Application of CRISPR / CAS13 to treat RNA virus and / or bacteria-induced diseases

Abstract

The present invention relates to a composition comprising a clustered regularly interspaced short palindromic repeats (CRISPR) system, the system comprising i) at least one CRISPR-associated protein 13 (Cas13) or a nucleotide sequence encoding said Cas13 protein fused to at least one nuclear localization signal (NLS) fused to at least one nuclear export sequence (NES), and ii) at least one guide RNA (gRNA) or a nucleotide sequence encoding said gRNA capable of hybridizing to one or more target RNA molecules. The present invention further relates to a composition comprising a clustered regularly interspaced short palindromic repeats (CRISPR) system, the system comprising: i) at least one CRISPR-associated protein 13 (Cas13) or a nucleotide sequence encoding said Cas13 protein, fused to at least one nuclear localization signal (NLS) or to at least one nuclear transport sequence (NES); and ii) at least one guide RNA (gRNA) or a nucleotide sequence encoding said gRNA, capable of hybridizing with one or more target RNA molecules, fused to at least one viral transport element. The present invention also relates to said composition for use in therapy. In particular, the present invention relates to said composition for use in a method for preventing or treating a viral or bacterial disease in a subject. The present invention further relates to a nucleic acid molecule comprising a nucleotide sequence encoding said Cas13 protein and / or said gRNA of said composition, a vector comprising said nucleic acid molecule, and a host cell comprising said vector or said nucleic acid molecule. The present invention further relates to a kit comprising said composition. The present invention also includes a method for producing said composition of the present invention.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to LU 102326, filed December 22, 2020, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to a composition comprising a clustered regularly interspaced short palindromic repeats (CRISPR) system, the system comprising: i) at least one CRISPR-associated protein 13 (Cas13) or a nucleotide sequence encoding said Cas13 protein, fused with at least one nuclear localization signal (NLS) fused to at least one nuclear export sequence (NES); and ii) at least one guide RNA (gRNA) or a nucleotide sequence encoding said gRNA, capable of hybridizing with one or more target RNA molecules.Furthermore, the present invention relates to a composition comprising a clustered regularly interspaced short palindromic repeats (CRISPR) system, the system comprising: i) at least one CRISPR-associated protein 13 (Cas13) fused to at least one nuclear localization signal (NLS) or to at least one nuclear export sequence (NES), or a nucleotide sequence encoding said Cas13 protein; ii) at least one guide RNA (gRNA) capable of hybridizing to one or more target RNA molecules, or a nucleotide sequence encoding said gRNA, fused to at least one viral transport element; The present invention also relates to said composition for use in therapy. In particular, the present invention relates to said composition for use in a method for preventing or treating a viral or bacterial disease in a subject. The present invention further relates to a nucleic acid molecule comprising a nucleotide sequence encoding said Cas13 protein and / or said gRNA of said composition, a vector comprising said nucleic acid molecule and a host cell comprising said vector or said nucleic acid molecule. Furthermore, the present invention relates to a kit comprising said composition. The present invention also includes a method for producing said composition of the present invention. [Background technology]

[0003] 2. Background of the Invention From a clinical perspective, the problem with the rapid spread of viruses or bacterial strains in general is that drug development takes too long to develop a therapeutic agent in a reasonable time. In particular, drug development is a key issue for the novel coronavirus SARS-CoV-2, which is a (+)-RNA virus of the Coronaviridae family and has caused over 1.000.000 deaths worldwide as of early October 2020. RNA viruses are also responsible for two other recent epipandemics and pandemics (SARS-CoV-1 and MERS-CoV). These two epidemics share with COVID-19 a combination of high virulence and an efficient route of spread via droplet infection (P. Anfinrud et al. (2020), N Engl J Med 382, ​​2061-2063 (Non-Patent Document 1); J. Chen (2020) Microbes Infect 22, 69-71 (Non-Patent Document 2)).

[0004] For this reason, the only available recourse is the "repurposing" of already approved drugs. For example, remdesivir, originally developed as a treatment for Ebola, is currently being discussed as a treatment for COVID-19 (J. Grein et al. (2020), N Engl J Med 382, ​​2327-2336). However, small molecule-based therapies such as antibodies and remdesivir use the tertiary structure of proteins as target structures, so approved inhibitors often cannot be applied to new viral or bacterial strains, and furthermore, viral or bacterial mutations may change the binding properties of the therapeutic agent.

[0005] The prokaryotic immune system CRISPR / Cas works differently from the immune system of higher eukaryotes. Instead of binding to protein antigens, the CRISPR / Cas system directly recognizes the genetic information of the phage at the ribonucleic acid level. By simply expressing a guide RNA (gRNA) complementary to the phage genome, an effector nuclease is directed to the phage genome and induces genome cleavage (F. Hille et al. (2018), Cell 172, 1239-1259 (Non-Patent Document 4)). There are six types of CRISPR / Cas systems divided into two classes (KS Makarova et al. (2020), Nat Rev Microbiol 18, 67-83 (Non-Patent Document 5)). In addition to Cas9, Cas13 has recently been discovered as a programmable DNAse. Unlike Cas9, Cas13 does not cleave DNA, but rather the RNA of phages that attack prokaryotic hosts (OO Abudayyeh et al. (2016), Science 353). The nuclease of the Cas13 effector is the HEPN (higher eukaryotic and prokaryotic nucleotide binding) domain, which is split within the protein and therefore inactive. Upon binding of the target RNA, a change occurs in the tertiary structure of the protein, which brings the separate nuclease domains into close proximity and activates them (OO Abudayyeh et al. (2016)).

[0006] Different approaches using Cas13 in the prior art have already been demonstrated in several model viruses, such as influenza A, and in artificial experimental systems in which synthetic SARS-CoV-2 sequences are transfected into human cells already expressing Cas13 (CA Freije et al. (2019), Mol Cell 76, 826-837 (Non-Patent Document 7), Abbott et al. (2020), Cell 181, 865-876 (Non-Patent Document 8)). Despite current attempts to develop vaccines against human pathogenic viruses and also bacterial infections, there remains a need to provide alternative or improved agents, compositions and methods for the treatment and / or prevention of diseases caused by human pathogenic viruses or bacteria. The technical problem underlying the present application is therefore to meet this need.

[0007] This technical problem is solved by providing the embodiments reflected in the claims, described herein and illustrated in the following examples and figures. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] P. Anfinrud et al. (2020), N Engl J Med 382, ​​2061-2063 [Non-Patent Document 2] J. Chen (2020) Microbes Infect 22, 69-71 [Non-Patent Document 3] J. Grein et al. (2020), N Engl J Med 382, ​​2327-2336 [Non-Patent Document 4] F. Hille et al. (2018), Cell 172, 1239-1259 [Non-Patent Document 5] KS Makarova et al. (2020), Nat Rev Microbiol 18, 67-83 [Non-Patent Document 6] OO Abudayyeh et al. (2016), Science 353 [Non-Patent Document 7] CA Freije et al. (2019), Mol Cell 76, 826-837 [Non-Patent Document 8] Abbott et al. (2020), Cell 181, 865-876 Summary of the Invention

[0009] The inventors have developed a novel CRISPR-Cas based approach for the treatment of viral diseases, preferably caused by RNA viruses such as COVID-19, and for the treatment of bacterial diseases, in which invasive or cell-replicating viral / bacterial mRNAs are bound by the so-called Cas13 protein in the presence of complementary gRNAs (CRISPR-associated RNAs) and degraded by the RNAse activity of Cas13, thus rendering them harmless. The novel CRISPR / Cas concept directly targets ribonucleic acid sequences instead of the tertiary structure of proteins and can be used as an antiviral / antibacterial therapy, specifically attacking viral and / or bacterial RNA sequences of SARS-CoV-2 directly. Such an approach is therefore modular and adaptable. The advantage of such an RNA-based therapeutic approach is that viral and similarly bacterial genomes can be used as targets, which means that potential therapeutics can be accessed much more easily and quickly than addressing the tertiary structure of proteins.

[0010] In particular, the present inventors were able to test a number of modifications to gRNA and Cas13 protein, and identified three components that increase the knockdown efficiency of Cas13 protein (see Figure 6, Figure 11 and Figure 14). The most important optimization step included the fusion of at least one nuclear localization signal (NLS) to Cas13 protein, which is further fused to at least one nuclear export signal (NES) (see Figure 1). In addition, it included the fusion of tRNA to support gRNA elongation (see Figures 1 and 2) and gRNA folding, and the fusion of a ribozyme to the gRNA (see Figure 3). The composition of the present invention that includes all three modifications may also be referred to as Cas13-IDG, as defined elsewhere herein. Furthermore, other modifications of Cas13 protein and / or gRNA at the RNA level, including any one of the following modifications, such as modified 5' and / or 3' UTR, post- or co-transcriptional addition of a 5' CAP structure, replacement of UTP with N1-methyl pseudo-UTP, or replacement of the two 5' and / or 3' terminal nucleotides with 2'-O-methyl-3' P-thioate, were found to further improve the knockdown efficiency of Cas13 protein (see Figures 10 and 15). Furthermore, the inventors also found that another modification of gRNA at the DNA level, including fusion of at least one viral transport element, preferably fusion of at least one constitutive transport element (CTE) or VARdm, most preferably fusion of at least one CTE with gRNA, further increases the knockdown efficiency of Cas13 protein (fused to at least one NLS fused to at least one NES as defined above) for DNA delivery (see Figure 16).

[0011] Although gRNA is transcribed in the cell nucleus, according to the inventors, it is necessary to first bind gRNA to activate Cas13d RNAse activity (see Figure 4). Ideally, this step can only take place in the cell nucleus. In contrast, viral or bacterial mRNA target molecules after viral or bacterial infection are essentially present in the cytosol. The fusion of the localization signal resulted in the localization of Cas13 protein to the cytoplasm and the stabilization of the protein in this compartment. In contrast to already published Cas13 variants, this step makes it possible for the first time to degrade cytosolic RNA instead of nuclear RNA. This step is essential, since the replication of many RNA viruses, such as coronaviruses, takes place exclusively in the cytosol. However, when the compositions of the present invention are delivered as an RNA or protein-based system rather than as a DNA-based system as described above, the novel compositions containing the above modifications are also of great importance for viruses that are located in the nucleus and cytosol, such as the Orthomyxoviridae family, preferably influenza viruses, in which case a balanced localization of the Cas13 protein is required in both cellular compartments, which is achieved by fusion of the Cas13 protein to at least one NLS fused to at least one NES.

[0012] In summary, this highly unconventional and non-obvious approach has proven to be the most suitable among several strategies tested to direct the translated Cas13 protein into the nucleus to load the gRNA and then transport it to the cytosol, or vice versa, resulting in an increased efficiency of degradation of the desired target structure, and thus holds great potential in diagnostics and therapeutics (see Figures 6 and 7).

[0013] Thus, in a first aspect, the present invention relates to a composition comprising a clustered regularly interspaced short palindromic repeats (CRISPR) system, the system comprising i) at least one CRISPR-associated protein 13 (Cas13) or a nucleotide sequence encoding said Cas13 protein fused to at least one nuclear localization signal (NLS) fused to at least one nuclear export sequence (NES), and ii) at least one guide RNA (gRNA) or a nucleotide sequence encoding said gRNA capable of hybridizing to one or more target RNA molecules.

[0014] The present invention may also disclose compositions as defined elsewhere herein, in which a Cas13 protein is fused via a linker to at least one NLS fused to at least one NES.

[0015] The inventors have also demonstrated that the same effect, i.e., increased efficiency of degradation of a desired target structure, can be achieved by a composition comprising a clustered regularly interspaced short palindromic repeats (CRISPR) system, said system comprising: i) at least one CRISPR-associated protein 13 (Cas13) fused to at least one nuclear localization signal (NLS) or to at least one nuclear export sequence (NES), or a nucleotide sequence encoding said Cas13 protein; ii) at least one guide RNA (gRNA) capable of hybridizing to one or more target RNA molecules, or a nucleotide sequence encoding said gRNA, fused to at least one viral transport element; It has been found that this is achieved by the composition comprising: wherein the cytosolic knockdown by Cas13-NES or NLS is increased by fusing at least one viral transport element, preferably a constitutive transport element (CTE) or a minihelix of adenovirus VA1 RNA (VARdm), most preferably a CTE, to said gRNA (see FIG. 17).

[0016] Also encompassed herein are compositions as defined elsewhere herein, wherein the Cas13 protein is a Cas13d protein.

[0017] Also described herein are compositions as defined elsewhere herein, wherein the Cas13d protein is derived from the genus Ruminococcus, preferably Ruminococcus flavevaciens.

[0018] The present invention may also disclose compositions as defined elsewhere herein, wherein the gRNA has a length of at least about 23 nucleotides.

[0019] The present invention may also disclose compositions as defined elsewhere herein, wherein the gRNA has a length of about 26 to about 30 nucleotides.

[0020] Also described herein are compositions as defined elsewhere herein, in which the gRNA has at least about 80% complementary sequence identity to one or more target RNA molecules.

[0021] Also encompassed herein are compositions as defined elsewhere herein in which the gRNA is capable of hybridizing to the 5' and / or 3' untranslated regions of one or more target RNA molecules.

[0022] The present invention may also disclose compositions as defined elsewhere herein, in which the nucleotide sequence encoding the gRNA is fused to a tRNA or a ribozyme.

[0023] The present invention may also disclose compositions as defined elsewhere herein, wherein the one or more target RNA molecules are viral or bacterial target RNA molecules.

[0024] The present invention may also disclose compositions as defined elsewhere herein for inactivating bacterial or viral ssRNA.

[0025] Also encompassed herein are compositions as defined elsewhere herein that are pharmaceutical compositions, preferably further comprising at least one pharma- ceutically acceptable carrier.

[0026] In a second aspect, the present invention also relates to a composition for use in therapy.

[0027] In a third aspect, the invention further relates to the composition for use in a method of preventing or treating a viral or bacterial disease in a subject comprising administering a therapeutically effective amount of the composition to the subject.

[0028] Further, the present invention may also disclose a composition for use as defined elsewhere herein, wherein the viral disease is caused by an RNA virus, preferably, the viral disease is any one of a coronavirus disease, influenza A, Ebola, measles, hepatitis C, tick-borne encephalitis (TBE), Venezuelan equine encephalitis (VEE) virus infection, dengue fever, yellow fever or Zika fever, and even more preferably, the viral disease is a COVID-19 disease.

[0029] In a fourth aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence encoding said Cas13 protein and said gRNA as defined elsewhere herein, a vector comprising a nucleic acid molecule as defined elsewhere herein, or a host cell comprising said nucleic acid molecule or said vector as defined elsewhere herein.

[0030] In a fifth aspect, the present invention also relates to a kit comprising a composition as defined elsewhere herein. The present invention may also disclose a kit as defined elsewhere herein, further comprising a delivery system and / or a label.

[0031] In a sixth aspect, the present invention also relates to a method for producing a composition of the invention, said composition being produced by a genetic engineering method, starting from a nucleic acid encoding a Cas13 protein and a gRNA as defined herein, thereby producing said composition; and optionally obtaining said produced composition. [Brief description of the drawings]

