Novel Cas13B orthologs CRISPR enzymes and systems

Abstract

The present invention provides systems, methods and compositions for targeting nucleic acids. In particular, the present invention provides non-naturally occurring or engineered RNA-targeting systems comprising novel RNA-targeting Cas13b effector proteins and at least one targeting nucleic acid component such as a guide RNA or crRNA.

Description

[0001] Related Applications and Incorporation by Reference

[0002] This application claims priority to U.S. Provisional Application No. 62 / 471,710 filed on March 15, 2017 and U.S. Provisional Application No. 62 / 566,829 filed on October 2, 2017.

[0003] Reference is made to PCT applications including those designated therein, inter alia, U.S. Application No. PCT / US2016 / 058302 filed October 21, 2016. Reference is made to U.S. Provisional Patent Application 62 / 245,270 filed October 22, 2015, U.S. Provisional Patent Application 62 / 296,548 filed February 17, 2016, and U.S. Provisional Patent Applications 62 / 376,367 and 62 / 376,382 filed August 17, 2016. Further reference is made to U.S. Provisional 62 / 471,792 filed March 15, 2017, and U.S. Provisional 62 / 484,786 filed April 12, 2017. Mentioned Smargon et al. (2017), “Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28,” Molecular Cell 65, 618-630 (February 16, 2017) doi:10.1016 / j.molcel.2016.12.023. Epub January 5, 2017 and Smargon et al. (2017), “Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28,” bioRxiv 092577; doi:https: / / doi.org / 10.1101 / 092577. Epub December 9, 2017. Each of the foregoing applications and literature citations is hereby incorporated herein by reference.

[0004] Indeed, this document and all documents cited or referenced herein or documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference as if each individual document was specifically and individually indicated to be incorporated by reference.

[0005] Statement Regarding Federally Funded Research

[0006] This invention was made with government support under Grant Nos. MH100706 and MH110049 awarded by the National Institutes of Health. The government has certain rights in this invention. Field of the Invention

[0007] The present invention generally relates to systems, methods and compositions for controlling gene expression involving sequence targeting (such as perturbation of gene transcripts or nucleic acid editing), which can use vector systems related to clustered regularly interspaced short palindromic repeats (CRISPR) and its components. Background of the Invention

[0009] CRISPR-CRISPR-associated (Cas) systems of bacterial and archaeal adaptive immunity are some such systems that show extreme diversity in protein composition and genomic locus structure. CRISPR-Cas system loci have more than 50 gene families, and there are no strict universal genes to indicate the rapid evolution and extreme diversity of locus structure. So far, a comprehensive cas gene identification has been performed on approximately 395 maps of 93 Cas proteins using a multi-pronged approach. The classification includes signature gene maps as well as locus structure signatures. A new classification method for CRISPR-Cas systems is proposed, in which these systems are roughly divided into two categories: Class 1 with multi-subunit effector complexes and Class 2 with single-subunit effector modules exemplified by Cas9 proteins. Novel effector proteins associated with Class 2 CRISPR-Cas systems may be developed as powerful genome engineering tools, and are very important for the prediction of putative novel effector proteins and their engineering and optimization. Novel Cas13b orthologs and their uses are desirable.

[0010] Citation or identification of any document in this application does not constitute an admission that such document is available as prior art to the present invention. Summary of the invention

[0011] Effector proteins include two subgroups, VI-B1 type and VI-B2 type, and include RNA programmable nucleases, RNA interference and members that can participate in bacterial adoptive immunity against RNA phages. The Cas13b system can include a large single effector (about 1100 amino acids in length) near the CRISPR array, and one or none of two small putative auxiliary proteins (about 200 amino acids in length). Based on nearby small proteins, the system is divided into two loci A and B. In the species of each locus, it is conservative to have no additional proteins to 25 kilobase pairs upstream or downstream of the array. With a few exceptions, the CRISPR array includes a forward repeat sequence of 36 nucleotides in length and a spacer sequence of 30 nucleotides in length. The forward repeat sequence is usually very conservative, especially at the end, where the GTTG / GUUG at the 5' end is complementary to the CAAC at the 3' end. This conservatism implies a strong base pairing of the RNA ring structure, which may interact with one or more proteins in the locus. A motif search complementary to the direct repeats revealed no candidate tracrRNAs near the array, possibly indicating a single crRNA, similar to that found in the Cpf1 locus.

[0012] In an embodiment of the present invention, the VI-B type system comprises a novel Cas13b effector protein and an optional small auxiliary protein encoded upstream or downstream of the Cas13b effector protein. In certain embodiments, the small auxiliary protein enhances the ability of the Cas13b effector to target RNA.

[0013] The present invention provides a non-naturally occurring or engineered composition comprising

[0014] i) certain novel Cas13b effector proteins, and

[0015] ii) crRNA,

[0016] wherein the crRNA comprises a) a guide sequence, which is capable of hybridizing to a target RNA sequence, and b) a direct repeat sequence,

[0017] Thus, a CRISPR complex comprising a Cas13b effector protein complexed with a guide sequence hybridized to a target RNA sequence is formed. The complex can be formed in vivo or ex vivo and introduced into a cell or contacted with RNA; or it can be formed in vivo.

[0018] In some embodiments, the non-naturally occurring or engineered compositions of the invention may comprise an accessory protein that enhances the activity of a Type VI-B CRISPR-Cas effector protein.

[0019] In certain such embodiments, the accessory protein that enhances the activity of the Cas13b effector protein is a csx28 protein. In such embodiments, the Type VI-B CRISPR-Cas effector protein and the Type VI-B CRISPR-Cas accessory protein may be from the same source or from different sources.

[0020] In some embodiments, the non-naturally occurring or engineered compositions of the invention comprise an accessory protein that represses the activity of the Cas13b effector protein.

[0021] In certain such embodiments, the auxiliary protein that represses the activity of the Cas13b effector protein is a csx27 protein. In such embodiments, the VI-B type CRISPR-Cas effector protein and the VI-B type CRISPR-Cas auxiliary protein may be from the same source or from different sources. In certain embodiments of the invention, the VI-B type CRISPR-Cas effector protein is from Table 1. In certain embodiments, the VI-B type CRISPR-Cas auxiliary protein is from Table 1.

[0022] In some embodiments, the non-naturally occurring or engineered compositions of the invention comprise two or more crRNAs.

[0023] In some embodiments, the non-naturally occurring or engineered compositions of the invention comprise a guide sequence that hybridizes to a target RNA sequence in a prokaryotic cell.

[0024] In some embodiments, the non-naturally occurring or engineered compositions of the invention comprise a guide sequence that hybridizes to a target RNA sequence in a eukaryotic cell.

[0025] In some embodiments, the Cas13b effector protein comprises one or more nuclear localization signals (NLS).

[0026] The Cas13b effector protein of the present invention is, or is, or comprises, or consists essentially of, or consists of, or relates to, or is related to: such a protein from Table 1 or as shown by Table 1. The present invention is intended to provide, or is related to, or is related to, or comprises, or consists essentially of, or consists of: a protein from Table 1 or as described by Table 1 comprising a mutation or alteration thereof as shown herein. Table 1Cas13b effector proteins are discussed in more detail herein in conjunction with Table 1.

[0027] In some embodiments of the non-naturally occurring or engineered compositions of the invention, the Cas13b effector protein is associated with one or more functional domains. The association can be performed by direct linkage of the effector protein to the functional domain, or by association with the crRNA. In a non-limiting example, the crRNA comprises an added or inserted sequence that can be associated with the target functional domain, including, for example, an aptamer or nucleotide that binds to a nucleic acid binding adapter protein.

[0028] In certain non-limiting embodiments, the non-naturally occurring or engineered compositions of the invention comprise a functional domain that cleaves a target RNA sequence.

[0029] In certain non-limiting embodiments, the non-naturally occurring or engineered compositions of the invention comprise a functional domain that modifies the transcription or translation of a target RNA sequence.

[0030] In some embodiments of the compositions of the invention, the Cas13b effector protein is associated with one or more functional domains; and the effector protein contains one or more mutations in the HEPN domain, so that the complex can deliver epigenetic modifiers or transcriptional or translational activation or repression signals. The complex can be formed in vitro or ex vivo and introduced into cells or contacted with RNA; or it can be formed in vivo.

[0031] In some embodiments of the non-naturally occurring or engineered compositions of the invention, the Cas13b effector protein and the accessory protein are from the same organism.

[0032] In some embodiments of the non-naturally occurring or engineered compositions of the invention, the Cas13b effector protein and the accessory protein are from different organisms.

[0033] The present invention also provides a Type VI-B CRISPR-Cas vector system, comprising one or more vectors, wherein the vector comprises:

[0034] a first regulatory element operably linked to a nucleotide sequence encoding a Cas13b effector protein, and

[0035] A second regulatory element operably linked to a nucleotide sequence encoding crRNA. In certain embodiments, the vector system of the present invention further comprises a regulatory element operably linked to a nucleotide sequence of a Type VI-B CRISPR-Cas accessory protein.

[0036] When appropriate, the nucleotide sequences encoding the Type VI-B CRISPR-Cas effector protein and / or encoding the Type VI-B CRISPR-Cas accessory protein are codon-optimized for expression in eukaryotic cells.

[0037] In some embodiments of the vector system of the present invention, the nucleotide sequences encoding the Cas13b effector protein and the accessory protein are codon-optimized for expression in eukaryotic cells.

[0038] In some embodiments, the vector system of the present invention is contained in a single vector.

[0039] In some embodiments of the vector systems of the invention, one or more vectors comprise a viral vector.

[0040] In some embodiments of the vector systems of the invention, the one or more vectors include one or more retroviral, lentiviral, adenoviral, adeno-associated, or herpes simplex viral vectors.

[0041] The present invention provides a delivery system for a Cas13b effector protein and one or more nucleic acid components configured to deliver a non-naturally occurring or engineered composition, the non-naturally occurring or engineered composition comprising:

[0042] i) Cas13b effector protein, and

[0043] ii) crRNA,

[0044] wherein the crRNA comprises a) a guide sequence that hybridizes to a target RNA sequence in a cell, and b) a direct repeat sequence,

[0045] Among them, Cas13b effector protein forms a complex with crRNA.

[0046] The guide sequence specifically binds to the target RNA sequence.

[0047] Thereby forming a CRISPR complex comprising a Cas13b effector protein complexed with a guide sequence hybridized to a target RNA sequence. The complex can be formed in vitro or ex vivo and introduced into a cell or contacted with RNA; or it can be formed in vivo.

[0048] In some embodiments of the delivery system of the invention, the system comprises one or more vectors or one or more polynucleotide molecules comprising one or more polynucleotide molecules encoding a Cas13b effector protein and one or more nucleic acid components of a non-naturally occurring or engineered composition.

[0049] In some embodiments, the delivery system of the present invention comprises a delivery vehicle comprising one or more liposomes, one or more particles, one or more exosomes, one or more microvesicles, a gene gun, or one or more viral vectors.

[0050] In some embodiments, the non-naturally occurring or engineered compositions of the invention are used in therapeutic treatment methods or in research programs.

[0051] In some embodiments, the non-naturally occurring or engineered vector systems of the invention are used in therapeutic treatment approaches or in research programs.

[0052] In some embodiments, the non-naturally occurring or engineered delivery systems of the invention may be used in therapeutic treatment approaches or in research programs.

[0053] The present invention provides a method for modifying the expression of a target gene of interest, the method comprising contacting the target RNA with one or more non-naturally occurring or engineered compositions, the non-naturally occurring or engineered compositions comprising

[0054] i) Cas13b effector protein, and

[0055] ii) crRNA,

[0056] wherein the crRNA comprises a) a guide sequence that hybridizes to a target RNA sequence in a cell, and b) a direct repeat sequence,

[0057] Among them, Cas13b effector protein forms a complex with crRNA.

[0058] The guide sequence specifically binds to the target RNA sequence in the cell.

[0059] Thereby forming a CRISPR complex comprising the Cas13b effector protein complexed with the guide sequence hybridized to the target RNA sequence,

[0060] The expression of the target locus of interest is thereby modified. The complex can be formed in vivo or ex vivo and introduced into a cell or contacted with RNA; or can be formed in vivo.

[0061] In some embodiments, the method of modifying the expression of a target gene of interest further comprises contacting the target RNA with an auxiliary protein that enhances the activity of the Cas13b effector protein.

[0062] In some embodiments of the method of modifying the expression of a target gene of interest, the auxiliary protein that enhances the activity of the Cas13b effector protein is the csx28 protein.

[0063] In some embodiments, the method of modifying the expression of a target gene of interest further comprises contacting the target RNA with an auxiliary protein that represses the activity of the Cas13b effector protein.

[0064] In some embodiments of the method of modifying the expression of a target gene of interest, the auxiliary protein that represses the activity of the Cas13b effector protein is a csx27 protein.

[0065] In some embodiments, the method of modifying the expression of a target gene of interest comprises cleaving the target RNA.

[0066] In some embodiments, methods of modifying the expression of a target gene of interest include increasing or decreasing the expression of a target RNA.

[0067] In some embodiments of the methods of modifying expression of a target gene of interest, the target gene is in a prokaryotic cell.

[0068] In some embodiments of the methods of modifying expression of a target gene of interest, the target gene is in a eukaryotic cell.

[0069] The invention provides cells comprising a modified target of interest, wherein the target of interest is modified according to any of the methods disclosed herein.

[0070] In some embodiments of the invention, the cell is a prokaryotic cell.

[0071] In some embodiments of the invention, the cell is a eukaryotic cell.

[0072] In some embodiments, modification of the target of interest in a cell results in:

[0073] The cell comprises altered expression of at least one gene product;

[0074] The cell comprises altered expression of at least one gene product, wherein expression of at least one gene product is increased; or

[0075] The cell comprises altered expression of at least one gene product, wherein expression of the at least one gene product is reduced.

[0076] In some embodiments, the cell is a mammalian cell or a human cell.

[0077] The present invention provides cell lines comprising cells disclosed herein or cells modified by any of the methods disclosed herein.

[0078] The invention provides a multicellular organism comprising one or more cells disclosed herein or one or more cells modified according to any of the methods disclosed herein.

[0079] The present invention provides plant or animal models comprising one or more cells disclosed herein or one or more cells modified according to any of the methods disclosed herein.

[0080] The invention provides gene products from the cells or cell lines or organisms or plants or animal models disclosed herein.

[0081] In some embodiments, the amount of gene product expressed is greater or less than the amount of gene product from a cell that does not have altered expression.

[0082] The present invention provides an isolated Cas13b effector protein comprising or consisting essentially of or consisting of or as shown in Table 1. Table 1 Cas13b effector protein As discussed in more detail herein in conjunction with Table 1. The present invention provides an isolated nucleic acid encoding a Cas13b effector protein. In some embodiments of the present invention, the isolated nucleic acid comprises a DNA sequence and further comprises a sequence encoding a crRNA. The present invention provides an isolated eukaryotic cell comprising a nucleic acid encoding a Cas13b effector protein. Therefore, herein, "Cas13b effector protein" or "effector protein" or "Cas" or "Cas protein" or "RNA targeting effector protein" or "RNA targeting effector protein" or similar expressions should be understood with reference to Table 1 and can be read as Table 1 Cas13b effector protein CRISPR system; and reference to guide RNA or sgRNA should be read in conjunction with the discussion of Cas13b system crRNA herein, such as it is sgRNA in other systems, which can be considered as or similar to the crRNA in the present invention.

[0083] The present invention provides a method for identifying a suitable guide sequence for the Cas13b effector protein of the present invention (such as Table 1), the method comprising:

[0084] (a) Select a set of essential genes in an organism

[0085] (b) Designing a library of targeted guide sequences that can hybridize to the regions (coding regions of these genes and 5' and 3' UTRs of these genes)

[0086] (c) generating a randomized guide sequence that does not hybridize to any region within the genome of the organism as a control guide

[0087] (d) preparing a plasmid comprising the RNA-targeting protein and the first resistance gene, and a guide plasmid library comprising said library of targeting guides and said control guide and the second resistance gene,

[0088] (e) co-introducing the plasmid into the host cell

[0089] (f) introducing said host cell into a selective medium for said first and second resistance genes

[0090] (g) Sequencing of genes essential for host cell growth

[0091] (h) determining the significance of depletion of cells transformed with the targeted guide by comparing the depletion of the cells to a control guide; and

[0092] (i) Determining the need for a suitable guide sequence based on the exhausted guide sequence.

[0093] In one aspect of such methods, a PFS sequence for a suitable guide sequence for an RNA targeting protein is determined by comparing sequences targeted by the guide in depleted cells. In one aspect of such methods, the method further comprises comparing guide abundance for different conditions in different replicates. In one aspect of such methods, control guides are selected because they are determined to show limited deviation in guide depletion in replicates. In one aspect of such methods, the significance of depletion is determined as: (a) more depletion than the most depleted control guide; or (b) depletion greater than two times the standard deviation of the mean depletion + control guides. In one aspect of such methods, the host cell is a bacterial host cell. In one aspect of such methods, the step of co-introducing the plasmid is performed by electroporation and the host cell is an electrocompetent host cell.

[0094] Cas13b

[0095] The present invention provides a method for modifying a sequence associated with or at a target locus of interest, the method comprising delivering a non-naturally occurring or engineered composition comprising a Cas13b effector protein and one or more nucleic acid components to the locus, wherein the effector protein forms a complex with the one or more nucleic acid components, and after the complex binds to the target locus, the effector protein induces modification of a sequence associated with or at the target locus of interest. In a preferred embodiment, the modification is the introduction of a strand break. In a preferred embodiment, the sequence associated with or at the target locus of interest comprises RNA or consists of RNA.

[0096] The present invention provides a method for modifying a sequence associated with or at a target locus, the method comprising delivering a non-naturally occurring or engineered composition comprising a Cas13b effector protein, an optional small auxiliary protein, and one or more nucleic acid components to the locus, wherein the effector protein forms a complex with the one or more nucleic acid components, and after the complex binds to the target locus, the effector protein induces modification of a sequence associated with or at the target locus. In a preferred embodiment, the modification is the introduction of a strand break. In a preferred embodiment, the sequence associated with or at the target locus comprises RNA or consists of RNA.

[0097] The present invention provides a method for modifying a sequence associated with or at a target target locus, the method comprising delivering a non-naturally occurring or engineered composition comprising a Cas13b locus effector protein and one or more nucleic acid components to a sequence associated with or at a locus, wherein the Cas13b effector protein forms a complex with the one or more nucleic acid components, and after the complex binds to the target locus, the effector protein induces modification of a sequence associated with or at a target target locus. In a preferred embodiment, the modification is the introduction of a strand break. In a preferred embodiment, the Cas13b effector protein forms a complex with a nucleic acid component; advantageously, an engineered or non-naturally occurring nucleic acid component. The induction of modification of a sequence associated with or at a target target locus can be guided by a Cas13b effector protein-nucleic acid. In a preferred embodiment, a nucleic acid component is CRISPR RNA (crRNA). In a preferred embodiment, a nucleic acid component is a mature crRNA or guide RNA, wherein the mature crRNA or guide RNA comprises a spacer sequence (or guide sequence) and a forward repeat (DR) sequence or a derivative thereof. In a preferred embodiment, the spacer sequence or its derivative comprises a seed sequence, wherein the seed sequence is important for identifying and / or hybridizing to the sequence at the target locus. In a preferred embodiment of the present invention, crRNA is a short crRNA that can be associated with a short DR sequence. In another embodiment of the present invention, crRNA is a long crRNA that can be associated with a long DR sequence (or double DR). Various aspects of the present invention relate to Cas13b effector protein complexes with one or more non-naturally occurring or engineered or modified or preferred nucleic acid components. In a preferred embodiment, the nucleic acid component comprises RNA. In a preferred embodiment, the nucleic acid component of the complex may include a guide sequence connected to a forward repeat sequence, wherein the forward repeat sequence includes one or more stem loops or a preferred secondary structure. In a preferred embodiment of the present invention, the forward repeat may be short DR or long DR (double DR). In a preferred embodiment, the forward repeat may be modified or include one or more protein binding adapters. In a preferred embodiment, one or more adapters may be included, such as a portion of an optimized secondary structure. Such adapters may be able to bind to phage coat proteins. The bacteriophage coat protein can be selected from the group consisting of QP, F2, GA, fir, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, Mil, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, 4>Cb5, 4>Cb8r, 4>Cbl2r, 4>Cb23r, 7s and PRR1. In a preferred embodiment, the bacteriophage coat protein is MS2.The invention also provides nucleic acid components of the complex that are 30 or more, 40 or more, or 50 or more nucleotides in length.

[0098] The present invention provides a method for genome editing or modifying a sequence associated with a target locus or at a target locus, wherein the method includes introducing a Cas13b complex into any desired cell type, prokaryotic or eukaryotic cell, so that the Cas13b effector protein complex effectively interferes with the RNA in a eukaryotic or prokaryotic cell. In a preferred embodiment, the cell is a eukaryotic cell, and the RNA is transcribed from a mammalian genome or is present in a mammalian cell. In a preferred method for RNA editing or genome editing in human cells, the Cas13b effector protein may include, but is not limited to, a specific species of the Cas13b effector protein disclosed herein.

[0099] The present invention also provides a method for modifying a target locus of interest, the method comprising delivering a non-naturally occurring or engineered composition comprising a Cas13b effector protein and one or more nucleic acid components to the locus, wherein the Cas13b effector protein forms a complex with the one or more nucleic acid components, and when the complex binds to the target locus, the effector protein induces modification of the target locus of interest. In a preferred embodiment, the modification is the introduction of a strand break.

[0100] In such methods, the target locus of interest may be contained within an RNA molecule. In such methods, the target locus of interest may be contained within an RNA molecule in vitro.

[0101] In such methods, the target locus can be contained in an RNA molecule in a cell. The cell can be a prokaryotic cell or a eukaryotic cell. The cell can be a mammalian cell. The modification introduced into the cell by the present invention can make it possible to change the cell and the cell progeny to improve the production of biological products such as antibodies, starch, alcohol or other desired cell outputs. The modification introduced into the cell by the present invention can make the cell and the cell progeny contain changes in the biological products produced by the change.

[0102] Mammalian cells can be non-human mammals, such as primates, bovines, ovines, porcines, canines, rodents, Leporidae such as monkeys, cattle, sheep, pigs, dogs, rabbits, rats or mice. Cells can be non-mammalian eukaryotic cells, such as birds (e.g., chickens), vertebrates (e.g., salmon) or shellfish (e.g., oysters, whitefish, lobsters, shrimps). Cells can also be plant cells. Plant cells can belong to monocots or dicots, or to crops or cereals, such as cassava, corn, sorghum, soybeans, wheat, oats or rice. The plant cell can also belong to algae, trees or production plants, fruits or vegetables (e.g., trees such as citrus trees, such as orange, grapefruit or lemon trees; peach or nectarine trees; apple or pear trees; nut trees such as almond or walnut trees or pistachio trees; nightshade; Brassica; lettuce; spinach: peppers, cotton, tobacco, asparagus, carrots, cabbage, broccoli, cauliflower, tomatoes, eggplant, peppers, lettuce, spinach, strawberries, blueberries, raspberries, blackberries, grapes, coffee, cocoa, etc.).

[0103] The present invention provides a method for modifying a target locus of interest, the method comprising delivering a non-naturally occurring or engineered composition comprising a Cas13b effector protein and one or more nucleic acid components to the locus, wherein the effector protein forms a complex with the one or more nucleic acid components, and after the complex binds to the target locus, the effector protein induces modification of the target locus of interest. In a preferred embodiment, the modification is the introduction of a strand break.

[0104] In such methods, the target locus of interest may be comprised within an RNA molecule. In a preferred embodiment, the target locus of interest comprises or consists of RNA.

[0105] The present invention also provides a method for modifying a target locus of interest, the method comprising delivering a non-naturally occurring or engineered composition comprising a Cas13b effector protein and one or more nucleic acid components to the locus, wherein the Cas13b effector protein forms a complex with the one or more nucleic acid components, and when the complex binds to the target locus, the effector protein induces modification of the target locus of interest. In a preferred embodiment, the modification is the introduction of a strand break.

[0106] Preferably, in such methods, the target locus of interest may be contained in an RNA molecule in vitro. Also preferably, in such methods, the target locus of interest may be contained in an RNA molecule in a cell. The cell may be a prokaryotic cell or a eukaryotic cell. The cell may be a mammalian cell. The cell may be a rodent cell. The cell may be a mouse cell.

[0107] In any of the described methods, the target locus of interest can be a genomic or epigenomic locus of interest. In any of the described methods, the complex can be delivered with multiple guides for multiplexed use. In any of the described methods, more than one protein can be used.

[0108] In other aspects of the invention, the nucleic acid component may include a CRISPR RNA (crRNA) sequence. Since the effector protein is a Cas13b effector protein, the nucleic acid component may include a CRISPR RNA (crRNA) sequence and may generally not include any trans-activating crRNA (tracrRNA) sequence.

[0109] In any of the described methods, the effector protein and nucleic acid components can be provided by one or more polynucleotide molecules encoding protein and / or nucleic acid components, and wherein the one or more polynucleotide molecules are operably configured to express protein and / or nucleic acid components. The one or more polynucleotide molecules may contain one or more regulatory elements that are operably configured to express protein and / or nucleic acid components. The one or more polynucleotide molecules may be contained in one or more vectors. In any of the described methods, the target locus of interest can be a target genome, epigenome, or transcriptome locus. In any of the described methods, the complex can be delivered with multiple guides for multiple uses. In any of the described methods, more than one protein can be used.

[0110] In any of the described methods, the strand break can be a single strand break or a double strand break. In a preferred embodiment, a double strand break can refer to the break of two parts of an RNA, such as the two parts of an RNA formed when a single stranded RNA molecule folds onto itself, or a putative double helix formed by an RNA molecule containing self-complementary sequences allows part of the RNA to fold and pair with itself.

[0111] The regulatory element may include an inducible promoter. The polynucleotide and / or vector system may include an inducible system.

[0112] In any of the described methods, one or more polynucleotide molecules may be included in the delivery system, or one or more vectors may be included in the delivery system.

[0113] In any of the methods described, the non-naturally occurring or engineered composition can be delivered via liposomes, nanoparticle-containing particles, exosomes, microvesicles, a gene gun, or one or more viral vectors.

[0114] The invention also provides a non-naturally occurring or engineered composition having the features as discussed herein or as defined in any of the methods described herein.

[0115] Thus, in certain embodiments, the present invention provides a non-naturally occurring or engineered composition, such as a composition that is particularly capable of or configured to modify a target locus of interest, the composition comprising a Cas13b effector protein and one or more nucleic acid components, wherein the effector protein forms a complex with the one or more nucleic acid components, and when the complex is bound to the target locus, the effector protein induces modification of the target locus of interest. In certain embodiments, the effector protein may be a Cas13b effector protein.

[0116] In another aspect, the present invention also provides a non-naturally occurring or engineered composition, such as a composition that can be configured to modify a target locus of interest, comprising: (a) a guide RNA molecule (or a combination of guide RNA molecules, such as a first guide RNA molecule and a second guide RNA molecule) or a nucleic acid encoding a guide RNA molecule (or one or more nucleic acids encoding a combination of guide RNA molecules); (b) a Cas13b effector protein. In certain embodiments, the effector protein can be a Cas13b effector protein.

[0117] In another aspect, the present invention also provides a non-naturally occurring or engineered composition comprising: (I.) one or more CRISPR-Cas system polynucleotide sequences comprising (a) a guide sequence capable of hybridizing to a target sequence in a polynucleotide locus, (b) a tracr partner sequence, and (c) a tracr RNA sequence; and (II.) a second polynucleotide sequence encoding a Cas13b effector protein, wherein when transcribed, the tracr partner sequence hybridizes to the tracrRNA sequence, and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence, and wherein the CRISPR complex comprises a Cas13b effector protein complexed with the following sequences: (1) a guide sequence hybridized to the target sequence and (2) a tracr partner sequence hybridized to the tracrRNA sequence. In certain embodiments, the effector protein may be a Cas13b effector protein.

[0118] In certain embodiments, tracrRNA may not be required. Therefore, in certain embodiments, the present invention also provides a non-naturally occurring or engineered composition comprising: (I.) one or more CRISPR-Cas system polynucleotide sequences, comprising (a) a guide sequence capable of hybridizing with a target sequence in a polynucleotide locus, and (b) a forward repeat sequence; and (II.) a second polynucleotide sequence encoding a Cas13b effector protein, wherein when transcribed, the guide sequence directs the sequence-specific binding of the CRISPR complex to the target sequence, and wherein the CRISPR complex comprises a Cas13b effector protein compounded with the following sequence: (1) a guide sequence hybridized with the target sequence, and (2) a forward repeat sequence. Preferably, the effector protein can be a Cas13b effector protein. Without limitation, the applicant assumes that in such cases, the forward repeat sequence may include a secondary structure sufficient to load crRNA onto the effector protein. As an example and not limitation, such secondary structures may include stem loops (e.g., one or more stem loops) within the forward repeat, consisting essentially of or consisting of.

[0119] The present invention also provides a vector system comprising one or more vectors comprising one or more polynucleotide molecules encoding components of a non-naturally occurring or engineered composition, the composition being a composition having the characteristics defined in any of the methods described herein.

[0120] The present invention also provides a delivery system comprising one or more vectors or one or more polynucleotide molecules comprising one or more polynucleotide molecules encoding components of a non-naturally occurring or engineered composition having the features as discussed herein or as defined in any of the methods described herein.

[0121] The present invention also provides a non-naturally occurring or engineered composition for use in a therapeutic method of treatment, or one or more polynucleotides encoding the components of the composition, or a vector or delivery system comprising one or more polynucleotides encoding the components of the composition. The therapeutic method of treatment may include gene or genome editing, or gene therapy.

