathogenic bacterium). Additionally or alternatively, the first MOI targets and kills or reduces the growth of a second bacterium (e.g. a pathogenic bacterium). Pathogenic bacterium are well known to those skilled in the art, and are described for example in the pathogen priority list by WHO see (https: / / iris.who.int / server / api / core / bitstreams / la41ef7e-dd24-4ce6-a9a6-1573562e7f37 / content) and CDC (see https: / / www.cdc.gov / antimicrobial-resistance / data-research / threats / update-2022.html), which are incorporated herein by reference for its description of pathogenic bacterial species and strains. Other undesirable bacteria (second target bacterium) which may not be on the prioritized pathogen lists but still associated with human disease and which could be targeted include those listed in Table 2 and their associated diseases. Thus in any

[0573] Table 2: Undesirable bacteria which may be second target bacterium

[0574]

[0575]

[0576]

[0577]

[0578] In all of these examples, the target cell of the peptide product of a) is a donor bacterium which comprises a conjugative or mobilizable plasmid which comprises the one or more exogenous nucleic acids for expression of the first MOI, and the donor bacterium or conjugative or mobilizable plasmid comprises the one or more nucleic acid(s) described elsewhere herein (in particular of part a), part b), part c) and / or part d). The conjugative plasmid may be as described elsewhere herein. In particular, the conjugative plasmid is transmissible to the second bacterium.

[0579] Exogenous genes for the conversion of a second MOI to a first MOI

[0580] The one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) for the production of a first MOI may encode one or more protein(s) for the conversion of a second MOI (B) to the first MOI (A).

[0581] The one or more exogenous nucleic acids encoding one or more protein(s) may be comprised by one or more operons, in particular by one operon.

[0582] Where a second MOI is converted to a first MOI, the first MOI may be selected from a therapeutic molecule (e.g. as described elsewhere herein) and a beneficial or therapeutic metabolite.

[0583] For example, the therapeutic molecule may be a prodrug, and the protein encoded by the one or more exogenous nucleic acids is an enzyme which converts the prodrug to the active therapeutic drug. In another example, the therapeutic molecule is a peptide or fusion protein, and the protein encoded by the one or more exogenous nucleic acids cleaves the peptide or fusion protein to release a desired sub-unit of the original peptide or fusion protein.

[0584] Examples of small molecule therapeutics include beneficial metabolites. Beneficial metabolites include, without limitation, L-3,4-dihydroxyphenylalanine (L-DOPA), butyrate, propionate, acetate, GLP1, GLP2, IL10, IL22, indole, tryptophan, indole-3-acetic acid (IAA), arginine, tryptophan, kynurenine and kynurenic acid.

[0585] Alternatively, the second MOI may be a detrimental metabolite. In this embodiment, the first MOI is either neutral or does not cause the detrimental effect of the second MOI to the cell, organism or microbiome. Thus, reducing levels of detrimental metabolites may be achieved by the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) described herein.

[0586] Such detrimental metabolites include leucine, isoleucine, valine, chenodeoxycholic acid (CDCA), oxalate, uric acid, propionate and methionine. In a particular embodiment, the first MOI is a beneficial metabolite or a therapeutic metabolite.

[0587] L-DOPA (a first MOI), for example, can be made from tyrosine (second MOI) using tyrosine 3-hydroxylase (EC 1.14.16.2); or from L-phenylalanine (second MOI) using two enzymes, phenylalanine 4-hydroxylase (EC 1.14.16.1) and tyrosine 3-hydroxylase. Methods for gene insertion into bacteria are provided in W02022 / 013407A1 (Danmarks Tekniske Universitet), which is incorporated herein by reference in its entirety.

[0588] Genes for producing butyrate (first MOI) from crotonyl-CoA (second MOI) are described for example in WO2017 / 136792A2 and WO2017 / 074566A1 (Synlogic Operating Company, Inc.), which are both incorporated herein by reference in their entirety. Genes may include one or more (for example all) of the genes from butyrate production pathway from Peptodostridium difficile 630 (bcd2, etfB3, etfA3, thiAl, hbd, crt2, pbt, and buk); or trans-2-enoynl-CoA reductase (te ) from Treponema denticoia, in combination with thiAi, hbd, crt2, pbt, and buk ovn Peptodostridium difficile 630; or a thioesterase tesB) from E. Coii'vn combination with bcd2, etfB3, etfA3, thiAl, hbd, crt2 from Peptodostridium difficile 630; or terfrom Treponema denticoia, in combination with thiAi, hbd, crt2, pbt, and buk from Peptodostridium difficile 630 and tesZJfrom E. coli. ikc\ of the intermediate substrates of these reactions could be the second MOI, and the number of exogenous genes could be reduced if the recipient bacterium is able to produce the intermediate substrate itself.

[0589] WO2017 / 074566A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety, discloses genes for the production of gut barrier enhancer molecules, such as propionate, acetate, GLP2, IL-22, IL-10, indole and tryptophan (each first MOIs).

[0590] WO2017 / 123418A1, WO2021 / 242897A1 and WO2023 / 225667A2 (all Synlogic Operating Company, Inc.), each of which is incorporated herein by reference in its entirety, disclose genes for the production of IAA (first MOI) from tryptophan (second MOI), and deletions in trpR and tnaA genes to increase the production of IAA, also see other embodiments disclosed herein for further genes and configurations for the production of IAA from various molecules.

[0591] W02016 / 090343A1 and W02020 / 232063A1 (both Synlogic Operating Company, Inc), each of which is incorporated herein by reference in its entirety, disclose methods of converting glutamate (second MOI) to arginine (first MOI) in an eight-step enzymatic process involving N-acetylglutamate synthetase (ArgA), N -acetylglutamate kinase (ArgB), N-acetylglutamate phosphate reductase (ArgC), acetylornithine aminotransferase (ArgD), N-acetylornithinase (ArgE), carbamoylphosphate synthase (encoded by carA and carB), ornithine transcarbamylase (TI / pTand ArgI), argininosuccinate synthase ArgG, and argininosuccinate lyase (ArgH). Ornithine acetyltransferase (ArgJ) is bifunctional and can be used instead of both N-acetylglutamate synthetase (ArgA) and N-acetylornithinase (ArgE). All of the genes encoding these enzymes are subject to repression by arginine via its interaction with ArgR to form a complex that binds to the regulatory region of each gene and inhibits transcription. Thus, the PMS may encode a nucleic acid inhibitor or peptide molecule which reduces or prevents arginine-mediated repression, for example by preventing binding of arginine (first MOI) to ArgR or by preventing ArgR binding to the regulatory region of each gene (known as an "ARG box"), or by arginine binding to N-acetylglutamate synthetase.

[0592] WO2016 / 210384A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety, discloses genes for the production of various metabolic or satiety effector molecules, including bile salt hydrolase, n-acyl-phophatidylethanolamine (NAPE), n-acyl-ethanolamines (NAE), ghrelin receptor antagonist, peptide YY3-36, acholecystokinin (CCK), CCK58, CCK33, CCK22, CCK8, bombesin, gastrin releasing peptides (GRP), neuromedin B (P), glucagon, GLP-1, GLP-2, apolipoprotein A-IV, amylin, somatostatin, enterostatin, oxyntomodulin, pancreatic peptides, short-chain fatty acids, butyrate, propionate, acetate, serotonin receptor agonists, nicotinamide adenine dinucleotides (NAD), nicotinamide mononucleotides (NMN), nucleotide riboside (NR), nicotinamide, nicotinic acid (NA), but in particular examples tryptophan, kynurenine, kynurenic acid, indole and IAA, as well as transporters for importing these molecules into the cell.

[0593] In an embodiment, the second MOI may be a detrimental metabolite.

[0594] WO2016 / 201380A1, WO2021 / 188618A1 and WO2021 / 146394A1 (all Synlogic Operating Company, Inc.), each of which is incorporated herein by reference in its entirety, disclose genes for the reduction of branched chain amino acids, such as leucine, isoleucine, valine (second MOIs), as well as various mutations in exporters to reduce export of the second MOIs, and importers to increase import of the second MOIs into the cell for increased degradation.

[0595] Further, W02020 / 257610A1 and W02020 / 257707A1 (both Synlogic Operating Company, Inc. and Ginko Bioworks, Inc.) each of which is incorporated herein by reference in its entirety, disclose genes for the reduction of leucine (second MOI), converting it into isopentanol (first MOI) and including importers to increase import of the second MOI into the cell for increased degradation.

[0596] WO2022 / 169909A2 (Novome Biotechnologies, Inc.), incorporated herein by reference in its entirety, describes Bacteriodes strains which have been engineered to convert CDCA (second MOI) to ursodeoxycholic acid, UDCA (first MOI). Chenodeoxycholic acid, CDCA is detrimental metabolite which is elevated in diseases such as IBS, whereas UDCA is generally considered tolerable for humans. Two enzymes 7a-HSDH from E. co / / Nissle 1917 and 70-HSDH from Coiinseiia aerofaciens ATCC 25986 are used to convert CDCA into UDCA.

[0597] Oxalate accumulation in urine causes hyperoxaluria. Elevated urinary oxalate has been linked to recurrent calcium oxalate kidney stones, kidney damage, and eventually end-stage renal disease. WO2020 / 123483A1 (Novome Biotechnologies, Inc.), incorporated herein by reference in its entirety, describes various oxalate-metabolising enzymes and proteins (e.g. oxalate decarboxylase (OXDC), EC 4.1.1.2, e.g. from O. formigenes, oxalate decarboxylase; oxalate oxidase (OXO), EC 1.2.3., e.g. from Hordeum vulgare, oxalate oxidoreductase (OOR), EC 1.2.7.10, e.g. from Moorella thermoacetica, ATCC 39073; oxalate-CoA ligase / oxalyl-CoA synthetase (OXS), EC 6.2.1.8, e.g. from A. thaliana or S. cerevisiae, formyl-CoA:oxalate CoAtransferase (FCOCT), EC 2.8.3.16, e.g. from E. coir, acetyl-CoA:oxalate CoA-transferase (ACOCT), EC 2.8.3.19, e.g. from E. coir, succinyl-CoA:oxalate CoA-transferase (SCOCT), EC 2.8.3.2, e.g. from Cupriavidus oxaiaticus, oxalyl-CoA decarboxylase (OXC), EC 4.1.1.8, e.g. from A. tha / iana or ’ cerevisiae, and oxalyl-CoA reductase / glyoxylate: NADP+ oxidoreductase (OXR), EC 1.2.1.17, e.g. from Methyiobacterium extorquens) for the degradation of oxalate (second MOI) into various products such as formate (first MOI), oxalyl-CoA (first MOI), formyl-CoA (first MOI), or glyoxylate (first MOI).

[0598] Importers of oxalate (e.g. oxalate:formate antiporter (OxIT)) and exporters of formate may further be included in the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element).

[0599] WO2017 / 040719A1, WO2021 / 146397A1 and W02022 / 204406A1 (all Synlogic Operating Company, Inc.), and each incorporated herein by reference in their entirety, disclose various genes, such as frc, ScAAE3, YfdE, Oxc x the degradation of oxalate (second MOI), optionally in combination with OxIT importer of oxalate.

