Alteration of microbial populations and modification of microbiota

Abstract

To provide the use of a host modifying (HM) CRISPR / Cas system for inhibiting bacterial population growth or altering the relative proportions of subpopulations in a mixed bacterial population.SOLUTION: (I) a nucleic acid sequence encoding a Cas nuclease; (ii) a host cell target sequence and an engineered host modifying (HM) CRISPR array comprising a spacer sequence and repeats encoding a HM-crRNA comprising a sequence that hybridises to the host cell target sequence to guide Cas to the host cell target and modify the target sequence; (iii) a tracrRNA sequence or DNA sequence expressing same; (iv) a nucleic acid vector that is divided between the host cell and a nucleic acid vector that transforms the host cell; Whereby the HM-crRNA guides the Cas to the target and modifies the host CRISPR / Cas system in the host cell; wherein the target sequence is modified by the Cas, whereby the host cell is killed or host cell proliferation is reduced.SELECTED DRAWING: Figure 12

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention provides a method for inhibiting the growth of a bacterial population, inhibiting the growth of a first bacterial and a second bacterial subpopulation in a mixed bacterial population, and The present invention relates to a method for modifying the relative ratio, a nucleic acid array therefor, and a vector containing said array. The present invention provides an engineered system for host cell nucleic acid modification, components of the system, and an industrial and their applications in pharmaceuticals. The present invention relates to, for example, environmental, food and beverage uses. The present invention is particularly useful for treating microorganisms for industrial or domestic systems. Methods for treating microbiologically influenced corrosion (MIC) or biofouling of substrates or fluids in water The present invention also relates to processed fluids and vectors for use in the method. In one embodiment, the method uses horizontal propagation of the array. For this purpose, an array composed of mobile genetic elements (MGEs) and a vector containing the array are Tar also provided. [Background technology]

[0002] Background of the Invention Bacterial population growth inhibition and alteration of the relative proportions of coexisting bacterial species have been demonstrated in a wide range of industrial and other applications. It is applied to the treatment of waterways, drinking water or other environmental facilities in facilities and in the treatment of human and non-human Reduction of infectious diseases or rebalancing of intestinal or oral microflora in human animals, e.g., livestock. In recent years, the number of people with various weight or obesity profiles has increased. Analysis of the relative proportions of gut bacteria in humans or the impact of bacteria in diseases such as Crohn's disease There is also interest in exploring the possibilities of sound.

[0003] Although bacterial innate immune mechanisms against phages are abundant, bacterial adaptive immunity has been widely reported. The system is a CRISPR / Cas system. The system is found in various types of prokaryotic and eukaryotic cells, ranging from bacteria to animal and plant cells. It has been used for precise modification of nucleic acids in bacteria and Prokaryotes, such as archaea, use CRISPR / Cas (Cycloheximide / CRISPR) technology to encode short inverted synonymous repeats (RIRs). at) insertions that encode an adaptive immune system called CRISPR-associated against mobile invaders such as viruses (e.g., bacteriophages) and plasmids Bacteriophages (or phages) are the most abundant life forms on Earth. , estimated to be over 10 times more numerous than their bacterial prey (see Seed et al. (2013)). The constant threat of food generates a wide range of bacterial immune mechanisms, which in turn generate diverse phage immunity. This has resulted in disease-evasion strategies and a dynamic co-evolutionary arms race.

[0004] Host immunity is mediated by the uptake of invader DNA sequences in memory loci (CRISPR arrays), which The formation of guide RNA from the locus and its position next to the protospacer adjacent motif (PAM) It is based on the degradation of the cognate intercalator DNA (protospacer) that is placed in the target region. See No. 5424. The host CRISPR array consists of different elements: the repeats are identical, The immediately 5' leader (promoter) of one or more repeat-spacer-repeat units with different spacers Acquisition of spacer sequences from invading viral or plasmid nucleic acids This allows the host defense system to insert new spacers into the CRISPR array (each spacer is a replica). (adjacent to the target) to deal with future invasion by viruses or plasmids Recently acquired spacers can act as a memory for the host array reader. -It has been observed that insertion tends to occur immediately after

[0005] CRISPR loci and their associated genes (Cas) are responsible for the expression of phages and It has been reported that HIV-1 deficiency virus (HIV)-associated pathogens (HIV-1) confer adaptive immunity against HIV-1 deficiency virus (HIV-1) and other invasive genetic elements (Heler et al. (2014) )). A fundamental requirement of any immune system is to deal more efficiently with recurrent infections. The ability of the CRISPR-Cas immune system to build memories of past infections is key. The reactive function is to memorize the DNA sequence of the invading molecule and transfer it to the CRISPR antigen in the form of a 'spacer'. Transcription of the spacer depends on the ability of the leukocyte to integrate between the repeat sequences. A, which protects cells from infection by RNA-guided Cas nucleases. By acquiring a new spacer, CRISPR-Cas The immune system adapts rapidly to novel threats and is therefore 'adapted' (i.e., vector This is called a sequence spacer acquisition.

[0006] Seed et al. (2013) demonstrated that phage-encoded CRISPR / Cas systems are essential for the replication of bacterial hosts. reported a remarkable transformation of events that was used to counteract the phage-inhibitory chromosomal island. Successful lytic infection by CRISPR requires sequence identity between the CRISPR spacer and the target chromosomal island. In the absence of such targeting, phage-encoded CR The ISPR / Cas system allows efficient targeting of chromosomal islands for the restoration of phage replication. Bondy-Denomy et al. (2014) 2012) was an early example of a gene mediating inhibition of the CRISPR / Cas system. Five different 'anti-CRISPR' genes were used to inhibit bacteriophage infection. Mutations in the phage anti-CRISPR gene discovered in the genome of Domonas aeruginosa The functional CRISPR / Cas system renders bacterial infection impossible, and CRISPR / Cas Addition of the same gene to the genome of the targeting phage is achieved using the CRISPR / Cas system. Makes avoidance possible.

[0007] Premature RNA is transcribed from the CRISPR array and then matures to form crRNA. Some CRISPR / Cas systems also express trans-activating RNA (tracrRNA). It also contains a sequence encoding a repeat in the premature crRNA that hybridizes to the repeat in the premature crRNA. Pre-crRNA can be formed, which allows further processing to mature or The structure of the cRNA is a type involving the CRISPR / Cas system (type I, II or III).

[0008] CRISPR-associated (cas) genes are often associated with CRISPR arrays. Extensive comparative genomic analysis has identified many different cas genes, spanning 40 bacterial and archaeal species. Initial analysis of the fungal genome revealed the presence of 45 cas gene families, with two genes, cas1 It was suggested that only Cas1 and Cas2 may exist universally. It is believed to be essential for acquiring new spacers to the array, and therefore, This is an important mechanism for the development of resistance to invading nucleic acids from the host. Nunez et al. (2015) The Cas1-Cas2 complex is the minimal mechanism for catalyzing spacer DNA acquisition, and Cas The importance of CRISPR repeats in providing sequence and structural specificity for l-Cas2-mediated adaptive immunity It was reported that the importance of

[0009] The CRISPR / Cas system also detects the presence of neighboring nucleotides in the invader nucleotide sequence. Express a nuclease (e.g., Cas9) for cleavage of the phospho-cognate recognition motif (PAM). The PAM recognition of nucleases is specific to each type of Cas nuclease. The PAM of the invader sequence can be located immediately 3' of the protospacer sequence, and the nuclease , which generally cleaves 3-4 nucleotides upstream (5') of the PAM. Differing among ISPR-Cas systems and evolutionarily related to cas1 and leader sequences Fineran et al. (2014) reported that the invaders were located in the protospacer or its adjacent P By creating point mutations in the AM region (the "seed region"), the target gene of Escherichia coli K12 was Type IE CRISPR-Cas can evade immunity, but the host acquires ("priority") Integrating new spacers in a positive feedback process involving "mixing" To date, PAM has been shown to rapidly restore immunity to many type I and type II viruses. The effects of mutations in the protospacer have been well characterized in type I and type II systems. has been documented (see references 5, 14, 23, 46, and 47 in Fineran et al. (2014)). Fineran et al. (2014) found that their results, in agreement with previous studies, indicated that PAM and seed distribution It was concluded that this demonstrated the important role of columns.

[0010] Semenova et al. (2011) explored the role of seed sequences in Escherichia coli subtypes. For CRISPR / Cas systems, the requirement for crRNA matching is the sequence immediately following the PAM. We conclude that mutations in the seed region are critical for the regulation of crRNA-guided Ca2+ transcription. By decreasing the binding affinity of the scade complex to protospacer DNA, C It was observed to abolish RISPR / Cas-mediated immunity.

[0011] The stages of CRISPR immunity in each of the three major types of adaptive immunity are as follows: be. (1) Acquisition involves recognition of the invading DNA by Cas1 and Cas2 and opening of the protospacer. Begins with a cleft; (2) a protospacer sequence is ligated to the direct repeats adjacent to the leader sequence; and Call (3) Single-stranded extension repairs CRISPR and doubles the direct repeat.

[0012] The crRNA processing and interference steps are divided into three major CRISPR system types The primary CRISPR transcript is cleaved by Cas and In Type I systems, Cas6e / Cas6f bind directly to the ribosomal RNA and produce rRNA. Cleaved at the junction of ssRNA and dsRNA formed by a hairpin loop in Type II systems use transactivating (tracr) RNA to transduce dsRNA. The type III system forms a nucleotide sequence that is cleaved by Cas9 and RNase III. Use a Cas6 homolog that does not require the hairpin loop in the direct repeat for cleavage. In Type II and Type III systems, secondary trimming occurs at the 5' or 3' end. This is followed by the production of mature crRNA, which then binds to the Cas protein. Forms an interference complex. In type I and type II systems, crRNA and PAM Base pairing between the two cleavage sites leads to degradation of the invading DNA. Type III systems are responsible for successful degradation. In type III-A systems, no PAM is required, and base pairing is achieved in type III-B systems. It occurs not in the DNA targeted by the stem, but rather between the crRNA and the mRNA. Summary of the Invention

[0013] Description of the invention First aspect of the present invention The inventors have proposed a method for identifying microbiota (human, animal or environmental microbiota) using one or more of the following characteristics: For the first time, we have investigated the inhibition of population growth of specific bacterial strains in mixed communities of naturally occurring bacteria. I'm sure it was shown. · Targeting wild-type cells; Use of wild-type endogenous Cas nuclease activity; · Targeting essential antibiotic resistance genes; where the target is the wild-type sequence Inhibition of population growth by

[0014] We have demonstrated this with a mixed bacterial population with the following characteristics: Targeting mixed populations of human microbiota (e.g. gut microbiota) species; · where the population includes three species; · Involves selective killing of one of these species while sparing cells of the other species; Targets for cell growth inhibition in the presence of other phylogenetically related species that are preserved by such inhibition getting; Cells in a mixed population containing target Firmicutes and non-Firmicutes species targeting growth inhibition; - specific Firmicutes strains while preserving different Firmicutes species in mixed populations Targeting cell proliferation inhibition; Cells of specific Gram-positive bacterial strains while preserving different Gram-positive species in mixed populations targeting growth inhibition; Targeting pathogenic (in humans) bacterial species while sparing commensal human gut bacterial species ; · Targeting pathogenic bacterial species while sparing probiotic human gut bacterial species; · Targeting cell growth inhibition in mixed bacterial populations on surfaces; At least one bacterial species, either alone or mixed with multiple other bacterial species in a community Achieving 10-fold growth inhibition; and Achieving at least 10-fold growth inhibition of two different strains of a specific bacterial species.

[0015] The ability to utilize endogenous Cas activity in wild-type cells is essential for the development of cellular signaling pathways in organisms (e.g., humans and animals). It is extremely useful for the in situ treatment of host cell infections in humans, animals, and the environment. or wild-type (i.e., unengineered or pre-modified) such as plant microbiota. Treatment of bacterial populations can also be addressed using the present invention. The ability to exert selective growth inhibition is essential for tackling bacterial populations such as human, animal, or plant microbiota. This property is also useful for treating diseases such as asthma, asthma, and asthma in humans, and for addressing the environmental microbiome. For example, administered to a human or animal subject for any treatment or prevention disclosed herein. Bacterial cell transplants for or production of herbicidal or insecticidal compositions comprising the product bacterial population of the present invention The present invention is also useful for the production of selective killing of various bacteria in a mixed population. The ratio can be selectively varied to produce a pharmaceutical, herbicide or insecticide, or For example, a modified bacterial population can be produced in which a pharmaceutical or pesticide is produced. or may be implanted intranasally into an animal recipient to effect such treatment or prevention.

[0016] In the working examples below, growth inhibition is measured by measuring the bacterial population (Gram-positive phagocytic leukemia) on a solid surface. A >10-fold population growth inhibition was achieved. The present invention provides a method for inhibiting the growth of antibiotic-resistant bacteria. where the target sequence is the sequence of an antibiotic resistance gene. Co-administration of the leutide sequence with antibiotics may be effective. This may be more effective in humans or animals. provide treatment or prevention of host cell infection in a subject and / or administer to a human or animal This can allow for a reduction in the therapeutically effective antibiotic dose administered to humans and animals. This is useful in light of the increasing concerns about antibiotic overuse in the population and the emergence of resistance. The present invention also relates to the treatment of industrial or medical fluids, surfaces, devices or containers (e.g., food consumer goods, cosmetics, personal health care products, petroleum or petroleum products) or water treatment They also find ex vivo and in vitro applications for the treatment of tracts, water, beverages, food or cosmetics. wherein the host cell is present in a fluid, surface, device, container, waterway, water, beverage, food or cosmetic The present invention also addresses corrosion, biofilm and bio-attachment. The first aspect therefore provides the following concept:

[0017] Host modification to alter the relative proportions of primary and secondary bacterial subpopulations in a mixed bacterial population (HM) Use of a CRISPR / Cas system, wherein the second bacterium comprises a host cell; For each host cell, the system includes elements (i) to (iv). (i) at least one nucleic acid sequence encoding a Cas nuclease; (ii) HM-crRNA encoding spacer sequence (HM-spacer) and repeats A host cell target sequence and an engineered host-modified (HM) CRISPR array, The crRNA guides the Cas to a target in the host cell and modifies the target sequence in the host cell. those containing sequences that hybridize with the target sequence; (iii) any tracrRNA sequence or DNA sequence expressing a tracrRNA sequence; (iv) wherein the elements of the system comprise a host cell and at least one transformant that transforms the host cell. The nucleic acid vector is split between the HM-crRNA and the Cas gene, allowing it to target and guide the host. Modifying the host CRISPR / Cas system in host cells; and The target sequence is then modified by Cas, thereby killing or apoptotically targeting the host cell. Cell proliferation is inhibited.

[0018] A host cell for the use of claim 1 for the modification of a target nucleotide sequence in a bacterial host cell. 1. A modified (HM) CRISPR / Cas system, comprising: (i) at least one nucleic acid sequence encoding a Cas nuclease; (ii) HM-crRNA encoding spacer sequence (HM-spacer) and repeats A host cell target sequence and an engineered host-modified (HM) CRISPR array, The crRNA guides the Cas to a target in the host cell and modifies the target sequence in the host cell. those containing sequences that hybridize with the target sequence; (iii) any tracrRNA sequence or DNA for expressing a tracrRNA sequence array; (iv) wherein the elements of the system comprise a host cell and at least one vector capable of transforming the host cell. The nucleic acid vectors are split between the HM-crRNA and the Cas, which guides the HM-crRNA to target the target. modifies the host CRISPR / Cas system in the host cell; A system including elements (i) to (iv).

[0019] This means that we have demonstrated at least a 10-fold increase in the selective host cell population in mixed and non-mixed cell populations. The mixture is exemplified by examples showing inhibition of cell proliferation. and mimic the combination of strains.

[0020] Use of wild-type endogenous Cas nuclease activity of bacterial host cell populations to inhibit population growth wherein each host cell contains an endogenous CRIS having wild-type Cas nuclease activity. The use includes transforming a population of host cells, wherein each transformant has a PR / Cas system. The recombinant host cell is a recombinant host cell that expresses host-modified (HM) cRNA or guide RNA (gRNA) in the host cell. and transformed with a modified nucleotide sequence to provide HM-cRNA or gR NA contains a host cell targeting protospacer sequence and a hybridization sequence to guide endogenous Cas to target the target. wherein the cRNA or gRNA comprises a hybridizable sequence to the wild-type nuclease. It is homologous to the endogenous Cas nuclease of the host cell, which has enzyme activity, and after host cell transformation , the growth of the population is inhibited.

[0021] Host-modified (HM) CRISPR / Cas systems for killing or reducing proliferation of bacterial host cells Use of the system (if desired, use in accordance with the use in the immediately preceding paragraph), for each host cell , the system is (i) at least one nucleic acid sequence encoding a Cas nuclease; (ii) HM-crRNA encoding spacer sequence (HM-spacer) and repeats A modified host-modified (HM) CRISPR array, wherein the HM-crRNA binds the Cas. hybridize with a host cell target sequence to guide the target to the host cell and modify the target sequence; containing sequences to be adjusted; (iii) any tracrRNA sequence or DNA sequence expressing a tracrRNA sequence; (iv) wherein the components of the system comprise a host cell and at least one transformant that transforms the host cell. The nucleic acid vector is split between the HM-crRNA and the Cas gene, allowing it to target and guide the host. modifying target sequences in host cells; wherein the Cas nuclease is endogenous to the host cell, and wherein the target sequence is and thereby killing or reducing host cell proliferation. be It includes elements (i) to (iv).

[0022] Therefore, the HM-cRNA guides the Cas to the target in the host cell and The nucleic acid sequence can hybridize with a host cell target sequence to modify the nucleic acid sequence. Alternatively, the HM-crRNA and tracrRNA may be combined with a single guide RNA (gRNP). A).

[0023] By utilizing endogenous Cas nucleases, embodiments of the present invention provide nuclease activity (pretreatment of host cells to activate or enhance nuclease activity) (No genetic modification is required.) Therefore, for example, Cas nucleases can be used to transfect wild-type genes in host cells. For example, nucleases can be encoded by endogenous Cas nucleases in the host cell. without inactivating the repressor of the Casase (or Cas nuclease gene) Therefore, the present invention provides an efficient Cas-mediated cell It can address wild-type bacterial populations without the need for prior manipulation to result in death or reduced growth. Therefore, populations can be compared to their wild-type environment (e.g., waterways or human or animal mammals). When the cells are in the microbiome, they can be exposed to cRNA.

[0024] For example, the first bacterium is a Bacteroidetes (e.g., Bacteroides) cell. The second bacterium is a Firmicutes cell. The method can be used, for example, to analyze a gut microbiota population (e.g., For example, ex vivo or in vivo) to alter the rate of growth, which can be used to, for example, increase body weight. and for the treatment or prevention of hypertension or obesity (e.g., wherein the first bacterium is a Firmicutes cells).

[0025] The first aspect also provides: a mixed bacterium comprising a subpopulation of a first bacterium and a subpopulation of a second bacterium; a method for modifying the relative proportions of said subpopulations in a population, wherein a first bacterium is mutated by a phage The second bacterium is a host cell (e.g., a Bacteroidetes cell) that is infected with the phage. the vector nucleic acid is not transfected with the host cell (or is not a Bacteroidetes bacterium), and the method and then mixed with the mixed population in one or more steps to allow bacterial growth in the mixed population. and combining a number of vectors, wherein the relative ratio of the first bacterium to the second bacterium is altered. , wherein each vector is a phage target nucleotide sequence for modifying the phage target nucleotide sequence in a cell. Engineered phage-modified (PM) CRISPR arrays for introduction into phage-infected host cells Including A, (a) wherein the PM-CRISPR array comprises one or more sequences for expression of PM-crRNA. and a promoter for transcription of the sequence in a phage-infected host cell; and (b) Here, PM-crRNA delivers Cas (e.g., Cas nuclease) to infected host cells. The phage target sequence can then be hybridized to the phage target sequence to modify the target sequence.

[0026] In a second aspect, the present invention provides: Host-modified (HM) CRISPR / Cas for modification of target nucleotide sequences in host cells 1. A system (e.g., for use in a first mode), comprising: (i) at least one nucleic acid sequence encoding a Cas nuclease; (ii) HM-crRNA encoding spacer sequence (HM-spacer) and repeats A modified host-modified (HM) CRISPR array, wherein the HM-crRNA binds the Cas. It is capable of hybridizing with a host target sequence to guide it to a target in a host cell and modify the target sequence. containing sequences that can be used; (iii) any tracrRNA sequence or DNA for expressing a TracrRNA sequence array; (iv) wherein said elements of the system comprise a host cell and at least one gene capable of transforming the host cell. The nucleic acid vector is split between the HM-crRNA and the Cas gene, allowing it to target and guide the host. modifying target sequences in host cells It includes elements (i) to (iv), wherein, optionally, element (i) is endogenous to the host cell.

[0027] The second aspect also provides the following: a method of the first aspect for modifying the genome of a phage, 1. A modified phage-modified (PM) CRISPR array for use in (a) wherein the PM-CRISPR array comprises one or more sequences for expression of PM-crRNA. and a promoter for transcription of the sequence in a phage-infected host cell; and (b) Here, PM-crRNA directs Cas (e.g., Cas nuclease) into infected host cells. can hybridize to a phage genome target sequence to guide and modify the target sequence .

[0028] For example, the phage may be a Bacteroidetes (e.g., Bacteroides) phage, e.g., c It is the rAss phage. For example, the arrays are functional in the host cell CRISPR / Cas system. This is beneficial for increasing the selectivity of the array to the desired cells in the bacterial mixture. This also includes one or more Cas proteins (and It is not necessary to include bulky nucleotide sequences encoding the tracrRNA and / or tracrRNA. This simplifies the production of arrays and vectors comprising the arrays of the invention, as they may be easily replicated. Alternatively, the array may contain cognate Cas9 coding sequences and, optionally, cognate tracrRN. A coding sequence is provided.

[0029] In a third aspect, the present invention provides: Engineered nucleic acids for the modification of bacterial host cells containing endogenous CRISPR / Cas systems A vector, (a) for use in a CRISPR / Cas system or for use in accordance with the present invention For expression of multiple different crRNAs (e.g., single guide RNAs, i.e., gRNAs) and (b) lacking a nucleic acid sequence encoding a Cas nuclease; wherein a first of said crRNAs is capable of hybridizing to a first nucleic acid sequence of said host cell; and A second of the crRNA is capable of hybridizing to a second nucleic acid sequence of the host cell, wherein the second sequence the sequence is different from the first sequence; and (c) the first sequence contains an antibiotic resistance gene (or its RNA), and the second sequence contains an anti-antibiotic resistance gene; sex genes (or RNA thereof), and optionally, these genes are different; (d) the first sequence contains an antibiotic resistance gene (or its RNA) and the second sequence contains an essential or pathogenic gene; containing the proto-gene (or its RNA); (e) The first sequence contains an essential gene (or its RNA), and the second sequence contains an essential or pathogenic gene. or containing the offspring (or its RNA) (f) the first sequence contains a virulence gene (or its RNA), and the second sequence contains an essential or virulence gene; Contains a gene (or its RNA).

[0030] The third aspect also provides: a nucleic acid vector for use in the method of the invention; - (e.g., a plasmid, a phage, or a phagemid), the vector being a C Contains RISPR arrays.

[0031] In a fourth aspect, the present invention provides: The genome of a host bacterial cell (e.g., a pathogenic bacterial cell as described above) or a host cell Modified CRIS for modifying a target sequence in the genome of a virus (e.g., a phage) A nucleic acid vector (e.g., a plasmid, virus, phage, or dimide), (a) Here, the CRISPR array is used to measure the expression of crRNA (e.g., provided as gRNA). and a promoter for transcription of the sequence in a host cell; (b) where the crRNA guides Cas (e.g., Cas nuclease) in the host cell. and capable of hybridizing with a target sequence to modify the target sequence; (c) Here, the array is a transmembrane array that can be horizontally transferred between a first bacterium and a second bacterial cell of a different species. It consists of poson.

[0032] In a fifth aspect, the present invention provides: Modified CRISPR nucleic acid vectors containing or consisting of mobile genetic elements (MGEs) wherein the MGE is a gene that is inserted into the genome of a host cell (e.g., a pathogenic bacterial cell) or into a host cell. Origin of transfer (or iT) and CRISPR arrays, (a) wherein the CRISPR array comprises one or more sequences for expression of crRNA and a host cell a promoter for transcription of the sequence in the cell; (b) where the crRNA guides Cas (e.g., Cas nuclease) in the host cell. and capable of hybridizing with a target sequence to modify the target sequence; (c) wherein the vector (i) is inserted between a first nucleic acid and a second nucleic acid locus in a first host cell, where each locus is a transfectant. (ii) the location of the target sequence in the host cell; or (iii) the location of the target sequence in the host cell. Between the host cell and a second host cell (wherein the target sequence is contained in the first and / or second host cell) It can be transmitted in

[0033] In a sixth aspect, the present invention provides: Controlling microbially affected corrosion (MIC) or biofouling of substrates in industrial or domestic systems a method for detecting a surface of a substrate, wherein the surface of the substrate is a first microbial species that mediates MIC or biofouling of the substrate; contacting a population of first host cells of the species, and the method (i) contacting the population with a plurality of vectors capable of transforming or transducing cells, and The vector contains a CRISPR array, which allows the CRISPR array to be introduced into the host cell. Here, (a) Each CRISPR array contains one or more nucleotide sequences for expression of a crRNA and a promoter for transcription of the sequence in the host cell; and (b) Each crRNA guides a Cas (e.g., a Cas nuclease) into the host cell and targets the target. To modify the sequence (e.g., to cleave the target sequence), the target sequence is hybridized with the target sequence in the host cell. and the target sequence is a gene sequence that mediates host cell viability; and (ii) expressing the cRNA in the presence of Cas in a host cell, thereby The target sequence is modified to reduce host cell viability and control the MIC or biofouling of the substrate. bring This includes:

[0034] In other embodiments, the following is provided: Microorganisms of substrates contained in crude oil, gas or petrochemical recovery, processing, storage or transportation equipment A method for controlling biofouling (MIC) or biofouling, wherein the surface of a substrate is treated with a first host cell. The first host cell contacts a population of cells, where the first host cell mediates MIC or bioadhesion of the substrate. species of sulfur- or sulfate-reducing bacteria (SRB), extracellular polymeric substance-producing bacteria (EPSB), and acid-producing bacteria (APB), sulfur or sulfide oxidizing bacteria (SOB), iron oxidizing bacteria (IOB), manganese oxidizing bacteria (MOB), ammonia-producing bacteria (AmPB) or acetogenic bacteria (AcPB), The surface and cell populations are selected from the group consisting of seawater, freshwater, fracking fluid, or well fluid. The method is selected (i) combining the cell population and the vector into a liquid and a plurality of vectors capable of transforming or transducing the first host cell; The vectors are contacted by mixing, each vector containing a CRISPR array, This allows the CRISPR array to be introduced into the host cell, where it (a) Each CRISPR array contains one or more sequences for expression of crRNA and a host cell. a promoter for transcription of the sequence; (b) Each crRNA guides a Cas (e.g., a Cas nuclease) into the host cell and targets the target. hybridize with a target sequence in a host cell to modify the target sequence (e.g., cleave the target sequence). soybean, and the target sequence is a gene sequence that mediates host cell viability; (c) wherein each sequence of (a) is a sequence encoding a nucleic acid sequence encoding a nucleic acid sequence that is capable of expressing and producing each crRNA in a first host cell. for the CRISPR repeat, where R1 is the first CRISPR repeat and R 1' is the second CRISPR repeat, R1 or R1' is optional, and S1 is the first host A nucleic acid sequence comprising or consisting of a nucleotide sequence that is 80% or more identical to a target sequence of a cell. a CRISPR spacer, and (ii) expressing the cRNA in the presence of Cas in a host cell, thereby The target sequence is modified to reduce host cell viability and control the MIC or biofouling of the substrate. bring This includes:

[0035] Other embodiments provide the following: A vector for use in the method, wherein the first cell is a sulfate-reducing bacterial (SRB) cell. and the vector is a SR B. one or more CRISPR arrays for targeting, wherein each array comprises (a) to As defined in (c).

[0036] In other embodiments, the following is provided: A method for controlling microbial biofouling of a fluid, wherein the fluid is a first agent that mediates said biofouling. the method includes a population of first host cells of a microbial species, (i) contacting the population with a plurality of vectors capable of transforming or transducing cells, and The vector contains a CRISPR array, which allows the CRISPR array to be introduced into the host cell. Here, (a) Each CRISPR array contains one or more nucleotide sequences for the expression of crRNA and and a promoter for transcription of the sequence in the host cell; and (b) Each crRNA guides a Cas (e.g., a Cas nuclease) into the host cell and targets the target. hybridize with a target sequence in a host cell to modify the target sequence (e.g., cleave the target sequence). made in soybean, wherein the target sequence is a gene sequence that mediates host cell viability; and (ii) expressing the cRNA in the presence of Cas in a host cell, thereby Modification of target sequences, resulting in reduced host cell viability and control of biofouling This includes:

[0037] For example, the following may be provided: To control bacterial biofouling in the ballast water of ships or vessels; a method for controlling the biofouling of a first host cell population of a first microbial species, wherein the water contains a first host cell population of a first microbial species that mediates the biofouling; The method includes (i) contacting the population with a plurality of vectors capable of transforming or transducing cells, and The vector contains a CRISPR array, which allows the CRISPR array to be introduced into the host cell. Here, (a) Each CRISPR array contains one or more sequences for expression of crRNA and a host cell. comprising a promoter for transcription of the sequence; and (b) Each crRNA guides a Cas (e.g., a Cas nuclease) into the host cell and targets the target. hybridize with a target sequence in a host cell to modify the target sequence (e.g., cleave the target sequence). soybean, and the target sequence is a gene sequence that mediates host cell viability; and (ii) expressing the cRNA in the presence of Cas in a host cell, thereby Modification of target sequences, resulting in reduced host cell viability and control of biofouling This includes:

[0038] Other embodiments involve the use of ballast seawater (e.g., seawater samples or is seawater in a container), where the ballast water is obtained or obtained by the method. The ballast water may be contained in a ship, vessel, marine container, or drilling rig. vectors for use in the preparation of cholera (e.g., Vibrio, e.g., O1 or or O139), Escherichia coli, or Enterococcus cells, and the vector and one or more CRISPR arrays for cell targeting, wherein each array is a As defined in (a) and (b).

[0039] The present invention also relates to the use of this sixth aspect or other applications such as medical uses or food or beverage processing. Also provided are vectors and CRISPR arrays suitable for use in the application. Provided are vectors for introducing CRISPR arrays into bacterial host cells. So, here the bacteria can be transmitted through water, (a) The CRISPR array includes sequences for expression of crRNA and sequences in the host cell. a promoter for transcription of (b) The crRNA guides Cas (e.g., Cas nuclease) into the host cell to target the gene. capable of hybridizing to a host cell target sequence for modification of the sequence (e.g., cleavage of the target sequence) and the target sequence is a nucleotide sequence that mediates host cell viability; (c) wherein the sequence of (a) comprises the sequence R1-S1-R1' for expression and production of crRNA. wherein R1 is a first CRISPR repeat and R1' is a second CRISPR repeat. R1 or R1' is optional, and S1 is a nucleic acid sequence that is 80% or more identical to the host cell target sequence. A first CRISPR spacer comprises or consists of a nucleotide sequence.

[0040] Also provided is a water or food treatment composition comprising a plurality of such vectors. A medicament for the treatment or prevention of bacterial infections (e.g., Vibrio cholerae infections) in humans. The present invention also relates to bacterial populations, compositions, Food and beverages are also provided, for example, the food or beverage is a dairy product.

[0041] In a seventh aspect, the present invention provides: In a first aspect, A method for modifying an expressible gene encoding a first Cas, the method comprising: (a) A guide RNA (gRNA1) and a Cas gene are combined with the first Cas gene expressed from the gene. Combined in the presence of, and (b) gRNA1 is linked to the sequence of the Cas gene (e.g., its promoter or the first Cas code). The first Cas is hybridized to the gene (a coded DNA sequence) to guide the first Cas to the gene, Cas modifies Cas genes This includes:

[0042] For example, a first nucleic acid vector or combination of vectors for use in the methods There, here (a) The first vector or the vector of the combination guides the first Cas to the first site (CS1) is complementary to PS1 to modify the intended protospacer sequence (PS1). a guide RNA (gRNA1, e.g., a single gRNA) that guides the first Cas1 gene, The cognate PAM (P1) or expressible sequence is involved in the tracrRNA and gR encoding the crRNA that forms NA1; and (b) PS1 and P1 are the sequences of the first expressible Cas-encoding gene, and PS1 is the It can be modified with CS1 by one Cas.

[0043] These aspects of the invention are directed to methods for regulating Cas activity, e.g., in cells or in vitro. The present invention provides a method for targeting Cas-encoding genes to limit Cas activity. The present invention also provides a method for the transient regulation of Cas, e.g., in cells. High stringency to reduce the chance of off-target Cas cleavage in the modified genome Applications may also be useful in settings where it is desirable to For gene therapy or gene targeting of organisms, off-target effects are minimized or eliminated For example, in the modification of human, animal or plant cells. as modifications for gene therapy or for the treatment or prevention of a disease or condition in humans When used for the desired modification of human cells (e.g., iPS cells) to be administered to patients, High stringency is required. This disclosure is provided as part of the methods and products of the invention. These applications are provided.

