A bacterial platform for delivering gene editing systems into eukaryotic cells

Abstract

A bacteria-mediated gene editing delivery platform that uses invasive, non-pathogenic bacteria to deliver gene editing cargoes, including CRISPR / Cas systems, to eukaryotic cells. The bacteria contain a prokaryotic expression cassette encoding the gene editing cargo.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 856,055, filed on June 01, 2019. Technical Field

[0003] The present invention relates to the delivery of gene editing systems to eukaryotic cells using a non-pathogenic bacterial delivery platform. Background Art

[0004] Gene editing is a form of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. The editing of a specific sequence in the genome can be used for research purposes, and by targeting mutations to specific genes or inserting functional genes into an organism and targeting them to replace defective genes associated with a pathogenic phenotype or a genetic disease for use in gene therapy. A common method for gene editing is to use engineered nucleases or synthetic nucleases. An example is CRISPR (clustered regularly spaced short palindromic repeats) / Cas or CRISPR / Cas systems, which are rapidly advancing the field of genome editing. Summary of the invention

[0005] The present invention provides a bacteria-mediated gene editing delivery platform, which uses invasive non-pathogenic bacteria to deliver gene editing cargoes including but not limited to CRISPR / Cas systems to eukaryotic cells, wherein the bacteria contain a prokaryotic promoter associated with a prokaryotic expression cassette encoding the gene editing cargo.

[0006] In one aspect, the present invention provides a method for producing and delivering a gene editing system including a nuclease-based gene editing system to a eukaryotic cell. The method includes the step of invading eukaryotic cells with invasive non-pathogenic bacteria. The bacteria contain qidongzi, and the plasmid has a prokaryotic expression cassette encoding a gene editing system. The prokaryotic expression cassette can be operated under the control of one or more prokaryotic promoters, such as T7, lacUV5, gapA, T5, recA, Ptac, Patac, pA1, lac, Sp6, araBad, trp, and hybrid promoters. These prokaryotic promoters can be inducible, constitutive, or a combination thereof. It is expected that in the case of multiple promoters on one or more plasmids in bacteria, different promoter types can be used based on the relative activity of the promoter in the system. By using different types of prokaryotic promoters, transcription can be customized to control the level of transcripts. By using prokaryotic promoters in bacteria, transcription of gene editing system components occurs in bacteria, rather than in target eukaryotic cells.

[0007] In a second aspect, the present invention provides a composition comprising bacteria for modifying the genome of a eukaryotic cell. The bacteria have a prokaryotic vector. The prokaryotic vector (or vector) may be a plasmid or multiple plasmids that use a prokaryotic promoter to control transcription from a plasmid. One or more plasmids may encode a CRISPR / Cas system having (a) at least one CRISPR-related (Cas) enzyme, (b) at least one tracr sequence, and (c) at least one CRISPR RNA (crRNA). The crRNA may have (i) at least one CRISPR direct repeat region and (ii) at least one spacer sequence that can hybridize with a target sequence in a eukaryotic cell. The plasmid encoding the CRISPR / Cas system may optionally include a nuclear localization signal sequence to facilitate nuclear transfer of the gene editing system to the nucleus of a eukaryotic cell. The bacteria are engineered to be invasive to eukaryotic cells. In this second aspect of the present invention, the bacteria produce a programmable nuclease system for directing CRISPR complex activity in the nucleus of a eukaryotic cell.

[0008] In a third aspect, the present invention provides a composition comprising the use of a bacterial delivery system to produce and deliver a gene editing system (gene editing cargo), so that invasion factors, invasin proteins (encoded by the inv gene) and listeriolysin O (LLO) proteins (encoded by the hlyA gene) can be used to enter eukaryotic cells. The bacterial system is engineered to produce and / or deliver the gene editing system to eukaryotic cells. The composition comprising the use of bacterial cells to produce and / or deliver the gene editing system may contain one or more prokaryotic plasmids with one or more prokaryotic promoters, one or more eukaryotic plasmids with one or more eukaryotic promoters, or any combination thereof. In this third aspect of the invention, the bacterial system used is able to attach to eukaryotic cells by expressing invasin proteins and escape from eukaryotic endosomes by expressing LLO proteins, thereby allowing the gene editing cargo to be released into the cytoplasm of eukaryotic cells and, where appropriate, transferred to the nucleus of eukaryotic cells.

[0009] In a fourth aspect, the present invention provides a composition for in vitro, in vivo or ex vivo engineering of eukaryotic cells to insert, remove or replace genes. For in vitro use, the bacterial system engineered to produce and deliver the gene editing system can be used to insert, remove or replace the target region on the eukaryotic cell genome and modify the target DNA in vitro. For example, the CRISPR motif or PAM sequence present on the target DNA in vitro (in living cells outside the living system) can be recognized by the CRISPR / Cas gene editing system produced by the prokaryotic plasmid and delivered to the eukaryotic cell by the bacterial cell. In another embodiment, the CRISPR motif or PAM sequence on the target DNA in vitro is recognized by the CRISPR / Cas gene editing system, which is transcribed and translated from the eukaryotic plasmid inside the target eukaryotic cell after bacterial cell delivery. These genome modification experiments can be performed in vitro, including but not limited to primary cells, immortalized cells, dividing and non-dividing cells, and hematopoietic stem cells / progenitor cells. These in vitro gene editing applications may include, but are not limited to, the purpose of studying diseases, the pathogenesis of specific gene modifications, infectious agents, genetic diseases, functional genomic screening, chromosome drawing, construction of disease models (including cancer models), and are used together with in vitro selection libraries, mismatch detection nuclease analysis, high-throughput atlas analysis, diagnosis, and related next-generation sequencing activities. For ex vivo genome editing and modification, eukaryotic cells are separated from living systems, and a bacterial system engineered to produce and deliver a gene editing system is used to insert, remove, or replace the target region on the isolated eukaryotic cell genome, and to modify the target DNA in vitro. For example, CRISPR motifs or PAM sequences present in vitro (in cells separated from living systems) can be recognized by CRISPR / Cas gene editing systems produced by prokaryotic plasmids and delivered in vitro to eukaryotic cells by bacterial cells. In another embodiment, CRISPR motifs or PAM sequences present in ex vivo DNA are recognized by CRISPR / Cas gene editing systems transcribed and translated from eukaryotic plasmids inside target eukaryotic cells after bacterial cell delivery. In some cases, the present invention is used to achieve treatment success, wherein the separated cells are processed and returned to the target tissue to be transplanted back into the living system from which the eukaryotic cells are derived. An example of ex vivo (or in vitro) application is for chimeric antigen receptor (CAR) T cell therapy, wherein T cells derived from the patient's blood are extracted and the genome of the separated T cells is edited using the present invention to express an artificial receptor targeting a specific tumor antigen. For in vivo genome editing and modification, the bacterial system engineered to produce and deliver the gene editing system can be used to insert, remove or replace the target region on the eukaryotic cell genome to achieve specific genome modifications of DNA in a living system. In some cases, the present invention is used to deliver the gene editing system to target tissues (e.g., eyes, muscles, lungs, brain, nasal cavity, gastrointestinal tract, heart, skin, oral cavity, genital tissues, or any combination thereof).For example, the CRISPR motif or PAM sequence present in vivo can be recognized by the CRISPR / Cas gene editing system produced by the prokaryotic plasmid and delivered to the target eukaryotic cell by the bacterial cell in vivo. In another embodiment, the CRISPR motif or PAM sequence present in the DNA in vivo is recognized by the CRISPR / Cas gene editing system, which is transcribed and translated from the eukaryotic plasmid inside the target eukaryotic cell after being delivered to a specific tissue by the bacterial cell. These in vivo gene editing applications may include but are not limited to studying the pathogenesis of diseases, specific gene modifications, infectious agents, genetic diseases, developing gene knock-ins and knock-out animals (including the generation of transgenic embryos), for the purpose of treating diseases, preventing diseases and treating rare genetic diseases.

[0010] In a fifth aspect, the present invention provides a composition comprising the use of a bacterial delivery system to produce and deliver a gene editing system (gene editing cargo), wherein the elements comprising the gene editing system are expressed from a recombinant plasmid, expressed from a bacterial chromosome (integrated into a bacterial chromosome), or expressed using any combination thereof. Similarly, the invasion factors (inv and hlyA) required to enter eukaryotic cells and escape from eukaryotic cell endosomes are expressed from recombinant plasmids, expressed from bacterial chromosomes, or expressed using any combination thereof. Any component of the gene editing system and any factor required for bacterial invasion can be expressed in combination from one or more recombinant plasmids, from bacterial chromosomes, or from any combination thereof.

[0011] In a sixth aspect, the invention provides a plasmid containing one or more of a Cas gene, an NLS, a CRISPR direct repeat region, a spacer sequence (or an insertion site for a spacer sequence), and a tracr sequence. These elements can be expressed from a plasmid, for example, by one or more prokaryotic promoters when transforming bacterial cells. In an advantageous embodiment, the plasmid contains a spacer sequence (or an insertion site for a spacer sequence) and a prokaryotic or eukaryotic promoter. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] For a more complete understanding of the present invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings, in which:

[0013] Figure 1 is a graphic representation of a bacteria-mediated gene editing delivery system that uses invasive, non-pathogenic bacteria to deliver gene editing cargoes such as CRISPR / Cas systems to eukaryotic cells. The bacteria may contain a prokaryotic expression cassette encoding the gene editing cargo.