[0122] The present invention also provides methods and compositions, wherein one or more amino acid residues of the effector protein may be modified, such as an engineered or non-naturally occurring Cas13b effector protein of Table 1 protein, or comprising Table 1 protein, or consisting of Table 1 protein, or consisting essentially of Table 1 protein. In one embodiment, the modification may include mutations of one or more amino acid residues of the effector protein. One or more mutations may be in one or more catalytically active domains of the effector protein. Compared to the effector protein lacking the one or more mutations, the effector protein may have reduced or eliminated nuclease activity. The effector protein may not direct the cleavage of an RNA chain at the target locus of interest. In a preferred embodiment, one or more mutations may include two mutations. In a preferred embodiment, one or more amino acid residues are modified in the Cas13b effector protein, for example, an engineered or non-naturally occurring Cas13b effector protein. In certain embodiments of the present invention, the effector protein comprises one or more HEPN domains. In a preferred embodiment, the effector protein comprises two HEPN domains. In another preferred embodiment, the effector protein comprises a HEPN domain at the C-terminus of the protein and another HEPN domain at the N-terminus. In certain embodiments, one or more mutations or two or more mutations may be in the catalytically active domain of the effector protein, which comprises a HEPN domain, or a catalytically active domain homologous to a HEPN domain. In certain embodiments, the effector protein comprises one or more of the following mutations: R116A, H121A, R1177A, H1182A (wherein the amino acid position corresponds to the amino acid position of the 29th group protein derived from Bergeyella zoohelcum ATCC 43767). The technician will understand that the corresponding amino acid positions in different Cas13b proteins can be mutated to the same effect. In certain embodiments, one or more mutations completely or partially eliminate the catalytic activity of the protein (e.g., changes in cleavage rate, changes in specificity, etc.). In certain embodiments, the effector protein as described herein is a "dead" effector protein, such as a dead Cas13b effector protein (i.e., d Cas13b). In certain embodiments, the effector protein has one or more mutations in HEPN domain 1. In certain embodiments, the effector protein has one or more mutations in HEPN domain 2. In certain embodiments, the effector protein has one or more mutations in HEPN domain 1 and HEPN domain 2. The effector protein may comprise one or more heterologous functional domains. The one or more heterologous functional domains may include one or more nuclear localization signal (NLS) domains. The one or more heterologous functional domains may include at least two or more NLS domains.One or more NLS domains may be located at or near the end of the effector protein (e.g., Cas13b effector protein), and if two or more NLS, each of the two NLS may be located at or near the end of the effector protein (e.g., Cas13b effector protein). One or more heterologous functional domains may include one or more transcriptional activation domains. In a preferred embodiment, the transcriptional activation domain may include VP64. One or more heterologous functional domains may include one or more transcriptional repression domains. In a preferred embodiment, the transcriptional repression domain includes a KRAB domain or a SID domain (e.g., SID4X). One or more heterologous functional domains may include one or more nuclease domains. In a preferred embodiment, the nuclease domain includes Fokl.

[0123] The present invention also provides that one or more heterologous functional domains have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription inhibition activity, transcription release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity and nucleic acid binding activity. At least one or more heterologous functional domains can be at or near the amino terminus of the effector protein and / or at least one or more heterologous functional domains are at or near the carboxyl terminus of the effector protein. One or more heterologous functional domains can be fused to the effector protein. One or more heterologous functional domains can be tethered to the effector protein. One or more heterologous functional domains can be connected to the effector protein through a linker portion.

[0124] In certain embodiments, a Cas13b effector protein as described herein can be associated with a locus comprising a short CRISPR repeat of between 30 and 40 bp long, more typically between 34 and 38 bp long, even more typically between 36 and 37 bp long, e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bp long. In certain embodiments, the CRISPR repeat is a long repeat or double repeat of between 80 and 350 bp long, such as between 80 and 200 bp long, even more typically between 86 and 88 bp long, e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 bp long.

[0125] In certain embodiments, the protospacer sequence adjacent motif (PAM) or PAM-like motif guides the effector protein (e.g., Cas13b effector protein) complex as disclosed herein to bind to the target target genome. In some embodiments, PAM can be 5'PAM (i.e., upstream of the 5' end of the protospacer sequence). In other embodiments, PAM can be 3'PAM (i.e., downstream of the 5' end of the protospacer sequence). In other embodiments, both 5'PAM and 3'PAM are required. In certain embodiments of the present invention, PAM or PAM-like motifs may not be required to guide the binding of effector proteins (e.g., Cas13b effector proteins). In certain embodiments, 5'PAM is D (i.e., A, G, or U). In certain embodiments, 5'PAM is D for Cas13b effector proteins. In certain embodiments of the invention, cleavage at a repeat sequence can produce a crRNA (e.g., a short or long crRNA) comprising a full-length spacer sequence flanked at the 5' end by a short nucleotide (e.g., 5, 6, 7, 8, 9 or 10 nt or longer if double repeat) repeat sequence (this can be referred to as a crRNA "tag") and at the 3' end by the remainder of the repeat sequence. In certain embodiments, targeting by the effector protein described herein may require a lack of homology between the crRNA tag and the target 5' flanking sequence. This requirement may be similar to the requirement further described in Samai et al. "Co-transcriptional DNA and RNA Cleavage during Type III CRISPR-Cas Immunity" Cell 161, 1164-1174, May 21, 2015, where the requirement is considered to distinguish true targets on invading nucleic acids from the CRISPR array itself, and where the presence of a repeat sequence will result in complete homology to the CRISPR tag and prevent autoimmunity.

[0126] In certain embodiments, the Cas13b effector protein is engineered and may include one or more mutations that reduce or eliminate nuclease activity, thereby reducing or eliminating RNA interference activity. Mutations may also be made at adjacent residues, for example, at amino acids near amino acids involved in nuclease activity. In some embodiments, one or more putative catalytic nuclease domains are inactivated, and the effector protein complex lacks cleavage activity and functions as an RNA binding complex. In a preferred embodiment, the resulting RNA binding complex may be connected to one or more functional domains as described herein.

[0127] In certain embodiments, one or more functional domains are controllable, ie, inducible.

[0128] In certain embodiments of the present invention, the guide RNA or mature crRNA comprises a forward repeat sequence and a guide sequence or a spacer sequence, consisting essentially of or consisting of it. In certain embodiments, the guide RNA or mature crRNA comprises a forward repeat sequence connected to a guide sequence or a spacer sequence, consisting essentially of or consisting of it. In a preferred embodiment of the present invention, the mature crRNA comprises a stem loop or an optimized stem loop structure or an optimized secondary structure. In a preferred embodiment, the mature crRNA comprises a stem loop or an optimized stem loop structure in a forward repeat sequence, wherein the stem loop or the optimized stem loop structure is important for cleavage activity. In certain embodiments, the mature crRNA preferably comprises a single stem loop. In certain embodiments, the forward repeat sequence preferably comprises a single stem loop. In certain embodiments, the cleavage activity of the effector protein complex is modified by introducing mutations that affect the structure of the stem loop RNA duplex. In a preferred embodiment, mutations that maintain the stem loop RNA duplex can be introduced to thereby maintain the cleavage activity of the effector protein complex. In other preferred embodiments, mutations that destroy the RNA duplex structure of the stem loop can be introduced to thereby completely eliminate the cleavage activity of the effector protein complex.

[0129] As provided herein, the CRISPR system can utilize crRNA or similar polynucleotides comprising a guide sequence, wherein the polynucleotide is a mixture of RNA, DNA, or RNA and DNA, and / or wherein the polynucleotide comprises one or more nucleotide analogs. The sequence can constitute any structure, including but not limited to the structure of natural crRNA, such as a bulge, a hairpin, or a stem-loop structure. In certain embodiments, the polynucleotide comprising a guide sequence forms a duplex with a second polynucleotide sequence, which can be an RNA or DNA sequence.

[0130] In certain embodiments, the method utilizes chemically modified guide RNA. Examples of guide RNA chemical modification include, but are not limited to, incorporation of 2'-O-methyl (M), 2'-O-methyl 3' thiophosphate (MS) or 2'-O-methyl 3' thio PACE (MSP) at one or more terminal nucleotides. Compared to unmodified guide RNA, this chemically modified guide RNA may include increased stability and increased activity, although the on-target specificity is unpredictable compared to the off-target specificity. (See, Hendel, 2015, Nat Biotechnol. 33 (9): 985-9, doi: 10.1038 / nbt.3290, published online on June 29, 2015). Chemically modified guide RNA also includes, but is not limited to, RNA with thiophosphate linkage and locked nucleic acid (LNA) nucleotides, wherein the RNA includes a methylene bridge between the 2' and 4' carbons of the ribose ring.

[0131] The present invention also provides nucleotide sequences encoding effector proteins, which are codon-optimized for expression in eukaryotic organisms or eukaryotic cells in any of the methods or compositions described herein. In embodiments of the present invention, the codon-optimized effector protein is any Cas13b effector protein discussed herein, and is codon-optimized for operability in a eukaryotic cell or organism, such as such cells or organisms as mentioned elsewhere herein, for example, but not limited to, yeast cells or mammalian cells or organisms, including mouse cells, rat cells and human cells or non-human eukaryotic organisms, such as plants.

[0132] In certain embodiments of the present invention, at least one nuclear localization signal (NLS) is attached to the nucleic acid sequence encoding the Cas13b effector protein. In a preferred embodiment, at least one or more C-terminal or N-terminal NLSs are attached (therefore the nucleic acid molecules encoding the Cas13b effector protein can include encoding NLSs so that the expressed product has an attached or connected NLS). In a preferred embodiment, for optimal expression and nuclear targeting in eukaryotic cells, preferably human cells, a C-terminal NLS is attached. The present invention also encompasses methods for delivering a variety of nucleic acid components, wherein each nucleic acid component is specific to different target loci, thereby modifying a plurality of target loci. The nucleic acid components of the complex may include one or more protein-binding nucleic acid aptamers. One or more aptamers are capable of binding to phage coat proteins. The bacteriophage coat protein may be selected from the group consisting of Qβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCbl2r, φCb23r, 7s and PRR1. In a preferred embodiment, the bacteriophage coat protein is MS2. The present invention also provides a nucleic acid component of a complex having a length of 30 or more, 40 or more, or 50 or more nucleotides.

[0133] In another aspect, the invention provides a eukaryotic cell comprising a modified target locus of interest, wherein the target locus of interest has been modified according to any of the methods described herein. In another aspect, a cell line of the cell is provided. In another aspect, a multicellular organism comprising one or more of the cells is provided.

[0134] In certain embodiments, modification of the target locus of interest can result in: a eukaryotic cell comprising an altered expression of at least one gene product; a eukaryotic cell comprising an altered expression of at least one gene product, wherein expression of the at least one gene product is increased; a eukaryotic cell comprising an altered expression of at least one gene product, wherein expression of the at least one gene product is decreased; or a eukaryotic cell comprising an edited genome.

[0135] In certain embodiments, the eukaryotic cell can be a mammalian cell or a human cell.

[0136] In further embodiments, the non-naturally occurring or engineered compositions, vector systems, or delivery systems as described herein can be used for: site-specific gene knockout; site-specific genome editing; RNA sequence-specific interference; or multiple genome engineering.

[0137] Also provided is a gene product from a cell, cell line or organism as described herein. In certain embodiments, the amount of the gene product expressed can be greater than or less than the amount of the gene product from a cell of the genome without expression or editing. In certain embodiments, the gene product can be altered compared to a gene product from a cell of the genome without expression or editing.

[0138] In another aspect, the present invention provides a method for identifying novel nucleic acid modifying effectors, comprising: identifying a putative nucleic acid modifying locus from a group of nucleic acid sequences encoding a putative nucleic acid modifying enzyme locus, wherein the putative nucleic acid modifying enzyme locus is within a defined distance from a conserved genomic element of the locus, the locus comprises at least one protein exceeding a defined size limit, or both; grouping the identified putative nucleic acid modifying loci into subgroups comprising homologous proteins; identifying a final group of candidate nucleic acid modifying loci by selecting nucleic acid modifying loci from one or more subgroups based on one or more of the following: comprising subgroups of loci with putative effector proteins with low domain homology to known protein domains relative to loci in other subgroups; subgroups containing putative proteins with minimal distance to conserved genomic elements relative to loci in other subgroups; subgroups of loci with large effector proteins that have the same orientation as putative neighboring accessory proteins relative to large effector proteins in other subgroups; subgroups containing putative effector proteins with lower existing nucleic acid modification classifications relative to other loci; containing loci with lower proximity to known nucleic acid modification loci relative to other subgroups; and the total number of candidate loci in each subgroup.

[0139] In one embodiment, the set of nucleic acid sequences is obtained from a genomic or metagenomic database, such as a genomic or metagenomic database comprising prokaryotic genomic or metagenomic sequences.

[0140] In one embodiment, the defined distance from the conserved genomic element is between 1 kb and 25 kb.

[0141] In one embodiment, the conserved genomic elements include repetitive elements, such as a CRISPR array. In a specific embodiment, the defined distance from the conserved genomic elements is within 10 kb of the CRISPR array.

[0142] In one embodiment, the defined size limit of the protein contained in the putative nucleic acid modification (effector) locus is greater than 200 amino acids, or more specifically, the defined size limit is greater than 700 amino acids. In one embodiment, the putative nucleic acid modification locus is between 900 and 1800 amino acids.

[0143] In one embodiment, a nucleic acidome repeat or pattern finding analysis (such as PILER-CR) is used to identify conserved genomic elements.

[0144] In one embodiment, the grouping step of the methods described herein is based at least in part on the results of a domain homology search or a HHpred protein domain homology search.

[0145] In one embodiment, the defined threshold is a BLAST nearest neighbor cutoff of 0 to le-7.

[0146] In one embodiment, the methods described herein further comprise a filtering step that includes only loci with putative proteins between 900 and 1800 amino acids.

[0147] In one embodiment, the methods described herein further include experimental validation of the nucleic acid modification function of the candidate nucleic acid modification effectors, comprising generating a set of nucleic acid constructs encoding nucleic acid modification effectors and performing one or more biochemical validation assays, such as by using PAM validation in colonies, in vitro lysis assays, test methods, experiments in mammalian cells, PFS validation, or a combination thereof.

[0148] In one embodiment, the methods described herein further comprise preparing a non-naturally occurring or engineered composition comprising one or more proteins from the identified nucleic acid modifying loci.

[0149] In one embodiment, the identified locus comprises Class 2 CRISPR effectors, or the identified locus lacks Cas1 or Cas2, or the identified locus comprises a single effector.

[0150] In one embodiment, the single large effector protein is greater than 900 or greater than 1100 amino acids in length, or comprises at least one HEPN domain.

[0151] In one embodiment, at least one HEPN domain is near the N- or C-terminus of the effector protein, or is located at an internal location of the effector protein.

[0152] In one embodiment, a single large effector protein comprises HEPN domains at the N- and C-termini of the protein, and two HEPN domains within the protein.

[0153] In one embodiment, the identified loci also comprise one or two putative small accessory proteins within 2 kb to 10 kb of the CRISPR array.

[0154] In one embodiment, the small accessory protein is less than 700 amino acids. In one embodiment, the small accessory protein is 50 to 300 amino acids in length.

[0155] In one embodiment, the small accessory protein comprises multiple predicted transmembrane domains, or comprises four predicted transmembrane domains, or comprises at least one HEPN domain.

[0156] In one embodiment, the small accessory protein comprises at least one HEPN domain and at least one transmembrane domain.

[0157] In one embodiment, the locus does not comprise additional protein beyond 25 kb from the CRISPR array.

[0158] In one embodiment, the CRISPR array comprises a direct repeat sequence comprising about 36 nucleotides in length. In a specific embodiment, the direct repeat comprises GTTG / GUUG at the 5' end, which is reverse complementary to CAAC at the 3' end.

[0159] In one embodiment, the CRISPR array comprises a spacer sequence comprising about 30 nucleotides in length.

[0160] In one embodiment, the identified locus lacks a small accessory protein.

[0161] The present invention provides a method for identifying novel CRISPR effectors, comprising: a) identifying sequences in a genomic or metagenomic database encoding a CRISPR array; b) identifying one or more open reading frames (ORFs) in said selected sequences within 10 kb of the CRISPR array; c) selecting loci based on the presence of putative CRISPR effector proteins with a size between 900-1800 amino acids, d) selecting loci encoding putative accessory proteins of 50-300 amino acids; and e) identifying loci encoding putative CRISPR effectors and CRISPR accessory proteins, and optionally classifying them based on structural analysis.

[0162] In one embodiment, the CRISPR effector is a Type VI CRISPR effector. In one embodiment, step (a) comprises i) comparing sequences in a genomic and / or metagenomic database with at least one pre-identified seed sequence encoding a CRISPR array and selecting a sequence comprising the seed sequence; or ii) identifying the CRISPR array based on a CRISPR algorithm.

[0163] In one embodiment, step (d) comprises identifying a nuclease domain. In one embodiment, step (d) comprises identifying a RuvC, HPN and / or HEPN domain.

[0164] In one embodiment, there are no ORFs encoding Cas1 or Cas2 within 10 kb of the CRISPR array.

[0165] In one embodiment, the ORF in step (b) encodes a putative accessory protein of 50-300 amino acids.

[0166] In one embodiment, the putative novel CRISPR effector obtained in step (d) is used as a seed sequence for further comparison of genomic and / or metagenomic sequences and subsequent selection of target loci as described in steps a) to d) of claim 1. In one embodiment, the pre-identified seed sequence is obtained by a method comprising: (a) identifying CRISPR motifs in a genomic or metagenomic database, (b) extracting multiple features in the identified CRISPR motifs, (c) classifying CRISPR loci using unsupervised learning, (d) identifying conserved locus elements based on the classification, and (e) selecting putative CRISPR effectors suitable as seed sequences therefrom.

[0167] In one embodiment, features include protein elements, repeat structures, repeat sequences, spacer sequences, and spacer maps. In one embodiment, the genome and metagenomic databases are bacterial and / or archaeal genomes. In one embodiment, the genome and metagenomic sequences are obtained from the Ensembl and / or NCBI genome databases. In one embodiment, the structural analysis in step (d) is based on secondary structure prediction and / or sequence alignment. In one embodiment, step (d) is achieved by clustering the proteins encoded by the remaining loci and performing manual curation on the obtained clusters.

[0168] Therefore, it is not an object of the present invention to include in the present invention any previously known product, process for making the product, or method of using the product, and the applicant reserves the right and hereby discloses a disclaimer of any previously known product, process, or method. It should also be noted that the present invention is not intended to include within the scope of the present invention any product, process, or method of making a product or method of using a product that does not comply with the written description and enabling requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), and the applicant reserves the right and hereby discloses a disclaimer of any previously described product, process for making a product, or method of using a product. In the practice of the present invention, it may be advantageous to comply with Article 53(c) of the EPC and Articles 28(b) and (c) of the EPC Rules. Nothing herein shall be construed as a promise.

[0169] It should be noted that in this disclosure, and particularly in the claims and / or paragraphs, terms such as “comprises,” “comprised,” “comprising,” and the like may have the meanings assigned to them by U.S. patent law; for example, they may refer to “includes,” “included,” “including,” and the like; and terms such as “consisting essentially of” and “consists essentially of” have the meanings assigned to them by U.S. patent law, for example, they allow for elements not expressly listed, but exclude elements that exist in the prior art or that affect the basic or novel characteristics of the invention.

[0170] These and other embodiments are disclosed or are obvious from and encompassed by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0171] The novel features of the invention are particularly pointed out in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and the accompanying drawings which set forth illustrative embodiments in which the principles of the invention are utilized:

[0172] Figure 1A to Figure 1B A tree alignment of Cas13b orthologs is shown.

[0173] FIG. 2A to FIG. 2C A tree alignment of C2c2 and Cas13b orthologs is shown.

[0174] Figure 3 Exemplary results of testing the activity of Cas13b orthologs in E. coli are shown, whereby the introduction of Cas13b from B. zoohelcum was compared to the introduction of an empty vector.

[0175] Figure 4 A general comparison of the specific RNA cleavage activities obtained using the different orthologs of Table 1 is shown.

[0176] Figure 5 An alignment of different Cas13b orthologs as provided in Figures 1 and 2 is shown.

[0177] FIG. 6A to FIG. 6B (A) shows the original spacer sequence design of the MS2 phage drop plaque assay for testing RNA interference and identifying PFS. (B) shows a schematic diagram of RNA interference assay. The target sequence is placed in a frame at the start position of the transcription bla gene that confers ampicillin resistance, or in a non-transcribed region on the opposite strand of the same target plasmid. The target plasmid is co-transformed with a bzCas13b plasmid or an empty vector that confers chloramphenicol resistance and is plated on a double selection antibiotic plate. Exhausted colonies are identified, and the corresponding targets are sequenced to identify PFS.

[0178] Figure 7 Shown is a heat map of normalized PFS scores for safely depleted spacers from orthologs 1, 13, and 16 in the absence and presence of the csx27 accessory protein.

[0179] Figure 8 Shown is a heat map of normalized PFS scores for safely depleted spacers from orthologs 2, 3, 8, 9, 14, 19, and 21 in the absence and presence of the csx28 accessory protein. Fig. 9 A heat map of normalized PFS scores for safe-depletion spacers from orthologs 5, 6, 7, 10, 12, and 15 is shown.

[0180] FIG. 10A to FIG. 10BBShown are heat maps of normalized PFS scores and derived PFS of safely depleted spacers from different orthologs.

[0181] Fig.11 An overview of luciferase interference data for Cas13b orthologs with lower activity in mammalian cells using the tested guide sequences is provided.

[0182] Fig.12 An overview of luciferase interference data for Cas13b orthologs that showed low to moderate activity in mammalian cells using the tested guide sequences is provided.

[0183] Fig.13 An overview of luciferase interference data for Cas13b orthologs that showed significant activity in mammalian cells using the tested guide sequences is provided.

[0184] FIG. 14A to FIG. 14B An overview of luciferase interference data for some Cas13b orthologs that showed significant activity in mammalian cells using the tested guide sequences is provided.

[0185] Fig.15 An overview of luciferase interference data for Cas13b orthologs that showed significant activity in mammalian cells using the tested guide sequences and comparison with C2c2 activity is provided.

[0186] Fig.16 Composite data for orthologs with significant activity in eukaryotic cells using the same guide sequence are shown.

[0187] FIG. 17A to FIG. 17G Shown are the paralogous effects of Cas13b orthologs in mammalian cells using two different reporter genes (G-luciferase and C-luciferase).

[0188] Fig.18Shown is the characterization of high activity Cas13b orthologs knocked down for RNA.A) Schematic diagram of conventional Cas13 locus and corresponding crRNA structure.B) Luciferase knockdown of 19 Cas13a, 15 Cas13b and 7 Cas13c orthologs is evaluated using two different guide sequences.The orthologs marked with two guide sequences that are effectively knocked down are marked with their host organism names.C) PspCas13b and LwaCas13a are compared to knock down activity by tiling guide sequences for Gluc and measuring luciferase expression.D) PspCas13b and LwaCas13a are compared to knock down activity by tiling guide sequences for Clue and measuring luciferase expression.E) Expression levels of all genes detected in the RNA-seq library of non-targeted control (x-axis) contrast Gluc targeting conditions (y-axis) in log2 (transcripts per million (TPM)) values. Shown is the average of three biological replicates. Gluc transcript data points are marked. F) Expression levels of all genes detected in the RNA-seq library of non-targeted control (x-axis) versus Gluc targeting condition (y-axis) for PspCas13b (blue) and shRNA (black) in log2 (transcripts per million (TPM)) values. Shown is the average of three biological replicates. Gluc transcript data points are marked. G) According to the transcriptome-wide analysis in E and F, the number of significant off-targets was knocked down from LwaCas13a, PspCas13b and shRNA Gluc.

[0189] Fig.19 Engineered dCas13b-ADAR fusions for RNA editing are shown. A) Schematic diagram of RNA editing by dCas13b-ADAR fusion protein. B) Schematic diagram of Cypridina luciferase W85X target and targeting guide sequence design. C) Quantification of luciferase activity recovery using Cas13b-dADAR1 (left) and Cas13b-ADAR2-cd (right) of tiled guide sequences of 30, 50, 70 or 84nt in length. D) Schematic diagram of the target site for targeting Cypridina luciferase W85X. E) Sequencing quantification of A->I editing of 50nt guide sequences targeting Cypridina luciferase W85X.

[0190] Fig. 20Measured sequence flexibility for RNA editing by REPAIRv1 is shown. A) Schematic diagram of screening to determine the protospacer flanking site (PFS) preference for RNA editing by REPAIRv1. B) Distribution of RNA editing efficiency for all 4-N PFS combinations at two different editing sites. C) Quantification of the editing percentage of REPAIRv1 at Clue W85 among all possible 3-base motifs. D) Heat map of 5' and 3' base preferences for RNA editing at Clue W85 for all possible 3-base motifs.

[0191] Fig.21 Correction of disease-associated mutations using REPAIRv1 is shown. A) Schematic diagram of target and guide sequence design for targeting AVPR2 'N / '&G>N. B) 878G>A mutation in AVPR2 was corrected at different percentages using REPAIRv1 with three different guide sequence designs. C) Schematic diagram of target and guide sequence design for targeting FANCC 1517G>A. D) 1517G>A mutation in FANCC was corrected at different percentages using REPAIRv1 with three different guide sequence designs. E) Quantification of editing percentages of 34 different disease-associated G>A mutations using REPAIRv1. F) Analysis of all possible G>A mutations that can be corrected as annotated by the ClinVar database. G) The distribution of editing motifs for all G>A mutations in ClinVar is shown relative to the editing efficiency of REPAIRv1 per motif as quantified on Gluc transcripts.

[0192] Fig. 22 Characterization specificity of REPAIRv1 is shown. A) Schematic diagram of KRAS target site and guide sequence design. B) Quantification of editing percentages of tiled A7MS'-targeted guide sequences. Editing percentages are shown at on-target and nearby adenylate sites. For each guide sequence, the region of duplex RNA is represented by a red rectangle. C) Transcriptome-wide important RNA editing sites through REPAIRv1 with Clue-targeted guide sequences. On-target Clue sites (254A>G) are highlighted in orange. D) Transcriptome-wide important RNA editing sites through REPAIRv1 with non-targeted guide sequences.

[0193] Fig.23Rational mutagenesis of ADAR2 to improve the specificity of REPAIRv1 is shown. A) Quantification and specificity scores of luciferase signal recovery by various dCas13-ADAR2 mutants drawn along the contact schematic diagram between key ADAR2 deaminase residues and dsRNA targets. Specificity score is defined as the ratio of luciferase signal between targeted guide sequence and non-targeted guide sequence conditions. B) Quantification and specificity scores of luciferase signal recovery by various dCas13-ADAR2 mutants. C) The target editing score and the number of significant off-targets of each dCas13-ADAR2 mutant are measured by transcriptome-wide sequencing of mRNA. D) Important RNA editing sites of the transcriptome range of REPAIRv1 and REPAIRv2 are used to target the pre-termination site in Clue. The on-target Clue site (254A>G) is highlighted in orange. E) RNA sequencing reads around the on-target Clue editing site (254A>G) highlight the difference of off-target editing between REPAIRv1 and REPAIRv2. All A>G edits are highlighted in red, while sequencing errors are highlighted in blue. F) RNA editing by REPAIRv1 and REPAIRv2 using guide sequences targeting out-of-frame UAG sites in endogenous KRAS and PPIB transcripts. On-target editing scores are shown as horizontal bars to the right of each condition row. The duplex region formed by the guide RNA is shown by a red outline box.

[0194] Fig.24 Bacterial screening and PFS determination of Cas13b orthologs for in vivo efficiency are shown. A) Schematic diagram of bacterial assays for determining the PFS of Cas13b orthologs. Cas13b orthologs with β-lactamase targeting spacers are co-transformed with β-lactamase expression plasmids and double selected. B) Quantification of the interference activity of Cas13b orthologs targeting β-lactamase as measured by colony forming units (cfu). C) PFS identification of Cas13b orthologs as determined from bacterial assays by depletion sequences.

[0195] Fig.25Optimization and further characterization of mismatch specificity of Cas13b knockdown are shown. A) The first 2 Cas13a and the first 4 Cas13b orthologs fused with various nuclear localization and nuclear export tags are used to measure the Gluc knockdown using two different guide sequences. B) KRAS knockdown is measured for LwaCas13a, RanCas13b, PguCas13b and PspCas13b with four different guide sequences, and compared with shRNA controls matched with four positions. C) Schematic diagram of single mismatch and double mismatch plasmid libraries for evaluating the specificity of LwaCas13a and PspCas13b knockdown. Each possible single mismatch and double mismatch is present in the target sequence and in 3 positions flanking the 5' end and 3' end of the target site. D) The exhaustion level of the transcript with the indicated single mismatch is plotted as a heat map of LwaCas13a and PspCas13b conditions. E) Depletion levels of transcripts with the indicated double mismatches are plotted as a heat map for LwaCas13a and PspCas13b conditions.

[0196] Fig.26 Characterization of design parameters for dCas13-ADAR2 RNA editing is shown. A) Knockdown efficiency of Gluc targeting wild-type Cas13b and catalytically inactive H133A / H1058A Cas13b (dCas13b). B) Quantification of luciferase activity recovery tested by tiling Clue targeting guide sequence by dCas13b fused to wild-type ADAR2 catalytic domain or highly active E488Q mutant ADAR2 catalytic domain. C) Guide sequence design and sequencing quantification of A->I editing of 30nt guide sequence targeting sea firefly luciferase W85X. D) Guide sequence design and sequencing quantification of A->I editing of 50nt guide sequence targeting PPIB. E) Effect of linker selection on luciferase activity recovery of REPAIRv1. F) Effect of base recognition opposite to targeting adenylate on luciferase activity recovery of REPAIRv1.

[0197] Fig. 27 The clinVar motif distribution of G>A mutations is shown. The number of each possible triplet motif observed in the ClinVar database for all G>A mutations.