[0044] The present invention also addresses the problem of limited insertion capacity of vectors, particularly viral vectors. nothing.

[0045] Therefore, in an eighth aspect of the present invention there is provided: Foreign DNA sequences greater than 1.4 kb encoding elements of the CRISPR / Cas system A nucleic acid vector comprising the sequence, wherein the sequence is capable of infecting a host cell (any cell herein, e.g. , human, animal, or bacterial or archaeal host cells) Modified arrays or sequences for expression of rRNA or gRNA (as desired) as described herein), wherein the array or modified sequence is cRNA or does not contain a nucleotide sequence encoding a Cas nuclease that is cognate to the gRNA or the gRNA, Optionally, at least two, three, or four of the cRNAs or gRNAs are exogenous DNA. is coded by

[0046] A nucleic acid vector comprising a foreign DNA sequence of more than 1.4 kb or more than 4.2 kb. wherein the exogenous DNA encodes one or more elements of a CRISPR / Cas system and Modified architecture for expressing one or more HM-crRNAs or gRNAs in host cells a sequence or sequence (e.g., any such described herein), wherein the exogenous sequence The columns represent nucleotides encoding Cas nucleases that are cognate to the cRNA or gRNA. and optionally at least two different cRNAs or gRNAs are It is coded by A.

[0047] Here, in all forms, for example, the cRNA may comprise one or more single guide RNAs (gRNAs). A), in which case the "CRISPR array" is one or more CRISPR arrays encoding the gRNAs. Therefore, the sequence may refer to a nucleotide sequence capable of expressing the gRNA inside the cell. It can be expressed in a host cell for expression.

[0048] This invention is primarily described for bacteria, but can be applied mutatis mutandis to archaea. It is possible.

[0049] Any feature of one aspect herein may be used in combination with, for example, a different aspect of the invention. It is possible that such combinations may be encompassed by one or more claims of this application. [Brief explanation of the drawings]

[0050] [Figure 1] Xylose-inducible system.

[0051] [Figure 2] ST1-CRISPR array.

[0052] [Figure 3] Spot assay on TH agar for the strains used in this experiment. All strains were grown on TH agar for 20 hours at 37°C. Serial dilutions of overnight cultures were performed in duplicate for E. coli, L. lactis, and Streptococcus mutans, and in triplicate for both Streptococcus thermophilus strains to count individual colonies.

[0053] [Figure 4] Selective growth of Streptococcus thermophilus, Streptococcus mutans, Lactococcus lactis, and Escherichia coli under various culture conditions. Tetracycline could not be used for the selective growth of Streptococcus thermophilus LMD-9. However, 3 g l-1 of PEA was shown to selectively grow Streptococcus thermophilus LMD-9 while restricting the growth of Escherichia coli.

[0054] [Figure 5] Construction of two xylose-inducible cassettes (center and right) based on the wild-type Bacillus megaterium operon (left). (Xie et al. 2013)

[0055] [Figure 6] Characterization of the xylose-inducible cassette in Streptococcus thermophilus LMD-9 in the plasmid pBAV1KT5-XylR-mCherry-Pldha. A clear fluorescence response is observed with increasing amounts of xylose.

[0056] [Figure 7] Engineering a CRISPR array in pBAV1KT5-XylR-mCherry-Pldha+XylA. The array contains two spacer sequences targeting Streptococcus thermophilus genes under the inducible xylose promoter and tracrRNA under the strong constitutive promoter P3A.

[0057] [Figure 8] Transformation efficiency of Streptococcus thermophilus LMD-9 with the plasmids pBAV1KT5-XylR-CRISPR-Pldh+Xyl A (left) and pBAV1KT5-XylR-CRISPR-PXylA (right).

[0058] [Figure 9]Schematic of the xylose-inducible CRISPR device. Xylose induction leads to expression of the CRISPR array targeting polIII and tetA in the Streptococcus thermophilus LMD-9 genome. A complex is formed with Cas9 along with the constitutively expressed tracrRNA. This complex introduces double-strand breaks in the tetA and polIII genes in the Streptococcus thermophilus LMD-9 genome, resulting in restricted cell viability.

[0059] [Figure 10] Growth inhibition of Streptococcus thermophilus DSM 20617(T) with plasmid pBAV1KT5-XylR-CRISPR-PXylA (left) or pBAV1KT5-XylR-CRISPR-Pldha+XylA (right). Uninduced (top panel) and induced (bottom panel). Pictures were taken after 63 hours of incubation. Colony counts in the lower left corner (top row: >1000, >1000; bottom row: 336, 113).

[0060] [Figure 11] Maximum likelihood phylogenetic tree of 16S sequences from Streptococcus thermophilus, Lactococcus lactis, and Escherichia coli.

[0061] [Figure 12]Selective growth inhibition of Streptococcus thermophilus in co-cultures of E. coli, Lactococcus lactis, and Streptococcus thermophilus harboring the pBAV1KT5-XylR-CRISPR-PxylA or pBAV1KT5-XylR-CRISPR-PldhA+Xyl plasmids. No difference in growth was observed in E. coli harboring the pBAV1KT5-XylR-CRISPR-PxylA or pBAV1KT5-XylR-CRISPR-PldhA+Xyl plasmids (center column). However, Streptococcus thermophilus (selective growth on TH agar supplemented with 2.5 g l-1 PEA, last column) shows, as expected, transformation efficiency between the pBAV1KT5-XylR-CRISPR-PxylA (strong) and pBAV1KT5-XylR-CRISPR-PldhA + XylA (weak) plasmids. We thus demonstrate selective growth inhibition of the target Streptococcus thermophilus subpopulation in a mixed population of cells. Colony counts are shown in the lower left corner (top row: >1000, >1000, 68; bottom row: >1000, >1000, 32). DETAILED DESCRIPTION OF THE INVENTION

[0062] Detailed Description Inhibition of microbial population growth and alteration of microbial ratios The present invention relates to the Bacteroidetes phylum (e.g., Bacteroides), Phascolytic fungi, and the like in the human microbiota. Alterations in bacterial populations, such as alterations in the ratio of gram-micutes and / or gram-positive or gram-negative bacteria inhibiting bacterial growth or altering the relative proportions of the first and second bacterial subpopulations in a mixed bacterial population; For example, methods, uses, systems, and allergens for the modification of the human or animal microbiome. a) cRNA, gRNA, and vectors. For example, the first to third forms described herein The present invention relates to, for example, a host bacterial cell, such as a Bacteroidetes cell or a phylum cell. It involves modification of one or more target nucleotide sequences in a micutes cell.

[0063] In both humans and germ-free mice, two major intestinal phyla, Bacteroidetes and Família, There are numerous papers pointing out that various levels of Cutes are associated with obesity. The authors speculate that carbohydrate metabolism may be an important factor. The biota was more heavily enriched in Firmicutes bacteria and less in Bacteroidetes. They observed that this bacterial mixture was probably due to the microorganisms in the lean individuals (which had the opposite ratio). They speculate that they extract energy more efficiently from a given diet than other organisms. In a study, the relative abundance of Bacteroidetes increased as obese individuals lost weight. Furthermore, when the microbiota of obese mice was transferred to germ-free mice, these mice became lean. Mice were found to gain more fat than controls who received microbiota. h, PJ, RE Ley, MA Mahowald, V. Magrini, ER Mardis, and JI Gordon. 2006, “An obesity-associated gut microbiome with increased capacity for energy See "harvest", Nature 444:1027-1131.

[0064] concept The present invention provides the following concepts involving host cell targeting. 1. Host-modifying (HM) CRISPR / Cas systems for killing or reducing proliferation of bacterial host cells and for each host cell, the system (i) at least one nucleic acid sequence encoding a Cas nuclease; (ii) HM-crRNA encoding spacer sequence (HM-spacer) and repeats A modified host-modified (HM) CRISPR array, wherein the HM-crRNA binds the Cas. hybridize with a host cell target sequence to guide the target to the host cell and modify the target sequence; containing sequences to be adjusted; (iii) any tracrRNA sequence or DNA sequence expressing a tracrRNA sequence; (iv) wherein the components of the system comprise a host cell and at least one transformant that transforms the host cell. The nucleic acid vector is split between the HM-crRNA and the Cas gene, allowing it to target and guide the host. Modifying target sequences in host cells It includes elements (i) to (iv), wherein the Cas nuclease is endogenous to the host cell, and wherein the target sequence is The enzyme is then modified to kill the host cell or reduce host cell proliferation.

[0065] Concept 1 also offers the following: Host modification to alter the relative proportions of primary and secondary bacterial subpopulations in a mixed bacterial population (HM) Use of a CRISPR / Cas system, wherein the second bacterium is contained in a host cell and each For host cells, the system (i) at least one nucleic acid sequence encoding a Cas nuclease; (ii) HM-crRNA encoding spacer sequence (HM-spacer) and repeats A modified host-modified (HM) CRISPR array, wherein the HM-crRNA binds the Cas. hybridize with a host cell target sequence to guide the target to the host cell and modify the target sequence; containing sequences to be adjusted; (iii) any tracrRNA sequence or DNA sequence expressing a tracrRNA sequence; (iv) wherein the components of the system comprise a host cell and at least one transformant that transforms the host cell. The nucleic acid vector is split between the HM-crRNA and the Cas gene, allowing it to target and guide the host. Modifying target sequences in host cells It includes elements (i) to (iv), wherein optionally the Cas nuclease is endogenous to the host cell, and The target sequence is then modified by Cas, thereby killing or inhibiting the host cell. Cell proliferation is reduced.

[0066] Concept 1 also improves the relative proportions of primary and secondary bacterial subpopulations in a mixed bacterial population. a method of transforming a mixed population of host-modified (HM) Cs into a second bacterium, the second bacterium comprising a host cell, the method comprising: Combined RISPR / Cas systems, thereby killing or transforming the second bacterial host cell reduces proliferation of the cells, thereby altering the ratio, Here, for each host cell, the system (i) at least one nucleic acid sequence encoding a Cas nuclease; (ii) HM-crRNA encoding spacer sequence (HM-spacer) and repeats A modified host-modified (HM) CRISPR array, wherein the HM-crRNA binds the Cas. hybridize with a host cell target sequence to guide the target to the host cell and modify the target sequence; containing sequences to be adjusted; (iii) any tracrRNA sequence or DNA sequence expressing a tracrRNA sequence; (iv) wherein the components of the system comprise a host cell and at least one transformant that transforms the host cell. The nucleic acid vector is split between the HM-crRNA and the Cas gene, allowing it to target and guide the host. Modifying target sequences in host cells It includes elements (i) to (iv), wherein optionally the Cas nuclease is endogenous to the host cell, and The target sequence is then modified by Cas, thereby killing or inhibiting the host cell. Cell proliferation is reduced.

[0067] Concept 1 also offers: Host modification to alter the relative proportions of primary and secondary bacterial subpopulations in a mixed bacterial population (HM) Use of a CRISPR / Cas system, wherein the second bacterium is a target protospacer. and for each host cell, the system comprises a plurality of host cells comprising a sequence as defined above. The system further comprises at least one enzyme encoding a Cas nuclease. and another nucleic acid sequence, wherein said element (ii) and said Cas encoding sequence form a host cell. (i) HM-crRNA guides Cas to modify the target sequence in the host cell; wherein the Cas nuclease is endogenous to the host cell, and wherein the target sequence is The resulting modification results in the death of the host cell or a decrease in host cell proliferation.

[0068] In some embodiments, the growth of the first bacterium is not inhibited or the growth inhibition of the host cell is , at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x of growth inhibition of the first cells, 10x, 50x, 100x or 1000x. Growth inhibition is expressed as fold inhibition or percent inhibition. In other instances, inhibition can be calculated as a percentage of the culture surface area (as described herein). The optical absorbance (e.g., OD) is measured in a sample using a spectrophotometer. 600 ) is planned Measure at the beginning and end of the crRNA / gRNA treatment period (expressed as fold inhibition or (See the description herein of such periods when determined as percentages). For example, the increase in absorbance of the host cell sample (absorbance at the start of a predetermined period and absorbance at the end of that period) The absorbance (comparison of absorbance at time of exposure) was less than that of the control sample (not exposed to the cRNA or gRNA). For example, the increase in the former is at least 10 times, 100 times, 1000 times, or 1000 times greater than the latter. 0 or 100,000 times less (e.g., OD 600 For example, the inhibition of growth The determination (i.e., at the end of the predetermined period) is made during mid-logarithmic growth of each sample (e.g., 6-7 hours after the start of the scheduled period).

[0069] For example, the host cell may be a member of an organism or the environment (e.g., waterway microbiota, aquatic microbiota, human or animal intestinal microbiota, human or animal oral microbiota, human or animal vaginal microbiota, human or animal or animal skin or hair microbiota or human or animal axillary microbiota) A community contains a primary bacterium that is mutualistic or commensal with an organism or the environment. The second bacterium comprises the host cell, wherein the host cell is harmful to an organism or the environment (e.g., a pathogen). In some embodiments, the population is ex vivo.

[0070] The ratio of the first bacterial subpopulation to the second bacterial subpopulation increases.

[0071] Concept 1 also provides uses for inhibiting host cell proliferation, as further described below.

[0072] 2. For modification of a target nucleotide sequence in a host cell (e.g., for use in Concept 1) A host-modified (HM) CRISPR / Cas system, wherein the system (i) at least one nucleic acid sequence encoding a Cas nuclease; (ii) HM-crRNA encoding spacer sequence (HM-spacer) and repeats A modified host-modified (HM) CRISPR array, wherein the HM-crRNA binds the Cas. It is capable of hybridizing with a host target sequence to guide it to a target in a host cell and modify the target sequence. containing sequences that can be used; (iii) any tracrRNA sequence or DNA for expressing a tracrRNA sequence array; (iv) wherein the elements of the system comprise a host cell and at least one vector capable of transforming the host cell. The HM-crRNA guides Cas to target the target. modifying a target sequence in a host cell; It includes elements (i) to (iv), wherein, optionally, element (i) is endogenous to the host cell.

[0073] Alternatively, the HM-crRNA and tracrRNA may be combined with a single guide RNA (gRNP). A).

[0074] By utilizing endogenous Cas nucleases, embodiments of the present invention provide nuclease activity (i.e., to activate or enhance nuclease activity) (without requiring prior genetic modification of the cells). Therefore, for example, Cas nucleases can be The nuclease is encoded by a wild-type gene in the host cell. For example, Without inhibiting the endogenous Cas nuclease (or Cas nuclease gene) repressor , active to achieve cell killing or reduced proliferation. Therefore, the present invention provides an efficient C wild-type cells without the need for prior manipulation to result in AS-mediated cell death or reduced proliferation. Therefore, it is possible to analyze the population in a way that allows the population to adapt to its natural environment (e.g., waterways). or human or animal microbiomes), exposure to cRNA can.

[0075] For example, the second bacterium is a Bacteroidetes (e.g., Bacteroides) cell. The second bacterium is a Firmicutes cell. used to alter the ratio of microbiota populations (e.g., ex vivo or in vivo), which for example, for the treatment or prevention of weight gain or obesity (e.g., where the second bacterium is a (These are Firmicutes cells).

[0076] For example, uses, methods, systems, vectors, modified nucleotide sequences, cRNA or or gRNAs may be used, for example, to treat or improve obesity, diabetes, IBD, GI tract conditions, or oral conditions. or for the therapeutic or prophylactic use of the microbiota of humans or non-human animals, including mixed populations, for prophylaxis. This is for rebalancing.

[0077] For example, the microbiota described herein can be a human or animal microbiome (e.g., gut, The microbiota of the vagina, scalp, axilla, skin, bloodstream, pharynx or oral microbiome.

[0078] For example, the microbiota described herein is axillary microbiota, and the uses, methods, systems, vectors, The target, modified nucleotide sequence, cRNA or gRNA may be used to prevent or control body odor in humans. is for mitigation.

[0079] For example, the host cell population or mixed population may be used in beverages or water for human consumption (e.g., water in food or drinking water.

[0080] For example, uses, methods, systems, vectors, modified nucleotide sequences, cRNA or or gRNAs may be used to reduce pathogenic infections or rebalance the gut or oral microbiota, e.g. For the treatment or prevention of obesity or disease in humans or animals. , system, vector, modified nucleotide sequence, cRNA or gRNA in the intestine for knockdown of Clostridium difficile bacteria in the microbiota.

[0081] For example, the first bacterium is a Bacteroides bacterium and the second bacterium is a Firmicutes or The pathogenic bacteria, e.g., enterobacteria. For example, the host cell or the second bacterium, e.g., streptococcus aureus, Leptococcus (e.g., Thermophilus and / or Pyogenes), Bacillus, Lactobacillus Bacillus, Listeria, Clostridium, Helicobacter and Staphylococcus For example, the mixed population may be comprised of Bacteroides and Firmicutes cells. and metronidazole (MTZ)-resistant C. difficile strain 630 subpopulations. wherein the host cell comprises said Clostridium difficile cell.

[0082] For example, a host cell population, mixed population, or system may contain a defined population of its gut or oral microbiota. Compositions for administration to humans or non-human animals for rehydration or rebalancing (e.g., drinking water) in food, mouthwash or food products.

[0083] For example, products or systems for use or methods, vectors, modified nucleosides, The cRNA or gRNA may be administered via mucosal, intestinal, oral, nasal, rectal, vaginal, ocular or is for administration to humans or non-human animals by buccal administration.

[0084] Any of the forms described herein, e.g., mixed populations (arrays, gRNA, crRNA, or (before combining with the modified sequence) may be used to identify the microbiota of a human or animal subject, e.g., Intestinal or any other microbiota or any microbiome disclosed herein For example, in this case, the product of use of the present invention is a human biota sample, as disclosed herein. or a modified microbiota population useful in the treatment or therapy of an animal subject.

[0085] 3. One or more vectors contain a Cas (e.g., Cas9) nuclease-encoding sequence Concept 2 system, which lacks

[0086] 4. Each host cell is of a strain or species found in the human microbiota, and optionally The host cells are a mixture of cells of different strains or species, where the different cells are human (e.g., human in the gut) and probiotic, commensal, or mutualistic Enterobacteriaceae or bacteria The use, method or system of any of the preceding concepts, for example, the host cell is a -micutes cells, for example, Streptococcus cells.

[0087] 5. Bacteria in mixed bacterial populations (e.g., in humans, such as in the human microbiota) Any of the above concepts for modifying the proportion of bacteria in the phylum Iodes (e.g., Bacteroides). Use, method or system of.

[0088] 6. Concentration of microbial communities to increase the relative ratio of Bacteroidetes to Firmicutes Concept 5. Use, method or system.

[0089] 7. The Cas nuclease integrates into the cell's endogenous type II CRISPR / Cas system. A use, method or system of any of the above concepts is provided by

[0090] 8. Any of the above uses, methods or concepts, wherein element (iii) is endogenous to the host cell. system.

[0091] 9. The target sequence is contained in an antibiotic resistance gene, a virulence gene, or an essential gene of the host cell. any use, method or system of any of the preceding concepts.

[0092] 10. The array is included in an antibiotic composition, wherein the array is combined with an antibiotic. Any use, method or system of the above concepts.

[0093] 11. Alternatively, the HM-crRNA and tracrRNA are a single guide RNA (gRNA) The use, method or method of any of the above concepts, comprising, for example, providing a vector is a system.

[0094] 12. The host cell is then allowed to cleave the deoxyribonucleotides with free ends encoding the HM sequence of interest (HM-DNA). The system comprises a ribonucleic acid strand and / or a sequence encoding HM-DNA, The HM-DNA is then integrated into the host genome (e.g., chromosomal or episomal sites). 1 each homologous to one or more sequences within or adjacent to the target sequence for insertion A use, method or system of any of the preceding concepts, comprising one or more sequences.

[0095] 13. Engineered Bacterial Host Cell Modification Containing Endogenous CRISPR / Cas Systems A nucleic acid vector, the vector comprising (a) a CRISPR / Cas system, method or use according to any of the preceding concepts; Nucleic acid sequences expressing multiple different crRNAs (e.g., gRNAs) for use in Contains; and (b) optionally lacking a nucleic acid sequence encoding a Cas nuclease; wherein a first of said crRNAs is capable of hybridizing to a first nucleic acid sequence of said host cell; and A second of the crRNA is capable of hybridizing to a second nucleic acid sequence of the host cell, wherein the second sequence the sequence is different from the first sequence; and (c) the first sequence contains an antibiotic resistance gene (or its RNA), and the second sequence contains an anti-antibiotic resistance gene; sex genes (or RNA thereof), and optionally, these genes are different; (d) the first sequence contains an antibiotic resistance gene (or its RNA) and the second sequence contains an essential or pathogenic gene; containing the proto-gene (or its RNA); (e) The first sequence contains an essential gene (or its RNA), and the second sequence contains an essential or pathogenic gene. or containing the offspring (or its RNA) (f) the first sequence contains a virulence gene (or its RNA), and the second sequence contains an essential or virulence gene; Contains a gene (or its RNA).

[0096] 14. Operable with vector-encoded cRNA (e.g., single guide RNA) A Concept 13 vector in a host cell comprising one or more Cas that are operable.

[0097] 15. The concept, wherein the HM-CRISPR array contains multiple copies of the same spacer. any use, method, system or vector of

[0098] 16. Use of any of the preceding concepts, wherein the vector contains multiple HM-CRISPR arrays. Use, method, system or vector.

[0099] 17. Each vector is a plasmid, cosmid, virus, virion, phage, or phagemi Any use, method, system or base of any of the above concepts, Kutar.

[0100] 18. The system or vector comprises a nucleic acid sequence encoding a crRNA (e.g., gRNA). containing two, three or more copies, where the copies are part of a host cell sequence (e.g., pathogenic, the same spacer sequence for targeting a gene sequence (e.g., a resistance or essential gene sequence), Any use of the concept, method, system or vector.

[0101] 19. Use of Concept 18 where copies are split between two or more vector CRISPR arrays , method, system or vector.

[0102] 20. A bacterial host cell comprising a system or vector according to any of the preceding concepts.

[0103] 21. Concept 2, combined with an antibiotic (e.g., a beta-lactam antibiotic) Any of ~20 systems, vectors, or cells.

[0104] 22. Each host cell is a Staphylococcus, Streptococcus, Pseudomonas, Salmonella Nella, Listeria, Escherichia coli, Desulfovibrio or Clostridium host Any use, method, system, vector or cell of the preceding concepts, which is a cell. For example, each host cell may be a Firmicutes cell, e.g., Staphylococcus, Streptococcus, or the like. Coccus, Listeria or Clostridium cells.

[0105] For example, each CRISPR array is designed to regulate the expression and production of each crRNA in the host cell. (i) comprising a sequence R1-S1-R1' for (e.g., contained in a single guide RNA), R1 is the first CRISPR repeat, R1' is the second CRISPR repeat, and R1 or R1' is arbitrary, and (ii) S1 is a nucleotide sequence that is 95% or more identical to the target sequence. A first CRISPR spacer comprising or consisting of:

[0106] For example, R1 and R1' are the first and second CRISPR arrays of the second host cell type, respectively. and the second repeat sequence are at least 95% identical to the first repeat sequence. For example, R1 and R1' are For example, the first (5'-most) and second (first repeat) CRISPR arrays of the host cell of the species. repeats) and at least 95% (e.g., 96%, 97%, 98%) of the repeat sequence, respectively. %, 99% or 100%) are identical. For example, R1 and R1' are identical in the host cell. To modify targets in the target region, type II Cas9 nucleases (e.g., Streptococcus typhimurium) are used. Staphylococcus thermophilus, Streptococcus pyogenes or Staphylococcus aureus It is functional using urea (Cas9).

[0107] An alternative concept 1 use of the present invention, as shown in the working experimental example, is to provide. Use of wild-type endogenous Cas nuclease activity of bacterial host cell populations to inhibit population growth wherein each host cell contains an endogenous CRIS having wild-type Cas nuclease activity. The use includes transforming a population of host cells, wherein each transformant has a PR / Cas system. The recombinant host cell is a recombinant host cell that expresses host-modified (HM) cRNA or guide RNA (gRNA) in the host cell. and transformed with a modified nucleotide sequence to provide HM-cRNA or gR NA contains a host cell targeting protospacer sequence and a hybridization sequence to guide endogenous Cas to target the target. wherein the cRNA or gRNA comprises a hybridizable sequence to the wild-type nuclease. It is homologous to the endogenous Cas nuclease of the host cell, which has enzyme activity, and after host cell transformation , the growth of the population is inhibited.

[0108] In the working examples below, inhibition is measured using a bacterial population (Gram-positive Firmicutes) on a solid surface. A >10-fold inhibition of host cell population proliferation was achieved. The present invention targets biologic resistance genes and essential genes. In some embodiments, the target sequence is an antibiotic resistance gene sequence. Co-administration of the nucleotide sequence with an antibiotic may be effective. This may be due to the more complete human or provide treatment or prevention of host cell infection in animal subjects and / or human or animal This may allow for a reduction in the therapeutically effective antibiotic dose administered to human and animal populations. This is useful in light of the increasing concern about antibiotic overuse and resistance development in the community. .

[0109] Demonstration of the ability of the present invention to inhibit host cell growth on a surface will be provided by the present invention in human or animal subjects. Diseases or conditions mediated or caused by the microbiota disclosed herein in In some embodiments, it is important and desirable to treat or prevent such microflora. is generally administered to the tissue of interest (e.g., intestine, oral cavity, lung, axilla, eye, vagina, anus, ear, nose, or pharynx). tissues), and therefore we can determine the surface microbiota bacterial species (Streptococcus The demonstration of growth inhibitory activity of the compounds exemplified herein is believed to support this utility.

[0110] For example, wild-type host cell endogenous Cas9 or cfp1 activity is used. The nucleotide sequence may not be combined with an exogenous Cas nuclease-encoding sequence.

[0111] For example, the host cell is a wild-type (e.g., unengineered) bacterial cell. In this case, the host cell has been engineered (e.g., by chromosomally introducing an exogenous nucleotide sequence). or endogenous nucleotide sequences, for example, on the host cell chromosome or plasmid (to modify the gene), wherein the host cell is Contains endogenous CRISPR / Cas systems with live Cas nuclease activity. For example, bacterial colony formation of the host cells is inhibited after the transformation. Growth is inhibited after the transformation, e.g., the host cell is killed after the transformation.

[0112] The "cognate" crRN guides the endogenous Cas to its target in the host cell. A subject skilled in the art will understand that this is an Cas guides such as these are commonly used to control CRISPR / Cas activity in bacterial cells, e.g., endogenous Wild-type CRISPR in bacterial cells with an active wild-type CRISPR / Cas system Understand that this is a characteristic of R / Cas activity.

[0113] By "wild-type" Cas activity, it is understood by those skilled in the art that endogenous Cas activity is is not an engineered Cas or the cells have been engineered to derepress endogenous Cas activity. This is intended to be an indication that Cas nuclease activity is naturally suppressed in certain cells. This is in contrast to bacteria (i.e., those with no wild-type Cas nuclease activity or those with none of the present invention). This is in contrast to, for example, the fact that endogenous Cas activity inhibits cell population growth. (Applicable to in situ wild-type host cell approaches that can be used for this purpose).

[0114] For example, the inhibition of growth of a host cell population may be observed in cells of the host cell population that have not been exposed to the modified nucleotide sequence. At least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or For example, growth inhibition can be measured by measuring the growth of a second sample of host cells (either alone or in admixture). At least 2, 3, 4, 5, 6, 7 times higher than the number of colonies in the bacterial population Low bacterial colonization of the first sample (single or mixed bacterial populations) of 8x, 9x, or 10x host cells wherein the first cell is transformed with the modified nucleotide sequence. The second sample is exposed to the modified nucleotide sequence, while the second sample is not exposed to the modified nucleotide sequence. In some embodiments, the colony count is determined by the 12 hour, 24 hour, or 30 minute time points after the first sample is exposed to the modified sequence. In one embodiment, colonies are determined after 1 hour, 36 hours, or 48 hours. In vitro, they are grown on solid agar (e.g., petri dishes). Thus, growth inhibition indicates that the target sequence is not present. Decreased proliferation of cells or populations containing IgG (<1% compared to non-treated, i.e., control sample proliferation) This can be demonstrated by a complete elimination of such proliferation (00% proliferation) or by a complete elimination of such proliferation. It is understood that, for example, propagation of a host cell population can be achieved over a predetermined period of time (e.g., by increasing the number of host cells). After combining the cRNA or gRNA, mix the cRNA or gRNA in a 500-ml tube for at least 10 min (24 or 48 hours). %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% reduction i.e., the growth of the host cell population has not been exposed to the cRNA or gRNA. from the growth of a control host cell population maintained under the same conditions for the predetermined period of time. At least such a percentage less. For example, the percentage reduction in proliferation is colony counts in samples of each population at a later time point (e.g., mid-log phase for control samples) For example, after exposing a test population to crRNA or gRNA at time 0, , samples of the test and control populations are taken, and each sample is plated on an agar plate. Incubate under the same conditions for a specified period of time. At the end of the period, count the number of colonies in each sample. Count the number of test colonies and obtain the percentage difference (i.e., divide the number of test colonies by the number of control colonies, Multiply it by 100 and subtract the result from 100 to get the percentage growth reduction). The fold increase is calculated by dividing the number of control colonies by the number of test colonies.

[0115] Inhibition of population growth may therefore be indicated by a decrease in the growth of host cell numbers in the population. This is due to cell killing by nucleases and / or nuclease deletion on the target protospacer sequence. downregulation of host cell growth (division and / or cell proliferation) by the action of cleavage enzymes In the treatment or prevention embodiments disclosed herein, a human or animal subject The host cell burden of the antibody is reduced, thereby treating (e.g., alleviating or eliminating) the disease or condition. or prevent (i.e., reduce or eliminate the subject's risk of developing a disease or condition) .

[0116] The present invention includes beverages and foodstuffs (or devices for producing, processing or storing them). Wild-type bacteria found naturally in the environment (e.g., in water or waterways, cooling or heating equipment) useful for targeting wild-type bacterial populations in the human or animal microbiota. Therefore, the present invention provides a method for pre-modifying host cells to make them susceptible to death or growth inhibition. are useful in situations where it is not possible or desirable to (When in situ treatment of microflora at other locations is desired). The invention provides a method for the treatment or prevention of a disease or condition caused or mediated by a host cell. It is useful for the ex vivo production of pharmaceuticals for administration to human or animal subjects, where the pharmaceuticals are Containing a mixed bacterial population (e.g., obtained from the feces or intestinal microbiota of one or more human donors) This is a product for the use or method of the invention, where the population is a species or species of host cell. The strain contains a subpopulation of bacteria of a different species or strain. This subpopulation of cells does not contain the target and therefore Thus, for example, the method is not modified by the use or method disclosed herein. For the production of a pharmaceutical for the treatment or prevention of a metabolic or GI condition or disease, This reduces the proportion of specific Firmicutes subpopulations in the colony, preserving the Bacteroidetes phylum. In this way, the present invention provides such a use or such treatment in humans or animals. A prophylactic modified bacterial transplant (e.g., modified fecal transplant) medicine can be provided. For example, a method can include: Medical use (e.g., for metabolic conditions (e.g., obesity or diabetes) or GI tract conditions (e.g., or any such condition described in (1) or cancer (e.g., GI tract cancer) For cosmetic or personal hygiene use (e.g., topical application to the human axilla or other relevant locations on the human body) for the reduction of axillary or other body odor by applying a topical antiperspirant to a human (e.g., for topical use in humans) or in vitro modification of one or more microbiota of a modified collection of bacteria for administration to an animal. In other examples, the array, crRNA, gRNA or modified nucleic acid may be used. The nucleic acid sequence is administered to a human or animal, and the host cell is maintained in the human or animal, e.g. , human or animal microbiota (e.g., gut microbiota or any other type disclosed herein) In this way, they are mediated or triggered by host cells. For example, transformation can be performed in vitro to treat or prevent a disease or condition. Optionally, the array, crRNA, gRNA or modified nucleotide sequence is transfected into a host cell. For example, the nucleic acid may be an RNA (e.g., a copy of a gRNA). In other instances, the nucleic acid encodes a crRNA or gRNA for expression in a host cell. It is the DNA that encodes it.

[0117] Thus, for example, the present invention provides a method for detecting and / or treating a bacterial infection mediated or induced by host cells of a subject's microbiota. The method comprises administering to a subject a drug or drug product that is present in the microbiota of a human or animal subject for the treatment or prevention of a disease or condition that may result from the administration of the drug or drug product. Host cell modified (HM) cRNA or guide RNA in a population of wild-type bacterial host cells A modified nucleotide sequence to provide a cRNA or gRNA The RNA hybridizes with the host cell targeting protospacer sequence to guide Cas to the target. a cRNA or gRNA that contains a solvable sequence, wherein the cRNA or gRNA has wild-type nuclease activity Cas nucleases are homologous to endogenous host cell Cas nucleases, which are involved in the transformation of host cells in a population. Thereafter, proliferation of the population is inhibited and the disease or condition is treated or prevented.