[0600] WO2021 / 173808A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety, discloses genes such as uricase, rasburicase, aegA or y / for the degradation of uric acid (second MOI), optionally in combination with an importer of uric acid, such as ygfY, uacT or ygfU.

[0601] WO2017 / 023818A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety, discloses genes for the degradation of propionate (second MOI) and genes which are importers of propionate.

[0602] Dietary methionine (second MOI) is converted to cysteine using several different enzymes, and mutations in one or more of these enzymes leads to homocystinuria. WO2021 / 163421A1 (Synlogic Operating Company, Inc.), incorporated herein by reference in its entirety, discloses genes for the degradation of methionine using methionine gamma lyase (MGL) or methionine decarboxylase (MDC) from various sources, along with increasing expression of the methionine importer, metNIQ, and decreasing or preventing expression of the methionine exporter yjeH.

[0603] In the case of first MOIs which are peptides or proteins, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) may further comprise a nucleic acid encoding a signal peptide as described herein. For all first MOIs, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) may further comprise a nucleic acid encoding one or more exporter(s) of the first MOI (e.g. the therapeutic protein molecule or metabolite). In any embodiment herein, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) may further comprise an exogenous nucleic acid encoding one or more exporter(s) of the first MOI (e.g. the therapeutic protein molecule or metabolite). In a particular embodiment, the nucleic acid encodes an exporter of the first MOI.

[0604] The one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) may further comprise a nucleic acid encoding one or more importer(s) of the second MOI (e.g. a protein molecule or substrate for the production of the first MOI). In any embodiment herein, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) may further comprise an exogenous nucleic acid encoding one or more importer(s) of the second MOI (e.g. a protein molecule or substrate for the production of the first MOI). In a particular embodiment, the nucleic acid encodes an importer of the second MOI.

[0605] Where the first MOI is produced from a second MOI, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) may further comprise a nucleic acid encoding one or more importer(s) of any further substrate which is converted by one of the protein(s) for the conversion of the second MOI to the first MOI (e.g. a substrate used in the pathway for the production of the first MOI). In any embodiment herein, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) may further comprise an exogenous nucleic acid encoding one or more importer(s) of any further substrate which is converted by one of the protein(s) for the conversion of the second MOI to the first MOI (e.g. a substrate used in the pathway for the production of the first MOI). In a particular embodiment, the nucleic acid encodes an importer of the any further substrate.

[0606] In one particular embodiment, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) comprises an exogenous nucleic acid encoding one or more protein(s) for the conversion of a second MOI to the first MOI and an exporter of the first MOI. The exogenous nucleic acid may further encode an importer of a further substrate which is converted by one of the protein(s) for the conversion of the second MOI to the first MOI.

[0607] In one embodiment, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) comprises an exogenous nucleic acid encoding one or more protein(s) for the conversion of a second MOI to the first MOI and an importer of the second MOI. The exogenous nucleic acid may further encode an importer of a further substrate which is converted by one of the protein(s) for the conversion of the second MOI to the first MOI.

[0608] In one embodiment, the one or more exogenous nucleic acids (e.g. comprised by the target cell or a transmissible element) comprises an exogenous nucleic acid encoding one or more protein(s) for the conversion of a second MOI to the first MOI and an exporter of the first MOI and an importer of the second MOI. The exogenous nucleic acid may further encode an importer of a further substrate which is converted by one of the protein(s) for the conversion of the second MOI to the first MOI.

[0609] In one embodiment, the one or more exogenous nucleic acids does not comprise any reporter genes. In one embodiment, the one or more exogenous nucleic acids does not comprise any antibiotic resistance genes.

[0610] Promoters

[0611] The promoters described herein can be used for the expression of any of the one or more nucleic acid(s), any nucleotide sequences, exogenous nucleic acids or any other expressible nucleotide described herein.

[0612] The promoter may be a promoter which is naturally occurring in (i.e. endogenous to) the target cell. Alternatively, the promoter may be one which is naturally occurring in (i.e. endogenous to) any transmissible element (e.g. plasmid, conjugative plasmid, phage or phagemid) comprising any of the one or more nucleic acid(s), nucleotide sequences, or exogenous nucleic acids described herein.

[0613] In other embodiments, the promoter is exogenous (i.e. heterologous) to the target cell or transmissible element, for example a promoter which is engineered into the target cell or transmissible element (e.g. plasmid, conjugative plasmid, phage or phagemid). The promoter may be one which is not naturally operably connected to the sequence in nature.

[0614] In an example, the promoter is a eukaryotic promoter. In another example, the promoter is a virus (e.g. phage, AAV or lentivirus) promoter. The promoter, in one example, is not an E coH or Klebsiella promoter. In another example, the promoter is an animal promoter (optionally a mammalian or human promoter). In another example, the promoter is a plant promoter. In another example, the promoter is a fungus promoter (optionally a yeast promoter). In another example, the promoter is an insect promoter. In another example, the promoter is a synthetic promoter. In another example, the promoter is a viral, AAV or lentiviral promoter.

[0615] Each element of the one or more nucleic acid(s) may be under the control of a different promoter. Where the one or more exogenous nucleic acids (e.g. either for the production of a first MOI or for the conversion of a second MOI to a first MOI) comprises more than one expressed product (for example more than one of an enzyme, an importer and / or an exporter), each element of the exogenously-added nucleic acid(s) encoding said expressed product may be under the control of a different promoter. In other embodiments, all the expressed proteins from the one or more exogenous nucleic acids (e.g. either for the production of the first MOI or for the conversion of a second MOI to a first MOI) are comprised by a single operon under the control of a single promoter. In another example, all of the one or more nucleic acid(s) are comprised by a single operon under the control of a single promoter. The promoters may be any of the promoters described elsewhere herein, and may be exogenous or endogenous to the target cell. In particular the promoter is a constitutive promoter (as described elsewhere herein).

[0616] Where there is more than one operon, each operon is under the control of a different promoter, in particular a constitutive promoter (as described elsewhere herein).

[0617] Constitutive promoters may be particularly useful for the expression of the one or more nucleic acid(s) described herein, providing sustained and high level production of the first, second, third, fourth... nucleotide sequences. Equally, constitutive promoters may be particularly useful for expression of the one or more exogenous nucleic acids (e.g. either for the production of a first MOI or for the conversion of a second MOI to a first MOI).

[0618] Thus, in one embodiment, each nucleic acid of the one or more nucleic acid(s) is under the control of a constitutive promoter (which may be the same or different). Each nucleic acid of the one or more nucleic acid(s) may be under the control of a single constitutive promoter. Each exogenous nucleic acid of the one or more exogenous nucleic acids may be under the control of a constitutive promoter (which may be the same or different). All exogenous nucleic acids of the one or more exogenous nucleic acids may be under the control of a single constitutive promoter. The constitutive promoter(s) may be any of the constitutive promoters described herein.

[0619] In one example, the tRNA / aaRS pair (e.g. expressed from the second and third nucleotide sequences of the one or more nucleic acid(s)) are comprised by a nucleic acid under the control of a single constitutive promoter. Additionally or alternatively, all components of the peptide product required for functionality (e.g. a nuclease, any supporting proteins, such as Cascade proteins, and any crRNA or gRNA - e.g. as expressed from the first, fourth, fifth, sixth... nucleotide sequences of the one or more nucleic acid(s) described herein) may be under the control of a single constitutive promoter. In an alternative, any nucleotide sequence encoding a crRNA or gRNA (e.g. as expressed from the fourth nucleotide sequence of the one or more nucleic acid(s) described herein) is under the control of a different constitutive promoter to the peptide product (and any supporting proteins, such as Cascade proteins and the like - e.g. as expressed from the first, fifth, sixth... nucleotide sequences of the one or more nucleic acid(s) described herein).

[0620] In some examples, it may be desirable to express higher levels of an RNA product (e.g. any tRNA or crRNA or gRNA as described elsewhere herein) than of the component protein product (e.g. aaRS or CRISPR / Cas system respectively). Thus, in one embodiment, an RNA product (e.g. any tRNA or crRNA or gRNA as described elsewhere herein) may be under the control of a stronger promoter than the component protein product (e.g. aaRS or CRISPR / Cas system respectively), and / or may be expressed in multiple copies (e.g. 2, 3, 4 or 5 copies), from the nucleic acid. In one embodiment, the components of the one or more exogenous nucleic acids for the production of the first MOI are each under the control of one or more constitutive promoter(s) (e.g. any of the constitutive promoters described herein). The components of the one or more exogenous nucleic acids for the production of the first MOI may all be under the control of a single constitutive promoter(s) (e.g. any of the constitutive promoters described herein).

[0621] In one example, the promoter is a promoter which is based on the sequence of a tac promoter. Tac promoters are based on a combination of promoters from the trp and lac operons, see de Boer, eta / ., PNAS, 80(1), 21-25, 1983. doi:10.1073 / pnas.80.1.21, which is incorporated herein by reference in its entirety. Several tac-based promoters have been reported in the art, see e.g. Zhang et al., Microb. Cell Fact, 16:84, 2017, doi: 10.1186 / S12934-017-0700-2, which is incorporated herein by reference in its entirety. In one example, the promoter is a tac promoter. In one example, the promoter is a Pc-tga promoter. In one example, the promoter comprises a nucleotide sequence of Seq ID No: 18.

[0622] The promoter may be a BolA promoter, e.g. a pBolA promoter comprising the nucleotide sequence of SEQ ID No: 14. The promoter may be a p70a promoter, e.g. a p70a promoter comprising the nucleotide sequence of SEQ ID No: 15. BolA promoters are known to be active under stress conditions in bacteria, and thus may result in expression of the kill switch components even when cells are not actively dividing (e.g. in the stationary phase or when in a biofilm).

[0623] Constitutive promoters and variants are well known in the art and include, but are not limited to, BBa_J23100, a constitutive E. colics promoter (e.g. an osmY promoter (International Genetically Engineered Machine (iGEM) Registry of Standard Biological Parts Name BBa_J45992; BBa_J45993)), a constitutive E. colic2promoter (e.g. htpG heat shock promoter (BBa_J45504)), a constitutive E. co / id70promoter (e.g. lacq promoter (BBa_J54200; BBa_J56015), E. coliQve. N& QE) phosphate sensing operon promoter (BBa_J64951), GlnRS promoter (BBa_K088007), lacZ promoter (BBa_Kl 19000; BBa_Kl 19001), M13K07 gene I promoter (BBa_M13101), M13K07 gene II promoter (BBa_M13102), M13K07 gene III promoter (BBa_M13103), M13K07 gene IV promoter (BBa_M13104), M13K07 gene V promoter (BBa_M13105), M13K07 gene VI promoter (BBa_M13106), M13K07 gene VIII promoter (BBa_M13108), M13110 (BBa_M13110)), a constitutive Bacillus subtiiis cd promoter (e.g. promoter veg (BBa_K143013), promoter 43 (BBa_K143013), PhaG (BBa_K823000), PiepA(BBa_K823002), Pveg(BBa_K823003)), a constitutive Bacillus subtilised promoter (e.g. promoter etc (BBa_K143010) or promoter gsiB (BBa_K143011)), a Salmonella promoter (e.g. Pspv2 from Salmonella (BBa_Kl 12706), Pspv from Salmonella (BBa_Kl 12707)), a bacteriophage T7 promoter (e.g. T7 promoter (BBa_I712074; BBa_I719005; BBa_J34814; BBa_J64997; BBa_K113010; BBa_K113011; BBa_K113012; BBa_R0085; BBa_R0180; BBa_R0181; BBa_R0182; BBa_R0183; BBa_Z0251; BBa_Z0252; BBa_Z0253)), and a bacteriophage SP6 promoter (e.g. SP6 promoter (BBa_J64998)). In another embodiment, the promoter is a constitutive Pc promoter comprising the nucleotide sequence of SEQ ID No: 19.