[0014] FIG. 2 is a series of schematic diagrams of plasmid systems used to genetically engineer bacteria to produce and deliver gene editing systems in one or more embodiments of the present invention ( Figure 2A , Figure 2B and Figure 2C ). Figure 2A Schematic diagram of a putative sequence-length CRISPR-mediated gene editing plasmid (pSiCRISP) containing at least one prokaryotic promoter, an origin of replication, a selectable marker, a crRNA leader, at least one spacer / crRNA homologous to a target DNA gene of interest, tracr / tracrRNA, CRISPR direct repeat sequences, a nuclear localization signal (NLS), and a Cas gene. Figure 2B is a schematic diagram of the putative sequence length invasion plasmid (pSiVEC), which contains at least one prokaryotic promoter, an origin of replication, a selectable marker, at least one invasion factor for receptor-mediated endocytosis (invasion factor A), and at least one invasion factor for endosomal escape (invasion factor B). Figure 2C Schematic diagram of a putative sequence-length CRISPR-mediated gene editing plasmid (pSiCRISP) containing at least one eukaryotic promoter, an origin of replication, a selectable marker, a crRNA leader, at least one spacer / crRNA homologous to a target DNA gene of interest, tracr / tracrRNA, CRISPR direct repeats, a nuclear localization signal (NLS), and a Cas gene.

[0015] Figure 3 are three images of FEC19 E. coli co-transformed with pSiCRISP_GFP and pSiVEC ( Figure 3 A. Figure 3 B and Figure 3 C), by using specific primer pairs for PCR ( Figure 3 A) 424bp NLS, ( Figure 3 B) 727bp Cas9 and ( Figure 3 C) PCR amplification of the 201 bp inv gene expression cassette was used to perform PCR screening on the FEC19 E. coli to verify the presence of pSiCRISP_GFP and pSiVEC components.

[0016] Figure 4 It is a graph showing the proportion of A549 cells expressing GFP. DETAILED DESCRIPTION

[0017] Gene editing is a form of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. The editing of specific sequences in the genome can be used for research purposes, and for gene therapy by targeting mutations to specific genes or inserting functional genes into organisms and targeting them to replace defective genes associated with pathogenic phenotypes or genetic diseases. A common method for gene editing is to use engineered nucleases or synthetic nucleases. An example is the CRISPR (clustered regularly spaced short palindromic repeats) system, which is rapidly advancing the field of genome editing.

[0018] CRISPR / CRISPR-associated proteins (Cas) have been used to edit the genomes of various model organisms such as bacteria, yeast, Caenorhabditis elegans, Drosophila melanogaster, plants, zebrafish, and mouse and human cells (Mali et al. "Cas9 as a versatile tool for engineering biology" Nat. Methods. 10: 957-963 (2013)). CRISPR / Cas systems have been delivered to these model organisms using a range of delivery mechanisms and vectors. More specifically, CRISPR / Cas systems have been delivered to eukaryotic cells using numerous delivery methods including viral vectors and non-viral vectors.

[0019] Despite advances in the field, efficient delivery of gene editing nuclease systems, including CRISPR / Cas systems, to target cells in vitro and in vivo remains a challenge. There are multiple disadvantages associated with current CRISPR / Cas system delivery strategies that are hampering the clinical translation of gene editing applications and therapies in human and animal medicine. We have developed a novel bacterial delivery platform for efficiently delivering any gene editing nuclease system, including a CRISPR / Cas system, to target eukaryotic cells, thereby providing cellular uptake and, where appropriate, nuclear translocation of the gene editing nuclease system without host genome integration for transient gene editing applications.

[0020] The present invention provides a novel bacterial delivery platform for gene editing systems including CRISPR systems to provide tissue-specific delivery and cellular internalization of genome editing components in any eukaryotic cell (dividing and non-dividing) without eukaryotic cell genome integration.

[0021] Gene editing methods are generally characterized by using a nuclease-based system (i.e., CRISPR / Cas) to effectively solve various gene editing applications, including prevention of infectious diseases, chronic non-infectious diseases, and other genetic diseases. Typically, these nucleases produce site-specific double-strand breaks at the desired position in the genome. The induced double-strand breaks are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations of the genome. An example of a nuclease-based gene editing system is the CRISPR system. Other examples include, but are not limited to, transcription activator-like effector nucleases (TALEN), meganucleases, zinc finger nucleases (ZFN), ARC nucleases, nucleases based on the Argonaute system, TtAgo nuclease systems, and other homing endonucleases.

[0022] The CRISPR system consists of two important components. The first component is a single guide RNA oligonucleotide (sgRNA) or a guide RNA (gRNA), which represents the fusion of CRISPR RNA (crRNA) and tracrRNA. The second component is the Cas nuclease. These two components are combined to represent sgRNA / gRNA and CRISPR-related proteases / nucleases. When the protospacer-adjacent motif (PAM) is present in the genomic locus on the target DNA molecule, the Cas nuclease is guided to the PAM locus by gRNA, and site-specific double-strand breaks occur in DNA to induce stable gene editing events. This gene editing event may lead to permanent gene knockout (gene removal) or knock-in (gene insertion).

[0023] There are 3 main types of CRISPR / Cas systems: I, II, and III. The three types differ in their gene editing mechanisms, although all three are thought to be applicable to eukaryotic cells. The most commonly used CRISPR / Cas system in eukaryotes is type II, which requires a single Cas protein, Cas9. Similar to CRISPR / Cas, other gene editing systems provide desired gene mutations or genome modification events.

[0024] For clinical and research applications, these gene editing nuclease proteins and gene editing systems are difficult to deliver. For example, due to the large size of Cas9 protein, the positive charge of Cas9 and the strong negative charge of sgRNA, it is difficult for existing systems to effectively co-deliver Cas9 / sgRNA complexes to host eukaryotic cells and pass through the cell membrane. Cas9, sgRNA and Cas9 / sgRNA complexes cannot pass through the lipid bilayer of the cell membrane without a delivery vector. One method of implementing CRISPR / Cas gene editing is to use plasmids to deliver Cas9 protein and sgRNA. sgRNA is integrated into the plasmid together with the sgRNA scaffold and the Cas9 box. The plasmid can also include a nuclear localization signal (NLS) for the gene editing components delivered to the nucleus. For example, sgRNA and Cas9 protein may require NLS to allow both to enter the nucleus from the cytoplasm for transcription. The present invention uses a plasmid with a prokaryotic promoter to drive the expression of a gene editing system located in a bacterial cell for delivery to eukaryotic cells. In the present invention, a plasmid expressed by a prokaryotic promoter-driven gene editing system (i.e., direct repeats, tracr / tracrRNA, spacer, Cas9, and NLS) is transformed into a non-pathogenic bacterial cell. In addition, bacterial cells are engineered to be invasive so that they can enter eukaryotic cells by receptor-mediated endocytosis. In certain embodiments, the combined construct of bacteria and plasmids together with the gene editing cargo expressed under the control of a prokaryotic promoter constitutes a bacterial-mediated gene editing delivery system to be delivered to eukaryotic cells.

[0025] Compared with other gene editing delivery methods, the bacterial-mediated gene editing delivery platform taught herein provides many advantages. First, the system facilitates transient delivery. The bacterial delivery platform rapidly achieves the desired editing event, thereby minimizing off-target editing, improving DNA specificity, and reducing any adverse consequences due to prolonged CRISPR / Cas9 or other nuclease editing systems in cells.

[0026] Second, the system is non-immunogenic. The bacterial cells are engineered to evade antigen presenting cell recognition, and the system does not induce immune or other cytokine responses in the host. In contrast, antibodies can be formed against other delivery vehicles such as nanoparticles. Liposomes and viral vectors can induce immune responses.

[0027] Third, the system is non-integrative. In some embodiments, the expression of the gene editing components is derived only from prokaryotic promoters. This inhibits host genome integration and provides controlled delivery of CRISPR / Cas9 or other nuclease editing systems, and eliminates the risk of carcinogenesis and other unwanted side effects to the host. However, it is expected that the system can use plasmids with eukaryotic promoters and use non-pathogenic bacteria as delivery vectors for plasmids, which are then transcribed in target cells.

[0028] Fourth, the system is highly stable. The delivery platform is not inhibited by exposure to serum, proteases, and nucleases, allowing the bacterial vector to remain stable until it reaches its target site. Unlike other non-viral vectors, our bacterial delivery platform is not eliminated by phagocytic clearance, which further contributes to its stability.

[0029] Fifth, the system enables robust payload delivery. Despite the large size of the enzyme, the bacterial delivery platform is able to efficiently deliver Cas enzymes, such as Cas9. Other non-viral vectors have difficulty delivering sufficient concentrations of Cas9 to target tissues, but the system taught herein works through receptor-mediated endocytosis to enable efficient cellular internalization and endosomal release of bacterial vectors containing the delivered gene editing system.

[0030] Sixth, the system provides a large therapeutic window. The bacterial delivery platform can be administered over a range of doses to achieve gene editing effects without causing toxicity.

[0031] Seventh, the system can achieve effective delivery. The delivery platform can effectively enter eukaryotic cells through receptor-mediated endocytosis and effectively escape from the host endosome to facilitate the delivery of the gene editing system.

[0032] So far, it has been a challenge to effectively deliver gene editing and CRISPR / Cas systems to target cells in vivo. Current CRISPR / Cas delivery strategies are mainly based on mechanical methods (electroporation, hydrodynamic injection, microinjection) and viral vector delivery (lentivirus, adenovirus, adeno-associated virus). For clinical applications in animals or human patients, many of these methods cannot be easily converted. Non-viral delivery methods, such as liposomes and nanoparticles, can also be used. However, in order to obtain therapeutic efficacy, CRISPR / Cas systems need to be delivered more effectively and more specifically to avoid adverse side effects and safety issues caused by systemic administration, transport to undesirable tissues and host integration.