[0118] For example, the modified nucleotide sequence can be used to construct a HM-CRISPR array as defined herein. For example, the modified nucleotide sequence encodes a single guide RNA. The modified nucleotide sequence may be a guide RNA (e.g., a single guide RNA) or a crR For example, the modified sequence may be contained in a bacteriophage that can infect a host cell. where transformation includes transduction of a host cell with a bacteriophage. The phage can be a bacteriophage described herein, e.g., a modified nucleic acid The nucleic acid sequence is contained in a plasmid (e.g., a conjugative plasmid) that can transform a host cell. The plasmid may be any of the plasmids described herein, e.g., a modified nucleoside. The peptide sequence is contained in a transposon that can be transferred within and / or between host cells. The sposon may be a transposon as described herein.

[0119] Any use or method of the present invention may comprise a step of producing cRNA or gRNA in a cell. For example, the modified nucleotide sequence may be transformed into a host cell with a nucleic acid vector. A vector or nucleic acid containing the code sequence is introduced into a mixed bacterial population (e.g., human or animal microbiota). orally, intravenously, topically, ophthalmically, intranasally, by inhalation, or rectally to humans or animals, including as part of administration, auricular administration, vaginal administration, or any other route of administration disclosed herein or otherwise, The administration transforms host cells with the vector or nucleic acid.

[0120] For example, the host cell population is ex vivo. For example, the mixed population is a human or animal subject. and to treat or prevent host cell infection in a subject.

[0121] For example, the first bacterium and the second bacterium are included in a microbial community, where the bacteria are mutualistic. For example, the community may be a human or animal microbiota, for example, the community may be a human or In animals (e.g., as used herein, systems, modified sequences, vectors, or cells is a treatment for infecting a host cell of a community in a human or animal, e.g., where the host cell The cells mediate antibiotic resistance or adverse diseases or conditions in humans or animals. The species used in the working examples below (Escherichia coli, Lactobacillus subtilis) Streptococcus lactis and Streptococcus thermophilus) are common in the human and animal intestines. The examples also show mixed Gram-positive and It also addresses targeting in gram-negative bacterial populations. Populations of Mycutes (Streptococcus thermophilus) and Enterobacteriaceae (Escherichia We also worked on populations of Enterobacteriaceae (Bacillus subtilis, Bacillus subtilis, and Bacillus coli), both of which are found in the human microbiota. Examples include Salmonella, Yersinia pestis, Klebsiella, Shigella, Proteus, Entamoeba The bacteria are Terobacter, Serratia and Citrobacter.

[0122] For example, methods, uses, modified nucleotide sequences, arrays, crRNA, gRNA, The vector or system is for the treatment of host cell infection in human gut microbiota populations. and optionally the population also includes human commensal enterobacteria and / or Enterobacteriaceae. and bacteria, for example, where the host cell and commensal cell (first bacterium and second bacterium) are human. They survive mutualistically in the intestinal microbiota.

[0123] For example, the use or system may be adapted to produce a mixture of bacteria, including Bacteroidetes bacteria and other bacteria. This is to change the proportion of Bacteroidetes in the population. against one, more, or all Firmicutes (e.g., against Streptococcus) In this case, the host cell is able to express the target-containing phagocytic phylum C. For example, the population may be a microbe contained in a human or animal subject. and the bacterial population of the present invention, and the method, use, modified nucleotide sequence, vector or system The method includes (i) treating a control infection with the included host cells (e.g., included in a mixed population); (ii) treating or preventing a condition or disease mediated by said host cells in a subject; (iii) (iv) the reduction of human body odor caused or mediated by cells; or (iv) the improvement of human personal hygiene. For example, the modified nucleotide sequences, arrays, crRNA, gR The NA or vector is for use in such a system or use of the invention. do.

[0124] For example, the condition or disease is a metabolic or digestive disease or condition, e.g., obesity, IBD For example, the condition or disease may be cancer, e.g., bronchitis, IBS, Crohn's disease, or ulcerative colitis. For example, a solid tumor or GI cancer (e.g., gastric cancer), liver cancer, or pancreatic cancer. Resistance or hyporesponsiveness to antibiotics (eg, any of the antibiotics disclosed herein).

[0125] For example, the cell may be modified with element (ii) in the production of said HM-crRNA in the cell. Alternatively, one or more of the vectors may be inserted into the host cell. Nucleotides encoding such RNase III for expression in vivo Contains code arrays.

[0126] For example, essential genes (including targets) encode DNA polymerases in cells. An example is shown below.

[0127] Use, system, vector or cell, e.g., array, cRNA or gRNA NGG, NAG, NGA, NGC, NGGNG, NNGRRT or NNAGAAW Pro spacer adjacent motif (PAM), e.g., AAAGAAA or TAAGAAA P It contains a sequence that can hybridize to the host cell targeting protospacer sequence adjacent to the AM ( These sequences are shown 5' to 3'. In one embodiment, the PAM is a protospacer. For example, Cas is located immediately adjacent to the 3' end of the Ser sequence in Staphylococcus aureus. Streptococcus thermophilus or Streptococcus pyogenesCas For example, Cas is Cpf1 and / or PAM is TTN or CTA. be.

[0128] For example, the modified nucleotide sequence, crRNA, gRNA or array may be an antibiotic For example, where the target is contained in an antibiotic resistance gene, and where the antibiotic is In some embodiments, the host cell is sensitive to an antibiotic. Eliminate infection in the presence of host cells (e.g., in humans or manufacturing containers / devices containing populations) Although the cells may be insufficiently susceptible to antibiotic use for the treatment of the disease, antibiotics may be used in the treatment of the disease. Further killing or growth inhibition is achieved using Cas modification (e.g., targeted cleavage) according to the method described above. This can slow or reduce host cell subpopulation size or proliferation.

[0129] The present invention provides a method for antibiotic (first antibiotic) treatment of infection of host cells in a human or animal subject. Uses for, systems, arrays, crRNAs, gRNAs, modified nucleotide sequences and a vector or cell, wherein the vector or cell is a vector containing an antibiotic resistance gene (for resistance to a first antibiotic). (for example, a Cas targeting system or vector in a host cell, The system, array, crRNA, gRNA, modified nucleotide sequence, and vector are This involves administering a vector or cell and an antibiotic. The gene is down-regulated, i.e. That is, expression of the protein product encoded by the gene is reduced or eliminated in the host cell. , thereby downregulating antibiotic resistance. Infection is reduced or prevented in the subject. For example, antibiotics, systems, arrays, crRNA, gRNA, modified nucleosides, In other instances, administration is sequential. (e.g., systems, arrays, crRNA, gRNA, modified nucleotide sequences, vectors) This feature of the present invention is advantageous in that, for example, the antibiotic alone does not In some cases, the use of steroids to enhance antibiotic treatment in a subject is not efficient enough to treat such host cell infections. The antibiotic may be any of the antibiotics disclosed herein, e.g., tetracycline. It can be phosphorus.

[0130] For example, each modified nucleotide sequence or vector can be used in the CRISPR array or a sequence encoding the crRNA or gRNA, and further comprising an antibiotic resistance gene (e.g., (e.g., kanamycin resistance), where the HM-crRNA or gRNA confers antibiotic resistance It does not target genes. For example, the target sequence may be contained in an antibiotic resistance gene of the host cell. wherein the antibiotic is different from the first antibiotic (e.g., kanamycin). In this way, the system The modified sequence or vector may target the host without targeting itself. Exposing the host cells to the first antibiotic results in the production of cells containing the first antibiotic resistance gene. However, the modified sequence or vector has a survival advantage in the presence of the first antibiotic. Positive selection pressure can be used to promote the retention of the vector in the target cell (e.g., by modified sequences or vectors). (when the untransformed host cell is not resistant to the first antibiotic). host cells to promote maintenance of the cRNA or gRNA coding sequence in the host cells. Alternatively, the mixed population may be treated with an antibiotic (e.g., kanamycin) and the modified sequence or vector. or the system, modified sequence, array or vector of the present invention. is provided in combination with said antibiotic.

[0131] For example, the cRNA or gRNA encoding sequence or element (ii) is operable in the host cell species. under an activatable constitutive promoter (e.g., a strong promoter) or under an inducible promoter For example, element (iii) may be a constitutive promoter or an inducible promoter operable in the host cell type. Available under promoter.

[0132] For example, each host cell is a Gram-positive cell. In another example, each host cell is a Gram-negative cell. They are sex cells.

[0133] For example, the method, use, system, modified sequence or vector may be used to inhibit the growth of the human gut microbiota. for the treatment of host cell infection in a population, optionally the population containing human commensal enterobacteria. including (i.e., enteric bacteria that are commensal with humans).

[0134] Methods, Uses, Systems, Arrays, crRNAs, gRNAs, Modified Sequences or Vectors For example, the host cell may be contained in a mixed bacterial population contained in a human or animal subject, and the method, use, The system, array, crRNA, gRNA, modified sequence, or vector for: (i) (ii) treating an infection in a subject with host cells contained in a mixed population; (iii) treating or preventing a condition or disease mediated by said host cell; or (iv) for the reduction of mediated human body odor or for human personal hygiene.

[0135] Methods, Uses, Systems, Arrays, crRNAs, gRNAs, Modified Sequences or Vectors - e.g., in vitro treatment of industrial or medical fluids, solid surfaces, devices or containers (e.g., food, consumer goods, cosmetics, personal health care products, petroleum or petroleum products) or waterways, water, beverage, food or cosmetic processing, where host cells are present in fluids, surfaces, equipment, components contained in or on containers, waterways, water, beverages, food or cosmetics.

[0136] The present invention may also be obtained by the use or method of any of the concepts herein. An ex vivo mixed population of bacteria is provided.

[0137] For example, the product of the mixed population or use or method may be In a container. For example, the container may be, for example, in an inhaler or a syringe or IV needle. A connected sterile container.

[0138] For example, the product population of a use or method may be administered to a human or animal to define its microbiome. It is useful to administer it to the patient for implantation.

[0139] The present invention relates to food for human or non-human animal consumption, including mass products for use or methods. Provide food or drink.

[0140] Here, for example, Bacteroides in any form, concept, or aspect is a bacterium, such as a caccae. , Capirosus, Cellulosiliticus, Coprocola, Coprophilus, Coprosuis, Di Stasonis, Dray, Eggersii, Faesis, Fine Goldie, Fluxus, Radilis, Intestinalis, Melaninogenicus, Nordii, Olaciplenus, O Rallis, Overtus, Pectinophilus, Plebius, Starcoris, Thetaiotao A species selected from Micron, Uniformis, Vulgatus and Xylanisolvens. For example, Bacteroides is thetaiotaomicron, e.g., where host cells Alternatively, the mixed population may be an ex vivo or in vitro gut microbiota population. The chief cells, first or second bacterial subpopulations may be composed of multiple different Bacteroidetes species or multiple bacteria. Cteroides species (e.g., Bacteroides thetaiotaomicron and Bacteroides fragilis) or Bacteroides and Prevotella species. For example, Prevotella includes bergensis, bivia, buccae, buccalis, copri, melaninoji Selected from: Enica, Orris, Ruminicola, Tannerae, Timonensis and Belloralis Alternatively, the host cell, the first or second bacterium is a Firmicutes cell. For example, the host cells, first or second subpopulations may be Anaeroturuncus, Acetozoa Erobacterium, Acetylomaculum, Acetivibrio, Anaerococcus, Anaerov Film, Anaerosinus, Anaerostipes, Anaerovorax, Butyrivibrio, Clostridium, Capracoccus, Dehalobacter, Diarrister, Dorea, Ente Lococcus, Ethanoligenes, Faecalis bacterium, Fusobacterium, Lassiliba Acta, Guggenheimera, Hesperia, Lachnobacterium, Lachnospira, Lactobacillus Chilus, Leuconostoc, Megamonas, Mollie, Mitsuokera, Oribacterium, O. Xobacter, Papilibacter, Proprionispira, Pseudobutyrivibrio, Pseudobutyrivibrio Dramibacter, Roseburia, Ruminococcus, Sarcina, Seinonella, Chateauworthi A, Sporobacter, Spirobacterium, Streptococcus, Subdoligranulum, Selected from Syntrophococcus, Thermobacillus, Turicibacter and Weissella comprising or consisting of one or more Firmicutes, e.g., a host cell or a first or second A second subpopulation may consist of Clostridial cells (and optionally other subpopulations may consist of bacteria). roides (e.g., thetaiotaomicron) cells). For example, host cells or One or the second subpopulation consists of Enterococcus cells (and optionally other subpopulations) Cteroides (e.g., Thetaiotaomicron) cells). For example, host cells or The first or second subpopulation comprises Ruminococcus cells (and optionally other subpopulations). Bacteroides (e.g., thetaiotaomicron) cells), e.g., host cells or The first or second subpopulation comprises streptococcal cells (and optionally other subpopulations). The population consists of Bacteroides (e.g., Thetaiotaomicron) cells). For example, the host The cells or first or second subpopulations comprise (and optionally contain) Faecalibacterium cells. Other subpopulations consist of Bacteroides (e.g., Thetaiotaomicron) cells). For example, Faecalibacterium is Faecalibacterium prausnitzii (e.g. , A2-165, L2-6, M21 / 2 or SL3 / 3).

[0141] For example, the host cell or the first or second subpopulation may be selected from the group consisting of Anaeroturuncus, Acetoanae Lobacterium, Acetimicamacrum, Acetivibrio, Anaerococcus, Anaerophilus Rum, Anaerosinus, Anaerostipes, Anaerovorax, Butyrivibrio, Rostridium, Capracoccus, Dehalobacter, Diarrister, Dorea, Enterococcus Coccus, Ethanoligenes, Faecalis, Fusobacterium, Lassibacterium Tarr, Guggenheimera, Hesperia, Lachnobacterium, Lachnospira, Lactobacillus Rus, Leuconostoc, Megamonas, Mollie, Mitsuokera, Oribaterium, Oki Sobacter, Papyribacter, Proprionispira, Pseudobutyrivibrio, Pseudo Ramibacter, Roseburia, Ruminococcus, Sarcina, Seinonella, Chateauworthia , Sporobacter, Spirobacterium, Streptococcus, Subdoligranulum, 1 selected from Enterococcus, Thermobacillus, Turicibacter and Weissella For example, a host cell or a first or second Firmicutes The second subpopulation comprises (and optionally comprises) Clostridium (e.g., difficile) cells. Other subpopulations consist of Bacteroides (e.g., Thetaiotaomicron) cells). For example, the host cells or the first or second subpopulations comprise Enterococcus cells (and Other subpopulations consist of Bacteroides (e.g., Thetaiotaomicron) cells). For example, the host cells or the first or second subpopulation may comprise Ruminococcus cells (and Optionally, another subpopulation comprises Bacteroides (e.g., Thetaiotaomicron) cells. For example, the host cells or the first or second subpopulation may consist of Streptococcus cells (and and optionally subpopulations of Bacteroides (e.g., Thetaiotaomicron) and / or or Enterobacteriaceae (e.g., Escherichia coli) cells). For example, host cells or The first or second subpopulation comprises Faecalibacterium cells (and optionally other subpopulations). The population consists of Bacteroides (e.g., Thetaiotaomicron) cells). For example, the host The cells or first or second subpopulations are Streptococcus cells (optionally Streptococcus The other subpopulations consist of bacteria (Cas thermophilus and / or pyogenes cells), Iodes (e.g., Thetaiotaomicron) and / or Enterobacteriaceae (e.g., Escherichia It consists of Chia coli (chia coli) cells.

[0142] The products for the uses or methods of the present invention may, in some embodiments, be mucosal, intestinal, oral , intranasal, rectal, vaginal, ocular, or buccal administration to humans or non-human animals. This is because.

[0143] Optionally, the host cell or the first or second subpopulation bacteria is Bacteroides fragilis. It is a bacterium and its population is found in water.

[0144] The arrays, systems, modified sequences, vectors or suitable beverages containing gRNA of the present invention The ingredients include, for example, probiotic drinks, such as adapted Yakult™, Actimel™, ), Kevita™, Activia™, Jarrow™ or similar beverages for human consumption. .

[0145] Phage sequence target In one embodiment of the invention, the target sequence is a sequence of a phage that infects a host bacterial cell. The desired modification of the phage genome achieved by the present invention can be achieved by phage killing or knockout. down, but rather to the desired phage genes or regulatory elements in the host cell. It can be activating (e.g., the phage is associated with increased host cell viability or proliferation). (e.g., when expressing a desired protein or other product). Alternatively, modifications can be made, e.g., to The use of inducible Cas targeted to the phage target site by In one embodiment, the present invention provides a method for the regulation of gene expression by Cas in a host cell. It provides for modification of the phage target site by cleavage with a nuclease. For example, this may be useful for a variety of reasons: A. Target site for mutation to activate or inactivate it (e.g., anti-host gene) for gene knockdown or inactivation of genes or for phage target integration into the host chromosome due to host cell death); B. For large sequence deletions of or including a target sequence (e.g., by using the present invention in phage genomes) When used with the first and second PM-crRNAs targeting spatial sites of the ribosomal RNA, Cleavage of the nucleotide sequence results in the deletion of the phage nucleic acid between the cleavage sites; C. To insert a desired PM-DNA sequence into the host cell genome (e.g., to insert a desired PM-DNA sequence into the host cell genome). Providing one or more PM-crNA guided cleavage in the host nucleic acid for homologous recombination insertion of A by doing so).

[0146] The present invention provides the following aspects. 1. Altering the relative proportions of a first bacterial subpopulation and a second bacterial subpopulation in a mixed bacterial population comprising the first bacterial subpopulation and the second bacterial subpopulation. wherein the first bacterium is a host cell (e.g., a Bacteroidetes host cell). wherein the first bacterium is optionally infected with a phage and the second bacterium is infected with the phage. The method involves the use of a vector nucleic acid (e.g., a vector that is not infected (or is not a Bacteroidetes phylum)). For the introduction of PM-containing transposons into host cells, a mixed population and multiple and allowing the bacteria to grow in a mixed population, wherein the first bacterium and The relative proportions of the second bacteria are altered; wherein each vector is capable of modifying a target nucleotide sequence (e.g., of the phage) in a cell. Engineered phage-modified (PM) CRISPR arrays for introduction into host cells for include, (a) Here, PM-CRISPR arrays are each designed for expression of one or more PM-crRNAs. and a promoter for transcription of the sequence in a host cell; and (b) where PM-crRNA targets Cas (e.g., Cas nuclease) in the host cell. It can guide and hybridize with a target sequence to modify the target sequence.

[0147] Phage sequences are used to inactivate genes required for phage viability, growth, or infectivity. By targeting, in one embodiment, the present invention provides a method for targeting a host cell infected with a phage. Providing arrays with a positive selective advantage that can enhance uptake and retention by host cells When host cells are killed or growth is reduced, the relative abundance of the first bacterium versus the second bacterium in the population increases. The present invention relates to, for example, the treatment of a disease or condition in a human or animal subject. The present application provides a group of such products for use as therapeutic or preventative (risk reduction) medicines. The disease or condition may be any of the diseases or conditions disclosed herein. For example, a single guide RNA (gRNA) provides the crRNA. Each vector contains an expressible gRNA encoding such a gRNA. It contains a modified nucleotide sequence.

[0148] In an example using PM-array, the target sequence is Bacteroides thetaiotaomicus Optionally, the target sequence is not contained in Bacteroides fragilis. is advantageous, for example, when the modification cleaves or otherwise renders the target sequence non-functional, This allows the ratio of Bacteroides thetaiotaomicron host cells to the total number of cells in a mixed population, e.g. When Bacteroides fragilis is the gut microbiota population described here, Bacteroides fragilis can cause abscesses in some settings. Related and therefore, for example, for the treatment or prevention of obesity or diabetes or IBD, For example, the present invention, which is useful for rebalancing the gut microbiota, reduces this risk. This reduces the number of Bacteroides thetaiotaomicron cells and simultaneously alters their ratio (Bacteroides thetaiotaomicron cell ratio). rate increase).

[0149] The promoter (or HM- or PM-array) is operable in the host cell For example, the promoter may be a viral or phage promoter, e.g., a T7 promoter. In other examples, the promoter is a bacterial promoter (e.g., a promoter specific to the host cell species). (Promoter of).

[0150] 2. The first bacterium is Bacteroides (e.g., Thetaiotaomicron or Fragilis), Ristipes, Alkaliflex, Parabacteroides, Tannerella, Xylanibacter and / or Prevotella bacteria.

[0151] 3. The second bacterium is a Firmicutes bacterium (e.g., the first bacterium is a Bacteroidetes bacterium or or Bacteroides).

[0152] 4. The ratio of the first bacterial subpopulation to the second bacterial subpopulation increases, i.e., after applying the method , larger than before, the method of any of the preceding aspects.

[0153] 5. Mixed populations can colonize or repopulate humans or non-human animals with their gut or oral microbiota. The composition comprises a composition (e.g., a drink, mouthwash, or food) for administration for the purpose of For example, where the mixed population is in vitro or in vivo in a human or non-human animal. , The method of embodiment 4.

[0154] 6. The ratio of the first bacterial subpopulation to the second bacterial subpopulation is reduced, i.e., after applying the method , smaller than the previous, method of embodiment 1, 2 or 3.

[0155] 7. The mixed population is contained in drinks or water for human consumption (e.g., waterways or drinking water); The method of embodiment 6.

[0156] 8. Each vector is a plasmid, a phage (e.g., a packaged phage) or a phage The method of any preceding aspect, wherein the

[0157] 9. Each vector is a phage (e.g., a packaged phage) and the vector nucleic acid is transferred to a host By transducing the phage vector nucleic acid into the cell, i.e., the host cell by the phage vector The method of embodiment 8, wherein the phage is introduced into the host cell by infection of the cell. For example, the phage is It contains one or more transposons.

[0158] 10. Each vector is a plasmid and the vector nucleic acid is transformed from bacteria containing the vector. The method of embodiment 8, wherein the plasmid is introduced into the host cell by recombinant or horizontal plasmid transfer. The vector may contain one or more transposons described herein. For example, the vector may contain It is a non-Bacteroidetes phylum or a non-Bacteroidetes species.

[0159] Additionally or alternatively, the bacterium containing the vector may be a non-Firmicutes species. For example, the bacterium containing the vector may be a Lactobacillus species (e.g., Lactobacillus spp., Lactobacillus acidophilus (e.g., Lactobacillus spp ... , La-5, La-14 or NCFM), brevis, bulgaricus, plantarum, La Mnosus, Fermentum, Caucasus, Helveticus, Lactis, Reuteri and casei, e.g., Bifidobacterium spp. (e.g., Bifidum, breve, longum or infantis), Streptococcus thermophilus and and Enterococcus faecium. For example, the bacterium is Lactobacillus acidophilus or Lactis.

[0160] 11. In the method of any of the preceding aspects for genome modification of the Bacteroidetes phage. Modified Bacteroidetes Phage-Modified (PM) CRISPR Array for Use in There was, (a) wherein the PM-CRISPR array comprises one or more sequences for expression of PM-crRNA. and a promoter for transcription of said sequence in a Bacteroidetes phage-infected host cell. and (b) where PM-crRNA is expressed in infected host cells as a Cas (e.g., Cas nuclease) The Bacteroidetes phage genome target sequence and the hybridization were used to guide the enzyme and modify the target sequence. It can be hybridized.

[0161] 12. The method of any one of aspects 1 to 10, comprising the PM-CRISPR array of aspect 11. A nucleic acid vector (e.g., a plasmid, phage, or phagemid) for use.

[0162] In a general embodiment of the present invention, there is another embodiment 12 provided. The genome of a host bacterial cell (e.g., a pathogenic bacterial cell as described above) or a host cell HM-C modified to modify a target sequence in the genome of a virus (e.g., a phage) A nucleic acid vector (e.g., a plasmid, virus, phage or phagemid), (a) wherein the CRISPR array comprises one or more sequences for expression of crRNA and a host cell a promoter for transcription of the sequence in the cell; and (b) where the crRNA guides Cas (e.g., Cas nuclease) in the host cell. and can hybridize with a target sequence to modify the target sequence.

[0163] The promoter is operable in the host cell. For example, the promoter may be a viral or a phage promoter, for example, a T7 promoter. The motor is a bacterial promoter (eg, a promoter of the host cell type).

[0164] For example, the array may be comprised in a transposon as described herein. For example, the plurality of arrays may be contained in a carrier bacterium, such as a phage or a host cell. and a plurality of arrays are provided for targeting one or more target nucleotide sequences, wherein the plurality of arrays are , a bacterial cell described herein, e.g., a carrier, a first recipient, or a second recipient. For example, the carrier cells may be used in the manufacture of beverages (e.g., beverages intended for human consumption) described herein. For example, the array or carrier bacteria can be found in human or for administration to a human or non-human animal for the treatment or prevention of infection in the animal. For example, where the host cell is pathogenic, e.g., the array or carrier bacteria is a pathogenic agent for humans or The present invention relates to an intestinal inflammatory disease (IBD) treatment for the treatment or prevention of obesity, diabetes, or IBD in a human or non-human animal. This is to administer it to

[0165] 13. The array or vector is transfected into bacterial cells for human or non-human animal consumption, e.g., 13. The array or vector of embodiment 11 or 12, which is contained in a probiotic cell.

[0166] 14. The vector is used in or in combination with a mixed population. whereby the vector nucleic acid is transformed (e.g., from a third bacterium to a first bacterial host) by horizontal plasmid vector or transposon transfer into host cells) or transduction (e.g. introduced into a host cell by, for example, phage vector infection of a first bacterial host cell; The method, array or vector of any preceding aspect.

[0167] 15. The or each array or vector is a human or non-human animal intestinal commensal or mutualist. Any of the preceding embodiments, contained in a live bacterial cell (e.g., a vehicle bacterial cell described herein). Thus, the cells may be commensal or transgenic with humans or non-human animals. It is a mutualistic intestinal bacterial species.

[0168] 16. Each vector is designed to infect Firmicutes host cells or Bacteroidetes phage. A plasmid containing an origin of replication that is operable with a host cell (e.g., a Bacteroides cell). , phage or phagemid, optionally commensal or 16. The method or vector of any of embodiments 12 to 15, wherein the method or vector is operable in a bacterial cell or a mutualistic bacterial cell. For example, the origin of replication is oriT or any other origin of replication described herein. .

[0169] 17. Each vector is (1) a human or non-human animal commensal or between (2) a mutualistic bacterial cell and a phage-infected cell, which is a Bacteroides cell, or (3) a phage-infected cell. Human or non-human animal commensal or mutualistic bacterial cells that are not Firmicutes cells (4) sequences capable of horizontal transfer between target sequence-containing Firmicutes cells (e.g., 16. A plasmid or phagemid comprising a transposon as described in any one of claims 12 to 16. Any of the methods or vectors.

[0170] 18. Each vector is (1) a phage-infected cell, which is a Bacteroides cell, and (2) a human or nonhuman (3) Target sequence-containing fragments between bacterial cells suitable for probiotic administration to the human animal intestine -micutes cells and (4) suitable for probiotic administration to the intestine of humans or non-human animals Plasmid or phagemid sequences capable of horizontal transfer between bacterial cells (e.g., 18. The method or vector of any of embodiments 12 to 17, wherein the transposon is a transposon as described above.

[0171] 19. Commensal, mutualistic or probiotic species include Lactobacillus species (e.g., dophilus (e.g., La-5, La-14, or NCFM), brevis, bulgaricus, Plantarum, Rhamnosus, Fermentum, Caucasus, Helveticus, Lacty reuteri or casei, e.g., L. casei shirota), Bifidobacterium species (e.g. , bifidum, breve, longum or infantis), Streptococcus sa and Enterococcus faecium. ~ Any of 18 methods or vectors.

[0172] 20. The method of claim 19, wherein the promoter is a phage-infected Bacteroidetes host cell and a a commensal, mutualistic or probiotic bacterial cell as defined above or a compound according to any one of aspects 15 to 19 Firmicutes cells containing any of the defined target sequences and commensal, mutualistic or protozoal organisms The method of any preceding aspect, wherein the sequence is capable of being transcribed in a probiotic bacterial cell. The array or vector. For example, the promoter may be a viral or bacterial promoter, e.g. For example, the promoter may be a host cell promoter, e.g., a T7 promoter. The promoter of the host CRISPR / Cas array.

[0173] 20. The modification results in (i) cleavage of the target sequence, (ii) transcriptional downregulation of the target sequence-containing gene, or (iii) target or (iv) the addition, deletion, or substitution of nucleic acid sequences at the target. The method, array or vector of any preceding aspect or any use thereof.

[0174] 21. Bacteroidetes phages include crAss phage, GB-124 phage, and GA- 17 phage, HB-13 phage, H16-10 phage, B40-8 phage, and Bacteroides fragilis phage ATCC51477-B1. Any method, array, or vector. Nat Commun. 2014 Jul 24;5:4498. doi: 10. 1038 / ncomms5498, "A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes", Dutilh BE et al. crA The approximately 97 kbp genome of ss phage is publicly available and has more information than all other known phages combined. It is 6-fold more abundant in available metagenomes and is more abundant in virus-like particle (VLP)-derived metagenomes and total comprised up to 90% and 22% of all reads of the combined community metagenome, respectively, and were included in the public data This accounts for 1.68% of all human fecal metagenomic sequencing reads in the database. Using a co-occurrence profiling approach, Dutilh et al. Unique carbohydrate-binding domains encoded in protein homologs and phage genomes We predicted a Bacteroides host for this phage, consistent with the inclusion of α- and β-actin.

[0175] 22. The target sequence is responsible for host cell infectivity, phage lysogenic or lytic cycle or phage Contains phage genes necessary for viability, e.g., essential genes or coat protein genes. The method, array or vector of any preceding aspect, or any use thereof, .

[0176] 23. The target sequence is a BACON (Bacteroidetes-associated carbohydrate-binding) domain-encoding sequence (e.g., where the host is a Bacteroides host) or The method, array, or vector of any of the preceding aspects, further comprising: 11):2421-6, doi:10.1016 / j.febslet.2010.04.045. Epub 2010 Apr 21, “Mining metage nomic data for novel domains: BACON, a new carbohydrate-binding module”, Mello See L et al. The presence of BACON domains in phage structural proteins has been shown to be a key factor in the development of slime This should explain the proposed bacteriophage adhesion to the model. Upon infection, the phage expresses a capsid-displaying carbohydrate-binding domain (e.g., an immunoglobulin-like fold Binds to mucin glycoproteins that make up the intestinal mucus layer via the BACON domain This promotes more frequent interactions between the phages and the bacteria they infect.

[0177] 25. CRISPR arrays are designed to encode sequences R1- S1-R1', (i) wherein R1 is the first CRISPR repeat and R1' is the second CRISPR repeat; R1 or R1' is optional, (ii) S1 comprises or consists of a nucleotide sequence that is 95% or more identical to the target sequence. The first CRISPR spacer, The method, array or vector of any of the preceding aspects or any use herein, for example: The target sequence may comprise a protospacer or may be a Cas sequence when the array of the invention is in a host cell. and the protospacer sequence immediately adjacent to the cognate protospacer adjacent motif (PAM). wherein the Cas is also cognate to the crRNA expressed from the array. In some embodiments, the Cas is endogenous to the cell. In other embodiments, the Cas is exogenous to the cell. is provided, for example, by the vector of the present invention.

[0178] 26. R1 and R1' are similar to or slightly different from the repeat sequences of the CRISPR array in cells homologous to the host cell. at least 95% (e.g., 96%, 97%, 98%, 99% or 100%) identical, The method, array or vector of embodiment 25.

[0179] 27. R1 and R1' are resistant to Thetaiotaomicron and fragilis (e.g., Bacteroides Crisp of Bacteroides species selected from Bacteroides fragilis (NCTC 9343) Repeat sequences of R arrays (e.g., Type II-C arrays) and at least 95% (e.g., 96 %, 97%, 98%, 99% or 100%) identical to the repeat sequence, and a functional CRISPR / Cas system, e.g., in the species Bacteroides. 26. The method, array, or vector of embodiment 25, wherein

[0180] 28. R1 and R1' are the first and second CRISPR arrays of the species, e.g., the host cell of the species. The first (5'-most) and second (the repeat immediately 3' of the first repeat) repeat sequences each contain at least 95 96%, 97%, 98%, 99% or 100% identical to the method of embodiment 27. The method, array, use, or vector, e.g., the array is a Type II-C array. The array or vector further comprises R2-S2-R2', where spacer S2 is a spacer It may be the same as pacer S1 or different (e.g., different sequences in the host cell or phage genome). (for targeting of target sites) where R2 and R2' are functional in the host cell. and optionally identical to R1. For example, each of R1, R1', R2 and R2' These are Bacteroides fragilis CRISPR repeats.

[0181] 29. (iii) Each of R1 and R1' is a CRISPR array (e.g., The repeats are identical to those of the Type II-C array, where the species are Caccae, Capirosus, Cellulosilyticus, Coprocola, Coprophilus, Coprosuis, Distasonis, Do Ray, Eggersii, Faesis, Fine Goldie, Fluxus, Fragilis (e.g. fragilis NCTC 9343), intestinalis, melaninogenicus, and Norde Ii, Olasiplenus, Oralis, Overtus, Pectinophilus, Plebius, S Turcoliths, Thetaiotaomicrons, Uniformis, Vulgatus and Xylanisols and (iv) wherein the host cell is selected from the group consisting of the repeat sequences and functional C Bacteroides cells of a species selected from the group, e.g., comprising a RISPR / Cas system 26. The method, array, use or vector of embodiment 25, wherein the selected species is (e.g., the same species as the selected species in (iii)).