[0624] One way for measuring the strength of activity is by measuring the Anderson score of any given promoter. The activity of the reporters is measured by the relative fluorescence of the promoter when used in the control plasmid EX-Ptet-S-rbsRFP-P "RFP reporter" (see http: / / parts.igem. Org / Part: BBa_J61002) in strain TGI grown in LB media to saturation. BBa_J23119 is the "consensus" promoter sequence and the strongest member of the family. The Nhel and Avril restriction sites present within these promoter parts make them a scaffold for further modification. For more information, see http: / / parts.igem. Org / Part: BBa_J23114.

[0625] Thus, in one embodiment, the constitutive promoter is a strong constitutive promoter (for example a promoter having an Anderson Score (AS) of AS >0.4, such as >0.5). In another embodiment, the promoter has an Anderson score of between 0.1 and 0.4 or between 0.1 and 0.5. The promoter may be a promoter of any of SEQ ID Nos:81 to 100.

[0626] Table 3: Anderson Promoter Collection

[0627]

[0628]

[0629] a: also shown in the Anderson Catalog, see http: / / parts.igem.org / Promoters / Catalog / Anderson

[0630] b: Strength is the Anderson Score (AS), e.g. a strength of 1 is a AS of 1. Reported activities of the promoters are given as the relative fluorescence of plasmids in strain TGI grown in LB media to saturation. A suitable plasmid is EX-Ptet-S-rbsRFP-P "RFP reporter" as described at http: / / parts.igem. Org / Part: BBa_J61002; insertion of a promoter element between Xbal and Spel sites results in a RFP reporter.

[0631] Whilst it is generally expected that a constitutive promoter will be used for the expression of the peptide product (e.g. for expression of the first nucleotide sequence, and optionally any fourth, fifth, sixth, seventh and / or eighth nucleotide sequence), an inducible promoter may be useful for other components of the system, in particular for expression of the one or more exogenous nucleic acids. The requirement of a NNAA-inducible kill switch may still be useful in these embodiments, as not all inducible promoters are capable of being completely turned off, and residual expression may still occur. Some inducible systems may be inadvertently induced, for example if the inducer is a molecule or environmental condition which is present more frequently than desired. If the production of the first MOI comes at a fitness cost to the target cell, inducible promoters for the one or more exogenous nucleic acids may also ensure genetic stability of the target cell before the expression of the first MOI is desired.

[0632] Thus, as an alternative to a constitutive promoter, an inducible promoter may be used, for example for the expression of the one or more exogenous nucleic acids (e.g. either for the production of a first MOI or for the conversion of a second MOI to a first MOI). Each exogenous nucleic acid (e.g. either for the production of a first MOI or for the conversion of a second MOI to a first MOI) may be under the control of an inducible promoter (which may be the same or different). All exogenous nucleic acids (e.g. either for the production of a first MOI or for the conversion of a second MOI to a first MOI) may be under the control of a single inducible promoter. The inducible promoter(s) may be any of the inducible promoters described herein.

[0633] The inducible promoter may be active only under certain environmental conditions. For example, the inducible promoter may be active under environmental conditions which are specific to the gut of a subject. In one embodiment, the inducible promoter is active under environmental conditions which are specific to the upper gastrointestinal tract of a subject (e.g. bile acids). In one embodiment, the inducible promoter is active under environmental conditions which are specific to the lower gastrointestinal tract of a subject (e.g. anaerobic conditions). In one embodiment, the inducible promoter is active under the low oxygen or anaerobic conditions which are specific to gut (e.g. the upper gastrointestinal tract and / or the lower gastrointestinal tract) of a subject.

[0634] In another embodiment, the inducible promoter is a temperature sensitive promoter, such as one which is active under physiological temperatures (e.g. approximately 35 to 39 °C, for example approximately 36 to 38 °C, such as approximately 37 °C). For a discussion on the use of this type of promoter (in this example, in a kill switch setting), see https: / / wyss.harvard.edu / news / kill-switches-for-engineered-microbes-gone-rogue / and the "cryodeath" system which is described in more detail in Stirling et al., Mol. Cell, 68, 686-697. e683, 2017, which is incorporated herein in its entirety.

[0635] In an example, the inducible promoter is selected from an aTc (anhydrotetracycline)-inducible Tet promoter, an IPTG or lactose-inducible Lac promoter, a benzoic acid-inducible XylS / Pm promoter, an arabinose-inducible Ara promoter, a rhamnose-inducible Rha promoter, a bile acid-inducible BetA promoter, temperature controlled promoters and porphyrin-inducible promoters, and synthetic derivatives thereof.

[0636] Temperature controlled promoters are described, for example, in Villaverde et al., "Fine regulation of cI857-controlled gene expression in continuous culture of recombinant Escherichia coli by temperature', Appl. Environ. Microbiol., 59(10), 1993, 3485-3487, doi: 10.1128 / aem.59.10.3485-3487.1993, which is incorporated herein by reference in its entirety.

[0637] Porphyrin-inducible promoters are described for example in W02020 / 252370A1 (Novome Biotechnologies, Inc.), which is incorporated herein by reference in its entirety.

[0638] In another embodiment, the promoter is active only in the presence of certain molecules present in the local physiological environment (such as molecules present only in the gut). These inducible promoters may therefore turn on and off production and secretion of the first MOI a when in the desired location (such as the gut). Such inducible promoters are described herein.

[0639] Other promoters of interest are ones which have been designed to be active when the bacterial cell is in a certain state, for example when it is a "stress-phase active". These promoters are useful for the expression of any of the one or more nucleic acid(s), any nucleotide sequences and of the one or more exogenous nucleic acids. Such stress-phase active promoters (SPAs) are described in PCT / EP2024 / 055870, which is incorporated herein in its entirety and for its disclosure relating to SPAs, in particular for any of promoters of SEQ ID Nos:l to 10 disclosed therein (Seq ID Nos: 14, 20 to 23, 25 to 29 in Table 4 hereinbelow) or any promoters in claims 77 to 86 therein.

[0640] Thus, in one embodiment the promoter is a promoter selected from a ReiB, BoiA, Hya, YiaG and a RpoH promoter. The promoter may be a promoter selected from a ReiB promoter sequence, o70; a BoiA promoter sequence, oS, o70; a Hya promoter sequence, oS, o70; a YiaG promoter sequence, oS; a RpoH promoter sequence Pl, o70; a RpoH promoter sequence P2, aS; a RpoH promoter sequence P3, o24; a RpoH promoter sequence P4, o70; a RpoH promoter sequence P5, o70; and a RpoH promoter sequence P6, o54. The promoter may be a promoter having a nucleotide sequence selected from any one of Seq ID Nos: 14, 20 to 23, 25 to 29, or a nucleotides sequence having 90% (or 95%) homology thereto.

[0641] In Example 2, promoters of SEQ ID Nos: 101, 102 and 103 are used. The promoter comprised by the sequence of SEQ ID No: 104 is also used. Thus, the promoter may be a promoter of SEQ ID No: 101, 102, or 103. The promoter may be a promoter comprised by the nucleic acid sequence of SEQ ID No: 104.

[0642] Target sequences and target cell engineering

[0643] The target cell comprises a target sequence which is targeted, and optionally modified, by the peptide product. Examples of the target sequences for different peptide products are described elsewhere herein.

[0644] Target sequences may be endogenous to the target cell, or may be engineered into the target cell. Any component of a target sequence (e.g. cleavage sequences, PAMs, protospacers, spacers and the like, all as described elsewhere herein) may be engineered into the target cell. In some examples, the entire target sequence is introduced into the target cell.

[0645] Particularly, (in relation to CRISPR / Cas-based peptide products), the target sequence may comprise a PAM sequence 5' of the or a protospacer sequence. In an example, the PAM is immediately adjacent to the protospacer sequence.

[0646] There is therefore provided a method of modifying a target cell to comprise a target sequence wherein the target sequence comprises any of the target sequences described elsewhere herein (e.g. one or more of a cleavage sequence, a PAM, a protospacer, a spacers and the like), said method comprising introducing one or more modifications into the cell to produce the target sequence; and further comprising the step of introducing into the cell one or more nucleic acid sequence(s) as described elsewhere herein, or one or more vectors described elsewhere herein, or a transmissible element or plasmid as described elsewhere herein. The method may further comprise the step of introducing into the target cell the one or more exogenous nucleic acids.

[0647] There is also provided a method of modifying a target cell to comprise a target sequence wherein the target sequence comprises, in 5' to 3' orientation:

[0648] a) a nuclease cleavage site nucleotide sequence (as described elsewhere herein); b) optionally a nuclease spacer nucleotide sequence (as described elsewhere herein); c) a PAM sequence (as described elsewhere herein); and

[0649] d) a protospacer sequence (as described elsewhere herein),

[0650] and optionally wherein sequences a) to d) are each immediately adjacent to each other, said method comprising introducing one or more modifications into the target cell to produce the target sequence; and introducing into the cell the one or more exogenous nucleic acids (as described elsewhere herein), and optionally introducing the one or more nucleic acid(s) as described elsewhere herein).

[0651] In one embodiment, one or more of a) to d) are non-native (i.e. exogenous) sequence(s) to the cell. In another embodiment, all of a) to d) are non-native (i.e. exogenous) sequences to cell. In another embodiment, a) and / or b) are non-native (i.e. exogenous) sequences to cell.

[0652] In one embodiment, the nuclease cleavage site nucleotide sequence may be an I-TevI cleavage site nucleotide sequence as described elsewhere herein.

[0653] In one embodiment, the nuclease spacer nucleotide sequence is present. In another embodiment, the nuclease spacer nucleotide sequence is absent.

[0654] In one embodiment, the nuclease spacer nucleotide sequence is an I-TevI spacer nucleotide sequence as described elsewhere herein.

[0655] In a particular example, the one or more nucleic acid(s) are introduced into the chromosome of the target cell using genetic engineering techniques. The genetic engineering technique may include or consist of homologous recombination, site-specific recombination, transposition, or non-homologous recombination, in particular homologous recombination.

[0656] Genetic engineering techniques are well-known to those skilled in the art and include homologous recombination and other recombination technologies.

[0657] In another example, the one or more exogenous nucleic acids are introduced into the chromosome of the target cell using genetic engineering techniques. The genetic engineering technique may include or consist of homologous recombination, site-specific recombination, transposition, or non-homologous recombination, in particular homologous recombination.

[0658] In an example, the one or more nucleic acid(s) and the one or more exogenous nucleic acids are introduced into the chromosome of the target cell using genetic engineering techniques, and the target sequence is produced by introducing one or more modifications into the cell. The genetic engineering technique may include or consist of homologous recombination, site-specific recombination, transposition, or non-homologous recombination, in particular homologous recombination.