[0032] [Figure 1] Knockdown efficiency of different Cas13 protein and gRNA variants. Each variant targeted co-transfected firefly luciferase and was normalized to a non-targeting gRNA. Fusion of an NLS-NES tandem signal to Cas13d improved efficiency compared to NLS-only variants. Extension of the gRNA further improved efficiency. All conditions were compared to the latest generation miRNA / RNA interference (Fellmann et al., 2013 in Cell reports). [Diagram 2] Characterization of different gRNA lengths of Cas13d-NLS-NES to target co-transfected nanoluciferase. The optimal gRNA length ranges from 26 bp to 30 bp. [Diagram 3] Fusion of either tRNA (MHV68 M1-7) or ribozyme (HDV) to the 30 bp gRNA increases the knockdown efficiency of co-transfected Cas13d-NLS-NES on nanoluciferase. [Figure 4]Different strategies to transport gRNA from the nucleus to the cytosol are compared. Knockdown efficiency against co-transfected nanoluciferase was measured for different Cas13d protein localizations and different gRNA expression strategies. Polymerase III-driven gRNA remains in the nucleus. Therefore, nuclear-localized Cas13d (NLS) is superior to cytosolic Cas13d (NES). The tandem fusion (NLS-NES) maximizes knockdown efficiency since it picks up the gRNA in the nucleus and is active against the cytosolic mRNA encoding nanoluciferase. Western blots confirmed that gRNA present in the same compartment as Cas13d stabilizes the protein. A complementary strategy of transporting gRNA by expression from a polymerase II-driven promoter does not improve efficiency. [Diagram 5] Knockdown efficiency of different Cas13d protein and gRNA variants. Each variant was targeted to co-transfected firefly luciferase and normalized to a non-targeting gRNA. Co-transfected nanoluciferase is measured in the same well but is not directly targeted by the gRNA, providing a readout of target RNA-induced collateral activity. Fusion of an NLS-NES tandem signal improved efficiency compared to NLS-only variants. Extension of the gRNA further improved efficiency. All conditions were compared to the latest generation miRNA / RNA interference. [Figure 6A] Schematic of the assay for the development of an optimized Cas13 system. [Figure 6B] Comparison of different Cas13 systems with enhanced Cas13-IDG (NLS-NES, 30 bp gRNA, 3'tRNA). Knockdown efficiency was measured by co-transfected nanoluciferase. [Figure 6C] Partial cytosolic localization of Cas13d-NLS-NES compared with Cas13d-NLS. [Figure 6D] Predicted mechanism of effect of Cas13d on cellular translation through direct degradation of coding mRNA and secondary cleavage at specific sites in 28S rRNA. [Figure 6E] Cas13d-NLS-NES targeted to co-transfected mRuby3 and subsequent analysis of ribosomal RNA on a bioanalyzer chip. [Figure 7A] Expected mechanism of action of Cas13-based antiviral strategies. Four levels of viral inhibition are achieved by targeting the viral genome directly, associated genomic copies, subgenomic mRNAs, or by inhibiting viral protein translation. [Figure 7B] Efficient knockdown of the artificially expressed viral protein RNA-dependent RNA polymerase (RdRp) (HA-tagged) confirmed by Western blot, with collateral effects confirmed by co-transfecting the unrelated mRuby3 protein. [Figure 7C] HEK293T cells transfected with ACE2, Cas13d-NLS-NES and gRNA were infected with SARS-CoV-2 and viral load was quantified by RT-qPCR after 72 hours. [Figure 8] Global cellular protein translation was measured for cells transfected with active or inactive Cas13d-NLS-NES, gRNA and target RNA. [Figure 9] Agarose gel of RNA extracted from cells transfected with either Cas13d-NLS-NES, target RNA and complementary gRNA, showing 28S rRNA cleavage products. The same 28S rRNA cleavage products are seen in vitro for purified 80S ribosomes incubated with either purified Cas13d, gRNA and target RNA. [Figure 10](A) Improvement of knockdown efficiency of Cas13 RNA delivery by optimizing different aspects of the transfected Cas13 mRNA or gRNA. Efficiency was measured by targeting co-transfected nanoluciferase and normalized to non-targeting gRNA. (B) Expression levels of Cas13 from mRNA were improved by modification of the UTR, 5' CAP structure, or replacement of UTP with N1-methyl pseudo-UTP. [Figure 11] (A) Illustration of Cas13d variants fused to different localization signals. (B) Knockdown efficiency of Cas13d variants with different localizations for co-transfected nanoluciferase target RNAs (crRNA 1 and crRNA 2). A balanced set of nuclear and cytosolic localization signals in Cas13d-IDG maximizes knockdown efficiency for the two crRNAs. [Figure 12] (A) Representative images showing the localization of Cas13d protein variants fused to different localization signals. (B) Quantification of the cytosolic / nuclear distribution of different Cas13d variants for 100 cells. [Figure 13] Stability of Cas13d variants fused to different localization signals analyzed by Western blot for polymerase II-driven gRNA (cytosol) and polymerase III-driven gRNA (nucleus). The complexed protein / gRNA is most stable in conditions where both components localize to the same compartment. [Figure 14]Comparison of knockdown efficiency of semi-cytosolic Cas13-NLS-NES and nuclear-localized Cas13-NLS against the Venezuelan equine encephalitis (VEE) replicon for gRNAs targeting different structural elements, as measured by reduction of mGreenLantern expression. Self-replicating VEE RNA consists of viral untranslated regions (UTRs), nonstructural proteins (nsp), interferon inhibitory proteins (B18R, E3L), mGreenLantern (mGL) reporter and puromycin resistance gene. Cas13d-NLS-NES strongly enhances knockdown of cytosolic replicating VEE. [Figure 15] (A) Sequential analysis of SARS-CoV-2-GFP replication for treatment conditions with Cas13d-IDG and different gRNAs targeting viral structural elements (NT: non-targeted, CDS: coding sequence, UTR: untranslated region). Chemically modified 3' UTR gRNA is the most efficient at inhibiting viral replication. (B) Targeting of different SARS-CoV-2 variants with Cas13d-IDG. Knockdown efficiency was analyzed by RT-qPCR for two viral target genes in biological sextuplicates and technical duplicates. [Figure 16] Comparison of knockdown efficiency of two gRNAs targeting nanoluciferase co-transfected for NES or tandem NLS-NES fused to Cas13. Additionally, either unmodified or CTE-fused gRNAs were tested. The combination of NLS-NES and CTE-gRNA maximizes knockdown efficiency. [Figure 17](A) Illustration of different crRNA transport strategies. Polymerase III (pol III)-driven crRNA remains in the nucleus, polymerase II (pol II)-driven crRNA is transported to the cytosol, and crRNA fused to a constitutive transport element (CTE) is transported similarly to crRNA fused to the minihelix of adenoviral VA1 RNA (VARdm). (B) Comparison of knockdown efficiency of different crRNA transport motifs on nanoluciferase target RNA (crRNA 1 and crRNA 2) in conjunction with either cytosolic (NES) or nuclear (NLS) Cas13d protein. crRNA fused to a CTE transport motif is the most efficient in driving Cas13d-based knockdown in the cytosol. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Detailed Description of the Invention The present invention is described in detail below, but it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, which may vary. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0034] In the following, the elements of the present invention are described. Although these elements are described with specific embodiments, it should be understood that they can be combined in any manner and in any number to produce further embodiments. The various described examples and preferred embodiments described throughout this specification should not be construed as limiting the present invention to only the embodiments explicitly described. This description should be understood as supporting and encompassing embodiments that combine the explicitly described embodiment with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of all elements described herein should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0035] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated member, integer or step or group of members, integers or steps, but not the exclusion of any other member, integer or step or group of members, integers or steps, although in some embodiments such other member, integer or step or group of members, integers or steps may be excluded, i.e., the subject matter is constituted by the inclusion of a stated member, integer or step or group of members, integers or steps. As used herein, the term "comprising" may be interchangeable with "containing" or "including," or, as used herein, with the term "having." As used herein, "consisting of" excludes any element, step or ingredient not specified.

[0036] The terms "a" and "an" and "the" and similar references used in the context of describing the present invention (especially in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein.

[0037] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better illustrate the invention and do not otherwise impose limitations on the scope of the claimed invention. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0038] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. The term "at least one" refers to 1, 2, 3 or more, such as 4, 5, 6, 7, 8, 9, 10 and more. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0039] The terms "less than" or "greater than" or "below" in the order do not include a specific number.

[0040] The term "and / or" wherever used in this specification includes the meaning of "and", "or" and "all or any other combination of the elements connected by this term".

[0041] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claimed element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0042] The term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0043] The term "about" means plus or minus 20%, preferably plus or minus 10%, more preferably plus or minus 5%, and most preferably plus or minus 1%.

[0044] Throughout the description and claims of this specification, the singular encompasses the plural, unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood to contemplate the plural as well as the singular, unless the context requires otherwise.

[0045] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0046] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing in this specification should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that the material incorporated by reference is inconsistent with or inconsistent with this specification, this specification will supersede any such material.

[0047] The contents of all publications and patent documents cited herein are incorporated by reference in their entirety.

[0048] A better understanding of the invention and its advantages will be gained from the examples given for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0049] CRISPR composition Cas13 protein The present invention relates to compositions comprising a CRISPR system as described elsewhere herein. Such CRISPR-Cas systems stand for "clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated proteins". They are based on an adaptive defense mechanism evolved by bacteria and archaea to protect them from invading viruses, bacteria and plasmids, and rely on small RNAs for sequence-specific detection and silencing of foreign ribonucleic acids. CRISPR / Cas systems consist of CRISPR arrays consisting of cas genes organized in operons and genomic targeting sequences (called spacers) interspersed with identical repeats (Bhaya et al. (2011) Annu Rev Genet 45:273-297; Barrangou R and Horvath P (2012) Annu Rev Food Sci Technol 3:143-162). Target recognition by guide RNA (gRNA) directs silencing of the foreign sequence by Cas proteins functioning in a complex with gRNA.

[0050] More specifically and generally, CRISPR-Cas or CRISPR system collectively refers to the transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeat sequences" and tracrRNA-processed partial direct repeat sequences in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or "RNAs" as that term is used herein (e.g., RNAs that guide Cas, e.g., CRISPR RNA and trans-activating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)), or other sequences and transcripts from a CRISPR locus. In general, CRISPR systems are characterized by elements (also referred to as protospacers in the context of endogenous CRISPR systems) that promote the formation of a CRISPR complex at the site of a target sequence.

[0051] In the present invention, the composition comprising the particular system is related to the CRISPR-Cas13 system. In the present invention, the composition comprising the CRISPR system comprises at least one (one or more) clustered regularly interspaced short palindromic repeats (CRISPR) associated (Cas) protein, which is a Cas13 protein. Thus, the composition comprises at least one (one or more), such as one, two, three, four, five or more Cas13 proteins as defined elsewhere herein. CRISPR-Cas13 is an RNA targeting and editing system based on the bacterial immune system that defends bacteria against viruses and bacteria. Cas13 is an effector protein in the type VI CRISPR-Cas system that targets and cleaves invasive ribonucleic acids from viruses and bacteria. The CRISPR-Cas13 system is similar to the CRISPR-Cas9 system. However, unlike Cas9, which targets DNA, Cas13, an RNase, targets / detects and cleaves / degrades single-stranded RNA (ssRNA). Thus, Cas13 protein is sometimes referred to as a (type VI) RNA-targeted effector protein. The Cas13 enzyme has two higher eukaryotic and prokaryotic nucleotide-binding (HEPN) endo-RNase domains that mediate precise RNA cleavage with a preference for targets with protospacer adjacent sites (PFS) observed biochemically and in bacteria. Examples of RNA-targeted effector proteins include C2c2 (now known as Cas13a), Cas13b, Cas13c and Cas13d. Cas13 was first discovered in L. shahii, a species of Leptotrichia bacteria, when researchers were searching for previously unidentified CRISPR systems. Since the Cas13 protein was identified in 2016 by the research group of Feng Zhang (Broad Institute, MIT), four different subtypes (Cas13a-d) have been described and intensively studied to date.Although their sequences are very different, they have two so-called HEPN domains (higher eukaryotic and prokaryotic nucleotide binding domains) as common features, which are involved in RNAse activity.These domains of Cas13 protein mediate precise RNA cleavage, preferentially targeting with protospacer adjacent site (PFS).In contrast, Cas13 protein does not show activity against DNA, like the aforementioned Cas9 protein.

[0052] In one embodiment, Cas13 protein comprises at least one HEPN domain, including but not limited to HEPN domains known in the art and domains recognized as HEPN domains by comparison with consensus sequence motifs.In certain embodiments, Cas13 protein comprises a single HEPN domain.In certain other embodiments, Cas13 protein comprises two HEPN domains.In another embodiment, Cas13 protein comprises one or more HEPN domains comprising RxxxxH motif sequence.RxxxxH motif sequence can be derived from HEPN domains known in the art, but not limited thereto.RxxxxH motif sequence further comprises motif sequence created by combining parts of two or more HEPN domains. As mentioned above, the consensus sequence can be derived from the sequences of orthologs disclosed in U.S. Provisional Patent Application No. 62 / 432,240, entitled "Novel CRISPR Enzymes and Systems," U.S. Provisional Patent Application No. 62 / 471,710, entitled "Novel Type VI CRISPR Orthologs and Systems," filed March 15, 2017, and U.S. Provisional Patent Application entitled "Novel Type VI CRISPR Orthologs and Systems," filed April 12, 2017, designated as Attorney Docket No. 47627-05-2133.

[0053] The composition of the present invention targets RNA molecules, where at least one Cas13 protein forms a complex with at least one gRNA, and at least one gRNA directs the complex to one or more target RNA molecules, thereby targeting one or more target RNA molecules, so the composition comprising the CRISPR system can also be referred to as a composition comprising a ribonucleic acid detection system.The composition not only targets RNA molecules via the Cas13 Rnase, but also subsequently cleaves the target RNA molecules, thereby degrading the RNA molecules, so the composition comprising the CRISPR system can also be referred to as a composition comprising a ribonucleic acid degradation system.

[0054] In one embodiment, at least one Cas13 protein or the nucleotide sequence encoding said protein is Cas13a protein or its functional variant, or its homolog or orthologue. In another embodiment, at least one Cas13 protein or the nucleotide sequence encoding said protein is Cas13b protein or its functional variant, or its homolog or orthologue. In yet another embodiment, at least one Cas13 protein or the nucleotide sequence encoding said protein is Cas13c protein or its functional variant, or its homolog or orthologue.The Cas13a protein is expressed in the genera Leptotrichia, Listeria, Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, and others. or the Cas13a protein may be derived from an organism selected from the group consisting of Leptotrichia shahii, Leptotrichia wadei, Listeria seeligeri, Lachnospiraceae bacterium, Leptotrichia spp. ... bacterium, Clostridium aminophilum, Carnobacterium gallinarum, Paludibacter propionicigenes, Listeria weihenstephanensis, Rhodobacter capsulatus, preferably Leptotrichia wadii.In another embodiment, the Cas13a protein may be selected from an amino acid sequence having at least 80% sequence identity to any of the sequences set forth in Tables 4 and 5 of US20200165594, which is incorporated herein by reference. In certain embodiments, Cas13b is from an organism selected from Bergeyella, Prevotella, Porphyromonas, Bacteroides, Alistipes, Riemerella, Capnocytophaga, Flavobacterium, Myroides, Chryseobacterium, Paludibacter, Psychroflexus, Phaeodactylibacter Sinomicrobium, Reichenbachiella, preferably Prevotella, even more preferably Prevotella sp. P5-125. In another embodiment, the Cas13b protein is or comprises an amino acid sequence having at least 80% sequence identity to any of the sequences set forth in Table 6 of US20200165594, which is incorporated herein by reference. In certain embodiments, the Cas13c protein is one disclosed in U.S. Provisional Patent Application No. 62 / 525,165, filed June 26, 2017, and PCT Application No. US 2017 / 047193, filed August 16, 2017.

[0055] In a preferred embodiment of the invention, the Cas13 protein or the nucleotide sequence encoding said protein is Cas13d or a functional variant thereof, or a homologue or orthologue thereof. The Cas13d protein may be derived from an organism of a genus selected from the group consisting of Leptotrichia, Listeria, Corynebacter, Stellera, Legionella, Treponema, Filifactor, Eubacterium, Ruminococcus, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavivora, Flavobacterium, Sphaerocheta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Pervibaculum, Staphylococcus, Nitratiphracter, Mycoplasma, Campylobacter and Lachnospira, preferably from Eubacterium or Ruminococcus, most preferably from Ruminococcus. Cas13d from Ruminococcus flavefaciens, preferably from Ruminococcus flavefaciens XPD3002, has been shown to be the most efficient at degrading target RNA. Thus, in an even more preferred embodiment, the Cas13d protein or the nucleotide sequence encoding said protein is derived from the genus Ruminococcus, most preferably from Ruminococcus flavefaciens. Derived means that the derived protein is primarily based on the wild-type protein, in the sense that it has a high degree of sequence homology with the Cas13d from Ruminococcus, and preferably from Ruminococcus flavefaciens, herein, and thus provides the same function as said wild-type protein, but is mutated (modified) in some way as known in the art or as described herein. The Cas13d protein from the genus Ruminococcus, preferably from Ruminococcus flavefaciens, may also be referred to as a functional variant as defined elsewhere herein.Also included and most preferred herein are compositions as defined herein, wherein the Cas13 protein is from the genus Ruminococcus, preferably from Ruminococcus flavefaciens.

[0056] In certain embodiments, the functional variant of the Cas13d protein as defined elsewhere herein has at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, more preferably at least about 85%, even more preferably at least about 90% sequence homology or identity, such as at least about 95%, at least about 96%, at least about 97%, at least about 98% or even at least about 99% amino acid sequence identity, to the wild-type amino acid sequence of the Cas13d protein of Ruminococcus flavefaciens (SEQ ID NO. 1). Thus, the present invention provides a Cas13 protein or a nucleotide sequence encoding the protein, comprising the sequence of SEQ ID NO: 1 and at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, Also included are compositions as defined elsewhere herein that are Cas13d proteins comprising an amino acid sequence having 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or even 100% sequence identity.

[0057] As used herein, a "functional variant" of a protein refers to a variant of such a protein that retains at least part of the activity of the protein. A functional variant may include a mutation, including polymorphisms, which may be an insertion, deletion or substitution, as known to those skilled in the art. Functional variants also include fusion products of such proteins with another, usually unrelated, nucleic acid, protein, polypeptide or peptide. A functional variant may be naturally occurring or artificially created. Advantageous embodiments may include engineered or non-naturally occurring Cas13 proteins, as defined herein. Thus, a functional variant of a Cas13 protein described herein retains the biological activity of the Cas13 protein from which it is derived. Generally, a Cas13 protein variant has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or even at least about 99% amino acid sequence identity to the Cas13 protein from which it is derived.

[0058] The terms "orthologue" (also referred to herein as "ortholog") and "homologue" (also referred to herein as "homolog") are well known in the art. As further guidance, a "homolog" of a protein, as used herein, is a protein of the same species that performs the same or similar function as the protein to which it is a homolog. Homologous proteins may be, but are not necessarily, structurally related, or are only partially structurally related. An "orthologue" of a protein, as used herein, is a protein of a different species that performs the same or similar function as the protein to which it is an ortholog. Orthologous proteins may be, but are not necessarily, structurally related, or are only partially structurally related. In certain embodiments, a homolog or orthologue of a Cas13 protein, such as Cas13a, b, c or d, referred to herein has at least 80%, more preferably at least 85%, even more preferably at least 90%, such as at least 95% sequence homology or identity with a Cas13 protein, such as Cas13a, b, c or d. In further embodiments, a homolog or orthologue of a Cas13 protein, such as Cas13a, b, c or d, referred to herein has at least 80%, more preferably at least 85%, even more preferably at least 90%, such as at least 95% sequence identity with a wild-type Cas13 protein, such as Cas13a, b, c or d. Each embodiment referring to a Cas13 protein as defined herein may also be applicable to a functional variant thereof or a homolog or orthologue thereof.

[0059] "Identity" or "sequence identity" refers to a characteristic of sequences that measures their similarity or relationship. The term "sequence identity" or "identity" as used herein refers to the percentage of identical residues in the longer of the two sequences after (homology) alignment of the sequence of the polypeptide of the present invention with the sequence in question. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100. Percentage of sequence identity can be determined herein, for example, using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf. Altschul, SF et al. (1997) Nucl. Acids Res.25, 3389-3402). In this embodiment, the percentage of homology is based on the alignment of the entire polypeptide sequence including each sequence (matrix: BLOSUM 62; gap cost: 11.1; cutoff value set to 10-3). It is calculated as the ratio of the number of resulting "positives" (homologous amino acids) in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0060] In certain embodiments, a protospacer adjacent motif (PAM) or PAM-like motif directs the binding of the Cas13 protein complex disclosed herein to the target locus of interest. In some embodiments, the PAM can be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (i.e., located downstream of the 5' end of the protospacer). The term "PAM" can be used interchangeably with the terms "PFS" or "protospacer adjacent site" or "protospacer adjacent sequence" as referred to elsewhere herein.

[0061] The improved efficiency of degrading target RNA molecules, mentioned elsewhere in this specification, is due to the specific design of the system of the invention. The inventors have been able to show that the cellular localization of Cas13 protein has a decisive influence on the overall composition degradation efficiency, and as mentioned above, the presence of Cas13 in both the cell nucleus and the cytoplasm is necessary. The novel composition comprising said specific CRISPR system uses an inventive tandem combination of NLS (nuclear localization signal) and NES (nuclear transport sequence). At the protein level, a 90% reduction of the viral SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) could thus be detected, as shown by the examples (see also Figures 1 and 4).

[0062] Therefore, the Cas13 protein of the CRISPR system contained in the composition of the present invention is always fused with at least one (e.g., one, two, three, four, five, six or more) nuclear localization signal (NLS), which is fused with at least one (e.g., one, two, three, four, five, six or more) nuclear transport sequence (NES).This modification to the system refers to the most important modification.The NES is a short targeting peptide that contains four hydrophobic residues in a protein, which targets the protein, i.e., Cas13, for transport from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport, for example, when the composition containing the system is delivered as a DNA-based system.The NLS targets the protein, i.e., Cas13, located in the cytoplasm for transport to the nucleus, for example, when the composition containing the system is delivered as an RNA- or protein-based system as defined elsewhere herein.