[0198] FIG. 28A to FIG. 28B RNA binding by dCas13b truncations is shown. Various N-terminal and C-terminal truncations of dCas13b are depicted. RNA binding is indicated in the presence of ADAR-dependent RNA editing, as measured by recovery of luciferase signal, comparing the activity using targeted and non-targeted guide sequences. Amino acid positions correspond to those of the Prevotella species P5-125 Cas13b protein.

[0199] Fig.29 Comparison of other programmable ADAR systems with the dCas13-ADAR2 editor is shown. A) Schematic diagram of two programmable ADAR schemes: BoxB-based targeting and full-length ADAR2 targeting. In the BoxB scheme (top), the ADAR2 deaminase domain (ADAR2 DD (E488Q)) is fused to a small bacterial viral protein called λN(AN), which specifically binds to a small RNA sequence called BoxB-A. A guide RNA containing two BoxB-A hairpins can then guide ADAR2 DD (E488Q),-XN for site-specific editing. In the full-length ADAR2 scheme (bottom), the dsRNA binding domain of ADAR2 binds to the hairpin in the guide RNA, allowing programmable ADAR2 editing. B) Using the Clue-targeting guide sequence and the non-targeting guide sequence through BoxB-ADAR2 DD (E488Q). The on-target Clue site (254A>G) is highlighted in orange. C) Transcriptome-wide important RNA editing sites by ADAR2 using guide sequences targeting Clue and non-targeting guide sequences. The on-target Clue site (254A>G) is highlighted in orange. D) Transcriptome-wide important RNA editing sites by REPAIRv1 using guide sequences targeting Clue and non-targeting guide sequences. The on-target Clue site (254A>G) is highlighted in orange. E) For the targeting guide sequence against Cluc, BoxB-ADAR2 DD Quantification of the percentage of on-target editing for (E488Q), ADAR2, and REPAIRv1. F) Overlap of off-target sites between different targeting and non-targeting conditions of the programmable ADAR system.

[0200] Fig.30 The efficiency and specificity of dCas13b-ADAR2 mutants are shown. A) Clue-targeted and non-targeted guide sequences by dCas13b-ADAR2 DD Quantification of the restoration of luciferase activity in the (E488Q) mutant. B)

[0201] Relationship between the ratio of on-target and non-targeted guide sequences and the number of RNA editing off-targets, as quantified by transcriptome-wide sequencing. C) For dCas13b-ADAR2 DD (E488Q) mutant, quantification of transcriptome-wide off-target RNA editing sites and on-target Clue editing efficiency.

[0202] Fig.31dCas13b-ADAR2 is shown DD Transcriptome-wide specificity of RNA editing of the (E488Q) mutant. A) Using a Clue-targeted guide sequence to target the target gene via dCas13b-ADAR2 DD Transcriptome-wide significant RNA editing sites in the (E488Q) mutant. The on-target Clue site (254A>G) is highlighted in orange. B) Using a non-targeting guide sequence to modify RNA editing via dCas13b-ADAR2 DD Transcriptome-wide essential RNA editing sites in the (E488Q) mutant.

[0203] Fig.32 Shown in dCas13b-ADAR2 DD Characterization of motif bias in off-target (E488Q) editing. A) For each dCas13b-ADAR2 DD (E488Q) mutant, showing the motif present in all A>G off-target edits in the transcriptome. B) For REPAIRv1 using targeted and non-targeted guide sequences, the distribution of off-target A>G edits per motif identity is shown. C) For REPAIRv2 using targeted and non-targeted guide sequences, the distribution of off-target A>G edits per motif identity is shown.

[0204] Fig.33 Further characterization of REPAIRv1 and REPAIRv2 off-targets is shown. A) Histogram of the number of off-targets per transcript for REPAIRv1. B) Histogram of the number of off-targets per transcript for REPAIRv2. C) Variant effect predictions for REPAIRv1 off-targets. D) Distribution of potential oncogenic effects of REPAIRv1 off-targets. E) Variant effect predictions for REPAIRv2 off-targets. F) Distribution of potential oncogenic effects of REPAIRv2 off-targets.

[0205] Fig.34 RNA editing efficiency and specificity of REPAIRv1 and REPAIRv2 are shown. A) Quantification of the editing percentage of KRAS using AXES'-targeting guide sequence 1 at targeting adenylate and adjacent sites for REPAIRv1 and REPAIRv2. B) Quantification of the editing percentage of KRAS using AXAS'-targeting guide sequence 3 at targeting adenylate and adjacent sites for REPAIRv1 and REPAIRv2. C) Quantification of the editing percentage of PPIB using / X / 7>-targeting guide sequence 2 at targeting adenylate and adjacent sites for REPAIRv1 and REPAIRv2.

[0206] Fig.35A display of all potential codon changes using the A>G RNA editor is shown. A) Table of all potential codon conversions enabled by A>I editing. B) Codon table showing all potential codon conversions enabled by A>I editing. C)

[0207] FIG. 36A to FIG. 36B The effect of Csx proteins on RNA interference is shown. A) Comparison of Cas13b and Cas13b+Csx27; B) Comparison of Cas13b and Cas13b+Csx28.

[0208] FIG. 37A to FIG. 37F Transcript knockdown by Cas13b and Cas13b+Csx28 was compared via luciferase assay. A) Pin Cas13b (WP_036860899); B) Pbu Cas13b (WP_004343973); C) Rin Cas13b (WP_004919755); D) Pau Cas13b (WP025000926); E) Pgu Cas13b (WP_039434803); F) Pig Cas13b (WP_053444417)

[0209] The drawings herein are for illustration purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION

[0210] In general, CRISPR-Cas or CRISPR systems collectively refer to transcripts and other elements involved in the expression of CRISPR-associated ("Cas") genes or directing the activity of the genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-pairing sequences (in the case of endogenous CRISPR systems, "direct repeats" and tracrRNA-processed partial direct repeats are covered), guide sequences (also referred to as "spacers" in the case of endogenous CRISPR systems), or as used herein, "one or more RNAs" (e.g., one or more RNAs to guide Cas (such as Cas9), e.g., CRISPR RNA and trans-activating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)), or other sequences and transcripts from the CRISPR locus. In general, CRISPR systems are characterized by elements (also referred to as protospacers in the case of endogenous CRISPR systems) that promote the formation of CRISPR complexes at target sequence sites. When the CRISPR protein is a Class 2 VI-B type effector, tracrRNA is not required. In the engineered system of the present invention, the forward repeat may cover a naturally occurring sequence or a non-naturally occurring sequence. The forward repeat of the present invention is not limited to the length and sequence of the naturally occurring sequence. The length of the forward repeat may be 36nt, but the forward repeat may be varied to be longer or shorter. For example, the forward repeat may be 30nt or longer, such as 30-100nt or longer. For example, the length of the forward repeat may be 30nt, 40nt, 50nt, 60nt, 70nt, 70nt, 80nt, 90nt, 100nt or longer. In some embodiments, the forward repeat of the present invention may include a synthetic nucleotide sequence inserted between the 5' end and the 3' end of the naturally occurring forward repeat sequence. In certain embodiments, the inserted sequence may be self-complementary, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% self-complementary.

[0211] In addition, the direct repeats of the present invention may include nucleotide insertions, such as aptamers or sequences that bind to adapter proteins (so as to associate with functional domains). In certain embodiments, one end of the direct repeats comprising such insertions is approximately the first half of the short DR, and the end is approximately the second half of the short DR.

[0212] In the case of forming a CRISPR complex, a "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, forward repeats may be identified via in silico by searching for repeat motifs that meet any or all of the following criteria: 1. Present in a 2Kb window of the genomic sequence flanking the type II CRISPR locus; 2. A span of 20 to 50 bp; and 3. An interval of 20 to 50 bp. In some embodiments, 2 of these criteria may be used, such as 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.

[0213] In embodiments of the present invention, the terms guide sequence and guide RNA, i.e., RNA capable of guiding the Cas13b effector protein to the target locus, can be used interchangeably as in documents cited herein such as WO 2014 / 093622 (PCT / US2013 / 074667). Typically, a guide sequence (or spacer sequence) is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and guide sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, when optimally aligned using a suitable alignment algorithm, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or greater. Optimal alignment can be determined with the aid of 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 www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the length of the guide sequence (or spacer sequence) is about or greater than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides. In some embodiments, the length of the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides. Preferably, the guide sequence is 10-40 nucleotides long, such as 20-30 or 20-40 nucleotides long or longer, such as 30 nucleotides long or about 30 nucleotides long. In certain embodiments, for Cas13b effectors, the guide sequence is 10-30 nucleotides long, such as 20-30 or 20-40 nucleotides long or longer, such as 30 nucleotides long or about 30 nucleotides long. In certain embodiments, the guide sequence is 10-30 nucleotides long, such as 20-30 nucleotides long, such as 30 nucleotides long.The ability of the guide sequence to guide the sequence-specific binding of the CRISPR complex to the target sequence can be evaluated by any suitable assay. For example, components of the CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, can be provided to a host cell with the corresponding target sequence, such as by transfection with a vector encoding the components of the CRISPR sequence, and then the preferential cleavage within the target sequence is assessed, such as by the Surveyor assay as described herein. Similarly, the cleavage of the target polynucleotide sequence can be evaluated in a test tube by providing a target sequence, components of the CRISPR complex (including a guide sequence to be tested and a control guide sequence different from the test guide sequence), and comparing the binding or cleavage rate at the target sequence between the test and control guide sequence reactions. Other assays are possible and will be thought of by those skilled in the art.

[0214] The present invention provides specific Cas13b effectors, nucleic acids, systems, vectors and methods of use. All VI-Bs are distinguishable from VI-A (Cas13b) by structure and also by the position of the HEPN domain. It is almost impossible to separate the Cas13b lacking a small auxiliary protein from the Cas13b with a small auxiliary protein, except that the VI-B2 locus appears to have a more conservative small auxiliary protein.

[0215] As used herein, the terms Cas13b-sl accessory protein, Cas13b-sl protein, Cas13b-sl, Csx27, and Csx27 protein are used interchangeably, and the terms Cas13b-s2 accessory protein, Cas13b-s2 protein, Cas13b-S2, Csx28, and Csx28 protein are used interchangeably.

[0216] In a classical CRISPR-Cas system, the degree of complementarity between a guide sequence and its corresponding target sequence can be about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%; the guide or RNA or crRNA can be about or greater than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length; or the guide or RNA or crRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or fewer nucleotides in length; and advantageously the tracr RNA is 30 or 50 nucleotides in length. However, one aspect of the present invention is to reduce off-target interactions, for example, to reduce the interaction of the guide with a target sequence having low complementarity. Indeed, in the examples shown, the present invention relates to mutations that enable the CRISPR-Cas system to distinguish between a target sequence and an off-target sequence having greater than 80% to about 95% complementarity, such as 83%-84% or 88-89% or 94-95% complementarity (e.g., distinguishing a target with 18 nucleotides from an off-target with 1, 2 or 3 mismatches). Therefore, in the context of the present invention, the degree of complementarity between the guide sequence and its corresponding target sequence is greater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or 99% or 99.5% or 99.9%, or 100%. The off-target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% of the complementarity between the sequence and the guide, advantageously the off-target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% of the complementarity between the sequence and the guide.

[0217] In a particularly preferred embodiment according to the present invention, the guide RNA (capable of guiding Cas to the target locus) may comprise (1) a guide sequence capable of hybridizing to a target locus (a polynucleotide target locus, such as an RNA target locus) in a eukaryotic cell; (2) a direct repeat (DR) sequence) located in a single RNA, i.e., sgRNA (arranged in a 5' to 3' orientation) or crRNA.

[0218] In certain embodiments, the wild-type Cas13b effector protein has RNA binding and cleavage functions.

[0219] In certain embodiments, the Cas13b effector protein may have a DNA cutting function. In these embodiments, a method may be provided based on the effector protein provided herein, the method comprising inducing one or more mutations in a eukaryotic cell as discussed herein (in vitro, i.e., in an isolated eukaryotic cell), the method comprising delivering a vector as discussed herein to a cell. One or more mutations may include the introduction, deletion or replacement of one or more nucleotides at each target sequence of a cell via one or more guide RNAs or sgRNAs or crRNAs. Mutations may include the introduction, deletion or replacement of 1-75 nucleotides at each target sequence of the one or more cells via one or more guide RNAs or sgRNAs or crRNAs. Mutations may include the introduction, deletion or replacement of 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or 75 nucleotides at each target sequence of the one or more cells via one or more guide RNAs or sgRNAs or crRNAs. The mutation may include the introduction, deletion or substitution of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or 75 nucleotides at each target sequence of the one or more cells via one or more guide RNAs or sgRNAs or crRNAs. The mutation includes the introduction, deletion or substitution of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or 75 nucleotides at each target sequence of the one or more cells via one or more guide RNAs or sgRNAs or crRNAs. The mutation may include the introduction, deletion or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or 75 nucleotides at each target sequence of the one or more cells via one or more guide RNAs or sgRNAs or crRNAs. The mutation may include the introduction, deletion or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides at each target sequence of the one or more cells via one or more guide RNAs or sgRNAs or crRNAs.

[0220] In order to minimize toxicity and off-target effects, it will be important to control the concentration of the Cas13bmRNA and guide RNA delivered. The optimal concentration of Cas13b mRNA and guide RNA can be determined by testing different concentrations in cells or non-human eukaryotic animal models and using the modification degree of the potential off-target genomic locus for deep sequencing analysis. Alternatively, in order to minimize toxicity levels and off-target effects, Cas13b nickase mRNA (such as Streptococcus pyogenes Cas9 with D10A mutation) can be delivered together with a pair of guide RNAs targeting sites of interest. Guidance sequences and strategies for minimizing toxicity and off-target effects can be as in WO 2014 / 093622 (PCT / US2013 / 074667); or by mutations as herein.

[0221] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands (if applicable) in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from) the target sequence.

[0222] The nucleic acid molecule encoding Cas13b is advantageously codon-optimized. In this case, an example of a codon-optimized sequence is a sequence optimized for expression in a eukaryote (e.g., human (i.e., optimized for expression in humans), or for another eukaryote, animal, or mammal as discussed herein; see, for example, WO 2014 / 093622 (PCT / US2013 / 074667) SaCas9 human codon-optimized sequence. Although this is preferred, it should be understood that other examples are possible, and codon optimization for host species other than humans or for codon optimization for specific organs is known. In some embodiments, the enzyme coding sequence encoding Cas is codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells can be those of a specific organism or derived from a specific organism, such as a mammal, including but not limited to humans, or non-human eukaryotes or animals or mammals as discussed herein, such as mice, rats, rabbits, dogs, livestock, or non-human mammals or primates. In some embodiments, the process of modifying the germline genetic identity of humans and / or the process of modifying the genetic identity of animals, which may not bring any substantial medical benefits to humans or animals, and the animals produced by such processes can be excluded. In general, codon optimization refers to the process of modifying nucleic acid sequences for enhancing expression in host cells of interest, which is by replacing at least one codon (e.g., about or greater than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. Each species exhibits a specific bias to certain codons of specific amino acids. Codon bias (differences in codon usage between organisms) is often related to the translation efficiency of messenger RNA (mRNA), and it is believed that the efficiency then depends in particular on the properties of the codons translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of the selected tRNA in the cell generally reflects the most frequently used codons in peptide synthesis. Therefore, genes can be adjusted based on codon optimization for optimal gene expression in a given organism. Codon usage tables are readily available, for example, in the "Codon Usage Database" available at www.kazusa.orjp / codon / , and these tables can be adapted in many 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 for codon optimization of specific sequences for expression in specific host cells are also available, such as 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 Cas correspond to the most frequently used codons for a specific amino acid.

[0223] In certain embodiments, the method as described herein may include providing a Cas13b transgenic cell, in which one or more nucleic acids encoding one or more guide RNAs are provided or introduced, and the one or more nucleic acids are operably connected to a regulatory element of a promoter comprising one or more genes of interest in the cell. The term "Cas13b transgenic cell" as used herein refers to such a cell, such as a eukaryotic cell, in which the Cas13b gene has been integrated on the genome. The nature, type or source of the cell is not particularly limited according to the present invention. Moreover, the manner in which the Cas13b transgenic is introduced into the cell may vary and may be any method as known in the art. In certain embodiments, the Cas13b transgenic cell is obtained by introducing the Cas13b transgenic into a separated cell. In certain other embodiments, the Cas13b transgenic cell is obtained by separating cells from a Cas13b transgenic organism. For example, but not limited to, the Cas13b transgenic cell as mentioned herein may be derived from a Cas13b transgenic eukaryotic organism, such as a Cas13b knock-in eukaryotic organism. Reference is made to WO 2014 / 093622 (PCT / US13 / 74667), which is incorporated herein by reference. The methods of U.S. Patent Publication Nos. 20120017290 and 20110265198 assigned to Sangamo BioSciences, Inc. for targeting the Rosa locus can be modified to utilize the CRISPR Cas system of the present invention. The methods of U.S. Patent Publication No. 20130236946 assigned to Cellectis for targeting the Rosa locus can also be modified to utilize the CRISPR Cas system of the present invention. By way of another example, reference is made to Platt et al. (Cell; 159(2):440-455(2014)), which describes Cas9 knock-in mice, which are incorporated herein by reference. The Cas13b transgene can also include a Lox-Stop-polyA-Lox (LSL) box, thereby facilitating Cas13b expression inducible by the Cre recombinase. Alternatively, Cas13b transgenic cells can be obtained by introducing the Cas13b transgene into isolated cells. Delivery systems for transgenics are well known in the art. For example, the Cas13b transgene can be delivered in, for example, eukaryotic cells by means of vectors (e.g., AAV, adenovirus, slow virus) and / or particles and / or particles as described elsewhere herein.

[0224] The skilled person will understand that cells such as Cas13b transgenic cells as referred to herein may contain genomic alterations, such as one or more oncogenic mutations, in addition to an integrated Cas13b gene or mutations resulting from the sequence-specific action of Cas13b when complexed with RNA capable of guiding Cas13b to the target locus, for example but not limited to those described in Platt et al. (2014), Chen et al. (2014) or Kumar et al. (2009).

[0225] In some embodiments, Cas13b sequence is fused with one or more nuclear localization sequences (NLS), such as fused with about or more than about 1,2,3,4,5,6,7,8,9,10 or more NLS.In some embodiments, Cas13b comprises about or more than about 1,2,3,4,5,6,7,8,9,10 or more NLS at or near the amino terminus, comprises about or more than about 1,2,3,4,5,6,7,8,9,10 or more NLS at or near the carboxyl terminus, or these combinations (for example, zero or at least one or more NLS at the amino terminus, and zero or one or more NLS at the carboxyl terminus).When there is more than one NLS, each NLS can be selected independently of one another so that a single NLS can exist more than one copy and / or exist in combination with one or more copies with one or more other NLS.In a preferred embodiment of the present invention, Cas13b comprises up to 6 NLS. In some embodiments, an NLS is considered to be proximal to the N-terminus or C-terminus when the proximal amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N-terminus or C-terminus.Non-limiting examples of NLSs include NLS sequences derived from: the NLS of the SV40 virus large T antigen, which has the amino acid sequence PKKKRKV (SEQ ID NO: X); an NLS from a nucleoplasmin (e.g., a nucleoplasmin bipartite NLS, which has the sequence KRPAATKKAGQAKKKK) (SEQ ID NO: X); a c-myc NLS, which has the amino acid sequence PAAKRVKLD (SEQ ID NO: X) or RQRRNELKRSP (SEQ ID NO: X); an hRNPAl M9 NLS, which has the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: X); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: X) from the IBB domain of importin-α; the sequences VSRKRPRP (SEQ ID NO: X) and PPKKARE D (SEQ ID NO: X) of myoma T protein; ID NO: X); the sequence of human p53 POPKKKPL (SEQ ID NO: X); the sequence of mouse c-abl IV SALIKKKKKMAP (SEQ ID NO: X); the sequences of influenza virus NS1 DRLRR (SEQ ID NO: X) and PKQKKRK (SEQ ID NO: X); the sequence of hepatitis virus delta antigen RKLKKKIKKL (SEQ ID NO: X); the sequence of mouse Mxl protein REKKKFLKRR (SEQ ID NO: X); the sequence of human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: X); and the sequence of steroid hormone receptor (human) glucocorticoid RKCLQAGMNLEARKTKK (SEQ ID NO: X). Typically, one or more NLSs have sufficient strength to drive Cas to accumulate in the nucleus of eukaryotic cells in a detectable amount. Typically, the strength of nuclear localization activity can be derived from the number of NLSs in Cas, the specific NLS used, or a combination of these factors. Detection of accumulation in the nucleus can be performed by any suitable technique. For example, a detectable marker can be fused to Cas to visualize the position within the cell, such as in combination with a means for detecting the position of the nucleus (e.g., a stain specific to the nucleus, such as DAPI). The nucleus can also be isolated from the cell, and the contents of the nucleus can then be analyzed by any suitable method for detecting proteins, such as immunohistochemistry, Western blot, or enzyme activity assay.Nuclear accumulation can also be determined indirectly, such as by an assay for the effect of CRISPR complex formation (e.g., an assay for DNA cleavage or mutation at the target sequence, or an assay for gene expression activity that is altered by CRISPR complex formation and / or Cas enzyme activity) compared to a control not exposed to Cas or the complex or exposed to a Cas lacking the one or more NLSs.

[0226] In certain aspects, the present invention relates to vectors, for example, for delivering or introducing Cas13b and / or RNA (i.e., guide RNA) capable of guiding Cas13b to a target locus in a cell, and also for propagating these components (e.g., in prokaryotic cells). As used herein, "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, a phage, or a cosmid, into which another DNA fragment can be inserted in order to achieve replication of the inserted fragment. Generally, a vector is capable of replication when associated with an appropriate control element. In general, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid already connected thereto. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, not comprising a free end (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other polynucleotide species known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop, into which additional DNA fragments 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 of viral origin 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 the virus for transfection into host cells. Some vectors can replicate autonomously in the host cells into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (e.g., non-additional mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, and are therefore replicated together with the host genome. In addition, some vectors can instruct the expression of genes operatively connected to them. Such vectors are referred to herein as "expression vectors". Expression vectors commonly used in recombinant DNA technology are often in the form of plasmids.

[0227] Recombinant expression vector can comprise nucleic acid of the present invention, and its form is suitable for expressing this nucleic acid in host cell, which means that recombinant expression vector comprises one or more regulating and controlling elements, and described regulating and controlling elements can be selected according to the host cell to be used for expression, and is operably connected with the nucleotide sequence to be expressed.In recombinant expression vector, "operably connected" is intended to represent that the nucleotide sequence of interest is connected with regulating and controlling elements in a manner that allows the expression of this nucleotide sequence (for example, in an in vitro transcription / translation system, or in a host cell when the vector is introduced into a host cell).About recombination and cloning method, with reference to U.S. Patent Application No. 10 / 815,730 announced with US2004-0171156 A1 on September 2, 2004, its content is incorporated herein by reference in its entirety.

[0228] The vector may include a regulatory element, such as a promoter. The vector may include a Cas13b coding sequence and / or a single, but may also include at least 2, 3, 8, 16, 32, 48, or 50 guide RNA (e.g., crRNA) coding sequences, 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 RNA (e.g., crRNA). In a single vector, there may be a promoter for each RNA (e.g., crRNA), advantageously when there are up to about 16 RNAs (e.g., crRNAs); and when a single vector provides more than 16 RNAs (e.g., crRNAs), one or more promoters may drive the expression of more than one of the RNAs (e.g., crRNAs), for example, when there are 32 RNAs (e.g., sgRNAs or crRNAs), each promoter may drive the expression of two RNAs (e.g., sgRNAs or crRNAs), and when there are 48 RNAs (e.g., sgRNAs or crRNAs), each promoter may drive the expression of three RNAs (e.g., sgRNAs or crRNAs). Based on simple algorithms and mature cloning schemes and the teachings of the present disclosure, those skilled in the art can easily implement the present invention for RNAs (e.g., sgRNAs or crRNAs) for suitable exemplary vectors such as AAV and suitable promoters such as U6 promoters (e.g., U6-sgRNAs or -crRNAs). For example, the packaging of AAV is limited to A skilled person can easily assemble 12-16 (e.g., 13) U6-sgRNA or crRNA boxes in a single vector. This can be assembled by any suitable means, such as by the golden gate strategy (http: / / www.genome-engineering.org / taleffectors / ) for TALE assembly. A skilled person can also use a tandem guide strategy to increase the number of U6-sgRNA or -crRNA by about 1.5 times, for example, from 12-16 (e.g., 13) to about 18-24 (e.g., about 19) U6-sgRNA or -crRNA. Therefore, a skilled person can easily reach about 18-24 (e.g., about 19) promoter-RNAs, such as U6-sgRNA or -crRNA in a single vector (e.g., AAV vector). A further method to increase the number of promoters and RNAs (e.g., sgRNAs or crRNAs) in a vector is to use a single promoter (e.g., U6) to express an array of RNAs (e.g., sgRNAs or crRNAs) separated by a cleavable sequence. And a further method to increase the number of promoter-RNAs (e.g., sgRNAs or crRNAs) in a vector is to express an array of promoter-RNAs (e.g., sgRNAs or crRNAs) separated by a cleavable sequence in an intron of a coding sequence or gene; and in this case, it is advantageous to use a polymerase II promoter, which can increase expression and can transcribe long RNAs in a tissue-specific manner (see, e.g., nar.oxfordjoumals.org / content / 34 / 7 / e53.short, www.nature.com / mt / joumal / vl6 / n9 / abs / mt2008144a.html). In an advantageous embodiment, AAV can package U6 tandem sgRNAs targeting up to about 50 genes. Thus, based on the knowledge in the art and the teachings of the present disclosure, a skilled artisan can readily prepare and use vectors, such as a single vector, expressing multiple RNAs or guides or sgRNAs or crRNAs under control or operably or functionally linked to one or more promoters—particularly with respect to the number of RNAs or guides or sgRNAs or crRNAs discussed herein, without any undue experimentation.

[0229] Guide RNA, such as sgRNA or crRNA coding sequence and / or Cas13b coding sequence, can be functionally or operably connected to regulatory elements, and thus regulatory elements drive expression.Promoter can be a constitutive promoter and / or a conditional promoter and / or an inducible promoter and / or a tissue-specific promoter.Promoter can be selected from 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, P-actin promoter, phosphoglycerol kinase (PGK) promoter and EF1a promoter. Advantageous promoter is that promoter is U6.

[0230] In one aspect of the present invention, the novel RNA targeting system of the present application, also referred to as RNA or RNA-targeted CRISPR system, is based on the Cas13b protein identified herein, which does not require the production of custom proteins to target specific RNA sequences, but rather a single enzyme can be programmed by an RNA molecule to recognize a specific RNA target, in other words, the RNA molecule can be used to recruit the enzyme to a specific RNA target.

[0231] In some embodiments, one or more elements of the nucleic acid targeting system are derived from a specific organism comprising an endogenous CRISPR RNA targeting system. In certain embodiments, CRISPR RNA targeting systems are found in true bacteria and Ruminococcus. In certain embodiments, the effector protein comprises targeted and collateral ssRNA cleavage activity. In certain embodiments, the effector protein comprises a dual HEPN domain. In certain embodiments, the effector protein lacks the corresponding domain of the Helical-1 domain of Cas13a. In certain embodiments, the effector protein is smaller than the previously characterized Class 2 CRISPR effector with a median size of 928aa. This median size is 190aa (17%) smaller than Cas13c, more than 200aa (18%) smaller than Cas13b, and more than 300aa (26%) smaller than Cas13a. In certain embodiments, the effector protein does not require a flanking sequence (e.g., PFS, PAM).

[0232] In certain embodiments, the effector protein locus structure includes a WYL domain containing an accessory protein (thus indicated after the three amino acids conserved in the initial confirmed group of these domains; see, e.g., WYL domain IPR026881). In certain embodiments, the WYL domain accessory protein comprises at least one helix-turn-helix (HTH) or ribbon-helix-helix (RHH) DNA binding domain. In certain embodiments, the WYL domain containing the accessory protein increases both the targeting and side branch ssRNA cleavage activity of the RNA targeting effector protein. In certain embodiments, the WYL domain containing the accessory protein comprises an N-terminal RHH domain, and a pattern of predominantly hydrophobic conserved residues, including an invariant tyrosine leucine pair corresponding to the original WYL motif. In certain embodiments, the WYL domain containing the accessory protein is WYL1. WYL1 is a single WYL domain protein primarily associated with Ruminococcus.

[0233] In other exemplary embodiments, the VI type RNA targeting Cas enzyme is Cas13d. In certain embodiments, Cas13d is Eubacterium inertia DSM 15702 (EsCas13d) or Ruminococcus N15.MGS-57 (RspCas13d) (see, e.g., Yan et al., Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector PositivelyModulated by a WYL-Domain-Containing Accessory Protein, Molecular Cell (2018), doi.org / 10.1016 / j.molcel.2018.02.028). RspCas13d and EsCas13d do not have flanking sequence requirements (e.g., PFS, PAM).

[0234] The nucleic acid targeting system, vector system, vector and composition described in this article can be used for various nucleic acid targeting applications, changing or modifying the synthesis of gene products, such as proteins, nucleic acid cutting, nucleic acid editing, nucleic acid splicing; transport of target nucleic acid, tracking of target nucleic acid, separation of target nucleic acid, visualization of target nucleic acid, etc.

[0235] In advantageous embodiments, the present invention encompasses the Cas13b effector proteins with reference to Table 1. Table 1 Cas13b effector proteins are discussed in more detail herein in conjunction with Table 1.