[0659] In another example, the one or more nucleic acid(s) may be introduced as part of a transmissible element (e.g. conjugative plasmid, phage or phagemid), a vector or a plasmid as described elsewhere herein.

[0660] The one or more exogenous nucleic acids may be introduced as part of a transmissible element (e.g. conjugative plasmid, phage or phagemid) as described elsewhere herein. The one or more exogenous nucleic acids may be introduced as part of a vector or a plasmid as described elsewhere herein.

[0661] In an example, the one or more nucleic acid(s) and the one or more exogenous nucleic acids are introduced into the target cell as part of a transmissible element (e.g. conjugative plasmid, phage or phagemid), a vector or a plasmid as described elsewhere herein, and the target sequence is produced by introducing one or more modifications into the target cell.

[0662] In some embodiments, the target sequence is comprised by the same nucleotide sequence as the one or more exogenous nucleic acids, such that when the target sequence is targeted by the peptide product of part a) (in the presence of the NNAA), expression of the one or more exogenous nucleic acids is reduced or prevented.

[0663] In particular embodiments, the target sequence is comprised by the chromosome of the cell.

[0664] In an example, the target sequence is in an essential gene. Blocking or preventing transcription of an essential gene in a target cell (e.g. bacteria) can be used to kill the target cell whilst leaving other, non-target cells unaffected.

[0665] In one embodiment, the peptide product may cut the target sequence, and result in death of the cell, stopping the expression of the one or more exogenous nucleic acids. Thus, in one embodiment, the target sequence is comprised by the chromosome of the cell, and the modification cuts the DNA and results in the death of the cell.

[0666] In another embodiment, the peptide product may modify the target sequence which is comprised by a gene essential to growth or maintenance of the cell, or where the target sequence is comprised by the one or more exogenous nucleic acids, and results in a reduction of growth of the cell, reducing the expression of the one or more exogenous nucleic acids.

[0667] In another embodiment, the peptide product modifies the target sequence which is comprised by the one or more exogenous nucleic acids, and results in a reduction of expression of the one or more exogenous nucleic acids.

[0668] Thus, in one embodiment, the target sequence is comprised by the chromosome of the cell, and the peptide product modifies the target sequence and the modification results in the death of the cell. The modification may be double-stranded cutting of the chromosome.

[0669] In another embodiment, the target sequence is comprised by the chromosome of the cell, and the peptide product modifies the target sequence and the modification results in a reduction in growth of the cell. The modification may be single-stranded cutting of the chromosome.

[0670] In another embodiment, the target sequence is comprised by the chromosome of the cell, and the peptide product modifies the target sequence and the modification prevents or reduces expression of the one or more exogenous nucleic acids comprised by the cell. The modification may be single-stranded cutting of the chromosome.

[0671] In other embodiments, the target sequence is comprised by a plasmid within the cell. This alternative embodiment is useful when the cell has been engineered to include a plasmid or vector which comprises the one or more exogenous nucleic acids (e.g. for production of the first MOI). In this embodiment, the peptide product may cut the target sequence in the plasmid or vector, and result in destruction of the plasmid or vector, also reducing or preventing the expression of the one or more exogenous nucleic acids.

[0672] Thus, in one example, the target sequence is comprised by a plasmid within the target cell, and the peptide product modifies the target sequence and the modification prevents or reduces expression of the one or more exogenous nucleic acids comprised by the cell. The modification may be double-stranded cutting of the plasmid which prevents expression of the one or more exogenous nucleic acids by destroying the plasmid. The modification may be singlestranded cutting of the plasmid which reduces expression of the one or more exogenous nucleic acids.

[0673] In an alternative embodiment, the target sequence may be comprised within an antibiotic resistance gene comprised by a plasmid or vector within the cell, and the method further comprises administration of the antibiotic to which the resistance gene conferred protection, thereby resulting in death of the cell which comprised the antibiotic resistant-plasmid. In this embodiment, the one or more exogenous nucleic acids may be comprised by the chromosome of the cell or by a second plasmid within the cell.

[0674] In an example, the target sequence is within the one or more exogenous nucleic acids comprised by the target cell. In another example, the target sequence is immediately adjacent to the one or more exogenous nucleic acids (e.g. is immediately upstream of a promoter of the one or more exogenous nucleic acids). In another example, the target sequence is comprised by a promoter of a nucleic acid of the one or more exogenous nucleic acids.

[0675] In an example, the peptide product modifies the target sequence and the modification of the target sequence is cutting of the nucleic acid. In another example, the modification of the target sequence is double-stranded cutting of the target sequence.

[0676] In some embodiments, the cutting is of single stranded DNA or RNA, wherein the peptide produce comprises a nickase or RNA nuclease. The nickase, RNA nuclease and single stranded DNA or RNA cutting may be as described elsewhere herein.

[0677] In other particular embodiments, the modification is cutting of a double-stranded DNA, and the peptide product comprises a nuclease, such as an I-TevI nuclease. The nuclease and double-stranded DNA cutting may as described elsewhere herein. In some example, the modification results in death of the cell.

[0678] Transmissible elements

[0679] The one or more nucleic acid(s) (and optionally the one or more exogenous nucleic acids), as described elsewhere herein, may be comprised by a transmissible element. For example, the transmissible element may be a plasmid, a virus, a conjugative plasmid, a phage or a phagemid (e.g. a phagemid which is packaged within a phage particle).

[0680] In one embodiment, the one or more nucleic acid(s) (and optionally the one or more exogenous nucleic acids), as described elsewhere herein, are comprised by a vector. In another embodiment, the one or more nucleic acid(s) (and optionally the one or more exogenous nucleic acids), as described elsewhere herein, are comprised by a plurality of vectors. The plurality of vectors may be 2 vectors or 3 vectors. The plurality of vectors may be 4 vectors or 5 vectors. The vector(s) may be DNA comprised by a nanoparticle or gold particle.

[0681] The one or more vector(s) may comprise:

[0682] a) a nuclear localization sequence (NLS);

[0683] b) a phage packaging sequence (optionally a pacor rossite);

[0684] c) a plasmid origin of replication;

[0685] d) a plasmid origin of transfer;

[0686] e) a bacterial plasmid backbone;

[0687] f) a eukaryotic plasmid backbone;

[0688] g) a human virus (optionally AAV or lentivirus) structural protein gene;

[0689] h) a virus (optionally AAV or lentivirus) rep and / or cap sequence;

[0690] i) a selection marker or sequence encoding a selectable marker;

[0691] j) a eukaryotic promoter; or

[0692] k) a nucleotide sequence of a human, animal, plant or fungus gene.

[0693] The vector(s) may be comprised within a transposon which is capable of transfer into the target cell. The transposon may be a conjugative transposon. The transposon may be a Type I transposon. The transposon may be a Type II transposon.

[0694] Any of the vectors or transmissible elements may further comprise one or more exogenous nucleic acids, as described elsewhere herein.

[0695] The one or more nucleic acid(s) (and optionally the one or more exogenous nucleic acids), as described elsewhere herein, may be comprised by a plasmid. The plasmid may be comprised within a phage particle, e.g. a phagemid. The plasmid may be a conjugative plasmid. The plasmid may be comprised by a virus. There is provided a cell comprising a plasmid as described elsewhere herein. When the (target) cell is a eukaryotic, such as mammalian (e.g. human) cell, the transmissible element may be a virus (e.g. a retrovirus, such as a lentivirus, or an adenovirus), a viral vector (e.g. an adenoviral vector), or a nanoparticle (e.g. a lipid nanoparticle, or any form of encapsulated DNA which can be transferred to a cell). Other transmissible elements that can be used in eukaryotic cells include conjugative plasmids (see for example, Walter, Nat. Genet., 29(4), 375-376, 2001, doi:10.1038 / ng779, which is incorporated herein by reference in its entirety), M13-based engineered phage and phagemid (see Ranjbar et al., Iran J. Basic Med. Sci., 21(9), 884-888, 2018, doi: 10.22038 / IJBMS.2018.26191.6432, which is incorporated herein by reference in its entirety) and other engineered phage (see Horst et al., PNAS, 72(9), 3531-3535, 1975, doi:10.1073 / pnas.72.9.3531, which is incorporated herein by reference in its entirety). Particular viruses include Herpes simplex viruses (HSV) and adeno-associated viruses (AAV). For a review of delivery technology, see Howarth et al., Cell Biol. Toxicol., 26(1), 1-20, 2010, doi:10.1007 / s10565-009-9139-5, which is incorporated herein by reference in its entirety.

[0696] In particular embodiments, the transmissible element is a conjugative plasmid. A conjugative plasmid is a plasmid which, when comprised within a bacterial cell ("donor" or "host" cell) is able to be transferred to another bacterium ("recipient" cell) through the mechanism of bacterial conjugation. Bacterial conjugation is the unidirectional and horizontal transmission of genetic information ("horizontal gene transfer") from one bacterium to another. Conjugative plasmids generally fall into two classes: mobilizable plasmids and self-transmissible plasmids.

[0697] There is provided a conjugative plasmid comprising any of the one or more nucleic acid(s) (and optionally any of the one or more exogenous nucleic acids) described elsewhere herein.

[0698] In any embodiment, the conjugative plasmid is capable of being transferred to a recipient bacterial cell. In any embodiment, the conjugative plasmid is transferred to a bacterial cell (i.e. a recipient cell). The recipient cell may be any bacterial cell described elsewhere herein (e.g. a gramnegative bacterial cell). The recipient cell is usually the target cell. There is provided a recipient target cell comprising a conjugative plasmid as described herein.

[0699] Before transmission to a target cell, the conjugative plasmids are comprised within a donor (host) cell. Thus, there is provided a donor cell comprising a conjugative plasmid as described elsewhere herein. The donor (host) cell may be any bacterial cell described elsewhere herein (e.g. a gram-negative bacterial cell).

[0700] The conjugative plasmids described herein and donor (host) cells comprising such conjugative plasmids are useful as medicaments.

[0701] Mobilizable plasmids comprise at least an origin of transfer (oriT), a relaxase and other genetic information on the plasmid which is transferred to the recipient cell. They require helper functions provided by e.g. a second plasmid or the chromosome of the donor cell to effect the plasmid transfer. In one embodiment, the conjugative plasmid is a mobilizable plasmid. In one embodiment, the conjugative plasmid is a mobilizable plasmid comprising an origin of transfer (oriT) and a relaxase.

[0702] A self-transmissible plasmid, in addition to the genetic information on the plasmid which is transferred to the recipient cell, also contain all the molecular machinery needed for self-transfer (e.g. for pilus formation and initiation of gene transfer) on the same plasmid. In one embodiment, the conjugative plasmid is a self-transmissible plasmid. In one embodiment, the conjugative plasmid is a self-transmissible plasmid which comprises all of the molecular machinery necessary for self-transfer. In one embodiment, the conjugative plasmid comprises an oriT and encodes all proteins required to mobilise the plasmid for conjugative transfer between cells.

[0703] Engineered conjugative plasmids are described in more detail in WO2021 / 037732 (SNIPR Biome ApS), which is incorporated herein in its entirety. The features of such conjugative plasmids and bacterial cells comprising them as described in the claims as filed in WO2021 / 037732 are also incorporated herein by reference.