[0063] The at least one NLS fused to the at least one NES is associated with a (tandem) localization signal, which allows Cas13 protein to shuttle between the nucleus, where it can be activated by binding to the at least one gRNA, for example after viral or bacterial infection of the subject, and the cytoplasm, where the target RNA molecule can be located. Viruses that also enter the nucleus of cells, such as Orthomyxoviridae viruses, preferably influenza, provide target RNA molecules not only in the cytoplasm but also in the nucleus, where the composition again requires a localization signal as defined herein, when it can be delivered at the RNA or protein level to allow the system to shuttle between the cytoplasm and the nucleus and degrade the target RNA molecule in the nucleus as well.

[0064] The Cas13 protein as defined herein is preferably fused to said localization signal (in other words said at least one NLS fused to said at least one NES) via any peptide linker known to the skilled artisan of at least one amino acid in length. Thus, in this context, the phrase Cas13 protein as defined herein is linked to said at least one NLS fused to said at least one NES may also be used herein. This may also refer to indirect fusion or linkage, as a peptide linker is applied. Thus, the phrases "indirectly fused" or "indirectly linked" may also be used herein in this context. The linker is preferably 1-20 amino acids in length. More preferably, the linker is 1-15 amino acids in length, even more preferably, the linker is 1-10 amino acids in length, for example 1-5 amino acids in length. Even more preferably, the linker is 10, 9, 8, 7, 6, 5, 5, 3, 2, 1 amino acids in length. Preferably, the linker molecule is a linear or helical linker, and even more preferably, the linker is a helical linker. More preferably, the linker is a flexible linker, for example using the amino acids glycine and / or serine. In a particularly preferred embodiment of the present invention, 50% to 100%, in particular 60% to 100%, in particular 70% to 100%, in particular 80% to 100%, in particular 90% to 100%, in particular 100% of the amino acid residues of the linker molecule are glycine and serine residues, preferably forming an α-helical structure.

[0065] The fusion / linkage of the Cas13 protein to the localization signal preferably comprises a GlySer linker, such as GGGS. They can be used in triplicate ((GGGS)3), or in repeats of six, nine or even twelve or more, to provide the appropriate length, as required. The linker is used to engineer an appropriate amount of "mechanical flexibility". In a preferred embodiment, the linker is a GlySerThr (GST) linker as defined herein. The linkage of the Cas13 protein to the localization signal can be covalent. Thus, the present invention further includes that the Cas13 protein is covalently fused / linked to the at least one NLS fused to at least one NES as defined herein. The term "covalently linked / fused" in this context, and as used throughout the present invention, refers to a covalent bond that is typically formed by the sharing of electron pairs between atoms. According to the present invention and when the term "covalently fused / linked" is used, a covalent bond is formed between said Cas13 protein and a localization signal as defined herein by the use of a peptide linker as described above, or such a covalent bond is formed between said at least one NLS (or between each other if two or more NLSs are used) and then between said at least one NES (or between each other if two or more NESs are used), as defined elsewhere herein.

[0066] The localization signal comprising at least one NLS fused to at least one NES as defined elsewhere herein may be fused to the N-terminus or C-terminus of said Cas13 protein. Thus, in one embodiment, the localization signal comprising at least one NLS fused to at least one NES as defined elsewhere herein is fused to the C-terminus of said Cas13 protein. In another embodiment, the localization signal comprising at least one NLS fused to at least one NES as defined elsewhere herein is fused to the N-terminus of said Cas13 protein. In a preferred embodiment, the localization signal comprising at least one NLS fused to at least one NES as defined elsewhere herein is fused to the C-terminus of said Cas13 protein. According to the present invention, said at least one NLS of said signal may be fused directly to said at least one NES, meaning that the amino acid sequences (also called peptide sequences) are arranged one after the other without a linker as defined elsewhere herein. Thus, "direct fusion" or "directly fused" as used throughout the present invention refers to fusing the at least one NLS to the at least one NES without any linker, as defined elsewhere herein, thereby forming a localization signal comprising at least one NLS and at least one NES. The above definition of direct fusion can be applied in any context of direct fusion. Direct fusion or "directly fused" also refers to the fusion of the NLS or NES to each other when two or more NLSs or NESs are used in the localization signal. In this context, any arrangement is included in the present invention, regardless of whether the Cas13 protein is first fused to the at least one NLS or first fused to the at least one NES of the localization signal. Thus, the Cas13 protein as defined herein can be fused / linked to at least one NLS followed by at least one NES as first described, or vice versa.Similarly, when the localization signal uses one or more NLSs and one or more NESs, any arrangement between the localization signals (e.g., NLS-NES-NLS or NES-NES-NLS or NLS-NLS-NES-NES, etc.) is possible and is therefore included in the present invention. Non-limiting examples of NLSs include an NLS sequence derived from the NLS of the SV40 virus large T-antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 2), an NLS sequence derived from an extended form of the NLS of the SV40 virus large T-antigen having the amino acid sequence PPKKKRKVED (SEQ ID NO: 5), an NLS consensus sequence (also called synthetic NLS) having the amino acid sequence PAAKKKLD (SEQ ID NO: 3), a c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 6), or an amino acid sequence. TIFF2024500932000001.tif4128. Non-limiting examples of NES include, but are not limited to, the NES sequence derived from the HIV virus NES having the amino acid sequence LQLPPLERLTL (SEQ ID NO: 4). In a preferred embodiment, the at least one NLS fused to the at least one NES defined herein comprises the SV40 NLS having the amino acid sequence shown in SEQ ID NO: 5, the HIV NES having the amino acid sequence shown in SEQ ID NO: 4, and the NLS consensus sequence having the amino acid sequence shown in SEQ ID NO: 3 (see FIG. 11). In this regard, any arrangement of the at least one NLS mentioned (as well as two or more NLSs according to SEQ ID NO: 5 and / or two or more NLSs according to SEQ ID NO: 3) as well as the at least one NES defined (as well as two or more NESs according to SEQ ID NO: 4) is included herein. In an even more preferred embodiment, said SV40 NLS having the amino acid sequence as shown in SEQ ID NO: 5 is fused / linked to a Cas13 protein, preferably to the C-terminus of said Cas13 protein, followed / fused to said HIV NES having the amino acid sequence as shown in SEQ ID NO: 4, which is then followed / linked to said NLS consensus sequence (also called synthetic NLS) having the amino acid sequence as shown in SEQ ID NO: 3. Thus, a composition of the invention as defined herein preferably comprises two NLSs and one NES fused to a Cas13 protein as defined herein, and even more preferably, the Cas13 protein of the composition as defined herein is fused with two NLSs fused to one NES.

[0067] In some embodiments, the Cas13 proteins described herein can be fused to one or more peptide tags, including His-tags, GST-tags, or myc-tags. In some embodiments, the Cas13 proteins described herein can be fused to a detectable moiety, such as a fluorescent protein (e.g., green fluorescent protein or yellow fluorescent protein).

[0068] According to the present invention, the Cas13 protein may be introduced as a protein as defined above, but alternatively, the Cas13 protein may also be introduced in the form of a nucleotide sequence encoding the protein. In other words, it may be introduced in the form of a nucleic acid molecule comprising a nucleotide sequence encoding the protein. It will be understood that the nucleotide sequence encodes the Cas13 protein in an expressible form, such that expression results in a functional Cas13 protein. Means and methods for ensuring the expression of functional polypeptides are well known in the art. For the at least one Cas13 protein, at least one nucleotide sequence encoding the at least one Cas13 protein may also be used. For example, when two Cas13 proteins are included in the system, this means that one Cas13 protein is encoded by one nucleotide sequence and the other Cas13 protein is separately encoded by another nucleotide sequence. For example, the coding sequence may be included in a vector, such as a plasmid, a cosmid, a virus, a bacteriophage or another vector conventionally used, for example in genetic engineering. The coding sequence inserted into the vector can be, for example, synthesized by standard methods or isolated from natural sources. The coding sequence can be further ligated to transcriptional regulatory elements and / or other amino acid coding sequences. Such regulatory sequences are well known to those skilled in the art and include, but are not limited to, regulatory sequences that ensure the initiation of transcription, internal ribosome entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476) and optionally regulatory elements that ensure the termination of transcription and the stabilization of the transcript.Non-limiting examples of regulatory elements ensuring the initiation of transcription include a translation initiation codon, transcriptional enhancers such as the SV40 enhancer, insulators and / or promoters such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter (Rous sarcome virus), the lacZ promoter, the chicken β-actin promoter, the CAG promoter (a combination of the chicken β-actin promoter and the cytomegalovirus immediate early enhancer), the gail0 promoter, the human elongation factor lot-promoter, the AOX1 promoter, the GAL1 promoter CaM-kinase promoter, the lac, trp or tac promoters, the lacUV5 promoter, the autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedron promoter, or globin introns in mammalian and other animal cells. Non-limiting examples of regulatory elements that ensure transcription termination include V40-polyA site, tk-polyA site, or SV40, lacZ, or AcMNPV polyhedral polyadenylation signals, which will be included downstream of the nucleic acid sequence of the present invention. Further regulatory elements may include translation enhancers, Kozak sequences, and intervening sequences flanked by donor and acceptor sites for RNA splicing. In addition, elements such as origins of replication, drug resistance genes, or regulators (as part of an inducible promoter) may also be included. The nucleotide sequence encoding the Cas13 protein includes DNA, such as cDNA or genomic DNA, and RNA. Preferably, the embodiments describing "RNA" are directed to mRNA.

[0069] Thus, the present invention includes compositions comprising a CRISPR system comprising at least one DNA sequence encoding at least one Cas13 protein as defined herein and at least one DNA sequence encoding at least one gRNA as defined herein, which may be related to a DNA-based system. However, the present invention also includes compositions comprising a CRISPR system comprising at least one RNA sequence (mRNA sequence) encoding at least one Cas13 protein as defined herein and at least one gRNA as defined herein, which may be related to a RNA-based system.

[0070] The nucleotide sequence encoding the Cas13 protein, particularly Cas13d, is advantageously a codon-optimized CRISPR protein. An example of a codon-optimized sequence is in this case a sequence optimized for expression in a eukaryote, for example a human (i.e., optimized for expression in a human), or a sequence optimized for another eukaryote, animal or mammal discussed herein. This is preferred, but other examples are possible, and it will be understood that codon optimization for host species other than human, or codon optimization for a specific organ, is known. In some embodiments, the nucleotide sequence encoding the Cas13 protein is codon-optimized for expression in a specific cell, such as a eukaryotic cell. The eukaryotic cell may be of or derived from a specific organism, such as a plant or mammal, including but not limited to a human, or a non-human eukaryote or animal or mammal discussed herein, such as a mouse, rat, rabbit, dog, livestock, or a non-human mammal or primate. In general, codon optimization refers to the process of modifying a nucleotide sequence for enhanced expression in a host cell of interest by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons of the native sequence with a codon that is more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species show a particular bias for certain codons of certain amino acids. Codon bias (differences in codon usage between organisms) often correlates with the translation efficiency of messenger RNA (mRNA), which is believed to depend, among other things, on the properties of the codon being translated and the availability of certain transfer RNA (tRNA) molecules. The dominance of a selected tRNA in a cell is usually a reflection of the codon that is most frequently used in peptide synthesis. Thus, genes can be tailored to obtain optimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the "Codon Usage Database" available at kazusa.orjp / codon / , and these tables can be adapted in several ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms are also available for codon-optimizing a particular sequence for expression in a particular host cell, for example, Gene Forge (Aptagen; Jacobus, Pa.). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all codons) in the sequence encoding the Cas13 protein correspond to the most frequently used codon for a particular amino acid.

[0071] It will be easily understood by the skilled artisan that due to the degeneracy of the genetic code, more than one nucleotide sequence may code for the Cas13 protein, preferably the Cas13d protein, according to the present invention. The degeneracy arises because the triplet codon specifies 20 amino acids and a stop codon. As there are four bases available to code the genetic information, a triplet codon is required to generate at least 21 different codes. There are 43 possible triplet bases, resulting in 64 possible codons, meaning that some degeneracy must exist. As a result, some amino acids are coded for by more than one triplet, i.e., by up to six. The degeneracy mainly arises from the variation of the third position of the triplet. This means that the present invention can utilize nucleotide sequences that have different sequences but still code for the same Cas13 protein. The nucleotide sequences used according to the present invention can be of natural or (semi-) synthetic origin. Thus, the nucleotide sequences can be, for example, nucleotide sequences that have been synthesized according to conventional protocols of organic chemistry. Those skilled in the art are familiar with the preparation and use of said probes (see, for example, Sambrook and Russel "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, NY (2001)). Also according to the present invention, the nucleotide sequences used by the present invention may be nucleic acid mimicking molecules known in the art, such as synthetic or semi-synthetic derivatives and mixed polymers of nucleotide sequences. They may contain additional non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Nucleic acid mimicking molecules or derivatives according to the present invention include, but are not limited to, phosphorothioate nucleic acid, phosphoramidate nucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA).

[0072] gRNA When the CRISPR protein is a Cas13 protein, as in the present invention, tracrRNA is not required in the composition, as described elsewhere herein.However, the composition further comprises at least one guide RNA (gRNA) that can hybridize with one or more target RNA molecules as defined herein.

[0073] As used herein, the term "guide RNA", "gRNA" or "complementary crRNA" refers to a polynucleotide comprising any polynucleotide sequence that has sufficient complementarity with one or more target RNA molecules to hybridize with one or more target RNA molecules and direct sequence-specific binding of an RNA targeting complex comprising gRNA and CRISPR Cas13 protein to one or more target RNA molecules. In general, a gRNA can be any polynucleotide sequence that (i) can form a complex with a CRISPR protein, such as Cas13, and (ii) comprises a sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with a target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. As used herein, the term "gRNA capable of hybridizing" refers to a subsection of a gRNA that has sufficient complementarity to one or more target RNA molecules to hybridize with one or more target RNA molecules and mediate binding of a CRISPR complex to the target RNA. Generally, a guide sequence, which is part of a gRNA, is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence, as defined elsewhere herein, to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.

[0074] As used herein, the term "capable of forming a complex with Cas13 protein" refers to a gRNA having a structure that allows Cas13 protein to specifically bind to the gRNA, so as to form a complex that can bind to a target RNA molecule in a sequence-specific manner and exert a function on the target RNA. The structural components of the gRNA can include a direct repeat sequence and a guide sequence (or spacer). The sequence-specific binding to the target RNA is mediated by the "guide sequence", which is a part of the gRNA, being complementary to the target RNA.

[0075] In a further step, such at least one gRNA is optimized by the present inventors.Usually, 22-base complementary sequence is used as standard.As used herein, at least one gRNA or the nucleotide sequence encoding said gRNA contained in the composition of the present invention preferably comprises a length of at least about 23, for example at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 nucleotides.Therefore, the gRNA of the present invention is optimized by extending its length as a further modification to the novel composition disclosed herein.

[0076] The at least one gRNA or the nucleotide sequence encoding said gRNA contained in the composition of the present invention comprises, in a more preferred embodiment, a length of about 23 to about 30 nucleotides, such as 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, preferably about 24 to about 30 nucleotides, more preferably about 25 to about 30 nucleotides, most preferably about 26 to about 30 nucleotides. The inventors have been able to show that longer gRNAs, particularly those comprising a length of about 26 to about 30, about 27 to about 30, about 28 to about 30, about 29 to about 30 nucleotides, significantly increase the enzymatic activity of the Cas13 protein (see Figures 1 and 2). Preferably, at least one gRNA has a length of about 26, 27, 28, 29 or about 30 nucleotides.

[0077] In a preferred embodiment, i) at least one Cas13 protein or a nucleotide sequence encoding same, which is fused to at least one NLS which is fused to at least one NES; ii) at least one gRNA as defined herein having a length of about 26 to about 30 nucleotides, or a nucleotide sequence encoding same; Also contemplated herein is a composition comprising a CRISPR system comprising: i) at least one Cas13 protein or a nucleotide sequence encoding same, which is fused to at least one NLS which is fused to at least one NES; ii) at least one gRNA as defined herein having a length of about 27 to about 30 nucleotides, or a nucleotide sequence encoding same; Also contemplated herein is a composition comprising a CRISPR system comprising: i) at least one Cas13 protein or a nucleotide sequence encoding same, which is fused to at least one NLS which is fused to at least one NES; ii) at least one gRNA as defined herein having a length of about 28 to about 30 nucleotides, or a nucleotide sequence encoding same; Also contemplated herein is a composition comprising a CRISPR system comprising: i) at least one Cas13 protein or a nucleotide sequence encoding same, which is fused to at least one NLS which is fused to at least one NES; ii) at least one gRNA as defined herein having a length of about 29 to about 30 nucleotides, or a nucleotide sequence encoding same; Also contemplated herein is a composition comprising a CRISPR system comprising: i) at least one Cas13 protein or a nucleotide sequence encoding same, which is fused to at least one NLS which is fused to at least one NES; ii) at least one gRNA as defined herein having a length of about 26 nucleotides, or a nucleotide sequence encoding same; Also contemplated herein is a composition comprising a CRISPR system comprising: i) at least one Cas13 protein or a nucleotide sequence encoding same, which is fused to at least one NLS which is fused to at least one NES; ii) at least one gRNA as defined herein having a length of about 27 nucleotides, or a nucleotide sequence encoding same; Also contemplated herein is a composition comprising a CRISPR system comprising: i) at least one Cas13 protein or a nucleotide sequence encoding same, which is fused to at least one NLS which is fused to at least one NES; ii) at least one gRNA as defined herein having a length of about 28 nucleotides, or a nucleotide sequence encoding same; Also contemplated herein is a composition comprising a CRISPR system comprising: i) at least one Cas13 protein or a nucleotide sequence encoding same, which is fused to at least one NLS which is fused to at least one NES; ii) at least one gRNA as defined herein having a length of about 29 nucleotides, or a nucleotide sequence encoding same; Also contemplated herein is a composition comprising a CRISPR system comprising: i) at least one Cas13 protein or a nucleotide sequence encoding same, which is fused to at least one NLS which is fused to at least one NES; ii) at least one gRNA as defined herein having a length of about 30 nucleotides, or a nucleotide sequence encoding same; Compositions comprising a CRISPR system comprising the same are therefore also contemplated herein.

[0078] In a preferred embodiment, the at least one gRNA or the nucleotide sequence encoding the gRNA has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or even 100% complementary sequence identity to one or more target RNA molecules. The inventors have found that it is acceptable to have one mismatch with one or more target RNA molecules, which means at least about 95% complementary sequence identity to one or more target RNA molecules, depending on the length of the gRNA. Two adjacent mismatches cause a significant reduction in activity. If the two mismatches are not adjacent to each other, the effect on activity is significantly smaller. However, if there are three mismatches with one or more target RNA molecules, the at least one gRNA can no longer hybridize to one or more target RNA molecules. Preferably, the at least one gRNA or the nucleotide sequence encoding the gRNA has at least about 85% complementary sequence identity to one or more target RNA molecules. In some embodiments, the degree of complementarity between a guide sequence as part of a gRNA as defined elsewhere herein and its corresponding target sequence contained in a target RNA molecule, when optimally aligned using a suitable alignment algorithm, is about or greater than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more, preferably about 85%, or greater than about 85%.Optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0079] At least one (one or more) gRNAs can be used in the composition according to the present invention, such as one, two, three, four, five and more. Thereby, each different gRNA can bind to a viral or bacterial sequence in the genome. Simultaneous application of two or more different gRNAs addressing several regions of the viral / bacterial genome can also be applied by the present invention. This is a process called "multiplexing". Thus, efficient and sustained degradation can be ensured even if the virus / bacteria mutates.