[0236] Cas13b nuclease

[0237] The Cas13b effector protein of the present invention is such as a protein from or listed in Table 1, or is from or listed in Table 1, or comprises such as a protein from or listed in Table 1, or consists essentially of such as a protein from or listed in Table 1, or consists of such as a protein from or listed in Table 1, or relates to such as a protein from or listed in Table 1, or is related to such as a protein from or listed in Table 1. The present invention is intended to provide a protein from or listed in Table 1, or is related to a protein from or listed in Table 1, or is related to a protein from or listed in Table 1, or comprises a protein from or listed in Table 1, or consists essentially of a protein from or listed in Table 1, or consists of a protein from or listed in Table 1, including mutations or changes thereof as listed herein. Table 1Cas13b effector proteins are discussed in more detail herein in conjunction with Table 1.

[0238] Thus, in some embodiments, the effector protein may be an RNA binding protein, such as a death-Cas type effector protein, which may optionally be functionalized as described herein, for example with a transcriptional activator or repressor domain, an NLS or other functional domain. In some embodiments, the effector protein may be an RNA binding protein that cleaves single-stranded RNA. If the bound RNA is ssRNA, the ssRNA is completely cleaved. In some embodiments, the effector protein may be an RNA binding protein that cleaves double-stranded RNA, for example if it comprises two RNase domains. If the bound RNA is dsRNA, the dsRNA is completely cleaved. In some embodiments, the effector protein may be an RNA binding protein with nickase activity, i.e., it binds to dsRNA but only cleaves one of the RNA strands.

[0239] RNase function in CRISPR systems is known, for example, mRNA targeting of certain type III CRISPR-Cas systems has been reported (Hale et al., 2014, Genes Dev, Vol. 28, 2432-2443; Hale et al., 2009, Cell, Vol. 139, 945-956; Peng et al., 2015, Nucleic acids research, Vol. 43, 406-417), and provides significant advantages. Therefore, a CRISPR-Cas system, composition or method for targeting RNA via the effector protein of the present invention is provided.

[0240] The target RNA, i.e., the RNA of interest, is the RNA to be targeted by the present invention, which causes the effector protein to be recruited to and bind to the target site of interest on the target RNA. The target RNA can be any suitable form of RNA. In some embodiments, this can include mRNA. In other embodiments, the target RNA can include tRNA or rRNA.

[0241] Interfering RNA (RNAi) and microRNA (miRNA)

[0242] In other embodiments, the target RNA may include interfering RNA, i.e., RNA involved in RNA interference pathways, such as shRNA, siRNA, etc. In other embodiments, the target RNA may include microRNA (miRNA). Controlling interfering RNA or miRNA can help reduce off-target effects (OTEs) produced in these methods by reducing the life span of the interfering RNA or miRNA in vivo or in vitro.

[0243] If the effector protein and a suitable guide are selectively expressed (e.g. spatially or temporally under the control of a suitable promoter, such as a tissue or cell cycle specific promoter and / or enhancer), this can be used to "protect" the cell or system (in vivo or in vitro) from RNAi in these cells. This may be useful in neighboring tissues or cells where RNAi is not desired, or for the purpose of comparing cells or tissues in which the effector protein and a suitable guide are and are not expressed (i.e., RNAi is not controlled and is controlled, respectively). The effector protein can be used to control or bind to molecules comprising or consisting of RNA, such as ribozymes, ribosomes or riboswitches. In embodiments of the invention, the RNA guide can recruit the effector protein to these molecules so that the effector protein can bind to them.

[0244] Ribosomal RNA (rRNA)

[0245] For example, azalide antibiotics such as azithromycin are well known. They target and destroy the 50S ribosomal subunit. In some embodiments, the effector protein of the present invention, together with a suitable guide RNA for targeting the 50S ribosomal subunit, can be recruited to the 50S ribosomal subunit and bind to it. Therefore, the present invention provides an effector protein of the present invention coordinated with a suitable guide for a ribosome (particularly a 50s ribosomal subunit) target. The use of this use effector protein coordinated with a suitable guide for a ribosome (particularly a 50s ribosomal subunit) target may include antibiotic use. In particular, antibiotic use is similar to the effect of azalide antibiotics such as azithromycin. In some embodiments, prokaryotic ribosomal subunits can be targeted, such as the 70S subunit in prokaryotes, the 50S subunit mentioned above, the 30S subunit, and the 16S and 5S subunits. In other embodiments, eukaryotic ribosomal subunits can be targeted, such as the 80S subunit, 60S subunit, 40S subunit, and 28S, 18S, 5.8S, and 5S subunits in eukaryotes.

[0246] The effector protein may be an RNA binding protein, optionally functionalized as described herein. In some embodiments, the effector protein may be an RNA binding protein that cleaves single-stranded RNA. In either case, but particularly where the RNA binding protein cleaves single-stranded RNA, ribosomal function may be modulated and particularly reduced or disrupted. This may apply to any ribosomal RNA and any ribosomal subunit, and the sequence of rRNA is well known.

[0247] It is therefore contemplated to control ribosomal activity by using the effector proteins of the invention in combination with appropriate guides to ribosomal targets. This may be done by cleaving or binding to the ribosome. In particular, it is contemplated to reduce ribosomal activity. This may be useful in determining ribosomal function in vivo or in vitro, but also as a means of controlling treatment based on ribosomal activity in vivo or in vitro. In addition, it is contemplated to control (i.e., reduce) protein synthesis in in vivo or in vitro systems, such control including antibiotics and research and diagnostic uses.

[0248] Riboswitch

[0249] A riboswitch (also known as an aptozyme) is a regulatory segment of a messenger RNA molecule that binds to a small molecule. This generally results in a change in the production of a protein encoded by the mRNA. Therefore, it is contemplated that riboswitch activity can be controlled by using an effector protein of the invention in conjunction with a suitable guide to the riboswitch target. This can be done by cutting or binding the riboswitch. In particular, it is contemplated that riboswitch activity is reduced. This may be useful in determining riboswitch function in vivo or in vitro, but also as a means of controlling treatment based on riboswitch activity in vivo or in vitro. In addition, it is contemplated that protein synthesis is controlled (i.e., reduced) in an in vivo or in vitro system. Such control of rRNA may include antibiotics and research and diagnostic uses.

[0250] Ribozyme

[0251] Ribozymes are RNA molecules with catalytic properties similar to enzymes (which are of course proteins). Because ribozymes, whether naturally occurring or engineered, contain or consist of RNA, they can also be targeted by the RNA-binding effector proteins of the present invention. In some embodiments, the effector protein can be an RNA-binding protein that cuts the ribozyme to inactivate it. It is therefore contemplated to control ribozyme activity by using the effector protein of the present invention in conjunction with a suitable guide to the ribozyme target. This can be done by cutting or binding the ribozyme. In particular, it is contemplated to reduce ribozyme activity. This may be useful in determining ribozyme function in vivo or in vitro, but also as a means of controlling treatment based on ribozyme activity in vivo or in vitro.

[0252] Gene expression, including RNA processing

[0253] Effector proteins can also be used with suitable guides for targeted gene expression, including control via RNA processing. Control of RNA processing can include RNA processing reactions, such as RNA splicing via targeted RNApol, including alternative splicing; viral replication (particularly satellite viruses, bacteriophages and retroviruses, such as HBV, HBC and HIV and other viruses listed herein), including viroids in plants; and tRNA biosynthesis. Effector proteins and suitable guides can also be used to control RNA activation (RNAa). RNAa leads to the promotion of gene expression, so control of gene expression can be achieved by destroying or reducing RNAa and thereby reducing the promotion of gene expression.

[0254] RNAi screening

[0255] By identifying gene products whose knockdown is associated with a phenotypic change, biological pathways can be interrogated and components identified via RNAi screens. Controls can also be exerted on or during these screens by using effector proteins and appropriate guides in the screen to eliminate or reduce the activity of RNAi and thereby restore the activity of the (previously perturbed) gene product (by eliminating or reducing perturbation / inhibition).

[0256] Satellite RNA (satRNA) and satellite viruses can also be processed.

[0257] Controlling with respect to RNase activity herein generally means reducing, negatively disrupting or knocking down or knocking out.

[0258] In vivo RNA applications

[0259] Inhibit gene expression

[0260] The target-specific RNases provided herein allow for very specific cleavage of target RNA. Since the genome is not modified, interference at the RNA level allows for regulation in a non-invasive manner both spatially and temporally.

[0261] Many diseases have been shown to be treatable by mRNA targeting. Although most of these studies involve the administration of siRNA, it is clear that the RNA targeting effector proteins provided herein can be applied in a similar manner.

[0262] Examples of mRNA targets (and corresponding disease treatments) are VEGF, VEGF-R1 and RTP801 (in the treatment of AMD and / or DME), caspase 2 (in the treatment of Naion), ADRB2 (in the treatment of intraocular pressure), TRPVI (in the treatment of dry eye syndrome), Syk kinase (in the treatment of asthma), Apo B (in the treatment of hypercholesterolemia), PLK1, KSP and VEGF (in the treatment of solid tumors), Ber-Abl (in the treatment of CML) (Burnett and Rossi Chem Biol. 2012, 19(1):60-71). Similarly, RNA targeting has been shown to be effective in treating RNA virus-mediated diseases such as HIV (targeting HIV Tet and Rev), RSV (targeting RSV nucleocapsid) and HCV (targeting miR-122) (Burnett and Rossi Chem Biol. 2012, 19(1):60-71).

[0263] It is further contemplated that the RNA targeting effector protein of the present invention can be used for mutation-specific or allele-specific knockdown. The guide RNA can be designed to specifically target a sequence containing a mutation or allele-specific sequence in the transcribed mRNA. This specific knockdown is particularly suitable for therapeutic applications involving disorders associated with mutations or allele-specific gene products. For example, most cases of familial hypolipoproteinemia (FHBL) are caused by mutations in the ApoB gene. This gene encodes two forms of apolipoprotein B protein: a short form (ApoB-48) and a longer form (ApoB-100). Several ApoB gene mutations that cause FHBL make both forms of ApoB abnormally short. Specific targeting and knockdown of mutated ApoB mRNA transcripts with the RNA targeting effector protein of the present invention can be beneficial for treating FHBL. As another example, Huntington's disease (HD) is caused by the expansion of CAG triplet repeats in the gene encoding huntingtin protein, which causes protein abnormalities. Specific targeting and knockdown of mutant or allele-specific mRNA transcripts encoding huntingtin protein using the RNA targeting effector proteins of the present invention may be beneficial for the treatment of HD.

[0264] It should be noted that in this context, and more generally for various applications described herein, it is conceivable to use RNA targeting effector proteins in a broken form. In fact, this can not only improve specificity, but also be conducive to delivery. In the sense that the two parts of the Cas13b enzyme basically contain functional Cas13b, Cas13b is broken. Ideally, the fracture should always leave the catalytic domain unaffected. The Cas13b can act as a nuclease, or it can be a dead Cas13b, which is essentially an RNA binding protein with little or no catalytic activity due to mutations usually in its catalytic domain.

[0265] Each half of the broken Cas13b can be fused with a dimerization partner. By way of example and not limitation, the use of a rapamycin-sensitive dimerization domain allows the generation of a chemically induced broken Cas13b for temporarily controlling the activity of Cas13b. Therefore, Cas13b can be made chemically inducible by breaking it into two fragments, and the rapamycin-sensitive dimerization domain can be used for the controlled reassembly of Cas13b. The two parts of the broken Cas13b can be considered to be the N' terminal part and the C' terminal part of the broken Cas13b. Fusion is usually at the breakpoint of Cas13b. In other words, the C' terminal of the N' terminal part of the broken Cas13b is fused to one of the two halves of the dimer, and the N' terminal of the C' terminal part is fused to the other half of the dimer.

[0266] In the sense that the rupture is newly generated, Cas13b does not necessarily have to be broken. The breakpoint is usually designed via computer simulation and cloned into the construct. The two parts of the fractured Cas13b, namely the N'terminal and C'terminal parts, form a complete Cas13b together, which preferably contains 70% or more wild-type amino acids (or nucleotides encoding them), preferably at least 80% or more, preferably at least 90% or more, preferably at least 95% or more and most preferably at least 99% or more wild-type amino acids (or nucleotides encoding them). Some pruning is possible, and mutants are envisioned. Non-functional domains can be completely removed. It is important that the two parts can be combined together, and the desired Cas13b function is restored or reconstructed. The dimer can be a homodimer or a heterodimer.

[0267] In certain embodiments, a Cas13b effector as described herein can be used for mutation-specific or allele-specific targeting, such as for mutation-specific or allele-specific knockdown.

[0268] The RNA targeting effector protein can be further fused to another functional RNase domain, such as a nonspecific RNase or Argonaute 2, which act synergistically to increase RNase activity or ensure further degradation of the message.

[0269] Regulating gene expression by modulating RNA function

[0270] In addition to directly affecting gene expression by cutting mRNA, RNA targeting can also be used to affect specific aspects of RNA processing in cells, which can allow more delicate regulation of gene expression. Generally, regulation can be mediated, for example, by interfering with the binding of proteins to RNA, such as blocking the binding of proteins or recruiting RNA binding proteins. In fact, regulation can be ensured at different levels such as the splicing, transport, positioning, translation and conversion of mRNA. Similarly, in the context of treatment, it is conceivable to solve (pathogenic) dysfunction at each level in these levels by using RNA-specific targeting molecules. In these embodiments, it is preferred in many cases that RNA targeting protein is "dead" Cas13b, which has lost the ability to cut RNA targets, but it retains the ability to bind to it, such as the mutant form of Cas13b described herein.

[0271] a) Alternative splicing

[0272] Many human genes express multiple mRNAs due to alternative splicing. Different diseases have been shown to be associated with abnormal splicing that causes loss of function or increased function of the expressed genes. Although some of these diseases are caused by mutations that cause splicing defects, some of them are not. A therapeutic option is to directly target the splicing mechanism. The RNA targeting effector protein described herein can, for example, be used to block or promote splicing, include or exclude exons, and affect the expression of specific isoforms and / or stimulate the expression of alternative protein products. Such applications are described in more detail below.

[0273] The RNA targeting effector protein bound to the target RNA can spatially block the access of splicing factors to the RNA sequence. The RNA targeting effector protein targeting the splicing site can block splicing at the site, optionally redirecting splicing to an adjacent site. For example, the RNA targeting effector protein bound to the 5' splicing site can block the recruitment of the U1 component of the spliceosome, which is conducive to skipping the exon. Alternatively, the RNA targeting effector protein targeting the splicing enhancer or silencer can prevent the trans-regulatory splicing factor from binding at the target site and effectively block or promote splicing. Exon exclusion can be further achieved by recruiting ILF2 / 3 to the pre-mRNA near the exon by the RNA targeting effector protein as described herein. As another example, a glycine-rich domain can be attached for the recruitment and exon exclusion of hnRNP A1 (Del Gatto-Konczak et al., Mol Cell Biol. 1999 Jan; 19 (1): 251-60).

[0274] In certain embodiments, through appropriate selection of gRNA, specific splice variants can be targeted while other splice variants are not targeted.

[0275] In some cases, RNA targeting effector proteins can be used to promote splicing (e.g., where splicing is defective). For example, RNA targeting effector proteins can be associated with effectors that can stabilize splicing regulatory stem-loops for further splicing. RNA targeting effector proteins can be linked to consensus binding site sequences for specific splicing factors in order to recruit proteins to target DNA.

[0276] Examples of diseases associated with abnormal splicing include, but are not limited to, paraneoplastic opsoclonus myoclonus ataxia (or POMA) caused by the loss of Nova protein, which regulates protein splicing that works in synapses, and cystic fibrosis caused by defective splicing of the cystic fibrosis transmembrane conductance regulator, which results in the production of non-functional chloride channels. In other diseases, abnormal RNA splicing results in gain of function. This is the case, for example, in myotonic dystrophy, which is caused by splicing defects due to CUG triplet repeat expansion (50 to >1500 repeats) in the 3'UTR of mRNA.

[0277] RNA targeting effector proteins can be used to include exons by recruiting splicing factors (e.g., U1) to the 5' splice site to promote the excision of introns around the desired exon. Such recruitment can be mediated by fusions with arginine / serine-rich domains that serve as splicing activators (Gravely BR and Maniatis T, Mol Cell. 1998(5):765-71).

[0278] It is envisioned that RNA targeting effector proteins can be used to block the splicing mechanism at the desired site, resulting in preventing exon recognition and the expression of different protein products. An example of a disease that can be treated is Duchenne muscular dystrophy (DMD), which is caused by mutations in the gene encoding dystrophin. Almost all DMD mutations can cause frameshifts, resulting in impaired translation of dystrophin. RNA targeting effector proteins can be paired with splicing junctions or exon splicing enhancers (ESEs) to prevent exon recognition, resulting in translation of partially functional proteins. This will convert the fatal Duchenne phenotype into a less severe Becker phenotype.

[0279] b) RNA modification

[0280] RNA editing is a natural process in which the diversity of gene products of a given sequence is increased by minor modifications in RNA. Typically, modification involves the conversion of adenosine (A) to inosine (I), resulting in an RNA sequence different from that encoded by the genome. RNA modification is typically ensured by ADAR enzymes, whereby the precursor RNA target is formed into imperfect double-stranded RNA by base pairing between exons and intron non-coding elements comprising adenosine to be edited. A classic example of AI editing is glutamate receptor GluR-B mRNA, whereby changes cause changes in the conductance properties of the channel (Higuchi M, et al. Cell. 1993; 75: 1361-70).

[0281] In humans, heterozygous loss of function in the ADAR1 gene leads to a skin disease, human pigmented genetic skin disease (Miyamura Y, et al. Am J Hum Genet. 2003; 73: 693-9). It is envisioned that the RNA targeting effector proteins of the present invention can be used to correct dysfunctional RNA modifications.

[0282] It is also contemplated that RNA adenosine methylase (N(6)-methyladenosine) can be fused to the RNA targeting effector protein of the present invention and targeted to the transcript of interest. This methylase causes reversible methylation, has a regulatory effect, and may affect gene expression and cell fate determination by regulating multiple RNA-related cellular pathways (Fu et al., Nat Rev Genet. 2014; 15(5): 293-306).

[0283] c) Polyadenylation

[0284] Polyadenylation of mRNA is important for nuclear transport, translation efficiency and stability of mRNA, all of which and the process of polyadenylation depend on specific RBPs. Most eukaryotic mRNAs receive a 3' poly (A) tail of about 200 nucleotides after transcription. Polyadenylation involves different RNA binding protein complexes, which stimulate the activity of poly (A) polymerase (Minvielle-Sebastia L et al., Curr Opin Cell Biol. 1999; 11: 352-7). It is envisioned that the RNA targeting effector protein provided herein can be used to interfere with or promote the interaction between RNA binding proteins and RNA.

[0285] An example of a disease associated with a defective protein involved in polyadenylation is oculopharyngeal muscular dystrophy (OPMD) (Brais B, et al. Nat Genet. 1998; 18: 164-7).

[0286] d) RNA export

[0287] After pre-mRNA processing, mRNA is exported from the nucleus to the cytoplasm. This is ensured by a cellular mechanism that involves the generation of a carrier complex, which is then transported through nuclear pores and releases the mRNA in the cytoplasm, followed by recovery of the carrier.

[0288] Overexpression of proteins that function in RNA export, such as TAP, has been found to increase export of transcripts that are otherwise inefficiently exported in Xenopus (Katahira J, et al., EMBO J. 1999; 18: 2593-609).

[0289] e) mRNA localization

[0290] mRNA localization can ensure spatial regulation of protein production. Transcript localization to a specific region of the cell can be ensured by a localization element. In a particular embodiment, it is envisioned that the effector proteins described herein can be used to target the localization element to the RNA of interest. The effector protein can be designed to bind to the target transcript and shuttle it to a location in the cell determined by its peptide signal tag. More specifically, for example, an RNA targeting effector protein fused to a nuclear localization signal (NLS) can be used to alter RNA localization.

[0291] Other examples of localization signals include the zip code binding protein (ZBP1) that ensures β-actin localization to the cytoplasm in several asymmetric cell types, namely the KDEL retention sequence (localized to the endoplasmic reticulum), the nuclear export signal (localized to the cytoplasm), the mitochondrial targeting signal (localized to mitochondria), the peroxisomal targeting signal (localized to peroxisomes), and the m6A marker / YTHDF2 (localized to p-bodies). Other approaches envisioned are to fuse RNA targeting effector proteins to proteins of known localization (e.g., membrane, synapse).

[0292] Alternatively, the effector protein according to the invention can be used, for example, for localization-dependent knockdown. By fusing the effector protein to a suitable localization signal, the effector is targeted to a specific cell compartment. Only target RNAs residing in this compartment will be effectively targeted, while the same target that would otherwise reside in a different cell compartment will not be targeted, thereby establishing localization-dependent knockdown.

[0293] f) Translation

[0294] The RNA targeting effector proteins described herein can be used to enhance or inhibit translation. Upregulation of translation is envisioned to be a very reliable method to control cellular circuits. In addition, for functional studies, protein translation screening may be superior to transcription upregulation screening, which has the disadvantage that transcription upregulation does not translate into increased protein production.

[0295] It is envisioned that the RNA targeting effector proteins described herein can be used to bring translation initiation factors (e.g., EIF4G) to the vicinity of the 5' untranslated repeat sequence (5'UTR) of the messenger RNA of interest to drive translation (as described by De Gregorio et al. EMBO J. 1999; 18(17): 4865-74 for non-reprogrammable RNA binding proteins). As another example, the cytoplasmic poly (A) polymerase GLD2 can be recruited to the target mRNA by the RNA targeting effector protein. This will allow for the targeted polyadenylation of the target mRNA, thereby stimulating translation.

[0296] Similarly, the RNA targeting effector protein envisioned herein can be used to block the translation repressor of mRNA, such as ZBP1 (Huttelmaier S, et al. Nature. 2005; 438: 512-5). By binding to the translation start site of the target RNA, translation can be directly affected.

[0297] Furthermore, fusing RNA-targeting effector proteins to proteins that stabilize mRNA, for example by preventing degradation by, for example, RNase inhibitors, can increase protein production of transcripts of interest.

[0298] It is envisioned that the RNA targeting effector proteins described herein can be used to inhibit translation by binding in the 5'UTR region of the RNA transcript and preventing ribosomes from forming and initiating translation.

[0299] Furthermore, RNA-targeting effector proteins can be used to recruit Caf1, a component of the CCR4-NOT adenylate enzyme complex, to target mRNAs, leading to deadenylation of target transcripts and inhibition of protein translation.

[0300] For example, the RNA targeting effector protein of the present invention can be used to increase or decrease the translation of therapeutically relevant proteins. Examples of therapeutic applications in which RNA targeting effector proteins can be used to downregulate or upregulate translation are amyotrophic lateral sclerosis (ALS) and cardiovascular disease. It is reported that the level of glial glutamate transporter EAAT2 is reduced in multiple EAAT2 mRNA abnormal transcripts in ALS motor cortex and spinal cord and in ALS brain tissue. The loss of EAAT2 protein and function is considered to be the main cause of ALS excitotoxicity. The restoration of EAAT2 protein levels and function may provide therapeutic benefits. Therefore, RNA targeting effector proteins can be beneficially used to upregulate the expression of EAAT2 protein, for example, by blocking translation repressors or stabilizing mRNA as described above. Apolipoprotein A1 is the main protein component of high-density lipoprotein (HDL), and ApoA1 and HDL are generally considered to have anti-atherosclerotic effects. It is envisioned that RNA targeting effector proteins can be beneficially used to upregulate the expression of ApoA1, for example, by blocking translation repressors or stabilizing mRNA as described above.

[0301] g) mRNA turnover

[0302] Translation is closely related to the mRNA stability of mRNA renewal and regulation. Specific proteins have been described to be related to the stability of transcripts (e.g., ELAV / Hu proteins in neurons, Keene JD, 1999, Proc Natl Acad Sci USA. 96: 5-7) and tristetraprolin (TTP). These proteins stabilize target mRNA (Peng SS et al., 1988, EMBO J. 17: 3461-70) by protecting information from degradation in the cytoplasm.

[0303] It is envisioned that the RNA targeting effector protein of the present invention can be used to interfere with or promote the activity of proteins used to stabilize mRNA transcripts, thereby affecting mRNA renewal. For example, the use of RNA targeting effector proteins to recruit human TTP to target RNA will allow translational inhibition and target degradation mediated by elements rich in adenylate-uridylate (AU-rich elements). AU-rich elements have been found in the 3'UTR of many mRNAs that encode proto-oncogenes, nuclear transcription factors, and cytokines and promote RNA stability. As another example, the RNA targeting effector protein can be fused with another mRNA stabilizing protein HuR (Hinman MN and Lou H, Cell Mol Life Sci 2008; 65: 3168-81) and recruited to the target transcript to extend its lifespan or stabilize short-lived mRNA.

[0304] It is also envisioned that the RNA targeting effector proteins described herein can be used to promote degradation of target transcripts. For example, m6A methyltransferases can be recruited to target transcripts to localize the transcripts to P-bodies, thereby resulting in degradation of the target.

[0305] As yet another example, an RNA targeting effector protein as described herein can be fused to the non-specific endonuclease domain PilT N-terminus (PIN) to recruit it to the target transcript and degrade it.

[0306] Patients with paraneoplastic neurological disease (PND)-associated encephalomyelitis and neuropathy are patients who produce autoantibodies against Hu proteins in tumors outside the central nervous system (Szabo A et al. 1991, Cell.; 67:325-33), which then cross the blood-brain barrier. It is envisioned that the RNA targeting effector proteins of the present invention can be used to interfere with the binding of autoantibodies to mRNA transcripts.

[0307] Patients with dystrophy type 1 (DM1), caused by (CUG)n expansion in the 3'UTR of the dystrophic myotonic protein kinase (DMPK) gene, are characterized by accumulation of such transcripts in the nucleus. It is envisioned that the RNA targeting effector protein of the present invention fused to an endonuclease targeting the (CUG)n repeat sequence can inhibit the accumulation of such aberrant transcripts.

[0308] h) Interaction with multifunctional proteins

[0309] Some RNA binding proteins can bind to multiple sites on numerous RNAs, thereby playing a role in a variety of processes. For example, the hnRNP A1 protein has been found to bind to exonic splicing silencer sequences, antagonize splicing factors, associate with telomere ends (thus stimulating telomere activity), and bind miRNAs to promote Drosha-mediated processing to affect maturation. It is contemplated that the RNA binding effector proteins of the present invention can interfere with the binding of RNA binding proteins at one or more locations.

[0310] i) RNA folding

[0311] RNA adopts a defined structure to perform its biological activity. The transition of conformations between alternative tertiary structures is crucial for most RNA-mediated processes. However, RNA folding may be associated with several problems. For example, RNA may have a tendency to fold into other incorrect conformations and unfold with other incorrect conformations, and / or the correct tertiary structure may not be sufficiently superior to other structures in thermodynamics. The RNA targeting effector protein of the present invention, especially the protein of the targeting RNA with cleavage defects or death, can be used to guide the folding of (m) RNA and / or obtain its correct tertiary structure.

[0312] Use of RNA-targeting effector proteins in regulating cellular states

[0313] In certain embodiments, Cas13b in a complex with crRNA is activated when bound to a target RNA and subsequently cleaves any nearby ssRNA targets (i.e., a "collateral" or "bystander" effect). Once Cas13b is triggered by a homologous target, it can cleave other (non-complementary) RNA molecules. Such promiscuous RNA cleavage can potentially cause cytotoxicity, or otherwise affect cell physiology or cell state.

[0314] Therefore, in certain embodiments, non-naturally occurring or engineered compositions, carrier systems or delivery systems as described herein are used for or used to induce cell dormancy. In certain embodiments, non-naturally occurring or engineered compositions, carrier systems or delivery systems as described herein are used for or used to induce cell cycle arrest. In certain embodiments, non-naturally occurring or engineered compositions, carrier systems or delivery systems as described herein are used for or used to reduce cell growth and / or cell proliferation, and in certain embodiments, non-naturally occurring or engineered compositions, carrier systems or delivery systems as described herein are used for or used to induce cell anergy. In certain embodiments, non-naturally occurring or engineered compositions, carrier systems or delivery systems as described herein are used for or used to induce apoptosis. In certain embodiments, non-naturally occurring or engineered compositions, carrier systems or delivery systems as described herein are used for or used to cell necrosis. In certain embodiments, non-naturally occurring or engineered compositions, carrier systems or delivery systems as described herein are used for or used to induce cell death. In certain embodiments, non-naturally occurring or engineered compositions, carrier systems or delivery systems as described herein are used for or used to induce programmed cell death.

[0315] In certain embodiments, the present invention relates to a method for inducing cell dormancy, comprising introducing or inducing a non-naturally occurring or engineered composition, carrier system, or delivery system as described herein. In certain embodiments, the present invention relates to a method for inducing cell cycle arrest, comprising introducing or inducing a non-naturally occurring or engineered composition, carrier system, or delivery system as described herein. In certain embodiments, the present invention relates to a method for reducing cell growth and / or cell proliferation, comprising introducing or inducing a non-naturally occurring or engineered composition, carrier system, or delivery system as described herein. In certain embodiments, the present invention relates to a method for inducing cell anergy, comprising introducing or inducing a non-naturally occurring or engineered composition, carrier system, or delivery system as described herein. In certain embodiments, the present invention relates to a method for inducing apoptosis, comprising introducing or inducing a non-naturally occurring or engineered composition, carrier system, or delivery system as described herein. In certain embodiments, the present invention relates to a method for inducing cell necrosis, comprising introducing or inducing a non-naturally occurring or engineered composition, carrier system, or delivery system as described herein. In certain embodiments, the present invention relates to a method for inducing cell death, comprising introducing or inducing a non-naturally occurring or engineered composition, carrier system, or delivery system as described herein. In certain embodiments, the invention relates to methods for inducing programmed cell death comprising introducing or inducing a non-naturally occurring or engineered composition, vector system, or delivery system as described herein.

