Vectors and methods

Abstract

The invention relates to vectors and methods. The present invention relates to vectors and methods for derepressing a Cas system in a host cell.

Description

[0001] This application is a divisional application of the invention patent application entitled “Carrier and Method”, which entered China with international application PCT / EP2018 / 066954 with international application date of June 25, 2018 and application number 201880054942.4. Technical Field

[0002] The present invention relates to vectors and methods for derepressing Cas or Cascade in host cells. Background Art

[0003] The IE type CRISPR-Cas system from E. coli encodes six Cas genes in two operons (casABCDE and cas3) required for CRISPR RNA processing and cleavage and degradation of target DNA. In E. coli strains such as K-12, the casABCDE operon is repressed by H-NS.

[0004] Gomaa et al. used a system consisting of two plasmids (pCasA-E and pCas3) that can inducibly express all six E. coli Cas genes. In addition, Gomaa et al. generated a third plasmid that encodes an altered version of the endogenous CRISPR1 array in E. coli K-12 that accommodates the insertion of engineered spacer sequences. The pCRISPR plasmid encoding the engineered, genome-targeted spacer was transformed into E. coli K-12 substrain BW25113 cells, which had been pre-engineered with T7 polymerase (BW25113-T7) and inducible expression of two Cas-expressing plasmids (pCasA-E and pCas3). In an alternative approach, the authors forced expression of chromosomally encoded Cas genes by deleting the hns gene.

[0005] Citorik et al. disclose the use of a conjugated plasmid or phage for delivering a nucleotide sequence encoding exogenous Streptococcus pyogenes Cas9 and a CRISPR locus into Escherichia coli.

[0006] US20160333348 (SNIPR Technologies Limited) discloses the use of active, endogenous Cas nucleases in bacteria, particularly for targeting species in mixed bacterial populations.

[0007] Although prior art, such as Gomaa et al., solves the problem of repressed endogenous Cas by introducing an exogenous CRISPR / Cas system, this requires the construction of multiple vectors to accommodate all sequences encoding components such as the CRISPR array (i.e., Cas3, CasA, B, C, D, and E). Therefore, the large number of exogenous sequences that need to be introduced requires many different vectors to enter the target bacterial cell, which reduces the likelihood of success and reduces the efficiency of the process. For example, targeting of natural human, animal, plant, or environmental microbial groups does not allow the target host cell to be pre-manipulated to equip it with exogenous sequences encoding one or more components of the exogenous CRISPR / Cas system. In addition, it is preferred to reduce the number of nucleic acid vectors that need to enter the target cell to achieve CRISPR / Cas activity (e.g., killing) in the host cell - preferably to one.

[0008] The most commonly used Cas9 (measured at 4.2 kilobases (kb)) comes from Streptococcus pyogenes. The length of the molecule imposes limits on how much genetic material a vector (such as a bacteriophage) can hold, creating an obstacle to using CRISPR in a variety of settings (see Ran et al.). The Streptococcus thermophilus Cas9 (UniProtKB-G3ECR1 (CAS9_STRTR)) nucleotide sequence has a size of 1.4 kb.

[0009] Solutions such as those using active, endogenous Cas nucleases (e.g., as described in US20160333348) are generally useful in the case where this is the naturally active Cas in the host. These solutions avoid the need to use bulky exogenous Cas sequences, but are expected to enable the use of naturally repressed endogenous Cas in the host, thereby addressing other naturally occurring bacterial species. It is also expected to be able to do this in wild-type cells (e.g., bacterial or archaeal cells), for example, for addressing microbiomes naturally found in humans, animals, or the environment.

[0010] References: Gomaa et al.; MBio.2014Jan 28;5(1):e00928-13.doi:10.1128 / mBio.00928-13, "Programmable removal ofbacterial strains by use ofgenome-targeting CRISPR-Cassystems"; Citorik et al., Nat Biotechnol. 2014Nov;32(11):1141-5.doi:10.1038 / nbt.3011.Epub 2014Sep 21, "Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Summary of the invention

[0011] To this end, the present invention provides:- In a first configuration, a nucleic acid vector for introduction into a host cell, wherein the cell comprises a CRISPR / Cas system that is repressed by a repressor in the cell, comprises (a) a nucleotide sequence encoding a derepressor capable of derepressing a CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) optionally, a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences each encoding a respective guide RNA (gRNA, e.g., a single guide RNA) in the cell; wherein each crRNA or gRNA is capable of directing Cas to modify a respective protospacer sequence of the host cell genome or to modify a protospacer sequence of an episome contained by the host in the presence of a derepressor.

[0012] In a second configuration, a nucleic acid vector for introduction into a bacterial or archaeal host cell, wherein the cell comprises an endogenous CRISPR / Cas system that is naturally repressed by a repressor in the cell, comprises (a) a nucleotide sequence encoding a derepressor capable of derepressing a CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) optionally, a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences each encoding a respective guide RNA (gRNA, e.g., a single guide RNA) in the cell; wherein each crRNA or gRNA is capable of directing Cas to modify a protospacer sequence of the host cell genome or to modify a protospacer sequence of an episome contained by the host in the presence of a derepressor; wherein the repressor is H-NS, StpA, LRP or CRP encoded by the cell genome or a homologue or orthologue or functional equivalent thereof; and The derepressor is LeuO or its homologue, orthologue or functional equivalent capable of forming a complex with H-NS; or a mutant of H-NS, StpA, LRP or CRP capable of forming a complex with H-NS, StpA, LRP or CRP repressor, respectively.

[0013] In a third configuration, a medicament, comprising a plurality of vectors according to any of the preceding configurations, optionally further comprising one or more medical drugs (e.g., anticancer drugs) or antibiotics (e.g., wherein the protospacer sequence is comprised by a host cell antibiotic resistance gene), is used to treat or prevent a disease or condition in a human or animal.

[0014] In a fourth configuration, a method for treating or preventing a disease or condition in a human or animal subject, the method comprising administering a vector or drug of any of the aforementioned configurations to the subject, wherein the subject's microbiome comprises a host cell modified by the cell's endogenous derepressed Cas, and carrying out the treatment or prevention.

[0015] In a fifth configuration, a method of killing a wild-type bacterial or archaeal cell (e.g., an E. coli or Salmonella cell), wherein the cell comprises an endogenous CRISPR / Cas system comprising nucleotide sequences encoding Cas3 and Cascade proteins, wherein Cas3 and / or Cascade are naturally repressed in the cell, the method comprising (a) derepressing the Cas3 and / or Cascade, and (b) introducing into the cell (i) a CRISPR array for producing one or more crRNAs in the cell; or (ii) one or more nucleotide sequences each encoding a respective guide RNA (gRNA, e.g., a single guide RNA); Each crRNA or gRNA guides Cas or Cascade to modify the respective original spacer sequence of the host cell genome or to modify the original spacer sequence of the episome contained in the host.

[0016] In a sixth configuration, a nucleic acid vector for introduction into a host cell, wherein the cell comprises a CRISPR / Cas system that is repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing a CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) for introducing the following sites: (i) a CRISPR array or CRISPR spacer sequence for producing one or more crRNAs in a cell; or (ii) a nucleotide sequence encoding a guide RNA (gRNA, e.g., a single guide RNA) in a cell; Wherein the crRNA or gRNA is capable of guiding Cas to modify the respective original spacer sequence of the host cell genome or modify the original spacer sequence of the episome contained in the host in the presence of a derepressor. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Regulators controlling the expression of spCas9 and a self-targeting sgRNA targeting ribosomal RNA subunit 16s are shown.

[0018] Figure 2 Showing specific targeting of E. coli strains by exogenous CRISPR-Cas systems. The sgRNA targeted the genome of a K-12-derived E. coli strain (e.g., E. coli TOP10), while another tested strain was unaffected.

[0019] Figure 3 Shown are spot assays with serial dilutions of individual bacterial species used in this study and mixed cultures in TH agar that did not induce the CRISPR-Cas9 system.

[0020] Figure 4 Dilution 10 is shown 3 Spot assay on different selective media. -1 TH of PEA is a selective medium for Bacillus subtilis alone. MacConkey supplemented with maltose is a selective and differential bacterial medium designed to selectively separate Gram-negative and enteric bacilli and differentiate them based on maltose fermentation. Thus, the TOP10ΔmalK mutant produces white colonies on the plate, while Nissle produces pink colonies; A is E. coliΔmalK, B is E. coli Nissile, C is B. subtilis, D is Lactococcus lactis, and E is a mixed culture; images of MacConkey- / B and E are shown in pink; images of MacConkey+ / B and E are shown in pink.

[0021] Figure 5 Selective growth of the bacteria used in this study on different media and selective plates is shown. DETAILED DESCRIPTION The present invention provides carriers, compositions, methods and uses comprising a variety of such carriers, in particular. The present invention can be used to target wild-type bacterial populations naturally found in the environment (e.g., in water or waterways, cooling or heating equipment, in or on agricultural plants, in soil), contained in beverages and foods (or equipment for manufacturing, processing or storing these), or contained in human or animal microbiota (e.g., in the intestine, in the lungs, or on the skin). Therefore, the present invention finds uses when it is impossible or undesirable for the host cell to be pre-modified to accept killing or growth inhibition (e.g., when it is desired to treat the microbiota of the intestinal tract or other parts of the subject in situ). In another application, the present invention finds uses for drugs (e.g., intestinal cell transplants) for in vitro production for administration to human or animal subjects for the treatment or prevention of diseases or conditions caused or mediated by host cells, wherein the drug comprises a modified mixed bacterial population (e.g., feces or intestinal microbiota obtained from one or more human donors), which is the product of the present invention's uses or methods, wherein the population comprises a bacterial subpopulation of a species or strain different from the host cell species or strain. The aforementioned subpopulation cells do not contain the original spacer target, and therefore are not modified by the use or method. Thus, for example, the method can be used to reduce the proportion of a specific subpopulation in a mixed population and retain the Bacteroidetes, for example, for producing a drug for treating or preventing a metabolic or GI condition (e.g., colitis) or disease disclosed herein. In this way, the present invention can provide a modified bacterial transplant (e.g., a modified fecal transplant) drug for such use or for the treatment or prevention in humans or animals. For example, the method can be used to modify one or more microbial populations in vitro to produce a modified bacterial collection for administration to humans or animals for medical purposes (e.g., treatment or prevention of metabolic conditions (such as obesity or diabetes) or gastrointestinal conditions (e.g., colitis, IBD, IBS, Crohn's disease, or any such condition mentioned herein) or cancer (e.g., gastrointestinal cancer or melanoma)) or for cosmetic or personal hygiene purposes (e.g., for topical use in humans, for example, for reducing axillary or other body odor by topical use in the axilla or other relevant parts of humans). In another example, the vector of the present invention is administered to a human or animal, and the host cell is contained by the human or animal, for example, by the microbial flora of the human or animal (such as the intestinal microbial flora or any other type of microbial flora disclosed herein). In this way, a disease or condition mediated or caused by the host cell can be treated or prevented. In one example, host cell transformation is implemented in vitro, and optionally, the vector is a plasmid or phagemid electroporated into the host cell; or, the vector is contained by a virus (e.g., a phage), which infects the host cell and introduces a derepressor and an array or gRNA-encoding sequence into the host cell. In one example, the nucleic acid is RNA (e.g., a copy of a gRNA).In another example, the vector is a DNA vector or an RNA vector.

[0023] In one example, the organism is a plant or an animal, such as a vertebrate (eg, any mammal disclosed herein or a human) or a crop or food plant.

[0024] In one example, the method, use, vector or composition is for medical or dental or ophthalmic use (eg, for treating or preventing an infection in an organism or limiting the spread of an infection in an organism).

[0025] In one example, the method, use, vector or composition is for cosmetic use (eg, for use in a cosmetic product (eg, cosmetics)), or for hygienic use (eg, for use in a hygienic product (eg, soap)).

[0026] In one example, the composition is any one of the following: In one example, the composition is a medical, ophthalmic, dental or pharmaceutical composition (e.g., contained in an anti-host vaccine). In one example, the composition is an antimicrobial composition, such as an antibiotic or antiviral substance, such as a drug, disinfectant or mouthwash. In one example, the composition is a cosmetic composition (e.g., a facial or body makeup composition). In one example, the composition is a herbicide. In one example, the composition is an insecticide (e.g., when the host is a Bacillus (e.g., Bacillus thuringiensis) host). In one example, the composition is a beverage (e.g., beer, wine or alcoholic beverage) additive. In one example, the composition is a food additive (e.g., wherein the host is an Escherichia coli, Salmonella, Listeria or Clostridium (e.g., Clostridium botulinum) host). In one example, the composition is a water additive. In one example, the composition is an additive for an aquatic animal environment (e.g., in a fish tank). In one example, the composition is a petroleum or petrochemical industry composition or is contained in such a composition (for example, when the host is a sulfate-reducing bacteria, such as a Desulfovibrio host). In one example, the composition is a petroleum or petrochemical additive. In one example, the composition is a chemical additive. In one example, the composition is a disinfectant (for example, a disinfecting device for human or animal use, such as for surgical or medical purposes, or for infant feeding). In one example, the composition is a personal hygiene composition for human or animal use. In one example, the composition is for environmental use, such as a composition for soil treatment or environmental purification (for example, from sewage, or from petroleum, petrochemicals or chemicals, such as when the host is a sulfate-reducing bacteria, such as a Desulfovibrio host). In one example, the composition is a plant growth promoter. In one example, the composition is a composition for petroleum, petrochemicals, metals or mineral extraction. In one example, the composition is a fabric treatment or additive. In one example, the composition is a hide, leather or suede treatment or additive. In one example, the composition is a dye additive. In one example, the composition is a beverage (e.g., beer or wine) brewing or fermentation additive (e.g., when the host is a Lactobacillus host). In one example, the composition is a paper additive. In one example, the composition is an ink additive. In one example, the composition is a glue additive. In one example, the composition is a composition against human, animal or plant parasites. In one example, the composition is an air additive (e.g., for air in or produced by an air conditioning device, e.g., where the host is a Legionella host). In one example, the composition is an antifreeze additive (e.g., where the host is a Legionella host).In one example, the composition is an eyewash or ophthalmic composition (e.g., contact lens solution). In one example, the composition is contained by a dairy product (e.g., the composition is in milk or a dairy product or is milk or a dairy product; for example, wherein the host is a Lactobacillus, Streptococcus, Lactococcus, or Listeria host). In one example, the composition is or is contained by a household or industrial cleaning product (e.g., wherein the host is an Escherichia coli, Salmonella, Listeria, or Clostridium (e.g., Clostridium botulinum) host). In one example, the composition is contained by a fuel. In one example, the composition is contained by a solvent (e.g., except water). In one example, the composition is a baking additive (e.g., a food baking additive). In one example, the composition is a laboratory reagent (e.g., for biotechnology or recombinant DNA or RNA technology). In one example, the composition is contained by a fiber retting agent. In one example, the composition is used in a vitamin synthesis process. In one example, the composition is an anti-crop or plant damage component (e.g., when the host is a saprophyte). In one example, the composition is an antiseptic compound, such as for preventing or reducing metal corrosion (e.g., when the host is a sulfate-reducing bacterium, such as a Desulfovibrio host, such as for reducing or preventing corrosion of oil extraction, processing or preservation equipment; metal extraction, processing or preservation equipment; or mineral extraction, processing or preservation equipment). In one example, the composition is an agricultural or farming composition or is contained in such a composition. In one example, the composition is a silage additive. The present invention provides a CRISPR array, a nucleotide sequence encoding a gRNA, a vector or multiple vectors described herein for use in any of the compositions described in this paragraph, or for any of the uses described in this paragraph, such as wherein the host cell is a bacterial or archaeal cell. The present invention provides a method for any of the applications described in this paragraph, wherein the method comprises combining the CRISPR array of the present invention, the nucleotide sequence encoding a gRNA, a vector or multiple vectors with a host cell (e.g., a bacterial or archaeal cell). In one embodiment, the host cell is not present in or on a human (or human embryo) or animal.

[0027] Any aspect of the invention, such as an array, carrier, composition, use or method, is for industrial or domestic use, or a method for such use. For example, it is used in agriculture, petroleum or petrochemical industry, food or beverage industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aerospace industry, automotive industry, biotechnology industry, medical industry, health industry, dental industry, energy industry, consumer products industry, pharmaceutical industry, mining industry, cleaning industry, forestry industry, fishery industry, leisure industry, recycling industry, cosmetics industry, plastic industry, pulp or paper industry, textile industry, clothing industry, leather or suede or hide industry, tobacco industry or steel industry or used in the above industries.

[0028] The present invention provides a nucleic acid vector for introduction into a host cell, wherein the cell comprises a CRISPR / Cas system that is repressed by a repressor in the cell, and the vector comprises (a) a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences each encoding a respective guide RNA (gRNA, e.g., a single guide RNA) in the cell; wherein each crRNA or gRNA is capable of directing Cas to modify a respective protospacer sequence of the host cell genome or to modify a protospacer sequence of an episome contained by the host in the presence of a derepressor.

[0029] The sequence of (a) is expressible because it can be operably linked to a promoter operable to express the derepressor in the cell.

[0030] Optionally, any host cell herein is a bacterial or archaeal cell. In one example, the cell is in stationary phase. In one example, the cell is in exponential phase. In one example, the cell is in lag phase. In one example, the cell is a wild-type cell or a naturally occurring cell, for example, comprised by a naturally occurring microbial group, for example, a human, an animal, a plant, soil, water, an ocean, a waterway or an environment. In one example, the cell is artificially genetically modified. In one example, the CRISPR / Cas system is artificially repressed and a derepressor removes or reduces the repression.

[0031] In one example, a variety of vectors of the present invention are introduced into a variety of host cells, wherein the host cell is comprised by a bacterial colony, such as in vitro, in vivo or in vitro. In one example, the host cell is comprised by a microbial colony, and the microbial colony is comprised by an organism or environment (e.g., waterway microbial colony, water microbial colony, human or animal intestinal microbial colony, human or animal oral microbial colony, human or animal vaginal microbial colony, human or animal skin or hair microbial colony or human or animal armpit microbial colony), and the colony comprises a first bacterium commensal or symbiotic with the organism or environment and a second bacterium comprising the host cell, wherein the host cell is harmful to the organism or environment (e.g., pathogenic). In one embodiment, the colony is in vitro. In one example, the ratio of the first bacterial subpopulation to the second bacterial subpopulation increases. In one example, the first bacterium is a bacterium of the genus Bacteroides (e.g., Bacteroides fragilis and / or Bacteroides thetaiotaomicron). Optionally, the genus Bacteroides comprises one, two, three or more species of Bacteroides selected from the group consisting of Bacteroides faecalis, Bacteroides pilosulae, Bacteroides cellulolyticus, coprocola, Bacteroides coprophilus, coprosuis, Bacteroides distachyon, Bacteroides dorei, Bacteroides elsdenii, faecis, finegoldii, fluxus, Bacteroides fragilis, Bacteroides enterica, Bacteroides melaninogenicus, nordii, oleiciplenus, Bacteroides oralis, Bacteroides ovalis, Bacteroides pectinophilus, Bacteroides coeruleus, Bacteroides faecalis, Bacteroides thetaiotaomicron, Bacteroides monomorphus, Bacteroides vulgaris and Bacteroides xylose degrading. For example, the genus Bacteroides is Bacteroides thetaiotaomicron or comprises Bacteroides thetaiotaomicron. For example, the genus Bacteroides is Bacteroides thetaiotaomicron or comprises Bacteroides thetaiotaomicron.

[0032] In one example, the host, the first or second cell is any bacterial species disclosed in US20160333348, GB1609811.3, PCT / EP2017 / 063593 and all U.S. equivalent applications. The disclosure of these species (particularly including Table 1 of PCT / EP2017 / 063593) is incorporated herein in its entirety and is used to include one or more disclosures therein that may be included in one or more claims herein.

[0033] In one example, the host cell or bacterial colony is carried by a beverage or water (e.g., waterway or drinking water) consumed by a person. In one example, the host cell or the colony is included by a composition (e.g., a medicine (e.g., a bacterial intestinal transplant), a beverage, a mouthwash or a food), and the composition is used to be applied to a human or non-human animal for forming a colony and rebalancing the intestinal tract or its oral microbiota (e.g., wherein the purpose of the medicine is to treat or prevent a disease or condition in a human or animal). In one example, the host cell or the colony is included on a solid surface or by a biofilm (e.g., a biofilm on an intestinal biofilm or an industrial device). In an example of the carrier, method or use, it is used for in vitro treatment of industrial or medical fluids, solid surfaces, devices or containers (e.g., food, consumer goods, cosmetics, personal health care products, oil or oil production); or for treating a waterway, water, beverage, food or cosmetics, wherein the host cell is included by a fluid, a surface, a device, a container, a waterway, water, a beverage, food or cosmetics.

[0034] In one example, the invention provides a container for medical or nutritional purposes, wherein the container comprises a colony or product of the purposes or method. For example, the container is a sterile container, such as an inhaler or is connected to a syringe or IV needle. In one example, the product colony of the purposes or method can be used to be applied to a human or animal to inhabit its microbial group to treat or prevent a disease or condition (e.g., a disease or condition described herein) in a human or animal. The invention provides: food or beverage for human or non-human animal consumption comprising a colony product for the purposes or method of the invention.

[0035] In one example, the carrier, composition is for administration to (or is administered to) a human or non-human animal by mucosal, enteral, oral, intranasal, rectal, vaginal, ocular or buccal administration.

[0036] Optionally, one or more vectors lack Cas (eg, Cas3 and / or Cas9) nuclease encoding sequences. In one example, the system comprises a repressed host cell Cascade, Cas3, CasCas9 or cpf1 activity.

[0037] In one example, the host cell is a wild-type (e.g., non-engineered) bacterial cell. In another example, the host cell is engineered (such as introducing an exogenous nucleotide sequence in a chromosomal manner or modifying an endogenous nucleotide sequence such as on the chromosome or plasmid of the host cell). In one example, after the vector is introduced into the host cell, the formation of the bacterial colony of the host cell is suppressed. In one example, after the introduction, the propagation of the host cell is suppressed. In one example, after the introduction, the host cell is killed.