[0080] In certain embodiments, at least one (one or more) gRNAs can / do bind to the coding strand of RNA. In certain embodiments, gRNAs can / do bind to the non-coding strand of RNA. In certain embodiments, gRNAs bind to viral / bacterial genomic RNA (positive or negative sense or coding or non-coding strand). In certain embodiments, gRNAs bind to RNA (positive or negative sense or coding or non-coding strand) transcribed from viral / bacterial genomic DNA.

[0081] In a preferred embodiment, said at least one(s) gRNA(s) can hybridize / bind or hybridize / bind to the 5' and / or 3' untranslated regions of said one or more target RNA molecules (see FIG. 7a). The term "non-coding region" / "non-coding sequence" may be used interchangeably with the term "untranslated region". These two regions are present in both the genome and all subgenomic mRNAs of every virus / bacteria, so the antiviral / antibacterial effect of Cas13 is strongest at these locations.

[0082] Moreover, the gRNA contained in the composition may be encoded by a nucleotide sequence. In other words, it may be introduced in the form of a nucleic acid molecule comprising a nucleotide sequence encoding the gRNA. The definitions and preferred embodiments described above for the nucleotide sequence encoding the Cas13 protein also apply mutatis mutandis to the nucleotide sequence encoding the gRNA.

[0083] As a further modification to the composition comprising the novel CRISPR system described elsewhere herein, the fusion of the nucleotide sequence encoding the gRNA with one tRNA proves to be advantageous as it supports gRNA folding (see FIG. 3). The tRNA is then fused directly to the 3' end of the nucleotide sequence encoding the gRNA, preferably as defined elsewhere herein when the nucleotide sequence is DNA. Thus, compositions in which the DNA nucleotide sequence encoding the gRNA is directly fused to a tRNA at the 3' end of the nucleotide sequence are also included herein. Any tRNA known to those skilled in the art may be used. The dominance of a selected tRNA in a cell is usually a reflection of the codons most frequently used in peptide synthesis. Thus, genes can be tailored to obtain optimal gene expression in a given organism based on codon optimization as defined elsewhere herein. Preferably, a tRNA derived from murine gamma-herpesvirus 68 (MHV68) having the amino acid sequence shown in SEQ ID NO: 8 is used for the fusion. In this context, and similarly with respect to fusion with a ribozyme, the definition of "derived from" as defined elsewhere herein may apply, and has at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or even at least about 99% RNA sequence identity with the tRNA or with the ribozyme from which it is derived. It is also encompassed by the present invention that said tRNA may be fused to said gRNA as defined elsewhere herein via a linker as defined herein.

[0084] Alternatively, said nucleotide sequence encoding said gRNA, preferably meaning a DNA sequence, can be directly fused to a ribonuclease enzyme (ribozyme for short) as defined herein. A "ribozyme" is an RNA molecule capable of catalyzing certain biochemical reactions, including RNA splicing in gene expression as known to those skilled in the art. For said fusion, natural or even synthetic ribozymes can be used herein. In a preferred embodiment, said ribozyme is derived from Hepatitis Delta Virus (HDV) with the amino acid sequence shown in SEQ ID NO: 9, as defined elsewhere herein. Contrary to the tRNA used and defined herein, which can be cleaved off by RNAse P and RNAse Z after a certain time, which results in a well-defined end of said gRNA, preferably a well-defined 3' end of said gRNA, the ribozyme used herein excises itself from said gRNA, but also results in a well-defined end of said gRNA, preferably a well-defined 3' end of said gRNA, both modifications leading to the efficiency improvements described herein.

[0085] The subject of the present invention is also an isolated ribozyme or ribozyme portion fused to said nucleotide sequence encoding said gRNA (see FIG. 3). Such a ribozyme comprises a motif that specifically and covalently fuses to said sequence, such that the ribozyme is specifically joined to the sequence. The ribozyme of the present invention may be a contiguous sequence or may be composed of two non-contiguous components that interact with each other to form a complete ribozyme. The two non-contiguous components are the ribozyme segment that specifically and covalently fuses to said sequence and the ribozyme segment that comprises the remaining ribozyme.

[0086] The ribozyme RNA specifically covalently fused to said nucleotide sequence is present in the ribozyme (is a component or segment thereof), which can be either a continuous sequence (the entire ribozyme is a continuous sequence) or composed of two non-contiguous components: one containing the ribozyme RNA specifically covalently fused to said sequence and one containing the remaining part of the ribozyme sequence. The remaining ribozyme sequence is the ribozyme sequence that, in combination with the components covalently fused to said sequence, constitutes the complete ribozyme. The ribozyme sequence or the segment covalently fused to said sequence can be as short as one nucleotide in length and can originate from any position within the ribozyme (e.g., the 5' end, an internal segment, the 3' end). In one embodiment, the ribozyme segment comprises 1 to about 18 nucleotides, e.g., from the first to about the 18th nucleotide (from the 5' end) of the ribozyme. In a further embodiment, the ribozyme segment is the first 13 to 18 nucleotides (from the 5' end) of the ribozyme. (e.g., the first 13, 14, 15, 16, 17 or 18 nucleotides from the 5' end). The other component is the remaining ribozyme sequence (the remaining part of the ribozyme required to form a functional ribozyme). The two ribozyme components interact to form a functional (complete) ribozyme. The 5' end of the ribozyme RNA may optionally contain three phosphate groups or an mRNA cap, such as 7-methylguanosine triphosphate. Thus, it is also included herein that the nucleotide sequence encoding the gRNA is covalently fused to a ribozyme RNA as defined herein. Preferably, a ribozyme as defined elsewhere herein is fused directly to the 3' end of a nucleotide sequence encoding the gRNA, as defined elsewhere herein, and preferably, the nucleotide sequence is DNA. Thus, it is also included herein that the DNA nucleotide sequence encoding the gRNA is fused directly to a ribozyme as defined elsewhere herein at the 3' end of the nucleotide sequence.

[0087] As mentioned above, the fusion of the tRNA and / or the ribozyme, which is a direct fusion with the DNA nucleotide sequence encoding the gRNA, may be covalent. The present invention therefore further includes that the nucleotide sequence (DNA sequence) encoding the gRNA is covalently (directly) fused with the tRNA and / or ribozyme. In this context, when the term "covalently fused" is used, a covalent bond is formed between the nucleotide sequence encoding the gRNA of the present invention and the tRNA or ribozyme as defined above. In these embodiments, the term "directly" again means that the nucleotide sequence encoding the gRNA and the tRNA and / or ribozyme are placed one after the other without a linker as defined elsewhere herein.

[0088] In a preferred embodiment, i) a nucleotide sequence encoding at least one Cas13 protein fused to at least one NLS fused to at least one NES; ii) a nucleotide sequence encoding at least one gRNA as defined herein, said gRNA having a length of about 26 to about 30 nucleotides, said nucleotide sequence encoding said gRNA also being fused to a tRNA or ribozyme as defined herein; Also contemplated herein is a composition comprising a CRISPR system comprising: i) a nucleotide sequence encoding at least one Cas13 protein fused to at least one NLS fused to at least one NES; ii) a nucleotide sequence encoding at least one gRNA as defined herein, said gRNA having a length of about 27 to about 30 nucleotides, said nucleotide sequence encoding said gRNA also being fused to a tRNA or ribozyme as defined herein; Also contemplated herein is a composition comprising a CRISPR system comprising: i) a nucleotide sequence encoding at least one Cas13 protein fused to at least one NLS fused to at least one NES; ii) a nucleotide sequence encoding at least one gRNA as defined herein, said gRNA having a length of about 28 to about 30 nucleotides, said nucleotide sequence encoding said gRNA also being fused to a tRNA or ribozyme as defined herein; Also contemplated herein is a composition comprising a CRISPR system comprising: i) a nucleotide sequence encoding at least one Cas13 protein fused to at least one NLS fused to at least one NES; ii) a nucleotide sequence encoding at least one gRNA as defined herein, said gRNA having a length of about 29 to about 30 nucleotides, said nucleotide sequence encoding said gRNA also being fused to a tRNA or ribozyme as defined herein; Also contemplated herein is a composition comprising a CRISPR system comprising: i) a nucleotide sequence encoding at least one Cas13 protein fused to at least one NLS fused to at least one NES; ii) a nucleotide sequence encoding at least one gRNA as defined herein, said gRNA having a length of about 26 nucleotides, said nucleotide sequence encoding said gRNA also being fused to a tRNA or ribozyme as defined herein; Also contemplated herein is a composition comprising a CRISPR system comprising: i) a nucleotide sequence encoding at least one Cas13 protein fused to at least one NLS fused to at least one NES; ii) a nucleotide sequence encoding at least one gRNA as defined herein, said gRNA having a length of about 27 nucleotides, said nucleotide sequence encoding said gRNA also being fused to a tRNA or ribozyme as defined herein; Also contemplated herein is a composition comprising a CRISPR system comprising: i) a nucleotide sequence encoding at least one Cas13 protein fused to at least one NLS fused to at least one NES; ii) a nucleotide sequence encoding at least one gRNA as defined herein, said gRNA having a length of about 28 nucleotides, said nucleotide sequence encoding said gRNA also being fused to a tRNA or ribozyme as defined herein; Also contemplated herein is a composition comprising a CRISPR system comprising: i) a nucleotide sequence encoding at least one Cas13 protein fused to at least one NLS fused to at least one NES; ii) a nucleotide sequence encoding at least one gRNA as defined herein, said gRNA having a length of about 29 nucleotides, said nucleotide sequence encoding said gRNA also being fused to a tRNA or ribozyme as defined herein; Also contemplated herein is a composition comprising a CRISPR system comprising: i) a nucleotide sequence encoding at least one Cas13 protein fused to at least one NLS fused to at least one NES; ii) a nucleotide sequence encoding at least one gRNA as defined herein, said gRNA having a length of about 30 nucleotides, said nucleotide sequence encoding said gRNA also being fused to a tRNA or ribozyme as defined herein; Compositions comprising a CRISPR system comprising the same are therefore also contemplated herein.

[0089] The guide RNA nucleotide sequence and / or the Cas nucleotide sequence can be functionally or operatively linked to a regulatory element, which thus drives the expression. The promoter can be a constitutive promoter and / or a conditional promoter and / or an inducible promoter and / or a tissue-specific promoter. Suitable promoters have already been described above and can further be selected from the group consisting of RNA polymerase, pol I, pol II, pol III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, (3-actin promoter, phosphoglycerol kinase (PGK) promoter, EF1α promoter and U6 promoter, TetOn / Off, 7SK, CAG, CBh, UbC or any tetracycline-dependent or interferon-dependent promoter.

[0090] The present invention also includes compositions comprising the CRISPR system as defined elsewhere herein, in which the nucleotide sequence encoding the Cas13 protein and / or gRNA as defined elsewhere herein may be further modified at the RNA level. In other words, the RNA sequence encoding the Cas13 protein and / or gRNA as defined herein may include modified UTRs (see FIG. 15), post-transcriptional or co-transcriptional addition of a 5' CAP structure, preferably post-transcriptional addition of a 5' CAP structure, replacement of UTP with N1-methyl pseudo-UTP, and / or replacement of the two 5' and / or 3' terminal nucleotides of said gRNA with 2'-O-methyl-3' P-thioate (see FIG. 10A). It is also included herein and in order to enhance expression and reduce possible toxicity that the nucleotide sequence encoding the Cas13 protein and / or guide RNA as defined elsewhere herein may be modified to include one or more modified nucleosides, for example with pseudo-U and / or 5-methyl-C, instead of or in addition to the above modifications. In a preferred embodiment, the present invention includes a composition comprising a CRISPR system as defined elsewhere herein, wherein the nucleotide sequence encoding a gRNA as defined elsewhere herein is further modified by substitution of the two 5' and / or 3' terminal nucleotides of said gRNA with 2'-O-methyl-3' P-thioate (also called 2'-O-methyl phosphorothioate).

[0091] The RNA can be stabilized by using modified UTR 5' and / or 3' UTR from β-globin polyA tail (a series of 120 or more adenines). Furthermore, the post-transcriptional or co-transcriptional 5-prime cap (5' CAP), a specially modified nucleotide on the 5' end of the RNA transcript of the present invention, protects the mRNA from exonuclease degradation and may also be important for nuclear export and initiation of translation. Another important advance can be seen in the preparation of chemically modified mRNA by replacing standard bases with modified bases, for example uridine with pseudouridine, thereby improving the half-life of the mRNA, the translation efficiency and its immunological profile. N1-methyl-pseudouridine is preferred because it provides more efficient translation and at the same time suppresses the innate immune response compared to unmodified mRNA. A final modification by which gRNA may be stabilized (substitution of the two 5' and / or 3' terminal nucleotides of the gRNA with 2'-O-methyl-3' P-thioate) includes that the 2'OH on the sugar of the RNA may be replaced by a methyl group, and that the phosphodiester bond in the RNA backbone may be modified by sulfur, thereby rendering the gRNA resistant to RNAse. Any combination of each of the above modifications to the RNA may also be included herein.

[0092] These modifications of the RNA (Cas13 mRNA and / or gRNA) improved the knockdown efficiency for RNA delivery of Cas13 protein (see FIG. 10B or FIG. 15). Thus, applying further modifications to the RNA encoding Cas13 protein and / or gRNA as defined herein, contained by the CRISPR system contained in the composition of the present invention, can lead to a further increase in the knockdown efficiency of Cas13 protein, with other modifications being used as further modifications applied to the system other than the fusion of at least one NLS fused to at least one NES, as described by the present invention. Thus, the RNA sequence of the CRISPR system comprised by the composition of the invention, which encodes the Cas13 protein and / or gRNA as defined herein, may further comprise (in addition to the other modifications of the system mentioned by the invention) any modification selected from the group consisting of modified UTRs as defined herein, such as modified nucleosides, post-transcriptional or co-transcriptional (preferably post-transcriptional) 5' CAP structures, substitution of UTP with N1-methyl pseudo-UTP and substitution of the two 5' and / or 3' terminal nucleotides of the gRNA with 2'-O-methyl-3' P-thioate, with pseudo-U and / or 5-methyl-C. In a particular embodiment, the RNA sequence of the CRISPR system comprised by the composition of the invention, which encodes the Cas13 protein and / or gRNA as defined herein, may further comprise (in addition to the other modifications of the system mentioned by the invention) a modified 3' UTR, in particular including a 3' UTR with SARS-CoV-2 N gene knockdown and / or RdRP gene knockdown (see FIG. 15).

[0093] Compositions comprising a CRISPR system as defined elsewhere herein are also included by the present invention, where the nucleotide sequence encoding the Cas13 protein and / or gRNA as defined elsewhere herein may be further modified at the DNA level, including fusing the nucleotide sequence encoding the gRNA, preferably DNA, with at least one viral transport element. The fusion of the nucleotide sequence encoding the gRNA with at least one viral transport element as defined herein has been found to be advantageous, as it further increases the knockdown efficiency (see FIG. 16). At least one viral transport element as defined herein is then fused to the 5'-end of the nucleotide sequence encoding the gRNA, preferably DNA, as defined elsewhere herein. In this context, fusing means that the nucleotide sequence encoding the gRNA and at least one viral transport element are fused via a linker, such as any nucleotide linker. Thus, compositions in which the DNA nucleotide sequence encoding the gRNA is fused to at least one viral transport element as defined herein at the 5'-end of the nucleotide sequence, preferably DNA, is also included herein. The term "at least one viral transport element" means that two, three, four, five or more viral transport elements as defined herein can be used. In a preferred embodiment, said at least one viral transport element is CTE or VARdm, preferably CTE, which is fused with said gRNA coding nucleotide sequence (preferably DNA sequence) as defined herein. In short, the combination of NLS-NES and CTE-gRNA maximizes knockdown efficiency. Such fusion to gRNA can also be performed at the protein level of said composition.

[0094] Furthermore, the inventors have also found that compositions comprising the novel CRISPR system defined elsewhere herein have additional RNAse activity in mammalian cells (see FIG. 5). This led to the conclusion that the modified system of the composition comprising the at least one Cas13 protein may further have non-specific RNAse activity, specifically directed against ribosomal RNA (rRNA), beyond its effect on the complementary target sequence hybridized by the at least one RNA / cleaved by the Cas13 protein. In other words, not only is the bound target RNA cleaved / degraded, but also adjacent RNAs (mainly rRNAs) are degraded, resulting in a bystander effect (so-called additional activity). The additional activity of the system comprised by the composition may be present in all types of mammalian cells. Examples of such mammalian cells can be mouse neuroblastoma (N2a), mouse myoblasts (C2C12), human glioblastoma (U87M), human hepatoma cells (HepG2), mouse mycoglial cells (BV-2), or primary cells.

[0095] The cleavage of viral or bacterial rRNA as an additional target other than one or more target RNA molecules defined herein by said system according to the present invention is further enhanced by applying the novel system modified herein.Therefore, the formation of essential structural proteins of viral or bacterial is further reduced by such additional activity of said system contained by composition.This is regarded as an additional mechanism for further slowing down the replication of virus in cells, for example, to reduce viral load (see Fig. 5).

[0096] Such concomitant RNAse activity of Cas13 protein mainly on rRNA is indicated by specific binding to 28S rRNA. In other words, the composition comprising the specific system defined herein can bind and cleave 28S rRNA. The reduction of ribosomal RNA experimentally demonstrated by the inventors may lead to a temporary translation block in cells due to the reduction of ribosome amount, which means that the virus or bacteria may be attacked at an additional level as well. (See FIG. 8). Thus, the system comprised in the composition may prefer a defined position of 28S ribosomal RNA, so that the Cas13 protein may first cleave its target RNA. Only when the target RNA is found, the Cas13 protein may be activated and also cleave the defined position of ribosomal RNA, which ultimately leads to a temporary inhibition of protein translation (FIG. 6D and E; also see FIG. 9).

[0097] CRISPR proteins such as Cas13 proteins and / or any of the present RNAs, e.g., guide RNAs, can be delivered using any suitable vector, e.g., plasmid or viral vector, e.g., adeno-associated virus (AAV), lentivirus, adenovirus or other viral vector types, or combinations thereof. Such proteins and one or more guide RNAs can be packaged into one or more vectors, e.g., plasmid or viral vector. In some embodiments, the vector, e.g., plasmid or viral vector, is delivered to the tissue of interest, e.g., by intramuscular injection, while in other cases, delivery is intravenous, transdermal, intranasal, oral, mucosal, or via other delivery methods. Such delivery can be via either single or multiple doses. Those skilled in the art will understand that the actual dosage delivered herein can vary widely depending on a variety of factors, such as the choice of vector, the target cell, organism or tissue, the general condition of the subject to be treated as mentioned elsewhere herein, the degree of transformation / modification desired, the route of administration, the method of administration, the type of transformation / modification desired, etc. Such dosages may further contain a carrier, as defined elsewhere herein.