[0316] The methods and uses as described herein can be therapeutic or preventive and can target specific cells, cell (sub) populations or cell / tissue types. Specifically, the methods and uses as described herein can be therapeutic or preventive and can target specific cells, cell (sub) populations or cell / tissue types expressing one or more target sequences, such as one or more specific target RNAs (e.g., ss RNAs). Without limitation, target cells can be, for example, cancer cells expressing specific transcripts, such as neurons of a given class, (immune) cells causing, for example, autoimmunity, or cells infected by specific (e.g., viral) pathogens, etc.

[0317] Therefore, in certain embodiments, the present invention relates to a method for treating a pathological condition characterized by the presence of undesirable cells (host cells), comprising introducing or inducing a non-naturally occurring or engineered composition, vector system or delivery system as described herein. In certain embodiments, the present invention relates to a non-naturally occurring or engineered composition, vector system or delivery system as described herein for treating a pathological condition characterized by the presence of undesirable cells (host cells). In certain embodiments, the present invention relates to a non-naturally occurring or engineered composition, vector system or delivery system as described herein, which is used to treat a pathological condition characterized by the presence of undesirable cells (host cells). It should be understood that, preferably, the CRISPR-Cas system targets a target that is specific to undesirable cells. In certain embodiments, the present invention relates to a non-naturally occurring or engineered composition, vector system or delivery system as described herein for treating, preventing or alleviating cancer. In certain embodiments, the present invention relates to a non-naturally occurring or engineered composition, vector system or delivery system as described herein, which is used to treat, prevent or alleviate cancer. In certain embodiments, the present invention relates to a method for treating, preventing or reducing cancer, comprising introducing or inducing a non-naturally occurring or engineered composition, vector system or delivery system as described herein. It should be understood that, preferably, the CRISPR-Cas system targets a target specific to cancer cells. In certain embodiments, the present invention relates to the use of a non-naturally occurring or engineered composition, vector system or delivery system as described herein for treating, preventing or reducing infection of a cell by a pathogen. In certain embodiments, the present invention relates to a non-naturally occurring or engineered composition, vector system or delivery system as described herein, which is used to treat, prevent or reduce infection of a cell by a pathogen. In certain embodiments, the present invention relates to a method for treating, preventing or reducing infection of a cell by a pathogen, comprising introducing or inducing a non-naturally occurring or engineered composition, vector system or delivery system as described herein. It should be understood that, preferably, the CRISPR-Cas system targets a target specific to cells infected by a pathogen (e.g., a target derived from a pathogen). In certain embodiments, the present invention relates to the use of a non-naturally occurring or engineered composition, vector system or delivery system as described herein for treating, preventing or reducing autoimmune disorders. In certain embodiments, the present invention relates to a non-natural or engineered composition, vector system or delivery system as described herein, which is used to treat, prevent or alleviate autoimmune disorders. In certain embodiments, the present invention relates to a method for treating, preventing or alleviating autoimmune disorders, comprising introducing or inducing a non-natural or engineered composition, vector system or delivery system as described herein. It should be understood that, preferably, the CRISPR-Cas system targets a target (e.g., a specific immune cell) that is specific to cells that cause autoimmune disorders.

[0318] Use of RNA-targeting effector proteins in RNA detection

[0319] It is further contemplated that the RNA-targeting effector protein can be used in a Northern blot assay. Northern blotting involves the use of electrophoresis to separate RNA samples by size. The RNA-targeting effector protein can be used to specifically bind to and detect a target RNA sequence.

[0320] The effector protein targeting RNA can be fused to a fluorescent protein (such as GFP) and used to track RNA localization in living cells. More specifically, the effector protein targeting RNA can be inactivated because it no longer cleaves RNA. In a particular embodiment, it is envisioned that the effector protein that targets RNA can be used to fall off, where the signal depends on the binding of two subproteins to ensure more accurate visualization. Alternatively, a fluorescent protein that falls off when multiple effector protein complexes targeting RNA are bound to the target transcript can be used. It is further envisioned that the transcript is targeted to multiple binding sites along the mRNA so that the fluorescent signal can expand the real signal and allow focus identification. As another alternative, the fluorescent protein can be reconstructed from the intein that falls off.

[0321] Effector proteins targeting RNA are suitable, for example, for determining the localization of RNA or specific splice variants, the level of mRNA transcripts, the upregulation or downregulation of transcripts, and disease-specific diagnosis. Effector proteins targeting RNA can be used to visualize nucleic acids in (living) cells using, for example, fluorescence microscopy or flow cytometry (such as fluorescence activated cell sorting (FACS), which allows high-throughput screening of cells and recovery of living cells after cell sorting). In addition, the expression levels of different transcripts can be evaluated simultaneously under stress (e.g., using molecular inhibitors to inhibit cancer growth or hypoxic conditions on cells). Another application is to track the localization of transcripts to synaptic connections during neural stimulation using two-photon microscopy.

[0322] In certain embodiments, the components or complexes according to the invention as described herein can be used in multiplexed error-robust fluorescence in situ hybridization (MERFISH; Chen et al., Science; 2015; 348(6233)), for example using a (fluorescently) labeled Cas13b effector.

[0323] In vitro apex labeling

[0324] Cellular processes depend on a network of molecular interactions between proteins, RNA, and DNA. Accurate detection of protein-DNA and protein-RNA interactions is key to understanding such processes. In vitro proximity labeling techniques use affinity tags combined with, for example, photoactivatable probes to label polypeptides and RNAs near in vitro target proteins or RNAs. After UV irradiation, the photoactivatable group reacts with proteins and other molecules adjacent to the tag molecules, thereby labeling them. The labeled interacting molecules can then be recovered and identified. The RNA targeting effector protein of the present invention can, for example, be used to target the probe to a selected RNA sequence.

[0325] These applications may also find application in animal models for disease-relevant applications or in vivo imaging of difficult-to-culture cell types.

[0326] Use of RNA-targeting effector proteins in RNA origami / in vitro assembly systems

[0327] Combinatorics

[0328] RNA origami refers to nanoscale folded structures for creating two-dimensional or three-dimensional structures using RNA as an integrated template. The folded structure is encoded in the RNA and the shape of the resulting RNA is therefore determined by the synthesized RNA sequence (Geary et al. 2014. Science, 345 (6198), pp. 799-804). RNA origami can be used as a scaffold for arranging other components such as proteins into a complex. The effector protein targeting RNA of the present invention can be used, for example, to target the target protein to RNA origami using a suitable guide RNA.

[0329] These applications may also find application in animal models for disease-relevant applications or in vivo imaging of difficult-to-culture cell types.

[0330] Use of RNA-targeting effector proteins in RNA isolation or purification, enrichment or depletion

[0331] It is further contemplated that, when complexed with RNA, the effector protein targeting RNA can be used to isolate and / or purify RNA. The effector protein targeting RNA can, for example, be fused to an affinity tag, which can be used to isolate and / or purify the effector protein complex targeting RNA. Such applications can, for example, be used to analyze gene expression profiles in cells.

[0332] In certain embodiments, it is envisioned that effector proteins targeting RNA can be used to target specific non-coding RNAs (ncRNAs), thereby blocking their activity, thereby providing useful functional probes. In certain embodiments, effector proteins as described herein can be used to specifically enrich for specific RNAs (including but not limited to increasing stability, etc.), or alternatively to specifically deplete specific RNAs (such as but not limited to, for example, specific splice variants, isoforms, etc.).

[0333] Exploration of lincRNA functions and other nuclear RNAs

[0334] The disadvantage of current RNA knockdown strategies (such as siRNA) is that they are mostly limited to targeting cytosolic transcripts because the protein machinery is cytosolic. The advantage of the RNA-targeting effector protein of the present invention (an exogenous system that is not necessary for cellular function) is that it can be used in any compartment in the cell. By fusing an NLS signal to the RNA-targeting effector protein, it can be directed to the nucleus, allowing targeting of nuclear RNA. For example, it is envisioned to probe the function of lincRNA.

[0335] Long intergenic noncoding RNAs (lincRNAs) are a vast area of ​​research that is not fully explored. Most lincRNAs have as yet unknown functions that can be studied for effector proteins using the targeting RNAs of the present invention.

[0336] Identification of RNA-binding proteins

[0337] Identifying proteins that bind to specific RNAs may help understand the role of many RNAs. For example, many lincRNAs are associated with transcriptional and epigenetic regulators to control transcription. Understanding which proteins bind to a given lincRNA can help elucidate the components in a given regulatory pathway. The RNA targeting effector protein of the present invention can be designed to recruit biotin ligase to specific transcripts in order to label locally bound proteins with biotin. The protein can then be pulled down and identified by mass spectrometry.

[0338] Assembly of complexes on RNA and substrate shuttling

[0339] The RNA targeting effector protein of the present invention can be further used to assemble complexes on RNA. This can be achieved by functionalizing the RNA targeting effector protein with a variety of related proteins (e.g., components of a specific synthetic pathway). Alternatively, a variety of RNA targeting effector proteins can be functionalized with these different related proteins and targeted to the same RNA or adjacent target RNA. Useful applications of assembling complexes on RNA are, for example, to promote substrate shuttling between proteins.

[0340] Synthetic Biology

[0341] The development of biological systems has a wide range of uses, including clinical applications. It is envisioned that the programmable RNA targeting effector proteins of the present invention can be used in fusion form to cleave proteins of toxic domains to achieve targeted cell death, for example using cancer-associated RNA as target transcripts. In addition, pathways involving protein-protein interactions can be affected in synthetic biological systems, for example, through fusion complexes with suitable effectors such as kinases or other enzymes.

[0342] Protein splicing: inteins

[0343] Protein splicing is a post-translational process in which an intermediate polypeptide (called an intein) catalyzes its own excision from the polypeptide (called an extein) that releases it, and the subsequent connection to these exteins. The assembly of two or more RNA targeting effector proteins described herein on a target transcript can be used to guide the release of the shed intein (Topilina and Mills Mob DNA. 2014 Feb 4; 5(1): 5), thereby enabling direct calculation of the amount of mRNA transcript present and the protein product (e.g., metabolic enzyme or transcription factor) subsequently released (for downstream activation of transcriptional pathways). This application may be of great significance in synthetic biology (see above) or large-scale bioproduction (producing products only under specific conditions).

[0344] Inducible self-inactivating delivery system

[0345] In one embodiment, the fusion complex comprising the RNA targeting effector protein of the invention and the effector component is designed to be inducible, such as light-inducible or chemically inducible. This inducibility enables the activation of the effector component at the desired time.

[0346] For example, light inducibility is achieved by designing a fusion complex in which the CRY2PHR / CIBN pairing is used for fusion. This system is particularly useful for light-induced protein interactions in living cells (Konermann S et al. Nature. 2013; 500: 472-476).

[0347] For example, chemical inducibility can be provided by designing a fusion complex in which the fusion is performed using a FKBP / FRB (FK506 binding protein / FKBP rapamycin binding) pairing. When using this system, rapamycin is used for protein binding (Zetsche et al. Nat Biotechnol. 2015; 33(2): 139-42 describes the use of this system for Cas9).

[0348] In addition, when the RNA targeting effector protein of the present invention is introduced into the cell in the form of DNA, it can be regulated by an inducible promoter, such as transcriptional activation controlled by tetracycline or doxycycline (Tet-On and Tet-Off expression systems), hormone-inducible gene expression systems, such as ecdysone-inducible gene expression systems and arabinose-inducible gene expression systems. When delivered in the form of RNA, the expression of the RNA targeting effector protein can be regulated by a riboswitch, which can sense small molecules such as tetracycline (as described in Goldfless et al, Nucleic Acids Res. 2012; 40(9): e64).

[0349] In one embodiment, the delivery of the RNA targeting effector proteins of the invention can be modulated to alter the amount of protein or crRNA in the cell, thereby changing the magnitude of the desired effect or any undesired off-target effects.

[0350] In one embodiment, the RNA targeting effector proteins described herein can be designed to be self-inactivating. When delivered into cells in the form of RNA (mRNA or replicating RNA therapeutics) (Wrobleska et al Nat Biotechnol. 2015 Aug; 33(8): 839-841), they can inactivate expression and subsequent effects by destroying their own RNA, thereby reducing residence time and potential adverse effects.

[0351] For further in vivo applications of RNA targeting effector proteins as described herein, reference is made to Mackay JP et al. (Nat Struct Mol Biol. 2011 Mar; 18(3): 256-61), Nelles et al. (Bioessays. 2015 Jul; 37(7): 732-9), and Abil Z and Zhao H (Mol Biosyst. 2015 Oct; 11(10): 2658-65), which are incorporated herein by reference. In particular, in certain embodiments of the present invention, preferably in certain embodiments, the following applications are envisioned by using a catalytically inactive Cas13b: enhancing translation (e.g., Cas13b-translation promoting factor fusion (e.g., eIF4 fusion)); inhibiting translation (e.g., gRNA targeting ribosome binding site); exon skipping (e.g., gRNA targeting splicing donor and / or acceptor site); exon inclusion (e.g., gRNA targeting a specific exon splicing donor and / or acceptor site to be included or fused to Cas13b or recruiting spliceosomal components (e.g., U1 snRNA); access RNA localization (e.g., Cas13b-flag fusions (e.g., EGFP fusions)); alter RNA localization (e.g., Cas13b-localization signal fusions (e.g., NLS or NES fusions)); RNA degradation (in which case, if Cas13b activity is relied upon, a catalytically inactive Cas13b is not used, or, to increase specificity, a shedding Cas13b can be used); inhibition of noncoding RNA function (e.g., miRNA), for example, by gRNA degradation or gRNA binding to a functional site (potentially titrated out at a specific site by relocalization via Cas13b-signal sequence fusions).

[0352] As described herein before and confirmed in the examples, Cas13b function is stable for 5' or 3' extension of crRNA and extension of crRNA loop. It is therefore envisioned that the MS2 loop and other recruitment domains can be added to crRNA without affecting the formation of the complex and the binding to the target transcript. This type of modification of crRNA intended to recruit various effector domains is applicable to various uses of the above-mentioned RNA targeting effector protein.

[0353] Cas13b is able to mediate resistance to RNA bacteriophages. Therefore, it is envisioned that Cas13b can be used, for example, to immunize animals, humans, and plants against RNA-only pathogens, including but not limited to Ebola and Zika viruses.

[0354] In certain embodiments, Cas13b can process (crack) its own array. This applies to both wild-type Cas13b proteins and mutant Cas13b proteins comprising one or more mutant amino acid residues described herein. It is therefore conceivable that multiple crRNAs designed for different target transcripts and / or applications can be delivered as a single pre-crRNA or as a single transcript driven by a promoter. The advantage of this delivery method is that it is substantially more compact, easier to synthesize and easier to deliver in a viral system. It should be understood that for orthologs of Cas13b herein, the exact amino acid position can vary, which can be appropriately determined by protein alignment, as known in the art, and as described elsewhere herein. Aspects of the present invention also encompass methods and uses of compositions and systems described herein in genome engineering, such as for changing or manipulating the expression of one or more genes or one or more gene products in prokaryotic or eukaryotic cells in vitro, in vivo or in vitro.

[0355] In one aspect, the present invention provides methods and compositions for regulating (e.g., reducing) the expression of a target RNA in a cell. In the subject method, a Cas13b system of the present invention is provided, which interferes with the transcription, stability and / or translation of RNA.

[0356] In certain embodiments, an effective amount of Cas13b system is used to crack RNA or inhibit RNA expression. In this regard, the system has a purpose similar to siRNA and shRNA, and therefore such methods can also be substituted. The method includes but is not limited to using the Cas13b system as a substitute for, for example, interfering ribonucleic acid (such as siRNA or shRNA) or its transcription template (such as DNA encoding shRNA). For example, by applying the Cas13b system to a mammal including a target cell, it is introduced into the target cell.

[0357] Advantageously, the Cas13b system of the present invention has specificity. For example, although interfering with ribonucleic acid (such as siRNA or shRNA) polynucleotide systems are plagued by design and stability problems and off-target binding, the Cas13b system of the present invention can be designed to have high specificity.

[0358] Destabilization of Cas13b

[0359] In certain embodiments, the effector proteins according to the invention described herein are associated with or fused to a destabilizing domain (DD). In some embodiments, the DD is ER50. In some embodiments, the corresponding stabilizing ligand of this DD is 4HT. Thus, in some embodiments, one of the at least one DD is ER50 and its stabilizing ligand is 4HT or CMP8. In some embodiments, the DD is DHFR50. In some embodiments, the corresponding stabilizing ligand of this DD is TMP. Thus, in some embodiments, one of the at least one DD is DHFR50 and its stabilizing ligand is TMP. In some embodiments, the DD is ER50. In some embodiments, the corresponding stabilizing ligand of this DD is CMP8. Thus, CMP8 may be an alternative stabilizing ligand for 4HT in the ER50 system. Although it is possible that CMP8 and 4HT can / should be used in competitive substances, certain cell types may be more susceptible to one or the other of these two ligands, and based on the present disclosure and the knowledge in the art, the skilled person can use CMP8 and / or 4HT.

[0360] In some embodiments, one or two DDs can be fused to the N-terminus of Cas13b, and one or two DDs can be fused to the C-terminus of Cas13b. In some embodiments, at least two DDs are associated with Cas13b, and the DDs are the same DDs, that is, the DDs are homologous. Therefore, both (or two or more) DDs can be ER50DDs. In some embodiments, this is preferred. Alternatively, two (or two or more) DDs can be DHFR50 DDs. This is also preferred in some embodiments. In some embodiments, the at least two DDs are associated with Cas13b, and the DDs are different DDs, that is, the DDs are heterologous. Therefore, one of the DDs can be ER50, and one or more or any other DD in the DDs can be DHFR50. It may be advantageous to have two or more heterologous DDs because a higher level of degradation control will be provided. The tandem fusion of more than one DD on the N-terminus or C-terminus may enhance degradation; and this tandem fusion can be, for example, ER50-ER50-Cas13b or DHFR-DHFR-Cas13b. It is envisioned that in the absence of both stabilizing ligands a high level of degradation will occur, in the absence of one stabilizing ligand and the presence of the other a moderate level of degradation will occur, and in the presence of both (or two of more) stabilizing ligands a low level of degradation will occur. This can also be controlled by having an N-terminal ER50 DD and a C-terminal DHFR50 DD.

[0361] In some embodiments, the fusion of Cas13b and DD includes a connexon between DD and Cas13b. In some embodiments, the connexon is a GlySer connexon. In some embodiments, DD-Cas13b also includes at least one nuclear export signal (NES). In some embodiments, DD-Cas13b includes two or more NES. In some embodiments, DD-Cas13b includes at least one nuclear localization signal (NLS). It can be that NLS is also included in addition to NES. In some embodiments, Cas13b includes a positioning (nuclear input or output) signal as a connexon between Cas13b and DD or as a part thereof, or is substantially composed of or composed of it. HA or Flag tags are also within the scope of the connexon of the present invention. Applicants use NLS and / or NES as connexon heads, and also use glycine serine connexons (the shortest is GS, as long as (GGGGS)3).

[0362] Destabilizing domains generally act to destabilize a variety of proteins; see, e.g., Miyazaki, J Am Chem Soc. Mar 7, 2012; 134(9): 3942-3945, which is incorporated herein by reference. CMP8 or 4-hydroxytamoxifen may be destabilizing domains. More generally, temperature-sensitive mutants of mammalian DHFR (DHFRts), which are destabilizing residues by the N-terminal rule, were found to be stable at permissive temperatures but unstable at 37°C. Addition of methotrexate, a high-affinity ligand for mammalian DHFR, to cells expressing DHFRts partially inhibited protein degradation. This is an important demonstration that small molecule ligands can stabilize proteins in cells that were otherwise targeted for degradation. Rapamycin derivatives were used to stabilize unstable mutants of the FRB domain of mTOR (FRB*) and restore function to the fusion kinase GSK-3β.6,7. This system demonstrates that ligand-dependent stability represents an attractive strategy for regulating the function of specific proteins in complex biological environments. The system for controlling protein activity may involve DD becoming functional due to dimerization of rapamycin-induced FK506 binding protein and FKBP12 during ubiquitin complementation. Mutants of human FKBP12 or ecDHFR protein can be engineered to be metabolically unstable in the absence of their high-affinity ligands (Shield-1 or trimethoprim (TMP) respectively). These mutants are some possible destabilizing domains (DD) that can be used to implement the present invention, and the instability of DD in the form of a fusion with Cas13b leads to Cas13b degradation of the entire fusion protein caused by the proteasome. Shield-1 and TMP bind and stabilize DD in a dose-dependent manner. Estrogen receptor ligand binding domain (ERLBD, residues 305-549 of ERS1) can also be engineered into a destabilizing domain. Since the estrogen receptor signaling pathway is involved in a variety of diseases, such as breast cancer, the pathway has been extensively studied, and many estrogen receptor agonists and antagonists have been developed. Therefore, ERLBD is known to be compatible with drugs. There are some ligands that bind to mutants of ERLBD rather than wild-type. By using one of these mutant domains encoding three mutations (L384M, M421G, G521R)12, ligands that do not interfere with the endogenous estrogen sensitivity network can be used to regulate the stability of ERLBD-derived DD. Another mutation (Y537S) can be introduced to further destabilize ERLBD and construct it into a potential DD candidate. This quadruple mutant is a favorable DD development. Mutant ERLBD can be fused to Cas13b, and its stability can be regulated or perturbed using a ligand, whereby Cas13b has DD.Another DD may be a 12-kDa (107 amino acids) tag based on a mutant FKBP protein and stabilized by a Shield 1 ligand. See, for example, Nature Methods 5, (2008). For example, the DD can be a modified FK506 binding protein 12 (FKBP12) that binds to and is reversibly stabilized by the synthetic, biologically inert small molecule Shield-1; see, e.g., Banaszynski LA, Chen LC, Maynard-Smith LA, Ooi AG, Wandless TJ. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell. 2006; 126:995-1004; Banaszynski LA, Sellmyer MA, Contag CH, Wandless TJ, Thorne SH. Chemical control of protein stability and function in living mice. Nat Med. 2008; 14:1123-1127; Maynard-Smith LA, Chen LC, Banaszynski LA, Ooi AG, Wandless TJ. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell. 2006; 126:995-1004; Banaszynski LA, Sellmyer MA, Contag CH, Wandless TJ, Thorne SH. Chemical control of protein stability and function in living mice. Nat Med. 2008; 14:1123-1127; Maynard-Smith LA, Chen LC, Banaszynski LA, Ooi AG, Wandless TJ. A directed approach for engineering conditional protein stability using biologically silent small molecules. The Journal of biological chemistry. 2007; 282: 24866-24872; and Rodriguez, Chem Biol. Mar 23, 2012; 19(3): 391-398 - all of which are incorporated herein by reference and can be employed in the practice of the present invention to select a DD associated with Cas13b in the practice of the present invention.It can be seen that the knowledge in the art includes many DDs, and DD can be fused to Cas13b, for example (advantageously through a linker), whereby DD can be stabilized in the presence of a ligand, and when the ligand is not present, DD may become unstable, whereby Cas 3b is completely unstable, or DD can be stable in the absence of a ligand, and when a ligand is present, DD may become unstable; DD enables Cas13b and therefore CRISPR-Cas13b complexes or systems to be regulated or controlled (it can be said to be turned on or off), thereby providing a means for, for example, regulating or controlling the system in an in vivo or in vitro environment. For example, when the protein of interest is expressed as a fusion with a DD tag, it is destabilized and rapidly degraded in the cell, such as by proteasome degradation. Therefore, the absence of a stable ligand causes D-related Cas to be degraded. When a new DD is fused to a protein of interest, its instability is imparted to the protein of interest, causing the entire fusion protein to rapidly degrade. The peak activity of Cas is sometimes beneficial for reducing off-target effects. Therefore, short bursts of high activity are preferred. The present invention can provide such peaks. In a sense, the system is inducible. In certain other senses, the system is repressed in the absence of a stabilizing ligand and derepressed in the presence of a stabilizing ligand.

[0363] Delivery or application according to the present invention can be carried out with liposome.Liposome is a spherical vesicle structure, composed of a single or multilayer lipid bilayer around an internal aqueous compartment and a relatively impermeable external lipophilic phospholipid bilayer.Liposome has received extensive attention as a drug delivery vehicle, because they have biocompatibility, nontoxicity, can deliver hydrophilic and lipophilic drug molecules, protect its load from being degraded by plasma enzymes, and can cross biological membranes and blood-brain barrier (BBB) ​​transport its load (see, for example, Spuch and Navarro, Journal of Drug Delivery, 2011 volumes, article number 469679, page 12, 2011, doi:10.1155 / 2011 / 469679 to view a review).Liposome can be made of several different types of lipids; However, phospholipids are most commonly used to produce liposomes as drug vehicles. Although liposome formation is spontaneous when the lipid film is mixed with an aqueous solution, the process can also be accelerated by applying force in the form of vibration using a homogenizer, ultrasonic generator, or extrusion equipment (see, e.g., Spuch and Navarro, Journal of Drug Delivery, Vol. 2011, Article No. 469679, p. 12, 2011, doi: 10.1155 / 2011 / 469679 for review).

[0364] Several other additives can be added to liposomes to modify their structure and characteristics. For example, cholesterol or sphingomyelin can be added to the liposome mixture to help stabilize the liposome structure and prevent the leakage of the internal cargo of the liposome. In addition, liposomes are prepared by hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol and dicetyl phosphate, and their average vesicle size is adjusted to about 50 and 100nm. (See, for example, Spuch and Navarro, Journal of Drug Delivery, 2011 volumes, article number 469679, page 12, 2011, doi:10.1155 / 2011 / 469679 for review). Liposomal preparations can mainly be composed of natural phospholipids and lipids such as 1,2-distearoyl-sn-glycerol-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholine and monosialoganglioside. Since such preparations consist only of phospholipids, liposome preparations encounter many challenges, one of which is instability in plasma. Several attempts have been made to overcome these challenges, particularly in the treatment of lipid membranes. One of the attempts focuses on the treatment of cholesterol. Adding cholesterol to conventional preparations slows down the rapid release of encapsulated bioactive compounds into plasma, or adding 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases stability (see, e.g., Spuc h and Navarro, Journal of Drug Delivery, Vol. 2011, Article No. 469679, p. 12, 2011, doi: 10.1155 / 2011 / 469679 for review). In a particularly advantageous embodiment, Trojan Horse liposomes (also known as Trojan Horses molecules) are desirable, and their scheme can be found in http: / / cshprotocols.cshlp.org / content / 2010 / 4 / pdb.prot5407.long . These particles allow the transgene to be delivered to the entire brain after intravascular injection. Without limitation, it is believed that neutral lipid particles with specific antibodies conjugated to the surface allow the blood-brain barrier to be crossed via endocytosis. Applicants assume that the CRISPR-Cas13b complex is delivered to the brain via intravascular injection using Trojan Horse liposomes, which will obtain whole-brain transgenic animals without embryonic manipulation. For in vivo administration in liposomes, about 1-5g of DNA or RNA can be considered.

[0365] In another embodiment, the nucleic acid targeting system or its components can be administered in liposomes such as stable nucleic acid-lipid particles (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005). Daily intravenous injections of about 1, 3, or 5 mg / kg / day of a specific nucleic acid targeting system targeted in SNALP are contemplated. Daily treatment can be over about three days, followed by weekly treatments for about five weeks. In another embodiment, SNALPs encapsulated with a specific nucleic acid targeting system administered by intravenous injection at a dose of about 1 or 2.5 mg / kg are also contemplated (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, May 4, 2006). The SNALP formulation may contain lipids 3-N-[(w-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol in a molar percentage of 2:40:10:48 (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, May 4, 2006). In another embodiment, it has been demonstrated that stable nucleic acid-lipid particles (SNALP) can effectively deliver molecules to highly vascularized HepG2-derived liver tumors, but not to poorly vascularized HCT-116-derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775-780). SNALP liposomes can be prepared as follows: D-Lin-DMA and PEG-C-DMA are formulated with distearoylphosphatidylcholine (DSPC), cholesterol and siRNA using a lipid / siRNA ratio of 25:1 and a cholesterol / D-Lin-DMA / DSPC / PEG-C-DMA molar ratio of 48 / 40 / 10 / 2. The size of the resulting SNALP liposomes is about 80-100 nm. In yet another embodiment, the SNALP may comprise synthetic cholesterol (Sigma-Aldrich, St Louis, MO, USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA), 3-N-[(w-methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxypropylamine and cationic 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (see, e.g., Geisbert et al., Lancet 2010; 375: 1896-905). A dosage of about 2 mg / kg total nucleic acid targeting system per dose administered, e.g., as an intravenous bolus, may be contemplated.In yet another embodiment, SNALP may comprise synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g., Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations for in vivo studies may comprise a final lipid / RNA mass ratio of about 9:1.