[0704] Thus, in one embodiment, the conjugative plasmid is devoid of a hypC2 nucleotide sequence, or a homologue thereof, for example a hypC2 nucleotide sequence of Seq ID No:39. The conjugative plasmid may comprise an OriT of an IncX plasmid. The conjugative plasmid may be an IncX plasmid. The conjugative plasmid may be a 010 plasmid.

[0705] The conjugative plasmid (or any other plasmid described herein) may be a plasmid based on any plasmid found in a bacterium disclosed herein. For example, the plasmid may be an Enterobacteriaceae plasmid. In one embodiment, the plasmid is an E. coli, Klebsiella, Salmonella, Erwinia, Shigella, Pantoea, Proteus or Citrobacter plasmid. In one embodiment, the plasmid may be from a genera selected from Bifidobacterium, Bacteroides, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus and Lactococcus. In one embodiment, the plasmid may be from a strain belonging to a genera selected from a Bifidobacterium genus or a Bacteroides genus. In one embodiment, the genera is a Bacteroides genus. In one embodiment, the genera is a Bifidobacterium genus. In one embodiment, the plasmid may be from a species which is selected from Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Levilactobacillus brevis, Ligilactobacillus salivarius, Limosilactobacillus reuteri and Lactococcus lactis. In one embodiment, the plasmid may be from a species which is selected from Bifidobacterium Iongum, Bifidobacterium breve, Bifidobacterium adoiescentis, Bacteroides uniformis, Bacteroides vuigatus and Bacteroides thetaiotaomicron. In one embodiment, the plasmid may be from a genus or species disclosed in Table 3. In an example, the conjugative plasmid is capable of replicating in a bacterial cell from any bacterial genus or species described herein. For example, the conjugative plasmid is capable of replicating in an E. coli, Klebsiella, Salmonella, Erwinia, Shigella, Pantoea, Proteus or Citrobacter host (donor) cell. In one embodiment, the conjugative plasmid is capable of replicating in a bacterial cell from a genera selected from Bifidobacterium, Bacteroides, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus and Lactococcus. In one embodiment, the conjugative plasmid is capable of replicating in a bacterial cell from a strain belonging to a genera selected from a Bifidobacterium genus or a Bacteroides genus. In one embodiment, the genera is a Bacteroides genus. In one embodiment, the genera is a Bifidobacterium genus. In one embodiment, the conjugative plasmid is capable of replicating in a bacterial cell from a species which is selected from Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Levilactobacillus brevis, Ligilactobacillus salivarius, Limosilactobacillus reuteri and Lactococcus lactis. In one embodiment, the conjugative plasmid is capable of replicating in a bacterial cell from a species which is selected from Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides thetaiotaomicron. In one embodiment, the conjugative plasmid is capable of replicating in a bacterial cell from a genus or species disclosed in Table 3.

[0706] In one embodiment the conjugative plasmid (or any other plasmid described herein) is capable of being hosted in an Enterobacteriaceae cell. In one embodiment, the plasmid is capable of being hosted in an E. coli, Klebsiella, Salmonella, Erwinia, Shigella, Pantoea, Proteus or Citrobacter ce\\. In one embodiment, the plasmid is capable of being hosted in a bacterial cell from a genera selected from Bifidobacterium, Bacteroides, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus and Lactococcus. In one embodiment, the plasmid is capable of being hosted in a bacterial cell from a strain belonging to a genera selected from a Bifidobacterium genus or a Bacteroides genus. In one embodiment, the genera is a Bacteroides genus. In one embodiment, the genera is a Bifidobacterium genus. In one embodiment, the plasmid is capable of replicating in a bacterial cell from a species which is selected from Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Levilactobacillus brevis, Ligilactobacillus salivarius, Limosilactobacillus reuteri and Lactococcus lactis. In one embodiment, the plasmid is capable of being hosted in a bacterial cell from a species which is selected from Bifidobacterium iongum, Bifidobacterium breve, Bifidobacterium adoiescentis, Bacteroides uniformis, Bacteroides vuigatus and Bacteroides thetaiotaomicron. In one embodiment, the plasmid is capable of being hosted in a bacterial cell from a genus or species disclosed in Table 3.

[0707] In one embodiment, the conjugative plasmid is capable of being conjugatively transferred to an Enterobacteriaceae cell. In one embodiment, the plasmid is capable of being conjugatively transferred to an E. coli, Klebsiella, Salmonella, Erwinia, Shigella, Pantoea, Proteus or Citrobacter cell. In one embodiment, the conjugative plasmid is capable of being conjugatively transferred to a bacterial cell from a genera selected from Bifidobacterium, Bacteroides, Lactobacillus, Lacticaseibacillus, Lactiplantibacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus and Lactococcus. In one embodiment, the conjugative plasmid is capable of being conjugatively transferred to in a bacterial cell from a strain belonging to a genera selected from a Bifidobacterium genus or a Bacteroides genus. In one embodiment, the genera is a Bacteroides genus. In one embodiment, the genera is a Bifidobacterium genus. In one embodiment, the conjugative plasmid is capable of being conjugatively transferred to a bacterial cell from a species which is selected from Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Lactobacillus gasseri, Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Levilactobacillus brevis, Ligilactobacillus salivarius, Limosilactobacillus reuteri and Lactococcus lactis. In one embodiment, the conjugative plasmid is capable of being conjugatively transferred to a bacterial cell from a species which is selected from Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bacteroides uniformis, Bacteroides vulgatus and Bacteroides thetaiotaomicron. In one embodiment, the conjugative plasmid is capable of being conjugatively transferred to a bacterial cell from a genus or species disclosed in Table 3.

[0708] The conjugative plasmid may be based on a bacterial conjugative plasmid selected from one of the following bacterial conjugative plasmid families: IncA, IncB / O (InclO), IncC, IncD, IncE, IncFl, IncF2, IncG, IncHIl, IncHI2, Incll, Incl2, IncJ, 10 IncK, IncL / M, IncN, IncP, IncQl, IncQ2, IncR, IncS, IncT, IncU, IncV, IncW, IncXl, IncX2, IncY, IncZ, ColEl, ColE2, ColE3, pl5A, pSClOl, IncP-2, IncP-5, IncP-7, IncP-8, IncP-9, Incl, Inc4, Inc7, Inc8, Inc9, Incll, Incl3, Incl4 and Incl8.

[0709] In one embodiment, the conjugative plasmid is a conjugative plasmid isolated from E. coli. In another embodiment, the conjugative plasmid is a 010 conjugative plasmid from E. coli (e.g. as shown in Example 3 herein).

[0710] In one embodiment, the donor (host) cell comprising a conjugative plasmid as described herein is an E.coli (such as a symbioflor E. coli, e.g. G6 / 7) host cell (e.g. as shown in Example 3 herein). In another embodiment, the donor (host) cell comprising a conjugative plasmid as described herein is an E. hormaechei host cell.

[0711] In one embodiment, the recipient cell is an E. coli recipient strain comprising a conjugative plasmid as described herein. The plasmid is introduced via conjugation from a donor (host) cell described herein (e.g. as shown in Example 3 herein). The E. coli recipient strain may be comprised by a native microbiome, e.g. in the gut of a subject. In another embodiment, the recipient cell is a klebsiella recipient strain comprising a conjugative plasmid as described herein. The plasmid is introduced via conjugation from a donor (host) cell described herein. The klebsiella recipient strain may be comprised by a native microbiome, e.g. in the gut of a subject.

[0712] In any embodiment described herein, the transmissible element may be capable of being introduced into the recipient bacterium without heat shock. In any embodiment described herein, the transmissible element may be capable of being introduced into a recipient bacterium without electroporation. In any embodiment described herein, the transmissible element may be capable of being introduced into a recipient bacterium without in vitro transformation techniques, including chemical induction (e.g. using calcium).

[0713] In one particular embodiment, there is provided a donor (host) bacterium comprising a conjugative plasmid as described elsewhere herein. In another particular embodiment, there is provided a recipient bacterium comprising a conjugative plasmid as described elsewhere herein.

[0714] As an alternative, the transmissible element is a phage particle. In one embodiment, the transmissible element is a phage or a phagemid (e.g. a phagemid which is packaged in a phage parti...

Claims

1. CLAIMS:

1. One or more nucleic acid(s) comprising:3.a) a first nucleotide sequence comprising a start codon, coding codons and a first stop codon, wherein the coding codons encode a peptide product which kills or inhibits the growth of a target cell, or which modifies a target sequence in a target cell to prevent or reduce expression of one or more exogenous nucleic acids comprised by the target cell, wherein the first nucleotide sequence comprises at least one mutation which,4.i. when the one or more nucleic acid(s) are expressed without the presence of a predefined non-natural amino acid, synthesis of the peptide product is truncated or rendered non-functional; and5.ii. when the one or more nucleic acid(s) are expressed in the presence of the non-natural amino acid produces the peptide product which is functional in the target cell; b) a second nucleotide sequence which expresses a tRNA which is capable of mediating the transfer of the non-natural amino acid into the peptide product of part a)ii. at a position corresponding to a codon which comprises the at least one mutation, and which tRNA is not a substrate for any natural aminoacyl-tRNA synthetase of the target cell; and6.c) a third nucleotide sequence encoding an aminoacyl-tRNA synthetase which is capable of adding the non-natural amino acid to the tRNA of part b).

2. The one or more nucleic acid(s) according to claim 1, wherein the mutation is the replacement of a coding codon with a non-natural quadruplet codon which results in a frameshift mutation, or the mutation is the insertion of a non-natural quadruplet codon between two coding codons which results in a frameshift mutation; and wherein8.in part a)i., when the one or more nucleic acid(s) are expressed without the presence of the predefined non-natural amino acid, synthesis of the peptide product is truncated or rendered non-functional by the frameshift mutation.

3. The one or more nucleic acid(s) according to claim 2, wherein the frameshift mutation results in a second stop codon being introduced into the first nucleotide sequence, such that in part a)i., when the one or more nucleic acid(s) are expressed without the presence of the predefined non- natural amino acid, synthesis of the peptide product is truncated at the amino acid encoded by the coding codon immediately preceding the second stop codon.

4. The one or more nucleic acid(s) according to claim 3, wherein the second stop codon is within the first 10% of coding codons (for example within the first five coding codons) after the quadruplet codon, and the quadruplet codon is within the first 10% of coding codons (for example within the first five coding codons) of the start codon.

5. The one or more nucleic acid(s) according to claim 3 or claim 4, wherein the peptide product comprises a catalytic domain and the second stop codon is introduced before the coding codon which encodes the first amino acid of the catalytic domain.

6. The one or more nucleic acid(s) according to any one of claims 2 to 5, wherein the non-natural quadruplet codon replaces a coding codon which encodes an amino acid residue which is a surface amino acid residue of the peptide product, or wherein the non-natural quadruplet codon is inserted between two coding codons which encode two amino acid residues on the surface of the peptide product.

7. The one or more nucleic acid(s) according to any one of claims 2 to 6, wherein in part a)i., when the one or more nucleic acid(s) are expressed without the presence of the predefined non-natural amino acid, the peptide product is rendered non-functional by the frameshift mutation.