[0033] Although viral vectors have been used to deliver CRISPR / Cas9 systems in vitro and in vivo, their fundamental drawbacks, such as the risk of oncogenicity due to host genome integration, limited insert size, stimulation of unwanted immune responses, and difficulty in large-scale production, severely limit their further applications. Host genome integration using viral vector delivery can result in high-level and prolonged expression of Cas9 protein or other nucleases, leading to higher off-target effects and strong immune responses in the host. Alternative non-viral delivery systems for CRISPR / Cas are urgently needed. Non-viral vectors represent another potential option for CRISPR / Cas9 delivery. These include, but are not limited to, liposomes, nanoparticles, exosomes, microvesicles, cell-penetrating peptides (CPPs), and other lipids, polymers, and lipid nanoparticles. Although they are useful in vitro, complex physiological conditions pose obstacles to the use of these non-viral delivery methods in vivo.

[0034] The present invention provides a system and method for delivering gene editing or CRISPR / Cas systems to eukaryotic cells, using invasive non-pathogenic bacteria containing prokaryotic expression cassettes to drive the expression of those genes prior to delivery. In other words, the bacteria used in the present invention are both invasive and non-pathogenic, and contain a plasmid driven by a prokaryotic promoter that imparts invasive properties to the bacteria, thereby allowing the bacteria to enter eukaryotic cells.

[0035] One advantage of using bacteria containing prokaryotic expression cassettes is that the necessary gene editing cargo (oligonucleotides and nuclease proteins) is expressed only by bacteria. They are produced by bacteria prior to delivery. Once the bacterial cells are taken up and enter the cytoplasm of the host eukaryotic cells, the plasmid containing the gene editing genes and proteins cannot be expressed in the eukaryotic cells due to polymerase restriction. This prevents these gene editing genes and proteins and any other plasmid DNA from integrating into the host's genome. This is an advantage because genomic integration is associated with harmful side effects, including the inability to control the expression of these genes, carcinogenesis, and toxicity (Kim S et al. 2014. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Research 24: 1012–1019). The gene editing event (targeted DNA modification) only needs to occur once per DNA copy to drive permanent gene modification in all cell populations. Therefore, in some cases, it is an advantage to use a delivery system to transiently introduce a gene editing system in which the gene editing components (i.e., Cas9 and sgRNA) are not integrated into the host cell's genome. With integrative gene editing methods (i.e., viral vector delivery), DNA mutations continue to occur in all DNA copies because the gene editing components (Cas9 and sgRNA) are integrated into the target cell genome. Avoiding integration into the target cell genome provides controlled expression of the gene editing components and further limits toxicity and off-target mutagenesis in the target cell.

[0036] The present invention uses invasion factors (such as inv and hlyA) that allow bacteria to enter eukaryotic cells and delivers nuclease-based gene editing goods (such as CRISPR, Talens, zinc fingers are examples) to bacteria of eukaryotic cells. The use of bacteria containing plasmids with eukaryotic promoters can produce a system in which plasmids with gene editing systems are delivered, and gene editing goods are manufactured in eukaryotic cells. The system using prokaryotic promoters implies that bacteria are transcription and expression sites of gene editing systems. The present invention covers two delivery routes (such as using prokaryotic promoters and eukaryotic promoters), but the system using prokaryotic promoters provides some significant advantages, including ensuring that the system is not integrated into target cells.

[0037] The present invention will find a wide range of applications. For example, in diagnosis, genomic mutations associated with genetic diseases or rejection markers before detecting organ transplantation are detected. The present invention can be used to treat diseases (infectious bacteria and viruses and non-infectious), wherein exogenous DNA from infectious agents present in eukaryotic cells is targeted for mutation, CAR-T applications for cancer treatment, prevention of disease in cases where DNA modification is associated with reduced infectious phenotypes, treatment of genetic diseases (i.e., cystic fibrosis, Huntington's disease, fragile X syndrome, sickle cell anemia, and familial hypercholesterolemia) in cases where DNA mutations are associated with unwanted genetic phenotypes, and whole genome genetic screening in cases where it is important to identify functional genes or gene sequences that affect physiological effects.

[0038] The present invention further provides a method for chromosome painting in living cells. The system can be used to perform CRISPR imaging as a method for detecting chromatin dynamics in living cells. Using a catalytically inactive dCas9 fused to a fluorescent marker such as green fluorescent protein (GFP) (a dead Cas9 without nuclease activity binds to DNA but cannot cleave the DNA to which it binds), researchers convert dCas9 into a customizable DNA marker that is compatible with fluorescence microscopy in living cells. Alternatively, CRISPR gRNAs can be fused to protein-interacting RNA aptamers that can recruit specific RNA binding proteins (RBPs) labeled with fluorescent proteins to visualize target genomic sites. Further applications include the ability to produce transgenic animals, in which animals are genetically modified to produce a target genotype or phenotype, which can be used, for example, for scientific research applications, such as the study of genetics and disease, or can be used to produce, for example, agricultural animals resistant to specific diseases or agricultural species (plants) resistant to specific diseases or conditions.

[0039] Figure 1 A graphical representation of a bacterial-mediated gene editing delivery system is provided, which uses invasive non-pathogenic bacteria to deliver gene editing cargoes such as CRISPR / Cas systems to eukaryotic cells. In an exemplary system, bacteria contain prokaryotic expression cassettes encoding gene editing cargoes. A representation of the ability of the system to generate CRISPR / Cas9 gene editing cargoes and deliver them to mammalian cells is described, which is provided by non-limiting examples. The figure illustrates the operation of a bacterial-mediated gene editing delivery system when it works through multiple steps in the process of delivering and achieving gene editing of eukaryotic cells.

[0040] First, all parts of the gene editing plasmid, namely the CRISPR-associated protein 9 (Cas9) sequence, the trans-activating CRISPR RNA (tracrRNA), at least one CRISPR direct repeat sequence, a nuclear localization signal (NLS), and at least one spacer sequence, are transcribed in bacteria (invasive bacterial delivery vector). This transcription can occur before entering the target eukaryotic cell. When inside the bacteria, tracrRNA hybridizes with the direct repeat sequence of crRNA and is then processed into a mature single guide RNA (sgRNA) containing a single spacer sequence, while the Cas9 RNA transcript is translated into an active enzyme. These components (Cas9 enzyme and mature sgRNA) generate the CRISPR / Cas9 gene editing system. Bacteria can optionally express the rnc gene of RNase III from a recombinant plasmid or a bacterial chromosome. Secondly, the bacterial vector that produces the CRISPR / Cas9 gene editing system targets mammalian cells through invasion factors (such as invasin protein) and enters the cell through receptor-mediated endocytosis (RME). Third, the endosome is used for the decomposition of the second invasion factor such as LLO released by the endosome, thereby releasing the sgRNA (mature crRNA: tracrRNA complex) and the Cas9 enzyme for transfer to the eukaryotic nucleus under the guidance of the NLS. Fourth, once entering the nucleus, the sgRNA guides Cas9 to the DNA target consisting of the prototype spacer and the downstream prototype spacer adjacent motif (PAM) through the heteroduplex formation between the crRNA and the prototype spacer DNA. Fifth, Cas9 mediates the cleavage of the target DNA upstream of the PAM to produce a double-strand break in the prototype spacer. In the sixth and final step, the target DNA is permanently modified. This figure describes an example process for adjusting this bacterial platform to generate, deliver and guide CRISPR complex activity in the nucleus of eukaryotic cells for targeted gene editing.

[0041] Figure 2A and 2B A schematic representation of a plasmid system used to genetically engineer bacteria to produce and deliver a gene editing system in one or more embodiments of the present invention is provided. Figure 2A Schematic diagram of a putative sequence-length CRISPR-mediated gene editing plasmid (pSiCRISP) containing at least one prokaryotic promoter, replication origin, selection marker, crRNA leader, at least one spacer / crRNA homologous to a target DNA gene of interest, tracr / tracrRNA, CRISPR direct repeats, NLS, and Cas genes. In addition to the pSiCRISP plasmid that provides the gene editing system, the system also requires components that allow bacteria to be invasive to eukaryotic cells. Figure 2BSchematic diagram of an invasion plasmid (pSiVEC) of putative sequence length, containing at least one prokaryotic promoter, an origin of replication, a selectable marker, at least one invasion factor for receptor-mediated endocytosis (invasion factor A) and at least one invasion factor for endosomal escape (invasion factor B). These two plasmid systems can be co-transformed into bacteria. In another embodiment of the invention, the components of these two plasmids can be combined into a single recombinant plasmid for bacterial transformation. In another embodiment of the invention, the components of these two plasmids can be expressed from the chromosome of the bacteria.

[0042] The following examples are provided for the purpose of illustrating various embodiments of the present invention and these examples are not intended to limit the present invention in any way. The following examples and the methods described herein represent preferred embodiments, are exemplary, and are not intended to limit the scope of the present invention. Those skilled in the art will appreciate its changes and other uses contained in the spirit of the present invention limited by the scope of the claims.