[0366] The safety of RNAi nanomedicines has been reviewed by Barros and Gollob of Alnylam Pharmaceuticals (see, for example, Advanced Drug Delivery Reviews 64 (2012) 1730-1737). Stable nucleic acid lipid particles (SNALPs) are composed of four different lipids: an ionizable lipid (DLinDMA) that is cationic at low pH, a neutral auxiliary lipid, cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. The particle is approximately 80 nm in diameter and is electrically neutral at physiological pH. During formulation, the ionizable lipid is used to condense the lipid with the anionic RNA during particle formation. When positively charged under increasingly acidic endosomal conditions, the ionizable lipid also mediates fusion of the SNALP with the endosomal membrane, thereby releasing the RNA into the cytoplasm. The PEG-lipid stabilizes the particles and reduces aggregation during formulation, subsequently providing a neutral hydrophilic exterior that improves pharmacokinetic properties. To date, two clinical programs have been initiated using SNALP formulations and RNA. Tekmira Pharmaceuticals recently completed a Phase I single-dose study of SNALP-ApoB in adult volunteers with elevated LDL cholesterol. ApoB is primarily expressed in the liver and jejunum and is essential for the assembly and secretion of VLDL and LDL. Seventeen subjects received a single dose of SNALP-ApoB (dose escalation across 7 dose levels). There was no evidence of liver toxicity (expected to be a potential dose-limiting toxicity based on preclinical studies). One of the two subjects who received the highest dose experienced flu-like symptoms consistent with immune system stimulation, so the decision was made to end the trial. Alnylam Pharmaceuticals similarly launched ALN-TTR01, which uses SNALP technology as described above and targets hepatocyte production of mutant and wild-type TTR to treat TTR amyloidosis (ATTR). Three ATTR syndromes have been described: familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC), both of which are caused by autosomal dominant mutations in TTR; and senile systemic amyloidosis (SSA) caused by wild-type TTR. A placebo-controlled, single-ascending dose Phase I trial of ALN-TTR01 in ATTR patients was recently completed. ALN-TTR01 was administered to 31 patients (23 study drug and 8 placebo) by 15-minute intravenous infusion at a dose range of 0.01 to 1.0 mg / kg (based on siRNA). Treatment was well tolerated with no significant increases in liver function tests. Of the 23 patients, 3 patients infused at ≥0.4 mg / kg experienced infusion-related reactions; all responded to slowing the infusion rate, and all remain on study.The two patients infused at the highest dose of 1 mg / kg experienced minimal and transient increases in serum cytokines IL-6, IP-10, and IL-1ra (expected based on preclinical and NHP studies). A decrease in serum TTR was observed at 1 mg / kg, which is the expected pharmacological effect of ALN-TTR01.

[0367] In another embodiment, SNALP can be prepared by dissolving, for example, in ethanol, cationic lipid, DSPC, cholesterol and PEG-lipid, for example, in a molar ratio of 40:10:40:10 (see Semple et al., Nature Niotechnology, Vol. 28, No. 2, February 2010, pp. 172-177). The lipid mixture is added to an aqueous buffer (50 mM citrate, pH 4), mixed until the final ethanol and lipid concentrations are 30% (volume / volume) and 6.1 mg / ml, respectively, and allowed to balance at 22°C for 2 minutes, then extruded. Using a Lipex extruder (Northern Lipids), hydrated lipids are extruded at 22°C through two overlapping pore sizes of 80 nm filter membranes (Nuclepore) until a vesicle (determined by dynamic light scattering analysis) of 70-90 nm in diameter is obtained. This generally requires 1-3 passes. siRNA (dissolved in 50mM citrate, pH 4 aqueous solution, containing 30% ethanol) was added to the pre-equilibrated (35°C) vesicles while mixing at a rate of about 5ml / min. After reaching a final target siRNA / lipid ratio of 0.06 (weight / weight), the mixture was incubated at 35°C for another 30 minutes for vesicle reconstitution and siRNA encapsulation. The ethanol was then removed and the external buffer was replaced with PBS (155mM NaCl, 3mM Na2HPO4, 1mM KH2PO4, pH 7.5) by dialysis or tangential flow diafiltration. siRNA was encapsulated in SNALP using a controlled stepwise dilution process. The lipid components of KC2-SNALP are DLin-KC2-DMA (cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti Polar Lipids), synthetic cholesterol (Sigma) and PEG-C-DMA, which were used in a molar ratio of 57.1:7.1:34.3:1.4. After forming the loaded particles, SNALP was dialyzed with PBS and sterilized by 0.2 μm filter filtration before use. The average particle size was 75-85 nm, and 90-95% of siRNA was encapsulated in lipid particles. The final siRNA / lipid ratio in the preparation for in vivo testing was about 0.15 (weight / weight). Immediately before use, the LNP-siRNA system containing Factor VII siRNA was diluted to an appropriate concentration in sterile PBS, and the preparation was administered intravenously with a total volume of 10 ml / kg through the lateral tail vein. This method and these delivery systems can be extrapolated to the nucleic acid targeting system of the present invention.

[0368] Other lipids

[0369] Other cationic lipids such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) can be used, for example, to encapsulate nucleic acid targeting systems or components thereof or nucleic acid molecules encoding them similar to siRNA (see, for example, Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533), and therefore can be used to practice the present invention. Preformed vesicles having the following lipid composition can be considered: amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol and (R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol) 2000)propyl carbonate (PEG-lipid) in a molar ratio of 40 / 10 / 40 / 10, respectively, and FVII siRNA / total lipid at a ratio of about 0.05 (weight / weight). To ensure a narrow size distribution in the range of 70-90 nm and a low polydispersity index of 0.11 ± 0.04 (n = 56), the particles can be extruded through an 80 nm membrane up to three times before adding the guide RNA. Particles containing the highly potent amino lipid 16 can be used, where the molar ratio of the four lipid components, 16, DSPC, cholesterol, and PEG-lipid (50 / 10 / 38.5 / 1.5) can be further optimized to enhance in vivo activity.

[0370] Michael SD Kormann et al. ("Expression of therapeutic proteins after delivery of chemically modified mRNA in mice: Nature Biotechnology, Vol. 29, pp. 154-157 (2011)) describe the use of lipid envelopes to deliver RNA. The use of lipid envelopes is also preferred in the present invention.

[0371] In another embodiment, lipids can be formulated with the RNA targeting system of the present invention (CRISPR-Cas13b complex, i.e., Cas13b compounded with crRNA) or its components or nucleic acid molecules encoding it to form lipid nanoparticles (LNPs). Lipids include but are not limited to DLin-KC2-DMA4, C12-200 and auxiliary lipids distearoylphosphatidylcholine, cholesterol, and spontaneous vesicle formation procedures can be used to formulate PEG-DMG with RNA targeting systems instead of siRNA (see, e.g., Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4; doi: 10.1038 / mtna.2011.3). The component molar ratio can be about 50 / 10 / 38.5 / 1.5 (DLin-KC2-DMA or C12-200 / distearoylphosphatidylcholine / cholesterol / PEG-DMG). In the case of DLin-KC2-DMA and C12-200 lipid particles (LNP), the final lipid: siRNA weight ratio can be about 12: 1 and 9: 1, respectively. The average particle size of the formulation can be about 80nm, and the coating efficiency is >90%. A dose of 3mg / kg can be considered. Tekmira has a portfolio of approximately 95 patent families in the U.S. and abroad covering various aspects of LNPs and LNP formulations (see, e.g., U.S. Pat. Nos. 7,982,027, 7,799,565, 8,058,069, 8,283,333, 7,901,708, 7,745,651, 7,803,397, 8,101,741, 8,188,263, 7,915,399, 8,236,943, and 7,838,658 and European Patent Nos. 1766035, 1519714, 1781593, and 1664316), all of which may be used and / or adapted for use with the present invention.

[0372] The RNA targeting system or its components or nucleic acid molecules encoding it can be encapsulated in PLGA microspheres such as those further described in U.S. published applications 20130252281 and 20130245107 and 20130244279 (assigned to Moderna Therapeutics) for delivery, which are related to the preparation of various aspects of compositions comprising modified nucleic acid molecules, which can encode proteins, protein precursors, or partially or completely processed forms of proteins or protein precursors. The molar ratio of the preparation can be 50:10:38.5:1.5-3.0 (cationic lipid: fusion lipid: cholesterol: PEG lipid). PEG lipids can be selected from, but not limited to, PEG-c-DOMG, PEG-DMG. Fusion lipids can be DSPC. See also Schrum et al., Delivery and Formulation of Engineered Nucleic Acids, U.S. published application 20120251618.

[0373] Nanomerics' technology addresses the bioavailability challenges of many therapeutics, including low molecular weight hydrophobic drugs, peptides, and nucleic acid-based therapies (plasmids, siRNA, miRNA). The technology has demonstrated significant advantages in specific routes of administration, including oral routes, transport across the blood-brain barrier, delivery to solid tumors, and ocular delivery. See, for example, Mazza et al., 2013, ACS Nano. 2013 Feb 26; 7(2): 1016-26; Uchegbu and Siew, 2013, J Pharm Sci. 102(2): 305-10 and Lalatsa et al., 2012, J Control Release. 2012 Jul 20; 161(2): 523-36.

[0374] U.S. Patent Publication No. 20050019923 describes cationic dendrimers for delivering bioactive molecules such as polynucleotide molecules, peptides and polypeptides and / or medicaments to mammals. The dendrimers are suitable for targeting and delivering bioactive molecules to, for example, liver, spleen, lungs, kidneys or heart (or even brain). Dendrimers are synthetic three-dimensional macromolecules that are progressively prepared from simple branched monomer units, and their properties and functions can be easily controlled and changed. Dendrimers are synthesized by repeatedly adding building blocks to a multifunctional core (divergent synthesis) or repeatedly adding building blocks toward a multifunctional core (convergent synthesis), and each addition of the three-dimensional shell of the building block will cause a new generation of dendrimers to form. Polypropylene imine dendrimers start from diaminobutane core, by adding acrylonitrile diMichael to primary amines, then hydrogenating nitrile, and adding twice the number of amino groups on the diaminobutane core. This causes the amino groups to double. Polypropylene imine dendrimers contain 100% protonatable nitrogen and up to 64 terminal amino groups (5th generation, DAB64). Protonatable groups are typically amine groups that can accept protons at neutral pH. The use of dendrimers as gene delivery agents is mainly focused on the use of polyamidoamines and phosphorus-containing compounds, where mixtures of amines / amides or N--P(O2)S are used as conjugation units, respectively, and no work has reported the use of lower generation polypropylene imine dendrimers for gene delivery. Polypropylene imine dendrimers have also been studied as pH-sensitive controlled release systems for delivering drugs and for encapsulating their guest molecules when chemically modified with peripheral amino acid groups. The cytotoxicity and interaction with DNA of polypropylene imine dendrimers and the transfection efficiency of DAB 64 have also been studied. U.S. Patent Publication No. 20050019923 is based on the following observations: Contrary to earlier reports, cationic dendrimers such as polypropylene imine dendrimers show suitable properties for targeted delivery of bioactive molecules such as genetic material, such as specific targeting and low toxicity. In addition, derivatives of cationic dendrimers also show suitable properties for targeted delivery of bioactive molecules. See also Bioactive Polymers (U.S. Publication Application No. 20080267903), which discloses that various polymers including cationic polyamine polymers and dendrimers have been shown to have antiproliferative activity and can therefore be used to treat diseases characterized by the occurrence of undesirable cell proliferation, such as neoplasms and tumors, inflammatory diseases (including autoimmune diseases), psoriasis and atherosclerosis. These polymers can be used alone as active agents, or as delivery vehicles for other therapeutic agents such as drug molecules or nucleic acids for gene therapy. In this case, the inherent anti-tumor activity of these polymers themselves can supplement the activity of the agent to be delivered. The disclosures of these patents can be used in combination with the teachings of this article for delivery of nucleic acid targeting systems or components thereof or nucleic acid molecules encoding them.

[0375] Supercharged Protein

[0376] Supercharged proteins are engineered or naturally occurring proteins with abnormally high positive or negative net theoretical charges and can be used to deliver nucleic acid targeting systems or components thereof or nucleic acid molecules encoding them. Both supernegatively and superpositively charged proteins exhibit significant resistance to thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Binding cargoes such as plasmid DNA, RNA or other proteins to these proteins can enable these macromolecules to be functionally delivered to mammalian cells in vitro and in vivo. David Liu's laboratory reported the production and characterization of supercharged proteins in 2007 (Lawrence et al., 2007, Journal of the American Chemical Society 129, 10110–10112).

[0377] The non-viral delivery of RNA and plasmid DNA into mammalian cells is valuable for both research and therapeutic applications (Akinc et al., 2010, Nat. Biotech. 26, 561-569). The purified +36 GFP protein (or other superpositively charged protein) is mixed with RNA in an appropriate serum-free medium and allowed to complex, and then added to the cells. The addition of serum at this stage will inhibit the formation of supercharged protein-RNA complexes and reduce the therapeutic effect. The following regimen was found to be effective for a variety of cell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106, 6111-6116). However, pilot experiments to change the protein and RNA dosage should be performed to optimize the procedure for a specific cell line. (1) One day before treatment, 1x 10 5 cells. (2) On the day of treatment, dilute purified +36GFP protein in serum-free medium to a final concentration of 200 nM. Add RNA to a final concentration of 50 nM. Vortex to mix and incubate at room temperature for 10 minutes. (3) During the incubation period, aspirate the medium from the cells and wash once with PBS. (4) After incubation with +36GFP and RNA, add the protein-RNA complex to the cells. (5) Incubate the cells with the complex at 37°C for 4 hours. (6) After incubation, aspirate the medium and wash three times with 20U / mL heparinized PBS. Incubate the cells with serum-containing medium for an additional 48 hours or longer, depending on the activity assay. (7) Analyze the cells by immunoblotting, qPCR, phenotypic assay, or other appropriate method.

[0378] +36 GFP was found to be an effective plasmid delivery agent in many cells. See also, for example, McNaughton et al., Proc. Natl. Acad. Sci. USA 106, 6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752 (2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011); Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, DB et al., Chemistry & Biology 19 (7), 831-843 (2012). The method of supercharged proteins can be used and / or adjusted to deliver the RNA targeting system of the present invention or its components or nucleic acid molecules encoding the same.

[0379] Cell Penetrating Peptides (CPP)

[0380] In yet another embodiment, cell penetrating peptides (CPPs) are contemplated for delivery of CRISPR Cas systems. CPPs are short peptides that promote cellular uptake of various molecular cargoes (from nanoscale particles to small chemical molecules and large DNA fragments). The term "cargo" as used herein includes, but is not limited to, therapeutic agents, diagnostic probes, peptides, nucleic acids, antisense oligonucleotides, plasmids, proteins, particles (including nanoparticles), liposomes, chromophores, small molecules, and radioactive substances. In various aspects of the present invention, the cargo may also include any component of the CRISPR Cas system or the entire functional CRISPR Cas system. Various aspects of the present invention also provide methods for delivering a desired cargo to a subject, comprising: (a) preparing a complex comprising a cell penetrating peptide of the present invention and a desired cargo, and (b) administering the complex orally, intraarticularly, intraperitoneally, intrathecally, intraarterially, intranasally, intraparenchymally, subcutaneously, intramuscularly, intravenously, transdermally, intrarectally, or topically to a subject. The cargo is associated with the peptide via covalent bonds or by non-covalent interactions by chemical bonding. The function of CPP is to deliver cargo into cells, a process that usually occurs through endocytosis, where cargo is delivered into the endosomes of living mammalian cells. Cell penetrating peptides have different sizes, amino acid sequences and charges, but all CPPs have a unique feature, namely the ability to transport plasma membranes and facilitate the delivery of various molecular cargoes into the cytoplasm or organelles. CPP transport can be divided into three main entry mechanisms: direct penetration of the membrane, endocytosis-mediated entry, and transport by forming temporary structures. CPPs have been widely used in medicine as drug delivery agents, as viral inhibitors, and as contrast agents for cell labeling in the treatment of different diseases (including cancer). Examples of the latter include carriers that act as GFP, MRI contrast agents, or quantum dots. CPPs have great potential as in vitro and in vivo delivery carriers for research and medicine. CPPs typically have the following amino acid composition: containing negatively charged amino acids with higher relative abundance, such as lysine or arginine, or having a sequence containing polar / charged amino acids and non-polar hydrophobic amino acids in an alternating pattern. These two types of structures are referred to as polycations or amphipathic, respectively. The third class of CPPs are hydrophobic peptides that contain only nonpolar residues, have a low net charge, or have hydrophobic amino acid groups that are critical for cellular uptake. One of the first CPPs discovered was the transactivating transcription activator (Tat) from human immunodeficiency virus 1 (HIV-1), which was found to be efficiently taken up from the surrounding medium by a variety of cultured cell types. Since then, the number of known CPPs has greatly increased, and small molecule synthetic analogs with more effective protein transduction properties have been generated. CPPs include, but are not limited to, penetratin, Tat (48-60), transportin, and (R-AhX-R4) (Ahx = aminocaproyl).

[0381] U.S. Patent No. 8,372,951 provides a CPP derived from eosinophil cationic protein (ECP), which exhibits high cell penetration efficiency and low toxicity. It is also provided that CPP is delivered to a vertebrate subject together with its payload. Other aspects of CPP and its delivery are described in U.S. Patent Nos. 8,575,305, 8; 614,194 and 8,044,019. CPP can be used to deliver CRISPR-Cas system or its components. Suresh Ramakrishna, Abu-Bonsrah KwakuDad, Jagadish Beloor et al., "Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA" (Genome Res, April 2, 2014, [Electronic version before printing]) also provide CPP can be used to deliver CRISPR-Cas system or its components, the document is incorporated by reference in its entirety, wherein it is demonstrated that the recombinant Cas9 protein conjugated with CPP and the guide RNA treatment of CPP complex can lead to endogenous gene destruction in human cell lines. In this paper, Cas9 protein is conjugated to CPP via a thioether bond, and guide RNA is complexed with CPP to form condensed negatively charged particles. The results show that simultaneous and sequential treatment of human cells, including embryonic stem cells, dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinoma cells, with modified Cas9 and guide RNA can lead to effective gene disruption, with reduced off-target mutations relative to plasmid transfection. CPP delivery can be used to practice the present invention.

[0382] Implantable Devices

[0383] In another embodiment, implantable devices are also contemplated for delivering RNA targeting systems or components thereof or nucleic acid molecules encoding them. For example, U.S. Patent Publication 20110195123 discloses an implantable medical device for local and long-term elution of drugs, including several types of such devices, treatment modes implemented, and implantation methods. The device includes a polymer substrate such as a matrix used as a device body and a drug, and in some cases also includes a scaffold material such as a metal or other polymer and a material that enhances visibility and imaging. The implantable delivery device can be advantageous for providing local and long-term release, wherein the drug is directly released into a lesion area such as a tumor, inflammation, a degenerated extracellular matrix (ECM), or for symptomatic purposes, or released into damaged smooth muscle cells, or for prevention. As disclosed above, a drug is RNA, and the system can be used for and / or is suitable for the nucleic acid targeting system of the present invention. In some embodiments, the implantation mode is an existing implantation procedure developed and used today for other treatments including brachytherapy and needle biopsy. In this case, the size of the new implant described in the present invention is similar to the original implant. Typically, several devices are implanted in the same treatment procedure. US Patent Publication 20110195123 provides a drug delivery implantable or insertable system, comprising a system suitable for use in a cavity such as the abdominal cavity and / or any other type of administration in which the drug delivery system is not anchored or attached, comprising a biostable and / or degradable and / or bioabsorbable polymer substrate, which may, for example, optionally be a matrix. It should be noted that the term "insertion" also includes implantation. As described in US Patent Publication 20110195123, the drug delivery system is preferably implemented as a "Loder". The polymer or multiple polymers are biocompatible, combined with a pharmaceutical agent and / or multiple pharmaceutical agents, enabling the release of the pharmaceutical agent at a controlled rate, wherein in some embodiments, the total volume of a polymer matrix such as a matrix is ​​optionally and preferably not greater than the maximum volume that allows the therapeutic level of the pharmaceutical agent to be reached. As a non-limiting example, such a volume is preferably 0.1m 3 Up to 1000mm 3In the range of, as required by the volume of the drug load. For example, when combined with a device whose size is determined by functionality (such as, but not limited to, a knee joint, an intrauterine contraceptive ring or a cervical ring, etc.), the Loder may be optionally larger. In some embodiments, the drug delivery system (for delivering the composition) is designed to preferably use a degradable polymer, wherein the main release mechanism is bulk erosion. Or in certain embodiments, a non-degradable or slowly degradable polymer is used, wherein the main release mechanism is diffusion rather than bulk erosion, so the outside acts as a membrane, and the inside acts as a drug reservoir, which is almost unaffected by the environment for a long time (such as about one week to about several months). A combination of different polymers with different release mechanisms may also be optionally used. Over a considerable period of time during the total drug release period, it is preferred to maintain the concentration gradient at the surface effectively constant, so the diffusion rate is effectively constant (referred to as "zero mode" diffusion). The so-called term "constant" refers to a diffusion rate preferably maintained above a lower threshold of the therapeutic effect, but it may still optionally have the characteristics of an initial burst release and / or may fluctuate, such as thereby increasing and decreasing to a certain extent. The diffusion rate is preferably maintained in this way for a long time, and it can be considered constant to a certain level to optimize the effective period of treatment, such as an effective silent period. The drug delivery system is optionally and preferably designed to protect nucleotide-based therapeutic agents from degradation, whether due to chemical properties or due to attacks by enzymes and other factors in the subject. The drug delivery system of U.S. Patent Publication No. 20110195123 is optionally associated with sensing and / or activation devices, which are operated when and / or after the device is implanted by a non-invasive and / or minimally invasive method of activation and / or acceleration / deceleration, the method, for example, optionally includes but is not limited to thermal heating and cooling, laser beams and ultrasound, including focused ultrasound and / or RF (radio frequency) methods or devices. According to some embodiments of U.S. Patent Publication 20110195123, the position for local delivery may optionally include a target position characterized by highly abnormal proliferation of cells, suppressed apoptosis, including tumors, activity and / or chronic inflammation and infection, including autoimmune disease states, degenerative tissues (including muscle and nerve tissue), chronic pain, degenerative positions, and fracture positions and other wound positions for enhancing tissue regeneration, as well as damaged myocardium, smooth muscle and striated muscle. The position or target position for implanting the composition preferably has a radius, area and / or volume small enough for targeted local delivery. For example, the diameter of the target position is optionally in the range of about 0.1mm to about 5cm. The position of the target position is preferably selected to achieve maximum therapeutic efficacy. For example, the composition of the drug delivery system (optionally together with the device for implantation as described above) is implanted in or near the tumor environment or the blood supply associated therewith.For example, the composition (optionally together with the device) is optionally implanted via the nipple within the vascular system in or near the pancreas, prostate, breast, liver, etc. The target location is optionally selected from, essentially consisting of, or consisting of the following locations (only as non-limiting examples, as optionally any location in the body may be suitable for implantation of Loder): 1. Degenerative sites in the brain, such as the basal ganglia, white matter and gray matter in Parkinson's disease or Alzheimer's disease; 2. Spine, such as amyotrophic lateral sclerosis (ALS); 3. Cervix, for prevention of HPV infection; 4. Active and chronic inflammatory joints; 5. Dermis, such as psoriasis; 6. Sympathetic and sensory nerve sites, for analgesic effects; 7. .Intraosseous implantation; 8. Sites of acute and chronic infection; 9. Intravaginal; 10. Inner ear auditory system, inner ear labyrinth, vestibular system; 11. Intratracheal 12. Intracardiac, coronary arteries, epicardium; 13. Bladder; 14. Biliary system; 15. Parenchymal tissue, including but not limited to kidney, liver, spleen; 16. Lymph nodes; 17. Salivary glands; 18. Gum; 19. Intra-articular (entering a joint); 20. Intra-ocular; 21. Brain tissue; 22. Ventricles; 23. Cavities, including the abdominal cavity (for example but not limited to ovarian cancer); 24. Intra-esophageal and 25. Intra-rectal.

[0384] Optionally, insertion of the system (e.g., a device containing the composition) is associated with injection of a material into the ECM at and near the target site to affect the local pH and / or temperature at and near the target site and / or other biological factors that affect drug diffusion and / or drug dynamics in the ECM. Optionally, according to some embodiments, the release of the agent can be associated with sensing and / or activation devices that operate during and / or after implantation of the device by non-invasive and / or minimally invasive and / or other methods of activation and / or acceleration / deceleration, including laser beams, radiation, thermal heating and cooling, and ultrasound, including focused ultrasound and / or RF (radio frequency) methods or devices and chemical activators.

[0385] According to the embodiments of U.S. Patent Publication 20110195123, which can be used to practice the present invention, the drug preferably comprises RNA, for example, for localized cancer cases in the breast, pancreas, brain, kidney, bladder, lung and prostate as described below. Although RNAi is used as an example, many drugs are suitable for encapsulation in Loder and can be used in conjunction with the present invention, as long as these drugs can be encapsulated in Loder substrates such as matrix, and such systems can be used and / or adjusted to deliver the nucleic acid targeting system of the present invention. As another example of a specific application, neurological and muscle degenerative diseases occur due to abnormal gene expression. Local delivery of RNA can have therapeutic properties to interfere with such abnormal gene expression. Local delivery of anti-apoptotic, anti-inflammatory and anti-degenerative drugs including small drugs and macromolecules can also optionally be therapeutic. In this case, Loder is used to extend release at a constant rate and / or by a dedicated device implanted separately.

[0386] All of these can be used and / or applied to the RNA targeting system of the present invention. The implantable device technology discussed herein can be used together with the teachings herein, so according to the present disclosure and the knowledge in the art, the CRISPR-Cas13b system or complex or its components or nucleic acid molecules encoding or providing components can be delivered via an implantable device.

[0387] Patient-specific screening methods

[0388] Nucleic acid targeting systems that target RNA can be used to screen patients or patient samples for the presence of specific RNA.

[0389] CRISPR effector protein mRNA and guide RNA

[0390] The CRISPR effector protein (Cas13b) or its mRNA (or more generally its nucleic acid molecule) and the guide RNA or crRNA may also be delivered separately, for example, the guide RNA or crRNA may be administered 1-12 hours (preferably about 2-6 hours) after the former is administered, or they may be delivered together. A second booster dose of guide RNA or crRNA may be administered 1-12 hours (preferably about 2-6 hours) after the initial administration.

[0391] Cas13b effector proteins are sometimes referred to herein as CRISPR enzymes. It should be understood that effector proteins are based on or derived from enzymes, so in some embodiments, the term "effector protein" certainly includes "enzymes". However, it should also be understood that, as required in some embodiments, effector proteins may have DNA or RNA binding activity, but not necessarily cutting or nicking activity, including death Cas effector protein functions.

[0392] Cellular targets include hematopoietic stem / progenitor cells (CD34+); human T cells; and the eye (retinal cells), such as photoreceptor precursor cells.

[0393] The method of the present invention may also include the delivery of a template. The delivery of the template may be carried out simultaneously or separately with the delivery of any or all CRISPR effector proteins (Cas13b) or guide sequences or crRNA, and may be carried out via the same or different delivery mechanisms.

[0394] Inducible system

[0395] In some embodiments, CRISPR effector protein (Cas13n) may form a component of an inducible system. The inducible nature of the system will allow the use of an energy form to control gene editing or gene expression in time and space. Energy forms may include, but are not limited to, electromagnetic radiation, acoustic energy, chemical energy, and thermal energy. Examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), small molecule dual hybrid transcriptional activation systems (FKBP, ABA, etc.), or light-inducible systems (photochrome, LOV domain, or cryptochrome). In one embodiment, the CRISPR effector protein may be part of a light-inducible transcriptional effector (LITE) to guide changes in transcriptional activity in a sequence-specific manner. The components of light may include CRISPR effector proteins, light-responsive cytochrome heterodimers (e.g., from Arabidopsis thaliana), and transcriptional activation / repression domains. Other examples of inducible DNA binding proteins and methods of using them are provided in US 61 / 736465 and US 61 / 721,283 and WO2014018423 A2, which are hereby incorporated by reference in their entirety.

[0396] Self-deactivation system

[0397] Once all copies of the RNA in a cell have been edited, there is no need for continued Cas13b effector protein expression or activity in that cell. Self-inactivating systems that rely on using Cas13b's RNA or crRNA as a guide target sequence can shut down the system by preventing Cas13b expression or complex formation.

[0398] Reagent test kit

[0399] On the one hand, the present invention provides a kit containing any one or more elements disclosed in the above methods and compositions. In some embodiments, the kit comprises one or more components of the vector system taught herein or the CRISPR / Cas13b system or complex taught herein, such as crRNA and / or Cas13b effector protein or mRNA encoding Cas13b effector protein, and instructions for using the kit. These elements may be provided alone or in combination, and may be provided in any suitable container such as a vial, bottle or tube. In some embodiments, the kit includes instructions in one or more languages, such as more than one language. The instructions may be specific to the applications and methods described herein. In some embodiments, the kit comprises one or more reagents for use in the process of utilizing one or more elements described herein. The reagent may be provided in any suitable container. For example, the kit may provide one or more reaction or storage buffers. The reagent may be provided in a form that can be used for a specific assay, or in a form that requires the addition of one or more other components before use (e.g., in a concentrated or lyophilized form). The buffer may be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the pH of the buffer is about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to the guide sequence for insertion into the vector so as to operably connect the guide sequence or crRNA sequence and the regulatory element. In some embodiments, the kit comprises a homologous recombination template polynucleotide. In some embodiments, the kit comprises one or more vectors and / or one or more polynucleotides as described herein. The kit can advantageously allow all elements of the system of the present invention to be provided.

[0400] The present invention has wide applications in, for example, gene therapy, drug screening, disease diagnosis and prognosis.

[0401] The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid", and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment; loci defined by linkage analysis; exons; introns; messenger RNA (mRNA); transfer RNA; ribosomal RNA; short interfering RNA (siRNA); short hairpin RNA (shRNA); micro RNA (miRNA); ribozymes; cDNA; recombinant polynucleotides; branched polynucleotides; plasmids; vectors; isolated DNA of any sequence; isolated RNA of any sequence, nucleic acid probes; and primers. The term also includes nucleic acid-like structures with synthetic backbones, see, for example, Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97 / 03211; WO 96 / 39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if any, may be imparted before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. As used herein, the term "wild type" is a term understood by those skilled in the art, and refers to a typical form of an organism, strain, gene or feature that exists in nature, which is different from a mutant or variant form. "Wild type" may be a baseline. As used herein, the term "variant" should be considered to refer to a quality that exhibits a pattern that is different from that found in nature. The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate artificial involvement. When referring to a nucleic acid molecule or polypeptide, these terms refer to a nucleic acid molecule or polypeptide that is at least substantially free of at least one other component that is naturally associated with the nucleic acid molecule or polypeptide in nature and exists in nature. "Complementarity" refers to the ability of one nucleic acid to form hydrogen bonds with another nucleic acid sequence through traditional Watson-Crick base pairing or other non-traditional types. The complementarity percentage represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90% and 100% complementary). "Fully complementary" means that all consecutive residues of a nucleic acid sequence will hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence.As used herein, "substantially complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or refers to hybridization of two nucleic acids under stringent conditions. As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence hybridizes primarily with the target sequence and does not substantially hybridize with non-target sequences. Stringent conditions are generally sequence dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes with its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Chapter II "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY. When referring to polynucleotide sequences, complementary or partially complementary sequences can also be envisioned. These sequences are preferably able to hybridize with a reference sequence under highly stringent conditions. Typically, in order to maximize the hybridization rate, relatively low stringency hybridization conditions are selected: specific thermal melting point (T. m ) is about 20℃ to 25℃ lower. m It is the temperature at which 50% of the specific target sequence hybridizes to a perfectly complementary probe in solution under defined ionic strength and pH.