[0098] In one embodiment herein, the delivery of the composition comprising the system based on the DNA level is via adenovirus, which may be a single booster dose. In another embodiment herein, the adenovirus is delivered via multiple doses. In a further embodiment herein, the delivery of the composition comprising the system based on the DNA level is via plasmid. In such a plasmid composition, the dosage should be a sufficient amount of plasmid to elicit a response. For example, a suitable amount of plasmid DNA in a plasmid composition can be about 0.1 to about 2 mg, or about 1 μg to about 10 μg per 70 kg individual. The plasmid of the present invention will generally include: (i) a promoter; (ii) a sequence encoding a Cas13 protein as defined herein, operably linked to the promoter; (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator downstream of (ii) and operably linked to (ii). The plasmid preferably also encodes the RNA components of the CRISPR complex, such as comprising the nucleotide sequence encoding the gRNA as defined herein, although one or more of these may alternatively be encoded on a different vector.In another further embodiment herein, the delivery of the composition comprising the system based on DNA level is via particles and / or nanoparticles as defined elsewhere herein.

[0099] In some embodiments, the compositions comprising the systems based on the RNA level (e.g., comprising the mRNA encoding the Cas13 protein and a pre-chemically synthesized gRNA) of the present invention, as well as on the protein level (including the Cas13 protein and a pre-chemically synthesized gRNA), are delivered in the form of liposomes or lipofectin formulations, etc., and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, which are incorporated herein by reference. Thus, delivery of the nucleotide sequence encoding the Cas13 protein and / or delivery of the gRNA of the present invention can be in the form of RNA, via liposomes, particles, microvesicles. For example, Cas13 mRNA and gRNA can be packaged into liposome particles for in vivo delivery.

[0100] Liposomes are spherical vesicular structures composed of a single or multilamellar lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes have attracted considerable attention as drug delivery vehicles because they are biocompatible and nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes to the blood-brain barrier (BBB). Liposomes can be made from several different types of lipids, however, phospholipids are most commonly used to make liposomes as drug carriers. Liposome transfection reagents such as Life Technologies' Lipofectamine and other commercially available reagents can effectively deliver RNA molecules to the liver.

[0101] Generally, a particle is defined as a small object that behaves as a whole unit with respect to its transport and properties. Particles are further classified by diameter. Coarse particles encompass the range of 2,500-10,000 nanometers. Fine particles are 100-2,500 nanometers in size. Ultrafine particles or nanoparticles are generally 1-100 nanometers in size. The basis for the 100 nm limit is the fact that the novel properties that distinguish particles from bulk materials are usually manifested at a critical length scale of less than 100 nm. Particle delivery systems within the scope of the present invention may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal particles. Thus, for example, any of the delivery systems described herein, including but not limited to lipid-based systems, liposomes, micelles, microvesicles, exosomes, or gene guns, may be provided as particle delivery systems within the scope of the present invention. In general, "nanoparticle" refers to any particle having a diameter of less than 1000 nm.

[0102] Similarly preferred means of RNA / protein delivery include delivery of RNA / protein via nanoparticles (Cho, S., Goldberg, et al. Advanced Functional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A., et al., Journal of Internal Medicine, 267: 9-21, 2010). In certain preferred embodiments, the nanoparticles of the present invention have a maximum dimension (e.g., diameter) of 500 nm or less. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension in the range of 25 nm to 200 nm. In other preferred embodiments, the particles of the present invention have a maximum dimension of 100 nm or less. In other preferred embodiments, the nanoparticles of the present invention have a maximum dimension in the range of 35 nm to 60 nm. Exosomes are endogenous nanovesicles that transport RNA and proteins and can deliver RNA to the brain and other target organs. Indeed, exosomes have been shown to be particularly useful in the delivery of siRNA, a system similar to the RNA targeting system. Further details of delivery systems for DNA delivery (AAV, lentivirus, etc.) or RNA / protein delivery (liposomes, particles, exosomes, etc.) are described in US20200165594, which is incorporated herein by reference.

[0103] CRISPR Cas13 mRNA and gRNA may be delivered separately. CRISPR Cas13 mRNA can be delivered before gRNA to allow time for CRISPR Cas13 protein to be expressed. CRISPR Cas13 mRNA can be administered 1-12 hours (preferably about 2-6 hours) before administration of gRNA. Alternatively, CRISPR Cas13 mRNA and gRNA can be administered together. Advantageously, a second booster dose of gRNA can be administered 1-12 hours (preferably about 2-6 hours) after the first administration of CRISPR Cas13 mRNA + gRNA.

[0104] The CRISPR Cas13 protein and gRNA may be delivered separately. Preferably, the CRISPR Cas13 protein and gRNA are delivered together. Prior to said delivery, the protein and the gRNA may be mixed and frozen as a complex until administration of the complex. Advantageously, a second booster dose of gRNA may be administered 1-12 hours (preferably about 2-6 hours) after the first administration of CRISPR Cas13 protein + gRNA.

[0105] target RNA molecule As already mentioned herein, said at least one gRNA can hybridize with one or more target RNA molecules. In the context of the formation of a CRISPR complex, "target sequence" refers to a sequence with which a guide sequence (part of a gRNA) is designed to have complementarity, as defined elsewhere herein, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. The target sequence can comprise an RNA polynucleotide. The term "target RNA molecule" refers to an RNA polynucleotide (target RNA) that is or comprises a target sequence. In other words, the target RNA can be a part of a gRNA, i.e., an RNA polynucleotide with which a guide sequence is designed to have complementarity as described herein, and to which an effector function mediated by a complex comprising a CRISPR effector protein and a gRNA is directed. In some embodiments, the target RNA molecule, and thus the target RNA sequence, is located in the nucleus and / or cytoplasm of a cell. Target RNA, i.e., the RNA of interest, is the RNA targeted by the present invention, which leads to the recruitment to the target site of interest on the target RNA and the binding of Cas13 protein at the target site of interest on the target RNA. The one or more target RNA molecules can be any suitable form of RNA. In some embodiments, it can include mRNA. In other embodiments, the one or more target RNA molecules can include tRNA or rRNA. In other embodiments, the one or more target RNA molecules can include miRNA. In other embodiments, the one or more target RNA molecules can include siRNA.

[0106] In some embodiments, the one or more target RNA molecules comprising the target sequence, or the target sequence itself, can be a sequence within an RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double-stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA) and small cytoplasmic RNA (scRNA). In some preferred embodiments, the one or more target RNA molecules comprising the target sequence, or the target sequence itself, can be a sequence within an RNA molecule selected from the group consisting of mRNA, pre-mRNA and rRNA. In some other preferred embodiments, the one or more target RNA molecules comprising the target sequence, or the target sequence itself, can be a sequence within an RNA molecule selected from the group consisting of ncRNA and lncRNA. In some other preferred embodiments, the target RNA molecule or molecules that comprise the target sequence, or the target sequence itself, may be a sequence within an mRNA molecule or a pre-mRNA molecule.

[0107] Thus, at least one gRNA capable of hybridizing with one or more target RNA molecules as defined elsewhere herein may also comprise hybridization with one or more RNA target sequences. Preferably, at least one gRNA that targets one or more target RNA molecules / RNA target sequences, to which it is to some extent complementary as defined elsewhere herein, focuses on the 5' and / or 3' untranslated regions / portions of said RNA molecules (mRNA molecules), as already defined elsewhere herein.

[0108] In a preferred embodiment, said one or more target RNA molecules are virus or bacterial target RNA molecules.In a particular embodiment, the virus is an RNA virus.In a further embodiment, the virus is a single-stranded or double-stranded RNA virus.In a further embodiment, the virus is a positive-sense RNA virus or a negative-sense RNA virus or an ambisense RNA virus. In further embodiments, the virus is a Retroviridae virus, a Lentiviridae virus, a Coronaviridae virus, a Picornaviridae virus, a Caliciviridae virus, a Flaviviridae virus, a Togaviridae virus, a Bomaviridae virus, a Filoviridae virus, a Paramyxoviridae virus, a Pneumoviridae virus, a Rhabdoviridae virus, a Arenaviridae virus, a Bunyaviridae virus, an Orthomyxoviridae virus, or a Deltavirus. In a particular embodiment, the virus is selected from the group consisting of lymphocytic choriomeningitis virus, coronavirus, HIV, SARS, Venezuelan equine encephalitis (VEE) virus, poliovirus, rhinovirus, hepatitis A, Norwalk virus, yellow fever virus, West Nile virus, hepatitis C virus, dengue virus, Zika virus, rubella virus, Ross River virus, Sindbis virus, chikungunya virus, Boma disease virus, Ebola virus, Marburg virus, measles virus, mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, human respiratory syncytial virus, rabies virus, Lassa virus, Hantavirus, Crimean-Congo hemorrhagic fever virus, influenza, and hepatitis D virus. In another particular embodiment, the virus is a DNA virus. In a further embodiment,The virus is a single-stranded or double-stranded DNA virus. In further embodiments, the virus is a positive-sense or negative-sense DNA virus or an ambisense DNA virus. In further embodiments, the virus is a Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae (including human herpesvirus and varicella zoster virus), Malocoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae (including African swine fever virus), Baculoviridae, Cicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae ae), Maseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae (including Simian virus 40, JC virus, and BK virus), Poxviridae (including cowpox and smallpox),The virus may be a Sphaerolipoviridae, Tectiviridae, Turriviridae, Dinodnavirus, Salterprovirus, or Rhizidovirus. In certain exemplary embodiments, the virus may be a retrovirus. Exemplary retroviruses may include one or more or any combination of viruses from the genera Alpharetrovirus, Betaretrovirus, Gammaretrovirus, Deltaretrovirus, Epsilonretrovirus, Lentivirus, Spumavirus, or the families Metaviridae, Pseudoviridae, Retroviridae (including HIV), Hepadnaviridae (including Hepatitis B virus), and Caulimoviridae (including Cauliflower Mosaic Virus). In certain embodiments, the virus is a drug-resistant virus and thus belongs to a multi-resistant virus as defined elsewhere herein. By way of example and not limitation, the virus may be a ribavirin-resistant virus. Ribavirin is a highly effective antiviral drug that attacks several RNA viruses. In a more preferred embodiment, the term "virus" as used throughout the present invention refers to coronaviruses, influenza A viruses, flaviviruses such as Ebola viruses, morbilliviruses, hepaciviruses, TBE viruses, dengue viruses, yellow fever viruses, and / or Zika viruses.

[0109] As for the bacterial target RNA molecule, it can comprise any RNA molecule defined herein from any bacterium that can be hybridized by gRNA defined herein.Because bacteria also comprise RNA, it is plausible to demonstrate that the composition comprising the CRISPR system can also be applied to target one or more bacterial RNA molecules.In certain embodiments, the bacterium is a drug-resistant bacterium, and therefore belongs to multi-resistant bacteria. By way of example, and without limitation, resistant bacteria include Staphylococcus aureus, Streptococcus pneumoniae, Koagulase-negative Staphylococcus, Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Serratia marcescens, Citrobacter spp., Pseudomonas aeruginosa, Acinetobacter baumannii, and Stenotrophomonas maltophilia. Such multi-resistant bacteria, as used herein, refer to a species of microorganism (such as a virus or bacteria) that contains antibacterial resistance to at least one antibacterial drug, as known to those of skill in the art.

[0110] Therefore, the present invention also includes that the composition defined herein is suitable for inactivating bacterial or viral single-stranded (ss) RNA.In this context, the term "inactivating" or "inactivate" or "inactivation" refers to a composition comprising a CRISPR system defined herein, in which said at least one Cas13 protein forms a complex with at least one gRNA, and at least one gRNA directs the complex to one or more target RNA molecules, and not only targets / detects one or more target RNA molecules, but also cleaves / degrades said target RNA molecules.Therefore, the terms "cleaving", "cleave", "cleavage" or "degrading", "degrade" or "degradation" can also be used interchangeably with the terms "inactivating", "inactivate" or "inactivation".

[0111] Pharmaceutical Compositions According to the present invention, the composition defined throughout the present invention may further comprise at least one carrier as defined elsewhere herein.The present invention also relates to the composition defined herein, which is a pharmaceutical composition.Accordingly, said pharmaceutical composition is used herein for therapeutic purposes.Furthermore, the present invention relates to the use of said composition disclosed herein above for the preparation of a pharmaceutical composition.

[0112] According to the present invention, the term "pharmaceutical composition" refers to a composition for administration to a patient, preferably a human patient. A pharmaceutical composition or formulation is usually in a form that allows the biological activity of the active ingredient to be effective and therefore can be administered to a subject for therapeutic applications as described herein. A pharmaceutical composition can be administered by inhalation, injection, infusion or orally. Thus, a pharmaceutical composition can be a composition for oral, parenteral, transdermal, intraluminal, intraarterial, intravenous, intrathecal and / or intranasal administration, or for direct injection into a tissue. A pharmaceutical composition can be administered to a subject in an appropriate dose. The administration regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, the particular compound administered, sex, time and route of administration, general health, and other drugs administered simultaneously.

[0113] The present invention may also include pharmaceutical compositions as defined herein, which further comprise at least one pharma- ceutically acceptable carrier.Thus, the therapeutic composition of the present invention further comprises a pharma- ceutically acceptable carrier, diluent or excipient.These terms can be used interchangeably.The pharma- ceutically acceptable carrier (also called excipient or diluent) includes any excipient / carrier / diluent that does not induce adverse side effects in the subject receiving the pharmaceutical composition by itself.

[0114] Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and lipid aggregates, such as oil droplets or liposomes. The carriers used in combination with the (pharmaceutical) composition of the present invention may be aqueous, forming an aqueous solution. An oil-based carrier solution containing the system of the present invention is an alternative to an aqueous carrier solution. The aqueous or oil-based solution further comprises a thickener to give the (pharmaceutical) composition the viscosity of a liniment, cream, ointment, gel, etc. Suitable thickeners are well known to those skilled in the art.

[0115] Pharmaceutically acceptable carriers according to the present invention include, by way of example and not limitation, diluents, disintegrants, binders, adhesives, wetting agents, polymers, lubricants, glidants, substances added to mask or neutralize unpleasant textures, tastes or odors, flavors, dyes, fragrances, and substances added to improve the appearance of the composition. Acceptable carriers include lactose, sucrose, starch powder, corn starch or derivatives thereof, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfate, gelatin, gum acacia, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine ​​hydrochloride, and the like. Examples of carriers suitable for soft gelatin capsules include vegetable oils, waxes, fats, semi-solid and liquid polyols. Carriers suitable for preparing solutions and syrups include, but are not limited to, water, polyols, sucrose, invert sugar and glucose. Carriers suitable for injection solutions include, but are not limited to, water, alcohols, polyols, glycerol and vegetable oils. Pharmaceutical compositions can further include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, buffers, coatings or antioxidants. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

[0116] Furthermore, the carrier of a (pharmaceutical) composition may refer to, for example, a diluent such as water, physiological saline, glycerol, ethanol, bacteriostatic water for injection (BWFI), Ringer's solution, dextrose solution, or an aqueous solution such as salts and / or buffers. Furthermore, substances necessary for formulation purposes may be included in said composition as acceptable carriers such as emulsifiers, stabilizers, surfactants and / or pH buffering substances known to those skilled in the art.

[0117] Said stabilizer / stabilizer may act as an osmotic tonicity modifier. The term "stabilizer" refers to an agent that improves or otherwise enhances the stability of a formulation, particularly the stability of a system contained in said (pharmaceutical) composition. Stabilizers that are osmotic tonicity modifiers may be non-reducing sugars, sugar alcohols or combinations thereof. The osmotic tonicity modifiers of the (pharmaceutical) composition of the present invention ensure that the osmotic tension, i.e., osmotic pressure, of the solution is essentially the same as that of normal physiological fluids, thus preventing swelling or rapid absorption of the composition after administration due to differences in ion concentration between the composition and physiological fluids. Preferably, the stabilizer / osmotic tonicity modifier is one or more non-reducing sugars, such as sucrose or trehalose, or one or more sugar alcohols, such as mannitol or sorbitol, and also preferred is a combination of a non-reducing sugar and a sugar alcohol.

[0118] In the (pharmaceutical) composition of the present invention, the addition of surfactants can be useful to reduce protein degradation during storage. Polysorbate 20 and 80 (Tween 20 and Tween 80) are well-established excipients for this purpose. Those skilled in the art will understand that the combination of the various components contained in the formulation can be made in any suitable order. Those skilled in the art should also understand that some of these chemicals may not be compatible in certain combinations and therefore can be easily replaced with different chemicals that have similar properties but are compatible in the relevant mixture.

[0119] The term "buffering agent" as used herein includes agents that maintain pH in a desired range. A buffer is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or a weak base and its conjugate acid. It has the property that the pH of the solution changes little when a small amount of a strong acid or strong base is added. Buffers are used in a wide variety of chemical applications as a means of keeping the pH at a nearly constant value. Generally, buffers when applied in the formulations of the present invention preferably stabilize the system contained in the (pharmaceutical) composition of the present invention.

[0120] Pharmaceutical compositions may also include one or more adjuvants. The term "adjuvant" is used according to its well-known meaning in relation to pharmaceutical compositions. Specifically, an adjuvant is an immunological agent that, when given by itself, has little, if any, desired immunogenic effect on the immune system, while altering and preferably enhancing the effect of such a composition. Suitable adjuvants can be, for example, inorganic adjuvants such as aluminum salts (e.g., aluminum phosphate, aluminum hydroxide), monophosphoryl lipid A, or organic adjuvants such as squalene or oil adjuvants, as well as virosomes.

[0121] The (pharmaceutical) composition of the present invention may be a liquid, preferably aqueous, composition. Further included herein are dried or frozen forms of the (pharmaceutical) composition defined herein. In this context, frozen form may refer to a composition at least about -20°C, including at least about -30°C, at least about -40°C, at least about -50°C, at least about -60°C, at least about -70°C, at least about -80°C or higher, etc. Preferably, the (pharmaceutical) composition is a liquid, even more preferably aqueous, composition. Thus, the (pharmaceutical) composition may be directly stored in liquid form for later use, may be stored in a frozen state and thawed before use, or may be prepared in a dried form, such as a lyophilized, air-dried or spray-dried form, for later reconstitution in liquid or other form before use. Thus, it is envisaged that the (pharmaceutical) composition described herein may be stored by any method known to the skilled artisan. Non-limiting examples include cooling, freezing, lyophilization, and spray drying of the formulation, with storage by cooling being preferred.