[0038] Optionally, each host cell is a strain or species found in the human microbiota, optionally wherein the host cell is mixed with cells of a different strain or species, wherein the different cells are Enterobacteriaceae or are probiotic, commensal or commensal bacteria with humans (e.g., in the human intestine). In one example, the host cell is an Escherichia coli or a Salmonella cell.

[0039] The present invention is optionally used to inhibit the growth of a bacterial population or to change the relative ratio of a first and a second bacterial subpopulation in a mixed population of bacteria, for example, to change a human or animal microbiome, such as to change the ratio of Bacteroidetes (e.g., Bacteroides genus, such as Bacteroides fragilis and / or Bacteroides thetaiotaomicron), Firmicutes and / or Gram-positive or -negative bacteria in a human microbiome. For example, one embodiment of the present invention provides:- An antimicrobial composition for use in a method of treating or preventing a disease in a human or animal subject, wherein the subject's intestine comprises a mixed bacterial population, the method comprising administering the antimicrobial agent to the subject to modify the mixed bacterial population comprised by the subject's intestine to favor commensal or commensal Bacteroidetes of the intestine population, thereby increasing the proportion of Bacteroidetes bacteria in the subject's intestine, wherein the treatment or prevention is achieved, wherein the composition comprises one or more vectors of the invention and the host cell comprises a CRISPR / Cas system that is naturally suppressed in the intestine population.

[0040] Utilizing the subject's commensal or symbiotic Bacteroidetes is advantageous for utilizing disease-modifying effects through the activity of endogenous commensals and symbionts already present in the intestine. This avoids the risks of administration of potentially pathogenic exogenous Bacteroidetes as taught in the art. In addition, it is possible to generate a more sustainable effect by utilizing the patient's own intestinal bacteria (e.g., by generating a niche in the patient's intestinal microbiome via targeting other intestinal bacteria, such as Gram-positive bacteria, such as Clostridium). The possibility of usefully utilizing endogenous patient bacteria also avoids the need to consider and maintain administration of preparations using bacterial preparations or extracts thereof.

[0041] Additionally, Bacteroidetes such as Bacteroides fragilis and Bacteroides thetaiotaomicron are strict anaerobes, which severely limits the production, storage and administration of compositions in anaerobic environments. The present invention avoids that by utilizing the patient's own Bacteroidetes, which are retained in the compatible anaerobic environment of the intestine.

[0042] Furthermore, the patient's own endogenous Bacteroides and the patient's immune system and other interacting factors in the intestine have co-evolved and matched to function effectively (e.g., stimulating a useful immune response for addressing the disease), and the present invention can take advantage of this by utilizing endogenous intestinal Bacteroides (e.g., for stimulating the subject's immune response). In addition, exogenously administered Bacteroides (or its peptides), which may be slightly clear (administration issues), are unnecessary and need to somehow find a way to effectively colonize the correct intestinal crypt location for beneficial use. In contrast, the effector bacteria in the present invention are already in position in the patient and are immediately available.

[0043] In one example, the method is used to treat or prevent inflammatory bowel disease (IBD). In one example, the method is used to treat or prevent obesity for medical purposes. In one example, the method is used to treat or prevent diabetes. In one example, the method includes increasing the relative ratio of Bacteroidetes compared to Firmicutes. In one example, the Bacteroidetes is Bacteroides fragilis and / or Bacteroides thetaiotaomicron. In one example, the Bacteroidetes is Bacteroides monomorpha.

[0044] In one example, the vector of the present invention is used for any method disclosed in US20160333348, GB1609811.3, PCT / EP2017 / 063593 and all US equivalent applications; in one example, the vector of the present invention is any vector disclosed in US20160333348, GB1609811.3, PCT / EP2017 / 0635933 and all US equivalent applications. The disclosures of US20160333348, GB1609811.3, PCT / EP2017 / 063593 and all US equivalent applications (including these specific disclosures) are incorporated herein in their entirety and are used to include one or more disclosures therein in one or more claims herein.

[0045] In one example, the vector or composition of the invention comprises a nucleotide sequence for expressing an endolysin for host cell lysis in a host cell, optionally wherein the endolysin is bacteriophage phi11, phage Twort, phage P68, phage phiWMY or phage K endolysin (e.g., MV-L endolysin or P-27 / HP endolysin).

[0046] The derepressor can act as an activator capable of activating the CRISPR / Cas system in a cell, wherein the activator activates the system in the presence of the repressor. For example, the repressor can bind to a nucleic acid of the system, such as a promoter, and when the repressor binds to an element of the system, the activator overrides repression and activates the system.

[0047] In one example, the protospacer sequence is comprised by the chromosome of the host cell, for example, wherein the sequence is comprised by an antibiotic resistance gene, a virulence gene or an essential gene of the host cell. An example provides a vector of the invention in combination with an antibiotic agent (e.g., a β-lactam antibiotic), for example, wherein the vector targets a protospacer sequence comprised by an antibiotic resistance gene, and the antibiotic resistance gene is comprised by the host cell genome or an episome (e.g., a plasmid comprised by the host cell). In one example, the episome is a plasmid, a transposon, a mobile genetic element or a viral sequence (e.g., a phage or prophage sequence).

[0048] In one example, the target sequence is a chromosomal sequence, an endogenous host cell sequence, a wild-type host cell sequence, a non-viral chromosomal host cell sequence, a non-endogenous sequence, and / or a non-phage sequence (i.e., one, multiple, or all of these), for example, a sequence is a wild-type host chromosomal cell sequence, such as an antibiotic resistance gene or an essential gene sequence contained by the host cell chromosome. In one example, the sequence is a host cell plasmid sequence, such as an antibiotic resistance gene sequence.

[0049] Optionally, the or each host cell protospacer sequence is adjacent to a NGG, NAG, NGA, NGC, NGGNG, NNGRRT or NNAGAAW protospacer sequence adjacent to a motif (PAM), e.g., AAAGAAA or TAAGAAA PAM (these sequences are written 5'-3'). In one embodiment, the PAM is adjacent to the 3' end of the protospacer sequence. In one example, Cas is Staphylococcus aureus, Streptococcus thermophilus or Streptococcus pyogenes Cas. In one example, Cas is Cpf1 and / or PAM is TTN or CTA.

[0050] Optionally, the system is a type I (e.g., IA, IB, IC, ID, IE or IF) CRISPR / Cas system. Optionally, the system is a type II CRISPR / Cas system. Optionally, the system is a type IIII CRISPR / Cas system. Optionally, the system is a type IV CRISPR / Cas system. Optionally, the system is a type V CRISPR / Cas system. Optionally, the system is a type VI CRISPR / Cas system.

[0051] Optionally, the CRISPR array comprises multiple copies of the same spacer for targeting the original spacer sequence. Optionally, one or more vectors of the present invention are provided, wherein the vector comprises multiple CRISPR arrays of the gRNA encoding sequence for host cell original spacer sequence targeting. Optionally, the or each vector comprises two, three or more copies of a nucleic acid sequence encoding crRNA (e.g., gRNA), wherein the copy comprises the same spacer sequence for targeting a host cell sequence (e.g., virulence, resistance or essential gene).

[0052] In one example, at least two target sequences are modified by Cas, such as an antibiotic resistance gene and an essential gene. Such multi-targeting can be used to reduce the evolution of escape mutations in host cells.

[0053] In one example, Cas is a wild-type endogenous host cell Cas nuclease. In one example, protospacer target modification or cleavage is performed by a dsDNA Cas nuclease (e.g., Cas9, e.g., spCas9 or saCas9), whereby cleavage is repaired by non-homologous end joining (NHEJ); alternatively, the Cas is an exonuclease or Cas3. In one example, Cas is a Cas nuclease for cleavage, a dead Cas (dCas) for interference, or a dCas (e.g., dCas3 or dCas9) conjugated to a transcriptional activator for activating the target.

[0054] In an example, the array, gRNA encoding sequence, or vector is not combined with a sequence encoding a Cas endonuclease that is naturally found in a cell with repeat sequences of the array or gRNA encoding sequence.

[0055] The tracrRNA sequence may be omitted from an array or vector of the invention, e.g., for a type of Cas system that does not use tracrRNA, or endogenous tracrRNA may be used together with crRNA encoded by the vector.

[0056] In one example, the host protospacer sequence comprises at least 5, 6, 7, 8, 9, 10, 20, 30 or 40 consecutive nucleotides.

[0057] In one example, the or each vector comprises an exogenous promoter functional for transcription of crRNA or gRNA in the host.

[0058] In one example, the or each array repeat region is identical (or has at least 90, 95 or 98% identity) to the repeat region in the host array contained in the CRISPR / Cas system of the host, wherein the vector array does not contain a PAM recognized by the Cas nuclease of the host CRISPR / Cas system. This applies to the repeat sequence of gRNA with necessary changes. This is advantageous because it only enables the CRISPR array to target the host target sequence using endogenous host Cas. This is effective because the array is designed for host machinery and therefore helps to function in the host cell. In addition, or, this enables the vector-encoded array sequence to be combined with endogenously encoded tracrRNA, because the CRISPR array repeat region will hybridize with endogenous tracrRNA for producing pre-crRNA and processing into mature crRNA hybridized with the host target sequence. The latter complex can then guide endogenous Cas nuclease (e.g., Cas3). Therefore, this embodiment provides the flexibility of simply constructing a vector (e.g., packaged virus or phage) containing a CRISPR array but not containing a tracrRNA- and / or Cas nuclease encoding sequence. This is more straightforward for vector construction and also frees up valuable space in the vector (e.g., virus or phage), which is a noteworthy and useful association with the capacity limitations of vectors, particularly viral vectors (e.g., phage). The additional space can be useful, for example, to enable more space to be included in the array, for example to target the host genome for modification, such as inactivating host genes or bringing in desired non-host sequences for expression within the host. Additionally or alternatively, the space can be used to include multiple CRISPR arrays in the vector. For example, these can be arrangements in which a first array is of a first CRISPR / Cas type (e.g., type II or type II-A), and a second array can be of a second type (e.g., type I or III or type II-B). Additionally or alternatively, the arrays can use different Cas nucleases within the host (e.g., one array can operate with a host Cas nuclease, and a second array can operate with an exogenous Cas nuclease (i.e., a vector-encoded nuclease) or a different host Cas). These aspects provide a mechanism for targeting in a host once a vector has been introduced, which is useful for reducing host resistance to the vector when the host needs to target a wider range of elements. For example, if the host is able to acquire new spacers based on the sequence of the first CRISPR array, the second CRISPR array can still function in the host to target the corresponding target sequence in the host cell. Therefore, this embodiment is useful for reducing host adaptation to the vector.

[0059] Optionally, the vector of the invention comprises 1, 2, 3, 4, 5, 6 or more CRISPR arrays or gRNA-encoding sequences of the invention, which comprise multiple (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 or more) copies of the spacer region that hybridizes to the host target sequence. This reduces the chance of losing all of these spacers by recombination in the host cell. In a further application of this aspect, a CRISPR array comprises a first array containing one or more (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or more) copies of a spacer and a second array containing one or more (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or more) copies of the same spacer, wherein the copies of the spacers in the first array are each flanked by a first repeat region, and the copies of the same spacers in the second array are each flanked by a second repeat region, wherein the first repeat region is different from the second repeat region. This has the following benefits: the first repeat sequence can be selected to be recognized by a first (e.g., derepressed) host Cas nuclease, and the second repeat sequence is recognized by a second Cas (e.g., vector-encoded or host's Cas) to reduce the chance of host adaptation involving more than one of the arrays.

[0060] Optionally, one or more vectors of the present invention are provided, wherein each vector is a plasmid, a cosmid, a virus, a virion, a phage, a phagemid or a prophage. For example, the present invention provides a plurality of phages comprising a plurality of vectors of the present invention, for example, wherein the vectors are the same. In one example, the vector is a viral vector. Viral vectors have a particularly limited capacity for insertion of exogenous DNA, so the viral packaging capacity needs to be considered. It is necessary to leave space for sequences encoding important viral functions, such as for expression of coat proteins and polymerases. In one example, the vector is a phage vector or an AAV or lentiviral vector. Phage vectors are useful in cases where the host is a bacterial cell. In one example, the vector is a virus capable of infecting an archaeal host cell.

[0061] Optionally, vector components (a) and (b) are comprised of a transposon capable of being transferred into and / or between host cells. The transposon may be a transposon as described in US20160333348, GB1609811.3, and all U.S. equivalent applications; the disclosures of these, including these specific transposon disclosures, are incorporated herein in their entirety and are used to include one or more disclosures thereof in one or more claims herein.

[0062] In one example, the or each carrier is provided by a nanoparticle or is provided in a liposome.

[0063] In one example, the transcription of one or more components of the CRISPR / Cas system is repressed. The transcription of one or more Cas sequences (e.g., Cas3, Cas9, or Cpf1) is repressed. For example, the transcription of one or more of CasA, B, C, D, and E of the type I CRISPR / Cas system is repressed, for example, CasA and / or Cas3 is repressed.

[0064] Optionally, the Cas modification of the host cell genome a. Kill host cells; b. reduce the growth or proliferation of cells or episomes; c. Increase the growth or proliferation of cells or episomes; d. reducing or preventing the transcription of a nucleotide sequence comprising the protospacer sequence or adjacent to the protospacer sequence; or e. increasing the transcription of a nucleotide sequence comprising the protospacer sequence or adjacent to the protospacer sequence.

[0065] In one example, the inhibition of host cell population growth is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times compared to the growth of the host cells not exposed to the vector of the present invention. For example, growth inhibition is shown by the following, that is, the number of colonies of the first host cell sample (alone or in a mixed bacterial population) is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower than the number of bacterial colonies of the second host cell sample (alone or in a mixed bacterial population), wherein the first sample of cells has been transformed with the vector, but the second sample has not been exposed to the vector. In one embodiment, the colony count is determined 12, 24, 36 or 48 hours after the first sample has been exposed to the vector of the present invention. In one embodiment, the colonies are grown in vitro (e.g., in a petri dish) on solid agar. Therefore, it should be understood that growth inhibition can be shown by a reduction in the growth of cells or colonies containing the target sequence (compared to the growth of untreated, i.e., control sample <100% growth), or such growth can be completely eliminated. In one example, the growth of the host cell colony is reduced by at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 95%, i.e., after a predetermined time period (e.g., 24 hours or 48 hours after the crRNA or gRNA combination in the host cell), i.e., the growth ratio of the host cell colony is not exposed to the carrier, but in addition, the growth of the control group host cell colony of the predetermined duration period is maintained under the same conditions. At least such a percentage. In one example, by comparing the number of colonies in each colony sample at the end of the time period (e.g., during the mid-exponential growth phase of the control group sample), the percentage reduction of growth is determined. For example, after the subject colony is exposed to the carrier at time zero, samples of the subject and control group colonies are taken, and each sample is plated on an agar plate, and the predetermined time is incubated under the same conditions. At the end of this period of time, the colony count and percentage difference of each sample are counted (i.e., the number of colonies in the subject group is divided by the number of colonies in the control group, and then multiplied by 100, and then the result is subtracted from 100 to obtain a reduction in growth percentage). The fold difference was calculated by dividing the number of colonies in the control group by the number of colonies in the test group.

[0066] Therefore, the inhibition of population growth can be indicated by a reduction in the proliferation of the number of host cells in the population. This may be due to the CRISPR / Cas system being de-repressed or activated and / or due to the killing of host cell proliferation (e.g., division and / or cell growth) by the down-regulation of the effect of the system on the target protospacer sequence in the host cell. In one embodiment of the methods, uses, treatments or preventions disclosed herein, the host cell burden of a human or animal subject or environment is reduced, and the disease or condition to be treated (e.g., reduced or eliminated) or prevented (i.e., the risk of a subject developing a disease or condition) is thereby reduced or eliminated or the environment is treated.

[0067] In one example, components (a) and (b) are alternatively comprised by different first and second vectors for introducing the vectors into a host cell where the Cas modification occurs.

[0068] In one example, the derepressor is a protein or RNA. For example, the derepressor is a silencing RNA (siRNA) that is complementary to a nucleotide sequence encoding a repressor contained in a host cell gene, for example, wherein the sequence is a sequence of an ORF or a regulatory element, such as a promoter or enhancer of a gene.

[0069] In one example, the repressor is an anti-CRISPR or anti-Cas (e.g., anti-Cas3 or anti-Cas9) protein, nucleic acid or RNA, for example, encoded by a prophage contained by a host cell. For example, the repressor is encoded by acr, acrIIA2 and acrIIA4, aca1 and aca2 genes or their orthologs, homologs or paralogs; and optionally, the de-repressor is an siRNA complementary to the de-repressor gene sequence in the host cell, thereby silencing the expression of the gene. In one example, the repressor is an AcrIIA protein, such as AcrIIA2 and / or AcrIIA4.

[0070] Because H-NS often works in combination with other nucleoid-associated proteins (NAPs), the binding of related regulatory proteins, such as StpA, LRP and FIS, has been analyzed (Luijsterburg et al., 2006; Dorman, 2009). The repressor can be H-NS (nucleoid structural protein), StpA, FIS, LRP ((leucine responsive regulatory protein) or CRP (cAMP receptor protein); or a direct homologue or paralogue, homologue or functional equivalent thereof, which acts as a repressor in the host cell. For example, the repressor can be a direct homologue or paralogue, homologue or functional equivalent of E. coli H-NS, StpA, FIS, LRP or CRP protein. In one example, the CRISPR / Cas system is repressed by more than one such repressor (e.g., H-NS and LRP; or H-NS and CRP).

[0071] The derepressor may be a mutant H-NS, StpA, LRP or CRP, which is capable of forming a complex with a H-NS, StpA, LRP or CRP repressor (e.g., a wild-type host or E. coli H-NS, StpA, LRP or CRP) in a host cell to prevent or reduce the repression of the CRISPR / Cas system. The derepressor may be a mutant H-NS, StpA, LRP or CRP, which is capable of forming a complex with a wild-type host or E. coli H-NS, StpA, LRP or CRP, respectively (e.g., in vitro or in a host cell or E. coli).

[0072] In one example, the repressor is H-NS or StpA, and the derepressor is LeuO.

[0073] In one example, the episome is a plasmid.

[0074] The following definitions apply:- Homologs A gene or protein that is related to a second gene or protein by descent from a common ancestral DNA sequence. The term homolog may apply to the relationship between genes or their protein products separated by the event of speciation (see orthologs) or by the event of gene duplication (see paralogs).

[0075] Orthologs ● Orthologs are genes or proteins in different species that have evolved from a common ancestral gene or protein through speciation. Usually, orthologs retain the same function during the process of evolution.

[0076] Paralogs ● Paralogs are genes or proteins that are related by duplication within a genome. Orthologs retain the same function during evolution, while paralogs evolve new functions, even if these are related to the original function.

[0077] A homolog of a repressor or derepressor itself has activity as a repressor or derepressor in a host cell. An ortholog of a repressor or derepressor itself has activity as a repressor or derepressor in a host cell. A paralog of a repressor or derepressor itself has activity as a repressor or derepressor in a host cell.

[0078] In one example, the cell is a bacterial or archaeal cell. In one example, the cell is contained by the environment, soil, plant, mammal, human, mouse, rat, pig, dog, primate, monkey, sheep, cattle, horse, cat, ruminant, livestock, insect or poultry (e.g., chicken or turkey), such as by its microbiome. The microbiome can be a plant leaf, plant stem, soil, intestinal tract, skin, oral cavity, lung, eye, ear, tongue, axilla, vagina, rectum, scrotum, penis or hair microbiome. In one example, the cell is a vertebrate, invertebrate, mammal, human, rodent, mouse, rat, fish or insect cell.

[0079] In one example, the host cell is an E. coli cell, such as selected from Shiga toxin-producing E. coli (STEC) (STEC may also be called verotoxin-producing E. coli (VTEC) or enterohemorrhagic E. coli (EHEC). This pathotype is the one most often heard in the news in connection with foodborne outbreaks); Enterotoxigenic Escherichia coli (ETEC); Enteropathogenic Escherichia coli (EPEC); Enteroaggregative Escherichia coli (EAEC); Enteroinvasive Escherichia coli (EIEC); and ●Diffusely attached Escherichia coli (DAEC).

[0080] The strain of Shiga toxin-producing E. coli O104:H4 that caused a large outbreak in Europe in 2011 is often referred to as EHEC. In North America, the most commonly identified STEC is E. coli O157:H7. In one example, the cell is E. coli O104:H4 or E. coli O157:H7.

[0081] It has been observed that endogenous CRISPR / Cas systems can be slightly derepressed and / or upregulated in the stationary phase in host cells, for example, when cells are densely packed, against phage invasion or plasmid horizontal transfer. In an example of the vector of the present invention, component (a) and / or (b) are operably linked to a promoter for expression in a stationary growth phase in a host cell. Densely packed cells may be present in a biofilm, and therefore, in an example of the vector of the present invention, component (a) and / or (b) are operably linked to a promoter for expression in a host cell comprised by a biofilm (e.g., in an environment or in a human or animal body, such as a lung or intestinal biofilm).

[0082] In one example of the vector of the invention, to also achieve protospacer targeting or alternatively in the exponential growth phase (e.g., where the endogenous CRISPR / Cas system can be repressed), components (a) and / or (b) are operably linked to a promoter for expression in the host cell in the exponential growth phase. In an example of the vector of the invention, components (a) and / or (b) are operably linked to a promoter for expression in the host cell in the lag phase.

[0083] The transcriptional regulator CsgD is critical for biofilm formation, controlling the expression of curli structural and export proteins as well as the diguanylate cyclase adrA (which indirectly activates cellulose production). Chirwa NT and Herrington MB, Microbiology. 2003 Feb; 149 (Pt 2): 525-35, "CsgD, a regulator of curli and cellulose synthesis, also regulates serine hydroxymethyltransferase synthesis in Escherichia coli K-12" explains that the homologous CsgD and AgfD proteins are members of the FixJ / UhpA / LuxR family and are proposed to regulate curli (thin aggregated fibers) and cellulose production in Escherichia coli and Salmonella enterica serovar Typhimurium, respectively. CsgD is proposed to upregulate glyA to facilitate curli synthesis. In examples of the vectors of the invention, components (a) and / or (b) are operably linked to a promoter for expression in a host cell, wherein the promoter is controlled by CsgD or AgfD (e.g., E. coli CsgD or AgfD). For example, this can be used to promote expression of vector components in host cells in a biofilm or to participate in the biogenesis of a biofilm. Optionally, the host cells are E. coli and Salmonella enterica serovar Typhimurium cells.