[0402] Generally, to require at least about 85% nucleotide complementarity of the hybridizing sequences, the ratio T is selected. m Highly stringent washing conditions are about 5°C to 15°C lower. To require at least about 70% nucleotide complementarity of the hybridizing sequences, a temperature lower than T is selected. m Moderately stringent washing conditions are about 15°C to 30°C lower. Highly permissive (very low stringency) washing conditions can be as low as 15°C lower than the T. m50 ℃ lower, thereby allowing high levels of mismatching between hybridization sequences. It will be appreciated by those skilled in the art that other physical and chemical parameters in the hybridization phase and the washing phase may also be changed to affect the results of detectable hybridization signals according to the specific homology level between the target sequence and the probe sequence. Preferred highly stringent conditions include incubation at 42 ℃ in 50% formamide, 5xSSC and 1% SDS, or incubation at 65 ℃ in 5xSSC and 1% SDS, and washing in 0.2xSSC and 0.1% SDS. "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding or any other sequence-specific manner. The complex may include two chains forming a double-stranded structure, three or more chains forming a multi-stranded complex, a single self-hybridizing chain, or any combination of these. The hybridization reaction may constitute a step in a broader process, such as the initiation of PCR, or the cleavage of polynucleotides with an enzyme. The sequence that can hybridize with a given sequence is referred to as the "complementary sequence" of the given sequence. As used herein, the term "genomic locus" or "locus" (multiple loci) is a specific position of a gene or DNA sequence on a chromosome. "Gene" refers to a segmented DNA or RNA encoding a polypeptide or RNA chain, which has a functional role in an organism and is therefore a genetic molecular unit in a living organism. For the purposes of the present invention, it can be considered that a gene includes a region that regulates the production of a gene product, regardless of whether such a regulatory sequence is adjacent to a coding sequence and / or a transcribed sequence. Therefore, a gene includes but is not necessarily limited to a promoter sequence, a terminator, a translation regulatory sequence (such as a ribosome binding site and an internal ribosome entry site), an enhancer, a silencer, an insulator, a boundary element, a replication origin, a matrix binding site, and a locus control region. As used herein, "expression of a genomic locus" or "gene expression" is a process in which information from a gene is used to synthesize a functional gene product. The product of gene expression is often a protein, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is a functional RNA. All known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea), and viruses - use the process of gene expression to produce functional products to survive. As used herein, "expression" of a gene or nucleic acid encompasses not only cellular gene expression, but also transcription and translation of nucleic acids in cloning systems and any other contexts. As used herein, "expression" also refers to the process by which a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcripts) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may be collectively referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in a eukaryotic cell.The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified; the modification being, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or non-natural or synthetic amino acids, including glycine and right-handed or left-handed optical isomers, as well as amino acid analogs and peptide mimetics. As used herein, the term "domain" or "protein domain" refers to a portion of a protein sequence that can exist and function independently of the rest of the protein chain. As described in various aspects of the invention, sequence identity is related to sequence homology. Homology comparisons can be made by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent (%) homology between two or more sequences, and can also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences.

[0403] As used herein, the term "wild type" is a term understood by those skilled in the art and refers to the typical form of an organism, strain, gene or trait that occurs in nature, as distinct from a mutant or variant form. "Wild type" can be a baseline.

[0404] As used herein, the term "variant" should be understood to mean the expression of a property with a pattern of deviation from natural occurrence. The terms "non-naturally occurring" or "engineered" are used interchangeably and represent human intervention. When referring to nucleic acid molecules or polypeptides, the term means that the nucleic acid molecules or polypeptides are at least substantially free of at least one other component that is naturally combined with them in nature and as found in nature. In all aspects and embodiments, whether or not they include these terms, it should be understood that, preferably, it can be optional, and therefore preferably is included or not preferably is not included. In addition, the terms "non-naturally occurring" and "engineered" can be used interchangeably, and therefore can be used alone or in combination, and one or the other can replace the mention of both together. In particular, "engineered" is preferred instead of "non-naturally occurring" or "non-naturally occurring and / or engineered".

[0405] Sequence homology can be generated by any of many computer programs known in the art (such as BLAST or FASTA, etc.). Suitable computer programs for performing this comparison are GCG Wisconsin Bestfit software packages (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include but are not limited to BLAST software packages (see Ausubel et al., 1999 ibid-Chapter 18), FASTA (Atschul et al., 1990, J.Mol.Biol., 403-410) and GENEWORKS comparison tool suite. BLAST and FASTA can be used for offline and online searches (see Ausubel et al., 1999 ibid, 7-58 pages to 7-60 pages). However, preferably, the GCG Bestfit program is used. Percent (%) sequence homology can be calculated on a continuous sequence, that is, one sequence is aligned with another sequence, and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such an ungapped alignment is performed only on a relatively small number of residues. Although this is a very simple and consistent method, it fails to take into account that, for example, an insertion or deletion in a pair of otherwise identical sequences may cause subsequent amino acids to be squeezed out of the alignment, and therefore, when performing a global alignment, it is likely to cause a large-scale reduction in percent homology. Therefore, most sequence comparison methods are designed to produce the best alignment taking into account possible insertions and deletions without excessively penalizing the overall homology or identity score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology or identity. However, these more complex methods provide for "gap penalties" for each gap that appears in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting a higher correlation between the two compared sequences - can obtain a higher score compared to a sequence with many gaps. "Affinify gap costs" are usually used, which allow the presence of a gap to bear a relatively high cost, while allowing each subsequent residue in the gap to bear a smaller penalty. This is the most commonly used gap scoring system. High gap penalties can certainly produce the best alignment with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, it is preferred to use default values ​​when using this software for sequence comparison. For example, when using the GCG Wisconsin Bestfit software package, the default gap penalty for amino acid sequences is -12 for each gap and -4 for each extension.Therefore, the calculation of maximum percentage homology ratio first requires to produce the best comparison under the situation that taking into account the gap penalty.The suitable computer program for carrying out this comparison is GCG Wisconsin Bestfit software package (Devereux etc., 1984Nuc.Acids Research 12 the 387th page).The example of other software outside executable sequence comparison includes but is not limited to BLAST software package (referring to Ausubel etc., 1999Short Protocols in Molecular Biology, the 4th edition-the 18th chapter), FASTA (Altschul etc., 1990J.Mol.Biol.403-410) and GENEWORKS comparison tool suite.BLAST and FASTA can all be used for off-line and online search (referring to Ausubel etc., 1999, Short Protocols inMolecular Biology, the 7-58th page to the 7-60th page).But, for some applications, preferably use GCG Bestfit program. A new tool called BLAST 2 Sequences can also be used to compare protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174 (2): 247-50; FEMS Microbiol Lett. 1999 177 (1): 187-8 and the National Center for Biotechnology Information on the National Institutes of Health website). Although the final percentage homology can be measured based on identity, the alignment process itself is usually not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is ​​usually used, which assigns scores to each pair comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. The GCG Wisconsin program usually uses a published default value or a custom symbol comparison table, if provided (see the user manual for further details). For some applications, it is preferred to use the default values ​​published by the GCG software package, or in other software, the default matrix, such as BLOSUM62. Alternatively, the percent homology can be based on an algorithm similar to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73 (1), 237-244), using DNASIS. TMThe multiple alignment feature in (Hitachi Software) is used for calculation. Once the software has produced the best comparison, it is possible to calculate the percentage homology, preferably the percentage sequence identity. The software usually performs this as a part of sequence comparison and generates a digital result. The sequence may also have a deletion, insertion or substitution of an amino acid residue, which produces a silent change, resulting in the production of a functionally equivalent substance. Intentional amino acid substitutions can be made based on the similarity of amino acid properties (such as, the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic properties of the residue), so it is useful to compile amino acids into functional groups together. Amino acids can be grouped together based only on the properties of their side chains. However, it is more useful to also include mutation data. For structural reasons, the amino acid groups derived from this may be conservative. These groups can be described in the form of a Venn diagram (Livingstone CD and Barton GJ (1993) "Protein sequence alignments: astrategy for the hierarchical analysis of residue conservation" Comput. Appl. Biosci. 9: 745-756) (Taylor WR (1986) "The classification of aminoacid conservation" J. Theor. Biol. 119; 205-218). Conservative substitutions can be made, for example, according to the following table describing the generally accepted Venn diagram groupings of amino acids (see also the discussion of conservative substitutions herein in conjunction with Table 1).

[0406]

[0407] References herein to conservative substitutions in conjunction with Table 1 are also referred to as referring to the same as conservative substitutions.

[0408] The terms "subject", "individual" and "patient" are used interchangeably herein and refer to vertebrates, preferably mammals, more preferably humans. Mammals include, but are not limited to, mice, monkeys, humans, farm animals, competitive animals and pets. Tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro are also covered.

[0409] The terms "therapeutic agent," "therapeutic capable agent," or "treatment agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect when administered to a subject. Beneficial effects include enabling a diagnostic determination; ameliorating a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally combating a disease, symptom, disorder, or pathological condition. As used herein, "treatment," "treating," "palliating," or "ameliorating" are used interchangeably. These terms refer to methods of obtaining beneficial or desired results, including, but not limited to, therapeutic benefit and / or prophylactic benefit. A therapeutic benefit means any treatment-related improvement in or effect on one or more of the diseases, disorders, or symptoms being treated. For prophylactic benefit, a composition may be administered to a subject at risk for developing a particular disease, disorder, or symptom, or to a subject who reports one or more physiological symptoms of a disease, even though the disease, disorder, or symptom may not yet be manifested. The term "effective amount" or "therapeutically effective amount" refers to the amount of an agent sufficient to achieve a beneficial or desired result. The therapeutically effective amount may vary depending on one or more of the following: the subject and the disease condition being treated, the subject's weight and age, the severity of the disease condition, the mode of administration, etc., and the therapeutically effective amount can be easily determined by one of ordinary skill in the art. The term also applies to doses that will provide an image for detection by any of the imaging methods described herein. The specific dose may vary depending on one or more of the following: the specific agent selected, the dosing regimen to be followed, whether it is administered in combination with other compounds, the time of administration, the tissue to be imaged, and the physical delivery system in which it is carried.

[0410] Unless otherwise indicated, the implementation of the present invention adopts the routine techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the technical scope of the art. Referring to Sambrook, Fritsch and Maniatis, MOLECULAR CLONING:A LABORATORY MANUAL, the 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., ed. (1987)); the series METHODS EN ENZYMOLOGY (Academic Press, Inc.): PCR 2:A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor ed. (1995)), Harlow and Lane, ed. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (RI Freshney, ed. (1987)). Several aspects of the present invention relate to a vector system comprising one or more vectors or multiple vectors themselves. The vector can be designed to express CRISPR transcripts (e.g., nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, a T7 promoter regulatory sequence and a T7 polymerase can be used, for example, to transcribe and translate a recombinant expression vector in vitro. Embodiments of the present invention include sequences (polynucleotides or polypeptides) that may contain homologous substitutions (substitution and substitution are used herein to represent the interchange of existing amino acid residues or nucleotides with alternative residues or nucleotides) that may occur (i.e., in the case of amino acids, like-for-like substitutions, such as substitutions of alkaline to alkaline, acidic to acidic, polar to polar, etc.). Non-homologous substitutions, i.e., substitutions from one class of residues to another, or alternatively involving the inclusion of non-natural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyridylalanine, thienylalanine, naphthylalanine and phenylglycine, may also occur.The variant amino acid sequence may include a suitable spacer group between any two amino acid residues that may be inserted into the sequence, the spacer group, in addition to amino acid spacers such as glycine or β-alanine residues, also includes an alkyl group such as a methyl, ethyl or propyl group. Those skilled in the art can well understand the changes in other forms, which relate to the presence of one or more amino acid residues in a peptoid form. For the avoidance of doubt, a "peptoid form" is used to refer to a variant amino acid residue in which the oc-carbon substituent is located on the nitrogen atom of the residue rather than on the oc-carbon. Methods for preparing peptides in peptoid form are known in the art, such as Simon RJ et al., PNAS (1992) 89 (20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13 (4), 132-134. Homology modelling: Corresponding residues in other Cas13borthologs can be identified by the methods of Zhang et al. 2012 (Nature; 490 (7421): 556-60) and Chen et al., 2015 (PLoS Comput Biol; 11 (5): el004248) - computational protein-protein interaction (PPI) methods that predict interactions mediated by domain-motif interfaces. PrePPI (predicted PPI) (a structure-based PPI prediction method) combines structural evidence with non-structural evidence using a Bayesian statistical framework. The method involves obtaining a pair of query proteins and using structural alignments to identify structural representatives corresponding to their experimentally determined structures or homology models. By considering global and local geometric relationships, the structural alignment is further used to identify structural neighbors and structural distant neighbors. Whenever two neighbors represented by the structure form a complex reported in the protein database, this defines a template for modeling the interaction between the two query proteins. The model of the complex is created by superimposing the representative structure on its corresponding structural neighbors in the template. This method is further described in Dey et al., 2013 (Prot Sci; 22: 359-66).

[0411] For the purposes of the present invention, amplification means any method using primers and a polymerase that can replicate the target sequence with reasonable fidelity. TM, T7 DNA polymerase, Klenow fragment of Escherichia coli DNA polymerase and reverse transcriptase) for amplification. The preferred amplification method is PCR. In certain aspects, the present invention relates to vectors. 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, a phage or a cosmid, into which another DNA segment can be inserted to achieve the replication of the inserted segment. Generally, when combined with appropriate control elements, the vector is able to replicate. Generally, the term "vector" refers to a nucleic acid molecule that is capable of transporting another nucleic acid that has been connected to it. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded or partially double-stranded; nucleic acid molecules that contain one or more free ends, no free ends (e.g., circular); nucleic acid molecules containing DNA, RNA or both; and other polynucleotide variants 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, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which a DNA or RNA sequence of viral origin is present in the vector for packaging into a virus (e.g., a retrovirus, a replication-defective retrovirus, an adenovirus, a replication-defective adenovirus, and an 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 host cells into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, certain vectors are capable of directing the expression of genes operably connected thereto. Such vectors are referred to herein as "expression vectors". Commonly used expression vectors useful in recombinant DNA technology are generally in the form of plasmids. Recombinant expression vectors can include the nucleic acid of the present invention in a form suitable for expressing nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory elements, which can be selected according to the host cell to be used for expression, and are operably connected to the nucleic acid sequence to be expressed. In a recombinant expression vector, "operably linked" is intended to mean that the target nucleotide sequence is linked to one or more regulatory elements in a manner that allows the nucleotide sequence to be expressed (e.g., in an in vitro transcription / translation system or in a host cell when the vector is incorporated into a host cell). With regard to recombination and cloning methods, reference is made to U.S. patent application Ser. No. 10 / 815,730, published as US2004-0171156 A1 on Sep. 2, 2004 (the contents of which are incorporated herein by reference in their entirety). Aspects of the invention relate to bicistronic vectors for guide RNA and wild-type, modified or mutant CRISPR effector proteins / enzymes (e.g., Cas13b effector proteins).Bicistronic expression vectors guide RNA and wild-type, modified or mutated CRISPR effector protein / enzyme (e.g., Cas13b effector protein) are preferred. Typically, especially in the present embodiment and wild-type, modified or mutated CRISPR effector protein / enzyme (e.g., Cas13b effector protein) is preferably driven by a CBh promoter. RNA can preferably be driven by a Pol III promoter (such as a U6 promoter). Ideally, the two are combined.

[0412] In some embodiments, a loop in a guide RNA or crRNA is provided. This can be a stem loop or a tetraloop. The loop is preferably GAAA, but is not limited to the sequence or is actually only 4bp in length. In fact, the preferred loop-forming sequence for a hairpin structure is 4 nucleotides in length, most preferably with the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. The sequence preferably includes a nucleotide triplet (e.g., AAA) and additional nucleotides (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG.

[0413] In implementing any method disclosed herein, suitable vectors can be introduced into cells or embryos by one or more methods known in the art, including but not limited to microinjection, electroporation, sonoporation, bioprojectiles, calcium phosphate mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nuclear transfection transfection, magnetofection, lipofection, piercing transfection, optical transfection, nucleic acid uptake enhanced by patented reagents, and delivery by liposomes, immunoliposomes, virosomes or artificial virosomes. In some methods, vectors are introduced into embryos by microinjection. One or more vectors can be microinjected into the nucleus or cytoplasm of the embryo. In some methods, one or more vectors can be introduced into cells by nuclear transfection.

[0414] Vectors can be designed to express CRISPR transcripts (e.g., nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells (such as E. coli), insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are further discussed in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, for example, a recombinant expression vector can be transcribed and translated in vitro using a T7 promoter regulatory sequence and T7 polymerase.

[0415] The vector can be introduced into a prokaryotic or eukaryotic cell and amplified in a prokaryotic or prokaryotic cell. In some embodiments, a prokaryotic organism is used to amplify a copy of a vector to be introduced into a eukaryotic cell or is used as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (for example, a plasmid is amplified as part of a viral vector packaging system). In some embodiments, a prokaryotic organism is used to amplify a copy of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. Protein expression in prokaryotic organisms is most commonly performed in Escherichia coli with a vector containing a constitutive or inducible promoter that directs the expression of a fusion protein or a non-fusion protein. Fusion vectors add many amino acids to the protein encoded therein, such as to the amino terminus of a recombinant protein. Such fusion vectors can be used for one or more purposes, such as: (i) increasing the expression of a recombinant protein; (ii) increasing the solubility of a recombinant protein; and (iii) assisting the purification of a recombinant protein by acting as a ligand in affinity purification. Typically, in a fusion expression vector, a proteolytic cleavage site is introduced at the junction of a fusion moiety and a recombinant protein so that the recombinant protein can be separated from the fusion moiety after the fusion protein is purified. Such enzymes and their homologous recognition sequences include factor Xa, thrombin and enterokinase. Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988.Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, NJ) fused with glutathione S-transferase (GST), maltose E binding protein or protein A to target recombinant protein, respectively. Examples of suitable inducible non-fusion Escherichia coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET lid (Studier et al., GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). In some embodiments, the vector is a yeast expression vector.Examples of vectors for expression in Saccharomyces cerevisiae include pYepSec1 (Baldari et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.) and picZ (InVitrogen Corp, San Diego, Calif.). In some embodiments, the vector drives protein expression in insect cells using a baculovirus expression vector. Baculovirus vectors that can be used to express proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39). In some embodiments, the vector can drive the expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the control function of the expression vector is generally provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma viruses, adenovirus 2, cytomegalovirus, simian virus 40, and other promoters disclosed herein and known in the art. For other suitable expression systems for prokaryotic and eukaryotic cells, see, e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Chapters 16 and 17. In some embodiments, the recombinant mammalian expression vector is capable of directing the nucleic acid to be preferentially expressed in a particular cell type (e.g., using tissue-specific regulatory elements for expressing the nucleic acid). Tissue-specific regulatory elements are known in the art.Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., 1987. Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43:235-275), in particular promoters of the T-cell receptor (Winoto and Baltimore, 1989. EMBO J. 8:729-733) and immunoglobulins (Baneiji et al., 1983. Cell 33:729-740; Queen and Baltimore, 1983. Cell 33:741-748), neuron-specific promoters (e.g., neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al., 1985. Science 230:912-916) and mammary gland-specific promoters (e.g., lactalbumin promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally regulated promoters are also contemplated, such as the mouse hox promoter (Kessel and Gruss, 1990. Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3:537-546). With respect to these prokaryotic and eukaryotic vectors, reference is made to U.S. Patent No. 6,750,059, the contents of which are incorporated herein by reference in their entirety. Other embodiments of the present invention may involve the use of viral vectors, with respect to which reference is made to U.S. Patent Application No. 13 / 092,085, the contents of which are incorporated herein by reference in their entirety. Tissue-specific regulatory elements are known in the art, and in this regard reference is made to US Pat. No. 7,776,321, the contents of which are incorporated herein by reference in their entirety.

[0416] In some embodiments, the regulatory element is operably connected to one or more elements of the CRISPR Cas13b system or complex or encodes the CRISPR Cas13b system or complex to drive the expression of the one or more elements of the CRISPR system. Typically, CRISPR (Regular Clustered Short Palindromic Repeats), also known as SPIDR (SPacer Interspersed Direct Repeats), constitutes a family of DNA loci that are usually specific to a particular bacterial species. The CRISPR locus contains different classes of interspersed short sequence repeats (Ishino et al., J. Bacteriol., 169: 5429-5433

[1987] ; and Nakata et al., J. Bacteriol., 171: 3553-3556

[1989] ) and related genes that are recognized in Escherichia coli. Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol., 10:1057-1065

[1993] ; Hoe et al., Emerg. Infect. Dis., 5:254-263

[1999] ; Masepohl et al., Biochim. Biophys. Acta 1307:26-30

[1996] ; and Mojica et al., Mol. Microbiol., 17:85-93

[1995] ). CRISPR loci are often distinguished from other SSRs by the structure of the repeat sequences, which are called short regularly interspaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33

[2002] ; and Mojica et al., Mol. Microbiol., 36:244-246

[2000] . Typically, the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences of essentially constant length (Mojica et al.,

[2000] , supra). Although the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequence of the spacers often vary from strain to strain (van Embden et al., J. Bacteriol., 182:2393-2401

[2000] ).CRISPR loci have been identified in more than 40 prokaryotes (see, e.g., Jansen et al., Mol. Microbiol., 43:1565-1575

[2002] ; and Mojica et al.,

[2005] ), including but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, and others. thanosarcina), Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeri a), Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter ter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

[0417] In general, as used in the present application, "RNA targeting system" refers to transcripts and other elements involved in the activity of expressing or guiding RNA targeting CRISPR-associated 13b ("Cas13b") genes (also referred to herein as effector proteins), including sequences encoding RNA targeting Cas (effector) proteins and guide RNAs (or crRNA sequences), with reference to Table 1 as discussed herein. In general, the RNA targeting system is characterized by promoting the formation of elements of RNA targeting complexes at the site of the target sequence. In the context of forming RNA targeting complexes, "target sequence" refers to an RNA sequence that is designed to have complementarity with the guide sequence (or guide sequence of a guide or crRNA), wherein hybridization between the target sequence and the guide RNA promotes the formation of RNA targeting complexes. Complete complementarity is not necessarily required, provided that there is sufficient complementarity to cause hybridization and promote the formation of RNA targeting complexes. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. In some embodiments, the target sequence may be in an organelle of a eukaryotic cell. A sequence or template that can be used to reorganize into a targeting locus containing a target sequence is referred to as an "editing template" or "editing RNA" or "editing sequence". In aspects of the invention, the exogenous template RNA may be referred to as an editing template. In one aspect of the invention, the recombination is homologous recombination. In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize with the target sequence and guide sequence-specific binding of a nucleic acid targeting complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or greater when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined with the aid of 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), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).In some embodiments, the length of the guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides. In some embodiments, the length of the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides. The ability of the guide sequence to direct the sequence-specific binding of the RNA targeting complex to the target sequence can be assessed by any suitable assay. The template polynucleotide can have any suitable length, such as a length of about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000 or more nucleotides. In some embodiments, the template polynucleotide is complementary to a portion of a polynucleotide comprising a target sequence. When optimally aligned, the template polynucleotide may overlap with one or more nucleotides (e.g., about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more nucleotides) of the target sequence. In some embodiments, when the template sequence and the polynucleotide comprising the target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence. In some embodiments, the RNA-targeting effector protein is part of a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the nucleic acid-targeting effector protein). In some embodiments, the CRISPR Cas13b effector protein / enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR Cas13b enzyme). Examples of protein domains that can be fused to effector proteins include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza virus hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). The effector protein targeting nucleic acid can be fused to a gene sequence encoding a protein or a fragment of a protein that binds to a DNA molecule or binds to other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusion, GAL4 DNA binding domain fusion, and herpes simplex virus (HSV) BP 16 protein fusion. Additional domains that may form part of a fusion protein comprising a nucleic acid targeting effector protein are described in US20110059502, which is incorporated herein by reference. In some embodiments, a labeled nucleic acid targeting effector protein is used to identify the location of a target sequence. In some embodiments, the CRISPR Cas13b enzyme may form a component of an inducible system. The inducible nature of the system will allow the use of an energy form for spatiotemporal control of gene editing or gene expression. The energy form may include, but is not limited to, electromagnetic radiation, acoustic energy, chemical energy, and thermal energy. Examples of inducible systems include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule dual hybrid transcriptional activation systems (FKBP, ABA, etc.), or light-inducible systems (phytochrome, LOV domains, or cryptochromes). In one embodiment, the CRISPR Cas13b enzyme may be a part of a light-inducible transcriptional effector (LITE) to guide changes in transcriptional activity in a sequence-specific manner. The components of light may include CRISPR enzymes, light-responsive cytochrome heterodimers (e.g., from Arabidopsis thaliana), and transcriptional activation / repression domains. Other examples of inducible DNA binding proteins and methods of use thereof are provided in US 61 / 736465 and US 61 / 721,283 and WO 2014 / 018423 and US8889418, US8895308, US20140186919, US20140242700, US20140273234, US20140335620, WO2014093635, which are hereby incorporated by reference in their entirety. In some aspects, the invention provides methods including delivering one or more polynucleotides, such as one or more vectors as described herein, one or more transcripts thereof and / or one or more proteins transcribed therefrom to a host cell. In some aspects, the invention also provides cells produced by such methods and organisms (such as animals, plants or fungi) comprising such cells or produced by such cells.In some embodiments, RNA targeting effector proteins combined with guide RNA or crRNA (and optionally compounded therewith) are delivered to cells. Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to apply nucleic acids encoding components of RNA targeting systems to cells in culture or in host organisms. Non-viral vector delivery systems include DNA plasmids, RNA (transcripts of vectors such as those described herein), naked nucleic acids, and nucleic acids compounded with delivery vehicles such as liposomes. Viral vector delivery systems include DNA and RNA viruses, which have free genes or integrated genomes after delivery to cells. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel and Feigner, TIBTECH 11:211-217 (1993); Mitani and Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer and Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology, Doerfler and Bohm (eds.) (1995); and Yu et al., Gene Therapy 1: 13-26 (1994). Non-viral delivery methods for nucleic acids include lipofection, nucleofection, microinjection, gene guns, viral particles, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, artificial viral particles, and reagent-enhanced uptake of DNA. Lipofection is described in, for example, U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355) and lipofection reagents are commercially available (e.g., Transfectam. TM and Lipofectin TM ). Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those of Felgner, WO 91 / 17424, WO 91 / 16024. Delivery can be to cells (eg, in vitro or ex vivo administration) or to target tissues (eg, in vivo administration).

[0418] Disease Model

[0419] The method of the present invention can be used to produce plants, animals or cells, which can be used to model and / or study target genetic or epigenetic disorders, such as models or disease models through target mutations. As used herein, "disease" refers to a disease, a disorder or an indication of a subject. For example, the method of the present invention can be used to produce a modification comprising one or more nucleic acid sequences associated with a disease, or a plant, animal or cell in which the expression of one or more nucleic acid sequences associated with a disease is changed. This nucleic acid sequence can encode or translate a disease-related protein sequence or can be a disease-related control sequence. Therefore, it should be understood that in embodiments of the present invention, plants, subjects, patients, organisms or cells can be non-human subjects, patients, organisms or cells. Therefore, the present invention provides plants, animals or cells or their progeny produced by the method of the present invention. Progeny can be clones of the plants or animals produced, or can be produced by sexual reproduction by hybridizing with other individuals of the same species to infiltrate other desired traits into their offspring. In the case of multicellular organisms, particularly animals or plants, cells can be in vivo or in vitro. In the case where the cell is in culture, if appropriate culture conditions are met and preferably, if the cell is suitably suitable for this purpose (e.g., stem cells), a cell line can be established. It is also envisioned that the bacterial cell line produced by the present invention. Therefore, a cell line is also envisioned. In some methods, the disease model can be used to study the effects of mutations of genes or gene products or more generally changes in the expression of genes or gene products (such as reduction) on the development and / or progression of animals or cells and diseases using measures commonly used in the study of diseases. Alternatively, this disease model is suitable for studying the effects of pharmaceutically active compounds on diseases. In some methods, the disease model can be used to evaluate the efficacy of potential gene therapy strategies. That is, disease-related RNA can be modified so as to display or inhibit or mitigate disease development and / or progression, and then test compounds for the effects of progression or inhibition or mitigation.

[0420] In the use of Table 1 Cas13b effector proteins and complexes thereof, nucleic acid molecules encoding them, and methods of using them, which can be used to practice the present invention, reference: Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O., Sanjana, NE., Hartenian, E., Shi, X., Scott, DA., Mikkelson, T., Heckl, D., Ebert, BL., Root, DE., Doench, JG., Zhang, F. Science Dec 12. (2013). [Epub ahead of print]; published in final edited form as: Science. 2014 Jan 3; 343(6166): 84-87. Shalem et al. relate to a new approach for interrogating gene function on a genome-wide scale. Their studies show that delivering a genome-wide CRISPR-Cas9 knockout (GeCKO) library targets 18,080 genes using 64,751 unique guide sequences that enable both negative and positive selection screening in human cells. First, the authors show the use of the GeCKO library to identify genes essential for cell survival in cancer and pluripotent stem cells. Next, in a melanoma model, the authors screened for genes whose loss was involved in resistance to Vemurafenib, a therapeutic agent that inhibits mutant protein kinase BRAF. Their studies showed that the top-ranked candidate genes included previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. The authors observed a high level of consistency and a high rate of hit confirmation between independent guide RNAs targeting the same gene, and thus confirmed the prospect of genome-wide screening using Cas9. Reference is also made to US Patent Publication No. US20140357530; and PCT Patent Publication No. WO2014093701, which are hereby incorporated herein by reference.