[0043] Example 1 - Generation of invasive bacteria containing a pSiCRISP plasmid capable of directing gene editing activity:

[0044] The pSiCRISP_GFP plasmid represents a CRISPR complex guide plasmid of crRNA expressing green fluorescent protein (GFP), which was synthesized and transformed into Escherichia coli (FEC19). FEC19 bacteria are additionally engineered to be invasive to eukaryotic cells by co-transformation with pSiVEC containing inv and hlyA genes, which are used for invasin-mediated receptor-mediated endocytosis and LLO-mediated endosomal release, respectively. The resulting FEC19 colonies containing pSiCRISP-GFP and pSiVEC were inoculated onto brain heart infusion (BHI) agar containing appropriate antibiotics for selection. The resulting FEC19 bacterial colonies (labeled A, B, C, D, E, F, and G) were screened by PCR to demonstrate the successful transformation of pSiCRISP and pSiVEC plasmids in FEC19 bacteria as a method of verifying the compositions of various aspects of the present invention. Specific primers were used in the PCR analysis to amplify the pSiCRISP_GFP sequence specific for the Cas9 gene and NLS and the pSiVEC sequence specific for the inv gene. Primers ( Figure 3 A) To verify the presence of NLS (screening in colonies A, D, E, F and G), primers that amplify a 727 bp product ( Figure 3 B) Verify the presence of the Cas9 gene (screening in colonies A, B, C, D, E, and F) and use primers that amplify a 201 bp product ( Figure 3C) Verify the presence of the inv gene (screening in colonies A, D, E, and F). A 1kb gradient marker is used as a reference in the figure. The Cas9 and NLS sequences in FEC19+pSiCRISP_GFP (FEC19 transformed with pSiCRISP_GFP) are both present in colonies D, E, and F. The inv sequence is present in all colonies tested. For each colony (colony D, E, and F) that is positive for inv as well as Cas9 and NLS by PCR, a single positive clone is sequence verified to confirm the presence of pSiCRISP_GFP and pSiVEC and propagated. This experiment demonstrates the production of Escherichia coli (FEC19) containing a gene editing plasmid (pSiCRISP) and a plasmid (pSiVEC) containing an invasion factor for target cell entry. Bacterial stocks of D, E, and F were produced in BHI medium containing appropriate antibiotics for selection and grown to an optical density equal to 1.0 measured at a wavelength of 600nm (OD600). These FEC19+pSiCRISP_GFP+pSiVEC stock solutions (D, E, F) were frozen at -80°C in 20% glycerol. A single frozen aliquot from each stock solution was thawed for plate counting. In short, 1 mL aliquots were centrifuged at 5000xg for 5 minutes, and the cells were then resuspended in 1 mL BHI. The resulting bacterial suspension was serially diluted and inoculated in triplicate on BHI agar containing antibiotics. The colony counts for each dilution were averaged to calculate the total colony forming unit (CFU) / mL, which represents the feasible concentration of the FEC19+pSiCRISP_GFP+pSiVEC stock solution. This system allows the quantitative live inoculum stock solution to be used directly for all future analyses.

[0045] Example 2: CRISPR-mediated gene editing in mammalian cell lines using FEC19+pSiCRISP+pSiVEC:

[0046] Human alveolar basal epithelial (A549) cells stably expressing GFP were maintained in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum, 2mM GlutaMAX, 100U / mL penicillin and 100g / mL streptomycin at 37°C and 5% CO2. Invasive (transformed with pSiVEC) FEC19 bacteria containing the pSiCRISP_GFP plasmid are able to enter A549 cells through RME and deliver the CRISPR-mediated gene editing cargo required to knock out the target gene GFP. The experiment included a positive control treatment (FEC19+pSiVEC_shGFP), in which the pSiVEC plasmid expressed a short hairpin RNA (shRNA) targeting GFP. This positive control treatment was included because knocking down GFP in A549 cells using shRNA targeting GFP is desirable and has been previously demonstrated. The invasion assay includes the following steps. First, A549 cells were seeded into black 24-well plates at a fixed concentration. On the day of bacterial challenge, four FEC19 stocks were thawed: 1) FEC19+pSiCRISP_GFP+pSiVEC, 2) FEC19+pSiCRISP_GFP (non-invasive), 3) FEC19+pSiCRISP_scramble (expressing scrambled / nonsense crRNA)+pSiVEC, and 4) FEC19+pSiVEC_shGFP (shRNA expressing GFP). FEC19 bacterial stocks were serially diluted 1:10 in DMEM, starting from 1x10 7 CFU / mL diluted to 1x10 5 CFU / mL. A549 cells were incubated with 1 mL of each FEC19 treatment for 2 hours and subsequently rinsed with DMEM to remove unbound FEC19 bacteria. GFP expression was quantified over a 72 hour time course using a Nexcelom Celigo, with T0 readings taken immediately before invasion. Figure 4 Compared with untreated A549 cells, the 7 The proportion of A549 cells expressing GFP after incubation with one of the four FEC19 treatments at 0, 24, 48, and 72 hours post-treatment.

[0047] Glossary

[0048] All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless the technical or scientific terms are defined differently herein.

[0049] The terms used herein are only for the purpose of describing specific embodiments and are not intended to limit any embodiment. As used herein, the singular forms "one", "an" and "said" are intended to also include plural forms unless the context clearly indicates otherwise. It will be further understood that when used in this specification, the terms "comprises" and / or "comprising" specify the presence of the features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or their groups. As used herein, the term "and / or" includes any and all combinations of one or more of the items listed in the relevant list. When used to define products, compositions and methods, "essentially consisting of..." refers to excluding other components or steps of any importance. Therefore, the composition consisting essentially of the components does not exclude trace contaminants and pharmaceutically acceptable carriers. "Consisting of..." should mean that trace elements of other components or steps are also excluded.

[0050] The terms "polynucleotide" and "nucleic acid" used interchangeably herein refer to polymeric forms of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. "Oligonucleotide" generally refers to a polynucleotide of about 5 to about 100 nucleotides of single-stranded or double-stranded DNA. However, for the purposes of this disclosure, there is no upper limit to the length of the oligonucleotide. Oligonucleotides are also referred to as "oligomers" or "oligos" and can be isolated from genes or chemically synthesized by methods known in the art. When applicable to the described embodiments, the terms "polynucleotide" and "nucleic acid" are understood to include single-stranded (e.g., sense or antisense) and double-stranded polynucleotides. The terms "nucleic acid", "polynucleotide" or "oligonucleotide" refer to DNA molecules, RNA molecules or their analogs. As used herein, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" include, but are not limited to, DNA molecules such as cDNA, genomic DNA or synthetic DNA, and RNA molecules such as guide RNA, messenger RNA or synthetic RNA. In addition, as used herein, the terms "nucleic acid" and "polynucleotide" include single-stranded and double-stranded forms.

[0051] "Genomic DNA" refers to the DNA of the genome of an organism, including but not limited to the genome of bacteria, viruses, fungi, archaea, plants or animals.

[0052] "Manipulation" of DNA includes binding, cleaving one strand, or cleaving (i.e., cleaving) both DNA strands, or includes modifying DNA or polypeptides associated with DNA (e.g., amidation, methylation, etc.). Manipulation of DNA can silence, activate, or modulate (increase or decrease) the expression of RNA or polypeptides encoded by the DNA.

[0053] "Gene editing" refers to the process of changing the genetic information present in the genome of a cell. This gene editing can be performed by manipulating genomic DNA, resulting in the modification of the genetic information. This gene editing may or may not affect the expression of the edited DNA.

[0054] As used herein, the term "guide RNA" generally refers to an RNA molecule (or a group of RNA molecules) that can bind to Cas protein and help target Cas protein to a specific position in a target polynucleotide (e.g., DNA). Guide RNA can include crRNA segments and tracrRNA segments. As used herein, the term "crRNA" or "crRNA segment" refers to an RNA molecule or part thereof including a guide sequence, a stem sequence, and an optional 5'-overhang sequence for a targeting polynucleotide. As used herein, the term "tracrRNA" or "tracrRNA segment" refers to an RNA molecule or part thereof including a protein binding segment (e.g., a protein binding segment can interact with CRISPR-associated proteins such as Cas9). The term "guide RNA" includes a single guide RNA (sgRNA), wherein the crRNA segment and the tracrRNA segment are located in the same RNA molecule. The term "guide RNA" also generally includes a group of two or more RNA molecules, wherein the crRNA segment and the tracrRNA segment are located in separate RNA molecules.

[0055] The term "modification" in the context of an oligonucleotide or polynucleotide includes, but is not limited to, (a) terminal modifications, such as 5' terminal modifications or 3' terminal modifications, (b) nucleobase (or "base") modifications, including substitution, insertion or removal of bases, (c) sugar modifications, including modifications at the 2', 3' and / or 4' positions, and (d) backbone modifications, including modification or substitution of phosphodiester bonds. The term "modified nucleotide" generally refers to a nucleotide having a modification to the chemical structure of one or more of the base, sugar and phosphodiester bond or backbone moiety, including the nucleotide phosphate.

[0056] As used more generally herein, "modification" refers to a part of a chemical part or chemical structure that is different from unmodified ribonucleotides, i.e., those found in adenosine, guanosine, cytidine, and uridine ribonucleotides. The term "modification" may refer to the type of modification. For example, "same modification" means that the modification type is the same, and "modified nucleotides are the same" means that the modified nucleotides have the same type of modification, while the bases (A, G, C, U, etc.) may be different. Similarly, a joint with "two modifications" is a guide RNA with two types of modifications, which may or may not be in the same nucleotide, and each type may appear in multiple nucleotides in the joint. Similarly, a joint polynucleotide with "three modifications" is a joint with three types of modifications, which may or may not be in the same nucleotide, and each type may appear in multiple nucleotides.

[0057] A "donor polynucleotide" is a nucleotide polymer or oligomer that is intended to be inserted into a target polynucleotide. The donor polynucleotide can be a natural or modified polynucleotide, an RNA-DNA chimera or a DNA fragment (single-stranded or double-stranded), or a PCR-amplified ssDNA or dsDNA fragment. A fully double-stranded donor DNA is advantageous because it can provide greater stability, as dsDNA fragments are generally more resistant to nuclease degradation than ssDNA.

[0058] The terms "xA", "xG", "xC", "xT" or "x(A,G,C,T)" and "yA", "yG", "yC", "yT" or "y(A,G,C,T)" refer to nucleotides, nucleobases or nucleobase analogs described in Krueger et al., "Synthesis and Properties of Size-Expanded DNAs: Toward Designed, Functional Genetic Systems", Acc. Chem. Res. 2007, 40, 141-150 (2007), the contents of which are incorporated herein by reference in their entirety.