[0182] 30. R1 and R1' are the target sequence modifications for the host Bacteroidetes or Phycomyces. 26. The method of embodiment 25, wherein the CRISPR / Cas system is functional with the CRISPR / Cas system of a neutrophil cell. For example, R1, R1', R2 and R2' are from the same bacterial species, For example, Bacteroides or Streptomyces such as Thetaiotaomicron or fragilis Type II streptococci such as L. thermophilus or L. pyogenes (e.g., Type II-C) CRISPR / Cas system repeats.

[0183] 31. R1 and R1' are from the phylum Bacteroidetes (e.g., Bacteroides or Prevotella) or CRISPR arrays in Firmicutes (e.g., Streptococcus) cells (e.g., repeat sequences) and at least 95% (e.g., 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 26. The method, array, use or vector of embodiment 25, wherein the vector is identical to the sequence of the first nucleotide sequence of the first nucleotide sequence of the first or second nucleotide sequence of the second or third ... -.

[0184] 32. Each of R1 and R1' is selected from SEQ ID NOs: 1 to 5 in Table 2, and 95% (e.g., 96%, 97%, 98%, 99% or 100%) identical, and optionally The first bacterial cell is, for example, a species or strain in Table 2 (e.g., a species or strain for the selected sequence). 26. The method, array, use or vector of embodiment 25, wherein the Bacteroides cell is a Bacteroides cell of any of the strains listed above. Tar.

[0185] 33. Each of R1 and R1' is selected from SEQ ID NOs: 6 to 11 in Table 2. 95% (e.g., 96%, 97%, 98%, 99% or 100%) identical, as desired. The first bacterial cell may be, for example, a species or strain in Table 2 (e.g., a species or strain for the selected sequence). 26. The method, array, use or vector of embodiment 25, wherein the cell is a Prevotella cell (a strain listed as a subtype of Prevotella). -.

[0186] 34. Each array contains one or more Cs that are functional with crRNA in a host cell and modify a target sequence. 3. The method, array, or vector of any preceding aspect, in combination with a nuclease. For example, the target sequence is immediately adjacent to a protospacer adjacent motif (PAM). and a pacer sequence, optionally wherein the PAM is a C Cas is a type II-C Cas nuclease. be.

[0187] 35. Each array is functional with crRNA in a host cell and modifies a target sequence. Any of the above-mentioned aspects is a combination of a nucleic acid sequence encoding the Cas nuclease. any method, array or vector.

[0188] 36. R1 and R1' are used to modify targets in host cells. creatinases (e.g., Streptococcus pyogenes, Streptococcus thermophilus) 25. The method of claim 25, wherein the method is functional with a recombinant human IgG1 vector (e.g., a recombinant human IgG1 vector or a recombinant human IgG1 vector), ... a method, an array, a use or a vector, optionally comprising the method, array or vector The vector is further according to embodiment 34 or 35, wherein the Cas is said Cas9.

[0189] 37. An ex vivo mixed population or a culture of bacteria obtained by the method of any one of embodiments 1 to 10 or 14 to 36. For example, the mixed population is in a container for medical or nutritional use. For example, the container is a sterile container.

[0190] 38. For therapeutic, preventative, or cosmetic weight loss in humans or non-human animals (e.g., 37. A composition for administration for cosmetic or nutritional use, comprising: Compositions containing mixed populations. For example, compositions may be administered orally, systemically, by inhalation, rectally, ophthalmically, bucally, or For example, the composition may be administered to the intestine or oral cavity of a human or non-human animal. It is for your use.

[0191] 39. A method for human or non-human animal consumption, comprising the mixed population of embodiment 37 or the composition of embodiment 38. Food or drink for.

[0192] 40. The food or beverage of embodiment 39, which is a nutritional supplement or probiotic drink or food. Fee.

[0193] 41. Treatment or prevention of Bacteroidetes infections in humans or non-human animals or drinking water an antibiotic composition for the prevention of polio, wherein the composition comprises an array or a compound according to any of embodiments 11 to 36. or a vector, optionally wherein the modifications are according to embodiment 21(iii) or (iv).

[0194] 42. Probiotics for increasing the proportion of intestinal Bacteroidetes in humans or non-human animals otic compositions (e.g., for the treatment of obesity, diabetes (e.g., type 1), or GI inflammatory conditions or for prophylaxis), wherein the composition comprises an array or vector of any of embodiments 11 to 36. optionally wherein the modification is according to embodiment 21(iii) or (iv).

[0195] 43. To assess the relative ratio of intestinal Bacteroidetes to Firmicutes in humans or animals, for example, obesity, diabetes (e.g., type 1 diabetes) or GI conditions (e.g., Crohn's disease) and the like for the treatment or prevention of diseases such as ulcerative colitis, IBD, IBS or ulcerative colitis. Or 42 compositions.

[0196] Alternatively, the "array" in any aspect of the invention may be a sequence of sequences of expression in a host cell. The modified nucleotide sequence encoding the HM-crRNA or gRNA for Any feature of the embodiments herein relating to arrays may therefore be modified in such a way. The modified sequences may be applied alternatively with modifications where necessary.

[0197] Mobile genetic elements and CRISPR systems 44. Host bacterial cells (e.g., Firmicutes or pathogenic bacterial cells as described above) targeted sequence modification of the genome of a virus (e.g., a phage) in a host cell Nucleic acid vectors (e.g., plasmids, viruses) containing modified CRISPR arrays for a plasmid, phage, or phagemid, (a) Wherein the CRISPR array comprises one or more sequences for expression of crRNA (e.g., g RNA) and a promoter for transcription of the sequence in the host cell; (b) where the crRNA guides Cas (e.g., Cas nuclease) in the host cell. and capable of hybridizing with a target sequence to modify the target sequence; (c) Here, the array is a transmembrane array that can be horizontally transferred between a first bacterium and a second bacterial cell of a different species. It consists of poson.

[0198] Optionally, the Cas nuclease is a wild-type endogenous Cas nuclease of the host cell. do.

[0199] 45. The array is for administration to a human or non-human animal and the first cell type is human or animal. the first cell type is non-pathogenic to the organism and the second cell type is pathogenic to humans or animals, wherein the array 45. The vector of embodiment 44 contained in a cell.

[0200] 46. ​​The first cell species is a species that is commensal or mutualistic with humans or animals, e.g., intestinal microorganisms. 46. ​​The vector of embodiment 45, which is a vector of the human species.

[0201] 47. When the first cell species is a Lactobacillus species (e.g., Lactobacillus acidophilus (e.g., La-5, La -14 or NCFM), brevis, bulgaricus, plantarum, rhamnosus, fir Menthum, Caucasus, Helveticus, Lactis, Reuteri or Casei, e.g. , casei shirota), Bifidobacterium species (e.g., Bifidum, Breve, Longum or infantis), Streptococcus thermophilus and Enterococcus 47. The vector of embodiment 45 or 46, wherein the vector is selected from the group consisting of S. faecium.

[0202] 48. If the vector is present in a beverage (e.g., a probiotic beverage) or food for human or animal consumption, 48. The vector of any one of aspects 44 to 47, contained in a food.

[0203] 49. The vector contains at least one repeat - spacer - for targeting the target sequence. repeat units, where the repeats are at least identical to the repeats of the CRISPR / Cas system of the host cell. are at least 95% (e.g., 96%, 97%, 98%, 99% or 100%) identical, and This allows the vector repeats to guide the Cas in the host system to modify the target nucleotide sequence. 49. The vector of any of embodiments 44 to 48, which is operable in a host cell to enhance expression of the vector.

[0204] 50. The method of embodiment 49, wherein the vector lacks a Cas (e.g., Cas nuclease) coding sequence. vector.

[0205] Targeting Nucleotide Sequences of a Host CRISPR / Cas System According to the Invention transfer of host cell resistance to the vector (e.g., an invading virus) or resistance expression or amplification For example, the present invention is useful for reducing the number of novel vectors (e.g., (ii) The activity of the endogenous CRISPR / Cas system is inhibited so that spacer acquisition is inhibited. Offers the advantages of targeting and knockdown.

[0206] A hallmark of mobilization is the presence of a cis-acting region (oriT) necessary for transfer. This region is The initiation site of DNA processing, where site- and strand-specific nicks initiate the transduction event The present invention uses mobile genetic elements (MGEs) as follows: Further embodiments are provided.

[0207] 1. Modified CRISPR nucleic acid vectors containing or consisting of mobile genetic elements (MGEs) wherein the MGE is the genome of a host cell (e.g., a pathogenic bacterial cell) or a host cell Origin of transfer (o) for targeted sequence modification of the genome of viruses (e.g., prophage) in riT) and CRISPR arrays, (a) wherein the CRISPR array comprises one or more sequences for expression of crRNA and a host cell a promoter for transcription of the sequence in the cell; (b) where the crRNA guides Cas (e.g., Cas nuclease) in the host cell. and capable of hybridizing with a target sequence to modify the target sequence; (c) wherein the vector (i) is inserted between a first nucleic acid and a second nucleic acid locus in a first host cell, where each locus is a transfectant. (ii) the location of the target sequence in the host cell; or (iii) the location of the target sequence in the host cell. Between the host cell and a second host cell (wherein the target sequence is contained in the first and / or second host cell) It is transmitted by.

[0208] Examples of MGEs are ICEs, transposons, plasmids and bacteriophages The origin of transfer (oriT) is the site of DNA transfer from the bacterial host containing it to the recipient organism during conjugation. oriT is a short sequence (e.g., up to 500 bp) required for transmission to the recipient. - found on the same DNA as the one being transmitted and transmitted along with that DNA. The origin consists of three functionally defined domains: a nicking domain, a transduction domain, and a termination domain. Contains the domain.

[0209] Optionally, the promoter is a promoter for the first and second (and optionally third) cells. Operative for transcription of the sequence.

[0210] Optionally, the target sequence is contained in the second cell. Optionally, the target sequence is not contained in the second cell. stomach.

[0211] For example, the first and second cells are of different bacterial species (e.g., intestinal, axillary, , species found in the vaginal or oral human microbiome populations). For example, the first and second The cells are ex vivo. In other examples, the first and second cells are in vivo or ex vivo. in vivo in the human gut, vaginal, axillary or oral microbiome.

[0212] 2. The vector of embodiment 1, wherein the MGE is or comprises an integrative and conjugative element (ICE). Alternatively, the MGE is a mobile MGE (i.e., a device that supports the MGE for movement). (Factors encoded by genes carried by the MGE can be used.) The term "mobility" in relation to MGEs and "connectivity" is readily apparent to a commercial audience.

[0213] ICEberg data provides examples of suitable ICEs and suitable sources of oriTs according to the present invention. For example, ICE is a database of group 1. a member of the ICE family, including an ICE selected from ∼28, or oriT are the oriTs of members of such families: 1=SXT / R391; 2=T n916;3=Tn4371;4=CTnDOT / ERL;5=ICEclc;6=IC EBs1;7=ICEHin1056;8=PAPI-1;9=ICEMlSym(R7 A);10=ICESt1;11=SPI-7;12=ICE6013;13=ICEK p1;14=TnGBS1;15=Tn5253;16=ICESa2603;17=I CEYe1;18=10270-RD.2;19=Tn1207.3;20=Tn1806 ;21=ICEA5632;22=ICEF-I / II;23=ICEAPG2;24=I CEM;25=10270-RD.1;26=Tn5801;27=PPI-1;28= ICEF-III. Family descriptions are available in the ICEberg database. For example, Tn9 The 16 families were selected from the list of families described by Roberts et al. (2009) (Trends Microbiol. 2009 Jun;17(6):251-8. doi: 10.1016 / j.tim.2009.03.002. Epub 2009 May 20; “A modular master on the move : the Tn916 family of mobile genetic elements”, Roberts A, Mullany P Members of the Tn916 family are defined by the following criteria: Roberts et al. It must have the general organization shown in Figure 1 and must be identical in sequence to the original Tn916 at the DNA level. and must have a structurally similar core region (conjugation and regulatory modules). The previously assigned Tn1549 and corresponding controls in this family should be described. Some conjugative transcripts, such as those with a high degree of protein homology, Spozons are an exception.

[0214] 3. The vector of embodiment 2, wherein the ICE is a transposon, e.g., a conjugative transposon. For example, MGEs are mobile transposons that are mobilizable in the presence of functional helper elements. wherein the transposon is optionally combined with said helper element.

[0215] 4. The vector is a plasmid, and optionally wherein the MGE is a vector contained in the plasmid. The vector of any of the preceding embodiments, wherein the transposon is a conjugative For example, the transposon is a mobile transposon (e.g., For example, a plasmid, e.g., encoded by a gene outside the transposon sequence of the plasmid Optionally, the transposon may be a Type I transposon. Optionally, the transposon is a type II transposon.

[0216] 5. The vector of any of the preceding embodiments, wherein oriT is functional in the first and second host cells. This is because, for example, the cells of a bacterial population may be different when the first and second cells are of different species. It is useful in promoting the spread and growth of bacteria.

[0217] 6. The vector of embodiment 5 when contained in a first cell, wherein the first cell is a second cell. a nucleotide sequence encoding a protein operable to deliver an MGE to a cell. , where the sequence is not included in the MGE. This is because the empty For example, this is useful to avoid the use of smaller M Allows for the construction of GE or the insertion of larger or more CRISPR arrays For example, to include multiple spacers for targeting each sequence in a host cell. or more CRIS to target different sequences in the first and second host cells. Allows insertion of PR arrays.

[0218] 7. The vector of embodiment 6, wherein the sequence is not contained in the vector. This is because the sequence This is useful to avoid the use of spaces in vectors or MGEs for The present study aims to develop a novel method for the construction of smaller vectors or MGEs for cell-to-cell transfer. allows for the insertion of larger or more CRISPR arrays, e.g., in host cells or to contain multiple spacers for targeting each sequence in the first and second host cells. and / or Cas proteins, e.g., Cas It contains more than one sequence encoding 9, allowing for the insertion of more CRISPR arrays. do.

[0219] 8. The vector of embodiment 6 or 7, wherein the sequence is contained in a conjugative transposon of the first cell. This involves the first and second host cells of the MGE of the present invention (e.g., human microbiome). outside the MGE to carry out conjugative transposition for horizontal transfer between different bacterial species in the This is useful because it allows the use of factors.

[0220] 9. The transposon is operable in trans to transmit the MGE to a second cell. A vector of embodiment 8. This is a vector for use in a first and second host cell (e.g., a human) of the MGE of the present invention. conjugative transfer for horizontal transfer between different bacterial species in the microbiome For example, the MG of the present invention is useful because it allows the use of factors outside the MG. The oriT of E is the same as the oriT contained in the conjugative transposon of the host cell. This means that the MGE of the invention can be used in combination with a first and second host cell (e.g., of different species, e.g., host cell-encoded proteins for horizontal transfer between bacterial cells of human microbiome species This is useful for enabling the MGE to operate with factors that are difficult to measure. This allows for the freeing up of space for CRISPR arrays and / or Cas genes. Let's say.

[0221] The term "transformer operable" means that the MGE (ICE) is For transfer of the vector (such as the entire vector) to a second cell, the vector nucleotide sequence Proteins expressed from host nucleotide sequences outside of the host cell (e.g., conjugative transcription factors of the host cell) This means that the protein expressed by the endonuclease gene is capable of horizontal transfer. Taste.

[0222] 10. The vector of any of the preceding embodiments when contained in a first cell, wherein The oriT of the MGE is identical to the oriT contained in the ICE of the first cell, where the ICE It is operable in trans to transmit the MGE to a second cell.

[0223] 11. If the vector oriT is from the phylum Bacteroidetes (e.g., Bacteroidae or Bacteroides oriT of any of the preceding embodiments, vector, which is a vector that is capable of transforming the first and / or second host cells, respectively, into the phylum Bacteroidetes (e.g. For example, Bacteroidae or Bacteroides) or Prevotella cells are useful. For example, the first cell may be a cell of such a species, and the second cell may be a Firmicutes cell. the target sequence is contained in the second cell but not in the first cell, thereby The R array directs Cas in the second cell to cleave the target sequence. For example, the target sequence is in essential genes or antibiotic resistance genes of the second cell (and with regard to the latter, The vector is then combined with an antibiotic or administered to a human or non-human animal in combination with an antibiotic. Optionally, the transposon is CTnDot or CT The nERL transposon and vector are combined with or without tetracycline. It is administered to humans or non-human animals in combination with tracycline.

[0224] 12. Vector oriT is CTnDot, CTnERL SXT / R391, Tn916 or the Tn4371 family transposon oriT. Vector of.

[0225] 13. The MGE contains the first and second terminal repeats and a CRISPR array between the repeats. The vector of any of the preceding embodiments.

[0226] 14. When MGE transfers a transposon copy to a second location, the first nucleic acid location is or (2) remaining in the first cell when transferred to the second cell, vectors, which are useful in promoting the growth and maintenance of MGEs in bacterial populations containing host cells. Alternatively, MGE can be used to transfer transposon copies to (i) the second location or (ii) not remaining in the first cell when transferred to the second cell.

[0227] 15. When contained in the first and / or second cells (e.g., the first and second cells a first and second copy of the vector contained in said vector), the vector of any of the preceding embodiments.

[0228] 16. The vector of embodiment 15, wherein the first and second cells are cells of different species. For example, The first cell may be a Lactobacillus cell (e.g., as described herein) and / or a second cell. Bacteroidetes (e.g., Bacteroides cells, e.g., as described herein) cells) or Firmicutes cells (e.g., cells as described herein). For example, for the treatment or prevention of GI conditions or diabetes or for the treatment of obesity. or for prophylactic administration of the human gut microbiome, where the first cells are Bacteroides Bacteroides cells (e.g., Bacteroidetes cells, e.g., as described herein) and Two of the cells are Firmicutes cells (eg, cells such as those described herein).

[0229] 17. The method of embodiment 15 or 16, wherein the first and second cells are bacterial or archaeal cells. Kutar.

[0230] 18. The first cell is non-pathogenic to humans (e.g., a commensal or mutualistic bacterial cell) and 18. The vector of embodiment 16 or 17, optionally wherein the second cell is a cell pathogenic to humans. In the method, the second cell is a cell that is non-pathogenic to humans. Pathogenic bacteria in the microbiome (e.g., intestinal, vaginal, axillary, or oral microbiome) Certain bacteria can exist in the human body without substantial pathogenicity, but are pathogenic in other parts of the human environment. Those skilled in the art will appreciate that the first The cell type can be maintained in or on a human and a second cell type can be maintained in or on a human. For example, CRISPR arrays have been shown to be effective in humans and The gene may then be used to modify the genome of a second cell in a human, either to kill it or to reduce cell viability or proliferation. For example, the target site is contained in a second cell and the site is modified by a Cas nuclease. cleaved, thereby inactivating or down-regulating the gene contained within the target site. The gene may be an essential gene of the second cell or an antibiotic resistance gene. For example, the gene may be a disease It is a primordial gene.

[0231] 19. The second cell (each host cell) is (i) a strain of, for example, methicillin-, vancomycin-, and tetanus-resistant Staphylococcus aureus cells resistant to antibiotics selected from icoplanin, (i i) For example, cephalosporins (e.g., ceftazidime), carbapenems (e.g., imipenem), penem or meropenem), fluoroquinolones, aminoglycosides (e.g., gentamicin, Pseudomonas aeruginosa resistant to antibiotics selected from the group consisting of isocin or tobramycin and colistin Domonas aeruginosa cells, (iii) Klebsiella resistant to carbapenems (e.g. (iv) e.g., erythromycin, clindamycin, beta-lamidopropyl betaine, selected from methicillin, macrolides, amoxicillin, azithromycin and penicillin Antibiotic-resistant streptococcus (e.g., pneumoniae or pyogenes) cells (v) for example, selected from ceftriaxone, azithromycin, and ciprofloxacin (vi) Salmonella (e.g., serovar Typhi) cells resistant to antibiotics, e.g., Ciprofloxacin, (vii) Shigella cells resistant to an antibiotic selected from loxacin and azithromycin; For example, isoniazid (INH), rifampicin (RMP), fluoroquinolones, amikacin Mycobacteria resistant to antibiotics selected from benzodiazepine, kanamycin, and capreomycin (viii) Enterococcus resistant to vancomycin, e.g., (ix) cells resistant to antibiotics selected from, for example, cephalosporins and carbapenems; Enterobacteriaceae, (x) e.g., trimethoprim, nitrofurantoin, cephalexin (xi) an Escherichia coli cell resistant to an antibiotic selected from fluconazole and amoxicillin; For example, resistant to antibiotics selected from fluoroquinolone antibiotics and carbapenems. (xii) Clostridium (e.g., difficile) cells, e.g., cefixime (e.g., oral cephalosporin), ceftriaxone (injectable cephalosporin), azithromycin Neisseria gonorrhoeae cells resistant to antibiotics selected from cyclosporine and tetracycline (xiii) an antibiotic selected from, for example, beta-lactams, meropenems, and carbapenems; Acinetobacter baumannii cells resistant to substances or (xiv) e.g., ciprofloxacin and azithromycin. The vector of any of the preceding embodiments or any use herein, wherein the cell is selected from Such species can be pathogenic to humans.

[0232] 20. The target site is contained in an antibiotic resistance gene in the second cell, where the antibiotic acts. The vector or use of embodiment 19, which is each antibiotic described in embodiment 19.

[0233] 21. The first cell is a Bacteroidetes (e.g., Bacteroidae or Bacteroides) cell. Lactobacillus (e.g., Lactobacillus acidophilus (e.g., La-5, La-14, or NC FM), Brevis, Bulgaricus, Plantarum, Rhamnosus, Fermentum, Coca Lactobacillus casei, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus reuteri or Lactobacillus casei, e.g., Lactobacillus casei shirota ), Bifidobacterium (e.g., Bifidum, Breve, Longum, or Infante) , Streptococcus thermophilus, Enterococcus faecium, Alice Tippes, Alkaliflex, Parabacteroides, Tannerella or Xylanibacter The vector of any one of embodiments 15 to 20, which is a cell.

[0234] 22. The first and / or second nucleic acid positions of (i) are from the phylum Bacteroidetes (e.g., Bacteroidetes or Bacteroides) cells contained in or (ii) the first and / or second host If the cells belong to the phylum Bacteroidetes (e.g., Bacteroidae or Bacteroides) or Prevotella The vector of any of the preceding embodiments, which is a Terra cell.

[0235] 23. The first cell is a Bacteroidetes (e.g., Bacteroidae or Bacteroides) cell. the second cell is a Firmicutes (e.g., Clostridium or Staphylococcus aureus) coccus) cells, for example, where the vector is used to treat or prevent a GI condition or diabetes. or to the human gut microbiome for the treatment or prevention of obesity The vector of embodiment 22, which is for

[0236] 24. The primary cell (or each primary cell) is environmentally tolerant in the environment (e.g., in the water or soil environment). and optionally the second cell (each host cell) is not permissive in the environment, 17 vector (or any use herein). Aquatic environments are readily apparent to those skilled in the art. For example, it may be a marine or waterway (e.g., lake, canal, river or reservoir) environment. For example, the water environment is drinking water or wastewater intended for human consumption. For example, the soil environment is soil in cultivated land. The soil may be from a mining site (e.g., a mineral or metal mining site).

[0237] By "permissive" and "non-permissive" one skilled in the art would mean a cell type that can be maintained in an environment. It is easy to see that the second cell type should be reduced in the environment. The PR array modifies the genome of the second cell to determine whether it will die in the environment or whether it will affect cell viability or The target site is modified to reduce proliferation. For example, the target site is contained in the second cell and the site is a Ca s nuclease, thereby inactivating or down-regulate, e.g., the gene is an essential gene or an antibiotic resistance gene in the second cell For example, the gene is a virulence gene.

[0238] For example, the environment can be the human microbiome, e.g., the oral microbiome or the gut microbiome. The microbiome or the bloodstream. For example, the environment is not the human environment. For example, the environment is not an environment for a non-human animal. In some embodiments, the environment is an air environment. In some embodiments, the environment is an agricultural environment. In some embodiments, the environment is a stony environment. An oil or petroleum recovery environment, such as an oil or petroleum rig or well. For example, the environment , the environment relating to food or beverages for human or non-human animal consumption.

[0239] For example, a vector, a system, a vector, an array, a crRNA, a gRNA, a method or Any use herein is an industrial use or the environment is an industrial environment, The industries include medical and healthcare, pharmaceuticals, human food, animal food, plant fertilizer, beverages, dairy, Meat processing, agriculture, livestock farming, poultry farming, fish and seafood farming, veterinary, oil, gas and petrochemicals, Water treatment, wastewater treatment, packaging, electronics and computers, personal healthcare and Toiletries, Cosmetics, Dental, Non-medical Dental, Ophthalmology, Non-medical Ophthalmology, Mineral Mining and Processing, Gold Metal mining and processing, quarrying, aviation, automobiles, railways, ships, space, environment, soil treatment, pulp and and paper, clothing manufacturing, dyes, printing, adhesives, air treatment, solvents, biological defense, vitamin supplements Supplement, cryopreservation, textile soaking and manufacturing, biotechnology, chemicals, industrial cleaning products, household Cleaning Products, Soaps and Detergents, Consumer Goods, Forestry, Fishing, Recreation, Recycling, Plastics , Leather, Leather Goods and Suede, Waste Management, Funerals and Funeral Services, Fuel, Construction, Energy The industry is in a field selected from the group consisting of the iron and steel, and tobacco industry fields.

[0240] 25. The above, combined with a nucleic acid (e.g., DNA) for incorporation at a modified target site. A vector according to any one of the preceding embodiments.

[0241] For example, the modification may be cleavage of the target site, allowing the nucleic acid (e.g., DNA) to undergo homologous recombination in the host cell. This allows for precise targeting of the host cell genome using the vectors of the invention. It is useful for making modifications.

[0242] 26. The nucleic acid for incorporation is a regulatory element or exon sequence, e.g., a human sequence, or A vector according to embodiment 25, comprising:

[0243] 27. Any of the preceding embodiments, combined with a transposase for mobilization of the MGE. Reka's vector.

[0244] 28. The vector or MGE comprises a toxin-antitoxin module operable with a first host cell. and optionally wherein the toxin-antitoxin module is operable with a cell other than the first cell. The vector of any of the preceding embodiments, comprising an antitoxin gene that is absent or has reduced activity. Tar.

[0245] 29. The vector or MGE comprises a toxin-antitoxin module operable with a second host cell. and optionally wherein the toxin-antitoxin module is operable with a cell other than the second cell. The vector of any of the preceding embodiments, comprising an antitoxin gene that is absent or has reduced activity. Tar.

[0246] 30. A toxin-antitoxin molecule capable of binding a vector or MGE to a first and second host cell. and optionally wherein the toxin-antitoxin module is a toxin-antitoxin module other than the first and second cells. The above-mentioned embodiment includes an antitoxin gene that is not operable or has reduced operability with the cell. The use of a toxin-antitoxin module in any of the vector embodiments provides a selective advantage, thus In addition, it is useful for MGE retention and diffusion. For example, the module is a Type I module, e.g. For example, the Hok-Sok module. For example, the module is a Type II module, e.g. For example, the HiCa-HicB module. For example, the module is a tad-ata type. For example, the module is a plasmid addiction module. For example, the first and / or second cells are Bacteroides cells and the module The module is a member of the Bacteroides species, e.g., Txe / YoeB family addi- ction module (see, for example, http: / / www.uniprot.org / uniprot / F0R9D1), RelE / StbE Family Addiction Module (e.g., http: / / www.uniprot.org / unip rot / F0R9A0), HigA family addiction module (e.g., http: / / www.u niprot.org / uniprot / D7J8V2 or http: / / www.uniprot.org / uniprot / D2ESD0), Re lE / StbE family addiction module (e.g., http: / / www.uniprot.org / The use of toxin-antitoxin in vectors or MGEs is vector-carrying cells other than the desired cells (e.g., the first and second and / or third bacterial cells) In this example, the MGE or vector may be useful in enabling the disruption of the bacterial a toxin-antitoxin module comprising a toxin gene and a cognate antitoxin gene, wherein the toxin and The expression of the antitoxin genes is separately controlled, for example, by different promoters. The toxin gene is constructed in the first, second (and third) cells so that the toxin is constantly produced. The antitoxin gene may comprise a promoter that is constitutively active in the first and / or second cells. Inducible by one or more factors (e.g., expressed proteins) in the cell, but different The promoter may include a promoter that is not inducible in non-target cells of a given strain or species. As we know, antitoxins are inherently less stable than toxins in bacterial toxin / antitoxin systems. Thus, vector delivery to cells that are not target cells (e.g., not the first and / or second cells) Transduction of the tRNA or MGE results in toxin expression in the absence or with low antitoxin activity. This, in turn, leads to the death of non-target cells. creating selective pressure for the incorporation and maintenance of the vector of the invention in the third cell; These contain the desired CRISPR array activity and are useful for detecting the activity of CRISPR in a population (e.g., gut microbiota). This also allows the array to be expanded across a population of target cells, so that the effect of the array can be controlled within the population. It also controls the spread of vectors or MGEs to non-target cells - in this regard, pressure to not take up the vector, and if they do, the recipient cells They do not survive, thereby limiting replication in non-target cells that carry the MGE and arrays.

[0247] 31. The first and second cells are of the same phylum (e.g., both bacterial cells) and the vector is d) in primary and / or secondary cells, but not in other cells of the same phylum, and (e) in other cells of the same order. (f) in other cells of the same class, but in the first and / or second cells (g) in the first and / or second cells, but not in other cells of the same eye (h) in the first and / or second cells, but not in other cells of the same family; In one cell and / or in a second cell, (i) in the first cell and not in other cells of the same genus (j) in the first cell and / or second cell, but not in other cells of the same species; or second cell, and (k) not in other cells of the same strain, but in the first cell and / or second cell. 2. The vector of any of the preceding embodiments, which is replicable or operable in a cell.

[0248] This provides selectivity of the vectors of the invention in the microbiome (e.g., (due to selective killing of second host cell types in mixed bacterial populations), where e.g., replication or requires the expression of specific proteins for its function (e.g., expression to produce crRNA). By manipulating the MGE or array (e.g., its promoter) as needed For example, the promoter may be operable with the first and / or second cell. or wherein the MGE is selected from promoters that are capable of expressing the MGE in a cell but not in other cells. One or more of the replication initiation sites is produced in the first and / or second cell but not in other cells These proteins are engineered to depend on proteins or other factors that are not produced in the normal environment.

[0249] 32. A first and second codon of any of the vectors of any of the preceding embodiments in a mixed population of cells. wherein the first vector is contained in a first cell and the second vector is contained in a second cell. The cells are of different species (e.g., different bacterial species) and one or more of the vector MGE Both are capable of transmission to a third cell (e.g., a bacterial cell), where the third cell type is either the first or second The two cells may be of the same species or of a different species than the first and second cell types. When the cell is a carrier (e.g., a non-pathogenic agent), it can be used to transport the microbiota in humans or animals. It is useful because it acts as a By horizontal transfer, the vehicle transfers the CRISPR array of the invention to a third cell, They can be propagated (either directly or via secondary cells, the latter of which act as a reservoir for the array). , the array then mediates Cas modification (e.g., cleavage) of the target sequence in a third cell, e.g., Third, essential or antibiotic resistance genes in the cell can be inactivated or downregulated. .

[0250] Generally, when the target sequence is contained in a cellular antibiotic resistance gene, the vector of the present invention - the modified sequence or array is administered to a human or animal in combination with (or in combination with) an antibiotic. This can be administered sequentially (either sequentially or sequentially) to kill or reduce proliferation of cells containing the target sequence. In this regard, the vector, modified sequence or array may be useful for administering antibiotics. wherein the target sequence is a sequence of a gene encoding resistance to said antibiotic. be.

[0251] Optionally, the mixed population includes a third cell.

[0252] For example, a plurality of first cells are provided, each comprising a vector of the invention. A plurality of second cells are provided, each comprising a vector of the invention. A plurality of first cells in combination with a plurality of second cells is provided, for example, a plurality of second cells. a plurality of first cells in combination with a plurality of third cells, At least two (or all) comprise a vector of the invention.

[0253] 33. Toxins that allow the vector or MGE to function with first, second, and third host cells -antitoxin modules, optionally wherein the toxin-antitoxin modules are first, second and and antitoxins that are inactive or have reduced activity (i.e., low activity) in cells other than the third cell. 33. The vector of embodiment 32, comprising a base gene.

[0254] 34. MGE is a conjugative transposon, and oriT functions in the first and second host cells. The MGE is a CRISPR array that contains the first and second terminal repeats and the CRISPR array between the repeats. wherein the first and second cells are bacterial cells, and the second cell is a human microbiota cell species ( for example, a pathogenic species), where the target site is contained in a second cell but not in a first cell. wherein the modification inactivates a gene or regulatory sequence comprising the target in the second cell. 3. The vector of any of the preceding embodiments, wherein the vector upregulates or downregulates a gene encoding a gene for which the gene is a nucleotide.

[0255] Usefully, the first cells thereby act as a carrier and reservoir for the arrays of the invention. This can be transmitted by horizontal transmission of MGEs.