[0122] In vivo therapeutic applications The present invention further refers to a composition as defined elsewhere herein for use as a medicament. Thus, the composition of the present invention can also be used for therapy, i.e., for the treatment of viral and / or bacterial diseases in a subject in need thereof, as defined herein for the diagnostic use of the composition of the present application. Thus, the composition of the present invention is particularly suitable for use in a method for preventing or treating viral and / or bacterial diseases in a subject in need thereof.

[0123] Thus, the present invention also provides a method for the prevention or treatment of viral and / or bacterial disease in a subject, said method comprising administering to a subject in need thereof a therapeutically effective amount of a composition of the present invention.

[0124] Thus, the term "treat", "treating" or "treatment" as used herein means to reduce (slow down), stabilize or inhibit, or at least partially alleviate or arrest the progression of symptoms associated with the respective disease. It therefore includes the administration of said composition comprising said system, preferably in pharmaceutical form, to a subject as defined elsewhere herein. Those in need of treatment include those already suffering from a disease, here a viral and / or bacterial disease as described elsewhere herein. Preferably, treatment reduces (slow down), stabilize or inhibit, or at least partially alleviate or arrest the progression of symptoms associated with the presence and / or progression of a disease or pathological condition. "Treat", "treating" or "treatment" refers to therapeutic treatment. In particular, in the context of the present invention, treating or treatment refers to the improvement of symptoms associated with said viral and / or bacterial disease as defined elsewhere herein in a subject in need thereof. In this context, the terms "treat", "treating" or "treatment" refer to antiviral and / or antibacterial therapies that directly attack the target (viral and / or bacterial) RNA molecule sequences of a particular virus / bacteria as defined herein. Thus, compositions comprising the system as defined herein may also be used as antiviral and / or antibacterial therapeutic agents.

[0125] The terms "prevent", "preventing", and "prevention" as used herein refer to prophylactic or preventative measures, where the subject is to prevent an abnormal condition, including a pathological condition, in an organism believed to lead to a defined disease, i.e., a viral and / or bacterial disease as defined herein. In other words, the term refers to a medical procedure aimed at preventing a disease, which means inhibiting a subject's likelihood of contracting any future viral and / or bacterial disease as defined herein. As used herein, such terms also refer to a reduction in the risk of acquiring or developing a given condition in a patient diagnosed with any viral and / or bacterial disease as defined herein. Thus, it also includes the administration of the composition comprising the system, preferably in pharmaceutical form, to a subject as defined elsewhere herein. Those in need of prevention include those who are prone to having a disease, such as a viral and / or bacterial disease as defined herein. In other words, those who are at risk of developing such disease and therefore will probably suffer from said disease in the near future.Therefore, the composition defined herein can be used as a preventive agent, not as a therapeutic agent, for the subject defined herein that is already infected.This can prevent Cas13 protein from being overwhelmed by viral RNA molecules.

[0126] As used herein, the term "subject" includes mammalian and non-mammalian subjects. Preferably, the subject of the present invention is a mammal, including humans, domestic and farm animals, non-human primates, and any other animals having mammary tissue. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a rat. In some embodiments, the mammal is a guinea pig. In some embodiments, the mammal is a rabbit. In some embodiments, the mammal is a cat. In some embodiments, the mammal is a dog. In some embodiments, the mammal is a monkey. In some embodiments, the mammal is a horse. In the most preferred embodiments, the mammal of the present invention is a human. Subjects also include human and veterinary patients.

[0127] The uses, kits and compositions described herein are generally applicable to both human and veterinary diseases. When a subject is a living human being that can be treated for a disease or condition described herein, it is also treated as a "patient". In some embodiments, the subject of the present invention is at risk of developing a viral and / or bacterial disease described herein. In some embodiments, the subject of the present invention is suffering from a viral and / or bacterial disease described herein. The term "suffering" as used herein means that the subject is no longer a healthy subject. The term "healthy" means that the respective subject does not have obvious or prominent features or symptoms of the respective disease. This further means that a subject suffering from said viral and / or said bacterial disease is a subject "in need" of the respective treatment with a composition comprising said system of the present invention. Those in need of treatment include those already suffering from a disease, as well as those susceptible to having the disorder or those in whom the disorder should be prevented (prophylaxis).

[0128] The composition comprising the system of the invention for use in a method for preventing or treating a subject suffering from a viral and / or bacterial disease as defined elsewhere herein is generally administered to the subject in a therapeutically effective amount. The therapeutically effective amount is sufficient to inhibit or alleviate symptoms of the viral and / or bacterial disease. "Therapeutic effect" or "therapeutically effective" means that the conjugate for use induces a biological or medical response in a tissue, system, animal or human that is desired by a researcher, veterinarian, physician or other clinician. The term "therapeutically effective" further refers to the inhibition of factors that cause or contribute to a disease or disorder. The term "therapeutically effective amount" includes that the amount of agent at the time of administration is sufficient to significantly improve the progression of the disease being treated or to prevent the onset of the disease. According to a preferred embodiment, the therapeutically effective amount is sufficient to alleviate or cure a viral and / or bacterial disease as defined herein.

[0129] The therapeutically effective amount will vary depending on the composition of the present invention, the disease and its severity, and also on the individual factors of the subject and / or how its administration (also called delivery) works. Therefore, the composition of the present invention will not result in therapeutically effective in all cases. Because the method disclosed herein cannot 100% safely predict whether a subject can respond to the detection system, as individual factors also play a role. It is expected that age, weight, general health, sex, diet, drug interactions, etc. may have a general effect on whether a compound for use in treating a subject suffering from the disease is therapeutically effective.

[0130] The term "administering" or "administered" or "administration" as used throughout the various aspects of the present invention means that a composition comprising the system as defined herein is given to the respective subject in an appropriate form and dosage and using an appropriate means. Administration of the composition according to the present invention can be performed by any method known in the art.

[0131] Administration of said composition or a therapeutically effective amount of said composition as defined elsewhere herein may be performed by inhalation, injection, infusion, or orally. Administration of a composition or a composition comprising said composition as defined elsewhere herein may be performed intraperitoneally, intravenously, intraarterially, subcutaneously, intramuscularly, parenterally, transdermally, intraluminally, intrathecally and / or intranasally or directly into a tissue.

[0132] If the composition is to be administered by infusion, it can be formulated and administered using an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration. If the composition is to be administered by inhalation, a suitable inhalation device known to those of skill in the art can be used.

[0133] In certain embodiments, the administration of the composition may comprise delivering to the subject through different methods based on the fact that the composition is delivered as a DNA, RNA or protein-based system.If it is delivered as a DNA-based system, in other words, if the composition is a nucleic acid molecule comprising the nucleotide sequence encoding Cas13 protein as defined herein and the nucleotide sequence encoding gRNA as defined herein, and the nucleic acid molecule is DNA, it may be administered as defined herein, using any vector described herein, such as adeno-associated virus (AAV) vector for AAV-mediated gene delivery, or adenovirus vector or lentivirus vector optimized for the expression of the at least one gRNA and the at least one Cas13 gene, and / or using particles and / or nanoparticles. If it is delivered as an RNA-based system, in other words if said composition is a nucleic acid molecule comprising a nucleotide sequence encoding a Cas13 protein as defined herein and a nucleotide sequence encoding a gRNA as defined herein, which may be administered as defined herein as said nucleic acid molecule being RNA, it may be administered using specific particles and / or nanoparticles and / or liposomes and / or exosomes comprising said composition as known to the skilled person, preferably via nanoparticles. If it is delivered as a protein-based system, in other words if said composition comprising at least one Cas13 protein as defined elsewhere herein and at least one gRNA as defined herein, which may be administered as defined herein, it may be administered using specific particles and / or nanoparticles and / or liposomes and / or exosomes comprising said composition as known to the skilled person, preferably via nanoparticles.In the case of RNA and protein-based systems, the at least one gRNA may already be chemically synthesized in the correct form prior to the delivery of the composition, whereas in the case of DNA-based systems, the gRNA may need to be transcribed within the cell.

[0134] The viral disease prevented or treated by the use of the composition may refer to a disease caused by a DNA virus, an RNA virus or a retrovirus as defined elsewhere herein, preferably a disease caused by an RNA virus. In a particular embodiment, the viral infection is caused by a double-stranded RNA virus, a positive-sense RNA virus, a negative-sense RNA virus or a combination thereof. In a more preferred embodiment, the viral disease prevented or treated by the use of the composition is any one of coronavirus disease, influenza A, Ebola, measles, hepatitis C, tick-borne encephalitis (TBE), Venezuelan equine encephalitis (VEE) virus infection, dengue fever, yellow fever or Zika fever, even more preferably a coronavirus or influenza disease. The "coronavirus" may be SARS-CoV, SARS-CoV2 or MERS or a related new zoonotic or mutant coronavirus. The coronavirus group consists of enveloped positive-stranded RNA viruses belonging to the Coronaviridae family and includes subtypes referred to as alpha-, beta-, gamma- and delta-coronaviruses. Alpha and beta affect mammals, while gamma affects birds and delta can affect both. The coronavirus family includes several well-known pathogenic members. The betacoronavirus family poses the greatest risk to humans so far and currently includes the most well-known viral targets, including severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and the most recent form to emerge, novel coronavirus (SARS-CoV-2). In a most preferred embodiment, the viral disease prevented or treated by use of the composition comprising the system is COVID-19 disease. In the case of COVID-19, the clinical spectrum of SARS-CoV-2 infection is broad and includes asymptomatic infection, mild upper respiratory tract disease, severe viral pneumonia with respiratory failure, and even death, with many patients likely hospitalized.

[0135] Since any bacterium also contains ssRNA, which can be targeted / detected and then also cleaved / degraded, thus inactivating said bacterial ssRNA, said therapeutic approach using said composition according to the invention can also be applied to treat or prevent any bacterial disease caused by bacteria, including drug-resistant bacteria as defined elsewhere herein.

[0136] In an alternative embodiment, the composition for use in the treatment of said viral and / or bacterial diseases of the present invention may be administered in combination with further therapeutic agents (drugs). Drugs or therapeutic agents useful in this context include, but are not limited to, drug-like molecules, proteins, peptides, and small molecules. Protein therapeutic agents include, but are not limited to, peptides, enzymes, antibodies, structural proteins, receptors and other cellular or circulating proteins, as well as fragments and derivatives thereof, and preferably, further therapeutic agents / drugs in the context of the present invention may be drugs for use in viral and / or bacterial diseases as described elsewhere herein, in particular drugs for combination therapy in said diseases. The combinations according to the present invention may be administered as a combined formulation or separately from each other.

[0137] Also included by the present invention is a method for preventing or treating a viral or bacterial disease in a subject, comprising administering to the subject a therapeutically effective amount of a composition as defined herein.Also included herein is the use of the composition as defined elsewhere herein for the manufacture of a medicament for therapeutic use in a viral or bacterial disease in a subject.The definitions and aspects made with respect to the first and second medical uses may be applied in this context as well.

[0138] As noted elsewhere herein, i) at least one Cas13 protein fused to at least one NLS or to at least one NES or a nucleotide sequence encoding said Cas13 protein; ii) at least one gRNA or a nucleotide sequence encoding said gRNA, capable of hybridizing to one or more target RNA molecules, fused to at least one viral transport element; Also included by the present invention are compositions comprising a CRISPR system comprising, wherein such an RNA transport element, such as CTE or VARdm, drives the transport of gRNA from the nucleus to the cytosol. Herein, the nucleotide sequence encoding the gRNA, preferably the nucleotide sequence being DNA, is fused to at least one viral transport element. At least one viral transport element as defined herein is fused to the 5'-end of the nucleotide sequence encoding the gRNA, preferably DNA, as defined elsewhere herein. In this context, fusing again means that the nucleotide sequence encoding the gRNA and at least one viral transport element are fused via a linker, such as any nucleotide linker. Thus, also included herein are compositions in which the DNA nucleotide sequence encoding the gRNA of the above composition (comprising at least one Cas13 protein or a nucleotide sequence encoding the protein fused to at least one NLS or at least one NES) is fused to at least one viral transport element as defined herein at the 5'-end of the nucleotide sequence, via a linker, such as any nucleotide linker. Again, the term "at least one viral transport element" refers to two, three, four, five or more viral transport elements as defined herein. In a preferred embodiment, said at least one viral transport element is a CTE or VARdm, preferably a CTE, which is fused as defined herein with the nucleotide sequence (preferably a DNA sequence) encoding said gRNA. The fusion of the nucleotide sequence encoding said gRNA with at least one viral transport element as defined herein proves to be advantageous, since it enhances the knockdown efficiency of said Cas13 protein fused to NLS or NES, preferably when viral CTE is fused to said gRNA, most preferably when viral CTE is fused to said gRNA and NES is fused to said Cas13 protein (see Figure 17).Fusion to such gRNAs can also be applied at the protein level of such compositions.

[0139] Any of the above definitions relating to a composition comprising, inter alia, a Cas13 protein fused to at least one NLS fused to at least one NES can be applied (in any combination) to a composition comprising, inter alia, a Cas13 protein fused to at least one NLS or to at least one NES, where applicable, and also comprising the fusion of at least one viral transport element to said gRNA.

[0140] nucleic acid molecule Further included by the present invention are nucleic acid molecules comprising a nucleotide sequence encoding a Cas13 protein and / or a nucleotide sequence encoding a gRNA, as defined elsewhere herein.This means that when such a nucleic acid molecule comprising a nucleotide sequence encoding said Cas13 protein is applied, said molecule encodes the Cas13 protein as defined herein, which is fused to at least one NLS, which is fused to at least one NES (also encoded by the nucleotide sequence), as described by the present invention.Furthermore, when said nucleic acid molecule comprising a nucleotide sequence encoding said gRNA is applied, said gRNA can be encoded by said molecule, as defined herein, for example with respect to said modification.

[0141] Nucleic acid molecules comprising nucleotide sequences encoding the Cas13 protein and / or the gRNA include DNA, such as cDNA or genomic DNA, and RNA. Preferably, embodiments describing "RNA" are directed to mRNA.

[0142] The present invention also relates to nucleic acid molecules as defined herein, comprising a nucleotide sequence encoding the Cas13 protein and / or gRNA described herein. Because the degeneracy of the genetic code allows for the substitution of certain codons with other codons that specify the same amino acid, the present invention is not limited to a specific nucleic acid molecule encoding Cas13 and / or gRNA as defined herein, but includes all nucleic acid molecules that comprise a nucleotide sequence encoding a functional Cas13 protein and / or gRNA as defined herein.

[0143] In some embodiments, a nucleic acid molecule, such as DNA or RNA, comprising a nucleotide sequence encoding a Cas13 protein and / or a gRNA disclosed in the present application may comprise nucleotide sequences "operably linked" to each other, i.e., as described above, a nucleotide sequence encoding at least one Cas13 protein described elsewhere herein (if more than one Cas13 protein is used, two or more nucleotide sequences encoding the Cas13 proteins may apply as well), a nucleotide sequence encoding at least one gRNA defined elsewhere herein (if more than one gRNA is used, two or more nucleotide sequences encoding the gRNAs may apply as well), and a nucleotide sequence encoding a localization signal defined elsewhere herein. The nucleotide sequences are operably linked to each other. In this regard, an operably linked link is a linkage in which a sequence element of one nucleotide sequence and a sequence element of another nucleotide sequence are connected in a manner that allows the expression of a detection system.

[0144] The present invention also includes nucleic acid molecules described elsewhere herein that contain additional mutations outside the indicated sequence positions of experimental mutagenesis. Such mutations are often tolerated or may even prove advantageous, for example, if they contribute to improved folding efficiency, serum stability, thermal stability, or ligand binding affinity.

[0145] The nucleic acid molecules disclosed in the present application can be "operably linked" to a regulatory sequence (or regulatory sequences) to allow expression of the nucleic acid molecule.

[0146] A nucleic acid molecule, such as DNA or RNA, is said to be "capable of expressing a nucleic acid molecule" or "capable of allowing expression of a nucleotide sequence" when it contains sequence elements that contain information for transcriptional and / or translational regulation and such sequences are "operably linked" to a nucleotide sequence encoding a Cas13 protein and / or gRNA as defined elsewhere herein. An operable linkage is one in which the regulatory sequence elements and the sequence to be expressed are connected in a manner that allows gene expression. The exact nature of the regulatory regions required for gene expression may vary from species to species, but generally these regions include promoters, which in prokaryotes include both the promoter itself, i.e., the DNA element that directs the initiation of transcription, and the DNA element that, when transcribed into RNA, signals the initiation of translation. Such promoter regions usually include 5' non-coding sequences involved in the initiation of transcription and translation, such as -35 / -10 boxes and Shine-Dalgarno elements in prokaryotes, or TATA boxes, CAAT sequences and 5'-capping elements in eukaryotes. These regions may also contain enhancer or repressor elements, as well as translation signal and leader sequences for targeting the native polypeptide to a particular compartment of the host cell.

[0147] In addition, the 3' non-coding sequences may contain regulatory elements involved in transcription termination, polyadenylation, etc. However, if these termination sequences do not function satisfactorily in a particular host cell, they may be replaced with signals which are functional in that cell.

[0148] Therefore, the nucleic acid molecule of the present invention can include a regulatory sequence such as a promoter sequence. In some embodiments, the nucleic acid molecule of the present invention includes a promoter sequence and a transcription termination sequence. Suitable promoters can be selected from the group consisting of RNA polymerase, pol I, pol II, pol III, T7, U6, H1, retrovirus Rous sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, (3-actin promoter, phosphoglycerol kinase (PGK) promoter, EF1α promoter and U6 promoter, TetOn / Off, 7SK, CAG, CBh, UbC or any tetracycline-dependent or interferon-dependent promoter.

[0149] vector The nucleic acid molecules of the invention can also be part of a vector or any other type of cloning vehicle, such as a plasmid, a phagemid, a phage, a baculovirus, a cosmid or an artificial chromosome, preferably a vector.

[0150] In certain aspects, the present invention includes vectors for, for example, delivery or introduction of Cas and / or RNA that can guide Cas to a target locus (i.e., guide RNA) into cells, as well as for propagating these components (e.g., in prokaryotic cells). As used herein, a "vector" is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted to cause replication of the inserted segment. Generally, a vector can replicate when associated with appropriate control elements. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules that include one or more free ends, nucleic acid molecules that do not include free ends (e.g., circular); nucleic acid molecules that include DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques. Another type of vector is a viral vector, in which a DNA or RNA sequence from a virus is present in the vector for packaging into a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus (AAV)). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell when introduced into the host cell, and are thereby replicated together with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". Common expression vectors useful in recombinant DNA techniques are often in the form of plasmids.