[0059] As used herein, the term "target polynucleotide" or "target" refers to a polynucleotide containing a target nucleic acid sequence. The target polynucleotide can be single-stranded or double-stranded, and in certain embodiments, is double-stranded DNA. In certain embodiments, the target polynucleotide is a single-stranded RNA. As used herein, "target nucleic acid sequence" or "target sequence" refers to a specific sequence or its complementary sequence that one wishes to bind, cut or cleave using a CRISPR / Cas system or other gene editing system.

[0060] The term "hybridization" or "hybridizing" refers to the process in which fully or partially complementary polynucleotide chains are brought together to form a double-stranded structure or region under suitable hybridization conditions, wherein the two constituent chains are connected by hydrogen bonds. As used herein, the term "partial hybridization" includes that a double-stranded structure or region comprises one or more protrusions or mispairings. Although hydrogen bonds are usually formed between adenine and thymine or adenine and uracil (A and T or A and U) or cytosine and guanine (C and G), other non-standard base pairs may also be formed (see, for example, Adams et al., The Biochemistry of the Nucleic Acids, 11th edition, 1992). It is expected that the modified nucleotides can form hydrogen bonds that allow or promote hybridization.

[0061] "Hybridizable" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA) comprises a nucleotide sequence that enables it to non-covalently bind (i.e., form Watson-Crick base pairs and / or G / U base pairs, "anneal" or "hybridize") to another nucleic acid in a sequence-specific antiparallel manner under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength (i.e., nucleic acid specifically binds to a complementary nucleic acid). As is known in the art, standard Watson-Crick base pairing includes: adenine (A) pairs with thymidine (T), adenine (A) pairs with uracil (U), and guanine (G) pairs with cytosine (C). In addition, it is also known in the art that for hybridization between two RNA molecules (e.g., dsRNA), guanine (G) base pairs with uracil (U). For example, in the case of base pairing of tRNA anticodons with codons in mRNA, G / U base pairing is part of the reason for the degeneracy (i.e., redundancy) of the genetic code. In the context of the present disclosure, guanine (G) of the protein-binding segment (dsRNA duplex) of the guide RNA molecule is considered to be complementary to uracil (U), and vice versa. Thus, when a G / U base pair can be formed at a given nucleotide position of the protein-binding segment (dsRNA duplex) of the guide RNA molecule, the position is not considered to be non-complementary, but rather complementary.

[0062] Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, EF, and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 thereof; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0063] Hybridization requires that two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions applicable to hybridization between two nucleic acids depend on the length and complementarity of the nucleic acid, which are variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the melting temperature (Tm) value of the nucleic acid hybrid with these sequences. For hybridization between nucleic acids with short complementary fragments (for example, complementarity on nucleotides of 35 or less, 30 or less, 25 or less, 22 or less, 20 or less or 18 or less), the position of mismatch becomes very important (see Sambrook et al., as above, 11.7-11.8). Generally, the length of hybridizable nucleic acids is at least about 10 nucleotides. The exemplary minimum length of hybridizable nucleic acids is: at least about 15 nucleotides; at least about 20 nucleotides; at least about 22 nucleotides; at least about 25 nucleotides; and at least about 30 nucleotides). In addition, it will be appreciated by those skilled in the art that the temperature and washing solution salt concentration can be adjusted as needed according to factors such as the length of the complementary region and the degree of complementarity.

[0064] It should be understood in the art that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid to be specifically hybridizable or hybridizable. In addition, the polynucleotide can hybridize on one or more sections so that the intervening section or adjacent section does not participate in the hybridization event (e.g., loop structure or hairpin structure). The polynucleotide can have at least 55%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100% sequence complementarity with the target region within the target nucleic acid sequence it targets. For example, an antisense nucleic acid wherein 18 of the 20 nucleotides of the antisense compound are complementary to the target region and thus specifically hybridize will represent 90% complementarity. In this example, the remaining non-complementary nucleotides can be clustered or interspersed with complementary nucleotides, and need not be adjacent to each other or adjacent to complementary nucleotides. The percent complementarity between specific segments of nucleic acid sequences within a nucleic acid can be determined using the BLAST programs (Basic Local Alignment Search Tool) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings and the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0065] The term "cleavage" or "cleaving" refers to the breaking of covalent phosphodiester bonds in the ribosyl phosphodiester backbone of a polynucleotide. The term "cleavage" or "cleaving" includes single-strand breaks and double-strand breaks. Double-strand cleavage may be the result of two different single-strand cleavage events. Cleavage may result in the generation of blunt ends or staggered ends.

[0066] The term "CRISPR-associated protein" or "Cas protein" refers to a wild-type Cas protein, a fragment thereof, or a mutant or variant thereof. The term "Cas mutant" or "Cas variant" refers to a protein or polypeptide derivative of a wild-type Cas protein, such as a protein having one or more point mutations, insertions, deletions, truncations, fusion proteins, or a combination thereof. In certain embodiments, the "Cas mutant" or "Cas variant" substantially retains the nuclease activity of the Cas protein. In certain embodiments, the "Cas mutant" or "Cas variant" is mutated so that one or both nuclease domains are inactivated. In certain embodiments, the "Cas mutant" or "Cas variant" has nuclease activity. In certain embodiments, the "Cas mutant" or "Cas variant" lacks some or all of the nuclease activity of its wild-type counterpart.

[0067] A synthetic guide RNA with "gRNA functionality" is a synthetic guide RNA with one or more functions of a naturally occurring guide RNA (e.g., association with a Cas protein, or a function performed by association of a guide RNA with a Cas protein). In certain embodiments, functionality includes binding to a target polynucleotide. In certain embodiments, functionality includes targeting a Cas protein or a gRNA:Cas protein complex to a target polynucleotide. In certain embodiments, functionality includes cutting a target polynucleotide. In certain embodiments, functionality includes acting within a gRNA:Cas system to cleave a target polynucleotide. In certain embodiments, functionality includes association or binding with a Cas protein. In certain embodiments, functionality is any other known function of a guide RNA in a CRISPR / Cas system (including an artificial CRISPR / Cas system with an engineered Cas protein) having a Cas protein. In certain embodiments, functionality is any other function of a natural guide RNA. Compared to a naturally occurring guide RNA, a synthetic guide RNA may have more or less gRNA functionality. In certain embodiments, a synthetic guide RNA may have greater functionality with respect to one characteristic and less functionality with respect to another characteristic.

[0068] As used herein, the term "segment" of a sequence refers to any portion of a sequence (e.g., a nucleotide subsequence or an amino acid subsequence) that is less than the entire sequence. A segment of a polynucleotide can be of any length, such as at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, or 500 or more nucleotides in length. A portion of a guide sequence can be about 50%, 40%, 30%, 20%, 10% of a guide sequence, such as one-third of a guide sequence or less, such as 7, 6, 5, 4, 3, or 2 nucleotides in length.

[0069] The term "derived from" in the context of a molecule refers to a molecule that is isolated or prepared using a parent molecule or information from the parent molecule. For example, Cas9 single mutant nickase and Cas9 double mutant null nuclease are derived from wild-type Cas9 protein.

[0070] In the context of two or more polynucleotides (or two or more polypeptides), the term "substantially identical" refers to sequences or subsequences having at least about 60%, at least about 70%, at least about 80%, at least about 90%, about 90-95%, at least about 95%, at least about 98%, at least about 99% or more nucleotide (or amino acid) sequence identity when compared and aligned for maximum counterparts using a sequence comparison algorithm or by visual inspection. Preferably, "substantial identity" between polynucleotides exists over a region of at least about 50 nucleotides in length, at least about 100 nucleotides in length, at least about 200 nucleotides in length, at least about 300 nucleotides in length, at least about 500 nucleotides in length of the polynucleotide, or over the entire length of the polynucleotide. Preferably, "substantial identity" between polypeptides exists over a region of at least about 50 amino acid residues in length, at least about 100 amino acid residues in length of the polypeptide, or over the entire length of the polypeptide.

[0071] As disclosed herein, multiple value ranges are provided. It should be understood that each intermediate value (up to one tenth of the lower limit unit) between the upper and lower limits of the range is also specifically contemplated. Each smaller range or intermediate value covered by the range is also specifically contemplated. The term "about" generally refers to plus or minus 10% of a specified number. For example, "about 10%" can represent a range of 9% to 11%, and "about 20" can represent 18-22. Other meanings of "about" can be clearly seen from the context, such as rounding, so, for example, "about 1" can also represent from 0.5 to 1.4.

[0072] A DNA sequence that "encodes" a specific RNA is a DNA nucleic acid sequence that is transcribed into RNA. A DNA polynucleotide can encode an RNA (mRNA) that is translated into a protein, or a DNA polynucleotide can encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, or guide RNA; also referred to as "non-coding" RNA or "ncRNA"). A "protein coding sequence" or a sequence encoding a specific protein or polypeptide is a nucleic acid sequence that is transcribed into mRNA (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of an appropriate regulatory sequence. The boundaries of the coding sequence are determined by the start codon at the 5' end (N-terminus) and the translation termination nonsense codon at the 3' end (C-terminus). The coding sequence may include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic nucleic acids. The transcription termination sequence is usually located at the 3' of the coding sequence.

[0073] As used herein, a "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. For the purposes of defining the present invention, a promoter sequence is bounded at its 3" terminus by a transcription start site and extends upstream (5' direction) to include the minimum number of bases or elements required to initiate transcription at a detectable level above background. Within the promoter sequence, a transcription start site will be found, as well as protein binding domains responsible for binding RNA polymerase. Various promoters, including inducible promoters, can be used to drive the vectors described in the present disclosure.

[0074] A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active ("ON") state), or it can be an inducible promoter (i.e., a promoter whose activity ("ON") or inactivity ("OFF") state is controlled by an external stimulus, such as the presence of a specific temperature, compound, or protein).