8. The one or more nucleic acid(s) according to any one of claims 2 to 7, wherein14.the non-natural quadruplet codon is AGGA; and15.the non-natural amino acid is homoglutamine; and16.the second nucleic acid of part b) encodes an AK514 homoglutamine tRNA; and the third nucleic acid of part c) encodes a homoglutamine tRNA-synthetase; and optionally, the AGGA replaces a coding codon which encodes an asparagine residue (AAT or AAC) or glutamine residue (CAA or CAG).

9. The one or more nucleic acid(s) according to claim 1, wherein the at least one mutation is the replacement of a coding codon with a second stop codon, or is the insertion of a second stop codon into the first nucleotide sequence after the start codon and before the stop codon, wherein the second stop codon is selected from an amber nonsense codon (TAG), an ochre nonsense codon (TAA) and an opal nonsense codon (TGA), and optionally is different from the first stop codon; and18.wherein in part a)i., when the one or more nucleic acid(s) are expressed without the presence of the predefined non-natural amino acid, synthesis of the peptide product is truncated at the amino acid residue encoded by the coding codon immediately preceding the second stop codon.

10. The one or more nucleic acid(s) according to any one of claims 1 to 9, wherein the expression in part a)i. is in vitro in media which does not contain the predefined non-natural amino acid, and the expression in part a)ii. is in vitro in media which contains the non-natural amino acid.

11. The one or more nucleic acid(s) according to claim 10, wherein the in vitro expression is measured in an assay measuring the number of colony forming units (CFU) of the target cell in the presence and absence of the non-natural amino acid, wherein a statistically significant decrease in the CFU in the presence of the non-natural amino acid indicates that the peptide product is functional for killing or inhibiting the growth of the target cell, for example the in vitro expression is measured in an assay as described in Example 2.1.3.

12. One or more nucleic acid(s) comprising:21.a) a first nucleotide sequence comprising a start codon, coding codons and a first stop codon, wherein the coding codons would encode a peptide product which kills or inhibits the growth of a target cell, or which modifies a target sequence in a target cell to prevent or reduce expression of one or more exogenous nucleic acids comprised by the target cell, except that at least one (e.g. one) of the coding codons which encodes a natural amino acid residue has been replaced with a second stop codon which is optionally different to the first stop codon, or except that a second stop codon has been inserted between two coding codons, or between the start codon and the first coding codon;22.b) a second nucleotide sequence which expresses a tRNA which is capable of mediating the transfer of a predefined non-natural amino acid into the peptide product of part a) at a position corresponding to the second stop codon, and which tRNA is not a substrate for any natural aminoacyl-tRNA synthetase of the target cell; and23.c) a third nucleotide sequence encoding an aminoacyl-tRNA synthetase which is capable of adding the non-natural amino acid to the tRNA of part b).

13. The one or more nucleic acid(s) according to any preceding claim, for use in biocontainment of a target cell whereby a functional peptide product of part a) is expressed in the presence of the predefined non-natural amino acid.

14. The one or more nucleic acid(s) according to any one of claims 9, 10 (when dependent on claim 9) or 11 (when dependent on claim 9), 12 or 13 (when dependent on claim 9 or claim 12), wherein the second stop codon is within the first 10% of coding codons (for example within the first ten or five coding codons) after the start codon, in particular immediately after the start codon.

15. The one or more nucleic acid(s) according to any one of claims 9 to 14, wherein the peptide product comprises a catalytic domain and the second stop codon replaces a coding codon for a residue before the catalytic domain, or the second stop codon is inserted before the coding codon which encodes the first amino acid of the catalytic domain.

16. The one or more nucleic acid(s) according to any one of claims 9 to 15, wherein the second stop codon replaces a coding codon which encodes an amino residue which is a surface amino acid residue of the peptide product, or wherein the second stop codon is inserted between two coding codons which encode two amino acid residues on the surface of the peptide product.

17. The one or more nucleic acid(s) according to any one of claims 9 to 16, wherein:28.the second stop codon is an amber nonsense codon (TAG); and29.the non-natural amino acid is O-methyl tyrosine (OMTyr); and30.the second nucleic acid of part b) encodes a tyrosyl tRNA; and31.the third nucleic acid of part c) encodes a tyrosyl tRNA synthetase.

18. The one or more nucleic acid(s) according to claim 17, wherein the TAG codon replaces a tyrosine coding codon (TAC or TAT).

19. The one or more nucleic acid(s) according to claim 17 or claim 18, wherein the TAG codon replaces a phenylalanine coding codon (TTT or TTC).

20. The one or more nucleic acid(s) according to any one of claims 17 to 19, wherein the tyrosyl tRNA of part b) is a J17 tyrosyl tRNA, for example a Methanococcus jannaschii 17 tyrosyl tRNA, optionally wherein the second nucleotide sequence comprises the nucleotide sequence of residues 97 to 173 of SEQ ID No:71.

21. The one or more nucleic acid(s) according to any one of claims 17 to 20, wherein the tRNA synthetase of part c) is a Methanococcus jannaschii tyrosyl tRNA synthetase (MjTyrRS), optionally wherein the third nucleotide sequence comprises the nucleotide sequence of SEQ ID No:72 or wherein the third nucleotide sequence encodes a tyrosyl tRNA synthetase comprising the amino acid sequence of SEQ ID No:73.

22. The one or more nucleic acid(s) according to any one of claims 9 to 16, wherein:37.the second stop codon is an ochre nonsense codon (TAA); and38.the non-natural amino acid is pyrrolysine; and39.the second nucleic acid of part b) encodes a pyrrolysyl tRNA; and40.the third nucleic acid of part c) encodes a pyrrolysyl tRNA synthetase.

23. The one or more nucleic acid(s) according to claim 22, wherein the TAA codon replaces a surface- exposed lysine coding codon (AAA or AAG), or replaces a surface-exposed arginine codon (AGA or AGG).

24. The one or more nucleic acid(s) according to claim 22 or claim 23, wherein the pyrrolysyl tRNA of part b) is a Methanosarcina barkeri or a Methanosarcina mazei tRNA,42.optionally wherein the second nucleotide sequence comprises the nucleotide sequence of SEQ ID No:36 or SEQ ID No:3025. The one or more nucleic acid(s) according to any one of claims 22 to 24, wherein the tRNA synthetase of part c) is a Methanosarcina barkeri or a Methanosarcina mazei tRNA synthetase, optionally wherein the third nucleotide sequence comprises the nucleotide sequence of SEQ ID No: 37 or SEQ ID No: 31 or wherein the third nucleotide sequence encodes a pyrrosyl tRNA synthetase comprising the amino acid sequence of SEQ ID No:38 or SEQ ID No:32.

26. The one or more nucleic acid(s) according to any one of claims 9 to 16, wherein:45.the second stop codon is an opal nonsense codon (TGA); and46.the non-natural amino acid is 5-hydroxy-L-tryptophan; and47.the second nucleic acid of part b) encodes a 5-hydroxytryptophan tRNA; and48.the third nucleic acid of part c) encodes a 5-hydroxytryptophan tRNA tRNA synthetase.

27. The one or more nucleic acid(s) according to claim 26, wherein the TGA codon replaces a tryptophan coding codon (TGG).

28. The one or more nucleic acid(s) according to claim 26 or claim 27, wherein the 5- hydroxytryptophan tRNA of part b) is a variant of a Bacillus subtilis tryptophan tRNA, optionally wherein the second nucleotide sequence comprises the nucleotide sequence of SEQ ID No:33.

29. The one or more nucleic acid(s) according to any one of claims 26 to 28, wherein the 5- hydroxytryptophan tRNA synthetase of part c) is a variant of a Bacillus subtilis tryptophan tRNA synthetase,52.optionally wherein the third nucleotide sequence comprises the nucleotide sequence of SEQ ID No:34 or wherein the third nucleotide sequence encodes a 5-hydroxytryptophan tRNA synthetase comprising the amino acid sequence of SEQ ID No:35.

30. The one or more nucleic acid(s) according to any preceding claim, wherein the peptide product of part a) is a nuclease or nickase (e.g. a nuclease) which kills or inhibits the growth of the target cell by modifying a target sequence comprised by the genome of the target cell, or which modifies the target sequence in the target cell to prevent or reduce expression of the one or more exogenous nucleic acids comprised by genome of the target cell.

31. The one or more nucleic acid(s) according to claim 30, wherein the nuclease or nickase is a CRISPR / Cas nuclease or nickase (e.g. nuclease).

32. The one or more nucleic acid(s) according to claim 31 wherein the nuclease or nickase is selected from a Type I, Type II, Type III or Type V nuclease or nickase (e.g. nuclease), optionally wherein the system is a Type I or a Type III CRISPR / Cas system in combination with at least one Cascade protein.

33. The one or more nucleic acid(s) according to claim 32, wherein the nuclease is a Type I nuclease selected from a Cas3' and Cas3, or a Cas3 (in particular a Cas3) nuclease, optionally in combination with a further one or more nucleotide sequence(s) encoding one or more of (in particular all of) a Casl, a Cas2, a Cas4, a Cas5, a Cas6 and a Cas7 protein,56.and wherein the one or more nucleic acid(s) further comprises:57.d) a fourth nucleotide sequence which expresses a gRNA or crRNA comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence.

34. The one or more nucleic acid(s) according to any one of claims 1 to 29, wherein the peptide product comprises a nuclease which is a Type I-C Cas3 nuclease in combination with at least one (in particular all of) Cascade protein selected from a Cas5, a Cas7 and a Cas8 protein, and at least one of the Cas3 nuclease and Cascade protein(s) are expressed from the first nucleotide sequence;59.and wherein the one or more nucleic acid(s) further comprises:60.d) a fourth nucleotide sequence which expresses a gRNA or crRNA comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence.

35. The one or more nucleic acid(s) according to claim 34, wherein the Type I-C Cas nuclease and the at least one (in particular all of) the Cascade proteins are selected from Cas5, a Cas7 and a Cas8 proteins are from Streptococcus mutans.

36. The one or more nucleic acid(s) according to claim 35, wherein the first nucleotide sequence encodes the Cas3 nuclease, and would encode an amino acid sequence of SEQ ID No: 106 or a homologue or orthologue thereof, except that at least one (e.g. one) of the coding codons which encodes an amino acid has been replaced with a second stop codon,63.(for example wherein at least one (e.g. one) of the coding codons which encodes a residue selected from F22, F34, F42 and F47 (in particular F22 and F34) has been replaced with TAG, and wherein the first stop codon is not TAG), and wherein the one or more nucleic acid(s) further comprises: a further nucleotide sequence encoding a Cas5 protein comprising the nucleotide sequence of SEQ ID No: 107 (or a nucleotide sequence having at least 90% identity thereto); a further nucleotide sequence encoding a Cas8 protein comprising the nucleotide sequence of SEQ ID No: 108 (or a nucleotide sequence having at least 90% identity thereto); and a further nucleotide sequence encoding a Cas7 protein comprising the nucleotide sequence of SEQ ID No: 109 (or a nucleotide sequence having at least 90% identity thereto).