[0151] A recombinant expression vector can include the nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory elements operably linked to the nucleotide sequence to be expressed, which can be selected based on the host cell used for expression. In a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory element in a manner that allows the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). For recombination and cloning methods, reference is made to US Patent Application Publication No. 10 / 815,730, published as US 2004-0171156 A1 on September 2, 2004, the contents of which are incorporated herein by reference in their entirety. Thus, the embodiments disclosed herein can include transgenic cells that include a CRISPR effector system. In certain exemplary embodiments, transgenic cells can function as individual discrete volumes. In other words, a sample containing the masking construct may be delivered to a cell, for example in a suitable delivery vehicle, and if the target is present in the delivery vehicle, the CRISPR effector is activated and a detectable signal is generated. The vector may include a regulatory element, for example, a promoter. The vector may include a sequence encoding Cas and / or a single sequence encoding a guide RNA, but may also include a sequence encoding at least 3 or 8 or 16 or 32 or 48 or 50 guide RNAs, such as 1-2, 1-3, 1-4 1-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNAs.In a single vector, when up to about 16 RNAs are present, there may advantageously be a promoter for each RNA; and when a single vector provides more than 16 RNAs, one or more promoters may drive the expression of two or more RNAs; for example, when 32 RNAs are present, each promoter may drive the expression of two RNAs, and when 48 RNAs are present, each promoter may drive the expression of three RNAs. A person skilled in the art can easily practice the invention with RNAs for suitable exemplary vectors such as AAV and suitable promoters such as the U6 promoter, by simple arithmetic and well-established cloning protocols and the teachings of this disclosure. For example, the packaging limit of AAV is about 4.7 kb. A single U6-gRNA (plus restriction sites for cloning) has a length of 361 bp. Therefore, a person skilled in the art can easily fit about 12-16, e.g., 13 U6-gRNA cassettes in a single vector. This can be assembled by any suitable means, such as the Golden Gate strategy used for TALE assembly (genome-engineering.org / taleffectors / ). Using the tandem guide strategy, one skilled in the art can increase the number of U6-gRNAs by approximately 1.5-fold, for example from 12-16, for example from 13, to approximately 18-24, for example, about 19 U6-gRNAs. Thus, one skilled in the art can easily reach approximately 18-24, for example, about 19 promoter-RNAs, such as U6-gRNAs, in a single vector, for example, an AAV vector. A further means to increase the number of promoters and RNAs in a vector is to use a single promoter (e.g., U6) to express an array of RNAs separated by cleavable sequences.And a further means to increase the number of promoter-RNAs in a vector is to express an array of promoter-RNAs separated by cleavable sequences in the coding sequence or introns of genes; and in this example, it is advantageous to use a polymerase II promoter, which can increase expression and allow transcription of long RNAs in a tissue-specific manner. (See, for example, nar.oxfordjournals.org / content / 34 / 7 / e53.short and nature.com / mt / journal / v16 / n9 / abs / mt2008144a.html). In one embodiment, an AAV can package U6 tandem gRNAs targeting up to about 50 genes. Thus, from the knowledge in the art and the teachings in this disclosure, one skilled in the art can easily make and use vectors, such as a single vector, that express multiple RNAs or guides under control or operatively or functionally linked to one or more promoters, particularly with respect to the number of RNAs or guides discussed herein, without undue experimentation.

[0152] In addition to the nucleic acid molecule containing the regulatory sequences described above and nucleotide sequences encoding the Cas13 protein and / or gRNA described herein, such cloning vehicles can contain replication and control sequences derived from a species compatible with the host cell used for expression, as well as a selection marker that confers a selectable phenotype on transformed or transfected cells. Many suitable cloning vectors are known in the art and are commercially available.

[0153] The cloning vectors comprising the nucleic acid molecules described herein and in particular the coding sequences of the Cas13 protein and / or gRNA as defined herein can be transformed into host cells capable of expressing the genes. Transformation can be carried out using standard techniques (Sambrook, J. et al. (1988) Molecular Cloning: A Laboratory Manual, 2nd Ed).

[0154] host cell Accordingly, the present invention is also directed to a host cell comprising the nucleic acid molecules or vectors described herein.

[0155] The transformed host cells are cultured under conditions suitable for the expression of the nucleotide sequence encoding the Cas13 protein and / or gRNA as defined herein. Suitable host cells can be prokaryotic cells, such as E. coli or Bacillus subtilis, or eukaryotic cells, such as Saccharomyces cerevisiae, Pichia pastoris, SF9 or High5 insect cells, immortalized mammalian cell lines, such as HeLa cells or CHO cells, or primary mammalian cells, preferably E. coli.

[0156] kit The present invention also relates to a kit comprising a composition as defined herein according to the present invention. Thus, when the kit comprises a composition, the composition may be provided in a vial or container, preferably also comprising at least one carrier as defined herein in the vial or container. Thus, when the kit comprises a pharmaceutical composition, the composition may be provided in a vial or container, preferably also comprising at least one pharma- ceutically acceptable carrier and / or adjuvant as defined herein. Furthermore, the kit may be accompanied by a notice in a format prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, reflecting the approval by the governmental agency of the manufacture, use or sale of the product for administration or diagnosis to humans. The kit may comprise a composition as defined herein, preferably in a vial or container, in a dried form, such as a lyophilized, air-dried or spray-dried form (powder form), for later reconstitution into a liquid form or other form before use. Furthermore, the kit may also comprise a composition, preferably in a vial or container, in a frozen state, to be thawed before use.

[0157] The kit according to the present invention may also comprise a delivery system. Delivery systems for transgenes are well known in the art. By way of example, a composition comprising said CRISPR system in the form of a Cas transgene, and therefore in the form of DNA, may be delivered to eukaryotic cells, for example, by a vector (e.g., AAV, adenovirus, lentivirus, plasmid) and / or by particles and / or nanoparticles as defined elsewhere herein. A composition comprising said CRISPR system in the form of RNA or at protein level may be delivered to eukaryotic cells, for example, by particles and / or nanoparticles and / or liposomes and / or exosomes, as also described elsewhere herein. Such a delivery system may also be included in one or more vials or containers of the kit defined above, or in an additional one or more vials or containers of the kit, preferably further comprising any excipient suitable for said delivery system to be mixed / contacted in said one or more vials or containers.

[0158] The kit according to the present invention may also include a label. Label as used herein may refer to a compound capable of targeting the system contained in the composition. The compound may also refer to an antibody, a bead coupled / coated with an antibody, such as a Dynabead coupled / coated with an antibody, preferably used in classical purification processes such as chromatography, when the composition can be produced at the protein level. Furthermore, the label may be an oligo-dT magnetic bead. Such a bead may be capable of binding to the polyA tail of full-length RNA, when the composition can be produced at the RNA level and then further purified as known to those skilled in the art.

[0159] In some embodiments, the label may also be included in one or more containers or vials of the kit defined above containing the composition, or in an additional one or more vials or containers of the kit, preferably further comprising any excipient suitable for the label to be mixed / contacted in said one or more vials or containers.

[0160] In vitro and in vivo applications The present invention also relates to a method for producing the composition of the present invention, in other words, a method for producing the CRISPR system contained by said composition. In this context, said composition is produced by genetic engineering methods, starting from a nucleic acid encoding Cas13 protein and / or gRNA as defined herein. Again, in this context, said encoded Cas13 protein is Cas13 protein fused to at least one NLS, which is fused to at least one NES, as described throughout the present invention. Thus, the end product of the production method provided is a composition of the present invention, which comprises said system in the form of a nucleic acid molecule comprising a nucleotide sequence encoding Cas13 protein and / or gRNA as defined elsewhere herein, wherein said nucleic acid molecule is DNA ("DNA-based system") or RNA ("RNA-based system"), or in the form of a protein-based system.

[0161] Thus, when the term "engineered nucleic acid encoding" is used herein with respect to a DNA-based system, it refers to the in vitro use of artificial gene synthesis (of the nucleotide sequence encoding the Cas13 protein and / or gRNA). Such synthesis refers to the methods used in synthetic biology to build and assemble genes de novo from nucleotides as known to those skilled in the art. When the term "engineered nucleic acid encoding" is used herein with respect to an RNA-based system, it refers to the in vitro use of RNA synthesis (of the Cas13 mRNA and / or the gRNA) as known to those skilled in the art. When the term "engineered nucleic acid encoding" is used herein with respect to a protein-based system, it should be divided to carry out the method in vivo or in vitro. In some embodiments and when referring to a protein-based system of the composition defined elsewhere herein, the method can be carried out in vivo, which means that the composition can be produced, for example, in a bacterial or eukaryotic host organism and then isolated from this host organism or a culture thereof by means known to those skilled in the art. When producing the composition in vivo, the nucleic acid molecule comprising the nucleotide sequence encoding the Cas13 protein and / or gRNA as defined herein is introduced into a suitable bacterial or eukaryotic host organism by recombinant DNA technology.To this end, the host cell is first transformed with a cloning vector comprising the nucleic acid molecule described herein using established standard methods.The host cell is then cultured under conditions that allow the expression of the heterologous DNA and thus the synthesis of the corresponding CRISPR system.The composition is then isolated from either the host cell or the culture medium.In other embodiments, when referring to a protein-based system, the nucleic acid encoding of the method can be carried out in vitro, which means that it is also possible to produce the composition of the present invention by using an in vitro translation system.

[0162] After encoding a nucleic acid as defined above, the method may further comprise the step of obtaining the produced composition.

[0163] In the case where the composition is produced at the DNA level (meaning the nucleotide sequence encoding said Cas13 protein and / or gRNA), the term "obtain" or "obtaining" used in this respect can be understood in that the produced composition is extracted from the use (reaction) buffer after nucleic acid encoding, for example by using artificial gene synthesis, and potentially purified before said composition can be used. Such a classical purification process of DNA-based systems can include the use of silica columns, as known to those skilled in the art. In the case where the composition is produced at the RNA level (meaning Cas13 mRNA and / or gRNA), the term "obtain" or "obtaining" used in this respect can be understood in that the produced composition is extracted from the use (reaction) buffer after nucleic acid encoding, for example by using RNA synthesis, and potentially purified before said composition can be used. Such a classical purification process of RNA-based systems can include the use of oligo-dT magnetic beads, as known to those skilled in the art. Such beads can bind to the polyA tail of the RNA of the components of the CRISPR system, where by-products can be separated and thus a better purity of the components can be achieved. If the composition is produced in vivo at the protein level (Cas13 protein and / or gRNA), the term "obtain" or "obtaining" used in this respect can be understood in that the produced composition is then isolated from the host organism or its culture by means known to the skilled person as described above. If the composition is produced in vitro at the protein level, the term "obtain" or "obtaining" can be understood in that the produced composition is extracted from the use (reaction) buffer after nucleic acid encoding, for example by using an in vitro translation system, and potentially purified before the composition can be used. Classical purification processes that can be used can refer to suitable chromatography as known to the skilled person.The terms "recover" or "recovering" may be used interchangeably herein with the terms "obtain" or "obtaining."

[0164] The above paragraphs and definitions relating to nucleic acid molecules, vectors, host cells, kits, and in vitro and in vivo applications, where applicable, also relate to a composition comprising a clustered regularly interspaced short palindromic repeats (CRISPR) system, the system comprising: i) at least one CRISPR-associated protein 13 (Cas13) fused to at least one nuclear localization signal (NLS) or to at least one nuclear export sequence (NES), or a nucleotide sequence encoding said Cas13 protein; ii) at least one guide RNA (gRNA) capable of hybridizing to one or more target RNA molecules, or a nucleotide sequence encoding said gRNA, fused to at least one viral transport element as defined elsewhere herein; The composition may also be applied to the composition comprising: EXAMPLES

[0165] Examples of the present invention material and method Cloning of key constructs The Cas13 coding sequence was ordered as a synthetic fragment (gBlock, IDT) and cloned into a pCAG backbone. Different localization sequences were fused to the C-terminus by ligation or Gibson assembly. The Cas13 gRNA scaffold sequence was ordered as a synthetic fragment (gBlock, IDT) and cloned into a U6 backbone. Different gRNAs were cloned into the scaffold by annealing complementary oligonucleotides (Metabion). HA-tagged SARS-CoV-2 was amplified by One-step RT-PCR (SuperScript IV One-step RT-PCR, Thermo Fisher) from a fragmented synthetic SARS-CoV-2 genome (Twist Bioscience). All luciferases used here were fused to a C-terminal degron derived from mouse ornithine decarboxylase to tightly couple RNA and protein levels. tRNA and ribozymes were ordered as synthetic fragments (gBlock, IDT) and added to the 3' end of the gRNA by Gibson assembly. The 5' and 3' β-globin stabilizing UTRs were amplified from HEK293T genomic DNA and cloned upstream or downstream of the Cas13 coding sequence.

[0166] Standard RNA preparation Templates for in vitro transcription were generated by linearization of plasmid templates. Digestions were purified (Monarch DNA Cleanup Kit, New England Biolabs) and subsequently used for mRNA synthesis (HiScribe T7 ARCA mRNA with tailing Kit, New England Biolabs). Synthesized RNA was purified (Monarch RNA Cleanup, New England Biolabs) and stored at -80°C. gRNA templates were generated by extension of overlapping oligonucleotides (Q5 Mastermix, New England Biolabs) and used directly for in vitro transcription. Synthesized gRNA was purified (Monarch RNA Cleanup, New England Biolabs) and stored at -80°C.

[0167] Cell lines and culture All experiments were performed in HEK293T cells. Cells were maintained at 37°C in a 7.5% CO2, H2O-saturated atmosphere in Advanced DMEM containing 10% FBS.

[0168] Knockdown efficiency measured by luciferase assay Cells were transfected with different Cas13 systems together with mRNA or DNA encoding nanoluciferase. 24–72 h after transfection, cells were lysed and nanoluciferase was measured (NanoGlo Assay, Promega). In experiments quantifying on-target and off-target activity, nanoluciferase was transfected together with firefly luciferase in the same well. 24–72 h after transfection, cells were lysed and both luciferases were measured independently (NanoGlo Dual Luciferase Assay, Promega).

[0169] Assessment of protein stability and SARS-CoV-2 RdRP knockdown Seventy-two hours after transfection with the different Cas13 variants, cells were lysed in M-PER buffer (Thermo Fisher) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher). Lysates were prepared in Laemmli Buffer (Sigma-Aldrich) and run on SDS PAGE (Thermo Fisher). Proteins were transferred to PVDF membranes overnight at 20 V, blocked, and incubated with either primary M2 anti-FLAG (Merck Millipore) antibody to stain Cas13 or HA antibody (Cell Signaling) to stain HA-tagged SARS-CoV-2 RdRP, and imaged with Amersham ECL Prime substrate (Sigma-Aldrich).

[0170] Cas13 protein localization Seventy-two hours after transfection with different Cas13 variants, cells were fixed with formalin, permeabilized, and incubated with primary M2 anti-FLAG (Merck Millipore) antibody. After incubation with secondary antibodies, cells were further stained with DAPI and imaged with an EVOS Cell Imaging System (Thermo Fisher).

[0171] SARS-CoV-2 inhibition Cells were transfected in 96-well plates with mRNA encoding ACE2 (20 ng), Cas13 NLS-NES (40 ng) and gRNA (40 ng). mRNA transfection was performed in MessengerMax (Thermo Fisher). 24 h post-transfection, cells were infected with SARS-CoV-2-GFP under BSL3 conditions. 72 h post-transfection, cells were lysed and virus was inactivated by heating in Trizol at 60°C. RNA was extracted by Trizol / chloroform extraction. Extracted RNA was then quantified by RT-qPCR (Luna Universal Probe One-Step Assay, New England Biolabs) with a CDC-approved diagnostic assay (for viral N gene, IDT).

[0172] Measurement of cellular translation Cells were transfected with Cas13, mRuby3, and gRNA targeting mRuby3. 72 hours after transfection, nascent protein translation was recorded and stained according to the manufacturer's protocol (Protein Synthesis Assay Kit, Cayman Chemical). Stained cells were analyzed by FACS.

[0173] Ribosome assay Cells were transfected with Cas13, gRNA, and target RNA. 72 hours after transfection, total RNA was extracted (Monarch Total RNA Miniprep Kit, New England Biolabs) and analyzed on an agarose gel. In comparison, purified 80S ribosomes were incubated with purified Cas13 protein (produced by in vitro translation: NEBExpress, New England Biolabs), in vitro transcribed gRNA, and in vitro transcribed target RNA (as described above) in NEB 4 buffer for 1 hour. The reaction was quenched by the addition of 0.5 M EDTA. The reaction was then incubated with Proteinase K for 1 hour at 37°C and then analyzed on an agarose gel.

[0174] Optimizing RNA delivery To stabilize Cas13 mRNA, a fragment of the 5' β-globin UTR and two copies of the β-globin 3' UTR were added to the Cas13 coding sequence. In addition, a genetically encoded 162 nucleotide polyA tail was added. UTP was replaced by N1-methyl pseudo-UTP (Jena Bioscience) and 5'-capping was performed post-transcriptionally by the Vaccina Capping System and 2'-O-methyltransferase (both New England Biolads). gRNAs were stabilized by replacing the two terminal nucleotides at the 5' and 3' ends with 2' O-methyl-phosphorothioates via chemical synthesis (IDT).

[0175] result Example 1: Characterization of optimal knockdown conditions for Cas13 To find the optimal composition of Cas13-based knockdown tools, Cas13d variants and miRNA-based RNA interference were directly compared in a firefly luciferase knockdown assay. The known Cas13d-NLS with a 22 bp gRNA resulted in moderate knockdown efficiency, which was improved by increasing the length of the gRNA to 30 bp and fusing the Cas13 protein to the NLS and NES together. This optimized system is even more efficient than the latest generation of RNA interference (Figure 1).

[0176] Originally, the optimal gRNA length for Cas13d was found to be 22 bp in an in vitro assay with purified components (Konermann et al. (2018), Cell 173(3): 665-676). We characterized the knockdown efficiency of different gRNA lengths in mammalian cell lines by targeting co-transfected nanoluciferase. In contrast to previous in vitro studies, the optimal gRNA length ranged from 26 to 30 bp (Figure 2).

[0177] We hypothesized that the addition of a tRNA that is processed by RNAse P / Z or a self-cleaving ribozyme to the 3' end of the gRNA could support gRNA folding. Furthermore, both motifs cleave and remove the U-stretch that is added to the gRNA during transcription termination of Pol III promoters (e.g., the U6 promoter). The addition of both motifs showed improved knockdown efficiency compared to unmodified gRNA (Figure 3).

[0178] Example 2: Utilizing different strategies to translocate Cas13d to the cytosol We compared the knockdown efficiency of Cas13d proteins with different localization signals in a luciferase-based assay. Because Pol III-driven gRNA is expressed and remains in the nucleus, Cas13d transported to the cytosol (NES) shows little activity. Furthermore, Western blot analysis showed that the protein is unstable when not bound to gRNA in the same compartment. The Cas13d variant transported to the nucleus (NLS) is active and stable because it resides in the same compartment as gRNA and forms a gRNA / Cas13d complex. For Cas13d fusion proteins consisting of both localization signals (NLS-NES), a shuffling mechanism was expected. The protein is imported into the nucleus (NLS), picks up the gRNA, and then transported back to the cytosol (NES), where the majority of luciferase mRNA is targeted for degradation. This NLS-NES strategy stabilized the protein in the cytosol and maximized knockdown efficiency. In this case, Cas13d-NES is the most active, so the complementary strategy in which the gRNA itself is transported to the cytosol by expression from a Pol II promoter also supports the hypothesis. A direct comparison of the two strategies shows that Cas13d-NLS-NES is the most efficient (Figure 4).