[0256] For example, MGE is a conjugative Bacteroidetes transposon, and oriT is the first and The Bacteroidetes oriT is functional in both the first and second host cells, and the MGE is the first and second termini. and a CRISPR array between the repeats, wherein the first and second cells Bacterial cells, the first cell is a Bacteroidetes cell and the second cell is a Firmicutes cell. a bacterial cell (e.g., a Clostridium or Staphylococcus cell), wherein the target site is a site contained in the second cell but not contained in the first cell, wherein the modification is Inactivate or downregulate the gene or regulatory sequence containing the target.

[0257] 35. The vector of embodiment 34 when contained in a first or second cell.

[0258] 36. The first and second cells are from a mixed bacterial cell population, e.g., a human or non-human animal (e.g., , dog, cat or horse) in the intestinal, vaginal, axillary or oral microbiota population, The vector of any of the preceding embodiments. As explained above, the population may be administered to a human or animal: It is useful to administer it to establish the microbiome.

[0259] 37. An ex vivo composition comprising a plurality of cells as defined in embodiment 22, wherein each The cell comprises the vector of any of embodiments 1 to 36. Alternatively, the composition can be administered in vivo, e.g. For example, in a non-human animal.

[0260] 38. A human or animal model comprising the vector of any one of embodiments 1 to 36 or the composition of embodiment 37. beverages or foodstuffs for human or non-human animal consumption. The beverages may be used, for example, to treat obesity or For example, daily or every two days by a human or animal to treat or prevent a GI condition. Or it may be, for example, a probiotic drink for weekly consumption.

[0261] 39. A composition comprising a plurality of Bacteroides cells, wherein each cell is a member of any one of embodiments 1 to 39. Contains any of the 36 vectors.

[0262] Usefully, the cells can be used to treat or inhibit obesity or GI conditions in humans or animals, for example. To prevent or mitigate the effects of microbial contamination on the human or animal microbiome (e.g., the gut microbiome), The arrays can act as vehicles and reservoirs for administration to a patient (eg, a group of patients).

[0263] 40. A mixed population of bacterial cells, comprising a first subpopulation of cells and a second subpopulation of cells, wherein the first cell comprises the vector of any of embodiments 1 to 36, wherein the vector is a first Horizontal transmission between the microbial community and a second cell subpopulation is possible. Such populations are important because bacteria are transmitted to humans or The human microbiome is designed to be colonized with one or more microbiomes of an animal (e.g., the gut microbiome). First (and optionally Second, cells are important for the development of new cell types, especially when these cells are involved in the development of a human or animal (e.g., gut microbiome) When the CRISPR array of the present invention is non-pathogenic in a pathogenic organism, it can be used as a carrier of the CRISPR array of the present invention. The microbiome can be any other microbiome or It may be a microbiota population.

[0264] 41. One or both of the first and second bacterial species are intestinal microorganisms of humans or non-human animals. and optionally the first bacterium is commensal or mutualistic with a human or animal. The population of embodiment 40. Advantageously, the first bacterium can be safely administered to a human or animal, and the They can act as vehicles for the delivery of the arrays of the invention to other cells of the post-microbiota.

[0265] 42. Mixed populations may be present in drinking water (e.g., watercourses or drinking water for human consumption) or soil. The population of embodiment 40, wherein the population is contained in water or soil. in heating, cooling or industrial systems or in drinking water storage containers It is useful for treating these.

[0266] For example, in any of the embodiments, the second cell is a cholera cell containing a target sequence, wherein the target When the target sequence is modified, the cells are killed or cell proliferation is reduced. The cells are present in water for human consumption (e.g., such water before or after processing for human consumption). For example, the vector may be administered to a human for the treatment or prevention of cholera in the human. The pharmaceutical composition is

[0267] 43. A composition comprising a plurality of vectors according to any one of embodiments 1 to 36 in vitro, e.g. The composition is mixed with a multi-species bacterial population in an industrial device or container (e.g., food, consumer goods, cosmetics, personal health care products, petroleum or petroleum products).

[0268] 44. Therapeutic or prophylactic colonization and transplantation of human or non-human animals with their microbiomes for the purpose of rebalancing or cosmetically altering humans or animals (e.g., cosmetic The vector, composition, food, beverage or pharmaceutical composition of any of the preceding embodiments for administration to a mammalian animal for the treatment of a disease (e.g., a mammalian animal), ... is a group.

[0269] 45. A method for modifying a target nucleotide sequence in a host cell, comprising: (1) Combine host cells and carrier cells; (a) Here, the carrier cell is a CRISPR nucleus containing a CRISPR array for targeted modification. Contains an acid vector, (b) wherein the CRISPR array comprises one or more sequences for expression of crRNA and a host a promoter for transcription of the sequence in the cell; (c) where the crRNA guides a Cas (e.g., a Cas nuclease) in the host cell. and capable of hybridizing to a target sequence to modify the target sequence; and (2) These cells are cultured together, where the vector is transferred from the carrier cells to the host cells. This allows the crRNA to hybridize with the target sequence to guide Cas in the host cell. and the target is modified A method comprising:

[0270] For example, the method is performed ex vivo. For example, the method is a cosmetic method and or as a preventative medical method.

[0271] 46. ​​The method of embodiment 45, wherein the vector is according to any one of embodiments 1 to 36.

[0272] 47. The host cells are cells of a human or non-human animal microbiome bacterial species, and are preferably 47. The method of embodiment 45 or 46, wherein the host cell is a cell of a pathogenic bacterial species. For example, any microbiome here can be found in the gut, vagina, axilla, scalp, skin or oral microbiome. Selected from the black biome.

[0273] 48. Carrier cells are commensal or mutualistic human or non-human animal microbiomes. 48. The method of any of embodiments 45 to 47, wherein the carrier cell is a bacterial species. is non-pathogenic to humans when administered, for example, intranasally, topically or orally.

[0274] In any form, concept, aspect, embodiment or example herein, the present invention is The vector, composition, array or population may be administered intranasally, topically or orally to a human or non-human animal. or for administration in the human or animal microbiome. Those skilled in the art will be able to determine the best route of administration depending on the microbiome of interest. For example, when the microbiome is the gut microbiome, administration can be intranasal or It can be oral or scalp. When the microbiome is in the oral cavity or pharynx, administration can be local. It can be oral.

[0275] 49. The host cells are from the gut microbiome bacterial species of humans or non-human animals. A method according to any one of embodiments 45 to 48.

[0276] 50. The ratio of the first bacterial and second bacterial host cell subpopulations is determined by the bacterial mixture containing the subpopulations. 1. A method of modifying a combined population, comprising: A: providing the first bacterium to a host cell; B: providing said second bacterium to a host cell, wherein the second cell is a cell of a different species or strain than the first cell; It is a cyst; C: The modified CRISPR array is introduced into a first bacterial host cell, where each CRISPR The array may comprise one or more sequences for expression of crRNA and sequences in the second host cell. a promoter for transcription, where the crRNA is a Cas (e.g. , Cas nuclease) to modify the target sequence contained in the second cell. capable of hybridizing to the sequence; D: Combining the first bacterial and second bacterial cells together to produce a mixed bacterial population; and E: Mixing occurs to allow horizontal transfer of the CRISPR array from the first bacterial cell to the second bacterial cell. The bacteria are grown in a combined population, where the target sequence in the second cell is Cas-modified, and This changes the relative ratio of the first bacterium to the second bacterium. A method comprising:

[0277] 51. The method of embodiment 50, wherein each CRISPR array is according to any of embodiments 1 to 26.

[0278] 52. Further, a first sample of the mixed population of step E is obtained, and optionally, a second sample of the first sample is obtained. comparing the proportion of cells in the sample to a proportion of a second cell in a second sample of cells, the second sample is the mixed population of bacterial cells used to provide the second cells in step B. and comparing the samples to see whether the proportion of second cells has increased or decreased after step E. The method of embodiment 50 or 51 shown.

[0279] 53. The second sample is a human or animal microbiome (e.g., gut, vagina, scalp, axilla) 53. The method of embodiment 52, wherein the sample is a sample of tissue (e.g., skin or oral cells).

[0280] 54. Human or animal microbiome (e.g., gut, vagina, scalp, axilla, skin, or mouth) Any of embodiments 50 to 53, wherein a sample of the luminal cells is used to provide the second cells in step B. That method.

[0281] 55. A recombinant, cultured population of first cells is used for step A, according to embodiments 50-54. A method according to any one of the above.

[0282] 56. Plasmids, ICE, or horizontal transposon transfer are used in step E, where each 50. The method of claim 50, wherein the plasmid, ICE, or transposon comprises a CRISPR array. Any of the 55 methods.

[0283] 57. For therapeutic or prophylactic rebalancing of the microbiota of humans or non-human animals, e.g. For example, for the treatment or prevention of obesity, diabetes, IBD, GI tract conditions, or oral conditions. The method of any one of aspects 50 to 56. The diabetes may be type I or type II. For example, prevention may involve medical intervention. For example, the prevention herein may be for non-medical, e.g., cosmetic or hygiene purposes. The microbiota is axillary microbiota, and the method is for preventing or reducing body odor in a human. For example, in this case, the method involves detecting the proliferation of host bacterial cells that mediate the development and / or persistence of human body odor. Downregulate proliferation or viability.

[0284] 58. Providing a third bacterial host cell of a different species or strain from the vehicle and host cell. wherein the third cell is either included in the mixed population in step E or combined with the outpopulation after step E.

[0062] Embodiment 50. ~57 any of the methods.

[0285] 59. The method of embodiment 58, wherein the third cell does not contain the target sequence.

[0286] In this way, a third cell can act as a carrier for the array and access the host cells containing the target sequences. Rays can be transmitted horizontally.

[0287] 60. The method of embodiment 58, wherein the third cell does not contain a target sequence for Cas modification.

[0288] 61. The carrier (and optionally also the third) cell is of the species described in embodiment 21, e.g. The method of any of embodiments 50 to 60, wherein the cell is, for example, a Bacteroides cell.

[0289] 62. The host cell is of the species according to embodiment 19 or a Firmicutes cell. The method of any one of embodiments 50 to 60.

[0290] 63. Each vector may be a plasmid, a phage (e.g., a packaged phage), or a phage. The vector, composition, foodstuff of any of the preceding embodiments, which is or comprises a dimid. beverage, group or method.

[0291] 64. The modification results in (i) cleavage of the target sequence, (ii) transcriptional downregulation of the gene containing the target sequence, or (iii) targeting or (iv) the addition, deletion, or substitution of a nucleic acid sequence at the target. The vector, composition, food, beverage, population or method of any preceding embodiment,

[0292] 65. Each target sequence is a sequence contained in a regulatory element or gene of a host cell, where the gene wherein the gene is an essential gene, a CRISPR gene or an antibiotic resistance gene, and optionally and the regulatory element is an element of such a gene. , food, drink, population or method. Alternatively, the gene is a pathogenicity gene.

[0293] 66. Each target sequence is a sequence contained in a phage genome, where the phage binds to a host cell. Included are the vectors, compositions, foods, beverages, populations or methods of any of the preceding embodiments. For example, if the target sequence is a phage promoter or promoter sequence that mediates host cell infectivity, phage lysogeny or lytic cycle or phage replication. Contains phage genes necessary for viability, e.g., essential genes or coat protein genes. It can be enjoyed.

[0294] For example, Bacteroidetes phages include crAss phage, GB-124 phage, and G A-17 phage, HB-13 phage, H16-10 phage, B40-8 phage, and and Bacteroides fragilis phage ATCC51477-B1. This includes, for example, the identification of bacteria in the Bacteroidetes phylum in the human or animal gut microbiome. In this way, it is useful to differentiate between Bacteroidetes and Firmicutes. The ratio of the two can be altered to increase the ratio of the former to the latter (e.g., obesity). For example, if the target sequence is BACON (Bacteroidetes-associated carbohydrate a microbial binding domain-encoding sequence (e.g., where the host is a Bacteroides host), or Included in the endolysin coding sequence.

[0295] 67. Each CRISPR array is designed for the expression and production of each crRNA in the host cell. comprising the sequence R1-S1-R1′, (i) wherein R1 is the first CRISPR repeat and R1' is the second CRISPR repeat; R1 or R1' is optional, (ii) S1 comprises or consists of a nucleotide sequence that is 95% or more identical to the target sequence. The first CRISPR spacer, The vector, composition, food, beverage, population or method of any of the preceding embodiments.

[0296] 68. R1 and R1' are the first and second CRISPR arrays of the second host cell type, respectively. 68. The vector, composition, or food of embodiment 67, which is at least 95% identical to the second repeat sequence. beverage, group or method.

[0297] 69. R1 and R1' are the CRISPR / Cas system of the host cell for modification of the target sequence. 69. The vector, composition, food, beverage, population or Or method.

[0298] 70. Each array functions with a respective crRNA in the host cell to modify the target sequence. The vector, composition of any of the preceding embodiments, in combination with one or more Cas nucleases. a product, food, drink, population, or method. The target sequence is a protospacer adjacent motif (PAM). It contains a protospacer sequence immediately adjacent to it.

[0299] 71. Each array is a sequence that controls the function of each crRNA in the host cell to modify the target sequence. Compositions, foods, and beverages in combination with nucleic acid sequences encoding the above Cas nucleases. , population or method.

[0300] 72. R1 and R1' are type II Cas9 targeting molecules for modification of a target in the host cell. Nucleases (e.g., Streptococcus pyogenes or Staphylococcus aureus) and optionally in the vector, composition, food, drink, The material, population or method further according to embodiment 70 or 71, wherein the Cas is said Cas9. 72. The vector, composition, food, drink, population or method of any of embodiments 67 to 71, .

[0301] 73. An ex vivo mixed population of bacteria obtained by the methods of embodiments 50 to 72.

[0302] 74. A therapeutic, prophylactic, or cosmetic use for a human or non-human animal, comprising the mixed population of embodiment 73. Administered to humans or non-human animals for weight loss (e.g., cosmetic weight loss) or nutritional use. A composition comprising:

[0303] 75. A human or non-human animal comprising the mixed population of embodiment 73 or the composition of embodiment 74. Food or drink for consumption.

[0304] 76. The food or beverage of embodiment 75, which is a nutritional supplement or probiotic drink or food. is a beverage.

[0305] 77. A human or non-human vector comprising the vector of any one of embodiments 1 to 36 and 63 to 72. Antibiotic compositions for treating or preventing bacterial infections in animals or drinking water or soil Finished product.

[0306] 78. A human or non-human vector comprising the vector of any one of embodiments 1 to 36 and 63 to 72. To increase the proportion of gut Bacteroidetes in animals (e.g., due to obesity, diabetes, or GI probiotic compositions for treating or preventing inflammatory conditions.

[0307] 79. In humans or animals, for example, for the treatment or prevention of obesity, diabetes, or GI conditions. 74. To increase the relative ratio of Enterobacteriaceae to Firmicutes in the , 77 or 78 compositions.

[0308] 80. The vector does not contain a Cas nuclease encoding sequence operable with the array; The vector, composition, food, beverage, population or method of any of the preceding embodiments. It is useful for securing space in the target (e.g., for host cell targeting). To allow for the insertion of large or more arrays into the array - this is Useful for targeting multiple genomic locations to mitigate the likelihood of developing resistance to ray (It is).

[0309] 81. The method of claim 1, wherein the MGE does not include a Cas nuclease encoding sequence operable with the array. The vector, composition, food, beverage, population or method of any of the above embodiments. It is useful for securing space in the host cell (e.g., for host cell targeting). To allow for the insertion of a type array or more arrays - this is the array of the present invention It is useful to target multiple genomic locations to mitigate the likelihood of resistance to For example, avoid including the large sequence encoding the Cas9 endonuclease. is possible.

[0310] 82. The array comprises Cas sequences found in cells of the same species or strain as the first and / or second cells. 82. The vector or composition of embodiment 80 or 81, which is operable with an endonuclease. For example, the array may be a mixture of cells of the same species or species as the host cell or a third cell. It can work with Cas endonucleases found in cells of various strains. It is useful for securing space in the target or MGE (e.g., host cell targeting). To allow for the insertion of larger or more arrays for loading - this , targeting multiple genomic locations to mitigate the likelihood of resistance to the arrays of the present invention. (This is useful for

[0311] 83. The first and second cells are bacterial cells of different species, where the second cell is a member of the human microbiota. species, and the first cells are of a non-pathogenic species in the human microbiota, The target sequence is not contained in the genome of the first cell, and the MGE is operable in both the first and second cells. riT, wherein the MGE is capable of horizontal transmission from a first cell to a second cell. Any of the vectors, compositions, foods, beverages or populations of the embodiments.

[0312] Alternatively, the following is provided: The carrier and host cells are bacterial cells of different species, wherein the host cells are human microbiota species wherein the carrier cells are of a non-pathogenic species in the human microbiota, The target sequence is not contained in the genome of the carrier cell, and the MGE functions in the carrier and host cells. It contains oriT, which can transfect MGE into host cells. 10. The method of any of the preceding embodiments.

[0313] 84. The vector is contained in a bacteriophage, and the bacteriophage is the first cell (carrier). 84. The vector of claim 83, which is capable of infecting and introducing MEG into a first (vehicle) cell. -, composition, food, beverage, group or method.

[0314] 85. The target sequence is included in the genome of a second (host) cell (e.g., an essential or antibiotic component of the genome). 85. The vector, composition, food, beverage of embodiment 83 or 84, Group or method.

[0315] 86. A second (host) cell type is pathogenic in the human microbiota, where the target sequence is 86. The vector of embodiment 85, wherein the vector is modified by cleavage of the target sequence or downregulation of the gene containing the target sequence. For example, the second (host) cell is a cell of embodiment 19. A cell that conforms to any of properties (i) to (xiv). For example, the second (host) cell is a firmic and the vector is a vector for the treatment or prevention of obesity in humans. is.

[0316] 87. The method of embodiment 83, 84 or 85, wherein the second (host) cell type is non-pathogenic in the human microbiota. or 85 vectors, compositions, foods, beverages, populations or methods.

[0317] 88. The second (host) cell is a Bacteroidetes or Prevotella cell, optionally MGEs can be horizontally transferred from a second (host) cell species to Firmicutes species in the human microbiota. A vector, composition, food, beverage, population or method according to any one of embodiments 83 to 87. The latter is a case where the target sequence is contained in Firmicutes but is not expressed in the first (vehicle) or second (host) cells. When not contained in cells, they are useful, for example, for the treatment or prevention of obesity in humans.

[0318] 89. MGEs can be horizontally transferred from a second (host) cell type to a third bacterial cell type in the human microbiota. wherein the third cell type is pathogenic in the human microbiota and comprises the target sequence. Any of the vectors, compositions, foods, beverages, populations, or methods of 83 to 88. For example, the first ( The second (vehicle) and second (host) cells do not contain the targeting sequence.

[0319] 90. An embodiment wherein the third cell is a cell according to any of features (i) to (xiv) of claim 19. The vector, composition, food, beverage, population or method of embodiment 89.

[0320] 91. The MGE encodes a Cas endonuclease that can operate with the repeat sequences of the array. The vector lacks a sequence encoding such a sequence (e.g., Cas9) outside the MGE. The vector, composition, food, beverage, population or method.

[0321] Any of the general characteristics are applicable to this aspect. Any feature of the embodiment or concept may be combined with this embodiment using MGE. stomach.

[0322] Therefore, the present invention provides the following features, numbered as paragraphs, which are incorporated herein by reference: This applies to any of the above aspects or any of embodiments 1 to 91 or any other aspect herein. 1. The target sequence is the nucleotide sequence of the host CRISPR / Cas system, and CRISPR / Cas system in host cells 51. The vector of any one of embodiments 44 to 50, which guides as to a target.

[0323] 2. The host CRISPR / Cas system is a type I, II, or III system and the target A system of this type in at least one, two or three additional host strains or species wherein the additional strain or species is different from the host. Fall 1 vector.

[0324] 3. If the target sequence is a Streptococcus species (e.g., Streptococcus thermophilus or or Streptococcus pyogenes) CRISPR / Cas system sequence , the vector of any of the preceding paragraphs.

[0325] 4. The vector of any of the preceding paragraphs, wherein the target sequence of the host CRISPR / Cas system but i. The CRISPR array leader or leader adjacent to the 5'-most nucleotide of the first repeat a promoter sequence (and optionally the 5'-most nucleotides of the repeat, e.g. , including the first three nucleotides at the 5′ end of the first repeat); ii. Up to 20 (e.g., 3, 5, 7, 9, 10, 12, 15, 20) nucleotides immediately 5' of the first repeat , 30 or 32) a sequence of consecutive nucleotides; iii. Up to 20 (e.g., 3, 5, 7, 9, 10, 12) of the 5'-most nucleotides of the first repeat , 15, 20, 30 or 32) a sequence of consecutive nucleotides or iv. Up to 20 (e.g., 3, 5, 7, 9, 10, 12, A sequence of 15, 20, 30 or 32) consecutive nucleotides (and optionally wherein the sequence is a spacer containing the 3'-most nucleotide, e.g., the 3'-end of the first repeat, containing the three nucleotides).

[0326] 5. If the array is a nucleic acid vector (e.g., a virus, virion, phage, phagemid, or is contained in a prophage), and i. the crRNA comprises or consists of the structure RSR, where R=CRISPR repeats and S = CRISPR spacer, where S is (5' to 3' direction) VH R or H R- V or where V = a sequence identical to the DNA sequence of the vector and H R = the DNA sequence of the repeats of the CRISPR array of the host cell CRISPR array; ii. where H R the sequence of is immediately adjacent to the sequence of V of the host CRISPR array; and iii. This is where Cas is host-targeted for modification of the host CRISPR array in cells. capable of hybridizing to the spacer of the host CRISPR array to guide Vector in paragraph 1, 2 or 3.

[0327] For example, V is the sequence of a phage vector coat protein coding sequence. In a study of bacterial resistance, Heler et al. Phage-resistant mutants showed a significant defect in phage adsorption (approximately 50%), and these were enveloped showed that these strains were most likely to carry rDNA resistance mutations.

[0328] 6. The first crRNA does not hybridize or does not substantially hybridize with nucleic acids present in the vector. Not a vector of paragraph 5. For example, the first crRNA hybridizes with V in the vector. hybridize with less intensity than the spacers in the host array. Hybridization tests are routine for those skilled in the art. Isolate or synthesize vector DNA in vitro and incubate it with crRNA For example, standard techniques using PCR can be used to determine the identity of the target gene. can be used to detect whether or not a reaction occurs (e.g., under the pH and temperature conditions found in the host cell). Tested under the following conditions.

[0329] 7. V = 1 or up to 40 (e.g., up to 15) contiguous nucleotides of vector DNA , the vector of paragraph 5 or 6. The direct substitution of PAM in the protospacer found in the target sequence The 5' seed sequence is essential for crRNA pairing and cleavage of the CRISPR / Cas system. This seed sequence is important for the function of the PAM. Contains nucleotides.

[0330] 8. The array is contained in a vector and comprises (in the 5' to 3' direction) a first repeat sequence, a first spacer sequence, and a second repeat sequence, wherein the spacer sequence hybridizes with the target sequence in the host cell. and hybridize (e.g., be identical or have greater than 90% identity), so that the array can be further a promoter for transcription of the repeat and spacer in a host cell comprising: The vector contains functional Cas and / or tracrRNA sequences in the host cell. Cas nuclease coding sequence and / or tracrRNA coding sequence a sequence complementary to the first or second repeat of the tracrRNA sequence, The method, array or vector of any preceding aspect or paragraph, comprising:

[0331] 9. The CRISPR array is contained in a vector, and includes (in the 5' to 3' direction) a first repeat sequence, a first a spacer sequence and a second repeat sequence, wherein the spacer sequence is a target sequence in a host cell. capable of hybridizing to (e.g., being identical to or having greater than 90% identity to) a target sequence , wherein the array further comprises a promoter for transcription of the repeat and spacer in a host cell. wherein the vector comprises a C does not contain a nuclease coding sequence and / or a tracrRNA coding sequence, wherein the tracrRNA sequence comprises a sequence complementary to the first or second repeat, M-CRISPR arrays deliver Cas (e.g., endogenous host Cas nuclei) to the host target site. In a host cell, optionally using a host tracrRNA to guide the The method, array or vector of any preceding aspect or paragraph, wherein the vector is functional.

[0332] 10. The repeats are identical to the repeats in the host array, wherein the CRISPR array of the invention Cas of the host CRISPR / Cas system (e.g., Cas nuclease, e.g., The method, array or vector of paragraph 8 or 9, which does not contain a PAM recognized by Cas9 The ability to omit the Cas sequence frees up space in the arrays of the invention.

[0333] An "essential gene" is a gene whose presence or expression is necessary to promote or sustain host cell growth or cell viability. Resistance genes are genes in the host whose presence or expression is necessary for the host to complete or partial resistance to an agent, e.g., an antibiotic, e.g., a beta-lactam antibiotic A virulence gene is a gene in the host whose presence or expression is necessary for the A gene in a host that is necessary for infectivity of an organism that can infect a host cell, e.g. , where the host is a pathogen (e.g., plants, animals, humans, livestock, pets, plants, birds, of fish or insects).

[0334] 11. CRISPR arrays transfect non-host cell Cas (e.g., host systems are type II or I) Type I system Cas when the host system is type II, and Type III when the host system is type I or III Type II system Cas or type III system when the host system is type I or II system Cas), optionally wherein the host cell is of the same type as the non-host Cas. The method, array or method of any preceding aspect or paragraph does not contain or express Cas. vector, which means that the CRISPR array can be delivered by itself (e.g., in a vector) or in a host. This is useful because it does not target vector-encoded Cas sequences.

[0335] 12. CRISPR array encodes tracrRNA sequence or tracrRNA sequence (e.g., on the same nucleic acid as the array), optionally The above-mentioned embodiment or the above-mentioned embodiment, wherein the NA sequence and HM-crRNA consist of a single guide RNA (gRNA). The paragraph can be either a method, an array or a vector.

[0336] 13. The CRISPR array is combined with a Cas or Cas-encoding sequence to produce the desired where the array is integrated into the host cell genome and the Cas is endogenous to the host cell or or an exogenous sequence. Vectors. For example, the Cas encoding sequence can be contained in, for example, a plasmid or virus, e.g. It is a foreign sequence that is being introduced from the phage into the host.

[0337] 14. CRISPR arrays can be used on plasmids, viruses, virions, phages, and phagemids. or the method of any preceding aspect or paragraph, wherein the nucleotide sequence of the prophage A phagemid is a packaged phage, array, or vector. A prophage is , a phage integrated into the host chromosome or episome in the cell.

[0338] 15. The CRISPR array is integrated into the host cell genome, e.g., into chromosomal or episomal nucleic acids. The method, array or vector of any preceding aspect or paragraph, wherein

[0339] In one example, the array comprises a transcription or transcription factor that acts on a target sequence or a gene that contains the target sequence. Combined with a dead Cas (e.g., dCas9) conjugated to a translation activator This is the case, for example, when the host is a microorganism, For example, to encode an antibiotic or to produce a food, beverage, pharmaceutical or other product according to the invention disclosed herein. For any other application, the desired exogenous protein for production in a host culture. For example, a desired gene, e.g., a gene previously engineered in a host cell, is used to encode the desired gene. These are useful for switching the expression of exogenous gene sequences.

[0340] 16. For example, to infect cells, e.g., with microorganisms, or for medical or dental use a virus (e.g., virion, phage, phagemid or prophage).

[0341] 17. A population of virions of paragraph 16, wherein the first and second virions are different arrays. leader or promoter and / or in the host cell or a different host strain for targeting different target sequences.

[0342] 18. A collection of CRISPR arrays, each array being described in any of the preceding aspects or paragraphs. Thus, where the first array contains a first promoter for crRNA transcription and the second array comprises a second promoter for crRNA transcription that is different from the first promoter, wherein each The promoter may be the same as or a homologue of the host promoter, and optionally wherein the first or both promoters are promoters of a host Cas (e.g., Cas1, 2, 9 or Csn 2) promoter or promoters identical to the host CRISPR array promoter, e.g. The first promoter may be an endogenous Cas nuclease promoter or an endogenous Cas1 or or Cas2 promoter or highly or constitutively expressed or essential in the host cell The promoter of an endogenous gene, which may be a virulence or resistance gene. The use of promoters exerts pressure to conserve host promoters during host evolution, This allows the host CRISPR / Cas defense system to target one or more promoters in the array. This reduces the likelihood of targeting.

[0343] 19. A collection of CRISPR arrays of the invention, wherein a first array comprises one or more CRISPR arrays. Pacer (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more) the first array contains more than one spacer (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more spacers), where the second The spacers of the array are identical to one or more spacers of the first array. Many such spaces are distributed across the array (or even across multiple arrays). Even if the host cell deletes some of the spacers, some HM-alleles remain. Homology-directed assembly of HM-array spacers increases the chances that the spacers remain in the host cell. Protection against deletion is also useful for circumventing host resistance by integrating different arrays. This is also enhanced by the use of different repeats flanked by identical copies of the spacer in

[0344] Therefore, the present invention provides the following: 20. The spacer (or the spacer) of the first array is adjacent to the first repeat that is identical, and The spacers (or the spacers) of the two arrays are adjacent to a second repeat that is identical, where the first A collection of paragraphs 18 or 19 in which the iteration differs from the second iteration.

[0345] 21. The method of claim 1, wherein the first repeat is identical to a repeat in the host cell CRISPR / Cas system. Drop 20 collectibles.

[0346] 22. Paragraph 20, in which the first repeat is different from the repeat in the host CRISPR / Cas system. Collectibles.

[0347] 23. The first and second arrays are derived from the same host cell or the same vector (e.g., a plasmid, Virus, virion, phage, phagemid or prophage) contained in paragraph 1 Any collection between 8 and 22.

[0348] 24. A method of preparing a nucleic acid sequence comprising: a first array contained in a first vector; and a second array contained in a second vector that does not contain the first array. A vector may be a plasmid or a virion (e.g., a vector contained in a vector containing a virus) phage (e.g., of the same phage type), Any of the collectibles from drop 18 to 22.

[0349] In one embodiment, the vector used in the method of the present invention is It is a vector contained in any of the arrays.

[0350] 25. The array, virus, virion, phage, phagemid, or protease inhibitor of any of the preceding paragraphs. A host cell containing a phage, population, or collection.

[0351] Any of the general characteristics (see below) may also apply to this embodiment.

[0352] One example of the present invention is to reduce the risk of host adaptation and resistance to the array by: To provide. CRISPR arrays of the present invention for modifying a target nucleotide sequence in a host cell or A vector comprising: a. wherein the host cell is a first endogenous promoter for transcription of the target sequence (first host promoter including tar); b. Here, the CRISPR array contains the crRNA-encoding sequence and the sequence for crRNA transcription. The crRNA optionally contains a single guide RNA (gRNA). Cas is a rare gene that is used to guide target sequences in host cells to modify the target sequence. capable of hybridizing with the primary target sequence; c. wherein the sequence of the first promoter is a second endogenous promoter that differs from the sequence of the first host promoter. The sequence of the host promoter.

[0353] For example, the promoter may be a promoter of an essential gene in the host. For example, phage) is used for CRISPR units - in this way, the host RNA must be expressed sufficiently (and the promoter must be switched on constantly or frequently) (Constitutively, if it is derived from a host gene that must be expressed.) The host Not readily adapted and therefore resistance does not readily develop. Opportunity for host adaptation (resistance) if desired. To reduce this, different essential promoters are used for different vector CRISPR units. It is possible to use a target in the host by the array (or a different array). Promoters of virulence, essential or resistance genes can be used. Resistance to phages To express the gene, the host must have an endogenous gene promoter and gene targeting site. (This may involve, for example, cell proliferation, viability, or the release of anti-host agents (e.g., antibiotics). There is also the risk of inactivating genes in this way (which may be within coding sequences essential for resistance to the substance). be.

[0354] Providing multiple copies of a nucleic acid sequence encoding crRNA according to the present invention, wherein The peptide contains the same spacer sequence for targeting a host cell sequence according to the present invention. The first step is to allow host removal of the useful targeting spacer from the vector (e.g., host cell homology). Multiple targeting spacers are advantageous for reducing the chance of targeted recombination. In order to provide an alternative method to avoid resistance, the present invention can be flexibly applied to the same or multiple HM-arrays. can be provided to.

[0355] Therefore, the present invention provides the following concept. 1. Host-modified (HM) CRISPR / C to modify target nucleotide sequences in host cells AS system (e.g., Type I, II, or III), comprising: (i) to (iv) (i) at least one nucleic acid sequence encoding a Cas nuclease (e.g., Cas9); (ii) Contains a spacer sequence (HM-spacer) and repeats encoding HM-crRNA A modified host-modified (HM) CRISPR array (e.g., an array described above), comprising: HM-c rRNA encodes the host target sequence to guide Cas into the host cell to modify the target sequence. those containing sequences hybridizable with the string; (iii) any tracrRNA sequence or DNA for expressing a tracrRNA sequence array; (iv) wherein the elements of the system are 2, 3 or more (e.g., 2, 3, 4, 5, 6, 7, 8 , 9, 10, 20, 30, 40, 50 or more copies of the nucleic acid sequence encoding the crRNA wherein the copies contain the same spacer sequence for targeting a host cell sequence. (e.g., host pathogenicity, resistance, or essential gene sequences or host vectors mediating adaptation) CRISPR / Cas system element sequence) A system containing elements of.