[0084] The gene rpoS (RNA polymerase, σS) encodes σ factor σ-38 (σ38 or RpoS), a 37.8 kD protein in Escherichia coli. σ factor is a protein that regulates transcription in bacteria. σ factor can be activated in response to different environmental conditions. rpoS is transcribed in the late exponential phase, and RpoS is the main regulator of stationary phase genes. RpoS is a central regulator of general stress response and acts in a retrospective and prospective manner: it not only allows cells to survive after environmental challenges, but also prepares cells for subsequent stress (cross protection). In an example of the vector of the present invention, component (a) and / or (b) is operably linked to a promoter for expression in a host cell, wherein the promoter is regulated by a σ factor (such as RpoS, for example, Escherichia coli RpoS). For example, this can be used to promote the expression of vector components in a host cell during the growth phase, wherein the σ factor and RpoS regulation are upregulated. Optionally, the host cell is an Escherichia coli and a Salmonella enterica serovar Typhimurium cell. The transcription of rpoS in E. coli is mainly regulated by the chromosomal rpoSp promoter. rpoSp promotes the transcription of rpoS mRNA and is induced in cells grown on rich medium after entering the stationary phase. Therefore, in one example, the or each promoter contained by the vector acts to upregulate the transcription of its component (a) or (b) in the host cell in the stationary phase, for example, the or each promoter is the rpoSp promoter.

[0085] As a defense mechanism, the bacterial host environment is hostile to invading pathogens, such as bacteriophages. Therefore, infection can be a stressful event for pathogenic bacteria, and the control of virulence genes can be temporally linked to the timing of infection by the pathogen. The discovery of RpoS-dependent virulence genes in Salmonella is consistent with RpoS being a general regulator of stress responses: the spv genes found on the virulence plasmid in this bacterium are under the control of RpoS and, interestingly, are required for growth in deep lymphoid tissues such as the spleen and liver. Thus, in one embodiment, the vector is a virus capable of infecting a host cell, e.g., a bacteriophage (e.g., an E. coli or Salmonella cell, and components (a) and / or (b) are operably linked to a promoter for expression in the host cell, wherein the promoter is regulated by RpoS (e.g., E. coli RpoS). Optionally, the host cell is comprised by a spleen or liver bacterial population comprised by a human or animal. Optionally, the vector is used for administration to the human or animal to treat or prevent a condition or disease, e.g., a spleen, liver or immune-related disease or condition. In one example, the or each promoter is a promoter of a virulence gene (e.g., a host cell virulence gene, e.g., an spv gene).

[0086] Optionally, the nucleotide sequence (a) comprises a constitutive promoter or a strong promoter for expressing the sequence in a host cell.

[0087] Optionally, the nucleotide sequence or array (b) comprises a constitutive promoter or a strong promoter for expressing the sequence or array in a host cell.

[0088] Optionally, (a) and (b) are comprised of the same operon or are under the control of a common expression operable in said cell (eg, the same promoter).

[0089] Optionally, the promoter is a strong and / or constitutive promoter for expression in the host cell.

[0090] Optionally, the host cell is a wild-type host cell, for example, where the vector is used in a natural environment or human or animal microbiome.

[0091] Optionally, the vector comprises an expressible htpG sequence, for example, wherein the host is an E. coli host, such as contained by a human or animal microbiome. HtpG increases the steady-state Cas3 protein level in E. coli at 37°C. Therefore, this embodiment is particularly useful for modifying a host cell, such as E. coli, contained by a human or animal (e.g., contained by its intestinal microbiome). In one example, the repressed Cas is Cas3.

[0092] In one embodiment, a nucleic acid vector for introduction into a host cell is provided, wherein the host cell contains a CRISPR / Cas system comprising Cascade and Cas3, wherein the Cascade is repressed in the host cell (e.g., by H-NS), and the vector comprises (i) an expressible nucleotide sequence encoding a derepressor of the Cascade repression (e.g., LeuO); and (ii) an expressible nucleotide sequence encoding Cas3, wherein the Cas3 is capable of functioning together with a derepressed Cascade in a host cell; wherein the nucleotide sequence is capable of being expressed in a host cell, wherein the derepressor derepresses or activates Cascade, wherein the Cascade and Cas3 function to modify the protospacer sequence of the host cell genome or the protospacer sequence of an episome contained in the host in the presence of the derepressor; or A nucleic acid vector for introduction into a host cell, wherein the host cell contains a CRISPR / Cas system comprising Cascade and Cas3, wherein the Cascade is repressed in the host cell (e.g., by H-NS), the vector comprising (i) an expressible nucleotide sequence encoding a derepressor of the Cascade repression (e.g., LeuO); and (ii) an expressible nucleotide sequence encoding Cas3, wherein the Cas3 is capable of functioning together with a derepressed Cascade in a host cell; The nucleotide sequence is capable of being expressed in a host cell, wherein the derepressor derepresses or activates Cascade, wherein the Cascade and Cas3 function to modify the original spacer sequence of the host cell genome or modify the original spacer sequence of an episome contained in the host in the presence of the derepressor.

[0093] Optionally, the nucleotide sequences of (i) and (ii) are comprised by the same operator or are under the control of a common expression control operable in said cell (eg, the same promoter).

[0094] Optionally, sequences (i) and (ii) and the CRISPR array or the sequence encoding the gRNA are comprised by two or more different vectors, which are used to be introduced into a host cell for expressing the derepressor, Cas3 and the array or gRNA together in the host cell.

[0095] One aspect provides:- A nucleic acid vector for introduction into a bacterial or archaeal host cell (optionally according to any other configuration, example, embodiment or aspect of the invention), wherein the cell comprises an endogenous CRISPR / Cas system that is naturally repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing a CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences each encoding a respective guide RNA (gRNA, e.g., a single guide RNA) in the cell; wherein each crRNA or gRNA is capable of directing Cas to modify a protospacer sequence of the host cell genome or to modify a protospacer sequence of an episome contained by the host in the presence of a derepressor; wherein the repressor is H-NS, StpA, LRP or CRP encoded by the cell genome, or a homolog, ortholog or functional equivalent thereof (e.g., a homolog, ortholog or functional equivalent of E. coli K12 H-NS, StpA, LRP or CRP); and The derepressor is LeuO or a homologue, orthologue or functional equivalent thereof (e.g., a homologue, orthologue or functional equivalent of E. coli K12 LeuO) that can form a complex with a repressor or E. coli K12 H-NS, StpA, LRP or CRP; or a mutant of H-NS, StpA, LRP or CRP that can form a complex with a repressor (e.g., a mutant of E. coli K12 H-NS, StpA, LRP or CRP).

[0096] In one example, the repressor inhibits, spatially blocks, or binds to one or more sequences of the CRISPR / Cas system (e.g., a promoter sequence, e.g., a promoter of a repressed Cas) to reduce or prevent transcription of Cas in a cell. For example, the promoter sequence is a Cas (e.g., CasA or Cas3) gene promoter. In one example, the repressor can compete with the H-NS, StpA, or CRP for binding to a type I CasA (cse1) promoter. In one example, the repressor can compete with the H-NS, StpA, or CRP for binding to a type I (e.g., IB or -F type) Cas3 promoter. For example, this can be determined in vitro using a standard competition assay such as surface plasmon resonance (SPR) or ELISA. In one example, the repressor inhibits, spatially blocks, or binds to σ 70 -dependent promoter sequence, P cispr1 Promoter sequence, P cas Promoter sequence and / or anti-P cas Promoter sequence, for example, wherein the host cell is an Escherichia coli cell.

[0097] In one example, the derepressor is a LysR-type regulatory protein. In one example, the derepressor can compete with LeuO for binding to a type I CasA (cse1) or Cas3 promoter (e.g., such a promoter from E. coli K12). For example, this can be determined in vitro using a standard competition assay (such as SPR or ELISA). In one example, the repressor is H-NS, and the derepressor is H-NS G113D .

[0098] In one example, the repressor binds to a CasA promoter comprised by a CRISPR array of a CRISPR / Cas system of a host cell species, wherein the cas is the repressor Cas, e.g., CasA or Cas3.

[0099] In Escherichia coli, σ 70-dependent promoter, which is approximately 50 bp upstream of the first (5'-most) nucleotide of the first CRISPR repeat from the type I array. DNA enzyme footprinting indicated the presence of a σ located between positions -40 and -90 70 -dependent promoter, which is referred to as Pcrispr1. Thus, in one example, the repressor binds to a σ comprised by the CRISPR array of the host cell's CRISPR / Cas system. 70 In one example, the repressor inhibits the σ binding sequence of the CRISPR array of the CRISPR / Cas system of the host cell. 70 RNA polymerase transcription.

[0100] In one example, the derepressor is an H-NS paralog, e.g., as discussed further below.

[0101] StpA, which is a paralog of H-NS with 58% amino acid identity (Zhang and Belfort, 1992), shows DNA-binding characteristics very similar to H-NS, generating the same large region of DNase I protection in its target binding site. Consistent with the generally higher affinity for DNA (Zhang et al., 1996), StpA achieves complete protection at slightly lower concentrations than H-NS. LRP and FIS cause weaker protection from DNase I cleavage. In one example, the repressor is a nucleoid-associated protein (NAP). In one example, the derepressor is a mutant of a nucleoid-associated protein (NAP), wherein the mutant competes with the NAP for binding to the target binding site of the NAP (e.g., an IGLB sequence), such as a binding site contained by a CRISPR array of a CRISPR / Cas system of a host cell species, wherein the Cas is the repressed Cas (e.g., Cas3 or CasA). In one example, the NAP is StpA, LRP, FIS, or H-NS. In one example, the repressor is H-NS, and the derepressor is LRP and / or FIS expressed from a vector, for example, by being under the control of a strong or constitutive promoter for expression in a host cell. In this way, the derepressor can be overexpressed to compete with H-NS for binding to a target binding site, but the derepressor may not repress or only weakly repress Cas or Cascade activity in a host cell. The promoter (or any other promoter herein) can be, for example, a bacterial constitutive promoter OXB17, OXB18, OXB19 or OXB20, preferably the latter, because it is the strongest (see http: / / www.oxfordgenetics.com / Products / Plasmids / Details / Bacterial / pSF-OXB18 / OG561). In one example, the promoter is a T7 promoter, and the vector encodes a T7 RNA polymerase for expression in a host cell.

[0102] Many factors that affect H-NS silencing and anti-silencing have been documented in the past (Navarre et al., 2007; Stoebel et al., 2008), including, for example, SlyA (Lithgow et al., 2007; Perez et al., 2008). Like some other related proteins, SlyA itself does not act as a regulator that interacts with DNA target sites, but forms a DNA-binding defective heterodimer with H-NS, thereby counteracting H-NS-mediated silencing. Therefore, in one example, the derepressor comprises SlyA. In one example, the derepressor comprises PhoP, PhoQ, Crp and / or Fnr. Various anti-silencing mechanisms have been observed, involving (i) protein-independent processes that function at the level of local DNA structure, (ii) DNA binding proteins such as Ler, LeuO, RovA, SlyA, VirB, and proteins associated with AraC, and (iii) regulatory mechanisms in which H-NS forms heterologous protein-protein complexes with full-length or partial paralogs such as StpA, Sfh, Hha, YdgT, YmoA, or H-NST. The RovA protein is a homolog of SlyA, which was initially identified as a positive regulator of inv (the gene encoding invasin), which responds to temperature and growth phase in Yersinia (Cathelyn et al., 2007). It is now known that RovA controls the transcription of regulators of genes that are repressed by H-NS proteins like inv. It has been proposed that the main function of RovA in Yersinia enterocolitica is to act as an antagonist of H-NS-mediated transcriptional silencing (Cathelyn et al., 2007). Thus, in one example, the derepressor comprises one, two, three or more of Ler, LeuO, RovA, SlyA, VirB, AraC, StpA, Sfh, Hha, YdgT, YmoA and H-NST; or their homologs, orthologs, paralogs or functional equivalents thereof.

[0103] The major virulence factors of Vibrio cholerae, the causative agent of Asian cholera, are encoded by genes within A+T-rich horizontally transmissible genetic elements (Davis & Waldor, 2003; McLeod et al., 2005; Murphy & Boyd, 2008). These genes are regulated by several environmental signals to ensure that their products are expressed when the bacteria reach the appropriate site in the host and that they are repressed elsewhere (Lee et al., 1999; Schild et al., 2007). The major virulence factors expressed by Vibrio cholerae are cholera toxin, CTX, and the toxin-coregulated cilia, Tcp (Skorupski & Taylor, 1997). H-NS silences the transcription of the genes encoding these major virulence factors by targeting their A+T-rich promoters (Nye et al., 2000). This silencing is countered by the ToxT regulatory protein, an AraC-like DNA binding protein that derepresses transcription of many virulence gene promoters in Vibrio cholerae (Yu & DiRita, 2002). It is believed that this mechanism involves not only displacement of H-NS, but also activation of ToxT transcription, which may be due to a direct interaction between ToxT and RNA polymerase (Hulbert & Taylor, 2002; Yu & DiRita, 2002). Therefore, in one example, the derepressor comprises ToxT (e.g., Vibrio cholerae ToxT). Therefore, in one example, the derepressor comprises AraC or a homologue, orthologue, paralogue or functional equivalent thereof. Examples of the latter are AppY, CfaD, GadW, GadX, HilC, HilD, PerA, RegA, Rns, UreR and VirF.

[0104] The ability to form nucleoprotein filaments with DNA plays an important role in H-NS-mediated transcriptional silencing. LeuO has been identified as a protein that can set limits for the polymerization of H-NS along genetic material. It is a LysR-like DNA-binding protein, which is identified as a transcriptional activator in a promoter relay that controls the expression of the leuABCD operon in Salmonella typhimurium (Chen & Wu, 2005; Chen et al., 2005; Fang & Wu, 1998). Therefore, in one example, the derepressor comprises LysR or its homologue, orthologue, paralogue or functional equivalent.

[0105] Other nucleoid-related proteins can antagonize the binding of H-NS to DNA. Experiments with magnetic tweezers and atomic force microscopy have shown that abundant HU proteins can compete with H-NS for the same binding sites in DNA, opening H-NS-concentrated promoter regions (van Noort et al., 2004). It has also been reported that Fis protein antagonizes H-NS repression, such as at rRNA gene promoters in those whose binding sites are distributed in H-NS (Schneider et al., 2003). In the later stages of growth, when Fis levels are low, H-NS represses rRNA gene promoters (Afflerbach et al., 1998). Nucleoid-related proteins HU and RpoS stress and stationary phase σ factors of RNA polymerase have been described as having positive regulatory effects at proU promoters repressed by H-NS in Escherichia coli (Manna & Gowrishankar, 1994), and more extensive overlaps between H-NS and RpoS regulators have been described (Barth et al., 1995). This may indicate a role for RpoS in overcoming H-NS-mediated repression in bacteria undergoing stress. Thus, in one example, the derepressor comprises HU, RpoS and / or Fis; or a homologue, orthologue, paralogue or functional equivalent thereof.

[0106] The protein group of interest consists of small polypeptides with homology to the oligomerization domain of H-NS. Those with the closest amino acid sequence similarity to this domain are members of the H-NST family, so called because they resemble H-NS truncations that lack the nucleic acid binding and linker domains (Williamson & Free, 2005). Genes encoding these truncations have been detected in the pathogenicity islands of various pathogenic enterobacteria, including enteropathogenic E. coli (EPEC) and uropathogenic E. coli. The protein H-NST (EPEC) from EPEC co-purifies with H-NS. This protein can interfere with the ability of H-NS to repress the proU operon in E. coli. Therefore, in one example, the de-repressor comprises H-NST; or a homologue, orthologue, paralogue or functional equivalent thereof; for example, wherein the repressor is H-NS or StpA.

[0107] Genes encoding small proteins that directly interact with H-NS are found in ancestral chromosomes and on islands acquired horizontally. The YmoA protein of Yersinia was originally identified as a regulator of virulence gene expression in Yersinia enterocolitica (Cornelis et al., 1991). It is related to the Hha protein originally found as a regulator of hemolysin gene expression in Escherichia coli, and the two proteins can functionally replace each other (Balsalobre et al., 1996; Mikulskis & Cornelis, 1994). Hha protein must interact with H-NS to play its role in hemolysin gene expression; YmoA also interacts with H-NS, and this relationship is utilized in separating H-NS protein from Yersinia (Nieto et al., 2000, 2002). The solution structure of YmoA has been resolved using nuclear magnetic resonance spectroscopy (McFeeters et al., 2007). This result helps to believe that YmoA (and Hha) should be regarded as an independent oligomerization domain of H-NS. Potentially, the proteins can oligomerize to produce YmoA-H-NS and Hha-H-NS heteromers. The absence of nucleic acid binding domains on the YmoA and Hha partners can result in the inability of the heteromers to participate in DNA-protein-DNA bridging, compromising (or at least modifying) the structure of the repressor complex. The discovery of paralogs of Hha-like proteins has added another layer of complexity. The ydgT gene encodes an Hha-like protein in Escherichia coli and Salmonella, and it can interact with H-NS and the H-NS paralog StpA protein (Paytubi et al., 2004). Thus, in one example, the derepressor comprises YmoA and / or Hha and / or ydgT; or a homolog, ortholog, paralog or functional equivalent thereof; for example, wherein the repressor is H-NS or StpA.

[0108] Not all H-NS paralogs are thought to act through direct protein-protein interactions with H-NS. The Ler DNA-binding protein is encoded by the LEE (site of enterocyte shedding) pathogenicity island of enterohemorrhagic Escherichia coli (EHEC) and EPEC. At 37°C, it activates transcription of the main virulence operon in the island by counteracting the silencing activity of H-NS (Barba et al., 2005; Bustamante et al., 2001; Haack et al., 2003; Umanski et al., 2002). Ler and H-NS are partial paralogs whose oligomerization domains are highly divergent coiled-coils; there is no evidence that Ler and H-NS form heterodimers. Instead, Ler is thought to displace H-NS (Haack et al., 2003). It also acts on gene expression outside of LEE (Elliott et al., 2000). For example, Ler counteracts the silencing activity of H-NS at the lpf operon in EHEC, which encodes long polar pili (Torres et al., 2007). Thus, despite its homology to H-NS, Ler acts more like VirB or SlyA. Thus, in one example, the derepressor comprises Ler; or a homolog, ortholog, paralog or functional equivalent thereof; for example, wherein the repressor is H-NS or StpA.

[0109] It is known that the gp5.5 protein from bacteriophage T7 binds and inactivates H-NS, thereby supporting the propagation of the phage (Liu and Richardson, 1993). Therefore, in one example, the derepressor comprises gp5.5; or a homologue, orthologue, paralogue or functional equivalent thereof; for example, wherein the repressor is H-NS or StpA.

[0110] Those skilled in the art may ask why the CRISPR-cas system is hidden. Because Cas proteins are involved in the integration of exogenous DNA spacers, cells must avoid erroneous integration of host DNA elements. The continuous expression of cas genes may of course be harmful to cells. Therefore, there must be a mechanism that silences cas gene expression until needed. Our data show that H-NS is at least an important component responsible for this control. Therefore, in one configuration, the present invention provides any vector disclosed herein (or the use of such a vector or composition or a method using such a vector or composition), except that the vector does not include a nucleotide sequence encoding crRNA or gRNA or does not include a CRISPR array, but wherein the vector encodes one or more of the de-repressors (for example, and the repressor is H-NS). In this configuration, the expression of the de-repressor in the cell de-represses or activates endogenous Cas expression (e.g., Cas3 and / or Cascade Cas, such as CasA) in the host cell, and this expression causes modification (e.g., cutting host DNA, such as chromosomal DNA), which kills cells or reduces cell growth or proliferation. For each derepressor, it may be advantageous to encode in the vector from a strong and / or constitutive promoter for expression in the host cell. High levels of anti-repressor expression can displace repressors such as H-NS and cause endogenous Cas to cut the chromosome or other DNA of the host cell, thereby killing the host cell or reducing its growth or proliferation.

[0111] For example, the vector does not include a CRISPR array for producing one or more crRNAs in the cell; and does not include one or more nucleotide sequences that each encode a respective guide RNA (gRNA, such as a single guide RNA) in the cell. For example, the vector does not encode crRNA or gRNA. This may be useful when derepression is sufficient to activate endogenous Cas nuclease activity in the host cell, thereby killing the host cell or inhibiting host cell growth or proliferation. For example, the repressor is H-NS or StpA, and the derepressor is LeuO or any other derepressor disclosed herein. For example, the expression of the derepressor is under the control of a strong and / or constitutive promoter for expression in the host cell.

[0112] In one embodiment, the present invention provides:- A method of treating or preventing a disease or condition in a human or animal subject, the method comprising administering a vector to the subject, wherein a host cell (e.g., Escherichia coli, Salmonella, or Salmonella enterica serovar Typhimurium) comprised by the subject's microbiome is modified by the cell's endogenous derepressed Cas, and carrying out the treatment or prevention; wherein (i) each cell comprises a CRISPR / Cas system (e.g., Cascade Cas, Cas3 or Cas9 is repressed) that is repressed by a repressor (e.g., H-NS and / or StpA) in the cell, (ii) the vector comprises a nucleotide sequence encoding a derepressor (e.g., LeuO) capable of derepressing the CRISPR / Cas system in the cell, wherein the sequence is expressible in the cell to produce the derepressor (e.g., under the control of a strong and / or constitutive promoter); and (iii) the vector does not have a CRISPR array for producing one or more crRNAs in the cell; and does not have one or more nucleotide sequences encoding respective guide RNAs (gRNAs, e.g., single guide RNAs) in the cell.