[0179] Example 3: Evaluation of Cas13d specificity For all Cas13 systems, in vitro experiments suggest a target RNA-based activation mechanism that uncontrols non-specific RNA degradation, called collateral RNA cleavage (Abudayyeh et al. (2016), Konermann et al. (2018)). Surprisingly, this non-specific RNA degradation is absent in all eukaryotic systems (Abudayyeh et al. (2017) Nature 550, 280-284, Konermann et al. (2018)). Eukaryotic transcriptomes are much more complex than prokaryotic transcriptomes (intron / exon structures, non-coding RNAs, small RNAs, etc.). We hypothesized that the differences observed in the Cas13 machinery between prokaryotes and eukaryotes may be directly related to differences in the activity of the Cas13 system. For moderately active Cas13 systems, collateral RNA cleavage can be monitored in the highly complex eukaryotic transcriptome. Therefore, we tested the highly active Cas13-IDG for its concomitant cleavage. As expected, when targeting firefly luciferase, the known Cas13d system, as well as miRNA, did not result in substantial inhibition of the co-transfected unrelated nanoluciferase. In the case of Cas13-IDG, an increase in the knockdown of nanoluciferase was measured along with an increase in the knockdown of firefly luciferase (Figure 5). This led us to conclude that Cas13-IDG shows the previously found characteristic of concomitant activity that can be exploited for viral inhibition by target transcripts.

[0180] Example 4: Efficient knockdown of heterologously expressed mRNA Prior art has given very different results regarding the efficiency of the individual systems, therefore we developed a luciferase-based assay that allows direct comparison of known Cas13 systems with several gRNAs and target RNAs in a 96-well format (Figure 6A). It was shown that Cas13d from Ruminococcus flavefaciens XPD3002 is the most efficient at degrading the target RNA. Based on this assay, we tested a number of modifications to the gRNA and Cas13 protein and were able to identify three modifications that increase the knockdown efficiency of Cas13d from 50% to more than 90% (Figure 6B). Optimization steps included elongation of the gRNA, fusion of a tRNA that supports gRNA folding, and fusion of nuclear and even cytoplasmic localization signals to the Cas13d protein. Fusing both localization signals together caused localization of Cas13d to the cytoplasm (Figure 6C). In contrast to published Cas13d variants, this step allows for the first time to degrade cytosolic RNA rather than nuclear RNA. The modified Cas13d composition containing each of the three modifications is designated Cas13-IDG.

[0181] In the course of research on Cas13-IDG, the inventors were able to demonstrate in mammalian cells the accessory activity of Cas13 composition, which was first described in prokaryotic systems. In contrast to the known accessory activity in vitro (experimental cell-free systems) and bacterial systems, Cas13-IDG appears to prefer to bind and cleave 28S ribosomal RNA at a defined position (Figure 6D, 6E), which ultimately leads to a transient inhibition of global cellular protein translation (Figure 8). By analyzing the effect of purified Cas13 on purified 80S ribosomes in an in vitro system, it was confirmed that 28S rRNA is directly cleaved by Cas13, not by endogenous cellular machinery (Figure 9).

[0182] Example 5: Inhibitory effect of Cas13-IDG on SARS-CoV-2 Based on the general optimization, and on the discovery of the effect of Cas13-IDG on cellular translation, we further developed these findings towards antiviral therapeutics. To do this, we used gRNAs complementary to different regions of the SARS-CoV-2 virus, focusing on the 5' and 3' parts of the virus. As these two regions are present in both the viral genome and all subgenomic mRNAs, the antiviral effect of Cas13 is likely strongest at these locations. In addition, it has recently become clear that after infection, viral RNA replicates in large quantities, exceeding the amount of endogenous RNA after a short time. Therefore, we expect four levels of viral inhibition when Cas13 is directed to the 5' or 3' viral UTR: 1) direct targeting of the viral genome, 2) targeting of copies of the viral genome, 3) degradation of viral mRNA, and 4) dramatic activation of Cas13-IDG in infected cells, thus with a strong concomitant effect on viral protein translation (Figure 7A).

[0183] We showed that using Cas13-IDG with gRNA targeting the artificially expressed RdRp protein of SARS-CoV-2 resulted in approximately 90% knockdown of the protein (Figure 7B). Based on this, we began to test the antiviral effect of Cas13-IDG on SARS-CoV-2 infected cells (under BSL-3 conditions). Initial results showed up to 80% viral reduction depending on the location of the gRNA (Figure 7C). In this experiment, the Cas13-IDG composition was delivered as RNA rather than DNA, as we anticipate that the transient effect of RNA will facilitate translation in preclinical and clinical models.

[0184] Example 6: Optimization of the RNA-based Cas13 delivery system To further enhance the knockdown of Cas13-IDG in the RNA-based delivery system, several modifications were tested by measuring the knockdown efficiency against co-transfected nanoluciferase mRNA. First, JetMessenger transfection reagent was shown to be preferable to MessengerMax. Addition of a portion of the 5'β-globin UTR and two copies of the 3'β-globin UTR increased the stability of Cas13 mRNA. Switching from the commonly used co-transcriptional ARCA-based 5'-capping to post-transcriptional capping with the Vaccina Capping System and 2'-O-methyltransferase further improved the knockdown efficiency. Replacement of UTP in Cas13 mRNA by N1-methyl pseudo-UTP increased the expression of Cas13 protein, which was shown by the Cas13-P2A-mRuby3 fusion construct (Figure 10B). In addition to improving Cas13 mRNA, we found that stabilizing the gRNA by replacing the two 5'- and 3'-terminal nucleotides with 2'-O-methyl phosphorothioate modified nucleotides further improved knockdown efficiency (Figure 10A).

[0185] Example 7: Illustration of Cas13d variants fused to different localization signals To induce nuclear / cytosolic shuttling, Cas13d was fused to different single or tandem localization signals (Figure 11A). Co-transfected nanoluciferase was targeted by two gRNAs combined with different localizing Cas13 variants. The combination of two NLS and one NES signal (Cas13d-NLS-NES / Cas13d-IDG) was found to be optimal, likely due to sufficient nuclear transport to pick up the gRNA and localization to the cytosol to efficiently target the primarily cytosolic nanoluciferase mRNA (Figure 11B).

[0186] Example 8: Localization of Cas13d protein variants fused to different localization signals Immunohistochemistry was performed to analyze the effect of different single or tandem localization sequences on Cas13d localization. Different fusion constructs were transfected and imaged in a high content analysis system (Figure 12A). Fluorescence intensity measurements of the cytosolic and nuclear fractions were then performed on 100 cells per construct. Analysis of the cytosolic to nuclear distribution revealed a gradual shift of protein localization to the cytosol, with a decrease in the NLS signal and an increase in the NES signal (Figure 12B). Cas13d-NES-NLS showed an intermediate localization, supporting the hypothesized shuttling mechanism.

[0187] Example 9: Stability of Cas13d variants fused to different localization signals To test whether the localization of gRNA and related proteins affects the stability of Cas13, Western blot analysis was performed. Therefore, Cas13 variants fused to different localization signals were transfected into HEK293T cells and then analyzed. Polymerase II-driven gRNA is transported to the cytosol, and therefore mainly stabilizes cytosolic Cas13 variants, while polymerase III-driven gRNA remains in the nucleus, and therefore preferably stabilizes nuclear Cas13 variants (Figure 13).

[0188] Example 10: Comparison of knockdown efficiency against Venezuelan equine encephalitis (VEE) replicons To study the effect of partial cytosolic Cas13d-NLS-NES on knockdown of cytosolic RNA, we targeted a self-replicating RNA derived from Venezuelan equine encephalitis (VEE) virus to mimic viral infection. VEE replicates exclusively in the cytosol without passing the nuclear barrier where conventional Cas13d-NLS is located. The mGreenLantern reporter gene was cloned onto a VEE replicon expression construct to measure the replication rate of the VEE replicon. The reporter replicon was transcribed in vitro and transfected into HEK293T cells. After 2 weeks of selection, different Cas13 constructs were transfected and their effects on VEE replication were analyzed by flow cytometry. For all gRNAs tested, Cas13d-NLS-NES outperformed Cas13d-NLS, therefore supporting that Cas13d-NLS-NES is more efficient in degrading cytosolic RNA compared to the previously described Cas13-NLS (Figure S14).

[0189] Example 11: Continuous analysis of SARS-CoV-2-GFP replication upon treatment conditions using Cas13d-IDG and different gRNAs SARS-CoV-2 is a positive-stranded RNA virus, which replicates exclusively in the cytosol, while Cas13d-NLS-NES efficiently degrades cytosolic RNA. To measure the effect of Cas13-NLS-NES on viral replication, HEK293T-ACE2 cells were transfected with Cas13d-NLS-NES-encoding mRNA together with different gRNAs and infected with SARS-CoV-2-GFP. SARS-CoV-2-GFP encodes an additional green fluorescent protein on the viral genome, allowing for the measurement of viral replication in time course experiments. Chemically modified gRNAs targeting the viral 3'UTR and gRNA pools targeting the viral 5'UTR and 3'UTR strongly inhibited viral replication (Figure 15A). To further validate these results, different SARS-CoV-2 variants (alpha and delta) were targeted by transfecting Cas13d-NLS-NES together with chemically modified gRNAs (Figure 15B). The viral load of SARS-CoV-2 was then measured by RT-qPCR for either the viral N or RdRp genes, confirming that the established system was able to inhibit SARS-CoV-2 variants without further modifications.

[0190] Example 12: Comparison of knockdown efficiency for two gRNAs targeting nanoluciferase co-transfected with NES or tandem NLS-NES fused to Cas13 While Cas13d-NLS-NES translocates to the nucleus to pick up the gRNA, the CTE-fused gRNA is transported to the cytosol alone. The combination of both transport systems may have additive effects. Therefore, two gRNAs targeting nanoluciferase, fused or not with a CTE motif, and either Cas13-NES or Cas13-NLS-NES were transfected into HEK293T cells. 48 hours after transfection, the knockdown efficiency was measured. Although both gRNA transport strategies performed similarly alone, the synergistic effect of both transport systems further maximized the efficiency (Figure 16).

[0191] Example 13: Illustration of different crRNA delivery strategies Conventional gRNAs are expressed by polymerase III promoters (e.g., U6 promoters) and therefore remain in the nucleus of expressing cells. To target cytoplasmic RNA, different gRNA transport strategies were utilized. Polymerase II-driven gRNAs (e.g., CAG promoters) are capped and polyadenylated, and therefore transported. Alternatively, viral RNA transport elements, such as CTE or VARdm, drive the transport of gRNAs from the nucleus to the cytosol (Figure 17A). These transport strategies were applied to two gRNAs, targeting co-transfected nanoluciferase, and compared for cytoplasmic or nuclear Cas13 protein (Figure 17B). As previously shown, conventional pol III-driven gRNAs are more efficient in combination with nuclear Cas13 systems. Pol II gRNA expression and fused viral transport motifs favor cytosolic Cas13 protein over nuclear Cas13 protein. gRNAs fused to viral CTE motifs are the most efficient in driving cytosolic knockdown by Cas13d-NES.

[0192] The present invention is further characterized by the following:

[0193] item 1. A composition comprising a clustered regularly interspaced short palindromic repeats (CRISPR) system, the system comprising: i) at least one CRISPR-associated protein 13 (Cas13) fused to at least one nuclear localization signal (NLS) fused to at least one nuclear export sequence (NES); ii) at least one guide RNA (gRNA) capable of hybridizing to one or more target RNA molecules; The composition comprising: 2. The composition of item 1, wherein the Cas13 protein is encoded by a nucleotide sequence. 3. The composition of item 1 or 2, wherein the gRNA is encoded by a nucleotide sequence. 4. The composition of any one of the preceding items, wherein the Cas13 protein is fused via a linker to at least one NLS fused to at least one NES. 5. The composition of any one of the preceding items, wherein the Cas13 protein is a Cas13d protein. 6. The composition of item 5, wherein the Cas13d protein is derived from the genus Ruminococcus, preferably Ruminococcus flavevaciens. 7. The composition of any one of the preceding items, wherein the gRNA has a length of at least about 23 nucleotides. 8. The composition of any one of the preceding items, wherein the gRNA has a length of about 26 to about 30 nucleotides. 9. The composition of any one of the preceding items, wherein the gRNA has at least about 80% complementary sequence identity to the one or more target RNA molecules. 10. The composition of any one of the preceding items, wherein the gRNA is capable of hybridizing to the 5' and / or 3' untranslated region of one or more target RNA molecules. 11. The composition of any one of items 3 to 10, wherein the nucleotide sequence encoding the gRNA is fused to a tRNA or a ribozyme. 12. The composition of any one of the preceding items, wherein the one or more target RNA molecules is a viral or bacterial target RNA molecule. 13. The composition of any one of the preceding items, which is for inactivating bacterial or viral ssRNA. 14. The composition of any one of the preceding items, which is a pharmaceutical composition. 15. The composition of item 14, further comprising at least one pharma- ceutically acceptable carrier. 16. The composition of any one of items 1 to 15 for use in therapy. 17. Any one of the compositions of items 1 to 15 for use in a method for preventing or treating a viral or bacterial disease in a subject, comprising administering to the subject a therapeutically effective amount of the composition of items 1 to 15. 18. The composition for use of item 17, wherein the viral disease is caused by an RNA virus. 19. The composition for use according to item 18, wherein the viral disease is any one of coronavirus disease, influenza A, Ebola, measles, hepatitis C, tick-borne encephalitis (TBE), dengue fever, yellow fever or Zika fever. 20. The composition for use according to item 19, wherein the viral disease is COVID-19 disease. 21. A nucleic acid molecule comprising a nucleotide sequence encoding a Cas13 protein and / or a gRNA as defined in any one of the preceding items. 22. A vector comprising the nucleic acid molecule of item 21. 23. A host cell comprising the vector of item 22 or the nucleic acid molecule of item 21. 24. A kit comprising any one of the compositions of items 1 to 15. 25. The kit of item 24, further comprising a delivery system and / or a label. 26. A method for producing a composition according to any one of items 1 to 15, comprising the steps of: a) a nucleic acid encoding a Cas13 protein and / or a gRNA as defined in any one of items 1 to 15 by genetic engineering methods, thereby producing said composition; optionally b) obtaining the composition produced in step a).

Claims

1. 1. A composition comprising a clustered regularly interspaced short palindromic repeats (CRISPR) system, the system comprising: i) at least one CRISPR-associated protein 13 (Cas13) fused to at least one nuclear localization signal (NLS) fused to at least one nuclear export sequence (NES), or a nucleotide sequence encoding said Cas13 protein; ii) at least one guide RNA (gRNA) capable of hybridizing to one or more target RNA molecules or a nucleotide sequence encoding said gRNA; The composition comprising:

2. The composition of claim 1, wherein the Cas13 protein is fused via a linker to at least one NLS fused to at least one NES.

3. The composition of claim 1 or 2, wherein the Cas13 protein is fused to two NLSs fused to one NES.

4. The composition of claim 3, wherein the two NLSs include an SV40 NLS having the amino acid sequence shown in SEQ ID NO:5 and an NLS consensus sequence having the amino acid sequence shown in SEQ ID NO:3, and the one NES includes an HIV NES having the amino acid sequence shown in SEQ ID NO:

4.

5. The composition of any one of claims 1 to 4, wherein the Cas13 protein is a Cas13d protein.

6. The composition of claim 5, wherein the Cas13d protein is derived from the genus Ruminococcus.

7. The composition of claim 5, wherein the Cas13d protein is derived from Ruminococcus flavevaciens.

8. The composition of any one of claims 1 to 7, wherein the gRNA has a length of at least about 23 nucleotides.

9. The composition of any one of claims 1 to 8, wherein the gRNA has a length of about 26 to about 30 nucleotides.

10. The composition of any one of claims 1 to 9, wherein the gRNA has at least about 80% complementary sequence identity to the one or more target RNA molecules.

11. The composition of any one of claims 1 to 10, wherein the gRNA is capable of hybridizing to the 5' and / or 3' untranslated region of one or more target RNA molecules.

12. The composition of any one of claims 1 to 11, wherein the nucleotide sequence encoding the gRNA is fused to a tRNA or a ribozyme.

13. The nucleotide sequence encoding the Cas13 protein and / or gRNA may contain the following modifications: Modified 5' and / or 3' UTRs, post- or co-transcriptional addition of a 5' CAP structure, replacement of UTP with N1-methylpseudo-UTP, or replacement of the two 5' and / or 3' terminal nucleotides with 2'-O-methyl-3' P-thioate The composition of any one of claims 1 to 12, comprising any one of:

14. The composition of any one of claims 1 to 13, wherein the nucleotide sequence encoding the gRNA is fused to at least one viral transport element.

15. The composition of claim 14, wherein at least one viral transport element is a constitutive transport element (CTE) or adenovirus VA1 RNA (VARdm).

16. The composition of any one of claims 1 to 15, wherein the one or more target RNA molecules is a viral or bacterial target RNA molecule.

17. The composition of any one of claims 1 to 16, for inactivating bacterial or viral ssRNA.

18. The composition of any one of claims 1 to 17, which is a pharmaceutical composition.

19. 20. The composition of claim 18, further comprising at least one pharma- ceutically acceptable carrier.

20. 20. The composition of any one of claims 1 to 19 for use in therapy.

21. 20. The composition of any one of claims 1 to 19 for use in a method of preventing or treating a viral or bacterial disease in a subject comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1 to 19.

22. 22. The composition for use according to claim 21, wherein the viral disease is caused by an RNA virus.

23. 23. The composition for use according to claim 22, wherein the viral disease is any one of a coronavirus disease, influenza A, Ebola, measles, hepatitis C, tick-borne encephalitis (TBE), Venezuelan equine encephalitis (VEE) virus infection, dengue fever, yellow fever or Zika fever.

24. 24. The composition for use according to claim 23, wherein the viral disease is COVID-19 disease.

25. A nucleic acid molecule comprising a nucleotide sequence encoding a Cas13 protein and a gRNA as defined in any one of claims 1 to 19.

26. A vector comprising the nucleic acid molecule of claim 25.

27. 27. A host cell comprising the vector of claim 26 or the nucleic acid molecule of claim 25.

28. A kit comprising the composition of any one of claims 1 to 19.

29. 30. The kit of claim 28, further comprising a delivery system and / or a label.

30. A method for producing a composition according to any one of claims 1 to 19, comprising: (a) starting from a nucleic acid encoding a Cas13 protein and a gRNA as defined in any one of claims 1 to 19 by genetic engineering methods, thereby producing said composition.

31. The method of claim 30, further comprising the step of: (b) obtaining the composition produced in step (a).

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