37. The one or more nucleic acid(s) according to claim 35, wherein the first nucleotide sequence encodes the Cas7 nuclease, and would encode an amino acid sequence of SEQ ID No: 109 or a homologue or orthologue thereof, except that at least one (e.g. one) of the coding codons which encodes an amino acid has been replaced with a second stop codon,65.(for example wherein at least one (e.g. one) of the coding codons which encodes F7 has been replaced with TAG, and wherein the first stop codon is not TAG),66.and wherein the one or more nucleic acid(s) further comprises: a further nucleotide sequence encoding a Cas3 nuclease comprising the nucleotide sequence of SEQ ID No: 106 (or a nucleotide sequence having at least 90% identity thereto); a further nucleotide sequence encoding a Cas5 protein comprising the nucleotide sequence of SEQ ID No: 107 (or a nucleotide sequence having at least 90% identity thereto); and a further nucleotide sequence encoding a Cas8 protein comprising the nucleotide sequence of SEQ ID No: 108 (or a nucleotide sequence having at least 90% identity thereto).

38. The one or more nucleic acid(s) according to any one of claims 1 to 29, wherein the peptide product comprises a nuclease which is a Type I-E Cas3 nuclease in combination with at least one (in particular all of) Cascade protein selected from a CasA, a CasB, Cas5, a Cas6, and a Cas7 protein, and at least one of the Cas3 nuclease and Cascade protein(s) are expressed from the first nucleotide sequence68.and wherein the one or more nucleic acid(s) further comprises:69.d) a fourth nucleotide sequence which expresses a gRNA or crRNA comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence.

39. The one or more nucleic acid(s) according to claim 38, wherein the Type I-E Cas nuclease and the at least one (in particular all of) the Cascade protein(s) selected from CasA, CasB, Cas5, Cas6, and Cas7 proteins are from Escherichia coli.

40. The one or more nucleic acid(s) according to claim 39, wherein the first nucleotide sequence encodes the Cas3 nuclease, and would encode an amino acid sequence of SEQ ID No: 110 or ahomologue or orthologue thereof, except that at least one (e.g. one) of the coding codons which encodes an amino acid has been replaced with a second stop codon,72.(for example wherein at least one (e.g. one) of the coding codons which encodes a residue selected from Y4, F17, F18, F20, Y35, Y49, Y63 and F64 has been replaced with TAG, and wherein the first stop codon is not TAG),73.and wherein the one or more nucleic acid(s) further comprises: a further nucleotide sequence encoding a CasA protein comprising the nucleotide sequence of SEQ ID No: 111 (or a nucleotide sequence having at least 90% identity thereto); a further nucleotide sequence encoding a CasB protein comprising the nucleotide sequence of SEQ ID No: 112 (or a nucleotide sequence having at least 90% identity thereto); a further nucleotide sequence encoding a Cas7 protein comprising the nucleotide sequence of SEQ ID No: 113 (or a nucleotide sequence having at least 90% identity thereto); a further nucleotide sequence encoding a Cas5 protein comprising the nucleotide sequence of SEQ ID No: 114 (or a nucleotide sequence having at least 90% identity thereto); and a further nucleotide sequence encoding a Cas6 protein comprising the nucleotide sequence of SEQ ID No: 115 (or a nucleotide sequence having at least 90% identity thereto).

41. The one or more nucleic acid(s) according to claim 38, wherein the first nucleotide sequence encodes the Cas7 protein, and would encode an amino acid sequence of SEQ ID No: 113, or a homologue or orthologue thereof, except that at least one (e.g. one) of the coding codons which encodes an amino acid has been replaced with a second stop codon,75.(for example wherein at least one (e.g. one) of the coding codons which encodes a residue selected from F4 and Y13 has been replaced with TAG, and wherein the first stop codon is not TAG),76.and wherein the one or more nucleic acid(s) further comprises: a further nucleotide sequence encoding a Cas3 nuclease comprising the nucleotide sequence of SEQ ID No: 110 (or a nucleotide sequence having at least 90% identity thereto); a further nucleotide sequence encoding a CasA protein comprising the nucleotide sequence of SEQ ID No: 111 (or a nucleotide sequence having at least 90% identity thereto); a further nucleotide sequence encoding a CasB protein comprising the nucleotide sequence of SEQ ID No: 112 (or a nucleotide sequence having at least 90% identity thereto); a further nucleotide sequence encoding a Cas5 protein comprising the nucleotide sequence of SEQ ID No: 114 (or a nucleotide sequence having at least 90% identity thereto); and a further nucleotide sequence encoding a Cas6 protein comprising the nucleotide sequence of SEQ ID No: 115 (or a nucleotide sequence having at least 90% identity thereto).

42. The one or more nucleic acid(s) according to claim 32, wherein the nuclease is a Type II Cas9 nuclease,and wherein the one or more nucleic acid(s) further comprises:78.d) a fourth nucleotide sequence which expresses a gRNA or crRNA (e.g. a gRNA), comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence.

43. The one or more nucleic acid(s) according to claim 42, wherein the first nucleotide sequence encoding the Cas9 nuclease would encode an amino acid sequence of SEQ ID No:40, or a homologue or orthologue thereof, except that at least one (e.g. one) of the coding codons which encodes an amino acid has been replaced with a second stop codon,80.for example wherein at least one (e.g. one) of the coding codons which encodes a residue selected from Y5, Y25, F32, Y812, Y814, Y815, Y823, Y836, F846, Y882, F897, F916, Y943, F966, F972, Y973, Y981 and Y988 has been replaced with TAG, and wherein the first stop codon is not TAG.

44. The one or more nucleic acid(s) according to claim 32, wherein the nuclease is a Type III CaslO nuclease, optionally in combination with a further one or more nucleotide sequence(s) encoding one or more of (in particular all of), a Cast, a Cas2, a Cas5, a Cas6, a Cas7 and a Casll protein, and wherein the one or more nucleic acid(s) further comprises:82.d) a fourth nucleotide sequence which expresses a gRNA or crRNA, comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence.

45. The one or more nucleic acid(s) according to claim 32, wherein the nuclease is a Type V Casl2 nuclease (e.g. Cpfl), optionally in combination with a further one or more nucleotide sequence(s) encoding a Cas4,84.and wherein the one or more nucleic acid(s) further comprises85.d) a fourth nucleotide sequence which expresses a gRNA or crRNA, comprising one or more repeat sequences and a first spacer sequence which hybridises to a first protospacer sequence comprised by the target sequence.

46. The one or more nucleic acid(s) according to claim 45, wherein the first nucleotide sequence encoding the Type V Casl2 nuclease is a Cpfl nuclease which would encode an amino acid sequence of SEQ ID No:41, or a homologue or orthologue thereof, except that at least one (e.g. one) of the coding codons which encodes an amino acid has been replaced with a second stop codon,87.for example wherein at least one (e.g. one) of the coding codons which encodes a residue selected from F896, Y925, Y926, F941, Y953, Y984, Y999, F1005, F1010, F1012, F1017, Y1024, Y1037, F1040, F1045, Y1055, F1061, and F1064 has been replaced with TAG, and wherein the first stop codon is not TAG.

47. The one or more nucleic acid(s) according to claim 30, wherein the nuclease is selected from a meganuclease, a zinc finger nuclease or a TALEN.

48. The one or more nucleic acid(s) according to any one of claims 1 to 30, wherein the peptide product comprises a base editor, optionally wherein the base editor further comprises a modified nuclease that is modified to be unable to perform DNA double strand breaks, while retaining its DNA binding capacity and is fused to a domain to perform base editing.

49. The one or more nucleic acid(s) according to claim 48, wherein the base editor kills or inhibits the growth of the target cell by modifying a target sequence comprised by the genome of the target cell, or wherein the base editor modifies the target sequence in the target cell to prevent or reduce expression of the one or more exogenous nucleic acids comprised by genome of the target cell.

50. The one or more nucleic acid(s) according to any preceding claim, wherein each nucleic acid sequence and / or each nucleic acid is under the control of a constitutive promoter, for example where the constitutive promoter is a strong constitutive promoter having an Anderson score >0.4 (e.g. >0.5); or a tac promoter, for example a Pc-tga promoter comprising the nucleotide sequence of SEQ ID No: 18, or a Pc promoter comprising the nucleotide sequence of SEQ ID No: 19; or a promoter of any of SEQ ID Nos:81 to 103; or a promoter comprised by the nucleic acid sequence of SEQ ID No: 104.

51. The one or more nucleic acid(s) according to any one of claims 1 to 49, wherein one, more or all of the nucleic acid(s) is under the control of a promoter selected from a Re! B, BoiA, Hya, YiaG and a RpoH promoter, such as a promoter selected from a Re / B promoter sequence, o70; a Bo / A promoter sequence, oS, o70; a Hya promoter sequence, oS, o70; a YiaG promoter sequence, oS; a RpoH promoter sequence Pl, o70; a RpoH promoter sequence P2, aS; a RpoH promoter sequence P3, o24; a RpoH promoter sequence P4, o70; a RpoH promoter sequence P5, o70; and a RpoH promoter sequence P6, o54, in particular a promoter having a nucleotide sequence selected from any one of Seq ID Nos: 14, 20 to 23, 25 to 29, or a nucleotide sequence having 90% (or 95%) homology thereto.

52. The one or more nucleic acid(s) according to any one of claims 1 to 29, 50 or 51 wherein the peptide product of part a) is a peptide toxin.

53. The one or more nucleic acid(s) according to claim 52, wherein the peptide toxin is selected from bacteriocins, lysins and toxic peptides of a toxin -antitoxin system.

54. The one or more nucleic acid(s) according to claim 52 or claim 53, wherein the first nucleotide sequence encoding the peptide toxin comprises a nucleotide sequence comprising SEQ ID No:76, or wherein the first nucleotide sequence encoding the peptide toxin encodes the amino acid sequence of SEQ ID No: 77, for example when expressed in the presence of OMTyr.

55. The one or more nucleic acid(s) according to any one of claims 1 to 29, 50 or 51 wherein the peptide product of part a) is selected from a cytosine deaminase (e.g. APOBEC, AID or bacterial cytosine deaminase), an adenine deaminase (e.g. TadA,), and Mom (phage p).

56. The one or more nucleic acid(s) according to claim 55, wherein the peptide product of part a) kills or inhibits the growth of the target cell by modifying (e.g. hypermutating or addition of methylcarbomyl or methyl moieties) a nucleotide sequence comprised by the genome (e.g. chromosome) of the target cell57. The one or more nucleic acid(s) according to any one of claims 33 to 51, wherein, in part d), the gRNA or crRNA first spacer sequence is from 25 to 39 nucleotides in length (e.g. 28 to 32 nucleotides in length), or is about 32 nucleotides in length (e.g. is 32 nucleotides in length).

58. The one or more nucleic acid(s) according to claim 57, wherein the spacer sequence comprises the nucleotide sequence of SEQ ID No:69.

59. The one or more nucleic acid(s) according to claim 57 or 58, wherein base pairs 1 to 28 of the first spacer are identical to the complement of nucleotides 1 to 28 of the first protospacer sequence which is immediately 5' of a PAM sequence in the target sequence.

60. The one or more nucleic acid(s) according to any one of claims 33 to 59, wherein, in part d) the first spacer is identical to the complement of the first protospacer in the target sequence.

61. The one or more nucleic acid(s) according to any one of claims 33 to 60, wherein, in part d) the gRNA or crRNA comprises two repeat sequences.

62. A vector, or a plurality of vectors comprising the one or more nucleic acid(s) as defined in any preceding claim or in any one of claims 68 to 82 or 85 to.