[0113] A method for treating or preventing a disease or condition in a human or animal subject, the method comprising administering a vector to the subject, wherein a host cell (e.g., Escherichia coli, Salmonella or Salmonella enterica serovar Typhimurium) comprised by the subject's microbiome is modified by an endogenous derepressed Cas of the cell, and carrying out the treatment or prevention; wherein (i) each cell comprises a CRISPR / Cas system repressed by a repressor selected from H-NS and / or StpA in the cell, wherein Cascade Cas, Cas3 or Cas9 is repressed, (ii) the vector comprises a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in the cell, wherein the sequence is expressible in the cell to produce the derepressor, which is under the control of a strong and / or constitutive promoter; and (iii) the vector does not have a CRISPR array for producing one or more crRNAs in the cell; and does not have one or more nucleotide sequences encoding respective guide RNAs (gRNAs, e.g., single guide RNAs) in the cell.

[0114] A method for killing a wild-type bacterial or archaeal cell (e.g., an E. coli or Salmonella cell), wherein the cell comprises an endogenous CRISPR / Cas system, the CRISPR / Cas system comprising nucleotide sequences encoding Cas3 and Cascade proteins, wherein Cas3 and / or Cascade are naturally repressed in the cell, the method comprising derepressing the Cas3 and / or Cascade without introducing into the cell (i) a CRISPR array for producing one or more crRNAs in the cell; or (ii) one or more nucleotide sequences encoding respective guide RNAs (gRNAs, e.g., single guide RNAs); or without engineering the host cell to encode crRNAs or guide RNAs. In one example, the method comprises introducing into the host cell a nucleic acid vector, wherein (i) the cell comprises a CRISPR / Cas system repressed by a repressor selected from H-NS and / or StpA in the cell, wherein Cascade Cas, Cas3 or Cas9 is repressed, (ii) the vector comprises a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in the cell, wherein the sequence is expressible in the cell to produce the derepressor, which is under the control of a strong and / or constitutive promoter; and (iii) the vector does not have a CRISPR array for producing one or more crRNAs in the cell; and does not have one or more nucleotide sequences encoding respective guide RNAs (gRNAs, e.g., single guide RNAs) in the cell.

[0115] A medicament comprising a plurality of nucleic acid vectors as described herein for introduction into a host cell, optionally further comprising one or more medical drugs (e.g., anticancer drugs) or antibiotics (e.g., wherein the protospacer sequence is comprised by a host cell antibiotic resistance gene), the medicament being used to treat or prevent a disease or condition in a human or animal; wherein (i) each cell comprises a CRISPR / Cas system repressed by a repressor selected from H-NS and / or StpA in the cell, wherein Cascade Cas, Cas3 or Cas9 is repressed, (ii) the vector comprises a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in the cell, wherein the sequence is expressible in the cell to produce the derepressor, which is under the control of a strong and / or constitutive promoter; and (iii) the vector does not have a CRISPR array for producing one or more crRNAs in the cell; and does not have one or more nucleotide sequences encoding respective guide RNAs (gRNAs, e.g., single guide RNAs) in the cell.

[0116] In one example, described or every kind of host cell (or the first and / or the second bacterium) is a gram-positive cell. In one example, described or every kind of host cell is an enterobacteriaceae, such as salmonella, pestis yersinia, klebsiella, shigella, proteus, enterobacter, serratia or citrobacter cell. Optionally, described or every kind of cell is an escherichia coli (e.g., escherichia coli K12) or salmonella (e.g., enteric salmonella typhimurium blood group variant) cell. Optionally, described or every kind of host cell (or the first and / or the second bacterium) is a gram-negative cell.

[0117] Optionally, the host (or the first and / or the second bacterium) is a Mycoplasma, Chlamydia, Spirochete or Mycobacterium tuberculosis. Optionally, the host (or the first and / or the second bacterium) is a Streptococcus (e.g., Streptococcus pyogenes or Streptococcus thermophilus) host. Optionally, the host (or the first and / or the second bacterium) is a Staphylococcus (e.g., Staphylococcus aureus, e.g., MRSA) host. Optionally, the host (or the first and / or the second bacterium) is an Escherichia coli (e.g., O157:H7) host. Optionally, the host (or the first and / or the second bacterium) is a Pseudomonas (e.g., Pseudomonas aeruginosa) host. Optionally, the host (or the first and / or the second bacterium) is a Vibrio (e.g., Vibrio cholerae (e.g., O139) or Vibrio vulnificus) host. Optionally, the host (or the first and / or the second bacterium) is a Neisseria (e.g., Neisseria gonorrhoeae or Neisseria meningitidis) host. Optionally, the host (or the first and / or the second bacterium) is a Bordetella (e.g., Bordetella pertussis) host. Optionally, the host (or the first and / or the second bacterium) is a Haemophilus (e.g., Haemophilus influenzae) host. Optionally, the host (or the first and / or the second bacterium) is a Shigella (e.g., Shigella dysenteriae) host. Optionally, the host (or the first and / or the second bacterium) is a Brucella (e.g., Brucella abortus) host. Optionally, the host (or the first and / or the second bacterium) is a Francisella host. Optionally, the host (or the first and / or the second bacterium) is a Xanthomonas host. Optionally, the host (or the first and / or the second bacterium) is an Agrobacterium host. Optionally, the host (or the first and / or the second bacterium) is an Erwinia host. Optionally, the host (or the first and / or the second bacterium) is a Legionella (e.g., Legionella pneumophila) host. Optionally, the host (or the first and / or the second bacterium) is a Listeria (e.g., Listeria monocytogenes) host. Optionally, the host (or the first and / or the second bacterium) is a Campylobacter (e.g., Campylobacter jejuni) host. Optionally, the host (or the first and / or the second bacterium) is a Yersinia (e.g., Yersinia pestis) host. Optionally, the host (or the first and / or the second bacterium) is a Borrelia (e.g., Borrelia burgdorferi) host. Optionally, the host (or the first and / or the second bacterium) is a Helicobacter (e.g., Helicobacter pylori) host. Optionally, the host (or the first and / or the second bacterium) is a Clostridium (e.g., Clostridium difficile or Clostridium botulinum) host. Optionally, the host (or the first and / or the second bacterium) is an Ehrlichia (e.g., Ehrlichia chaffeensis) host. Optionally, the host (or the first and / or second bacteria) is a Salmonella (e.g., Salmonella typhi or Salmonella enterica, e.g., Salmonella serotype Typhimurium, e.g., DT 104) host. Optionally, the host (or the first and / or second bacteria) is a Chlamydia (e.g., Chlamydia pneumoniae) host. Optionally, the host (or the first and / or second bacteria) is a Parachlamydia host.Optionally, the host (or the first and / or the second bacterium) is a Corynebacterium (e.g., Corynebacterium amycoticum) host. Optionally, the host (or the first and / or the second bacterium) is a Klebsiella (e.g., Klebsiella pneumoniae) host. Optionally, the host (or the first and / or the second bacterium) is an Enterococcus (e.g., Enterococcus faecalis or Enterococcus faecium, e.g., Linezolid-resistant) host. Optionally, the host (or the first and / or the second bacterium) is an Acinetobacter (e.g., Acinetobacter baumannii, e.g., multidrug-resistant) host.

[0118] Optionally, the derepressed Cas is Cas3. Optionally, the derepressed Cas is Cas9. Optionally, the derepressed Cas is Cascade Cas, for example, when the host cell is an E. coli cell. Cascade is also known as CRISPR-associated complex, which is used for antiviral defense.

[0119] Optionally, the CRISPR / Cas system is a type I (eg, type IB, type IE, or type IF) system.

[0120] Optionally, the protospacer sequence is comprised by an essential gene, virulence gene or antibiotic resistance gene comprised by the cell.

[0121] Optionally, the vector does not comprise a sequence from the group consisting of CasA, B, C, D and E (e.g., when the cell is an Escherichia coli cell) or CasABCDE12 (e.g., when the host cell is a Salmonella enterica serovar Typhimurium cell) nucleotide sequences, or wherein the vector does not comprise all sequences of said group.

[0122] Optionally, the vector does not comprise a sequence from the group consisting of Cas1, Cas2, Cas5 and Cas6 sequences.

[0123] Optionally, the vector does not comprise a sequence selected from the group consisting of vectors not comprising a Cas 3 nucleotide sequence.

[0124] Optionally, the vector of the present invention or any other aspect, it is used for the medical use of the disease or the patient's condition in the treatment or prevention of human or animal subjects, wherein the host cell is comprised by the subject. In one example, the host cell is a disease (i.e., infection of the subject by the host cell) or is related to or mediates a disease or condition (e.g., IBD, colitis, Crohn's disease, cancer, autoimmune disease or condition, obesity, diabetes or CNS disease or condition (e.g., Alzheimer's disease or Parkinson's disease)). Optionally, the vector of the present invention or any other aspect, it is used for the medical method of treating, preventing, diagnosing human or animal bodies.

[0125] Optionally, the vector or any other aspect of the invention is used to reduce the growth or proliferation of host cells in an environment (e.g., soil, a composition comprising the host cell and yeast cells), a human, an animal, or a plant microbiome. For example, this is useful when the microbiome exists naturally.

[0126] Optionally, the vector or any other aspect of the invention is used to kill various host cells or to reduce their growth or proliferation.

[0127] The or each host cell may be comprised by a microbiome (e.g., a gut microbiome or an environmental microbiome) comprising a plurality of such host cells and comprising one or more cells of a species or strain (e.g., a bacterial species or strain, or an archaeal species or strain) different from the species or strain of the host cell (e.g., a bacterial or archaeal host cell).

[0128] One aspect provides a medicament comprising a plurality of vectors according to the invention, optionally further comprising one or more medical drugs (e.g., anticancer drugs) or antibiotics (e.g., wherein the protospacer sequence is comprised by a host cell antibiotic resistance gene), for use in treating or preventing a disease or condition in a human or animal.

[0129] One aspect provides a method for treating or preventing a disease or condition in a human or animal subject, the method comprising administering a vector or medicament of the invention to the subject, wherein the subject's microbiome comprises a host cell modified by the cell's endogenous derepressed Cas, and carrying out the treatment or prevention.

[0130] One aspect provides a method for killing a wild-type bacterial or archaeal cell (e.g., an E. coli or Salmonella cell), wherein the cell comprises an endogenous CRISPR / Cas system, the CRISPR / Cas system comprising nucleotide sequences encoding Cas3 and Cascade proteins, wherein Cas3 and / or Cascade are naturally repressed in the cell, the method comprising (a) derepressing the Cas3 and / or Cascade, and (b) introducing into the cell (i) a CRISPR array for producing one or more crRNAs in the cell; or (ii) one or more nucleotide sequences each encoding a respective guide RNA (gRNA, e.g., a single guide RNA); Each crRNA or gRNA guides Cas or Cascade to modify the respective original spacer sequence of the host cell genome or to modify the original spacer sequence of the episome contained in the host.

[0131] Optionally, in step (b), a vector according to the present invention is introduced into a cell, thereby introducing (i) or (ii) into the cell.

[0132] Optionally, Cas3 transcription, expression or activity is derepressed.

[0133] Optionally, Cas transcription, expression or activity is derepressed, wherein the Cas is a Cascade Cas (e.g., Cas A, B, C, D or E).

[0134] Optionally, an expressible de-repressor sequence is introduced into the cell simultaneously or sequentially with the array or the sequence encoding the gRNA.

[0135] Optionally, the derepressor sequence and the array or gRNA encoding sequence are comprised by the same nucleic acid vector (eg, phagemid, phage or plasmid).

[0136] Optionally, the derepressor sequence and the array or gRNA encoding sequence are contained by the same operator or are under the control of a common expression control (e.g., the same promoter) operable in the cell. This facilitates the coordination of expression of these elements in the host cell.

[0137] Optionally, step (a) comprises expressing in the cell (i) LeuO or a homologue, orthologue or functional equivalent thereof capable of forming a complex with H-NS; or (ii) a mutant of H-NS, StpA, LRP or CRP capable of forming a complex with H-NS, StpA, LRP or CRP repressor, respectively.

[0138] Also provided are pharmaceutical compositions, foods, beverages, compositions for environmental remediation, pesticides, herbicides or cosmetics comprising one or more carriers according to the present invention.

[0139] One aspect provides a nucleic acid vector for introduction into a host cell, wherein the cell comprises a CRISPR / Cas system that is repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing a CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) for introducing the following sites: (i) a CRISPR array or CRISPR spacer sequence for producing one or more crRNAs in a cell; or (ii) a nucleotide sequence encoding a guide RNA (gRNA, e.g., a single guide RNA) in a cell; Wherein the crRNA (e.g., when comprised by a guide RNA) or gRNA is capable of directing Cas to modify the respective original spacer sequence of the host cell genome or to modify (e.g., cut or cause mutation thereof) the original spacer sequence of an episome contained by the host in the presence of a derepressor.

[0140] Optionally, site (b) is comprised by a CRISPR array, wherein the array is capable of accommodating one or more of the spacer sequences for targeting the respective original spacer sequences of the host cell. This can be used to prevent the development of resistance of the host cell to the vector.

[0141] Optionally, the array of (i) or the sequence of (ii) is under the control of a promoter that is a strong and / or constitutive promoter for expression in a host cell. A strong promoter can be used to maximize the opportunity for expression during any or different stages of growth or existence of a host cell population.

[0142] Optionally, the sequence of (a) is under the control of a promoter that is a strong and / or constitutive promoter for expression of the derepressor in a host cell.

[0143] Optionally, an expressible Cas3 sequence and / or an expressible htpG sequence is used for expression in a host cell. Upregulation of Cas3 by htpG may be beneficial in promoting modification of the target sequence.

[0144] Optionally, each of the crRNAs is operable with a Cas (e.g., a Cas nuclease, e.g., Cas9 or Cas3) in a host cell.

[0145] Optionally, the Cas is encoded by an endogenous nucleotide sequence of the host cell genome, wherein when derepressed, the Cas has nuclease activity.

[0146] Optionally, the Cas is encoded by a nucleotide sequence comprised by the vector or a different vector that can be introduced into a host cell for expressing the Cas therein.

[0147] Optionally, when the spacer of (i) or the sequence of (ii) has been inserted therein, then the vector is according to the vector of the present invention which can be introduced into a host cell.

[0148] BglJ-RcsB heteromers are known to activate the HNS-repressed leuO and bgl loci, and thus in one example, the vector encodes BglJ and / or RcsB for forming BglJ-RcsB heteromers in the host cell.

[0149] In one example, the protospacer is comprised of a gene encoding a protein that mediates quorum sensing of a host cell population (e.g., a BglJ or LuxI family gene). LuxI family proteins generate N-acyl homoserine lactone (AHL) quorum sensing signals, and therefore targeting these to reduce host cell population growth, viability or proliferation may be advantageous.

[0150] In the examples of promoters herein, the promoter is a constitutive promoter, which has no binding sites for H-NS or H-NS family members. A notable feature of a constitutive promoter is the high level of conservation of the classical TTGACA (-35) -17bp-TATAAT (-10) sequence, and thus in one example, the promoter comprises TTGACA and / or TATAAT sequences.

[0151] In the example of a promoter herein, the promoter is an H-NS promoter, for example, it comprises the sequence of an H-NS promoter of the host cell species. This can be used to provide expression of vector sequences in the cell at the same ratio and / or time as the H-NS repressor.

[0152] Hha and H-NS increase the inhibitory ability of H-NS. In one example, the vector encodes the inhibitor Hha / H-NS multimerization or YdgT / H-NS multimerization, which is used to express the inhibitor in a host cell.

[0153] In one example, the host cell is a Salmonella enterica serovar Typhimurium strain LT2 cell. It is proposed that the expression of the E-type (Cse) cas gene from Salmonella enterica may be regulated by H-NS and LeuO. For example, in Salmonella enterica serovar Typhimurium, the transcription of casA (STY3070) seems to be affected by H-NS and LeuO (Hernandez-Lucas et al., 2008), although the conservation of the intergenic region between the cas3 and casA in the strain is poor.

[0154] In one example, the host cell is an E. coli EPEC, EHEC, K12 or strain MG1655 cell.

[0155] Since H-NS is known to bind directly to incoming phage or plasmid DNA (Navarre et al., 2006; Navarre et al., 2007), this may result in redistribution of H-NS (Doyle et al., 2007; Dillon et al., 2010), allowing expression of Cascade genes due to reduced local concentrations of repressors. In one example, the vector comprises a H-NS or StpA binding site. In one example, the vector does not comprise a H-NS or StpA binding site.

[0156] Since leuO expression is negatively regulated by H-NS and positively regulated by LeuO itself (Hommais et al., 2001; Chen et al., 2005), this will further amplify the activation signal of cas gene transcription. In one example, the vector contains multiple sequences encoding LeuO. In the presence of LeuO protein, this can usefully amplify positive feedback.

[0157] The intergenic region (IGLB, for the intergenic region ygcL-ygcB) between the Cascade region, casA gene (ygcL) and cas3 gene (ygcB) of the host cell may contain a binding site for H-NS (e.g., in E. coli). Therefore, in one example, the vector encodes an inhibitor that inhibits the binding of the repressor to one or more IGLB regions of the host cell genome. For example, in E. coli, the following primers can be used to amplify the IGLB sequence -UP-IGLB (used as an upstream primer for cloning the IGLB region) 5'-TTG TTC TCC TTC ATATGC TCC GACATT TCT-3'(SEQ ID NO:1) DOWN-IGLB (used as downstream primer for cloning the IGLB region) 5'-CTT CGG GAATGATTG TTATCAATGACGATA-3'(SEQ ID NO:2) The casA-cas3 intergenic region (herein indicated as IGLB) contains Pcas, to which H-NS has strong binding affinity and an anti-cas3 (referred to as anti-Pcas) promoter located 80 bp upstream of Pcas, and produces antisense transcripts of unknown function (Pul et al., Mol Microbiol. 2010 Mar; 75 (6): 1495-512. doi: 10.1111 / j.1365-2958.2010.07073.x. Epub 2010 Feb 1, "Identification and characterization of E. coli CRISPR-cas promoters and their silencing by H-NS"). Both LeuO and H-NS bind to the IGLB fragment, as determined by electrophoretic mobility shift assay (EMSA). In one example of the present invention, the derepressor and / or repressor binds to the CRISPR / Cas system IGLB contained in the host cell genome. In one example of the invention, the derepressor and / or repressor binds to a CRISPR / Cas system Pcas (or a straight homologue or homologue) contained by the host cell genome. In one example of the invention, the derepressor and / or repressor binds to a CRISPR / Cas system anti-Pcas (or a straight homologue or homologue) contained by the host cell genome. The binding site is, for example, comprised by a type I CRISPR array of a host cell.

[0158] As an exemplary test of a derepressor, the derepressor binds to an IGLB sequence (which is contained by the host species genome) as determined by an electrophoretic mobility shift assay (EMSA), e.g., as described in Westra et al. 2010 (Westra et al., Mol Microbiol. 2010 Sep; 77(6): 1380-93. doi: 10.1111 / j.1365-2958.2010.07315.x. Epub 2010 Aug 18, "H-NS-mediated repression of CRISPR-based immunity in Escherichia coli K12 can be relieved by the transcription activator LeuO"). In an in vitro binding assay, pre-bound LeuO will prevent the cooperative binding of H-NS to the IGLB fragment. Consistent with this, when a derepressor is added to the H-NS / LeuO complex, pre-bound H-NS is released from the IGLB portion. To locate the binding region of LeuO or other derepressors within the IGLB sequence, DNase I footprinting analysis can be performed. As described previously, H-NS induces footprint extension after limited DNase I hydrolysis of IGLB DNA (Pul et al., 2010).

[0159] The derepressor can include an oligonucleotide that is complementary to a binding site of a repressor (e.g., H-NS) contained in the host cell genome, e.g., complementary to a Pcas promoter or an anti-cas promoter of the CRISPR / Cas system of the host cell, wherein transcripts can be initiated from a Pcas promoter or an anti-Cas promoter in the presence of the derepressor. In another example, the derepressor cannot bind to a LGLB DNA contained in the host genome.

[0160] Optionally, the derepressor is a dominant negative H-NS mutant protein G113D (Ueguchi et al., 1996; Pul et al., 2007). This H-NS mutant protein has lost its DNA-binding activity but is able to form heteromers with wild-type H-NS. The resulting heteromers have also lost their DNA-binding properties (Pul et al., 2005). For example, the derepressor is G113D or a functional equivalent thereof, for example, encoded in a vector by a nucleotide sequence under the control of a strong and / or constitutive promoter for expressing G113D or its equivalent in a host cell.

[0161] In one example, expression of the derepressor in the host cell is inducible, for example, where the derepressor is encoded by a vector nucleotide sequence under the control of an inducible promoter. For example, the induction may be physical (e.g., heat-induced), by light or by chemical means.

[0162] In one example, the method includes or the vector is used to derepress H-NS repression of RNA polymerase-promoter interaction in a CRISPR / Cas array in a host cell, wherein the Cas is the repressed Cas.

[0163] In one example, the repressor binds to the IGLB region of the CRISPR / Cas system of the host cell. In one example, the repressor binds to the promoter of the CRISPR / Cas system of the host cell. In one example, the repressor is an inhibitor of the RNA polymerase binding site of the CRISPR / Cas system of the host cell. In one example, the repressor is an inhibitor of RNA transcription from the CRISPR / Cas system of the host cell. In one example, the repressor is an inhibitor of RNA transcription from the transcription start site contained by the CRISPR / Cas system of the host cell, wherein the transcription start site is within 100, 50 or 30 nucleotides upstream of the first (most 5'-) nucleotide of the first CRISPR repeat of the CRISPR array of the system, or wherein the start site is within the leader region of the CRISPR array of the system. For example, the repressor is H-NH or StpA that binds to such regions.

[0164] In the E. coli IE type system, the PAM corresponds to the 5'-AWG-3' sequence located immediately upstream (5') of the protospacer. Thus, when the host cell is an E. coli cell, the protospacer is immediately 3' to the PAM, wherein the PAM is 5'-AWG-3', for example, AAG, AGG, GAG or ATG.

[0165] When the host cell is a Streptococcus thermophilus cell, the protospacer is immediately 3' or immediately 5' to the PAM, wherein the PAM is NNAGAAW or NGGNG. In one example, the PAM is 5'-AW-3', such as AA or AT or AG. Optionally, the PAM is the PAM of the CRISPR4 system of Streptococcus thermophilus. For example, the CRISPR array of the present invention or the nucleotide sequence encoding the guide RNA comprises a repeat sequence, wherein the repeat sequence is 5'-GTTTTTCCCGCACACGCGGGGGTGATCC-3' (SEQ ID NO:74).