[0356] For example, the system may encode four or more crRNAs containing the same spacer. This is advantageous for increasing the expression of the desired cRNA in the host. Furthermore, this avoids host resistance since more than one sequence is required for targeting. Offers great opportunities (especially 5, 10, 15, 20, 30, 40, 50 or 100+) (If there are many copies of the same array, e.g., The DNA strands are separated and the vectors containing them are then used to generate the desired cRNA coding sequence. Useful for reducing the chance of recombination between spacers or flanking repeats that can lead to deletions The chances for the host to delete all copies are distributed across many vector arrays. This is reduced by providing multiple copies of the desired spacer. Reduced (e.g., the number of copies in the first vector array and the number of copies in the second vector array) Many copies in - at least 2, 3, 4, 5, 6, 10 or more such Each of the desired spacers may be 2, 3, 4, 5, 6, 7, 8, 9, (including 10, 20, 30, 40, 50 or 100 or more copies).

[0357] 2. The components of the system are 4, 5, or 6 of the nucleic acid sequence encoding the crRNA containing the same spacer. ,Concept 1 systems containing 10, 15 or 20 or more copies.

[0358] 3. Concept 1 or 2, where copies are split into two or more nucleic acid vector CRISPR arrays. is a system of 2.

[0359] 4. The system comprises first and second HM-arrays, wherein the first and second vectors CR ISPR arrays are generated in the same host cells or using the same vector (e.g., plasmid, virus, Concept 3 cis- contained in a phage, phagemid, or prophage Tem.

[0360] 5. The first array is contained in a first vector and the second array is contained in a second vector that does not contain the first array. - (e.g., where a vector is a plasmid or a virion (e.g., a virus of the same type) of the same phage type) or phagemid (e.g., of the same phage type), 3 or 4 system.

[0361] 6. Any of the preceding concepts, wherein the repeats are identical to repeats in the host CRISPR array. system.

[0362] 7. What about Concepts 1-5, where the repeats are not identical to the repeats in the host CRISPR array? Either system.

[0363] 8. A system, vector, virus, virion, phage, or phage vector of any of the preceding concepts. A host cell containing the phagemid or prophage.

[0364] 9. Any system, vector, virus, virion, phage, or Antimicrobial compositions (e.g., antibiotics, e.g., medicines, disinfectants or mouthwashes).

[0365] Any of the general characteristics (see below) also apply to this concept.

[0366] Split CRISPR / CAS9 system This configuration allows for the use of targeting vectors that have limited capacity for carrying foreign sequences, e.g. This is advantageous for freeing up space in the virus or phage. Many targeting spacers or arrays can be included that are useful for circumventing primary resistance. This is because, for example, endogenous Cas endonucleases are used rather than being encoded in a vector. This is advantageous for the use of caspases – especially bulky Cas sequences such as sp or saCas9. Furthermore, adaptations, such as those that may be found in some exogenous Cas from non-host sources, The ability to reduce virus, e.g., phage genome size, is also It also favors host cell uptake (viral infection and / or maintenance in the host cell). In some instances, invasion of a host by a vector (e.g., a phage) results in the host C This may involve increased expression of CRISPR / Cas nucleases, which may upregulate host CRISPR / Cas activity. This is an advantage - in the host's attempt to fight off the invading nucleic acid. Also, for use in the arrays, vectors, systems and other aspects of this invention, When they contain one or more repeats recognized by the host Cas, they provide an endogenous Cas. The present invention is advantageous for providing one or more space targeting CRISPR arrays to a host. In the case where the host CRISPR array comprises a CRISPR cell (as in the first aspect of the invention), this may be in addition to the host CRISPR array itself. promotes the inactivation of CRI, akin to a "suicide" host cell, which then induces its own CRI It uses its own Cas nuclease for inactivation of the SPR system.

[0367] The invention therefore provides the following properties, numbered by way of example: 1. Host-modified (HM) CRISPR / C to modify target nucleotide sequences in host cells AS9 system (e.g., Type I, II, or III), comprising: (i) to (iv) (i) at least one nucleic acid sequence encoding a Cas nuclease (e.g., Cas9); (ii) Contains a spacer sequence (HM-spacer) and repeats encoding HM-crRNA an engineered host-modified (HM) CRISPR array (e.g., an array of the invention described above), The HM-crRNA guides the Cas to the target in the host cell and modifies the target sequence. those containing sequences that are hybridizable with host target sequences; (iii) any tracrRNA sequence or DNA for expressing a TracrRNA sequence array; (iv) wherein said elements of the system comprise a host cell and at least one gene capable of transforming the host cell. The nucleic acid vector is split between the HM-crRNA and the Cas gene, allowing it to target and guide the host. modifying target sequences in host cells; A system containing elements of.

[0368] "Split" here means that the vector splits one or more (but not all) of the system's elements. the host cell contains one or more (but not all) of the elements, and the vector is In some embodiments, a vector and a The vector and the host cell do not share any of the elements, e.g., the host cell contains element (i) and the vector contains element (ii), the vector contains element (iii), and / or the host cell contains element (iii). When the vector is within a host cell (e.g., integrated or episomal), vectors (e.g., prophage), which have transformed host cells It is intended that the nucleic acid be provided by a vector (and that the nucleic acid provided by such a vector is (Elements of the system that provide the trait are not to be construed as host cell elements in this case). The nucleic acid (e.g., chromosomal and episomal nucleic acid) of the transformed host is sequenced and compared to the same type of When the host is a microorganism, e.g., a bacterium or an archaea, e.g., This can be easily determined by comparing sequences from the same host (e.g., parent colony or clone). Cut.

[0369] Optionally, the system is a CRISPR / Cas9 system. The nuclease is a Type I Cas nuclease. Optionally, the nuclease of (a) is a type II Cas nuclease (e.g., Cas9). Optionally, the nuclease of (a) The ase is a type III Cas nuclease.

[0370] 2. The system of Example 1, wherein at least one of the elements is endogenous to the host cell.

[0371] 3. The system of Example 1 or 2, wherein element (i) is endogenous to the host cell.

[0372] 4. The system of any of Examples 1-3, wherein element (iii) is endogenous to the host cell.

[0373] 5. Host-modified (HM) CRISPR / Transcriptional Modifications for Modifying Target Nucleotide Sequences in Host Cells A Cas system (e.g., type I, II, or III), comprising: (a) to (e) a. at least one nucleic acid sequence encoding a Cas nuclease (e.g., Cas9); b. Contains a spacer sequence (HM-spacer) and repeats encoding HM-crRNA A modified host-modified (HM) CRISPR array, wherein the HM-crRNA is a sequence that can hybridize with a host target sequence to guide the Includes; c. Any tracrRNA sequence or DNA sequence for expressing a TracrRNA sequence column; d. wherein the elements of the system are separated into at least a first and a second nucleic acid vector; the first vector comprises element (a), while the second vector lacks element (a); and e. wherein the vectors can be co-transformed into a host cell simultaneously or sequentially, thereby producing HM -crRNA guides Cas to target and modify the target sequence in the host cell A system that includes elements that comply with

[0374] The above definition of "split" is, mutatis mutandis, the first and second vectors This applies to the present example.

[0375] In some embodiments, the tracrRNA sequence is not provided by a vector, but is endogenously the tracrRNA sequence of the CRISPR / Cas system in the host cell, where tra The crRNA encodes the mature crRNA to guide the Cas to its target in the host cell. It is then processed and can hybridize with HM-crRNA in the cell.

[0376] 6. The method of Example 5, wherein the first vector contains element (a) and the second vector contains elements (b) and (c). system.

[0377] 7. The first and / or second vectors each contain one, two, three or more further modified HM- The system of Example 5 or 6, comprising a CRISPR-array.

[0378] 8. One of the first and second vectors is a phagemid and the other vector is a helper phagemid. 8. The system of any of Examples 5 to 7, wherein the system is a phage.

[0379] 9. The crRNA and tracrRNA sequences are provided, for example, by a vector , any of the systems of the preceding examples (e.g., Examples 3 and 4) contained in a single guide RNA (gRNA). or 6).

[0380] 10. A system according to any of the preceding examples, wherein each vector has a limited capacity for inserting exogenous nucleic acid. Stem.

[0381] 11. One or more vectors are viruses (e.g., virions, packaged phages) Any of the preceding examples of a system, wherein the system is a vector (e.g., a vector, a phage, a phagemid, or a prophage).

[0382] 12. The host cell produces a deoxyribonucleic acid strand (H) with free ends that encode the HM sequence of interest. M-DNA) and / or wherein the system includes sequences encoding HM-DNA (e.g., integrated into the vector or host cell genome or its episome), wherein The HM-DNA is homologous to a sequence or sequences in or adjacent to the target sequence, respectively. The system of any of the preceding examples, including a sequence or sequences.

[0383] The strands are free ends, i.e., the strands are isolated from the host or vector so that they have one or two free ends. The DNA has unintegrated ends, i.e., the DNA is or 3' adjacent nucleotide.

[0384] 13. The target site is cleaved by Cas in the host cell (e.g., Cas nuclease is Cas9), HM-DNA host genome (e.g., chromosome or To insert the HM-DNA into the episomal site, 13. The system of Example 12, comprising first and second sequences that are homologous to 3' and 3'.

[0385] 14. The method of Example 13, wherein the insertion is by homologous directed recombination (HDR).

[0386] 15. The system of Example 13, wherein insertion is by non-homologous end joining (NHEJ).

[0387] 16. The HM sequence is placed in a regulatory element (e.g., a promoter, e.g., an endogenous promoter). (alternative inducible promoter), transcription inhibitory sequence, transcription enhancer sequence, tag or exogenous protein The sequences of Examples 12 to 15 which are or encode a protein or domain any system.

[0388] 17. A system comprising first and second HM-DNAs, wherein the sequence of the first HM-DNA is complementary to a sequence of the second DNA, thereby allowing the DNA to be integrated into the host cell genome (e.g., chromosomes) to form combinatorial HM-DNA for insertion into the target site (or into an episomal site). The systems of any of Examples 12-16, which can be combined in a host cell by co-directed recombination.

[0389] 18. One or more vectors deliver vector nucleic acid containing system elements into cells. Any of the preceding examples of a system capable of infecting a host cell to produce the virus.

[0390] 19. The system of any of the preceding examples, wherein the Cas nuclease is a nickase.

[0391] 20. The cell is a bacterium or archaea, and the Cas nuclease is endogenous to the bacterium or archaea. Any of the preceding examples of a system provided by a Type II CRISPR / Cas system. .

[0392] 21. One or more vectors are in a host cell and, if desired, integrated into the host DNA. 2. The system of any of the preceding examples,

[0393] 22. One or more vectors encode a Cas nuclease (e.g., aCas9). The system of any of the preceding examples, wherein the system lacks the sequence.

[0394] 23. Modified Microorganisms Containing Endogenous CRISPR / Cas Systems for Host Cell Infection a nucleic acid viral vector (e.g., a vector, virion, or packaged phage) And, (a) a plurality of CRISPR / Cas genes for use in a CRISPR / Cas system according to any of the preceding examples; comprising nucleic acid sequences for expressing different crRNAs; and (b) lacking a nucleic acid sequence encoding a Cas nuclease (e.g., Cas9); wherein a first of said crRNAs is capable of hybridizing to a first nucleic acid sequence of said host cell; and A second of the crRNA is capable of hybridizing to a second nucleic acid sequence of the host cell, wherein the second sequence the sequence is different from the first sequence; and (c) the first sequence is contained in an antimicrobial (e.g., antibiotic) resistance gene (or its RNA), and The two sequences are contained in antimicrobial resistance genes (or their RNA), and optionally, these genes is different; (d) the first sequence is contained in an antimicrobial resistance gene (or its RNA), and the second sequence is essential or containing a virulence gene (or its RNA); (e) The first sequence contains an essential gene (or its RNA), and the second sequence contains an essential or pathogenic gene. or containing the offspring (or its RNA) (f) the first sequence contains a virulence gene (or its RNA), and the second sequence contains an essential or virulence gene; containing the gene (or its RNA), vector.

[0395] 24. Modified vectors for transforming host cells containing endogenous CRISPR / Cas systems (Direct modification when the vector is derived from a modified vector that transforms the host) a nucleic acid vector (derived from the vector or isolated from the vector in a host cell), the vector described above, and (a') A plurality of vectors for use in a CRISPR / Cas system according to any of the preceding examples. comprising nucleic acid sequences for expressing different crRNAs; and (b') lacking a nucleic acid sequence encoding a Cas nuclease (e.g., Cas9); wherein a first of said crRNAs is capable of hybridizing to a first nucleic acid sequence of said host cell; and A second of the crRNA is capable of hybridizing to a second nucleic acid sequence of the host cell, wherein the second sequence the sequence is different from the first sequence; and the first and / or second sequences are target sequences of the host CRISPR / Cas system, The array of (c') Repetitive DNA or RNA sequences (e.g., as used herein) in the host CRISPR array. repeat is the 5'-most repeat (first repeat); (d') tracrRNA sequence or tracrRNA-encoding DNA sequence; (e') CRISPR array leader sequence; (f') Cas gene promoter (e.g., Cas1, Cas2, or Csn2 promoter) Tar); (g') CRISPR array leader promoter sequence or (h') Cas-encoding DNA or RNA sequence (e.g., where Cas is Cas9, Ca s1, Cas2, or Csn2), e.g., where the first crRNA is a host Cas One crRNA can target a gene sequence (or its RNA sequence), and the second crRNA can target a host It can target the Cas2 gene sequence (or its RNA sequence). is or contains vector.

[0396] 25. The first and / or second target sequences i. The CRISPR array leader or leader adjacent to the 5'-most nucleotide of the first repeat a promoter sequence (and optionally the 5'-most nucleotides of the repeat), e.g. , including the first three nucleotides at the 5′ end of the first repeat; ii. Up to 20 (e.g., 3, 5, 7, 9, 10, 12, 15, 20) nucleotides immediately 5' of the first repeat , 30 or 32) a sequence of consecutive nucleotides; iii. Up to 20 (e.g., 3, 5, 7, 9, 10, 12) of the 5'-most nucleotides of the first repeat , 15, 20, 30 or 32) a sequence of consecutive nucleotides or iv. Up to 20 (e.g., 3, 5, 7, 9, 10, 12, 15) amino acids immediately 3' of the first spacer 20, 30 or 32) consecutive nucleotide sequences (and optionally wherein the sequence is a first sequence) the 3'-most nucleotide of the first repeat, e.g., the 3'-most nucleotide of the first repeat, containing 3 nucleotides is or contains Example 24 vector.

[0397] 26. Each target sequence is included in or comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 44. 26. The vector of Example 24 or 25, which is the complement of

[0398] 27. The first crRNA comprises or consists of the structure RSR, where R=CRIS PR repeat and S = CRISPR spacer, where S is (in the 5' to 3' direction) V -H R or H R - V or where V = a sequence identical to the DNA sequence of the vector and BiH R = DNA sequence of repeats of the CRISPR array in the host cell CRISPR / Cas system where the first crRNA is for modification of the host CRISPR array in the cell. The spacer and hybridization of the host CRISPR array are used to guide Cas to the crRNA. Any of the vectors of Examples 24 to 26 that can be resized.

[0399] 28. The first crRNA does not substantially hybridize with nucleic acids present in the vector, e.g. For example, where the first crRNA does not hybridize with V in the vector or does not hybridize with V in the host allele. The vector of Example 27 hybridizes with lower intensity than the spacer of Example 2 The above discussion about determining this also applies to this example.

[0400] 29. V = 1 or up to 40 (e.g., up to 15) contiguous nucleotides of vector DNA. 27 or 28, e.g., V=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 consecutive nucleotides.

[0401] 30. i. The host CRISPR / Cas system can recognize the cognate PAM; j. where the vector DNA contains such a PAM immediately 3' of the protospacer sequence ; k, where V = 1 or up to 40 (e.g., up to 15) nucleotides of the protospacer Yes; and l. Here H R = a sequence identical to the contiguous sequence of repeats in the host CRISPR array, The vector in Example 29.

[0402] 31. The contiguous sequence of the repeat in the host array is at least 50% of the sequence of the host repeat (e.g. , including the 5'-most or 3'-most nucleotide of the host repeat), the vector of Example 30.

[0403] 32. V = 1 to 40 (e.g., up to 15) of the most 3' protospacer contiguous nucleotides and optionally the contiguous sequence of the repeat includes the 5'-most nucleotide of the host repeat. A vector of 0 or 31.

[0404] 33. V = 1 to 40 (e.g., up to 15) of the most 5' protospacer contiguous nucleotides Optionally, the contiguous sequence of the repeat includes the 3'-most nucleotide of the host repeat. is 31 vectors.

[0405] 34. R = repeat recognized by the host CRISPR / Cas system, Example 27-3 3 vectors. Alternatively, R = a vector recognized by the host CRISPR / Cas system. In this case, the vector is preferably homologous to R, i.e., the host A Cas nuclease (and optionally tracrR) that can function together with R in a cell. NA).

[0406] 35. The first sequence is in accordance with any of (c') to (h'), and the second sequence is a host-essential gene or a pathogenic gene. The vector of any of Examples 24 to 34, wherein the vector is selected from a gene or a resistance gene.

[0407] 36. To infect microbial host cells containing endogenous CRISPR / Cas systems, e.g. A modified nucleic acid viral vector for use in any of the systems set forth in 1 to 22 ( e.g., a virion or a packaged phage), wherein: a. the vector comprises a first nucleic acid sequence for expressing the first crRNA in a host; and hand b. wherein the first sequence comprises (in the 5' to 3' direction) R1a-S1-R1b, wherein R1a = first CRISPR repeat, where R1a is optional; R1b = second CRISPR repeats, and S1 = CRISPR spacer complementary to the host sequence (e.g., Example 23 or 24), where R1a and R1b are host Cas nucleases ( For example, recognized by a type II nuclease, such as Cas9; c. wherein the vector (i) encodes a Cas nuclease that recognizes the repeats of (b) (e.g., Cas 9) and / or (ii) a nucleic acid sequence encoding the crRNA sequence encoded by the first sequence The nucleic acid sequence encoding the tracrRNA sequence complementary to the sequence is absent.

[0408] For example, the vector is a nucleic acid vector contained in a phage.

[0409] 37. d. the vector comprises a second nucleic acid sequence for expressing the second crRNA in the host, The second crRNA is different from the first crRNA; e. wherein the second sequence comprises (in the 5' to 3' direction) R2a-S2-R2b, wherein R2a = first CRISPR repeat, where R2a is optional; R2b = second CRISPR repeats and S2 = CRISPR spacer complementary to the host sequence (e.g., Example 23 or 2 4), where R2a and R2b are host Cas nucleases (e.g., Recognized by type I or II nucleases (e.g., Cas6), The vector in Example 36.

[0410] Thus, for example, the first and second nucleic acid sequences may be contained on the same packaged phagemid. For example, on the same or different CRISPR arrays.

[0411] 38. The vector encodes a Cas (e.g., Cas6) that recognizes the repeats in (iii)(e). (iv) a nucleic acid sequence that is complementary to the crRNA sequence encoded by the second sequence; The vector of Example 37, which lacks a nucleic acid sequence encoding a tracrRNA sequence.

[0412] 39. For coinfecting microbial host cells containing endogenous CRISPR / Cas systems. A modified nucleic acid viral vector for use according to any of the systems of Examples 1 to 22 ( A collection of vectors, virions or packaged phage (e.g., as described above), the article comprises a first vector and a second vector; f. wherein the first vector is according to Example 36; g. wherein the second vector comprises a second nucleic acid sequence for expressing a second crRNA in a host. wherein the second crRNA is different from the first crRNA; h. wherein the second sequence comprises (in the 5' to 3' direction) R2a-S2-R2b, wherein R2a = first CRISPR repeat, where R2a is optional; R2b = second CRISPR repeat and S2 = CRISPR spacer complementary to the host sequence, where R2a and R2 b is a host Cas nuclease (e.g., a type I or II nuclease, e.g., Cas 6) is recognized.

[0413] For example, the first vector may be contained in a first packaged phagemid and the second vector may be contained in a second packaged phagemid. Included in the packaged phagemid.

[0414] 40. The second vector encodes a Cas (e.g., Cas9) that recognizes the repeats of (v)(b). and / or (vi) a nucleic acid sequence complementary to the crRNA sequence encoded by the first sequence. The collection of Example 39, comprising nucleic acid sequences encoding tracrRNA sequences.

[0415] For example, in this case, the Cas function is provided by the endogenous host system. - To secure space (e.g., insertion of more host-targeting HM-array spacers) ), simplifying vector and array construction.

[0416] 41. The second vector encodes a Cas (e.g., Cas6) that recognizes the repeat of (vii)(h). and (viii) a second sequence complementary to the crRNA sequence encoded by the nucleic acid sequence encoding the crRNA. The collection of Examples 39 or 40, which lacks a nucleic acid sequence encoding a tracrRNA sequence.

[0417] For example, in this case, Cas function is provided by the endogenous host system.

[0418] 42. The first and second vectors each contain a Cas (e.g., Cas) that recognizes the repeats of (ix)(b). 9) and (x) a Cas (e.g., Cas6) that recognizes the repeats of (h). and optionally wherein the collection recognizes repeats of (b) and (h). The collection of Example 39 contained in a host cell comprising one or more Cas.

[0419] 43. A third vector (e.g., a vector) further comprising (ix) and / or a nucleic acid sequence according to (x). on or phage).

[0420] 44. Each vector is packaged in a virion or phagemid or each virion or The collection of any of Examples 39-43, contained in a phage nucleic acid.

[0421] 45. The vector of any one of Examples 36 to 44, wherein R1a and R1b are contained in the same repeat sequence. Or collectibles.

[0422] 46. ​​The vector of any one of Examples 37 to 45, wherein R2a and R2b are contained in the same repeat sequence. Or collectibles.

[0423] 47. The repeat in (b) is different from the Cas nuclease that recognizes the repeat in (e). The vector or collection of any of Examples 37 to 46, which is recognized by an enzyme.

[0424] 48. If the host is harboring various types of CRISPR / Cas systems (e.g., Type I and Type II), Type II Systems; Type I and Type III Systems; Type II and Type III Systems or type I, II and III systems), or Collectibles.

[0425] 49. (b) Repeats are recognized by type II Cas nucleases, e.g., Cas9. , the vector or collection of any of Examples 36 to 48.

[0426] 50. (e) Repeats are synthesized by type I or III Cas nucleases, e.g., Cas6. As recognized, the vector or collection of any of Examples 37 to 49.

[0427] 51. Whether the vector is a virus, virion, phage, phagemid or prophage The vector or collection of any of Examples 23 to 50.

[0428] 52. Contains one or more Cas that are operable with the cRNA encoded by the vector The vector or collection of any of Examples 23-51 in a host cell.

[0429] 53. The vector or collection of any of Examples 23 to 52 in a host cell containing Cas9.

[0430] 54. In combination with HM-DNA (e.g., in a vector, plasmid, or host cell) integrated into the cellular genome or its episomes), where HM-DNA is 7. The vector or collection of any of Examples 23 to 53, as described in any of Examples 7.

[0431] 55. A nucleic acid sequence for expressing multiple different crRNAs, wherein the crRNAs are At least 3, 4, 5, 6, 7, 8, 9, 10, 15 in a host cell Can target 20, 30, 40, 50 or 100 DNA sequences The system, vector or collection of any of the preceding examples.

[0432] 56. Although not the Cas nuclease (e.g., Cas9), it mediates host-vector adaptation. The DNA sequence of the Cas nuclease (or its RNA sequence) can be targeted. a first crRNA or nucleic acid sequence encoding a first cRNA, and optionally in a host The first gene can target sequences (or their RNA) of resistance, virulence, or essential host genes. The system of any of the preceding examples includes a second crRNA or a nucleic acid sequence encoding a second cRNA. A program, vector, or collection.

[0433] 57. A nucleic acid sequence encoding two, three or more copies of crRNA, The copies contain the same spacer sequences for targeting host cell sequences (e.g., Cas nuclei mediate virulence, resistance or essential gene sequences or vector adaptation. a sequence of a host CRISPR / Cas system element that is not a CRISPR / Cas system element, any of the above examples of the system , vector or collection.

[0434] 58. The system of Example 57, wherein the copies are split between two or more vector CRISPR arrays. ctor or collectible.

[0435] 59. Vector repeats are identical to or in the host CRISPR array (e.g., For example, each vector repeat has at least 95% sequence identity to the host repeat), any of the above examples. any system, vector or collection.

[0436] 60. The vector repeats are not identical to or identical to the repeats in the host CRISPR array; The system, vector or collection of any of Examples 1 to 58.

[0437] 61. The same host cell or the same vector (e.g., a plasmid or virus or the first and second genes contained in the phage or prophage or phagemid) Any of the preceding examples of a system, vector, or collection, including a vector CRISPR array. .

[0438] 62. A method in which the first array is contained in a first vector and the second array is contained in a second vector that does not contain the first array. A vector may be contained in a vector (e.g., a plasmid or a virion (e.g., the same virus) of Example 61, which is a vector of the same phage type) or a phagemid (e.g., of the same phage type). A system, vector, or collection.

[0439] 63. A system, vector, collection, virus, virion, or phage according to any of the preceding examples. a host cell containing the vector, phagemid, or prophage.

[0440] 64. A system, vector, virus, virion, or phage according to any of Examples 1 to 62. Antimicrobial compositions (e.g., antibiotics, e.g., medicines, disinfectants or mouthwashes).

[0441] Batch conditioning of multiple microorganisms The present invention provides a method for co-conditioning and integrating hosts and viruses to promote co-evolution. and therefore conditioning of the host to viruses (e.g., phages) and vice versa. The present invention provides a method for producing a microorganism (e.g., a phage and / or bacterial population) comprising: Using repressible control of expression or activity of crRNA, the present invention provides a method for the production of desired spacer activity. in the presence or absence of a load imposed by spacer-guided Cas action in the host. For example, conditions that occur with or without antibiotic resistance gene targeting. intentionally shared, which can be switched on or off to allow for Evolution is controlled in a controllable manner. In this way, bacterial populations are regulated by phage-independent Use in situations where activation may be encountered (e.g., dairy or food manufacturing fermentations) or bacteria Kill or modulate the expression of phagocytic leukemia virus, e.g., for antibiotic resistance knockdown. This form can be further modified in some embodiments for use in the preparation of Therefore, this method intentionally suppresses the activity of the array antibiotic resistance gene inactivation during culture with the host. To control antibiotic resistance, the method comprises combining an antibiotic-resistant bacterial host with one or more vectors that target the host's antibiotic resistance genes. This allows for the cultivation of viruses, such as phages, containing the CRISPR array of the present invention. Therefore, resistant bacterial host populations can be used to propagate phages in culture (e.g., industrial culture media). In a culture container or plant, the phage and host co-evolve and multiply, thus Antibiotic resistance defects that interfere with the culturing ability of the bacteria (which would otherwise minimize phage expansion) They allow the proteins to be coordinated with each other without activating effects, while all other components of the desired phage are This still allows the enzyme to be adjusted to the culture host population. Sample testing, for example, on a laboratory scale using antibiotic-resistant host cell populations, but , an array of derepressed test filaments targeting antibiotic resistance genes in the host cell. This can be done using a phage. The presence and inhibition of synthesis, for example, of the tet system or a light-inducible system, are well known to those skilled in the art. It is well known.

[0442] Therefore, the present invention provides the following features, numbered as paragraphs: 1. A method for producing a microorganism, comprising: (a) Host CRISPR / Ca for nucleotide sequence targeting in host cells providing a host cell containing the s system; (b) providing a virus capable of infecting a host cell, wherein (i) the virus is a modified host cell that contains one or more modified host cells for targeted nucleotide sequence modification in the host cell; including modified (HM) CRISPR arrays (e.g., arrays described above); (ii) First, the HM-array guides Cas to a target in a host cell and modifies the target sequence. a spacer sequence (HM-spacer) capable of hybridizing to the first host target sequence to enhance a first HM-crRNA comprising a first target sequence, optionally containing a modification of the first target sequence; Reduces chief cell proliferation or viability; and (iii) the first HM-array is capable of reversibly suppressing the transcription of the first HM-crRNA; and / or the first HM-crRNA activity is repressible; (c) infecting a host cell with a virus to introduce one or more HM-CRISPR arrays into the cell; Dye; (d) Transcription of the first HM-crRNA and / or the first HM-crRNA activity in the cells suppress; (e) Infect host cells to produce a population of host cells (PH1) containing a population of viruses (PV1). Culturing the cells; and (f) Obtaining the viral population PV1 and / or cultured host cell population A method comprising:

[0443] For example, the first HM-crRNA guides Cas to a target in the host cell and an HM-spacer capable of hybridizing to a first host target sequence to modify the sequence; Here, the target sequence is the nucleotide sequence of the host CRISPR / Cas system, The first HM-crRNA modifies the host CRISPR / Cas system in the host cell The target sequence is then modified by the host CRISPR / Cas. Degrade or eliminate system functionality.

[0444] Alternatively, the modification enhances or prevents repression of the gene in the host. In embodiments, the gene is an essential gene, a virulence gene, or a resistance gene (e.g., an antibiotic In some embodiments, the modification is endogenous or exogenous to the host. For example, the host may be able to express an exogenous nucleotide sequence (e.g., a desired and a modified host cell comprising a desired Enhance or inhibit the expression of a protein. For example, the desired protein is an antibiotic. The host cell is a microorganism, e.g., a bacterial or archaeal cell. Thus, the method comprises: The antibiotic can be cultured to produce a viral population in which the antibiotic is expressed. This would otherwise be detrimental to the growth of the host cell population. One or more viruses in the group can be removed after isolating the first HM-crRNA suppressor, thereby enabling To provide a dynamic antibiotic viral composition, for the reduction of host cell growth or viability The present invention can therefore also be used in antimicrobial compositions. Also provided are such methods and such antibiotic compositions, including viruses capable of expressing the gene. Modifications to activate expression can be, for example, conjugated to transcriptional activators. This can be achieved by providing a Cas (e.g., Cas9) that encodes the first HM-crR. The activator is the cognate Cas for the NA, and the activator is the desired exogenous or endogenous gene. Modifications to inhibit expression include, for example, death Cas (e.g., dCas) 9), where the CAs are cognate Cass to the first HM-crRNA. and inhibit the transcription of desired exogenous or endogenous genes.

[0445] Repression of crRNA transcription or activity can be partial or complete (i.e., the presence of an array in the host). The activity may be due to the lack of crRNA activity or transcription from the Cas gene. crRN, which hybridizes with the cognate host sequence to guide it to the first host target site for This refers to A's ability.

[0446] For example, the virus is not so inhibited when introduced into the cell, and the method After infecting cells, they can be subjected to various methods, such as chemical, physical, mechanical, magnetic, or light, to induce inhibition. or other agent to perform step (d). The first HM-array contains a repressible promoter for transcription of the first HMcrRNA (HM-promoter). The promoter is repressed (e.g., For example, by binding a repressor agent, e.g., chemical or protein, to the promoter .

[0447] In other instances, the virus may be inhibited by, for example, chemical, physical, or mechanical means to cause inhibition. and (c) by the use of electrical, magnetic, optical or other inhibitors. In one embodiment, the first HM-array is capable of suppressing transcription of the first HMcrRNA. the first HM-array comprises a functional promoter (HM-promoter), before being introduced into the by binding to the promoter), where the first HM-array, which is then repressed, is introduced into the cell. will be done.

[0448] In certain embodiments, step (f) comprises isolating PV1. The process involved isolating PV1 or its virus from PH1 host cells.

[0449] 2. Further, after step (e) or (f), transcription and and / or derepression of the first HM-crRNA activity, and then optionally further host cells 10. The method of paragraph 1, comprising culturing the cells.

[0450] 3. A. Optionally, a host cell that is the same as the host cells in (a), (f) or the further cultured cells in paragraph 2. Obtaining a population of principal cells (PH2); B. Infecting host cells from A with viruses from population PV1; C. Transcription of primary HM-crRNA and / or primary HM-crRNA activity in cells suppress; D. Infected host cells are cultured to produce a population of host cells (PH3) containing a population of viruses (PV2). and E. Obtaining PV2 (or its viruses) and / or culturing host cell populations Any of the methods in the preceding paragraph, including:

[0451] 4. Further transcription and amplification of the first HM-crRNA in the viral population after step (D) or (E) and / or derepressing the first HM-crRNA activity, and then optionally inducing further host cells 4. The method of paragraph 3, comprising culturing the cells.

[0452] 5. Infecting virus population PV1 or PV2 on a further host cell or population (PH4) of host cells 2. Testing isolated samples of the two cells or populations, optionally wherein additional cell or population pH 4 is identical to the cells in (a), and the test involves adding a further cell or population PH4 to the sample virus. Infect the cells with the desired virion, wait a period of time to allow for some host cell proliferation, and then infect additional cells or populations. Whether a predetermined activity of PH4 (e.g., cell proliferation or viability) is modified (e.g., , inhibition of host cell proliferation or reduction of its viability * ) or occurring, and During this period, the virus is present in the cell or the cell is transcribing the first HM-crRNA and / or the method of any of the preceding paragraphs, wherein the first HM-crRNA activity is derepressed. * This is because the virus sample is added to cells seeded on a surface or to pH4. and can be tested using standard assays for plaque formation.

[0453] 6. All of the host cells are microbial cells (e.g., bacterial or archaeal cells) and the first target sequence Modification of the α-amyloid β-amyloid complex reduces host cell growth or viability, and this determination is crucial for antimicrobial activity. ** The occurrence of (5) Any of the methods set out in the preceding paragraph to determine the ** This can be determined using a standard plaque assay.