[0075] In the prior art, suitable promoters for expressing Cas proteins have been derived from viruses (and therefore can be referred to as viral promoters), and promoters for expressing Cas9 proteins in bacteria are also preferred. It is also possible to use a promoter known to drive Cas9 expression in one bacterium to drive the expression of Cas9 proteins derived from different bacteria (species). In this case, the promoter is referred to as heterologous relative to the Cas9 protein. The term "prokaryotic promoter" refers to a promoter that is active in bacteria to transcribe sequences. Bacteria can be further engineered to express bacteriophage RNA polymerase, thereby facilitating the use of phage promoters. In some aspects, the use of phage promoters can be beneficial for driving bacteria to express toxic genes. For example, the use of phage promoters and placing them upstream of toxic genes can make or force bacteria to produce large amounts of gene editing nucleases that may be toxic.

[0076] Invasive microorganisms

[0077] As used herein, the term "invasive" refers to the ability to deliver at least one molecule, such as RNA or a DNA molecule encoding RNA, to a target cell when it relates to a microorganism, such as a bacterium or a bacterial therapeutic particle (BTP). An invasive microorganism can be a microorganism that is able to penetrate a cell membrane, thereby entering the cytoplasm of the cell, and deliver at least a portion of its contents (e.g., RNA or DNA encoding RNA) to a target cell. The process of delivering at least one molecule to a target cell preferably does not significantly alter the invasion apparatus.

[0078] As used herein, the term "cross-kingdom" refers to a delivery system that uses bacteria (or another invasive microorganism) to produce the nucleic acid and deliver the nucleic acid intracellularly within the target tissue (i.e., across kingdoms: prokaryotes to eukaryotes, or across phyla: invertebrates to vertebrates) so that it can be processed without the need for host genome integration.

[0079] Invasive microorganisms include microorganisms that are naturally capable of delivering at least one molecule to a target cell (e.g., by passing through a cell membrane (e.g., a eukaryotic cell membrane) and entering the cytoplasm), as well as microorganisms that are naturally non-invasive and have been modified (e.g., genetically modified) to be invasive. In another preferred embodiment, microorganisms that are naturally non-invasive can be modified to be invasive by connecting bacteria or BTPs to "invasive factors" (also referred to as "entry factors" or "cytoplasm targeting factors"). As used herein, "invasive factors" are factors, such as proteins or a group of proteins, that make the bacteria or BTP invasive when expressed by non-invasive bacteria or BTPs. As used herein, "invasive factors" are encoded by "cytoplasm targeting genes". Invasive microorganisms have been generally described in the art, for example, U.S. Patent Publication Nos. US20100189691A1 and US20100092438 A1 and Xiang, S. et al., Nature Biotechnology 24, 697-702 (2006). Each of which is incorporated by reference in its entirety for all purposes.

[0080] In a preferred embodiment, as taught in the examples of the present application, the invasive microorganism is Escherichia coli. However, it is expected that other microorganisms may also be potentially suitable for serving as cross-border delivery vectors for delivering gene editing cargo. These non-toxic and invasive bacteria and BTPs will show invasiveness, or will be modified to show invasiveness, and may enter host cells through various mechanisms. In contrast to the uptake of bacteria or BTPs by professional phagocytes (which usually results in the destruction of bacteria or BTPs in special lysosomes), invasive bacteria or BTP strains have the ability to invade non-phagocytic host cells. Naturally occurring examples of such intracellular bacteria are Yersinia, Rickettsia, Legionella, Brucella, Mycobacterium, Helicobacter, Coxiella, Chlamydia, Neisseria, Burkolderia, Bordetella, Borrelia, Listeria, Shigella, (Shigella), Salmonella, Staphylococcus, Streptococcus, Porphyromonas, Treponema and Vibrio, but this property can also be transferred to other bacteria or BTPs, such as Escherichia coli, Lactobacillus, Lactococcus or Bifidobacterium, including probiotics (P. Courvalin, S. Goussard, C. Grillot-Courvalin, CR Acad. Sci. Paris 318, 1207 (1995)) through the transfer of invasion-related genes. Factors to be considered or addressed when evaluating other bacterial species as candidates for use as cross-kingdom delivery vectors include the pathogenicity or lack of pathogenicity of the candidate, the tropism of the candidate bacteria for the target cell, or the extent to which the bacteria can be engineered to deliver the gene editing cargo to the interior of the target cell, and any synergistic value that the candidate bacteria may provide by triggering the host's innate immunity.

[0081] A colony is defined as a visible group of microorganisms that all originate from a single parent cell, and thus colonies constitute genetically similar bacterial clones.

[0082] Promoter

[0083] Among the gene expression regulatory elements, promoters play a central role. Along the promoter molecule, the transcription machinery is assembled and transcription is initiated. This early step is often rate-limiting relative to the subsequent stages of protein production. Transcription initiation by promoters can be regulated in a variety of ways. For example, promoters can be induced by the presence of specific compounds or external stimuli, express genes at specific developmental stages, or express genes constitutively. Therefore, transcription of transgenics can be regulated by operably linking coding sequences to promoters with different regulatory properties. Therefore, regulatory elements such as promoters play a key role in improving the value of transgenic organisms.

[0084] As used herein, the term "promoter" refers to a polynucleotide molecule that participates in the recognition and binding of RNA polymerase and other proteins such as related sigma factors and activator proteins to initiate transcription of an operably connected gene. The promoter can be isolated from the 5' untranslated region (5'UTR) of the genomic copy of the gene. Alternatively, the promoter molecule can be artificially synthesized to produce or contain a modified DNA sequence. The promoter can be used as a regulatory element for regulating the expression of an operably connected transcribable polynucleotide molecule. The promoter itself may contain subelements that realize transcription of an operably connected gene, such as cis elements or enhancer domains.

[0085] In prokaryotes, the promoter consists of two short sequences located at positions -10 and -35 upstream of the transcription start site. The σ factor not only helps to enhance the binding of RNAP to the promoter, but also helps RNAP target those genes to be transcribed. The sequence at -10 is called the Pribnow box or -10 element, which usually consists of the six nucleotides TATAAT. The Pribnow box is absolutely necessary for the start of transcription in prokaryotes. The other sequence at -35 (the -35 element) usually consists of the six nucleotides TTGACA. Its presence allows very high transcription rates. The above two consensus sequences, although conserved on average, are not found intact in most promoters. On average, only 3 of the 6 base pairs of each consensus sequence are present in any given promoter. It is important to note that the above promoter sequences are only recognized by the σ-70 protein that interacts with prokaryotic RNA polymerase. Prokaryotic RNA polymerase and the complex with other σ factors recognize completely different core promoter sequences.

[0086] Many regulatory elements act in cis ("cis-elements") and are thought to affect DNA topology, thereby producing local conformations that selectively allow or restrict RNA polymerase access to the DNA template, or help to selectively open the double helix at the transcription start site. Cis-elements occur within the 5'UTR associated with a specific coding sequence, and are typically found in promoters and promoter regulatory sequences (inducible elements). Cis-elements can be identified using known cis-elements as target sequences or target motifs and using the BLAST program. Examples of cis-acting elements in the 5'UTR associated with a polynucleotide coding sequence include, but are not limited to, promoters and enhancers.

[0087] In prokaryotes, mRNA translation begins with UTG, GTG, or in rare cases with UUG, usually beginning with a sequence characteristic of a ribosome binding site (RBS; Shine and Dalgarno, PNAS 71:1342-1346, 1974). The RBS is rich in AG and is usually located 6-12 bp before the start codon. The RBS is considered essential for efficient mRNA translation in bacteria.

[0088] There are at least two types of information that can be used to predict promoter regions in genomic DNA sequences. First, promoters can be identified based on their sequence "content", such as transcription factor binding sites and various known promoter motifs. (Stormo, Genome Research 10: 394-397, 2000). These signals can be identified by computer programs that identify sites associated with promoters, such as TATA boxes and transcription factor (TF) binding sites. Second, promoters can be identified based on their "position", that is, their proximity to known or suspected coding sequences (Stormo). Prokaryotic promoters are typically present in a DNA region that extends about 1-500 base pairs in the 5' direction from the transcription or translation start codon of the coding sequence. Therefore, the promoter region can be identified by locating the translation start codon or transcription start site of the coding sequence and moving beyond the start codon in the 5' direction to locate the promoter region.

[0089] Promoter sequences can be analyzed for the presence of common promoter sequence features, such as the TATA box and other known transcription factor binding site motifs. These motifs are not always present in every known promoter, nor are they required for every promoter to function, but when present, they do indicate that the DNA segment is a promoter sequence.

[0090] The activity or intensity of the promoter can be measured according to the amount of mRNA, tRNA, dsRNA, miRNA, rRNA or protein accumulated specifically in a specific time period of cell growth containing a transgene. The production of commercially important proteins or proteins for catalytic reactions to produce commercially important compounds may require the production of mRNA with increased levels. The coding sequence of commercially important proteins can be identified by those skilled in the art and can be used together with the promoter of the present invention to affect the production and protein level of mRNA. When cells are grown under selective pressure after being transformed with, for example, a gene that allows the detoxification of compounds under the control of the promoter, the activity or intensity of the promoter can also be measured according to the enhanced cell growth.

[0091] A hybrid promoter is defined as a promoter generated by engineering bacteria or an expression cassette on a plasmid that has the -35 region of one promoter and the -10 region of another promoter.

[0092] Selectable marker

[0093] As used herein, the term "marker" refers to any transcribable polynucleotide molecule whose expression or lack can be screened or scored in some way. Marker genes used to implement the present invention include, but are not limited to, encoding β-glucuronidase (GUS described in U.S. Pat. No. 5,599,670, which is incorporated herein by reference), green fluorescent protein and its variants (GFP, RFP, etc. described in U.S. Pat. No. 5,491,084 and U.S. Pat. No. 6,146,826, all of which are incorporated herein by reference) or transcribable polynucleotide molecules of proteins that confer antibiotic resistance.