[0166] Optionally, the system comprises a repressed Cas and (a) the Cas comprises an amino acid sequence selected from SEQ ID NO: 58, 60, 66, and 68 or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% (e.g., at least 80%) identity to a selected sequence or is an ortholog or homolog thereof, and the ortholog or homolog is operable with a repeat sequence comprising a sequence selected from SEQ ID NO: 49-52 and a PAM comprising AWG (e.g., AAG, AGG, GAG, or ATG) or consisting thereof, wherein optionally the host cell is an Escherichia coli cell; or (b) the Cas comprises an amino acid sequence selected from SEQ ID NO: 56, 64, 70, and 72 or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% (e.g., at least 80%) identity to a selected sequence or is an ortholog or homolog thereof, and the ortholog or homolog is operable with a repeat sequence comprising SEQ ID NO: 53, wherein optionally the host cell is Salmonella enterica; or (c) the Cas comprises an amino acid sequence selected from SEQ ID NO: 62 or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% (e.g., at least 80%) identity to a selected sequence or is an ortholog or homolog thereof, and the ortholog or homolog is operable with a PAM comprising NNAGAAW, NGGNG, or AW (e.g., AA, AT, or AG) or consisting thereof, wherein optionally the host cell is a Streptococcus thermophilus cell.

[0167] Optionally, the system comprises a repressed Cas and the Cas is operable with: (a) a repeat sequence comprising a sequence selected from SEQ ID NO: 49-52 and a PAM comprising AWG (e.g., AAG, AGG, GAG, or ATG) or consisting thereof, wherein optionally the host cell is an Escherichia coli cell; (b) a repeat sequence comprising SEQ ID NO: 53, wherein the host cell is Salmonella enterica; or (c) a PAM comprising NNAGAAW, NGGNG, or AW (e.g., AA, AT, or AG) or consisting thereof, wherein optionally the host cell is a Streptococcus thermophilus cell.

[0168] Optionally, the system comprises a repressed Cas and the Cas is operable with: (a) a PAM comprising or consisting of AWG (e.g., AAG, AGG, GAG, or ATG), and optionally the Cas nuclease comprises an amino acid sequence selected from SEQ ID NOs: 58, 60, 66, and 68, or an amino acid sequence having at least 70, 80, 90, 95, or 98% identity to the selected sequence, wherein optionally the host cell is an E. coli cell; or (b) a PAM comprising or consisting of NNAGAAW, NGGNG or AW (e.g., AA, AT or AG), and optionally the Cas nuclease comprises an amino acid sequence selected from SEQ ID NO: 62, or an amino acid sequence having at least 70, 80, 90, 95 or 98% identity to the selected sequence, wherein optionally the host cell is a Streptococcus thermophilus cell.

[0169] Optionally, the target nucleotide sequence or protospacer comprises a sequence of at least 5, 6, 7, 8, 9 or 10 consecutive nucleotides immediately 3' to the PAM in the genome of the host cell. Optionally, the target nucleotide sequence or protospacer comprises a sequence of at least 5, 6, 7, 8, 9 or 10 consecutive nucleotides immediately 5' to the PAM in the genome of the host cell. In one example, the PAM is comprised by a chromosome or episome of the host cell.

[0170] Optionally, the repressor is (i) H-NS comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 19, 21, 23, 25 and 27, or an amino acid sequence having at least 70, 80, 90, 95 or 98% identity thereto; or (ii) StpA comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 31, 33 and 35, or an amino acid sequence having at least 70, 80, 90, 95 or 98% identity thereto.

[0171] Optionally, the derepressor is (iii) a LeuO comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13 and 15, or an amino acid sequence having at least 70, 80, 90, 95 or 98% identity thereto; or (iv) an LRP comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 39 and 41, or an amino acid sequence having at least 70, 80, 90, 95 or 98% identity thereto; or (v) a CRP comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 45 and 47, or an amino acid sequence having at least 70, 80, 90, 95 or 98% identity thereto.

[0172] In one example, the CRISPR / Cas system contained by a host cell (e.g., a mammalian, human, mouse, bacterial or archaeal cell) comprises a repressed Cas, such as Cas1, 2, 3, 9, A, B, C, D or E. For example, the Cas is encoded by an endogenous nucleotide sequence of the host cell, wherein the host cell is a bacterial or archaeal cell. In another example, the Cas is encoded by an exogenous nucleotide sequence contained by a cell (e.g., a cell that has been introduced by a vector (e.g., a vector of the present invention)).

[0173] In certain embodiments of the invention, the host cell is a bacterial or archaeal cell comprising an endogenous CRISPR / Cas system, wherein the system comprises a repressed Cascade or Cas (e.g., Cas1, 2, 3, 9, A, B, C, D or E), which is encoded by one or more endogenous nucleotide sequences of the host genome. Advantageously, the present invention can be used to de-repress Cascade or Cas, whereby Cascade or Cas can be used to modify (e.g., cut) a target protospacer sequence contained in the host genome (e.g., a chromosome or episomal protospacer sequence), for example to kill cells. This aspect relates to introducing one or more vectors encoding crRNA or guide RNA (e.g., a single guide RNA) that can operate with Cascade or Cas once Cascade or Cas has been de-repressed into a host cell. De-repression is implemented by means of a de-repressor of the present invention carried by one or more of the vectors, wherein the de-repressor is expressed in the host cell for de-repressing the repressed Cascade or Cas. Usefully, the ability to utilize the de-repressed endogenous Cas enables the omission of the corresponding Cas coding sequence on the vector of the present invention. This frees up valuable space on the vector (especially given that some Cas encoding sequences are large, e.g., the Streptococcus pyogenes Cas9 sequence is 4.2 kb, which approaches the packaging capacity of, e.g., phage vectors. For example, the free space enables the inclusion of more spacers and / or CRISPR arrays or sequences encoding gRNAs to enable multiplexing of cleavage of host sequences. This can be used to minimize the chances of the host evolving resistance to the vectors of the invention. Another advantage of utilizing endogenous Cas, rather than relying on exogenous Cas encoded by the vector, is that the endogenous Cas is native to the host cell machinery and is therefore likely to function efficiently once derepressed. Endogenous Cas expressed in E. coli cells, such as Streptococcus pyogenes Cas, may be inferior because it is a foreign protein and may not function efficiently with the E. coli machinery. Thus, optionally, the vector of the invention does not have a nucleotide sequence encoding a repressed Cas or Cascade; or the vector does not have any nucleotide sequence encoding Cas. Optionally, the method of the invention does not include introducing a nucleotide sequence encoding a repressed Cas into the host cell, or does not include introducing any nucleotide sequence encoding Cas into the host cell.

[0174] An example application of this configuration of the invention is to introduce one or more vectors of the invention into an E. coli cell (e.g., E. coli O157 H7 EDL933 (EHEC) cell) containing repressed Cas3 and / or Cascade (or its CasA) (wherein H-NS represses Cas and / or Cascade). The vector comprises (a) a nucleotide sequence encoding a derepressor (such as LeuO) capable of derepressing Cascade or Cas in the cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences encoding respective guide RNAs (gRNAs, such as single guide RNAs) in the cell; wherein each crRNA or gRNA is capable of guiding the Cas of Cas or Cascade to modify the respective original spacer sequence of the host cell genome or modify the original spacer sequence of the episome contained by the host in the presence of the derepressor. Component (b) comprises a CRISPR repeat sequence operable with a derepressed Cas or Cascade, for example, the repeat sequence comprises or consists of a sequence selected from SEQ ID NOs: 49-52. In one example, the derepressed Cas is a Cas3 comprising an amino acid sequence of SEQ ID NOs: 58 or 60. In one example, the derepressed Cas is a CasA comprising an amino acid sequence of SEQ ID NOs: 66 or 68. Optionally, the target nucleotide sequence or protospacer comprises a sequence of at least 5, 6, 7, 8, 9 or 10 consecutive nucleotides immediately 3' of a PAM in the genome of the host cell, wherein the PAM is selected from AWG, AAG, AGG, GAG and ATG.

[0175] An example application of this configuration of the invention is to introduce one or more vectors of the invention into a Salmonella enterica cell (e.g., a Salmonella enterica subsp. enterica serovar Typhimurium cell, such as a Salmonella enterica subsp. enterica serovar Typhimurium LT2 cell or a Salmonella enterica subsp. enterica serovar Typhimurium paratyphi A cell) containing a repressed Cas3 and / or Cascade (or its CasA) (wherein H-NS represses Cas and / or Cascade). The vector comprises (a) a nucleotide sequence encoding a derepressor (such as LeuO) capable of derepressing Cascade or Cas in the cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences encoding respective guide RNAs (gRNAs, such as single guide RNAs) in the cell; wherein each crRNA or gRNA is capable of directing the Cas of Cas or Cascade to modify the respective protospacer sequence of the host cell genome or modify the protospacer sequence of an episome contained by the host in the presence of the derepressor. Component (b) comprises a CRISPR repeat sequence operable with a derepressed Cas or Cascade, for example, the repeat sequence comprises or consists of SEQ ID NO: 53. In one example, the derepressed Cas is a Cas3 comprising an amino acid sequence of SEQ ID NO: 56 or 64. In one example, the derepressed Cas is a CasA comprising an amino acid sequence of SEQ ID NO: 70 or 72. Optionally, the target nucleotide sequence or protospacer comprises a sequence of at least 5, 6, 7, 8, 9 or 10 consecutive nucleotides immediately 3' of a PAM in the genome of the host cell, wherein the PAM is operable with Cas3.

[0176] Optionally, the vector is devoid of a nucleotide sequence encoding Cas or a nucleotide sequence encoding a repressed Cas of the system.

[0177] Optionally, in an alternative, the vector comprises component (a) but does not comprise component (b), wherein component (b) (and optionally also component (b)) is comprised by a second vector that is combined with the first vector; wherein optionally the vector does not have a nucleotide sequence encoding Cas or a nucleotide sequence encoding a repressed Cas of the system.

[0178] Optionally, the system comprises a repressed Cascade (e.g., CasA, B, C, D and E), and the derepressor is capable of derepressing the Cascade in a host cell (e.g., an E. coli or Salmonella cell), optionally wherein each vector lacks a nucleotide sequence encoding one or more Cas of the repressed Cascade.

[0179] Optionally, the system comprises a repressed CasA, Cas3 or Cas9, and the derepressor is capable of derepressing Cas in a host cell (eg, an E. coli or Salmonella cell), optionally wherein each vector is devoid of a nucleotide sequence encoding Cas.

[0180] aspect; Certain aspects of the invention are as follows: 1. A nucleic acid vector for introduction into a host cell, wherein the cell comprises a CRISPR / Cas system that is repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences encoding respective guide RNAs (gRNAs, e.g., single guide RNAs) in the cell; wherein each crRNA or gRNA is capable of directing Cas to modify the respective original spacer sequence of the host cell genome or to modify the original spacer sequence of an episome contained by the host in the presence of a derepressor.

[0181] 2. The vector of aspect 1, wherein transcription of one or more components of the CRISPR / Cas system is repressed.

[0182] 3. The vector of aspect 1 or 2, wherein transcription of one or more Cas sequences is repressed.

[0183] 4. The vector of any preceding aspect, wherein transcription of one or more of CasA, B, C, D and E of the type I CRISPR / Cas system is repressed.

[0184] 5. The vector of any preceding aspect, wherein transcription of Cas3 is repressed.

[0185] 6. A vector according to any of the preceding aspects, wherein the Cas modification of the host cell genome (a) Killing host cells; (b) reducing the growth or proliferation of cells or episomes; (c) increasing the growth or proliferation of cells or episomes; (d) reducing or preventing the transcription of a nucleotide sequence comprising the protospacer sequence or adjacent to the protospacer sequence; or (e) increasing the transcription of a nucleotide sequence comprising the protospacer sequence or adjacent to the protospacer sequence.

[0186] 7. The vector of any preceding aspect, wherein components (a) and (b) are alternatively comprised by different first and second vectors for introducing the vector into a host cell wherein the Cas modification occurs.

[0187] 8. The vector of any preceding aspect, wherein the derepressor is a protein or RNA.

[0188] 9. The vector of any preceding aspect, wherein the repressor is H-NS, StpA, LRP or CRP.

[0189] 10. The vector of any preceding aspect, wherein the derepressor is a mutant H-NS, StpA, LRP or CRP, which is capable of forming a complex with the H-NS, StpA, LRP or CRP repressor, respectively, in the host cell to prevent or reduce repression of the CRISPR / Cas system.

[0190] 11. The vector of any preceding aspect, wherein the repressor is H-NS or StpA, and the derepressor is LeuO.

[0191] 12. The vector of any preceding aspect, wherein the episome is a plasmid.

[0192] 13. The vector of any preceding aspect, wherein the cell is a bacterial or archaeal cell.

[0193] 14. The vector of any preceding aspect, wherein the nucleotide sequence (a) comprises a constitutive promoter or a strong promoter for expressing the sequence in a host cell.

[0194] 15. The vector of any preceding aspect, wherein the nucleotide sequence or array (b) comprises a constitutive promoter or a strong promoter for expressing the sequence or array in a host cell.

[0195] 16. The vector of any preceding aspect, wherein (a) and (b) are comprised by the same operator or are under the control of a common expression operable in said cell (eg, the same promoter).

[0196] 17. The vector of aspect 16, wherein the promoter is a strong and / or constitutive promoter for expression in a host cell.

[0197] 18. The vector of any preceding aspect, wherein the host cell is a wild-type host cell.

[0198] 19. The vector of any preceding aspect, wherein the vector comprises an expressible htpG sequence.

[0199] 20. The vector of any preceding aspect, wherein the cell contains a CRISPR / Cas system comprising Cascade and Cas3, wherein the Cascade is repressed in the host cell (e.g., by H-NS), wherein the vector comprises (i) an expressible nucleotide sequence encoding a derepressor of the Cascade repression (e.g., LeuO); and (ii) an expressible nucleotide sequence encoding Cas3, wherein the Cas3 is capable of functioning together with a derepressed Cascade in a host cell; The nucleotide sequence is capable of being expressed in a host cell.

[0200] 21. The vector of aspect 20, wherein the nucleotide sequences of (i) and (ii) are contained by the same operator or are under the control of a common expression control operable in said cell (eg, the same promoter).

[0201] 22. The vector of aspect 20 or 21, wherein sequences (i) and (ii) and the CRISPR array or the sequence encoding the gRNA are contained by two or more different vectors, which are used to introduce into a host cell for expressing the derepressor, Cas3 and the array or gRNA together in the host cell.

[0202] 23. A nucleic acid vector for introduction into a bacterial or archaeal host cell (optionally according to any preceding aspect), wherein the cell comprises an endogenous CRISPR / Cas system that is naturally repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences encoding respective guide RNAs (gRNAs, e.g., single guide RNAs) in the cell; wherein each crRNA or gRNA is capable of directing Cas to modify the original spacer sequence of the host cell genome or to modify the original spacer sequence of an episome contained by the host in the presence of a derepressor; wherein the repressor is H-NS, StpA, LRP or CRP encoded by the cell genome or a functional equivalent thereof; and wherein the derepressor is LeuO or a functional equivalent thereof capable of forming a complex with H-NS; or a mutant of H-NS, StpA, LRP or CRP capable of forming a complex with the H-NS, StpA, LRP or CRP repressor, respectively.

[0203] 24. The vector of aspect 23, wherein the cell is an Escherichia coli (eg, Escherichia coli K12) or a Salmonella (eg, Salmonella enterica serovar Typhimurium) cell.

[0204] 25. The vector of aspect 23 or 24, wherein the Cas is Cas3.

[0205] 26. The vector of any one of aspects 23 to 25, wherein the CRISPR / Cas system is a type I (e.g., type IE or IF) system.

[0206] 27. The vector of any one of aspects 23 to 26, wherein the protospacer sequence is comprised by an essential gene, a virulence gene or an antibiotic resistance gene comprised by the cell.

[0207] 28. The vector of any one of aspects 23 to 27, wherein the vector does not comprise a sequence from the group consisting of CasA, B, C, D and E nucleotide sequences, or wherein the vector does not comprise all sequences of said group.

[0208] 29. The vector of any one of aspects 23 to 28, wherein the vector does not comprise a sequence from the group consisting of Cas1, Cas2, Cas5 and Cas6 sequences.

[0209] 30. The vector of any one of aspects 23 to 29, wherein the vector does not comprise a Cas 3 nucleotide sequence.

[0210] 31. The vector of any preceding aspect for medical use in the treatment or prevention of a disease or condition in a human or animal subject, wherein the host cell is comprised by the subject.

[0211] 32. The vector of any preceding aspect for use in killing the host cell or for reducing its growth or proliferation in a human, animal or plant microbiome.

[0212] 33. A plurality of vectors according to any preceding aspect for use in killing a plurality of host cells or in reducing their growth or proliferation.

[0213] 34. The vector of any preceding aspect, wherein the or each host cell is comprised by a microbiome (e.g., an intestinal microbiome or an environmental microbiome) comprising a plurality of said host cells and comprising one or more cells of a species or strain different from that of the host cell.

[0214] 35. A medicament comprising a plurality of vectors according to any preceding aspect, optionally further comprising one or more medical drugs (e.g., anticancer drugs) or antibiotics (e.g., wherein the protospacer sequence is comprised by a host cell antibiotic resistance gene), the medicament being used to treat or prevent a disease or condition in a human or animal.

[0215] 36. A method for treating or preventing a disease or condition in a human or animal subject, the method comprising administering to the subject a vector or drug of any preceding aspect, wherein the subject's microbiome comprises a host cell modified by an endogenous derepressed Cas of the cell, and carrying out the treatment or prevention.

[0216] 37. A method for killing a wild-type bacterial or archaeal cell (e.g., an E. coli or Salmonella cell), wherein the cell comprises an endogenous CRISPR / Cas system comprising nucleotide sequences encoding Cas3 and Cascade proteins, wherein Cas3 and / or Cascade are naturally repressed in the cell, the method comprising (a) derepressing the Cas3 and / or Cascade, and (b) introducing into the cell (i) a CRISPR array for producing one or more crRNAs in the cell; or (ii) one or more nucleotide sequences encoding respective guide RNAs (gRNAs, e.g., single guide RNAs); Each crRNA or gRNA guides Cas or Cascade to modify the respective original spacer sequence of the host cell genome or to modify the original spacer sequence of the episome contained in the host.

[0217] 38. The method of aspect 37, wherein in step (b), the vector of any one of aspects 1 to 33 is introduced into a cell.

[0218] 39. The method of aspect 37 or 38, wherein Cas3 transcription, expression or activity is derepressed.

[0219] 40. The method of aspect 37, 38 or 39, wherein Cas transcription, expression or activity is derepressed, wherein the Cas is a Cascade Cas (e.g., CasA, B, C, D or E).

[0220] 41. The method of any one of aspects 37 to 40, wherein an expressible de-repressor sequence is introduced into the cell simultaneously or sequentially with the array or the sequence encoding the gRNA.

[0221] 42. The method of any one of aspects 37 to 41, wherein the derepressor sequence and the array or gRNA encoding sequence are comprised by the same nucleic acid vector (eg, phagemid, phage or plasmid).

[0222] 43. The method of any one of aspects 37 to 42, wherein the derepressor sequence and the array or sequence encoding the gRNA are contained by the same operator or are under the control of a common expression control operable in the cell (e.g., the same promoter).

[0223] 44. The method of any one of aspects 37 to 43, wherein step (a) comprises expressing in the cell (i) LeuO or a functional equivalent thereof capable of forming a complex with H-NS; or (ii) a mutant of H-NS, StpA, LRP or CRP capable of forming a complex with H-NS, StpA, LRP or CRP repressor, respectively.

[0224] 45. A pharmaceutical composition, a food, a beverage, a composition for environmental remediation, a pesticide, a herbicide or a cosmetic comprising one or more carriers according to any one of aspects 1 to 34.

[0225] 46. ​​A nucleic acid vector for introduction into a host cell, wherein the cell comprises a CRISPR / Cas system that is repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) for introducing the following sites: (i) a CRISPR array or CRISPR spacer sequence for producing one or more crRNAs in a cell; (ii) a nucleotide sequence encoding a guide RNA (gRNA, e.g., a single guide RNA) in a cell; Wherein the crRNA or gRNA is capable of guiding Cas to modify the respective original spacer sequence of the host cell genome or modify the original spacer sequence of the episome contained in the host in the presence of a derepressor.

[0226] 47. The vector of aspect 46, wherein site (b) is comprised by a CRISPR array, wherein said array is capable of accommodating one or more of said spacer sequences for targeting respective protospacer sequences of a host cell.

[0227] 48. The vector of aspect 46 or 47, wherein the array of (i) or the sequence of (ii) is under the control of a promoter which is a strong and / or constitutive promoter for expression in a host cell.

[0228] 49. The vector of aspect 46 or 47, wherein the sequence of (a) is under the control of a promoter, which is a strong and / or constitutive promoter for expressing the derepressor in a host cell.

[0229] 50. The vector of any one of aspects 46 to 49, comprising an expressible Cas3 sequence and / or an expressible htpG sequence for expression in a host cell.

[0230] 51. The vector of any one of aspects 46 to 50, wherein each of the crRNAs is operable with a Cas (e.g., a Cas nuclease) in a host cell.

[0231] 52. The vector of aspect 51, wherein the Cas is encoded by an endogenous nucleotide sequence of the host cell genome.

[0232] 53. The vector of aspect 51, wherein the Cas is encoded by a nucleotide sequence comprised by the vector or by a different vector that can be introduced into a host cell for expressing the Cas therein.

[0233] 54. The vector of any one of aspects 46 to 53, wherein when the spacer of (i) or the sequence of (ii) has been inserted therein, the vector is a vector according to any one of aspects 1 to 34.

[0234] 55. A method of treating or preventing a disease or condition in a human or animal subject, the method comprising administering a vector to the subject, wherein a bacterial or archaeal host cell (e.g., Escherichia coli, Salmonella, or Salmonella enterica serovar Typhimurium) comprised by the subject's microbiome is modified by the cell's endogenous derepressed Cas, and carrying out the treatment or prevention; wherein (i) each cell comprises a CRISPR / Cas system that is repressed by a repressor in the cell, (ii) the vector comprises a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (iii) the vector does not have a CRISPR array for producing one or more crRNAs in the cell; and does not have one or more nucleotide sequences encoding respective guide RNAs (gRNAs, e.g., single guide RNAs) in the cell.