[0454] 7. Paragraphs whose duration is at least 1, 5, 10, 30, 60 or 120 minutes. 5 or 6 ways.

[0455] 8. The cells of (a) and optionally PH1, PH2 and / or PH3 cells are cultured as a first target sequence. and wherein the additional cell or population of PH4 cells comprises the first target sequence. Any of the 7 methods.

[0456] 9. The cells of (a) and optionally PH1, PH2 and / or PH3 cells are cultured using a first antibiotic does not contain a gene that confers resistance to a disease, wherein the first target sequence is a target sequence of such a gene. a sequence, optionally wherein a further cell or population of PH4 cells comprises the gene; Any of methods 1 to 8.

[0457] 10. The cells of (a) and optionally PH1, PH2 and / or PH3 cells are cultured using the first antibiotic a gene that confers resistance to a substance, wherein the first target sequence is a target sequence of such a gene. A column, paragraphs 1 to 7, any of the following ways.

[0458] 11. All of the host cells are microbial cells (e.g., bacterial or archaeal cells) and the first target sequence is Modification of the sequence reduces host cell growth or viability or induces host cell resistance to antibiotics. Reduce by any of the methods in the preceding paragraph.

[0459] 12. All of the host cells are human, animal (e.g., non-human animal) or plant infectious disease pathogens. Any of the methods in the preceding paragraph.

[0460] 13. All of the host cells are, for example, Escherichia (e.g., Escherichia coli O157 :H7 or O104:H4), Shigella (e.g., Dysenteriae), Salmonella (e.g., , Typhi or Enterica, e.g., Serovar Typhimurium, e.g., DT104), E. Wisnia, Yersinia (e.g., pestis), Bacillus, Vibrio, Legionella (e.g., , pneumophila), Pseudomonas (e.g., aeruginosa), Neisseria (e.g., Gono re or meningitidis), Bordetella (e.g., pertussis), Helicobacter (e.g., pylori), Listeria (e.g., monocytogenes), Agrobacterium, Staphylococcus aureus, Ilococcus (e.g., aureus, e.g., MRSA), Streptococcus (e.g., pyogenes or thermophilus), Enterococcus, Clostridium (e.g., Di physeal or botulinum), Corynebacterium (e.g., Amycolatum), Mycobacterium T. tuberculosis, Treponema, Borrelia (e.g., Burgdorferi), Bacteria, Francisella, Brucella, Campylobacter (e.g., jejuni), Klebsiella (e.g., Pneumoniae), Frankia, Bartonella, Rickettsia, Shewanella, Serratia A, Enterobacter, Proteus, Providencia, Brochothrix, Bifidobacter Bacterium, Brevibacterium, Propionibacterium, Lactococcus, Lactobacillus Rous, Pediococcus, Leuconostoc, Vibrio (e.g., cholera, e.g., O 139 or vulnificus), Haemophilus (e.g., influenzae), Brucella (e.g., abortus), Francisella, Xanthomonas, Ehrlichia (e.g., chaffensi spp.), Chlamydia (e.g., pneumoniae), Parachlamydia, Enterococcus (e.g., faecalis or faecium (e.g., linezolid-resistant), Oenococcus and Reed Selected from species of Netobacter (e.g., baumannii, e.g., multi-drug resistant), same species Any of the methods in the preceding paragraph.

[0461] 14. The total number of host cells is, for example, methicillin, vancomycin resistance, and teicoplanin. 13. The Staphylococcus aureus cell of claim 13, wherein the Staphylococcus aureus cell is resistant to an antibiotic selected from the group consisting of: How to do it.

[0462] 15. If all of the host cells are infected with, for example, cephalosporins (e.g., ceftazidime), carbapenems, Nems (e.g., imipenem or meropenem), fluoroquinolones, aminoglycosides an antibiotic selected from the group consisting of gentamicin and tobramycin; 14. The method of claim 13, wherein the Pseudomonas aeruginosa cell is resistant to the substance.

[0463] 16. If all of the host cells are infected with, for example, carbapenem-resistant Klebsiella (e.g., P. pneumoniae), The method of claim 13, wherein the cells are erythrocytes.

[0464] 17. If all of the host cells are infected with, for example, erythromycin, clindamycin, beta-lactam selected from amoxicillin, macrolides, amoxicillin, azithromycin and penicillin Antibiotic-resistant Streptococcus (e.g., pneumoniae or pyogenes) cells The method of claim 13.

[0465] 18. All of the host cells contain, for example, ceftriaxone, azithromycin, and ciprofloxacin. Salmonella (e.g., serovar Typhi) cells that are resistant to antibiotics selected from the group consisting of fluoxacin and fluoxacin. 14. The method of claim 13.

[0466] 19. All of the host cells are selected from, for example, ciprofloxacin and azithromycin. 14. The method of claim 13, wherein the Shigella cells are resistant to the antibiotic.

[0467] 20. If all of the host cells are infected with, for example, isoniazid (INH), rifampicin (RMP), fluconazole, or other antiviral drugs, an antibiotic selected from oroquinolones, amikacin, kanamycin, and capreomycin; 14. The method of claim 13, wherein the Mycobacterium tuberculosis cells are resistant to the serovar.

[0468] 21. All of the host cells are, for example, vancomycin-resistant Enterococcus cells. 14. The method of claim 13.

[0469] 22. All of the host cells are infected with an antibiotic selected from, for example, cephalosporins and carbapenems. 14. The method of claim 13, wherein the Enterobacteriaceae cells are resistant to bioagents.

[0470] 23. All host cells are susceptible to, for example, trimethoprim, nitrofurantoin, cephalexin. and amoxicillin, 14. The method of claim 13.

[0471] 24. All of the host cells are selected from, for example, fluoroquinolone antibiotics and carbapenems. and wherein the selected antibiotic is a Clostridium (e.g., difficile) cell. 13 ways.

[0472] 25. All of the host cells are infected with, for example, cefixime (e.g., oral cephalosporins), From Riaxone (an injectable cephalosporin), azithromycin, and tetracycline 14. The method of claim 13, wherein the selected antibiotic-resistant Neisseria gonorrhoeae cells.

[0473] 26. All of the host cells are free from e.g., beta-lactams, meropenem, and carbapenems. 14. The method of claim 13, wherein the Acinetobacter baumannii cells are resistant to a selected antibiotic. Law.

[0474] 27. All of the host cells are selected from, for example, ciprofloxacin and azithromycin. 14. The method of claim 13, wherein the Campylobacter cells are resistant to the antibiotic.

[0475] 28. The method of any of the preceding paragraphs, wherein the host cell produces beta (β)-lactamase.

[0476] 29. The method according to the preceding paragraph, wherein the host cell is resistant to an antibiotic described in any one of paragraphs 14 to 27. Either way.

[0477] 30. A gene encoding a product that confers host cell resistance to the antibiotic, wherein the first target sequence The method in paragraph 29, which is an array of children.

[0478] 31. The first target sequence is the sequence of an antibiotic resistance gene (i.e., the host cell's resistance to the antibiotic). resistance, e.g., to confer methicillin resistance) and / or population PH1, population PH2 One, more or all of population PH3 and population PH4 are antibiotics or antibiotic-resistant. The method of any of the preceding paragraphs, wherein the antibiotic is resistant to the antibiotic (e.g., an antibiotic described in any of paragraphs 13 to 27).

[0479] 32. Derepressed viruses of the viral population PV1 or PV2 have antimicrobial activity. (e.g., antibacterial activity, such as when the virus is a phage), optionally wherein the host cell the cell or cells comprise a first target sequence described in paragraph 30, wherein modification of the first target enhances said anti-microbial The method of any of the preceding paragraphs, which provides biological activity.

[0480] 33. Cells of PH4 are resistant to antibiotics (e.g., antibiotics described in any of paragraphs 13 to 27). and the cells of (a) and PH2 are not resistant to the antibiotic, subject to paragraph 5 any of the methods of the preceding paragraphs, which avoid the risk of culturing and expanding antibiotic-resistant host cells. It can be done relatively safely without the need for drugs, which aids in the production of viruses for drug use (and and the risk of contamination and escape from drug manufacturing plants. However, testing for PH4 is being conducted in a collateral laboratory that is set up to use antibiotic-resistant host strains. or other facilities. When tested against PH4, the first HM-crRNA is: Modification of resistance genes in host cells is inhibited as possible with the HM-array of the present invention. It will be released.

[0481] 34. If the host CRISPR / Cas system is a Type I, II, or III system, the target sequence is from at least one, two, or three additional host strains or species of the same genus as the host cell of (a). nucleotide sequences conserved in systems of the type in any of the preceding paragraphs. Law.

[0482] 35. The method of any of the preceding paragraphs, wherein the virus is a phage or phagemid.

[0483] 36. (b) The viruses in (b) are Corticoviridae, Cystoviridae, Inoviridae, and Leviviridae. Viridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae or 36. The method of paragraph 35, wherein the virus is a Tectiviridae virus.

[0484] 37. (b) virus is transmitted from a phage, e.g., from a cell of the same strain as the cell in (a). 37. The method of paragraph 35 or 36, which occurs naturally in the induced phage.

[0485] 38. (b) The phage was subjected to selective pressure using phage-resistant bacteria. The method of paragraph 35, 36 or 37.

[0486] 39. In (b) (iv) the one or more HM-arrays guide the Cas to a second target in the host cell, and a second host target sequence comprising an HM-spacer capable of hybridizing to a second host target sequence to modify the sequence; The HM-array encodes two HM-crRNAs, where the second target sequence is a host CRI. The nucleotide sequence of the SPR / Cas system, which results in the second HM-crRNA , guiding Cas to a second target and activating the host CRISPR / Cas system in the host cell. wherein modification of the second target sequence reduces the function of the host CRISPR / Cas system. or eliminate; and (v) wherein the HM-array of (iv) comprises a second HM-array capable of hybridizing to a second host target sequence. (a) is active in cells for transcription of crRNA, Any of the methods in the preceding paragraph.

[0487] In some embodiments, the HM-arrays of (ii) and (iv) are the same HM-array. In some embodiments, they are different HM-arrays (e.g., different CRISPR / Cas type arrays, e.g., type I and II or type II and III or types I and III or different type II arrays).

[0488] 40. The method of paragraph 39, wherein any one or all of the cells of PH1 to PH4 contain the second target sequence. Law.

[0489] 41. The second target sequence is a genera or species of cells described in any of paragraphs 11 to 24, e.g., Streptococcus thermophilus or Streptococcus pyogenes or Sta The sequence of the CRISPR / Cas system of Phyllococcus aureus is identical to that of the CRISPR / Cas system of Phyllococcus aureus. 39 or 40 ways.

[0490] 42. The second target sequence is included in a sequence selected from the group consisting of SEQ ID NOs: 1 to 44, or Any method in paragraphs 39 to 41, which is its complement.

[0491] 43. The second target sequence is A. Repetitive DNA or RNA sequences in the host CRISPR array (e.g., where is the 5'-most repeat (first repeat); B. tracrRNA sequence or tracrRNA-encoding DNA sequence; CRISPR assay Ray reader array; C. Cas gene promoter (e.g., Cas1, Cas2, or Csn2 promoter) -); D. CRISPR array leader promoter sequence or E. Cas-encoding DNA or RNA sequences (e.g., where Cas is Cas9, Cas 1, Cas2 or Csn2) Any of the methods in paragraphs 39 to 42, including:

[0492] 44. The second target sequence is F. CRISPR array leader or leader adjacent to the 5'-most nucleotide of the first repeat a promoter sequence (and optionally including the 5'-most nucleotides of the repeat); G. A sequence of up to 20 contiguous nucleotides immediately 5' of the first repeat; H. A sequence of up to 20 contiguous nucleotides of the 5'-most nucleotide of the first repeat, or I. A sequence of up to 20 contiguous nucleotides immediately 3' of the first spacer repeat (and optionally where the sequence includes the first spacer most 3' nucleotide Any of the methods in paragraphs 39 to 43, including:

[0493] 45. J. The second HM-crRNA comprises or consists of the structure RSR, where R=CR ISPR repeat and S = CRISPR spacer, where S is the CRISPR repeat (5' to 3' direction) In)VH R or H R -V or, where V=(b) a DNA sequence of a virus and a small amount of a sequence that is at least 95%, 96%, 97%, 98% or 99% identical to H R =applicable is the DNA sequence of the CRISPR repeats of the host cell CRISPR / Cas system; K. Here H R The sequence of is immediately adjacent to the sequence of V in the host CRISPR / Cas system. border; and L. where the second HM-crRNA transduces the host CRISPR / Cas system in the cell. A host molecule is used to guide Cas to the spacer for modification (e.g., cleavage or inactivation). It can hybridize with the spacer of the main CRISPR / Cas system, Any of the methods set out in paragraphs 39 to 44.

[0494] 46. ​​V = 1 or up to 40 (e.g., up to 15) consecutive nucleotides of viral DNA. The method of paragraph 45.

[0495] 47. The second HM-crRNA does not substantially hybridize to the nucleic acid of the virus of (b). Any of the methods set out in paragraphs 39 to 46.

[0496] 48. a. The host CRISPR / Cas system can recognize the cognate PAM; b. where the viral nucleic acid in (b) contains such a PAM immediately 3' of the protospacer sequence Includes; c. Where V = 1 or up to 40 (e.g., up to 15) nucleotides of the protospacer Yes; and d. Here, H R = a sequence identical to the repeat sequence of the host CRISPR / Cas system Ru, Any of the methods in paragraphs 45 to 47.

[0497] 49. The contiguous sequence of the repeat in the host system is at least 50% of the sequence of the host repeat (e.g., 49. The method of paragraph 48, wherein the repeat comprises a nucleotide sequence 5' or 3' of the host repeat.

[0498] 50. V = 1 to 40 (e.g., up to 15) consecutive nucleotides of the 3' protospacer. and optionally, the contiguous sequence of the repeat includes the 5' nucleotide of the host repeat, paragraph 45 or There are 46 ways.

[0499] 51. V = 1 to 40 (e.g., up to 15) consecutive nucleotides of the 5' protospacer. Optionally, the contiguous sequence of the repeat includes the 3' nucleotide of the host repeat. How to do it.

[0500] 52. R = repeat recognized by the host CRISPR / Cas system, paragraph 45 Any of the 51 methods.

[0501] 53. Each HM-CRISPR contains (in the 5' to 3' direction) a first repeat sequence, a first spacer sequence, and a second repeat sequence, wherein the spacer sequence c is a repeat sequence that ... The array contains hybridizable sequences, repeats and spacers in a further host cell. and optionally, the (b) viral nucleic acid is transfected into the host cell. Cas to encode functional Cas and / or tracrRNA sequences in the cells a nuclease-encoding sequence and / or a tracrRNA-encoding sequence, Any of the preceding paragraphs, wherein the tracrRNA sequence comprises a sequence complementary to the first or second repeat. How to do it.

[0502] 54. Each HM-CRISPR array contains (in the 5' to 3' direction) a first repeat sequence, a first spacer sequence, and a second repeat sequence, wherein the spacer sequence c is a repeat sequence for each target sequence in the host cell. The array contains sequences that can hybridize to the repeats and sequences in additional host cells. a promoter for transcription of pacer, wherein the vector is capable of transfecting pacer in a host cell; Cas nuclease coding sequence and / or tr for encoding crRNA sequence does not contain the acrRNA coding sequence, where the tracrRNA sequence is in the first or second repeat wherein the HM-CRISPR array optionally comprises a sequence complementary to a host tra crRNA is used to target Cas (e.g., endogenous host Cas nuclease) to each host target site. The method of any of the preceding paragraphs, wherein the nucleic acid sequence is functional in the host cell to guide the nucleic acid sequence to the target site.

[0503] 55. The repeats are identical to repeats in the host CRISPR / Cas system, where each H The M-CRISPR array is a Cas-like protein of the host CRISPR / Cas system (e.g., Cas Paragraph 53 or 54 ways.

[0504] 56. Each HM-CRISPR array contains more than one copy of the HM-spacer (e.g., (e.g., at least 2, 3, or 4 copies), by any of the methods in the preceding paragraph.

[0505] 57. Encoding a second or third HM-crRNA (additional HM-crRNA), Additional HM-crRNA guides Cas to the target in the host cell. and optionally a nucleotide sequence capable of hybridizing to the target sequence of the host cell. Essential, virulence or resistance genes or essential components of the host cell's CRISPR / Cas system The method of any of the preceding paragraphs, wherein the raw nucleotide sequence is

[0506] 58. Each HM-CRISPR array is responsible for the production of each HM-crRNA in the host cell. a precursor comprising a CRISPR repeat sequence that is identical to an endogenous CRISPR repeat sequence of the host cell; Either way.

[0507] 59. (b) Virus encodes a Cas (non-host Cas) that is functional in (a) host cells. (e.g., where the non-host Cas is a Type I system Ca s, where the host system is type II or III, and the non-host Cas is type II system Cas, where the host system is type I or III, or non-host The primary Cas system is the type III Cas system, where the host system is type I or II. ), optionally wherein the host cell does not contain the same type of Cas as the non-host Cas. or not manifesting any of the methods in the preceding paragraph.

[0508] 60. (b) The virus contains a nucleotide sequence encoding the tracrRNA sequence, Optionally, the tracrRNA sequence and the first HM-crRNA are a single guide RNA ( The method of any of the preceding paragraphs, comprising:

[0509] 61. Any of the preceding paragraphs, wherein each HM-crRNA is contained in each single guide RNA (gRNA). In this way.

[0510] 62. The first HM-array is subjected to Cas cleavage at the first target sequence, the first target sequence (or the first Activation of the first target sequence (or the gene of the first target sequence), knockdown of the first target sequence (or the gene of the first target sequence) the method of any of the preceding paragraphs, operable to cause mutation of the first target sequence. .

[0511] 63. A virus, host cell or virus population obtained by any of the methods set out in the preceding paragraph. Thus, optionally, the population is the same as PV1 or PV2 or the virus is It comes from groups like this.

[0512] 64. A population of host cells (e.g., bacterial cells) obtained by the method of any of the preceding paragraphs, comprising: Optionally, the population herein is PH1, PH2, PH3, or PH4 or any of the preceding paragraphs. The cultured cell population is identical to that described in .

[0513] 65. The population does not contain nucleic acid of the virus in (b) or the first HM-array or the second HM-array 65. The host cell population of paragraph 64, which does not comprise an HM-array (e.g., as determined by PCR).

[0514] 66. For medical, dental or ophthalmic use (e.g., for the treatment of infections in living organisms or any of the viruses in paragraphs 63 to 65 of the Act on the Prevention and Control of Infectious Diseases (for the prevention or limitation of the spread of infection in living organisms); Host cell or population.

[0515] 67. Use in food, beverages, dairy products or cosmetics (e.g. cosmetic products, e.g. chemical Use in cosmetics) or hygiene products (e.g., in hygiene products, e.g., soap) a virus, host cell or population according to any of paragraphs 63 to 66 for use in composition.

[0516] 68. Any of paragraphs 63 to 67 for medical or dental therapeutic or prophylactic use Use of the composition, virus, host cell or population according to the invention.

[0517] 69. Use in cosmetics (e.g. cosmetic products, e.g. use in cosmetics) or hygiene Paragraph 63 for use in household products (e.g., in hygiene products, e.g., soap). - Use of a composition, virus, host cell or population according to any of claims 1 to 68.

[0518] 70. For microbial host cell modification (e.g., killing or sterilizing cells or cultures of microbial cells) (for reducing proliferation) the use of any of paragraphs 63 to 69, virus, host cell or population.

[0519] 71. The virus or viruses in the population contain holins and / or or an endolysin, optionally wherein the endolysin is expressed as phage phage 11, phagocytosis Phage Two, phage P68, phage Phi WMY, or phage K endolysin (e.g., MV-L endolysin or P-27 / HP endolysin), paragraphs 1 to 6 3 and 66-70 any method, virus or virus population.

[0520] 72. The virus or viruses in the population contain holins and / or The method of any of paragraphs 1 to 63 and 66 to 70, wherein the virus does not express an endolysin or an endolysin. or viral populations.

[0521] 73. A virus (e.g., a virus of (b)) or a virus in each of the populations is a host of (a) Antimicrobial functionality in cells, e.g., antibiotics, e.g., beta-lactam antibiotics ( For example, the antibiotics described in any of paragraphs 1 to 63 and 13 to 27. and any of 66-70 methods, viruses or virus populations.

[0522] Corrosion, biofilm and biofouling control The present invention relates to the treatment of microbial corrosion of substrates or fluids, particularly in industrial or domestic systems. The present invention also relates to a method for controlling microorganisms (MIC) or biofouling. ... Regarding treatment fluids and vectors.

[0523] Corrosion leads to the deterioration of materials such as metals (e.g., steel or iron), plastics, and stone It is a series of chemical, physical and (micro)biological processes that affect large social and economic Current corrosion control strategies based on chemical manufacturing products are in a severe environmental They are under increasing pressure from border regulations. Furthermore, they are rather inefficient and the drugs they use are This can be hindered by microbial (e.g., bacterial) resistance to the agent. There is an urgent need for sustainable corrosion control strategies. Corrosion occurs through different electrochemical reactions, It is influenced by a complex process of different microorganisms secreting proteins and metabolites that may have secondary effects. can be.

[0524] The importance of microbial corrosion processes is evident in the corrosion of stainless steels, nickel and zinc, used in industry and in the home. and aluminum-containing alloys as well as metals and alloys, concrete, asphalt This is evident from the fact that many substances, such as polymers, are easily degraded by microorganisms. Protective coatings, inhibitors, oils and emulsions are also subject to microbial degradation.

[0525] Microbiologically Influenced Corrosion (MIC) is a common cause of corrosion in hydrocarbon production and processing equipment, water distribution systems, ships, railways, Affects automotive and other types of metallic and non-metallic industrial and domestic systems In particular, MIC is a cost-effective alternative to hydrocarbon fuels, including production, transportation, and storage systems. It is known to cause considerable damage to infrastructure, often resulting in catastrophic environmental contamination. Approximately 40% of pipe corrosion is caused by microbiological corrosion and occurs every year in the production, transportation and storage of petroleum. Pipe biofilms cause huge economic losses due to incrustation on the walls. This process can result in a decrease in fluid velocity in the system. This results in environmental and productivity impacts.

[0526] MIC occurs in environments such as soil, freshwater, and saltwater and is responsible for 30 percent of all corrosion damage. The MIC is calculated based on the immobilization of microorganisms such as bacteria, the release of metabolites, and the normal It is caused by the formation of biofilms that induce or accelerate corrosion processes. The majority of bacterial groups are sulfur or sulfate-reducing bacteria (SRB), extracellular polymeric substance-producing bacteria (EPSB), , acid-producing bacteria (APB), sulfur or sulfide oxidizing bacteria (SOB); iron or manganese oxidizing bacteria (IOB), ammonia-producing bacteria (AmPB) and acetogenic bacteria (AcPB). Unit ribosomal RNA gene pyrosequencing survey of acetic acid-producing bacteria (Acetobacter and Gluconacetobacter) in environments exposed to fuel-grade ethanol and water. This indicates that the disease is widespread in the border area.

[0527] Microbial growth under environmental conditions directly or indirectly affects electrochemical reactions. -substrate interactions lead to initial adhesion and biofilm formation. Substrate binding, metabolite release, and biofilm formation result in electrochemical changes at the substrate surface. They affect the metal substrate, inducing or accelerating the corrosion process and thereby intervening in the MIC process. Bacterial biofilm formation on metals involves the following steps: I - The formation of organic and inorganic molecules on metals Adsorption-mediated film formation, which modifies load distribution on metallic surfaces and also It acts as a nutrient source for bacteria and promotes the adhesion of free-floating microorganisms present in the liquid; II - Microorganisms Adhesion and growth of colony-forming aerobic bacteria; III - Extracellular adhesion by some sessile bacteria. Production of polymeric substances (EPS); IV - In biofilms required for strict anaerobic bacteria Aerobic planktonic microbial cells consume oxygen through respiration, creating a local anaerobic environment. V - biofilm thickness, which may be favorable for the shielding of the outer layer. The EPS produced by the bacteria connects to the biofilm and supplements essential ions for growth. These are used as a means of attachment and help the bacteria develop differential aeration zones. Protects against biocides that interfere with corrosion mechanisms and, in cases of low nutrient availability, provides a nutrient source. The process of differential aeration corrosion occurs in the aerated (surrounding biofilm) and non-aerated areas. The uneven distribution of biofilm on metal substrates with an air zone (below the biofilm) Biofilm formation on metal surfaces reduces the oxygen content, creating a nearly completely anaerobic environment. Pseudomonas is the main EPS-producing genus.

[0528] Examples of MIC biocorrosion processes mediated by corrosive bacteria are: (A) freshwater, seawater Aerobic corrosion bacteria from industrial / domestic systems or storage tanks can build up on surfaces that are contaminated with contaminants. For equipment and pipelines in industrial or domestic systems with a protective film (B) EPS-producing bacteria attach to the equipment / pipeline walls and produce EPS, which (C) Corrosiveness of other groups to pipeline walls. Bacterial adhesion occurs, which releases its metabolites and leads to the development of microcolonies through cell division. The action of iron-oxidizing bacteria results in the accumulation of large amounts of ferric iron precipitates. sulfuric acid released by sulfur-oxidizing bacteria can cause environmental acid (D) Low oxygen concentrations and organic acids released by acid-producing bacteria promote hydrogen sulfide decomposition. It favors the adhesion and development of sulfate-reducing bacteria that produce H2S, thereby accelerating the corrosion process. (E) Corrosion of equipment / pipelines occurs, which is accompanied by micro-leaks. Partially shielded by iron precipitates and containing bacterial biofilms, H2S is released into the affected system. In addition, thick biofilms and sludge can cause serious health risks to those who operate the machines. The production of silage leads to biofouling and interferes with the functioning of the system.

[0529] Similarly, bacterial colonies may be present in fluids such as water reservoirs or reservoirs (e.g., drinking water or cooling systems). They can also reproduce in the water (in the system) and thereby mediate biofouling of the fluid. An example is the acidification of waterways or drinking water reservoirs.

[0530] The present invention addresses the issues of MIC and biofouling by providing the following aspects 1 and below: 1. Controlling microbial corrosion (MIC) or biofouling of substrates in industrial or domestic systems a method for controlling adhesion of a substrate to a surface of the substrate, the method comprising: in contact with a population of primary host cells of a species; (i) contacting the population with a plurality of vectors capable of transforming or transducing cells, and The vector contains a CRISPR array, which allows the CRISPR array to be introduced into the host cell. Here, (a) Each CRISPR array contains one or more nucleotide sequences for expression of a crRNA and a promoter for transcription of the sequence in the host cell; and (b) Each crRNA encodes a Cas (e.g., a Cas nuclease, e.g., Cas9 or Cp f1) into the host cell and induce the host to modify the target sequence (e.g., cleave the target sequence). capable of hybridizing to target sequences in cells; the target sequences are genes that mediate host cell viability. is a child array; and (ii) expressing the cRNA in the presence of Cas in a host cell, thereby The target sequence is modified to reduce host cell viability and control the MIC or biofouling of the substrate. bring A method comprising:

[0531] For example, the system may include a device (e.g., for use in an industrial process) and the surface may be a component of the device. For example, each array may be a surface of a modified array, such as any of the arrays disclosed herein. In one embodiment, the vector comprises a modified array of the vectors described herein. For example, biofouling can be caused by microbial biofilms and and / or sludge formation, growth, or maintenance. For example, the first host cell is sessile. For example, "control" of methods 1 or 4 (below) refers to the MIC or biofouling in a system. This includes preventing, reducing, or eliminating the MIC or reducing the spread of biofouling. Non-limiting examples of MIC or biofouling mediating factors are described above. Or maintenance may be, for example, a characteristic of cell viability. Thus, for example, the method may involve the growth of a host cell. The method reduces the growth and / or maintenance of the host cell, for example, by killing the host cell.

[0532] 2. The method of embodiment 1, wherein the host cells are comprised in a microbial biofilm that contacts the substrate.

[0533] 3. The surface and host cells are in a fluid such as an aqueous solution (e.g., seawater, freshwater, stored water, or The method of any preceding embodiment, wherein the suction power is in contact with a water source (e.g., a water source or drinking water).

[0534] Freshwater is found on the Earth's surface as ice sheets, ice caps, glaciers, icebergs, marshes, ponds, lakes, rivers and streams, and It is water that occurs naturally underground as groundwater in aquifers and underground streams. Generally characterized by low concentrations of dissolved salts and other total dissolved solids. The term specifically excludes seawater and brackish water, but includes mineral-rich waters such as iron springs. Water can be any of these types of freshwater. Potable water is water that is used for human or animal (e.g., livestock) consumption. The fluid is water. For example, the fluid is industrial cooling water (where the system is a cooling system). , wastewater (where the system is a wastewater treatment or storage system), drinking water (where the system is a drinking water treatment, storage, transportation or delivery system), paper water (where the system is a paper manufacturing or processing system), swimming pool water (where the system water treatment or storage system), fire extinguisher water (where system is a fire extinguishing system) or in industrial works in pipes, tanks, holes, ponds or ditches Selected from Chengshui.

[0535] 4. A method for controlling microbial biofouling of fluids in industrial or domestic systems. (e.g., for bacterial acidification control of a liquid in a reservoir or container), where the fluid a population of first host cells of a first microbial species that mediates the biofouling, and the method (i) contacting the population with a plurality of vectors capable of transforming or transducing cells; each vector containing a CRISPR array, whereby the CRISPR array is transferred to a host is introduced into cells, where (a) Each CRISPR array contains one or more sequences for expression of crRNA and a host cell. comprising a promoter for transcription of the sequence; and (b) Each crRNA guides a Cas (e.g., a Cas nuclease) into the host cell and targets the target. hybridize with a target sequence in a host cell to modify the target sequence (e.g., cleave the target sequence). soybean, and the target sequence is a gene sequence that mediates host cell viability; and The method further comprises expressing the cRNA in the presence of Cas in a host cell, thereby transforming the host cell. and modifying target sequences in the cells, resulting in reduced host cell viability and control of biofouling. This includes:

[0536] For example, the fluid is a liquid. For example, the fluid is a gaseous fluid.

[0537] system: An example system for any of the embodiments is selected from the group consisting of: Petrochemical recovery, processing, storage or transportation systems, hydrocarbon recovery, processing, storage or transportation systems crude oil recovery, processing, storage or transportation systems; natural gas recovery, processing, storage or transport systems (e.g., oil wells, oil rigs, oil drilling equipment, oil pumping systems) , oil pipelines, gas drilling rigs, gas extraction equipment, gas pumping equipment, gas pipelines tankers, oil tankers, gas tankers, oil or gas storage units), water processing water reservoirs (e.g., drinking water reservoirs), air or water conditioning (e.g., cooling or heating) equipment, such as refrigerant lines, condensers or heat exchangers, medical or Surgical equipment, environmental (e.g., soil, waterways, or air) treatment equipment, papermaking, or recycling equipment, power generation equipment, e.g., thermal or nuclear power generation equipment, fuels (e.g., hydrocarbon fuels, e.g. , petroleum, diesel or LPG) storage equipment, mining or metallurgical, mineral or fuel recovery systems systems, such as mining or extraction equipment, engineering systems, marine equipment, cargo or goods storage equipment (e.g. shipping containers), food or beverage manufacturing, processing or packaging equipment, cleaning equipment (e.g. laundry appliances, e.g., washing machines or dishwashers), food serving (e.g., domestic or commercial food serving) equipment , farm equipment, construction (e.g., architecture, infrastructure, or road construction) equipment, aviation equipment, aircraft Space equipment, transportation equipment (e.g., motor vehicles (e.g., automobiles, large trucks, or light trucks) , train, aircraft (e.g., airplane) or sea or waterway transport medium (e.g., ship or boat, submarines or hovercraft), packaging equipment, e.g., consumer goods packaging equipment or food or beverage packaging equipment food packaging equipment, electronic equipment (e.g., computers or mobile phones or electronic components thereof) or electronics manufacturing or packaging equipment, dental equipment, industrial or domestic piping (e.g., subsea pipe) or storage container (e.g., water tank or fuel tank (e.g., gasoline tank) tanks, e.g., gasoline tanks for transport media), underground installations, buildings (e.g., residences or businesses) or commercial facilities or factories or power plants), roadways, bridges, agricultural equipment, factory systems, crude oil or or natural gas exploration equipment, business systems, and home systems.

[0538] For example, the system may be used in agriculture, the oil or petroleum industry, the food or beverage industry, the clothing industry, Packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aerospace industry, automobile industry Automotive industry, biotechnology industry, medical industry, healthcare industry, dental industry, energy industry, consumer goods Industry, pharmaceutical industry, mining industry, cleaning industry, forestry industry, fishing industry, entertainment industry, recycling industry , cosmetics industry, plastics industry, pulp or paper industry, textile industry, clothing industry, leather Selected from the group consisting of product or suede or animal leather industry, tobacco industry and steel industry For example, the surface or fluid is used in the selected industry or industries. For example, systems are used in the crude oil industry for the treatment of surfaces or fluids in the equipment used. For example, systems are used in the natural gas industry. The system is used in the oil industry. For example, the system is used to transport marine containers, platforms, or drilling equipment (e.g., oil or gas platforms or drilling equipment for use at sea) In one embodiment, such a system may be anchored at sea. For example, fixed in the sea for a period of one month, two months, three months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 3 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 2 It may be fixed at 1 month, 22 months, 23 months, 24 months or more (e.g., consecutive months). In an embodiment, such a system is fixed in the oceans of a country or state, e.g. It is fixed in seawater, not temporarily, for example, in the waters of the country for 1 month, 2 months, 3 months, 4 months , 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months , 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months , 22 months, 23 months, 24 months or more (e.g., consecutive months).