[0094] Useful antibiotic resistance markers, including those encoding proteins that confer resistance to kanamycin (nptII), hygromycin B (aph IV), streptomycin or spectinomycin (aad, spec / strep), and gentamicin (aac3 and aacC4) are known in the art. An advantageous positive selection marker is hisD (histidinol dehydrogenase). The hisD gene of Salmonella typhimurium encodes histinol dehydrogenase, which catalyzes the two-step NAD'-dependent oxidation of L-histidinol to L-histidine. In a medium lacking histidine and containing histinol, only cells expressing the hisD product can survive.

[0095] The term "selectable marker" also includes genes encoding secretable markers, the secretion of which can be detected as a means of identifying or selecting transformed cells. Examples include markers encoding secretable enzymes that can be catalytically detected. Other possible selectable marker genes will be apparent to those skilled in the art.

[0096] In a specific embodiment, the selectable marker used can be hisD, green fluorescent protein (GFP), neomycin phosphotransferase II (nptII), luciferase (LUX) or an antibiotic resistance coding sequence (aadA). In certain embodiments, the selectable marker is a kanamycin, streptomycin and / or spectinomycin resistance marker. Examples of coding sequences providing tolerance to antibiotics and herbicides can be found in, for example, U.S. Patent Application Publications 20080305952 and 20080280361, which are incorporated herein by reference.

[0097] Cell transformation

[0098] The term "transformation" refers to the introduction of nucleic acids into a recipient host. The term "host" refers to a prokaryotic cell, particularly a bacterial cell. As used herein, the term "transformed" refers to a cell or organism into which an exogenous polynucleotide molecule, such as a construct, has been introduced. The introduced polynucleotide molecule can be integrated into the genomic DNA of the recipient cell or organism so that the introduced polynucleotide molecule is inherited by subsequent offspring or can remain in the cytoplasm as a self-replicating unit. A "transgenic" or "transformed" cell or organism also includes the offspring of the cell or organism. The term "transgenic" refers to a cell or other organism containing one or more heterologous polynucleic acid molecules.

[0099] There are many methods for introducing heterologous polynucleic acid molecules into cells. The method generally includes the steps of selecting a suitable host cell, transforming the host cell with a recombinant vector and obtaining the transformed host cell. Suitable methods for introducing heterologous polynucleic acid molecules into prokaryotes include freeze-thaw (heat shock), triparental mating and electroporation, etc. These methods are known to those skilled in the art of prokaryotic transformation.

[0100] Reagent test kit

[0101] Also provided are kits for implementing the methods of the present invention. "Kit" means any preparation (e.g., packaging or container) that includes at least one reagent, such as the gene-edited bacteria of the present invention. The kit can be promoted, distributed, or sold as a unit for implementing the methods of the present invention. In addition, the kit may also contain a package insert describing the kit and its method of use. Any or all of the kit reagents may be provided in a container that isolates it from the external environment, such as in a sealed container or pouch.

[0102] With respect to the compounds of the invention, the term "administering" and variants thereof (e.g., "administering" a compound) refers to introducing the compound into the system of a subject in need of treatment. When the compounds of the invention are provided in combination with one or more other active agents (e.g., AIV vaccines, etc.), "administering" and variants thereof are each understood to include both simultaneous and sequential introduction of the compound and other agents.

[0103] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0104] As used herein, the term "therapeutically effective amount" refers to the amount of an active compound or agent that causes a biological or medical response in a tissue, system, animal or human that is sought by a researcher, veterinarian, doctor or other clinician. With regard to viral infections, an effective amount includes an amount sufficient to prevent infectious diseases or mitigate the severity of the disease, as demonstrated by clinical disease, clinical symptoms, viral titers, or viruses shed from a subject, or as demonstrated by the ability to prevent or reduce transmission between animals. In some embodiments, an effective amount is an amount sufficient to delay clinical disease and / or symptom onset or prevent disease. In some embodiments, an effective amount is an amount sufficient to reduce viral titers and / or reduce viral shedding. An effective amount can be administered in one or more doses.

[0105] As used herein, "treatment" refers to obtaining a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, any one or more of the following: alleviating one or more symptoms, reducing the extent of viral infection, stabilizing (i.e., not aggravating) the viral infection state, preventing or delaying the spread of viral infection (e.g., shedding), preventing, delaying, or slowing the progression of viral infection, and / or maintaining body weight / weight gain. The methods of the present invention encompass any one or more of these therapeutic aspects.

[0106] A "pharmaceutically acceptable" component is one that is suitable for use with humans and / or animals without excessive adverse side effects (eg, toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0107] "Safe and effective amount" refers to the amount of a component sufficient to produce the desired therapeutic response without excessive adverse side effects (eg, toxicity, irritation, or allergic response) commensurate with a reasonable benefit / risk ratio when used in the manner of the present invention.

[0108] As used throughout the application, the terms "a" and "an" mean "at least one", "at least a first", "one or more" or "a plurality" of the referenced components or steps, unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0109] The term "and / or" wherever used herein includes the meanings of "and," "or," and "all or any other combinations of the elements connected by the term."

[0110] The above advantages as well as those apparent from the foregoing description are efficiently obtained.As certain changes could be made in the above constructions without departing from the scope of the invention, it is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

[0111] All references cited in this application are hereby incorporated by reference in their entirety to the extent not inconsistent herewith.

[0112] It will be seen that the advantages set forth above and those apparent from the foregoing description are efficiently obtained, and since certain changes may be made in the foregoing constructions without departing from the scope of the invention, it is intended that all matter contained in the foregoing description or shown in the accompanying drawings shall be interpreted as illustrative and not restrictive.

[0113] It is also to be understood that the following claims are intended to cover all generic and specific features of the invention described herein, and all statements of the scope of the invention which, as a matter of language, might be considered to fall between them.The invention has now been described.

Claims

1. A bacterium for delivering a gene editing system to a eukaryotic cell, wherein the bacterium is engineered to express at least one invasion factor to facilitate entry of the bacterium into the eukaryotic cell, wherein the bacterium comprises a plasmid encoding a gene editing system, the gene editing system comprising a CRISPR-associated enzyme, a nuclear localization signal, one or more CRISPR direct repeat regions, one or more spacer sequences, and one or more tracr sequences, and wherein the plasmid comprises a prokaryotic promoter to facilitate transcription of one or more elements of the encoded gene editing system; wherein the bacterium is Escherichia coli ( E. coli ), the invasion factors are invasin protein and listeriolysin O protein, and the spacer sequence is capable of hybridizing with a target sequence in a eukaryotic cell.

2. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 1, wherein the plasmid encodes more than one CRISPR-associated enzyme.

3. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 1, wherein the encoded CRISPR-associated enzyme is a Cas9 gene.

4. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 1, wherein the eukaryotic cell is an animal cell.

5. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 4, wherein the eukaryotic cell is a dividing cell or a non-dividing cell.

6. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 1, wherein the prokaryotic promoter is selected from T7, lacUV5, gapA, T5, recA, Ptac, Patac, pA1, lac , Sp6, araBad, trp or a hybrid promoter.

7. A bacterium for delivering a gene editing system to a eukaryotic cell, the bacterium being engineered to express at least one invasion factor, a CRISPR-associated enzyme, a nuclear localization signal, one or more CRISPR direct repeat regions, one or more spacer sequences, and one or more tracr sequences that facilitate bacterial entry into eukaryotic cells, wherein the bacterium is Escherichia coli, the invasion factor is an invasin protein and a listeriolysin O protein, and the spacer sequence is capable of hybridizing with a target sequence in a eukaryotic cell.

8. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 7, wherein one or more of an invasion factor that helps bacteria enter eukaryotic cells, the CRISPR-associated enzyme, the nuclear localization signal, the one or more CRISPR direct repeat regions, the one or more spacer sequences, or the one or more tracr sequences are expressed from the chromosome of the bacterium.

9. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 7, wherein the CRISPR-associated enzyme is expressed from the chromosome of the bacterium.

10. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 9, wherein the bacterium comprises a plasmid encoding one or more CRISPR direct repeat regions, one or more spacer sequences and one or more tracr sequences, and wherein the plasmid comprises a prokaryotic promoter to facilitate transcription of the one or more CRISPR direct repeat regions, one or more spacer sequences and one or more tracr sequences.

11. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 10, wherein the prokaryotic promoter is selected from T7, lacUV5, gapA, T5, recA, Ptac, Patac, pA1, lac , Sp6, araBad, trp or a hybrid promoter.

12. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 9, wherein the bacterium comprises a plasmid encoding one or more spacer sequences, and wherein the plasmid comprises a prokaryotic promoter to facilitate transcription of the one or more spacer sequences.

13. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 12, wherein the prokaryotic promoter is selected from T7, lacUV5, gapA, T5, recA, Ptac, Patac, pA1, lac , Sp6, araBad, trp or a hybrid promoter.

14. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 7, wherein the encoded CRISPR-associated enzyme is a Cas9 gene.

15. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 7, wherein the chromosome encodes more than one CRISPR-associated enzyme.

16. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 7, wherein the invasion factor is expressed from the chromosome of the bacterium.

17. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 7, wherein the CRISPR-associated enzyme and the invasion factor are expressed from the chromosome of the bacterium.