[0235] 56. A medicament comprising a plurality of nucleic acid vectors for introduction into a bacterial or archaeal host cell, optionally further comprising one or more medical drugs (e.g., anticancer drugs) or antibiotics (e.g., wherein the protospacer sequence is comprised by a host cell antibiotic resistance gene), the medicament being used to treat or prevent a disease or condition in a human or animal; wherein (i) each cell comprises a CRISPR / Cas system that is repressed by a repressor selected from H-NS and / or StpA in the cell, (ii) the vector comprises a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in the cell, wherein the sequence is expressible in the cell to produce the derepressor, optionally under the control of a strong and / or constitutive promoter; and The vector does not have a CRISPR array for producing one or more crRNAs in the cell; and does not have one or more nucleotide sequences encoding respective guide RNAs (gRNAs, e.g., single guide RNAs) in the cell.

[0236] 57. The method of aspect 55 or the medicament of aspect 56, wherein Cascade Cas, Cas3 or Cas9 is repressed.

[0237] 58. The vector, medicament, method or composition of any preceding aspect, wherein the system comprises a repressed Cas and (a) the Cas comprises an amino acid sequence selected from SEQ ID NOs: 58, 60, 66 and 68, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity to the selected sequence, or is an ortholog or homolog thereof, which is operable with a repeat sequence comprising a sequence selected from SEQ ID NOs: 49-52 and a PAM comprising or consisting of AWG (e.g., AAG, AGG, GAG or ATG), wherein optionally the host cell is an E. coli cell; or (b) the Cas comprises an amino acid sequence selected from SEQ ID NOs: 56, 64, 70 and 72, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity to the selected sequence, or an ortholog or homolog thereof, which is operable with a repetitive sequence comprising SEQ ID NO: 53, wherein optionally the host cell is Salmonella enterica; or (c) the Cas comprises an amino acid sequence selected from SEQ ID NO: 62, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity to the selected sequence, or is an ortholog or homolog thereof, which is operable with a PAM comprising or consisting of NNAGAAW, NGGNG or AW (e.g., AA, AT or AG), wherein optionally the host cell is a Streptococcus thermophilus cell.

[0238] 59. The vector, medicament, method or composition of any preceding aspect, wherein the system comprises a repressed Cas and the Cas can operate with: (a) comprising a repeat sequence of a sequence selected from SEQ ID NOs: 49-52 and a PAM comprising or consisting of AWG (e.g., AAG, AGG, GAG or ATG), wherein optionally the host cell is an E. coli cell; (b) comprising a repeat sequence of SEQ ID NO: 53, wherein the host cell is Salmonella enterica; or (c) a PAM comprising or consisting of NNAGAAW, NGGNG or AW (eg, AA, AT or AG), wherein optionally the host cell is a Streptococcus thermophilus cell.

[0239] 60. The vector, medicament, method or composition of any preceding aspect, wherein the system comprises a repressed Cas and the Cas can operate with: (a) a PAM comprising or consisting of AWG (e.g., AAG, AGG, GAG, or ATG), and optionally the Cas nuclease comprises an amino acid sequence selected from SEQ ID NOs: 58, 60, 66, and 68, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% (e.g., at least 80%) identity to the selected sequence, wherein optionally the host cell is an E. coli cell; or (b) a PAM comprising or consisting of NNAGAAW, NGGNG or AW (e.g., AA, AT or AG), and optionally the Cas nuclease comprises an amino acid sequence selected from SEQ ID NO: 62, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity to the selected sequence, wherein optionally the host cell is a Streptococcus thermophilus cell.

[0240] 61. The vector, medicament, method or composition of any one of aspects 58 to 60, wherein the protospacer comprises a sequence of at least 5, 6, 7, 8, 9 or 10 consecutive nucleotides immediately 3' to the PAM in the genome of the host cell.

[0241] 62. The vector, medicament, method or composition of any one of aspects 58 to 60, wherein the protospacer comprises a sequence of at least 5, 6, 7, 8, 9 or 10 consecutive nucleotides immediately 5' to the PAM in the genome of the host cell.

[0242] 63. The vector, medicament, method or composition of any preceding aspect, wherein the repressor is (i) an H-NS comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 19, 21, 23, 25 and 27, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity to the selected sequences; or (ii) StpA comprising an amino acid sequence selected from SEQ ID NOs: 29, 31, 33 and 35, or an amino acid sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identical to the selected sequence.

[0243] 64. The vector, drug, method or composition of any preceding aspect, wherein the derepressor is (iii) LeuO comprising an amino acid sequence selected from SEQ ID NO: 3, 5, 7, 9, 11, 13 and 15, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity thereto; or (iv) an LRP comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 39 and 41, or an amino acid sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identical to said selected sequence; or (v) a CRP comprising an amino acid sequence selected from SEQ ID NOs: 43, 45 and 47, or an amino acid sequence that is at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identical to said selected sequence.

[0244] 65. The vector, medicament, method or composition of any preceding aspect, wherein the vector lacks a nucleotide sequence encoding Cas or a nucleotide sequence encoding a repressed Cas of the system.

[0245] 66. A vector, a drug, a method or a composition according to any preceding aspect, wherein alternatively the vector comprises component (a) but does not comprise component (b), wherein component (b) is comprised by a second vector combined with the first vector; wherein optionally the vector does not have a nucleotide sequence encoding Cas or a nucleotide sequence encoding a repressed Cas of the system.

[0246] 67. The vector, medicament, method or composition of any preceding aspect, wherein the system comprises a repressed Cascade (e.g., CasA, B, C, D and E), and the derepressor is capable of derepressing the Cascade in a host cell (e.g., an E. coli or Salmonella cell), optionally wherein each vector lacks a nucleotide sequence encoding one or more Cas of the repressed Cascade.

[0247] 68. The vector, medicament, method or composition of any preceding aspect, wherein the system comprises a repressed CasA, Cas3 or Cas9, and the derepressor is capable of derepressing Cas in a host cell (e.g., an E. coli or Salmonella cell), optionally wherein each vector is devoid of a nucleotide sequence encoding Cas.

[0248] Reorganization The present invention also provides the following nucleic acid recombination method: 69. An in vitro method for implementing nucleic acid (e.g., DNA) recombination in a cell (e.g., a bacterial cell, such as an E. coli cell), wherein the cell comprises a CRISPR / Cas system repressed by a repressor, the method comprising (a) introducing a nucleic acid of interest (NOI) into cells (e.g., by electroporation); (b) introducing a vector of the invention (e.g., according to any of the above aspects or the following clauses) into a cell (e.g., by electroporation), wherein steps (a) and (b) are performed simultaneously or in any order; (c) expressing a derepressor and a crRNA or gRNA encoded by a vector in a cell, wherein the derepressor derepresses the CRISPR / Cas system, and the Cas nuclease of the system is guided by the crRNA or gRNA to modify (e.g., cleave) a protospacer sequence comprised by the NOI; and (d) optionally isolating the modified NOI.

[0249] The cell is a cell with recombination ability, for example, comprising rac prophage RecE / RecT or λRedαβδ. For example, the cell is a cell with recombination ability comprising a λred recombination system. In one example, the NOI is DNA (e.g., dsDNA or ssDNA). In another example, the NOI is RNA. The isolated modified NOI may also include modifications in addition to the modifications produced by Cas, for example, modifications performed before or after the modifications performed by Cas.

[0250] This aspect of the invention can be used to control the recombination method. For example, the initiation, timing and / or duration of Cas modification (e.g., Cas cleavage) can be controlled by the expression of a derepressor. For example, the NOI and the template nucleic acid or the insert nucleic acid or other components of the recombination method can be first introduced into the cell, and then the derepressor is expressed, thereby implementing the modification by Cas, and the template / insert nucleic acid is also used to modify or replicate the NOI modified by Cas. For example, the method includes introducing the second NOI into the cell simultaneously or sequentially with the introduction of the first NOI; expressing the derepressor; Cas cleaving the original spacer, thereby generating a recombination end in the first NOI; inserting a nucleotide sequence contained by the second NOI at or near the nick in the first NOI; and optionally generating a continuous modified NOI containing a sequence of the first NOI continuous with the sequence of the second NOI. In another embodiment, the method includes using the second NOI to retrieve the sequence of the first NOI at or near the nick. For suitable insertion or retrieval techniques that can be used in the methods of the present invention, see, for example, WO2017 / 118598, and it is therefore incorporated herein by reference.

[0251] 70. The method of aspect 69, wherein the nuclease is a dsDNA nuclease.

[0252] 71. The method of aspect 69, wherein the nuclease is a ssDNA nuclease.

[0253] 72. The method of aspect 69, wherein the nuclease is a nickase.

[0254] 73. The method of any one of aspects 69 to 72, wherein the nuclease is Cas9.

[0255] 74. The method of any one of aspects 69 to 72, wherein the nuclease is Cas3, and optionally the repressor represses Cascade (eg, CasA).

[0256] 75. The method of any one of aspects 69 to 74, comprising introducing the isolated modified NOI into a second cell (e.g., a non-human vertebrate, a mammal, a human, an animal (e.g., a cow, pig, sheep, goat, livestock, fish, salmon or horse), a rodent, a mouse, a rat or a zebrafish or an African clawed frog cell), and optionally obtaining progeny cells therefrom.

[0257] 76. The method of aspect 75, wherein the second cell is an embryonic stem cell (ES cell) or an induced pluripotent stem cell (iPS cell).

[0258] For example, the second cell is a non-human animal (eg, a mammal or non-human vertebrate) cell, such as a rodent, mouse, or rat cell.

[0259] 77. The method of aspect 76, comprising developing the second cell or descendant cell into a non-human animal (e.g., a cow, pig, sheep, goat, livestock, fish, salmon, horse, rodent, mouse, rat, zebrafish, or Java).

[0260] 78. The method of aspect 77, further comprising isolating a protein or nucleic acid (or a nucleotide sequence thereof) from the animal, for example, isolating an antibody, an antibody chain, an antibody variable region, or a nucleic acid thereof.

[0261] 79. The method of aspect 78, further comprising inserting the nucleic acid (or its nucleotide sequence) into an expression vector or a host cell for expressing a protein comprising an amino acid sequence (e.g., an antibody variable domain) encoded by the nucleic acid or its nucleotide sequence, expressing the protein and isolating the protein, and optionally formulating the isolated protein into a drug for use in humans or animals.

[0262] Optionally, the nucleic acid or sequence is mutated or fused to another nucleic acid or nucleotide sequence before, during or after insertion into an expression vector. For example, an antibody variable domain sequence can be operably linked to a nucleotide sequence encoding an antibody constant region in a vector for expression of antibody chains from the vector.

[0263] Terms : Certain clauses of the present invention are as follows:-1. A nucleic acid vector for introduction into a host cell, wherein the cell comprises a CRISPR / Cas system repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences encoding respective guide RNAs (gRNAs) in the cell; wherein each cRNA or gRNA is capable of directing Cas to modify a respective protospacer sequence of the host cell genome or to modify a protospacer sequence of an episome contained by the host in the presence of a derepressor.

[0264] 2. The vector of clause 1, wherein the nucleotide sequence (a) comprises a constitutive promoter or a strong promoter for expressing the sequence in a host cell.

[0265] 3. The vector of clause 1 or 2, wherein the nucleotide sequence or array (b) comprises a constitutive promoter or a strong promoter for expressing the sequence or array in a host cell.

[0266] 4. The vector of any preceding clause, wherein (a) and (b) are comprised by the same operator or are under the control of a common promoter operable in said cell.

[0267] 5. The vector of any preceding clause, wherein the host cell is a wild-type host cell.

[0268] 6. The vector of any preceding clause, wherein transcription of one or more Cas sequences is repressed, optionally wherein transcription of one or more of CasA, B, C, D and E of a type I CRISPR / Cas system is repressed.

[0269] 7. The vector of any preceding clause, wherein the Cas modification of the host cell genome (a) Killing host cells; (b) reducing the growth or proliferation of cells or episomes; (c) increasing the growth or proliferation of cells or episomes; (d) reducing or preventing the transcription of a nucleotide sequence comprising the protospacer sequence or adjacent to the protospacer sequence; or (e) increasing the transcription of a nucleotide sequence comprising the protospacer sequence or adjacent to the protospacer sequence.

[0270] 8. The vector of any preceding clause, wherein the repressor is H-NS, StpA, LRP or CRP.

[0271] 9. The vector of any preceding clause, wherein the derepressor is a mutant H-NS, StpA, LRP or CRP, which is capable of forming a complex with the H-NS, StpA, LRP or CRP repressor, respectively, in the host cell to prevent or reduce repression of the CRISPR / Cas system.

[0272] 10. The vector of any preceding clause, wherein the derepressor is LeuO or LysR or a functional equivalent thereof.

[0273] 11. The vector of any preceding clause, wherein the cell is a bacterial or archaeal cell.

[0274] 12. The vector of any preceding clause, wherein the vector comprises an expressible htpG sequence.

[0275] 13. The vector of any preceding clause, wherein the cell contains a CRISPR / Cas system comprising Cascade and Cas3, wherein the Cascade is repressed in the host cell, wherein the vector comprises (i) an expressible nucleotide sequence encoding a derepressor of said Cascade repression; and (ii) an expressible nucleotide sequence encoding Cas3, wherein the Cas3 is capable of functioning together with a derepressed Cascade in a host cell; The nucleotide sequence is capable of being expressed in a host cell.

[0276] 14. The vector of clause 13, wherein the nucleotide sequences of (i) and (ii) are under the control of one or more constitutive promoters operable in the cell.

[0277] 15. A nucleic acid vector for introduction into a bacterial or archaeal host cell (optionally according to any preceding clause), wherein the cell comprises an endogenous CRISPR / Cas system that is naturally repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences encoding respective guide RNAs (gRNAs) in the cell; wherein each cRNA or gRNA is capable of directing Cas to modify the original spacer sequence of the host cell genome or to modify the original spacer sequence of an episome contained by the host in the presence of a derepressor; in (c) the repressor is H-NS, StpA, LRP or CRP encoded by the cell genome or a functional equivalent thereof; (d) the nucleotide sequence (a) comprises a constitutive promoter or a strong promoter for expressing the sequence in a host cell; and (e) The nucleotide sequence or array (b) comprises a constitutive promoter or a strong promoter for expressing the sequence in a host cell.

[0278] 16. The vector of clause 15, wherein the derepressor is LeuO or a functional equivalent thereof; or a mutant of the repressor; or an siRNA complementary to a nucleotide sequence contained by the host cell encoding the repressor.

[0279] 17. The vector of any preceding clause, wherein the cell is an E. coli, Streptococcus or Salmonella cell, optionally an EHEC E. coli or a Salmonella enterica serovar Typhimurium cell.

[0280] 18. The vector of any preceding clause, wherein the protospacer sequence is a chromosomal sequence, an endogenous host cell sequence, a wild-type host cell sequence, a non-viral chromosomal host cell sequence, and is not an exogenous sequence and / or a non-phage sequence.

[0281] 19. The vector of any preceding clause, wherein the CRIPSR / Cas system comprises Cas3 and a repressed Cascade, and the derepressor is capable of derepressing Cascade in the cell, wherein the nucleotide sequence or array (b) comprises a CRISPR repeat sequence operable with the derepressed CRISPR / Cas system, the repeat sequence comprising or consisting of a sequence selected from SEQ ID NO: 49-52 or a sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity to the selected sequence.

[0282] 20. The vector of clause 19, wherein the Cas3 comprises an amino acid sequence selected from SEQ ID NO: 58 or 60, or a sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity to the selected sequence.

[0283] 21. The vector of clause 19 or 20, wherein the Cas3 is operable with a PAM comprising or consisting of the nucleotide sequence AWG.

[0284] 22. The vector of clause 19, 20 or 21, wherein the Cascade comprises a repressed CasA and the derepressor is capable of derepressing the CasA, wherein the CasA comprises an amino acid sequence selected from SEQ ID NO: 66 or 68, or a sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity to the selected sequence.

[0285] 23. The vector of any one of clauses 1 to 18, wherein the cell is a Salmonella enterica cell and the CRISPR / Cas system comprises a repressed E-type (Cse) Cas, wherein the derepressor is capable of derepressing the Cas, optionally wherein the derepressor is LeuO or a functional equivalent thereof.

[0286] 24. The vector of any one of clauses 1 to 18 and 23, wherein the CRIPSR / Cas system comprises Cas3 and a repressed Cascade, and the derepressor is capable of derepressing Cascade in the cell, wherein the nucleotide sequence or array (b) comprises a CRISPR repeat sequence operable with the derepressed CRISPR / Cas system, the repeat sequence comprising or consisting of SEQ ID NO: 53 or a sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity thereto.

[0287] 25. The vector of clause 24, wherein the Cas3 comprises an amino acid sequence selected from SEQ ID NO: 56 or 64, or a sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity to the selected sequence.

[0288] 26. The vector of clause 24 or 25, wherein the Cascade comprises a repressed CasA and the derepressor is capable of derepressing the CasA, wherein the CasA comprises an amino acid sequence selected from SEQ ID NO: 70 or 72, or a sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity to the selected sequence.

[0289] 27. The vector of any preceding clause, wherein the nucleotide sequence or array (b) comprises a CRISPR repeat sequence operable with a derepressed CRISPR / Cas system in a cell, the repeat sequence having at least 90% identity with a repeat sequence in a host array comprised by the CRISPR / Cas system of the cell, wherein the vector or sequence or array (b) does not comprise a PAM recognized by a Cas nuclease of the host CRISPR / Cas system.

[0290] 28. The vector of any preceding clause, wherein the vector does not comprise a sequence from the group consisting of Cas A, B, C, D and E nucleotide sequences, or wherein the vector does not comprise all sequences of said group.

[0291] 29. The vector of any one of clauses 1 to 28, wherein the vector does not comprise a sequence from the group consisting of Cas1, Cas2, Cas5 and Cas6 sequences.

[0292] 30. The vector of any preceding clause, wherein the vector does not comprise a Cas 3 nucleotide sequence.

[0293] 31. The vector of any preceding clause for medical use in the treatment or prevention of a disease or condition in a human or animal subject, wherein the host cell is comprised by the subject.

[0294] 32. A vector according to any preceding clause for medical use for killing said host cell or for reducing its growth or proliferation in a human or animal microbiome.

[0295] 33. The vector of clause 32, wherein the microbial population comprises a plurality of said host cells and comprises additional cells of a species or strain different from the species or strain of the host cells, wherein the additional cells do not comprise a protospacer sequence.

[0296] 34. A plurality of phage or phagemids comprising a plurality of vectors of any preceding clause, optionally wherein the vectors are identical.

[0297] 35. A plurality of bacteriophages or phagemids as claimed in clause 34 as appended to clause 33, wherein said phages are capable of infecting a host cell but are unable to infect further cells, or said phagemids are comprised of such phages.

[0298] 36. A medicament comprising a plurality of vectors, phages or phagemids according to any preceding clause, optionally further comprising one or more drugs or antibiotics, for use in treating or preventing a disease or condition in a human or animal.

[0299] 37. A method for treating or preventing a disease or condition in a human or animal subject, the method comprising administering to the subject a vector, a plurality of vectors or a drug of any preceding clause, wherein the subject's microbiome comprises a host cell modified by an endogenous derepressed Cas of the cell, and carrying out the treatment or prevention.

[0300] 38. The method of clause 37, wherein the method kills wild-type E. coli or Salmonella host cells.

[0301] 39. A nucleic acid vector for introduction into a host cell, wherein the cell comprises a CRISPR / Cas system that is repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) for introducing the following sites: (i) a CRISPR array or CRISPR spacer sequence for producing one or more crRNAs in a cell; (ii) a nucleotide sequence encoding a guide RNA (gRNA) in a cell; Wherein the cRNA or gRNA is capable of directing Cas to modify the respective original spacer sequence of the host cell genome or to modify the original spacer sequence of the episome contained in the host in the presence of a derepressor.

[0302] 40. The vector of clause 39, wherein insertion of (i) or (ii) into said site forms a vector according to any one of clauses 1 to 33.

[0303] 41. A medicament comprising a plurality of nucleic acid vectors for introduction into a bacterial or archaeal host cell, optionally further comprising one or more drugs or antibiotics, for treating or preventing a disease or condition in a human or animal; wherein (i) each cell comprises a CRISPR / Cas system that is repressed by a repressor selected from H-NS and / or StpA in the cell, (ii) the vector comprises a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in the cell, wherein the sequence is expressible in the cell to produce the derepressor, optionally under the control of a strong and / or constitutive promoter; and The vector lacks a CRISPR array for producing one or more crRNAs in the cell; and lacks one or more nucleotide sequences encoding respective guide RNAs (gRNAs) in the cell.

[0304] 42. The vector, phage, phagemid, medicament or method of any preceding clause, wherein Cascade Cas, Cas3 or Cas9 is repressed.

[0305] 43. The vector, phage, phagemid, medicament or method of any preceding clause, wherein the system comprises (a) a Cas comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 60, 66 and 68, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity to the selected sequence, or an ortholog or homolog thereof that is operable with a repeat sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 49-52 and a PAM comprising or consisting of AWG; or (b) a Cas comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 56, 64, 70 and 72, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity to the selected sequence, or an ortholog or homolog thereof that is operable with a repetitive sequence comprising SEQ ID NO: 53; or (c) Cas, comprising an amino acid sequence selected from SEQ ID NO: 62, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity to the selected sequence, or an ortholog or homolog thereof, which is operable with a PAM comprising or consisting of NNAGAAW, NGGNG or AW.

[0306] 44. The vector, phage, phagemid, drug or method of any preceding clause, wherein the system comprises (a) a repetitive sequence comprising a sequence selected from SEQ ID NO:49-52 (or a sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity with the selected sequence) and a PAM comprising or consisting of AWG; (b) a repetitive sequence comprising SEQ ID NO:53 (or a sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identity with the selected sequence); or (c) a PAM comprising or consisting of NNAGAAW, NGGNG or AW.