63. A transmissible element (e.g. a plasmid, virus, conjugative plasmid, phage or phagemid), comprising the one or more nucleic acid(s) as defined in any one of claims 1 to 61, or 68 to 82.

64. A plasmid (e.g. a conjugative plasmid or plasmid comprised by a phage particle) comprising the one or more nucleic acid(s) as defined in any one of claims 1 to 61, or 68 to 82.

65. A cell comprising the one or more nucleic acid(s) as defined in any one of claims 1 to 61, or 68 to 82, or a cell comprising a plasmid as defined in claim 64.

66. A cell according to claim 65, wherein the cell is a bacterial cell, in particular a bacterial cell of a species or strain as listed in Table 3.

67. A cell according to claim 65, wherein the cell is a prokaryotic cell or a yeast cell.

68. The cell according to any one of claims 65 to 67, wherein the cell further comprises one or more exogenous nucleic acids for the production of a first molecule of interest (MOI) in the cell; or the one or more nucleic acid(s) according to any one of claims 1 to 61, wherein the one or more exogenous nucleic acids comprised by the target cell are for the production of a first molecule of interest (MOI).

69. The cell or one or more nucleic acid(s) according to claim 68, wherein the one or more exogenous nucleic acids encodes the first MOI.

70. The cell or one or more nucleic acid(s) according to claim 68 or claim 69, wherein the first MOI is a therapeutic molecule, such as a peptide molecule (e.g. an antibody fragment, a hormone such as GLP-1, an interleukin, a cytokine, a chemokine, an eukaryotic growth factor, or an enzyme) or a small molecule (e.g. a metabolite such as L-DOPA, indole-3-acetic acid, butyrate etc).

71. The cell or one or more nucleic acid(s) according to any one of claims 68 to 70, wherein the one or more exogenous nucleic acids encodes one or more (e.g. one) exporter(s) of the first MOI from the cell.

72. The cell or one or more nucleic acid(s) according to any one of claims 68 to 71, wherein the one or more exogenous nucleic acids further encodes a sequence encoding a signal peptide for the secretion of the first MOI to the periplasm of the cell, to the cell surface of the cell, or to the extracellular space outside of the cell.

73. The cell or one or more nucleic acid(s) according to any one of claims 68 to 72, wherein the one or more exogenous nucleic acids encodes, in 5' to 3' direction, a promoter, a sequence encoding a signal peptide, at least one nucleic acid for the expression of the first MOI and optionally a further nucleic acid sequence encoding one or more (e.g. one) exporter(s) of the first MOI from the cell.

74. The cell or one or more nucleic acid(s) according to claim 68 or claim 69, wherein the first MOI is selected from:113.A. a nuclease or nickase, optionally as defined for the peptide product (including any Cascade proteins and gRNAs and / or crRNA) in any one of claims 30 to 46;114.B. a meganuclease, a zinc finger nuclease or a TALEN;115.C. a base editor, optionally as defined for the peptide product in claim 48;116.D. a peptide toxin, optionally as defined for the peptide product in any one of claims 52 to 54; and117.E. a cytosine deaminase (e.g. APOBEC, AID or bacterial cytosine deaminase), an adenine deaminase (e.g. TadA), and Mom (phage p).

75. The cell or one or more nucleic acid(s) according to claim 74, wherein the first MOI targets an antimicrobial resistance gene of a second bacterium (e.g. a pathogenic bacterium) and / or targets and kills or reduces the growth of a second bacterium (e.g. a pathogenic bacterium).

76. The cell or one or more nucleic acid(s) according to claim 74 or claim 75, wherein120.the target cell of the peptide product of a) is a donor bacterium which comprises a conjugative plasmid which comprises the one or more exogenous nucleic acids for expression of the MOI, or the target sequence targeted by the peptide product of a) is comprised by a conjugative plasmid comprised by the target cell, which is a donor cell, and the conjugative plasmid comprises the one or more exogenous nucleic acids for expression of the MOI;121.optionally wherein the conjugative plasmid is transmissible to the second bacterium of claim 75.

77. The cell or one or more nucleic acid(s) according to claim 68, wherein the one or more exogenous nucleic acids encodes one or more protein(s) for the conversion of a second MOI to the first MOI.

78. The cell or one or more nucleic acid(s) according to claim 77, wherein the one or more exogenous nucleic acids encoding one or more protein(s) is comprised by one or more operons (e.g. by one operon).

79. The cell or one or more nucleic acid(s) according to claim 78, wherein the one or more operons are under the control of a constitutive promoter, for example a promoter as defined in claim 50.

80. The cell or one or more nucleic acid(s) according to any one of claims 77 to 79, wherein the first MOI is selected from a therapeutic molecule (e.g. a peptide molecule) and a beneficial or therapeutic metabolite, in particular a beneficial or therapeutic metabolite, or alternatively, the second MOI is a detrimental metabolite.

81. The cell or one or more nucleic acid(s) according to any one of claims 77 to 80, wherein the one or more exogenous nucleic acids further encodes126.one or more (e.g. one) exporter(s) of the first MOI, and / or127.one or more (e.g. one) importer(s) of the second MOI, and / or128.one or more (e.g. one) importer(s) of any further substrate which is converted by one of the protein(s) for the conversion of the second MOI to the first MOI.

82. The cell according to any one of claims 69 to 81, which further comprises the or a target sequence which comprises a PAM sequence 5' of the or a protospacer sequence, optionally wherein the PAM is immediately adjacent to the protospacer sequence, or130.the one or more nucleic acid(s) according to any one of claims 77 to 81 wherein the target sequence comprises a PAM sequence 5' of the or a protospacer sequence, optionally wherein the PAM is immediately adjacent to the protospacer sequence.

83. A cell comprising a target sequence which comprises a nuclease cleavage site nucleotide sequence (e.g. a zinc finger or TALEN cleavage site), and further comprising132.the one or more nucleic acid(s) as defined in any one of claims 1 to 61, and133.the one or more exogenous nucleic acids as defined in any one of claims 68 to 82, and wherein the peptide product of part a) is a meganuclease, a zinc finger nuclease or a TALEN.

84. A cell comprising a target sequence which comprises, in 5' to 3' orientation:135.a) a PAM sequence; and136.b) a protospacer sequence,137.and further comprising138.the one or more nucleic acid(s) as defined in any one of claims 1 to 61, and139.the one or more exogenous nucleic acids as defined in any one of claims 68 to 82, and wherein the peptide product of part a) is a CRISPR / Cas nuclease or nickase (optionally as defined in any one of claims 30 to 46), and140.optionally wherein the target sequence comprises a PAM sequence 5' of the protospacer sequence, e.g. wherein the PAM is immediately adjacent to the protospacer sequence.

85. The cell according to claim 82 or claim 84, or the one or more nucleic acids according to any one of claims 33 to 60, wherein the protospacer sequence is comprised by the chromosome of the cell or target cell.

86. The cell or one or more nucleic acids according to claim 85, wherein the genome (e.g.142.chromosome) of the cell or target cell comprises a second protospacer sequence, and143.A. the gRNA or crRNA comprises a second spacer sequence which hybridises to the second protospacer sequence; or144.B. the one or more nucleic acid(s) comprises a further nucleotide sequence encoding a gRNA or crRNA comprising one or more repeat sequences and a second spacer sequence which hybridises to the second protospacer.

87. The cell or one or more nucleic acids according to claim 87, wherein the genome (e.g.146.chromosome) of the cell or target cell comprises a third protospacer sequence, and147.A. the gRNA or crRNA comprises a third spacer sequence which hybridises to the third protospacer sequence; or148.B. the one or more nucleic acid(s) comprises a further nucleotide sequence encoding a gRNA or crRNA comprising one or more repeat sequences and a third spacer sequence which hybridises to the third protospacer.

88. The cell according to any one of claims 82, or 84 to 87, or the one or more nucleic acids according to any one of claims 85 to 87, wherein the cell or target cell comprises more than one (for example 2 or 3) copies of the same protospacer sequence (and optionally the PAM sequence).

89. The one or more nucleic acids according to any one of claims 1 to 61 or 68 to 82 or 86 to 88, or the cell according to any one of claims 83, or 85 to 89, wherein the target sequence and / or protospacer sequence is an essential gene in the target cell.

90. A pharmaceutical composition (optionally an in vitro composition, or wherein the composition is comprised by a medical container) comprising (i) the one or more nucleic acids as defined in any one of claims 1 to 61 or 68 to 82 or 86 to 88, or (ii) the cell as defined in any one of claims 65 to 89, or (iii) the vector or transmissible element as defined in any one of claims 62 to 64, and a pharmaceutically acceptable excipient or carrier.

91. A pharmaceutical composition according to claim 90, which is formulated for oral or rectal administration, preferably oral administration, for example formulated as a capsule or coated tablet.

92. A pharmaceutical composition according to claim 90 or claim 91, which is a lyophilised formulation or is an encapsulated formulation to be released in the lower gut of a subject.

93. A biocontainment method for a target cell, said method comprising154.(I) providing a target cell comprising a target sequence as defined in any one of claims 76 or 82 to 89; and155.(II) exposing the target cell to the predefined non-natural amino acid to produce the peptide product of part a);156.whereby the target cell is killed or the growth of the target cell is inhibited (in particular whereby the target cell is killed).

94. A biocontainment method for a target cell comprising one or more exogenous nucleic acids (e.g.158.as defined in any one of claims 68 to 82), said method comprising159.(I) providing a target cell comprising a target sequence as defined in any one of claims 76 or 82 to 89 and the one or more exogenous nucleic acids as defined in any one of claims 68 to 72; and160.(II) exposing the target cell to the predefined non-natural amino acid to produce the peptide product of part a);161.whereby the expression of said one or more exogenous nucleic acids in the target cell is reduced or prevented.

95. A method of modifying target cells in a microbiome (e.g. gut of a subject), comprising163.(I) administering to said microbiome (i) a cell as defined in any one of claims 76 or 82 to 89, or (ii) a pharmaceutical composition as defined in any one of claims 90 to 92, and164.(II) subsequently administering to the microbiome the predefined non-natural amino acid to produce the peptide product of part a),165.thereby modifying a target sequence in the target cells (in particular whereby the modification of the target cell results in death of the target cell).

96. The method according to any one of claims 93 to 95, wherein the production of the peptide product of part a) modifies the target nucleic acid to modulate (e.g. up- or down-regulates) expression of a gene comprised by the cell, and wherein the growth of the cell is reduced.

97. The method according to claim 96, wherein the method results in death of the cell, optionally wherein the method is carried out on a population of cells, and the method results in death of at least (about) 70% (for example at least (about) 80%, (about) 85%, (about) 90%, (about) 92%,(about) 93%, (about) 94%, (about) 95%, (about) 96%, (about) 97%, (about) 98%, (about) 99%) of the cells in the population which comprise the target sequence.

98. The method according to claim 96, wherein the method is carried out on a population of cells, and the method results in a reduction in growth of at least (about) 70% (for example at least (about) 80%, (about) 85%, (about) 90%, (about) 92%, (about) 93%, (about) 94%, (about) 95%, (about) 96%, (about) 97%, (about) 98%, (about) 99%) of the cells in the population which comprise the target sequence.

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