18. A kit for preparing bacteria for delivering a gene editing system to eukaryotic cells, the kit comprising: A bacterium that has been engineered to express at least one invasion factor that facilitates bacterial entry into eukaryotic cells and a gene editing system comprising a CRISPR-associated enzyme, a nuclear localization signal, one or more CRISPR direct repeat regions, and one or more tracr sequences; and a plasmid encoding one or more spacer sequences and a promoter for controlling transcription of the gene editing system; The bacterium is Escherichia coli, the invasion factors are invasin protein and listeriolysin O protein, the spacer sequence is capable of hybridizing with a target sequence in eukaryotic cells, and the promoter is a prokaryotic promoter.

19. The kit for preparing bacteria for delivering a gene editing system to eukaryotic cells according to claim 18, wherein the prokaryotic promoter is selected from T7, lacUV5, gapA, T5, recA, Ptac, Patac, pA1, lac , Sp6, araBad, trp or a hybrid promoter.

20. The kit for preparing bacteria for delivering a gene editing system to eukaryotic cells according to claim 18, wherein the CRISPR-associated enzyme and / or the invasion factor are expressed from the chromosome of the bacteria.

21. A kit for preparing bacteria for delivering a gene editing system to eukaryotic cells according to claim 18, wherein the CRISPR-associated enzyme, the one or more CRISPR direct repeat regions and the one or more tracr sequences are expressed from the chromosome of the bacteria.

22. The kit for preparing bacteria for delivering a gene editing system to eukaryotic cells according to claim 18, wherein the plasmid is packaged separately from the bacteria.

23. A kit for preparing bacteria for delivering a gene editing system to eukaryotic cells, the kit comprising: A bacterium that has been engineered to express at least one invasion factor that facilitates bacterial entry into eukaryotic cells and a gene editing system, wherein the gene editing system comprises a CRISPR-associated enzyme, a nuclear localization signal, one or more CRISPR direct repeat regions, one or more spacer sequences, and one or more tracr sequences; and a plasmid having an insertion site for the one or more spacer sequences and a promoter for controlling the transcription of the spacer sequences; The bacterium is Escherichia coli, the invasion factors are invasin protein and listeriolysin O protein, the spacer sequence is capable of hybridizing with a target sequence in eukaryotic cells, and the promoter is a prokaryotic promoter.

24. The kit for preparing bacteria for delivering a gene editing system to eukaryotic cells according to claim 23, wherein the prokaryotic promoter is selected from T7, lacUV5, gapA, T5, recA, Ptac, Patac, pA1, lac , Sp6, araBad, trp or a hybrid promoter.

25. The kit for preparing bacteria for delivering a gene editing system to eukaryotic cells according to claim 23, wherein the plasmid further comprises the invasion factor, the CRISPR-associated enzyme, the one or more CRISPR direct repeat regions or the one or more tracr sequences.

26. A kit for preparing bacteria for delivering a gene editing system to eukaryotic cells according to claim 23, wherein the CRISPR-associated enzyme and / or the invasion factor are expressed from the chromosome of the bacteria.

27. A kit for preparing bacteria for delivering a gene editing system to eukaryotic cells according to claim 23, wherein the CRISPR-associated enzyme, the one or more CRISPR direct repeat regions and the one or more tracr sequences are expressed from the chromosome of the bacteria.

28. A kit for preparing bacteria for delivering a gene editing system to eukaryotic cells according to claim 23, wherein the plasmid is packaged separately from the bacteria, thereby separate packaging facilitates cloning of the gene editing sequence into the plasmid.

29. A kit for preparing bacteria for delivering a gene editing system to eukaryotic cells, the kit comprising: a non-pathogenic bacterium, wherein the bacterium is Escherichia coli; and One or more plasmids encoding an invasion factor, a gene editing system, and a promoter for controlling the transcription of the gene editing system, wherein the gene editing system comprises a CRISPR-associated enzyme, a nuclear localization signal, one or more CRISPR direct repeat regions, one or more spacer sequences, and one or more tracr sequences; The plasmid comprises a prokaryotic promoter to facilitate transcription of one or more elements of the encoded gene editing system, the invasion factor is an invasin protein and a listeriolysin O protein, and the spacer sequence is capable of hybridizing with a target sequence in a eukaryotic cell.

30. A non-therapeutic or non-diagnostic method for delivering a gene editing system to a target eukaryotic cell, comprising the following steps: Providing a bacterium according to any one of claims 1 to 17; and The target eukaryotic cell is contacted with the provided bacteria under conditions effective to allow the non-pathogenic bacteria to enter the target cell.

31. A non-therapeutic or non-diagnostic method for delivering a gene editing system to a target eukaryotic cell according to claim 30, wherein the target cell is contacted in vitro and the target cell is a cell selected from the group consisting of primary cells, immortalized cells, dividing cells, non-dividing cells and hematopoietic stem / progenitor cells.

32. A non-therapeutic or non-diagnostic method for delivering a gene editing system to a target eukaryotic cell according to claim 30, wherein the method is applied in gene editing applications, including functional genomic screening, chromosome painting, used together with in vitro selection libraries, mismatch detection nuclease analysis, high-throughput profiling, diagnostics and related next-generation sequencing activities.

33. A non-therapeutic or non-diagnostic method for ex vivo genome editing and modification of eukaryotic cells isolated from a living system, comprising the following steps: Isolation of eukaryotic cells from living systems; Providing a bacterium according to any one of claims 1 to 17; and The isolated eukaryotic cell is contacted with the provided bacteria under conditions effective to allow the non-pathogenic bacteria to enter the target cell, whereby the method facilitates the insertion, removal or replacement of a region of interest on the genome of the isolated eukaryotic cell and the ex vivo modification of the target DNA.

34. The non-therapeutic or non-diagnostic method of ex vivo genome editing and modification of eukaryotic cells isolated from a living system according to claim 33, further comprising the step of reintroducing the edited cells into a living multicellular organism.

35. The non-therapeutic or non-diagnostic method of ex vivo genome editing and modification of eukaryotic cells isolated from a living system according to claim 33, wherein the edited cells are returned to a target tissue selected from the group consisting of eye, muscle, lung, brain, nasal cavity, gastrointestinal tract, heart, skin, oral cavity, genital tissue, blood, or any combination thereof.

36. A non-therapeutic or non-diagnostic method for ex vivo genome editing and modification of eukaryotic cells and bacterial systems isolated from a living system according to claim 33, wherein the cell is introduced into the same organism or the same species from which it was isolated.

37. Use of edited T cells in the preparation of a medicament for a method of chimeric antigen receptor T cell therapy, wherein the method comprises the following steps: Isolation of T cells from living systems; Providing a non-pathogenic bacterium, wherein the bacterium is the bacterium according to any one of claims 1 to 17; contacting the isolated T cells with the provided bacteria under conditions effective to allow the non-pathogenic bacteria to enter the target cells, whereby the method facilitates the insertion, removal or replacement of the region of interest on the genome of the isolated eukaryotic cell and the ex vivo modification of the target DNA; and The edited T cells are reintroduced into a living multicellular organism.

38. A non-therapeutic or non-diagnostic method based on chimeric antigen receptor T cells, wherein the method comprises the following steps: Isolation of T cells from living systems; Providing a non-pathogenic bacterium, wherein the bacterium is the bacterium according to any one of claims 1 to 17; contacting the isolated T cells with the provided bacteria under conditions effective to allow the non-pathogenic bacteria to enter the target cells, whereby the method facilitates the insertion, removal or replacement of the region of interest on the genome of the isolated eukaryotic cell and the ex vivo modification of the target DNA; and The edited T cells are reintroduced into a living multicellular organism.

39. A method for producing non-pathogenic bacteria for delivering a gene editing system to eukaryotic cells, comprising the following steps: providing a plasmid comprising at least one prokaryotic expression cassette under the control of at least one prokaryotic promoter, wherein the plasmid comprises an insertion site for one or more spacer sequences, a CRISPR-associated enzyme, a nuclear localization signal, one or more CRISPR direct repeat regions, and one or more tracr sequences; Inserting the spacer sequence into the provided plasmid; providing a bacterium, wherein the bacterium has been engineered to express at least one invasion factor from a sequence on a chromosome of the bacterium; and transforming the bacteria with a plasmid comprising the one or more spacer sequences; The gene editing system comprises a CRISPR-associated enzyme, a nuclear localization signal, one or more CRISPR direct repeat regions, one or more spacer sequences, and one or more tracr sequences; The bacterium is Escherichia coli, the invasion factors are invasin protein and listeriolysin O protein, and the spacer sequence is capable of hybridizing with a target sequence in a eukaryotic cell.

40. A method for producing non-pathogenic bacteria for delivering a gene editing system to eukaryotic cells, comprising the steps of: providing a plasmid comprising at least one prokaryotic expression cassette under the control of at least one prokaryotic promoter, wherein the plasmid comprises an insertion site for one or more spacer sequences; Inserting the spacer sequence into the provided plasmid; providing a bacterium, wherein the bacterium has been engineered to express at least one invasion factor from a sequence on a chromosome of the bacterium; and transforming the bacteria with a plasmid comprising the one or more spacer sequences; The gene editing system comprises a CRISPR-associated enzyme, a nuclear localization signal, one or more CRISPR direct repeat regions, one or more spacer sequences, and one or more tracr sequences; wherein the bacterium is Escherichia coli, the invasion factors are invasin protein and listeriolysin O protein, and the spacer sequence is capable of hybridizing with a target sequence in a eukaryotic cell; Wherein the CRISPR-associated enzyme, nuclear localization signal, one or more CRISPR direct repeat regions or one or more tracr sequences are encoded on the chromosome of the bacterium.

41. The method of claim 39 or 40, wherein the prokaryotic promoter is selected from T7, lacUV5, gapA, T5, recA, Ptac, Patac, pA1, lac , Sp6, araBad, trp or a hybrid promoter.

42. The bacterium for delivering a gene editing system to a eukaryotic cell according to claim 1, wherein the eukaryotic cell is a plant cell.

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