[0307] 45. The vector, phage, phagemid, medicament or method of any preceding clause, wherein the system comprises a repressed Cas and the Cas is operable with: (a) a PAM comprising or consisting of AWG, and optionally the Cas nuclease comprises an amino acid sequence selected from SEQ ID NOs: 58, 60, 66 and 68, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity to the selected sequence; or (b) a PAM comprising or consisting of NNAGAAW, NGGNG or AW, and optionally the Cas nuclease comprises an amino acid sequence selected from SEQ ID NO: 62, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity to the selected sequence.

[0308] 46. ​​The vector, phage, phagemid, drug or method of any preceding clause, wherein the repressor comprises (i) an amino acid sequence selected from SEQ ID NO: 17, 19, 21, 23, 25 and 27, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity thereto; or (ii) an amino acid sequence selected from SEQ ID NO: 29, 31, 33 and 35, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity thereto.

[0309] 47. The vector, phage, phagemid, medicament or method of any preceding clause, wherein the derepressor comprises (i) an amino acid sequence selected from SEQ ID NO: 3, 5, 7, 9, 11, 13 and 15, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity thereto; or (ii) an amino acid sequence selected from SEQ ID NO: 37, 39 and 41, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity thereto; or (iii) an amino acid sequence selected from SEQ ID NO: 43, 45 and 47, or an amino acid sequence having at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% (e.g., at least 80%) identity thereto.

[0310] 48. The vector, phage, phagemid, medicament or method of any preceding clause, wherein the or each vector lacks a nucleotide sequence encoding Cas or a nucleotide sequence encoding a repressed Cas of the system.

[0311] Providing a sequence encoding a derepressor and a sequence encoding crRNA or gRNA on the same vector can be used to ensure that these are used to cause the means of modification of the host cell genome mediated by Cas are introduced into the cell at the same time. Therefore, this ensures that all cells in which derepression is achieved will also be provided with means of Cas-mediated modification. If separate vectors are used for various components, this will be difficult to ensure and control; some cells can accept derepressors, but not crRNA / gRNA, or vice versa. By ensuring that each component is delivered together, the present invention can be used to solve wild-type host cells, in which it is impossible to pre-modify the crRNA / gRNA encoding sequence with the genome. Such cells can be, for example, included by a microbial group of a human, an animal, or a natural environment (e.g., soil or waterway or water source). Therefore, the configuration of the present invention in which a sequence encoding a derepressor and a sequence encoding crRNA or gRNA are included by the same vector is a useful medical method implemented in humans or animals. These configurations are further useful because the potential for co-transfer of sequences into target host cells enables more predictable administration of each sequence, and therefore allows more reliable administration of medical or other uses for compositions to recipient host cells (e.g., contained by a microbial community). In addition, the configuration in which the sequence encoding the derepressor and the sequence encoding crRNA or gRNA are contained by the same vector allows, for example, co-controlling the expression of the sequence by a common promoter or by designing the vector, so that these sequences are contained by the same operon. For example, an inducible promoter can be used, in which the vector of the present invention has been introduced into a host cell, wherein an inducer (e.g., applied to a human or animal to which the vector has been applied or to which the vector has been applied) is provided to turn on a promoter for simultaneous expression of the derepressor and crRNA / gRNA.

[0312] In other embodiments, the use of a constitutive promoter is advantageous because it ensures the expression of derepressors and crRNA / gRNA in host cells, which then increases the chance that the derepressed Cas will be directed by the crRNA or gRNA of the present invention (to cut or otherwise modify the target protospacer), rather than by endogenously produced crRNA or gRNA. For medical purposes, for example, when the vector is administered to a human or animal subject (such as to its intestinal microbiome), it may be useful to ensure constitutive expression to control dosing and maximize the chance that the vector will be effective in reaching its target in vivo, rather than trying to rely on turning on activity by administering an inducer (where an inducible promoter is used instead), in the hope that the inducer reaches the target cell and is used to effectively induce and produce an effective level of derepressors and crRNA / gRNA in the microbiome at an effective dose. Similarly, a strong promoter can be used to increase the chance of derepression, and also increase the chance that the desired expression of the crRNA / gRNA of the present invention is high (and may also produce endogenously encoded crRNA or gRNA beyond the background level)). Thus, use of a strong promoter increases the chance that cleavage (or other modification) of the target protospacer will occur in the host cell.

[0313] It should be understood that the specific embodiments described herein are shown by way of illustration, rather than by way of limitation of the present invention. Without departing from the scope of the present invention, the main features of the present invention may be used in various embodiments. Those skilled in the art will recognize or be able to determine multiple equivalents of the specific processes described herein using only routine research. Such equivalents are considered to be within the scope of the present invention and are covered by the claims. All publications and patent applications mentioned in the specification indicate the technical level of the technicians in the field to which the present invention belongs. All publications and patent applications and all U.S. equivalent patent applications and patents are incorporated herein by reference, to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference. When used in conjunction with the term "comprising" in the claims and / or specification, the use of the word "a" or "an" may refer to "one", but it is also consistent with the meaning of "one or more", "at least one" and "one or more than one". The use of the term "or" in the claims is intended to mean "and / or" unless explicitly stated to refer only to alternatives, or the alternatives are mutually exclusive, but the invention supports a definition referring only to alternatives and "and / or." Throughout this application, the term "about" is used to indicate that the stated value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0314] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended, and do not exclude additional, unrecited elements or method steps.

[0315] As used herein, the term "or combinations thereof" or similar terms refers to all permutations and combinations of the enumerated items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in the specific context, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, explicitly included are combinations containing repetitions of one or more items or clauses, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. The skilled artisan will understand that there is generally no limit to the number of items or clauses in any combination unless otherwise apparent from the context.

[0316] Any part of the disclosure may be read in combination with any other part of the disclosure, unless otherwise apparent from the context.

[0317] According to the present invention, all compositions and / or methods disclosed and claimed herein can be formed and implemented without undue experimentation. Although the compositions and methods of the present invention are described with respect to preferred embodiments, it will be apparent to those skilled in the art that changes may be applied to the steps or the order of steps of the compositions and / or methods and methods described herein without departing from the concept, spirit and scope of the present invention. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0318] The invention is described in more detail in the following non-limiting examples. Example

[0319] Example 1: Medical Use The present invention provides various vectors, drugs and methods as described herein for treating, preventing or reducing bacterial infection of host cells (eg, reducing the spread or expansion of bacteria) in humans or animals.

[0320] In a first embodiment, the host cell modification (HM)-array and LeuO-encoding nucleotide sequences of the present invention are contained in a population of class I, II or III Staphylococcus packaged phages (Caudovirales or Myoviridae phages). The phage population is administered to patients with MRSA infection with or without methicillin or vancomycin. In one trial, the phage HM-array targets (i) a region of 20 nucleotides 3' of the leading promoter of the endogenous Staphylococcus aureus CRISPR array and (ii) a methicillin resistance gene in the host cell. When vancomycin is administered, a lower dose than usual is administered to the patient. It is expected that host cell infection will be knocked down, and resistance to phage drugs will not be established or resistance to phage drugs will be established at a lower rate or severity than usual. In other experiments, the design was identical, except that the phage in those experiments also targeted the essential S. aureus gene ftsZ (Liang et al., Int J Infect Dis. 2015 Jan; 30: 1-6. doi: 10.1016 / j.ijid.2014.09.015. Epub 2014 Nov 5, "Inhibiting the growth of methicillin-resistant Staphylococcus aureus in vitro with antisense peptide nucleic acid conjugates targeting the ftsZ gene"). LeuO is expressed in the host staphylococcal cells and derepresses the H-NS-repressed Cas in the host cells. The phage vector does not have any Cas-coding sequence, but the crRNA expressed from the HM-array works with the endogenous Cas encoded by the host genome.

[0321] Additional experiments will repeat the above experiments but administer bacteriophage K endolysin in addition to or instead of methicillin.

[0322] Example 2: Selective bacterial population growth inhibition in mixed consortia of different microbial population species We demonstrate selective growth inhibition of specific bacterial species in a mixed population of 3 species. We selected species found in the intestinal microbiota of humans and animals (Streptococcus thermophilus DSM 20617 (T), Lactobacillus lactis and Escherichia coli). We included two Gram-positive bacterial species (Streptococcus thermophilus and Lactobacillus lactis) to observe whether this would affect the selective killing ability of the former species; in addition, in order to increase the difficulty (and to simulate the situation of the microbiota more closely), Lactobacillus lactis was selected because it is a phylogenetic related species of Streptococcus thermophilus (as indicated by the high 16s ribosomal RNA sequence identity between the two species). Both Streptococcus thermophilus and Lactobacillus lactis are Firmicutes. In addition, in order to stimulate the microbiota, human commensal intestinal species (Escherichia coli) are included.

[0323] 1. Materials and Methods The method and strains described in Example 6 of US20160333348 were used (except that the selective medium was 2.5 gl -1 2-phenylethanol (PEA) supplemented TH medium).

[0324] 1.1 Preparation of electrocompetent Lactobacillus lactis cells An overnight culture of Lactobacillus lactis in TH medium supplemented with 0.5 M sucrose and 1% M glycine was diluted 100-fold in 5 ml of the same medium and grown at 30 °C to an OD of 600 The cell density was between 0.2 and 0.7 (about 2 hours after inoculation). The cells were collected at 7000 x g for 5 minutes at 4°C and washed 3 times with 5 ml of ice-cold washing buffer (0.5 M sucrose + 10% glycerol). After washing the cells, electroporation was performed in electroporation buffer (0.5 M sucrose, 10% glycerol and 1 mM MgCl 2 ) and suspended them to OD 600 Keep the cells in electroporation buffer at 4°C until use (within 1 hour) or aliquot 50 μl in microcentrifuge tubes (Eppendorf tubes), freeze them in liquid nitrogen and store at -80°C for later use.

[0325] Electroporation conditions for all species were as described in Example 6 of US20160333348.

[0326] 1.2 Activation of CRISPR arrays: consortium experiments Streptococcus thermophilus DSM 20617, Lactobacillus lactis MG1363 and Escherichia coli TOP10 were genetically transformed with plasmids containing a CRISPR array targeting DNA polymerase III and tetA of Streptococcus thermophilus. All cells were grown individually and co-cultured at 37°C for 3 hours after transformation to allow recovery to develop antibiotic resistance encoded in the plasmid. We decided to use transformation efficiency as a readout value for CRISPR-encoded growth inhibition. Therefore, after allowing cells to recover, the cultures were plated in TH medium, TH supplemented with PEA, and MacConkey agar supplemented with kanamycin, and induced by 1% xylose.

[0327] 2. Results 2.0 Phylogenetic distances between Lactobacillus lactis, Escherichia coli, and Streptococcus thermophilus The sequence similarity calculated in the 16S rRNA encoding DNA sequences of Streptococcus thermophilus and Lactobacillus lactis was determined to be 83.3%. The following 16S sequences were used: Escherichia coli: AB030918.1, Streptococcus thermophilus: AY188354.1, Lactobacillus lactis: AB030918. The sequences were aligned using needle (http: / / www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html) using the following parameters: -gapopen 10.0-gapextend 0.5-endopen 10.0-endextend 0.5-aformat3 pair-snucleotide1-snucleotide2. Figure 11 of US20160333348 shows the maximum likelihood phylogenetic tree of 16S sequences from Streptococcus thermophilus, Lactobacillus lactis and Escherichia coli.

[0328] 2.1 Growth conditions and selective media Streptococcus thermophilus and Lactobacillus lactis are commonly used in combination in many fermented foods and yogurts. We chose these strains because they are generally known to be intestinal microorganisms that form close relationships with the host, and previous characterization of the 16S ribosomal RNA region of Streptococcus thermophilus and Lactobacillus lactis has shown these organisms to be phylogenetically closely related (Ludwig et al., 1995). In parallel, we also evaluated the growth of Escherichia coli for our mixed population co-cultivation experiments because this organism is also common in intestinal microbial communities. We first set out to establish bacterial strains and cultivation protocols that supported the growth of all strains we planned to use for co-cultivation experiments. We found that all strains were able to support growth in TH liquid medium at 37°C (US20160333348 Figure 3 ).

[0329] To determine the cell number of different species, it is important to distinguish different bacteria from a mixed culture. Selective growth of E. coli can be achieved with MacConkey agar, however, there is no specific medium for the selective growth of S. thermophilus. PEA agar is a selective medium for separating Gram-positive bacteria (S. thermophilus) from Gram-negative bacteria (E. coli). In addition, different concentrations of PEA partially inhibit the growth of different Gram-positive bacterial species and strains, which allows selection between other Gram-positive bacteria used in this work. Using 2.5gl -1 The PEA was shown to selectively grow Streptococcus thermophilus while restricting the growth of Lactobacillus lactis and Escherichia coli.

[0330] All strains were transformed with plasmids using the pBAV1KT5 vector backbone with the kanamycin selection marker; we found that using 30 μg ml -1 Kanamycin-supplemented medium is sufficient to grow cells while maintaining the plasmid.

[0331] 2.3 Transformation and selective growth inhibition in mixed populations We transformed S. thermophilus, L. lactis, and E. coli with plasmids containing the CRISPR array and cultured them in consortia of all bacterial species combined in equal parts, which allowed us to determine whether we could induce cell death specifically in S. thermophilus. We used pBAV1KT5-XylR-CRISPR-P XylA or pBAV1KT5-XylR-CRISPR-P ldha+XylA Plasmids transform all species.

[0332] Figure 12 of US20160333348 shows that the xylA or pBAV1KT5-XylR-CRISPR-P ldhA+XylA Selective growth inhibition of S. thermophilus in co-cultures of E. coli, L. lactis, and S. thermophilus. xylA or pBAV1KT5-XylR-CRISPR-P ldhA+XylA No growth differences were observed between E. coli strains harboring the plasmid (middle column). However, S. thermophilus (when treated with 2.5 g L -1 Selective growth on PEA-supplemented TH agar (last column) shows that, as expected, pBAV1KT5-XylR-CRISPR-P xylA (strong) or pBAV1KT5-XylR-CRISPR-P ldhA+XylA(Weak) Decreased transformation efficiency between plasmids. We thus demonstrated selective growth inhibition of a target S. thermophilus subpopulation in a mixed population of cells.

[0333] Targeting Escherichia coli in a hybrid consortium by exploiting derepressed endogenous Cas An illustrative application of this embodiment of the present invention is to target E. coli cells comprised of a mixed bacterial population comprising at least 3 different bacterial species by introducing one or more vectors of the present invention into E. coli cells (e.g., E. coli O157 H7 EDL933 (EHEC) cells) comprising repressed Cas3 and / or Cascade (or its CasA) (wherein H-NS represses Cas and / or Cascade). The vector comprises (a) a nucleotide sequence encoding a derepressor (such as LeuO) capable of derepressing Cascade or Cas in the cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences encoding respective guide RNAs (gRNAs, e.g., single guide RNAs) in the cell; wherein each crRNA or gRNA is capable of directing the Cas of Cas or Cascade to modify the respective original spacer sequence of the host cell genome or modify the original spacer sequence of an episome contained by the host in the presence of the derepressor. Component (b) comprises a CRISPR repeat sequence operable with a repressed Cas or Cascade, for example, the repeat sequence comprises or consists of a sequence selected from SEQ ID NOs: 49-52. In one example, the derepressed Cas is a Cas3 comprising an amino acid sequence of SEQ ID NOs: 58 or 60. In one example, the derepressed Cas is a CasA comprising an amino acid sequence of SEQ ID NOs: 66 or 68. Optionally, the target nucleotide sequence or protospacer comprises a sequence of at least 5, 6, 7, 8, 9 or 10 consecutive nucleotides immediately 3' of a PAM in the genome of the host cell, wherein the PAM is selected from AWG, AAG, AGG, GAG and ATG.

[0334] Targeting Salmonella enterica in a hybrid consortium by exploiting derepressed endogenous CasAn illustrative application of this embodiment of the invention is to target Salmonella enterica cells comprised of a mixed bacterial population comprising at least 3 different bacterial species by introducing one or more vectors of the invention into Salmonella enterica cells (e.g., Salmonella enterica subsp. serovar Typhimurium cells, e.g., Salmonella enterica subsp. serovar Typhimurium LT2 cells or Salmonella enterica subsp. serovar Typhimurium paratyphi A cells) comprising repressed Cas3 and / or Cascade (or its CasA) (wherein H-NS represses Cas and / or Cascade). The vector comprises (a) a nucleotide sequence encoding a derepressor (such as LeuO) capable of derepressing Cascade or Cas in a cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences encoding respective guide RNAs (gRNAs, such as single guide RNAs) in the cell; wherein each crRNA or gRNA is capable of guiding Cas or Cascade's Cas to modify respective original spacer sequences of the host cell genome or modify the original spacer sequences of the episomes contained by the host in the presence of the derepressor. Component (b) comprises a CRISPR repeat sequence that can operate with the repressed Cas or Cascade, for example, the repeat sequence comprises SEQ ID NO: 53 or consists of it. In one example, the derepressed Cas is a Cas3 comprising an amino acid sequence of SEQ ID NO: 56 or 64. In one example, the derepressed Cas is a CasA comprising an amino acid sequence of SEQ ID NO: 70 or 72. Optionally, the target nucleotide sequence or protospacer comprises a sequence of at least 5, 6, 7, 8, 9 or 10 consecutive nucleotides immediately 3' to a PAM in the genome of the host cell, wherein the PAM is operable with Cas3.

[0335] Example 3: Vector-Encoded Lines for Selective Species and Strain Growth Inhibition in Mixed Bacterial Consortia SystemIn Example 2, we surprisingly determined the possibility of utilizing endogenous Cas nuclease activity in host bacteria for selective population growth inhibition in mixed consortia of different species. We next explored the possibility of using vector-encoded Cas activity instead for selective population growth inhibition in mixed consortia of different species. We demonstrated selective growth inhibition of specific bacterial species in mixed populations of three different species, and further included alternative strains of target bacteria. We could surprisingly show selective growth inhibition of target strains only of predetermined target species. In addition, the alternative strains were not targeted by the vector-encoded CRISPR / Cas system, which is desirable for establishing the fine specificity of such vector-borne systems in mixed bacterial consortia that mimic elements of the human or animal intestinal microbiota.

[0336] We chose species found in the gut microbiota of humans and animals (Bacillus subtilis, Lactobacillus lactis, and Escherichia coli). We included two strains of the human commensal gut species Escherichia coli. We thought it would be interesting to see if we could distinguish between closely related strains that still had sequence differences that we could use to target killing of one strain without killing the other. This is interesting because some strains of E. coli in the microbiota are desirable, while other strains may be undesirable (e.g., pathogenic to humans or animals) and therefore could be targets for Cas modification to knock down that strain.

[0337] 1. Materials and methods 1.1. Plasmids and strains Unless otherwise indicated, all strains were grown in Todd-Hewitt broth (TH) (T1438 Sigma-Aldrich) under aerobic conditions at 37° C. Strains were stored in 25% glycerol at −80° C.

[0338] The self-targeting sgRNA-Cas9 complex consists of theophylline riboswitch and AraC / P BADThe expression system is strictly regulated. Cas9 needs to be strictly regulated in order to be stably carried in Escherichia coli. The plasmid contains exogenous Cas9 from Streptococcus pyogenes and a single guide RNA (sgRNA) targeting the K-12 strain of Escherichia coli. Therefore, when the system is activated, the K-12-derived strain TOP10 is susceptible to double-stranded self-cleavage and subsequent death. Escherichia coli strains such as Nissle do not have the same target sequence, so they are not affected by sgRNA-Cas9 activity. See Table 9-11 in USSN 15 / 478,912 (filed on April 4, 2017, and incorporated herein by reference), which shows the sequence used. We select a target sequence (ribosomal RNA coding sequence) that is conserved in the target cell and exists in multiple copies (7 copies), which increases the chance of using a single gRNA design to cut the host cell genome in multiple places to promote killing.

[0339] Figure 1 Regulators controlling the expression of spCas9 and a self-targeting sgRNA targeting ribosomal RNA subunit 16s are shown.

[0340] 1.2 Differential growth media All strains were grown on TH medium at 37°C for 20 h. The selective medium for Bacillus subtilis was 3 g l -1 TH medium supplemented with 2-phenylethanol (PEA) of 1.5%. PEA was added to the medium and autoclaved at 121°C at 15 psi for 15 minutes. Agar plates were prepared by adding 1.5% (wt / vol) agar to the corresponding medium.

[0341] 1.3. Cloning Escherichia coli (One ThermoFisher TOP10 chemically competent cells) were used for all subcloning procedures. TM PCR was performed using the polymerase A. All PCR products were purified using Nucleospin according to the manufacturer's protocol. TM Gel and Macherey-Nagel TM Purification was performed with PCR Clean-up of 1X FD buffer. The purified fragment was digested with restriction enzyme DpnI in 1X FD buffer with 1 μL enzyme in a total volume of 34 μL. The digested reaction was purified again with Nucleospin gel and PCR Clean-up of Macherey Nagel following the manufacturer's protocol. Gibson assembly was performed in 10 μL reactions following the manufacturer's protocol (New England Biolab).

[0342] Plasmid DNA was prepared using a Qiagen kit according to the manufacturer's instructions. Modifications for Gram-positive strains included growing the bacteria in media supplemented with 0.5% glycine and lysozyme to promote cell lysis.

[0343] 1.4. Conversion 1.4.1 Electrocompetent E. coli cells and transformation Commercially available electrocompetent cells were used for cloning and experiments (One ThermoFisher TOP10 electrocompetent E. coli). Electroporation was performed using standard settings: 1800 V, 25 μF, and 200 Ω, using an Electro Cell Manipulator (BTX Harvard Apparatus ECM630). After the pulse, 1 ml of LB-SOC medium was added and the cells were incubated at 37°C for 1 hour. The transformed cells were plated on LB-agar containing the corresponding antibiotics.