[0539] For example, the substrate surface to be treated may be stainless steel, carbon steel, copper, nickel, brass, aluminum, For example, the substrate may be metal welded or For example, the surfaces may be metallic (e.g., steel or iron) or non-metallic (e.g., plastic). (stick, concrete, asphalt, wood, rubber or stone) surfaces. Metals are alloys (e.g., stainless steel, brass or nickel alloys, zinc alloys, copper alloys, nickel For example, the surface is an artificial polymer surface. For example, the surface is a substrate coating. For example, the substrate is in contact with soil, fresh water, or seawater. There are.

[0540] For example, the fluid may be drinking water, waterways, brackish water or a liquid fuel, such as gasoline or diesel. diesel (e.g. for automobiles or motorized transport), LPG, kerosene, alcohol (e.g. ethanol, methanol or butanol), liquid hydrogen or liquid ammonia), e.g. For example, the fuel is a stored liquid fuel. For example, the fluid is an oil or a non-aqueous solution. For example, the fluid refers to a watercourse or body of water, e.g., seawater, freshwater, drinking water, river, stream, pond, lake, reservoir, stored water water (e.g., from storage tanks or cooling systems), groundwater, well water, water in rock formations, soil water, For example, the liquid is seawater. For example, the substrate is any of the liquids described in this paragraph. For example, the fluid or liquid may be oil, water, hydraulic fracturing fluid, fuel, fuel, carbon dioxide, natural gas, oil / water mixture, fuel / water mixture, salt-containing water, ocean or seawater , brackish water, water source, lake, river, stream, marsh, pond, swamp, snow or meltwater, spring, groundwater, Selected from the group consisting of aquifers, sediments, and any material that is liquid at ambient temperature (e.g., at rtp). It is hydrophobic, but does not react with organic solvents such as hexane, benzene, toluene, chloroform, dichloromethane, etc. Ethyl ether, vegetable oil, petrochemical oil, crude oil, refined petrochemical products, volatile essential oils, fossil fuels Soluble in fuels, gasoline, hydrocarbon blends, jet fuel, rocket fuel, and biofuels For example, the fluid is an oil / water mixture.

[0541] As used herein, the term "Microbiologically Influenced Corrosion" or "MIC" refers to the system, unless otherwise specified. Some element (substrate) of the As used herein, the term "biofouling" refers to the process by which a substance is structurally damaged by a given action. Unless otherwise specified, do not use in contact with fluids (e.g., water or aqueous solutions or hydrocarbons or petrochemicals). The process by which microorganisms (e.g., bacteria and / or archaea) accumulate on the surface of a substrate that is harboring a fluid ( Microorganisms (e.g., bacteria and / or in water or aqueous solutions or hydrocarbons or petrochemicals) This also includes the unwanted accumulation and proliferation of bacteria (or archaea), i.e., "acidification" of the fluid. For example, bacteria can be found in ship or vessel ballast water, which is environmentally undesirable. The term "substrate" used in this paper refers to a substrate on which cells can adhere and on which a biofilm can form and grow. or any type of surface on which biofouling (e.g., ooze or sludge formation) can occur. The substrate may be a surface such as equipment surfaces in petrochemical, fuel, crude oil or gas piping systems. "Industrial" substrates or "non-industrial" substrates such as countertops or shower substrates or garden substrates (e.g. For example, it may be a domestic (e.g., home or office) substrate.

[0542] In an alternative to any of these embodiments, instead of a population of host bacterial cells, the population is a first species It is a population of archaeal cells.

[0543] 5. The method of embodiment 4, wherein the fluid is an aqueous solution (e.g., seawater, freshwater, stored water, or drinking water). method.

[0544] 6. The method includes mixing a fluid with a vector, thereby contacting the host cell with the vector. The method of any one of aspects 3 to 5, comprising, for example, introducing the vector into a liquid (optionally containing an antibiotic or and then contacting the mixture with the surface (embodiment 1) or the fluid of embodiment 4. It may be added to the fluid in which it is present.

[0545] 7. Each target sequence is a host cell pathogenic, resistance, or essential gene sequence, e.g., its exons or or a regulatory sequence. The resistance can be antibiotic resistance. For example, a host cell is contacted with the antibiotic and the vector to reduce host cell viability. To make.

[0546] 8. Embodiment 1, wherein modification of the target sequence results in host cell death and / or reduced host cell proliferation Any of the methods from 1 to 7. Proliferation is, for example, the expansion or spreading of cells in contact with a surface. .

[0547] 9. The method of any of embodiments 1-8, wherein the vectors comprise identical CRISPR arrays.

[0548] 10. The method of any of aspects 1-9, wherein the host cell is a bacterial or archaeal cell. Alternatively, Instead, the first cell is an algal cell.

[0549] 11. If the first host cell is a sulfate-reducing bacterial (SRB) cell (e.g., Desulfovibrio or Desulfovibrio). The method of any one of aspects 1 to 10, wherein the cells are desulfotomaculum cells. Tomaculum nigrificans, Desulfacinum infernum, Thermodesulf Obacteria mobile, Thermodesulforhabdus norvegicus, Archaeoglobus ·Fulgidus, Desulfomicrobium apisheronum, Desulfovibrio gabonensi Desulfovibrio longus, Desulfovibrio vietnamensis, Desulfobacter Desulfobacter vibrioides, Desulfococcus cetonicum, Desulfococcus halophyllum, Desulfobacter vibrioides and Desulfotomaculum thermocisternum cells. For example, the population may contain a mixture of two or more of these cell types.

[0550] 12. Surface or fluid oil, gas or petrochemical recovery, processing, storage or transportation equipment 12. The method of embodiment 11, comprising: Crude oil is one of the most important energy resources in the world. Crude oil is gasoline, petroleum, paraffin oil, lubricants, asphalt, household fuel oil, petroleum jelly and the petroleum refining and chemical industry, where oil is refined through various technological processes into consumer goods such as polymers. Petroleum-derived products are also commonly used in many other chemical processes. Alternatively, the fluid is the consumer product or the surface is such a consumer product. In contact with the product.

[0551] 13. Surfaces in contact with or flowing through seawater, fracking fluids, or well fluids 13. The method of embodiment 11 or 12, wherein the body is seawater, fracking fluid, or well fluid.

[0552] 14. Step (i) of the method provides a population of microbial cells of a second species (second host cells), the second cells the vector, wherein the vector is transferable from the second host cell to the first host cell; The second host cell is then combined with the first host cell, thereby introducing the vector into the first host cell. The method of any one of aspects 1 to 13, comprising: environmentally, industrially or domestically acceptable in the water or soil environment, and The cells are not tolerated in the environment.

[0553] 15. The first host cell is included in a mixture of microbial cells (e.g., a microbial cell) prior to contact with the vector. 14. The method of claim 14, wherein the mixture comprises cells of the second species.

[0554] 16. The second species is a Bacillus or a nitrate-reducing bacterium or a nitrate-reducing, sulfide-oxidizing bacterium (NRB). 16. The method of embodiment 14 or 15, wherein the plant is a species.

[0555] 17. NRB is a bacterium that contains Campylobacter, Nitrobacter, Nitrosomonas, and Thiomicrobacter. Spira, Sulfurospirillum, Sauraera, Paracoccus, Pseudomonas, and Desulfovibrio or at least one of said species. The method of embodiment 16, comprising both

[0556] 18. NRB is a fungus that causes Nitrobacter vulgaris, Nitrosomonas europaea, and Pseudomonas Solanum stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans The group consisting of Sulfurospirillum delleianum and Rhodobacter sphaeroides 18. The method of embodiment 17, wherein the

[0557] 19. The method further comprises inoculating the first host cell and the biocide simultaneously or sequentially with the vector. 19. The method of any of embodiments 1-18, comprising contacting the vector and the biocide. , are provided pre-mixed into the composition that will contact the host cells.

[0558] 20. The biocide is tetrakishydroxymethylphosphonium sulfate (THPS), chloraldehyde, chlorine monoxide, chlorine dioxide, calcium hypochlorite, potassium hypochlorite , sodium hypochlorite, dibromonitriloproprionamide (DBNPA), methylene Bis(thiocyanate) (MBT), 2-(thiocyanomethylthio)benzothiazole (TCM TB), bronopol, 2-bromo-2-nitro-1,3-propanediol (BNPD), Tributyltetradecylphosphonium chloride (TTPC), taurinamide and its Derivatives, phenols, quaternary ammonium salts, chlorine-containing agents, quinoacridinium salts, lactates amines, organic dyes, thiosemicarbazones, quinones, carbamates, ureas, salicylamides, Rubanilide, guanide, amidine, imidazoline, acetic acid, benzoic acid, sorbic acid, propyl acetic acid, boric acid, dehydroacetic acid, sulfurous acid, vanillic acid, p-hydroxybenzoic acid ester , isopropanol, propylene glycol, benzyl alcohol, chlorobutanol, Phenylethylic alcohol, formaldehyde, iodine and its solution, povidone-iodine , hexamethylenetetramine, oxythiolin, 1-(3-chloroallyl)-3,5,7- Triazo-1-azoniaadamantane chloride, taurolidine, taurultam, N-( 5-nitro-2-furfurylidene)-1-amino-hydantoin, 5-nitro-2-furfurylidene aldehyde semicarbazone, 3,4,4'-trichlorocarbanilide, 3,4',5-tribromo Mosalicylanilide, 3-trifluoromethyl-4,4'-dichlorocarbanilide, 8- Hydroxyquinoline, 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo so-7-(1-piperazinyl)-3-quinolinecarboxylic acid, 1,4-dihydro-1-ethyl -6-fluoro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid, peracid Hydrogen chloride, peracetic acid, sodium oxychlorosene, parachlorometaxylenol, 2,4,4 '-Trichloro-2'-hydroxydiphenol, thymol, chlorhexidine, benzyl chloride Cetylpyridinium chloride, silver sulfadiazine, silver nitrate, bromine, Ozone, isothiazolone, polyoxyethylene (dimethylimino)ethylene (dimethylimino) Ethylene dichloride, 2-(tert-butylamino)-4-chloro-6-ethyl ester Selected from the group consisting of methylamino-5'-triazine (terbuthylazine) and combinations thereof 20. The method of embodiment 19, wherein the biocide is tetrakishydroxymethylphosphonium Sulfate (THPS). For example, biocides are quaternary ammonium compounds.

[0559] 21. If the system is used in mining, shipping, oil, gas or petrochemical recovery or processing, or hydraulic fracturing , air or water heating or cooling, potable water production, storage or distribution, hydrocarbon transportation, and waste 21. The method of any one of aspects 1 to 20, wherein the method is utilized in an industrial means selected from the group consisting of water treatment.

[0560] 22. The surface is a surface of a device in the selected industry or where the fluid is a surface of a device in the selected industry. 22. The method of embodiment 21, wherein the fluid is contained in a device used in

[0561] 23. The surface is a surface of kitchen, bath or garden equipment or where the fluid is a surface of kitchen, bath or garden equipment. The method of any of aspects 1-22, wherein the method is included in a gardening device. For example, the device is used in a domestic setting. will be done.

[0562] 24. The fluid is a portable liquid contained in a container (e.g., a water tank or bottle) and the surface Aspects 1 to 23, when dependent on Aspect 3, are surfaces of a container in contact with a liquid. Either way.

[0563] 25. Each vector contains a mobile genetic element (MGE), where the MGE contains an origin of transfer (oriT) and and the CRISPR array, wherein the MGE is coupled to the first species host cell and the industrial or 25. Any of aspects 1 to 24, wherein the microbial host cell is transmissible to additional microbial host cells in a home system. For example, the additional cells may be grown environmentally in an environment (e.g., in a water or soil environment). , industrially or domestically tolerated, the first host cell is not tolerated in the environment.

[0564] 26. The method of embodiment 25, wherein oriT is functional in the first and further host cells.

[0565] 27. The first and further host cells are contained in a biofilm in a fluid in contact with the surface. 27. The method of embodiment 25 or 26, wherein the cells are contained in or wherein the fluid.

[0566] 28. Embodiment 25, wherein the additional cell is a cell of a species described in any one of embodiments 16 to 18. 26 or 27. For example, the MGE can be derived from the first host cell and / or the additional cell. It can be transmitted in the opposite direction.

[0567] 29. The method of any of embodiments 25-27, wherein the additional cells are cells of the first type.

[0568] For example, in this embodiment, the MGE is transferred between first cells of the population in the system. It is possible that if an MGE leaves a copy of itself during the process of transmitting to other cells, then this MGE, hence the propagation and spread of CRISPR arrays across cell populations in the system. This provides a means for diffusion, thereby dispersing the effect of target sequence modification of the array. , to cause the spreading of arrays in biofilms in contact with surfaces or in fluids. It is highly effective and useful as a biofilm penetrant, making conventional biocides the most The dosage can be reduced to below the appropriate level.

[0569] 30. Each MGE is, contains, or is an integrative and conjugative element (ICE). each vector is a phage capable of infecting a host cell of the first type, and each MGE is an intercellular 30. The method according to any one of aspects 25 to 29, wherein the nucleic acid is a phage nucleic acid capable of said transfer.

[0570] 31. The method of embodiment 30, wherein each ICE is a transposon, e.g., a conjugative transposon. Law.

[0571] 32. An MGE in which each vector is a plasmid, optionally according to any one of aspects 25 to 31. The method of any of aspects 1 to 31, comprising:

[0572] 33. The first and / or further cells are operable for the transmission of the MGE to other cells. Contains a nucleotide sequence that encodes a protein, where the sequence is not included in an MGE , The method of any one of aspects 25 to 32.

[0573] 34. The method of embodiment 33, wherein the sequence is not contained in a vector.

[0574] 35. The sequence is contained in a conjugative transposon of the first cell and / or the further cell. The method of embodiment 33.

[0575] 36. Transposons are used in trans for the transmission of MGE between primary and further cells. The method of embodiment 35, wherein the method is operable.

[0576] 37. The oriT of the MGE is contained in the ICE of the first cell and / or further cells or iT, where ICE is the first and second cellular pathway for the transmission of MGE between cells. The method of any one of aspects 25 to 36, wherein the method is operable with a transformer.

[0577] 38. Embodiment 2, wherein the vector oriT is the oriT of an SRB or NRB transposon. Any of the methods 5 to 37.

[0578] 39. Each MGE contains first and second terminal repeat sequences and the CRISPR array between the repeat sequences. The method of any of aspects 25 to 38, comprising:

[0579] 40. If the MGE transfers a copy of the CRISPR array to the additional host cell, (2) the genome of the first host cell when transferred to the first host cell; or (3) the genome of the additional host cell when transferred to the first host cell. The method of any of embodiments 25 to 39, wherein the copy is left in the cell. For example, the copy is left in the genome of the cell. They are contained in transposons or prophage from which transmission occurs.

[0580] 41. The first and further cells are bacterial cells of different species (e.g., SRB and and NRB or SRB and Bacillus cells), the method of any of embodiments 25 to 40.

[0581] 42. According to embodiment 30, combined with a transposase for mobilization of MGE. The method according to any one of aspects 25 to 41.

[0582] 43. Toxin-antitoxin in which the vector or MGE is operable in a host cell of said first species and optionally wherein the toxin-antitoxin module is inoperable in other species. 43. The method of any one of embodiments 1 to 42, wherein the antitoxin gene is a gene that has no or reduced activity. The embodiment of the present invention involves the production of a vector / MGE (and thus a C) in a first host cell containing a target sequence. This is useful for creating selective pressure that favors the retention of RISPR arrays.

[0583] 44. Toxin in which the vector or MGE is operable in the second or further cell and an antitoxin module, optionally wherein the toxin-antitoxin module is a second or further

[0023] Aspects 1 to 4 include an antitoxin gene that is inoperable or has reduced operability in cells that are infected with the virus. 3. Any of the methods of 3., which involves culturing a population of CRISPR arrays in second or additional cells. maintain their normal function (e.g., when such cells are present in a biofilm that also contains primary cells). , where the toxin-antitoxin module induces further killing (target sequence modification) in the first host cell. It is useful for providing a vector or The MGE is a toxin-antitoxin that is operable in a first host cell and in said second or further cells. Includes modules.

[0584] 45. The toxin-antitoxin module fails to function in cells other than the first and second or further cells. The method of embodiment 43 or 44, wherein the CRI is reduced or possible. Selection pressure on both the first and second (or further) cells to maintain the SPR array Usefully, this then allows for the identification of the MGE between cells in a mixed population. Provide a reservoir for horizontal propagation of the array (e.g., biofilm in contact with a surface) or population contained in a fluid).

[0585] 46. ​​The first and second cells (or first and further cells) are of the same phylum (e.g. (e.g., both bacterial cells), the vector (A) in the first cell and / or in other cells of the same phylum but not in other cells of the same phylum. or in a second (or further) cell, (B) but not in other cells of the same eye. In one cell and / or a second (or further) cell, (C) in other cells of the same class (D) in the same eye, but not in the first cell and / or second (or further) cells in the first cell and / or the second (or further) cell, but not in other cells (E) in the first cell and / or the second (or further) cell, but not in other cells of the same family. (F) in the first cell and / or the second cell, but not in other cells of the same genus; in the first, but not in second (or further) cells, or (G) in other cells of the same species capable of replicating or functioning in a cell and / or a second (or further) cell , The method of any one of aspects 25 to 45.

[0586] 47. Each MGE is a conjugative transposon, and oriT is the first and further (or Second) functional in the host cell, wherein the MGE has first and second terminal repeat sequences between the repeat sequences; and the CRISPR array, wherein the first and further (or second) cells are bacteria. cells, wherein the target site is contained in the first cell but not in the further (or second) cell. wherein said modification inactivates a gene or regulatory sequence contained in said target in the first cell. and / or downregulating the first host cell viability and the MIC or biofouling control. The method of any one of aspect 25 or aspects 26 to 46 when dependent on aspect 25, which results in

[0587] 48. Each CRISPR array is designed for the expression and production of each crRNA in a first host cell. and (i) wherein R1 is the first CRISPR repeat and R1' is the second CRISPR repeat; R1 or R1' is optional, (ii) S1 contains a nucleotide sequence that is 95% or more identical to a target sequence in the first host cell or a first CRISPR spacer consisting of the same; The method of any one of aspects 1 to 47.

[0588] 49. R1 and R1' are the first and second repeats of a CRISPR array of a first host cell type. at least 95%, 96%, 97%, 98% or 99% identical to the sequence, Method of 48. In some embodiments, both R1 and R1' are present.

[0589] 50. R1 and R1' are CRISPR vectors in the first species of host cell for modification of a target sequence. 50. The method of embodiment 48 or 49, wherein the method is functional with a / Cas system. ...

Claims

1. A composition comprising a nucleic acid vector containing a modified host-modified (HM) CRISPR array for use in the treatment or prevention of bacterial host cell-mediated diseases or conditions in human subjects by altering the relative ratio of subpopulations of first and second bacterial cells in a mixed bacterial population, wherein the second bacteria comprises host cells and the mixed population is included in the subject, The second subpopulation consists of different bacterial species from the first subpopulation. Each host cell has a host target sequence, If the host target sequence is included in the host cell's antibiotic resistance gene, pathogenicity gene, or essential gene, A modified host-modified (HM) CRISPR array includes a spacer sequence (HM-spacer) and repeats encoding HM-crRNA, wherein the HM-crRNA includes a sequence that can hybridize with a host target sequence. The vector or host cell further comprises at least one nucleic acid sequence encoding a Cas nuclease, The vector can transform the host cell, HM-crRNA guides Cas nucleases to target, modifying target sequences in host cells, thereby causing host cell death or suppression of host cell proliferation. The growth of the first subpopulation of bacterial species is not inhibited by the crRNA, and the first bacterial species contains a 16s ribosomal RNA-coding DNA sequence that is at least 80% identical to the 16s ribosomal RNA-coding DNA sequence of other bacterial species in the mixed population. composition.

2. The use of a nucleic acid vector comprising a modified host-modified (HM) CRISPR array to alter the relative ratio of primary and secondary bacterial subpopulations in a mixed ex vivo population of bacteria, wherein the secondary bacteria comprises host cells, The second subpopulation consists of different bacterial species from the first subpopulation. Each host cell has a host target sequence, If the host target sequence is included in the host cell's antibiotic resistance gene, pathogenicity gene, or essential gene, A modified host modification (HM) CRISPR array comprises a spacer sequence (HM-spacer) and repeats encoding HM-crRNA, wherein the HM-crRNA comprises a sequence that hybridizes with a host cell target sequence. The vector or host cell further comprises at least one nucleic acid sequence encoding a Cas nuclease, The vector can transform the host cell, HM-crRNA guides the Cas nuclease to its target, modifying the target sequence in the host cell, thereby causing the host cell to die or inhibiting host cell proliferation. The growth of the first subpopulation of bacterial species is not inhibited by the crRNA, and the first bacterial species contains a 16s ribosomal RNA-coding DNA sequence that is at least 80% identical to the 16s ribosomal RNA-coding DNA sequence of other bacterial species in the mixed population. use.

3. The use according to claim 2, for the treatment of industrial or ex vivo medical fluids, surfaces, apparatus or containers, or for the treatment of waterways, water, beverages, food or cosmetics, wherein the host cells are contained in or on the fluids, surfaces, apparatus, containers, waterways, water, beverages, food or cosmetics.

4. The use according to claim 2, which produces bacterial cultures for administration to humans to support the Bacteroidetes phylum, which exhibits commensalism or mutualism in humans.

5. The use according to claim 2 or 4, wherein the mixed population is an ex vivo gut microbiota population, and optionally the host cells are Firmicutes or Clostridium difficile cells.

6. (a) Each host cell is a Staphylococcus, Streptococcus, Pseudomonas, Salmonella, Listeria, Desulfovibrio, or Clostridium host cell; (b) The primary bacterium is probiotic, commensal, or mutually beneficial with humans (e.g., in the human gut); or (c) The first bacterium is Enterobacteriaceae, and the second bacterium is Firmicutes. The use described in any one of claims 2 to 5.

7. (a) Each host cell is a Staphylococcus, Streptococcus, Pseudomonas, Salmonella, Listeria, Desulfovibrio, or Clostridium host cell; (b) The primary bacterium is probiotic, commensal, or mutually beneficial with humans (e.g., in the human gut); or (c) The first bacterium is Enterobacteriaceae, and the second bacterium is Firmicutes. The composition according to claim 1.

8. (a) alteration of the proportion of Bacteroidetes bacteria in a mixed bacterial population, (b) reduction of the proportion of Firmicutes subpopulation (host cells) in a mixed bacterial population, (c) reduction of the proportion of first Firmicutes species (host cells) in a mixed population, wherein the mixed population includes second Firmicutes species whose growth is not inhibited by the cRNA, (d) reduction of the proportion of first Gram-positive bacterial species (host cells) in a mixed bacterial population, wherein the mixed population includes second Gram-positive bacterial species whose growth is not inhibited by the cRNA, (f) in a mixed bacterial population The use according to any one of claims 2 to 6 for a reduction in the proportion of primary bacterial human intestinal microbiota species (host cells, e.g., Firmicutes) in a mixed bacterial population, wherein the mixed population comprises different bacterial species, wherein the different species are human intestinal probiotic species whose growth is not inhibited by the cRNA, or (g) a reduction in the proportion of bacterial human intestinal microbiota species (host cells, e.g., Firmicutes) in a mixed bacterial population, wherein the mixed population comprises different bacterial species, wherein the different species are human intestinal commensal species whose growth is not inhibited by the cRNA.

9. (a) alteration of the proportion of Bacteroidetes bacteria in a mixed bacterial population, (b) reduction of the proportion of Firmicutes subpopulation (host cells) in a mixed bacterial population, (c) reduction of the proportion of first Firmicutes species (host cells) in a mixed population, wherein the mixed population includes second Firmicutes species whose growth is not inhibited by the cRNA, (d) reduction of the proportion of first Gram-positive bacterial species (host cells) in a mixed bacterial population, wherein the mixed population includes second Gram-positive bacterial species whose growth is not inhibited by the cRNA, (f) reduction of first bacterial human intestinal microbiota species (host cells, e.g., Firmicutes) in a mixed bacterial population. The composition according to any one of claims 1 and 7, wherein the mixed population comprises different bacterial species, wherein the different species is a human intestinal probiotic species whose growth is not inhibited by the cRNA, or (g) a reduction in the proportion of a bacterial human intestinal microbiota species (host cell, e.g., Firmicutes) in the mixed bacterial population, wherein the mixed population comprises different bacterial species, wherein the different species is a human intestinal commensal species whose growth is not inhibited by the cRNA, and wherein (a) to (g) are for the treatment or prevention of (i) a microbiota infection by the bacterial species whose proportion is reduced in a human subject, or (ii) a disease or condition mediated by the bacterial species whose proportion is reduced.

10. (a) To change the relative ratio of the first and second subpopulations in a mixed bacterial population, where the second bacterium is Firmicutes; (b) To increase the relative ratio of Bacteroides to Firmicutes; or (c) To change the ratio of Bacteroidetes bacteria in a mixed bacterial population, The use according to any one of claims 2 to 6 and 8.

11. (a) In order to change the relative ratio of the first and second subpopulations in a mixed population of bacteria, Here, the second bacterium is a Firmicutes; (b) To increase the relative ratio of Bacteroides to Firmicutes; or (c) To change the ratio of Bacteroidetes bacteria in a mixed bacterial population, The composition according to any one of claims 1, 7, and 9.

12. Alternatively, the use according to any one of claims 2 to 6, 8 and 10, wherein the HM-crRNA and tracrRNA are provided, for example, by a vector containing a single guide RNA (gRNA).

13. Alternatively, the composition according to any one of claims 1, 7, 9, and 11, wherein HM-crRNA and tracrRNA are included in a single guide RNA (gRNA).

14. The use according to any one of claims 2 to 6, 8, 10, and 12, wherein the growth of the host cell population is reduced by at least five times compared to the growth of the host cell population that has not been transformed with the HM array or the nucleotide sequence encoding the gRNA.

15. The composition according to any one of claims 1, 7, 9, 11, and 13, wherein the growth of the host cell population is reduced by at least five times compared to the growth of the host cell population that has not been transformed with the HM array or the nucleotide sequence encoding the gRNA.

16. The use according to any one of claims 2 to 6, 8, 10, 12, and 14, wherein the proliferation of host cell populations on the surface is inhibited.

17. The composition according to any one of claims 1, 7, 9, 11, 13, and 15, wherein the proliferation of host cell populations on the surface is inhibited.

18. The use according to any one of claims 2 to 6, 8, 10, 12, 14 and 16, wherein at least one nucleic acid sequence encoding a Cas nuclease is endogenous in the host cell.

19. The composition according to any one of claims 1, 7, 9, 11, 13, 15, and 17, wherein at least one nucleic acid sequence encoding a Cas nuclease is endogenous in the host cell.

20. The use according to any one of claims 2 to 6, 8, 10, 12, 14, 16, and 18, wherein the vector lacks a sequence encoding a Cas nuclease.

21. The composition according to any one of claims 1, 7, 9, 11, 13, 15, 17, and 19, wherein the vector lacks a sequence encoding a Cas nuclease.

22. The use according to any one of claims 2 to 6, 8, 10, 12, 14, 16, 18 and 20, wherein the vector is a plasmid vector, a viral vector, a phage vector or a phagemide vector.

23. The composition according to any one of claims 1, 7, 9, 11, 13, 15, 17, 19, and 21, wherein the vector is a plasmid vector, a viral vector, a phage vector, or a phagemid vector.

24. The use according to any one of claims 2 to 6, 8, 10, 12, 14, 16, 18, 20, and 22, wherein each host cell further comprises a tracrRNA sequence or a DNA sequence for expressing a tracrRNA sequence.

25. The composition according to any one of claims 1, 7, 9, 11, 13, 15, 17, 19, 21, and 23, wherein each host cell further comprises a tracrRNA sequence or a DNA sequence for expressing a tracrRNA sequence.

26. The composition according to any one of claims 1, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25, wherein the mixed population is contained in the target intestinal microbiota.

27. The composition according to any one of claims 1, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 26, wherein the mixed population comprises Streptococcus thermophilus, Lactococcus lactis, and Escherichia coli.

28. The use according to any one of claims 2 to 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24, wherein the mixed population comprises Streptococcus thermophilus, Lactococcus lactis, and Escherichia coli.

29. The composition according to any one of claims 1, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, and 27, wherein the Cas nuclease is a type I Cas nuclease, a type II Cas nuclease, or a type III Cas nuclease.

30. The use according to any one of claims 2 to 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 28, wherein the Cas nuclease is a type I Cas nuclease, a type II Cas nuclease, or a type III Cas nuclease.

31. A nucleic acid vector comprising nucleic acid sequences for expressing a plurality of different crRNAs (e.g., contained in gRNA), wherein the Cas nuclease is a type I Cas nuclease, and the crRNA is functional using the type I Cas nuclease; and Here, the first crRNA can hybridize with the first nucleic acid sequence of the host cell, and the second crRNA can hybridize with the second nucleic acid sequence of the host cell, where the second sequence differs from the first sequence; and (a) The first sequence is contained in the antibiotic resistance gene (or its RNA), and the second sequence is contained in the antibiotic resistance gene (or its RNA), and these genes may differ as desired; (b) The first sequence is included in the antibiotic resistance gene (or its RNA), and the second sequence is included in the essential or pathogenic gene (or its RNA); (c) The first sequence is included in an essential gene (or its RNA), and the second sequence is included in an essential or pathogenic gene (or its RNA); or (d) The first sequence is included in the pathogenic gene (or its RNA), and the second sequence is included in the essential or pathogenic gene (or its RNA), The composition according to any one of claims 1, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, and 29.

32. A nucleic acid vector comprising nucleic acid sequences for expressing a plurality of different crRNAs (e.g., contained in gRNA), wherein the Cas nuclease is a type I Cas nuclease, and the crRNA is functional using the type I Cas nuclease; and Here, the first crRNA can hybridize with the first nucleic acid sequence of the host cell, and the second crRNA can hybridize with the second nucleic acid sequence of the host cell, where the second sequence differs from the first sequence; and (a) The first sequence is contained in the antibiotic resistance gene (or its RNA), and the second sequence is contained in the antibiotic resistance gene (or its RNA), and these genes may differ as desired; (b) The first sequence is included in the antibiotic resistance gene (or its RNA), and the second sequence is included in the essential or pathogenic gene (or its RNA); (c) The first sequence is included in an essential gene (or its RNA), and the second sequence is included in an essential or pathogenic gene (or its RNA); or (d) The first sequence is included in the pathogenic gene (or its RNA), and the second sequence is included in the essential or pathogenic gene (or its RNA), The use according to any one of claims 2 to 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, and 30.

33. The composition according to any one of claims 1, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, 29, and 31, wherein the nucleic acid vector comprises an exogenous DNA sequence exceeding 1.4 kb, the exogenous DNA encoding one or more elements of the CRISPR / Cas system, and comprises a modified array for the expression of HM-crRNA or gRNA in a host cell, the exogenous sequence lacking a nucleotide sequence encoding a Cas nuclease that is cognate with the cRNA or gRNA; wherein at least two different cRNAs or gRNAs are encoded by the exogenous DNA (e.g., by at least two HM-CRISPR arrays).

34. The use according to any one of claims 2 to 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30 and 32, wherein the nucleic acid vector comprises an exogenous DNA sequence exceeding 1.4 kb, the exogenous DNA encoding one or more elements of the CRISPR / Cas system, and comprises a modified array for the expression of HM-crRNA or gRNA in a host cell, the exogenous sequence lacking a nucleotide sequence encoding a Cas nuclease that is cognate with the cRNA or gRNA; wherein at least two different cRNAs or gRNAs are encoded by the exogenous DNA (e.g., by at least two HM-CRISPR arrays).

35. The composition according to any one of claims 1, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, 29, 31, and 33, wherein the mixed population comprises Escherichia coli.

36. The use according to any one of claims 2 to 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, and 34, wherein the mixed population comprises Escherichia coli.

37. The composition according to any one of claims 1, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, 29, 31, 33, and 35, wherein the mixed population is a human microbiome and comprises Gram-negative and Gram-positive bacteria.

38. The use according to any one of claims 2 to 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, and 36, wherein the mixed population is a human microbiome and includes Gram-negative and Gram-positive bacteria.

39. The composition according to claim 37, wherein the primary bacterium is Escherichia coli.

40. The use according to claim 38, wherein the first bacterium is Escherichia coli.

41. The composition according to claim 37, wherein the host bacterium is Escherichia coli.

42. The use according to claim 38, wherein the host bacterium is Escherichia coli.

43. The composition according to any one of claims 1, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 26, 27, 29, 31, 33, 35, 37, 39 and 41, wherein the second bacterium is a Firmicutes cell.

44. The use according to any one of claims 2 to 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 30, 32, 34, 36, 38, 40 and 42, wherein the second bacterium is a Firmicutes cell.

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