[0344] 1.5. Activation of sgRNA-Cas9 in E. coli and consortia experiments E. coli TOP10 and Nissle (both with plasmids containing sgRNAs targeting ribosomal RNA coding sequences of K-12 derivative strains), as well as other bacteria, were grown overnight in 3 ml TH liquid medium. The next day, cells were diluted to ~OD 0.5 and then serially diluted 10-fold in TH medium and plated using 96-well replica plates (Mettler Toledo Liquidator TM 96), 4 μL volume of droplets were spotted on TH agar, TH agar with inducer (1% arabinose and 2 mM theophylline), TH agar supplemented with 2.5 g l-1 PEA, and MacConkey agar supplemented with 1% maltose. The plates were incubated at 37°C for 24 hours, and colony forming units (CFU) were calculated from triplicate measurements.

[0345] 2. Results 2.1 Specific targeting of E. coli strains using exogenous CRISPR-Cas9 system We first tested whether the system could discriminate between two E. coli strains by introducing the killing system into both E. coli TOP10 and Nissle.

[0346] 2.1 Targeting E. coli using the exogenous CRISPR-Cas9 system in mixed cultures Serial dilutions of overnight cultures were performed in duplicate for two E. coli strains, B. subtilis, L. lactis, and in triplicate for mixed cultures. All strains were grown at 37 °C for 20 h in selective plates with and without inducers. The inducible activated sgRNA-Cas9 of this system targeted K-12-derived strains while keeping other bacteria intact.

[0347] To determine the number of cells of different species and to determine the specific removal of species, it is important to distinguish different bacteria from a mixed culture. MacConkey agar selectively grows E. coli and PEA agar is a selective medium for separating Gram-positive bacteria (Bacillus subtilis) from Gram-negative bacteria (E. coli). In addition, we found that different concentrations of PEA partially inhibited the growth of other Gram-positive bacteria. 2.5gl -1 The PEA was shown to enable the selective growth of Bacillus subtilis while restricting the growth of Escherichia coli and Lactobacillus lactis.

[0348] Figure 2 Showing specific targeting of E. coli strains by an inducible, exogenous, vector-borne CRISPR-Cas system. The sgRNA targeted the genome of a K-12-derived E. coli strain (E. coli TOP10), while another E. coli strain tested was unaffected.

[0349] Figure 3 Shown are spot assays with serial dilutions of individual bacterial species used in this study and mixed cultures in TH agar that did not induce the CRISPR-Cas9 system.

[0350] Figure 4 Dilution 10 is shown 3 Spot assay on different selective media. -1 TH of PEA is a selective medium for Bacillus subtilis alone. MacConkey supplemented with maltose is a selective and differential bacterial medium designed to selectively separate Gram-negative and enteric bacilli and differentiate them based on maltose fermentation. Thus, the TOP10ΔmalK mutant produces white colonies on the plate, while Nissle produces pink colonies; A is E. coliΔmalK, B is E. coli Nissile, C is B. subtilis, D is Lactococcus lactis, and E is a mixed culture; images of MacConkey- / B and E are shown in pink; images of MacConkey+ / B and E are shown in pink. Figure 5Selective growth of the bacteria used in this study on different media and selective plates is shown. As can be seen, we clearly selectively killed the target E. coli strain in a mixed population ( Figure 5 coli" on the x-axis in the figure), while another related strain ("E. coli-Nissle") was not similarly killed. In this experiment, the killing of the target strain in the mixed population was 1000-fold.

[0351] Targeting Escherichia coli in a hybrid consortium by utilizing derepressed exogenous Cas An illustrative application of this embodiment of the invention is to target E. coli cells comprised of a mixed bacterial population comprising at least 3 different bacterial species by introducing one or more vectors of the invention into E. coli cells (e.g., E. coli O157H7EDL933 (EHEC) cells) comprising a repressed Cas9, such as spCas9 or stCas9 (where H-NS represses Cas). Cas9 is encoded by a nucleotide sequence that is comprised by a vector introduced into the host cell (e.g., on the same or different vector as the repressor encoding sequence). A vector is introduced into a host cell, the vector comprising (a) a nucleotide sequence encoding a derepressor (such as LeuO) capable of derepressing Cas9 in the cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences encoding respective guide RNAs (gRNAs, such as single guide RNAs) in the cell; wherein each crRNA or gRNA is capable of guiding Cas9 to modify respective original spacer sequences of the host cell genome or modify the original spacer sequences of the episomes contained by the host in the presence of the derepressor. Component (b) comprises a CRISPR repeat sequence operable with the repressed Cas9, for example, the Cas9 is Streptococcus pyogenes Cas9. Optionally, the target nucleotide sequence or original spacer comprises a sequence of at least 5, 6, 7, 8, 9 or 10 consecutive nucleotides immediately 5' of a PAM in the genome of the host cell, wherein the PAM is NGG. References [1] Zhang, XZ, & Zhang, YHP (2011). Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis. Microbial Biotechnology, 4(1), 98-105. http: / / doi.org / 10.1111 / j.1751-7915.2010.00230.x [2]Wegmann,U.,O’Connell-Motherway,M.,Zomer.A.,Buist,G.,Shearman,C.,Canchaya,C.,...Kok,J.(2007).Complete genome sequence of the prototype lacticacid bacterium Lactococcus lactis subsp.cremoris MG1363.Journal ofBacteriology,1N9(8),3256-70. http: / / doi.org / 10.1128 / JB.01768-06.

[0352]

[0353] The present invention relates to the following items.

[0354] 1. A nucleic acid vector for introduction into a host cell, wherein the cell comprises a CRISPR / Cas system that is repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in the cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences encoding respective guide RNAs (gRNAs) in the cell; wherein each cRNA or gRNA is capable of directing Cas to modify a respective protospacer sequence of the host cell genome or to modify a protospacer sequence of an episome contained by the host in the presence of the derepressor.

[0355] 2. The vector of item 1, wherein the nucleotide sequence (a) comprises a constitutive promoter or a strong promoter for expressing the sequence in the host cell.

[0356] 3. The vector of item 1 or 2, wherein the nucleotide sequence or array (b) comprises a constitutive promoter or a strong promoter for expressing the sequence or array in the host cell.

[0357] 4. The vector of any preceding item, wherein (a) and (b) are comprised by the same operon or are under the control of a common promoter operable in said cell.

[0358] 5. The vector of any preceding item, wherein the host cell is a wild-type host cell.

[0359] 6. The vector of any preceding item, wherein transcription of one or more Cas sequences is repressed, optionally wherein transcription of one or more of CasA, B, C, D and E of a type I CRISPR / Cas system is repressed.

[0360] 7. The vector of any of the preceding items, wherein the Cas modification of the host cell genome (a) Killing host cells; (b) reducing the growth or proliferation of cells or episomes; (c) increasing the growth or proliferation of cells or episomes; (d) reducing or preventing the transcription of a nucleotide sequence comprising the protospacer sequence or adjacent to the protospacer sequence; or (e) increasing the transcription of a nucleotide sequence comprising the protospacer sequence or adjacent to the protospacer sequence.

[0361] 8. The vector of any preceding item, wherein the repressor is H-NS, StpA, LRP or CRP.

[0362] 9. The vector of any of the preceding items, wherein the derepressor is a mutant H-NS, StpA, LRP or CRP, which is capable of forming a complex with the H-NS, StpA, LRP or CRP repressor in the host cell to prevent or reduce repression of the CRISPR / Cas system.

[0363] 10. The vector of any preceding item, wherein the derepressor is LeuO or LysR or a functional equivalent thereof.

[0364] 11. The vector of any preceding item, wherein the cell is a bacterial or archaeal cell.

[0365] 12. The vector of any preceding item, wherein the vector comprises an expressible htpG sequence.

[0366] 13. The vector of any preceding item, wherein the cell contains a CRISPR / Cas system comprising Cascade and Cas3, wherein the Cascade is repressed in the host cell, wherein the vector comprises (i) an expressible nucleotide sequence encoding a derepressor of said Cascade repression; and (ii) an expressible nucleotide sequence encoding Cas3, wherein the Cas3 is capable of functioning together with a derepressed Cascade in a host cell; The nucleotide sequence is capable of being expressed in a host cell.

[0367] 14. The vector of item 13, wherein the nucleotide sequences of (i) and (ii) are under the control of one or more constitutive promoters operable in the cell.

[0368] 15. A nucleic acid vector for introduction into a bacterial or archaeal host cell (optionally according to any of the preceding items), wherein the cell comprises an endogenous CRISPR / Cas system that is naturally repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in the cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences encoding respective guide RNAs (gRNAs) in the cell; wherein each cRNA or gRNA is capable of directing Cas to modify the original spacer sequence of the host cell genome or to modify the original spacer sequence of an episome contained by the host in the presence of a derepressor; in (c) the repressor is H-NS, StpA, LRP or CRP encoded by the cell genome or a functional equivalent thereof; (d) the nucleotide sequence (a) comprises a constitutive promoter or a strong promoter for expressing the sequence in a host cell; and (e) The nucleotide sequence or array (b) comprises a constitutive promoter or a strong promoter for expressing the sequence in a host cell.

[0369] 16. The vector of item 15, wherein the derepressor is LeuO or a functional equivalent thereof; or a mutant of the repressor; or an siRNA complementary to a nucleotide sequence encoding the repressor contained in a host cell.

[0370] 17. The vector of any preceding item, wherein the cell is an Escherichia coli, Streptococcus or Salmonella cell, optionally an EHEC Escherichia coli or a Salmonella enterica serovar Typhimurium cell.

[0371] 18. The vector of any preceding item, wherein the protospacer sequence is a chromosomal sequence, an endogenous host cell sequence, a wild-type host cell sequence, a non-viral chromosomal host cell sequence, and is not an exogenous sequence and / or a non-phage sequence.

[0372] 19. The vector of any preceding item, wherein the CRIPSR / Cas system comprises Cas3 and a repressed Cascade, and the derepressor is capable of derepressing Cascade in the cell, wherein the nucleotide sequence or array (b) comprises a CRISPR repeat sequence operable with the derepressed CRISPR / Cas system, the repeat sequence comprising or consisting of a sequence selected from SEQ ID NO: 49-52 or a sequence having at least 70% identity with the selected sequence.

[0373] 20. The vector of item 19, wherein the Cas3 comprises an amino acid sequence selected from SEQ ID NO: 58 or 60 or a sequence having at least 70% identity with the selected sequence.

[0374] 21. The vector of item 19 or 20, wherein the Cas3 is operable with a PAM comprising or consisting of the nucleotide sequence AWG.

[0375] 22. The vector of item 19, 20 or 21, wherein the Cascade comprises a repressed CasA, and the derepressor is capable of derepressing the CasA, and the CasA comprises an amino acid sequence selected from SEQ ID NO: 66 or 68 or a sequence having at least 70% identity with the selected sequence.

[0376] 23. The vector of any one of items 1 to 18, wherein the cell is a Salmonella enterica cell and the CRISPR / Cas system comprises a repressed E-type (Cse) Cas, wherein the derepressor is capable of derepressing the Cas, optionally wherein the derepressor is LeuO or a functional equivalent thereof.

[0377] 24. The vector of any one of items 1 to 18 and 23, wherein the CRIPSR / Cas system comprises Cas3 and a repressed Cascade, and the derepressor is capable of derepressing Cascade in the cell, wherein the nucleotide sequence or array (b) comprises a CRISPR repeat sequence that can operate with the derepressed CRISPR / Cas system, and the repeat sequence comprises or consists of SEQ ID NO: 53 or a sequence having at least 70% identity thereto.

[0378] 25. The vector of claim 24, wherein the Cas3 comprises an amino acid sequence selected from SEQ ID NO: 56 or 64 or a sequence having at least 70% identity with the selected sequence.

[0379] 26. The vector of item 24 or 25, wherein the Cascade comprises a repressed CasA, and the derepressor is capable of derepressing the CasA, and the CasA comprises an amino acid sequence selected from SEQ ID NO: 70 or 72 or a sequence having at least 70% identity with the selected sequence.

[0380] 27. The vector of any preceding item, wherein the nucleotide sequence or array (b) comprises a CRISPR repeat sequence operable with a derepressed CRISPR / Cas system in a cell, the repeat sequence having at least 90% identity with a repeat sequence in a host array comprised by the CRISPR / Cas system of the cell, wherein the vector or sequence or array (b) does not comprise a PAM recognized by a Cas nuclease of the host CRISPR / Cas system.

[0381] 28. The vector of any preceding item, wherein the vector does not comprise a sequence from the group consisting of CasA, B, C, D and E nucleotide sequences, or wherein the vector does not comprise all sequences of the group.

[0382] 29. The vector of any one of items 1 to 28, wherein the vector does not comprise a sequence from the group consisting of Cas1, Cas2, Cas5 and Cas6 sequences.

[0383] 30. The vector of any preceding item, wherein the vector does not comprise a Cas 3 nucleotide sequence.

[0384] 31. The vector of any preceding item, for medical use in treating or preventing a disease or condition in a human or animal subject, wherein the host cell is comprised by the subject.

[0385] 32. The vector of any preceding item, for medical use for killing the host cell or for reducing its growth or proliferation in a human or animal microbiome.

[0386] 33. The vector of item 32, wherein the microbial group comprises a plurality of said host cells, and comprises additional cells of a species or strain different from the species or strain of the host cells, wherein the additional cells do not comprise a protospacer sequence.

[0387] 34. A plurality of bacteriophages or phagemids comprising a plurality of vectors of any preceding item, optionally wherein the vectors are the same.

[0388] 35. A plurality of bacteriophages or phagemids according to item 34 as defined in item 33, wherein the phages are capable of infecting a host cell but are unable to infect other cells, or the phagemids are comprised of such phages.

[0389] 36. A medicament comprising a plurality of vectors, phages or phagemids according to any preceding item, optionally further comprising one or more drugs or antibiotics, for use in treating or preventing a disease or condition in a human or animal.

[0390] 37. A method for treating or preventing a disease or condition in a human or animal subject, the method comprising administering a vector, a plurality of vectors or a drug of any preceding item to the subject, wherein the subject's microbiome comprises a host cell modified by an endogenous derepressed Cas of the cell, and carrying out the treatment or prevention.

[0391] 38. The method of claim 37, wherein the method kills wild-type E. coli or Salmonella host cells.

[0392] 39. A nucleic acid vector for introduction into a host cell, wherein the cell comprises a CRISPR / Cas system that is repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in the cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) for introducing the following sites: (i) a CRISPR array or CRISPR spacer sequence for producing one or more crRNAs in the cell; (ii) a nucleotide sequence encoding a guide RNA (gRNA) in the cell; Wherein the cRNA or gRNA is capable of directing Cas to modify the respective original spacer sequence of the host cell genome or to modify the original spacer sequence of the episome contained in the host in the presence of a derepressor.

[0393] 40. The vector of item 39, wherein (i) or (ii) is inserted into the site to form a vector according to any one of items 1 to 33.

[0394] 41. A medicament comprising a plurality of nucleic acid vectors for introduction into a bacterial or archaeal host cell, optionally further comprising one or more drugs or antibiotics, for treating or preventing a disease or condition in a human or animal; wherein (a) each cell comprises a CRISPR / Cas system that is repressed by a repressor selected from H-NS and / or StpA in the cell, (b) the vector comprises a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in the cell, wherein the sequence is expressible in the cell to produce the derepressor, optionally under the control of a strong and / or constitutive promoter; and The vector lacks a CRISPR array for producing one or more crRNAs in the cell; and lacks one or more nucleotide sequences encoding respective guide RNAs (gRNAs) in the cell.

[0395] 42. The vector, phage, phagemid, medicament or method of any preceding item, wherein Cascade Cas, Cas3 or Cas9 is repressed.

[0396] 43. The vector, phage, phagemid, drug or method of any preceding item, wherein the system comprises (a) a Cas comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 60, 66 and 68, or an amino acid sequence having at least 80% identity to the selected sequence, or an ortholog or homolog thereof, which is operable with a repeat sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 49-52 and a PAM comprising or consisting of AWG; or (b) Cas comprising an amino acid sequence selected from SEQ ID NO: 56, 64, 70 and 72, or an amino acid sequence having at least 80% identity to the selected sequence, or an ortholog or homolog thereof that is operable with a repetitive sequence comprising SEQ ID NO: 53; or (c) Cas, comprising an amino acid sequence selected from SEQ ID NO: 62 or an amino acid sequence having at least 80% identity with the selected sequence, or an ortholog or homolog thereof, wherein the ortholog or homolog thereof is operable with a PAM comprising or consisting of NNAGAAW, NGGNG or AW.

[0397] 44. The vector, phage, phagemid, drug or method of any preceding item, wherein the system comprises (a) a repetitive sequence comprising a sequence selected from the group consisting of SEQ ID NOs: 49-52 (or a sequence having at least 70% identity thereto) and a PAM comprising or consisting of AWG; (b) a repetitive sequence comprising SEQ ID NO:53 (or a sequence having at least 70% identity to the selected sequence); or (c) a PAM comprising or consisting of NNAGAAW, NGGNG or AW.

[0398] 45. The vector, phage, phagemid, drug or method of any preceding item, wherein the system comprises a repressed Cas and the Cas can operate with: (a) a PAM comprising or consisting of AWG, and optionally the Cas nuclease comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 58, 60, 66 and 68, or an amino acid sequence having at least 80% identity to the selected sequence; or (b) a PAM comprising or consisting of NNAGAAW, NGGNG or AW, and optionally the Cas nuclease comprises an amino acid sequence selected from SEQ ID NO: 62, or an amino acid sequence with at least 80% identity to the selected sequence.

[0399] 46. ​​The vector, phage, phagemid, drug or method of any preceding item, wherein the repressor comprises (i) an amino acid sequence selected from SEQ ID NO: 17, 19, 21, 23, 25 and 27 or an amino acid sequence having at least 80% identity with the selected sequence; or (ii) an amino acid sequence selected from SEQ ID NO: 29, 31, 33 and 35 or an amino acid sequence having at least 80% identity with the selected sequence.

[0400] 47. The vector, phage, phagemid, medicament or method of any preceding item, wherein the derepressor comprises (i) an amino acid sequence selected from SEQ ID NO: 3, 5, 7, 9, 11, 13 and 15 or an amino acid sequence having at least 80% identity to the selected sequence; or (ii) an amino acid sequence selected from the group consisting of SEQ ID NOs: 37, 39 and 41, or an amino acid sequence having at least 80% identity to said selected sequence; or (iii) an amino acid sequence selected from the group consisting of SEQ ID NOs: 43, 45 and 47, or an amino acid sequence having at least 80% identity to said selected sequence.

[0401] 48. The vector, phage, phagemid, medicament or method of any preceding item, wherein the or each vector lacks a nucleotide sequence encoding Cas or a nucleotide sequence encoding a repressed Cas of the system.

[0402] 49. An in vitro method for carrying out nucleic acid recombination in a bacterial cell (optionally an E. coli cell), wherein the cell comprises a CRISPR / Cas system repressed by a repressor, the method comprising (a) introducing a target nucleic acid (NOI) into the cell; (b) introducing a vector according to any one of items 1 to 33 and 42 to 48 into said cell, wherein steps (a) and (b) are performed simultaneously or in any order; (c) expressing the derepressor and the crRNA or gRNA encoded by the vector in the cell, wherein the derepressor derepresses the CRISPR / Cas system, and the Cas nuclease of the system is guided by the crRNA or gRNA to modify (e.g., cleave) the protospacer sequence comprised by the NOI; and (d) optionally isolating the modified NOI.

[0403] 50. The method of item 49, which comprises introducing the isolated modified NOI into a second cell, and optionally obtaining progeny cells therefrom.

[0404] 51. The method of item 50, wherein the second cell is a non-human animal embryonic stem cell (ES cell) or an induced pluripotent stem cell (iPS cell).

[0405] 52. The method of item 51, which comprises developing the second cell or descendant cell into a non-human animal.

[0406] 53. The method of item 51, which further comprises isolating the protein or nucleic acid (or its nucleotide sequence) from the animal.

[0407] 54. The method of item 53, which further comprises inserting the nucleic acid or its nucleotide sequence into an expression vector or a host cell for expressing a protein comprising an amino acid sequence encoded by the nucleic acid or its nucleotide sequence, expressing the protein and isolating the protein, and optionally formulating the isolated protein into a drug for use in humans or animals.

Claims

1. A nucleic acid vector for introduction into a host cell, wherein the cell comprises a CRISPR / Cas system that is repressed by a repressor in the cell, the vector comprising (a) a nucleotide sequence encoding a derepressor capable of derepressing the CRISPR / Cas system in the cell, wherein the sequence is expressible in the cell to produce the derepressor; and (b) a CRISPR array for producing one or more crRNAs in the cell; and / or one or more nucleotide sequences encoding respective guide RNAs (gRNAs) in the cell; wherein each cRNA or gRNA is capable of directing Cas to modify a respective protospacer sequence of the host cell genome or to modify a protospacer sequence of an episome contained by the host in the presence of the derepressor.

2. The vector of claim 1, wherein the nucleotide sequence (a) comprises a constitutive promoter or a strong promoter for expressing the sequence in the host cell.

3. The vector of claim 1 or 2, wherein the nucleotide sequence or array (b) comprises a constitutive promoter or a strong promoter for expressing the sequence or array in the host cell.

4. The vector of any preceding claim, wherein (a) and (b) are comprised by the same operon or are under the control of a common promoter operable in the cell.

5. The vector of any preceding claim, wherein the host cell is a wild-type host cell.

6. The vector of any preceding claim, wherein transcription of one or more Cas sequences is repressed, optionally wherein transcription of one or more of CasA, B, C, D and E of a type I CRISPR / Cas system is repressed.

7. The vector of any preceding claim, wherein the Cas modification of the host cell genome (a) Killing host cells; (b) reducing the growth or proliferation of cells or episomes; (c) increasing the growth or proliferation of cells or episomes; (d) reducing or preventing transcription of a nucleotide sequence comprising or adjacent to said protospacer sequence; or (e) increasing transcription of a nucleotide sequence comprising or adjacent to the protospacer sequence.

8. The vector of any preceding claim, wherein the repressor is H-NS, StpA, LRP or CRP.

9. The vector of any preceding claim, wherein the derepressor is a mutant H-NS, StpA, LRP or CRP, which is capable of forming a complex with the H-NS, StpA, LRP or CRP repressor, respectively, in the host cell to prevent or reduce repression of the CRISPR / Cas system.

10. The vector of any preceding claim, wherein the derepressor is LeuO or LysR or a functional equivalent thereof.

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