[0421] The term "associated with" is used herein with respect to the association of a functional domain with a Cas13b effector protein or an adapter protein. It relates to how one molecule "associates" with respect to another molecule, such as between an adapter protein and a functional domain or between a Cas13b effector protein and a functional domain. In the case of such protein-protein interactions, such association can be viewed in terms of recognition in the manner in which an antibody recognizes an epitope. Alternatively, a protein may be associated with another protein via a fusion of the two, such as a fusion of one subunit with another subunit. Fusion typically occurs by adding the amino acid sequence of one to the amino acid sequence of the other, such as by splicing together the nucleotide sequences encoding each protein or subunit. Alternatively, this can essentially be viewed as a binding or direct linkage between two molecules, such as a fusion protein. In any case, a fusion protein may include a linker between two target subunits (i.e., between an enzyme and a functional domain or between an adapter protein and a functional domain). Therefore, in some embodiments, a Cas13b effector protein or an adapter protein associates with a functional domain by binding to it. In other embodiments, the Cas13b effector protein or adaptor protein is associated with the functional domain in that the two are fused together, optionally via an intermediate linker.

[0422] Cas13b effector protein complex can be used in plants

[0423] In some embodiments, the present invention includes a method for modifying a cell or an organism. The cell may be a prokaryotic cell or a eukaryotic cell. The cell may be a mammalian cell. The mammalian cell may be a non-human primate, a cattle, a pig, a rodent, or a mouse cell. The cell may be a non-mammalian eukaryotic cell, such as poultry, fish, or shrimp. The cell may also be a plant cell. The plant cell may be a crop, such as cassava, corn, sorghum, wheat, or rice. The plant cell may also be an algae, a tree, or a vegetable. The modification introduced into the cell by the present invention may be such that the cell and the progeny of the cell are changed to improve the production of biological products such as antibodies, starch, alcohol, or other desired cell outputs. The modification introduced into the cell by the present invention may be such that the cell and the progeny of the cell contain changes in the biological products produced by the changes. The system may include one or more different vectors. In one aspect of the present invention, the effector protein is codon-optimized to express the desired cell type, preferably a eukaryotic cell, preferably a mammalian cell or a human cell. The Cas13b system (e.g., single or multiple) may be used in conjunction with the latest advances in crop genomics. Such CRISPR systems can be used to perform efficient and cost-effective plant gene or genome or transcriptome interrogation or editing or manipulation, such as for rapid survey and / or selection and / or interrogation and / or comparison and / or manipulation and / or transformation of plant genes or genomes; for example, to generate, identify, develop, optimize or confer plant traits or characteristics or transform plant genomes. Therefore, the production of plants, new plants with new combinations of traits or characteristics, or new plants with enhanced traits can be improved. Such CRISPR systems can be used in site-directed integration (SDI) or gene editing (GE) or any near reverse breeding (NRB) or reverse breeding (RB) technology with respect to plants. Therefore, unless otherwise specified, animal cells mentioned herein may also be applied to plant cells with necessary changes; and, enzymes with reduced off-target effects and systems using such enzymes herein can be used in plant applications, including those mentioned herein. Engineered plants and their progeny modified by effector proteins (Table 1Cas13b) and suitable guides (crRNA) are provided. These may include disease-resistant or drought-resistant crops, such as wheat, barley, rice, soybeans, or corn; plants modified to remove or reduce the ability to self-pollinate (but which may instead optionally hybridize); and allergenic foods, such as peanuts and nuts, in which immunogenic proteins have been inactivated, destroyed, or disrupted by targeting via effector proteins and suitable guides. Any aspects of using the classical CRISPR-Cas system may be applicable to CRISPR systems that are Cas protein agnostic, such as the Cas13b effector protein system.

[0424] Curative treatment

[0425] According to the present disclosure, including therapeutic agents, assays and other applications, the system of the present invention can be applied to the field of previous RNA cutting technology without excessive experimentation because the present application provides a basis for informing engineering of the system. The present invention provides therapeutic treatments for diseases caused by overexpression of RNA, toxic RNA and / or mutant RNA (e.g., splicing defects or truncations). Expression of toxic RNA may be associated with the formation of nuclear inclusion bodies and delayed degenerative changes in the brain, heart or skeletal muscle. In the most intensively studied example (myotonic dystrophy), it appears that the main pathogenic effect of toxic RNA is to chelate binding proteins and impair the regulation of alternative splicing (Hum. Mol. Genet. (2006) 15 (suppl 2): ​​R162-R169). Geneticists are particularly concerned about myotonic dystrophy [dystrophic myotonia (DM)] because it produces an extremely wide range of clinical features. A partial list includes muscle wasting, cataracts, insulin resistance, testicular atrophy, cardiac conduction slowing, skin tumors, and effects on cognition. The classical form of DM, which is now referred to as DM type 1 (DM1), is caused by the expansion of CTG repeat sequences in the 3'-untranslated region (UTR) of DMPK, a gene encoding a cytoplasmic protein kinase.

[0426] The innate immune system detects viral infections primarily by recognizing viral nucleic acids inside infected cells, which is known as DNA or RNA sensing. In vitro RNA sensing assays can be used to detect specific RNA substrates. RNA targeting effector proteins can be used, for example, for RNA-based sensing in living cells. An example of application is diagnosis by sensing, for example, disease-specific RNA. The RNA targeting effector protein (Table 1Cas13b) of the present invention can be further used for antiviral activity, particularly for RNA viruses. Suitable guide RNAs that are selective for selected viral RNA sequences can be used to target the effector protein (Table 1Cas13b) to viral RNA. In particular, the effector protein can be an active nuclease that cleaves RNA (such as single-stranded RNA). It is expected that the enzyme system of the present invention targets RNA. The therapeutic dose of the above-mentioned RNA is about 0.1 to about 2 mg / kg, and the dose can be sequentially administered under the monitored response, and if repeated doses are required, up to about 7 to 10 doses / patient. Advantageously, samples are collected from each patient during the treatment regimen to determine the effectiveness of the treatment. For example, RNA samples can be separated and quantified to determine whether expression is reduced or improved. This diagnosis is within the knowledge of those skilled in the art.

[0427] Transcript Detection Methods

[0428] The effector protein (Table 1Cas13b) and system of the present invention can be used to specifically detect RNA in cells or other samples. In the presence of a target RNA target, the guide-dependent Cas13b nuclease activity may be accompanied by non-specific RNA enzyme activity for an incidental target. In order to utilize RNA enzyme activity, all that is needed is a detectably cleaved reporter substrate. For example, the reporter molecule may include RNA, labeled with a fluorescent reporter molecule (fluor) at one end and labeled with a quencher at the other end. In the absence of Cas13b RNA enzyme activity, the physical proximity of the quencher suppresses the fluorescence from fluor to a low level. When Cas13b target-specific cleavage is activated by the presence of a target RNA target and a suitable guide RNA, the RNA-containing reporter molecule is non-specifically cleaved, and fluor and the quencher are separated in space. When excited by light of an appropriate wavelength, this causes fluor to emit a detectable signal. In an exemplary assay method, Cas13b effectors, target-specific guide RNAs, and reporter molecules are added to a cell sample. Increased fluorescence indicates the presence of a target RNA target. In another exemplary method, a detection array is provided. Each position of the array is provided with a Cas13b effector, a reporter molecule, and a target-specific guide RNA. Depending on the assay to be performed, the target-specific guide RNA at each position of the array can be the same, different, or a combination thereof. For example, when one or more targets in a single source sample need to be tested, different target-specific guide RNAs can be provided. The same target-specific guide RNA can be provided at each position, for example, when multiple samples of the same target need to be tested.

[0429] In certain embodiments, Cas13b is provided or expressed in an in vitro system or cell transiently or stably, and is targeted or triggered to non-specifically lyse cell nucleic acids. In one embodiment, Cas13b is engineered to knock down ssDNA, such as viral ssDNA. In another embodiment, Cas13b is engineered to knock down RNA. The system can be designed so that the knockdown depends on the target DNA present in the cell or in vitro system, or is triggered by adding a target nucleic acid to the system or cell.

[0430] In one embodiment, the Cas13b system is engineered to non-specifically cleave RNA in a subpopulation of cells, and the subpopulation of the cell can be distinguished by the presence of abnormal DNA sequences, such as where the cleavage of abnormal DNA may be incomplete or invalid. In a non-limiting example, targeting is present in cancer cells and drives DNA translocations that transform cells. Subpopulations of cells that undergo chromosomal DNA and repair can survive, while non-specific incidental ribonuclease activity advantageously leads to cell death of potential survivors.

[0431] Recently, the incidental activity was used in a highly sensitive and specific nucleic acid detection platform called SHERLOCK, which can be used in many clinical diagnostics (Gootenberg, JS et al. Nucleic acid detection with CRISPR-Cas13a / C2c2. Science 356, 438-442 (2017)).

[0432] According to the present invention, the engineered Cas13b system is optimized for RNA endonuclease activity and can be expressed in mammalian cells and targeted to effectively knock down reporter molecules or transcripts in the cells.

[0433] The side effect of engineered Cas13b and isothermal amplification can provide CRISPR-based diagnostics, providing rapid DNA or RNA detection with high sensitivity and single-base mismatch specificity. Cas13b-based molecular detection platforms are used to detect specific virus strains, distinguish pathogenic bacteria, genotype human DNA, and identify cell-free tumor DNA mutations. In addition, in order to be independent of the cold chain and long-term storage, the reaction reagents can be lyophilized and easily reconstituted on paper for field applications.

[0434] The ability to rapidly detect nucleic acids with high sensitivity and single-base specificity on a portable platform can aid in disease diagnosis and monitoring, epidemiology, and general laboratory tasks. Although methods are used to detect nucleic acids, they have trade-offs among sensitivity, specificity, simplicity, cost, and speed.

[0435] Microbial clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systems contain programmable nucleases that can support CRISPR-based diagnosis (CRISPR-Dx). Cas13b can be reprogrammed with CRISPR RNA (crRNA) to provide a platform for specific DNA sensing. After identifying its DNA target, the activated Cas13b participates in the "incidental" cleavage of nearby non-targeted nucleic acids (i.e., RNA and / or ssDNA). The incidental cleavage activity of this crRNA programming allows Cas13b to detect the presence of specific DNA in vivo by triggering programmed cell death or by nonspecific degradation of labeled RNA or ssDNA. Here, a highly sensitive in vitro nucleic acid detection platform based on the incidental cleavage of nucleic acid amplification and Cas13b-mediated commercial reporter RNA is described, thereby allowing real-time detection of targets.

[0436] In certain exemplary embodiments, the Cas13b effector protein is from an organism identified in Table 1. In certain exemplary embodiments, the Cas13b effector protein is from an organism selected from the group consisting of: Bergeyella zooulcerans, Prevotella intermedia, Prevotella buccal, Porphyromonas gingivalis, Bacteroides pyogenes, ZOR0009, Prevotella MA2016, Riemerella anatipestifer, Prevotella aurantiaca, Prevotella saccharolyticus, Myroides odoratimimus CCUG 10230, Capnocytophaga canimans, Porphyromonas odontotrophicus, Prevotella P5-125, Flavobacterium gracile, Flavobacterium pseudoscent, Flavobacterium columnaris, or Porphyromonas COT-052OH4946. In another embodiment, one or more guide RNAs are designed to bind to one or more target RNA sequences that are diagnostic of a disease condition.

[0437] In certain exemplary embodiments, the RNA-based masking construct inhibits the generation of a detectable positive signal, or the RNA-based masking construct inhibits the generation of a detectable positive signal by masking the detectable positive signal or alternatively generating a detectable negative signal, or the RNA-based masking construct comprises a silencing RNA that inhibits the production of a gene product encoded by a reporter construct, wherein the gene product generates a detectable positive signal when expressed.

[0438] In another exemplary embodiment, the RNA-based masking construct is a ribozyme that produces a negative detectable signal, and wherein a positive detectable signal is produced when the ribozyme is inactivated. In an exemplary embodiment, the ribozyme converts a substrate to a first color and wherein the substrate converts to a second color when the ribozyme is inactivated. In another exemplary embodiment, the RNA-based masking agent is an aptamer that chelates an enzyme, wherein the enzyme produces a detectable signal by acting on a substrate after release from the aptamer, or the aptamer chelates a pair of agents that combine to produce a detectable signal when released from the aptamer.

[0439] In another exemplary embodiment, the RNA-based masking construct comprises a detectable ligand oligonucleotide and an RNA oligonucleotide to which the masking component is attached. In certain exemplary embodiments, the detectable ligand is a fluorophore and the masking component is a quencher molecule.

[0440] In another aspect, the present invention provides a method for detecting a target RNA in a sample, the method comprising: distributing a sample or a sample group into one or more individual discrete volumes, the individual discrete volumes comprising a CRISPR system comprising an effector protein, one or more guide RNAs, and an RNA-based masking construct; incubating the sample or the sample group under conditions sufficient to allow the one or more guide RNAs to bind to the one or more target molecules; activating the CRISPR effector protein via the one or more guide RNAs binding to the one or more target molecules, wherein the activated CRISPR effector protein modifies the RNA-based masking construct to produce a detectable positive signal; and detecting the detectable positive signal, wherein detection of the detectable positive signal indicates the presence of the one or more target molecules in the sample.

[0441] In another aspect, the present invention provides a method for detecting peptides in a sample, the method comprising: allocating a sample or a sample group into a set of individual discrete volumes, the individual discrete volumes comprising a peptide detection aptamer, a CRISPR system comprising an effector protein, one or more guide RNAs, and an RNA-based masking construct, wherein the peptide detection aptamer comprises a masked RNA polymerase site and is constructed to bind to one or more target molecules; incubating the sample or the sample group under conditions sufficient to allow the peptide detection aptamer to bind to the one or more target molecules, wherein the aptamer binds to the corresponding target molecule to expose the RNA polymerase binding site, facilitating RNA synthesis of the trigger RNA; activating the CRISPR effector protein via binding of the one or more guide RNAs to the trigger RNA, wherein the activated CRISPR effector protein is modified to modify the RNA-based masking construct to produce a detectable positive signal; and detecting the detectable positive signal, wherein detection of the detectable positive signal indicates the presence of one or more target molecules in the sample.

[0442] In certain exemplary embodiments, one or more guide RNAs are designed to bind to one or more target molecules that are diagnostic of a disease condition. In certain other exemplary embodiments, the disease condition is an infection, an organ disease, a blood disease, an immune system disease, a cancer, a brain and nervous system disease, an endocrine disease, a pregnancy or childbirth-related disease, a genetic disease or an environmentally acquired disease, a cancer, or a fungal infection, a bacterial infection, a parasitic infection, or a viral infection.

[0443] In certain exemplary embodiments, the RNA-based masking construct inhibits the generation of a detectable positive signal, or the RNA-based masking construct inhibits the generation of a detectable positive signal by masking a detectable positive signal or alternatively generating a detectable negative signal, or the RNA-based masking construct comprises a silencing RNA that inhibits the generation of a gene product encoded by a reporter construct, wherein the gene product generates a detectable positive signal when expressed, or the RNA-based masking construct is a ribozyme that generates a negative detectable signal, and wherein a positive detectable signal is generated when the ribozyme is inactivated. In other exemplary embodiments, the ribozyme converts a substrate to a first state and wherein the substrate converts to a second state when the ribozyme is inactivated, or the RNA-based masking agent is an aptamer, or an aptamer chelates an enzyme, wherein the enzyme generates a detectable signal by acting on a substrate after being released from the aptamer, or an aptamer chelates a pair of agents that combine to generate a detectable signal when released from the aptamer. In other embodiments, the RNA-based masking construct comprises an RNA oligonucleotide having a detectable ligand on a first end of the RNA oligonucleotide and a masking component on a second end of the RNA oligonucleotide, or the detectable ligand is a fluorophore and the masking component is a quencher molecule.

[0444] General information regarding CRISPR-Cas systems, components thereof, and delivery of such components, including methods, materials, delivery vehicles, vectors, particles, AAVs, and methods of making and using them (including with respect to amounts and formulations) is applicable to the practice of the present invention, see: U.S. Patent Nos. 8,999,641; 8,993,233; 8,945,839; 8,932,814; 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359; U.S. Patent Publication Nos. US2014-0310830 (U.S. Application Serial No. 14 / 105,031); US2014-0287938; Al (U.S. application serial number 14 / 213,991), US2014-0273234Al (U.S. application serial number 14 / 293,674), US2014-0273232Al (U.S. application serial number 14 / 290,575), US2014-0273231 (U.S. application serial number 14 / 259,420), US2014-0256046 Al (U.S. application serial number 14 / 226,274), US2014-0248702Al (U.S. application serial number 14 / 258,458), US2014-0242700 Al (U.S. application serial number 14 / 222,930), US2014-0242699 Al (U.S. application serial number 14 / 183,512), US2014-0242664 Al (U.S. application serial number 14 / 104,990), US2014-0234972Al (U.S. application serial number 14 / 183,471), US2014-0227787Al (U.S. application serial number 14 / 256,912), US2014-0189896 Al (U.S. application serial number 14 / 105,035), US2014-0186958 (U.S. application serial number 14 / 105,017), US2014-0186919 Al (U.S. application serial number 14 / 104,977), US2014-0186843Al (U.S. application serial number 14 / 104,900), US2014-0179770 Al (U.S. application serial number 14 / 104,837) and US2014-0179006Al (U.S. application serial number 14 / 183,486), US2014-0170753 (U.S. application serial number 14 / 183,429); European patents EP 2 784 162 B1 and EP 2 771 468 B1;European patent applications EP 2 771 468 (EP13818570.7), EP 2 764 103 (EP 13824232.6), and EP 2 784 162 (EP14170383.5); and PCT patent publications WO 2014 / 093661 (PCT / US 2013 / 074743), WO 2014 / 093694 (PCT / US2013 / 074790), WO 2014 / 093595 (PCT / US2013 / 074611), WO 2014 / 093718 (PCT / US2013 / 074825), WO 2014 / 093709 (PCT / US2013 / 074812), WO 2014 / 093622(PCT / US2013 / 074667), WO 2014 / 093635(PCT / US2013 / 074691), WO 2014 / 093655(PCT / US2013 / 074736), WO 2014 / 093712(PCT / US2013 / 074819), WO 2014 / 093701(PCT / US2013 / 074800), WO 2014 / 018423(PCT / US2013 / 051418), WO 2014 / 204723(PCT / US2014 / 041790)、WO 2014 / 204724(PCT / US2014 / 041800)、WO 2014 / 204725(PCT / US 2014 / 041803), WO 2014 / 204726(PCT / US2014 / 041804), WO 2014 / 204727(PCT / US2014 / 041806), WO 2014 / 204728 (PCT / US2014 / 041808), WO 2014 / 204729 (PCT / US2014 / 041809). Reference is also made to U.S. Provisional Patent Applications Nos. 61 / 758,468, filed on January 30, 2013, March 15, 2013, March 28, 2013, April 20, 2013, May 6, 2013, and May 28, 2013; 61 / 802,174; 61 / 806,375; 61 / 814,263; filed on January 30, 2013, March 15, 2013, March 28, 2013, April 20, 2013, May 6, 2013, and May 28, 2013, respectively.Reference is also made to U.S. Provisional Patent Application No. 61 / 836,123 filed on June 17, 2013. Reference is also made to U.S. Provisional Patent Application Nos. 61 / 835,931, 61 / 835,936, 61 / 836,127, 61 / 836,101, 61 / 836,080 and 61 / 835,973, each filed on June 17, 2013. Further reference is made to U.S. Provisional Patent Applications 61 / 862,468 and 61 / 862,355 filed on August 5, 2013; U.S. Provisional Patent Application 61 / 871,301 filed on August 28, 2013; U.S. Provisional Patent Application 61 / 960,777 filed on September 25, 2013 and U.S. Provisional Patent Application 61 / 961,980 filed on October 28, 2013. Further reference is also made to: PCT Patent Application Nos.: PCT / US2014 / 041803, PCT / US2014 / 041800, PCT / US2014 / 041809, PCT / US2014 / 041804, and PCT / US2014 / 041806, each filed on June 10, 2014 (6 / 10 / 14); PCT / US2014 / 041806, filed on June 11, 2014 8; and PCT / US2014 / 62558, filed October 28, 2014, and U.S. Provisional Patent Applications Serial Nos.: 61 / 915,150, 61 / 915,301, 61 / 915,267, and 61 / 915,260, filed December 12, 2013, respectively; 61 / 757,972 and 61 / 768,959, filed January 29, 2013 and February 25, 2013, respectively; 61 / 835,936, 61 / 836,127, 61 / 836,101, 61 / 836,080, 61 / 835,973 and 61 / 835,931, all filed on June 17, 2014; 62 / 010,888 and 62 / 010,879, both filed on June 11, 2014; 62 / 010,329 and 62 / 010,441, each filed on June 10, 2014; and 62 / 010,441, each filed on February 12, 2014. 61 / 939,228 and 61 / 939,242 filed on April 15, 2014; 61 / 980,012 filed on April 15, 2014; 62 / 038,358 filed on August 17, 2014; 62 / 054,490, 62 / 055,484, 62 / 055,460 and 62 / 055,487 filed on September 25, 2014, respectively; and 62 / 069,243 filed on October 27, 2014. Reference is also made to U.S. Provisional Patent Application Nos. 62 / 055,484, 62 / 055,460 and 62 / 055,487 filed on September 25, 2014;Reference is made to U.S. Provisional Patent Application Nos. 61 / 980,012, filed April 15, 2014; and 61 / 939,242, filed February 12, 2014. Reference is made to PCT Application No. ...

Claims

1. A non-naturally occurring or engineered composition comprising: i) a Cas13b effector protein represented by SEQ ID NO: 34, and ii) crRNA, wherein the crRNA comprises a) a guide sequence, which is capable of hybridizing to a target RNA sequence, and b) a direct repeat sequence, Thereby forming a CRISPR complex comprising the Cas13b effector protein complexed with the guide sequence hybridized to the target RNA sequence.

2. The non-naturally occurring or engineered composition of claim 1, comprising an auxiliary protein that enhances the activity of a Cas13b effector protein, wherein the auxiliary protein that enhances the activity of a Cas13b effector protein is a csx28 protein.

3. The non-naturally occurring or engineered composition of claim 1, comprising an auxiliary protein that suppresses the activity of a Cas13b effector protein, wherein the auxiliary protein that suppresses the activity of a Cas13b effector protein is a csx27 protein.

4. The non-naturally occurring or engineered composition of any one of claims 1 to 3, comprising two or more crRNAs.

5. The non-naturally occurring or engineered composition of claim 1, wherein the guide sequence hybridizes to a target RNA sequence in a prokaryotic cell.

6. The non-naturally occurring or engineered composition of claim 1, wherein the guide sequence hybridizes to a target RNA sequence in a eukaryotic cell.

7. The non-naturally occurring or engineered composition of claim 1, wherein the Cas13b effector protein comprises one or more nuclear localization signals (NLS).

8. The non-naturally occurring or engineered composition of claim 1, wherein the Cas13b effector protein is associated with one or more functional domains, wherein each of the one or more functional domains comprises one or more transcriptional activation domains, transcriptional repression domains, and / or nuclease domains.

9. The non-naturally occurring or engineered composition of claim 8, wherein the one or more nuclease domains is Fok1.

10. The non-naturally occurring or engineered composition of claim 8, wherein the functional domain comprises one or more transcriptional activation domains or transcriptional repression domains.

11. The non-naturally occurring or engineered composition of claim 10, wherein the one or more transcriptional activation domains is VP64.

12. The non-naturally occurring or engineered composition of claim 10, wherein the one or more transcriptional repression domains are KRAB domains or SID domains.

13. The non-naturally occurring or engineered composition of claim 12, wherein the SID domain is SID4X.

14. The non-naturally occurring or engineered composition of claim 8, wherein the functional domain cleaves the target RNA sequence.

15. The non-naturally occurring or engineered composition of claim 2 or 3, wherein the Cas13b effector protein and the accessory protein are from the same organism.

16. The non-naturally occurring or engineered composition of claim 2 or 3, wherein the Cas13b effector protein and the accessory protein are from different organisms.

17. A Cas13b vector system for providing a composition as claimed in claim 1, comprising one or more vectors, wherein the vector comprises: A first regulatory element operably linked to a nucleotide sequence encoding a Cas13b effector protein shown in SEQ ID NO: 34, and A second regulatory element is operably linked to a nucleotide sequence encoding the crRNA.

18. The vector system of claim 17, further comprising: a regulatory element operably linked to a nucleotide sequence of an auxiliary protein, wherein the auxiliary protein is a csx28 protein or a csx27 protein.

19. The vector system of claim 17, wherein the nucleotide sequence encoding the Cas13b effector protein is codon-optimized for expression in eukaryotic cells.

20. The vector system of claim 18, wherein the nucleotide sequences encoding the Cas13b effector protein and the accessory protein are codon-optimized for expression in eukaryotic cells.

21. The vector system of claim 17, which is contained in a single vector.

22. The vector system of claim 17, wherein the one or more vectors comprise a viral vector.

23. The vector system of claim 17 or 21, wherein the one or more vectors comprise one or more retroviral, lentiviral, adenoviral, adeno-associated viral, or herpes simplex viral vectors.

24. A delivery system configured to deliver a Cas13b effector protein and one or more nucleic acid components of a non-naturally occurring or engineered composition, the delivery system comprising: i) a Cas13b effector protein represented by SEQ ID NO: 34, and ii) crRNA, wherein the crRNA comprises a) a guide sequence that hybridizes to a target RNA sequence in a cell, and b) a direct repeat sequence, wherein the Cas13b effector protein forms a complex with the crRNA, wherein the guide sequence directs sequence-specific binding of the complex to the target RNA sequence, Thereby forming a CRISPR complex comprising the Cas13b effector protein complexed with the guide sequence hybridized to the target RNA sequence.

25. The delivery system of claim 24, comprising one or more vectors comprising one or more polynucleotide molecules encoding the Cas13b effector protein and one or more nucleic acid components of the non-naturally occurring or engineered composition, wherein the one or more vectors comprise one or more retroviral, lentiviral, adenoviral, adeno-associated viral, or herpes simplex viral vectors.

26. The delivery system of claim 24, comprising a delivery vehicle comprising one or more particles, one or more microvesicles, a gene gun, or one or more viral vectors.

27. The delivery system of claim 26, wherein the delivery vehicle is one or more liposomes.

28. The delivery system of claim 26, wherein the delivery vehicle is one or more exosomes.

29. Use of the non-naturally occurring or engineered composition of any one of claims 1 to 16, the carrier system of any one of claims 17 to 23, or the delivery system of any one of claims 24 to 28 in the preparation of a medicament.

30. Use of one or more non-naturally occurring or engineered compositions in the preparation of a medicament for modifying the expression of a target gene of interest, wherein the non-naturally occurring or engineered composition comprises i) a Cas13b effector protein represented by SEQ ID NO: 34, and ii) crRNA, wherein the crRNA comprises a) a guide sequence that hybridizes to a target RNA sequence in a cell, and b) a direct repeat sequence, wherein the Cas13b effector protein forms a complex with the crRNA, wherein the guide sequence directs sequence-specific binding of the complex to the target RNA sequence in the cell, thereby forming a CRISPR complex comprising the Cas13b effector protein complexed with the guide sequence hybridized to the target RNA sequence, The expression of the target locus of interest is thereby modified.

31. The use of claim 30, wherein the target RNA is contacted with the one or more non-naturally occurring or engineered compositions.

32. The use of claim 30, further comprising contacting the target RNA with an auxiliary protein that enhances the activity of the Cas13b effector protein, wherein the auxiliary protein that enhances the activity of the Cas13b effector protein is a csx28 protein.

33. The use of claim 30, further comprising contacting the target RNA with an auxiliary protein that suppresses the activity of the Cas13b effector protein, wherein the auxiliary protein that suppresses the activity of the Cas13b effector protein is a csx27 protein.

34. The method of claim 30, wherein modifying the expression of the target gene comprises cleaving the target RNA.

35. The method of claim 30, wherein modifying the expression of the target gene comprises increasing or decreasing the expression of the target RNA.

36. The method of claim 30, wherein the cell is a prokaryotic cell or a eukaryotic cell.

37. The use of claim 30, comprising modifying the expression of the target gene in vitro or in vivo.

38. A cell comprising a modified target of interest, wherein the target of interest has been modified by a non-naturally occurring or engineered composition as described in any one of claims 1 to 16, wherein the cell is a prokaryotic cell or a eukaryotic cell.

39. The cell of claim 38, wherein modification of the target of interest results in: The cell comprises altered expression of at least one gene product; The cell comprises an altered expression of at least one gene product, wherein expression of the at least one gene product is increased; or The cell comprises altered expression of at least one gene product, wherein expression of the at least one gene product is reduced.

40. The eukaryotic cell of claim 38 or 39, wherein the cell is a mammalian cell or a human cell.

41. A cell line comprising the cell according to claim 38 or 39, or a progeny thereof.

42. An isolated Cas13b effector protein represented by SEQ ID NO:

34.

43. An isolated nucleic acid encoding the Cas13b effector protein of claim 42.

44. The isolated nucleic acid of claim 43, which is DNA and further comprises a sequence encoding crRNA.

45. An isolated eukaryotic cell comprising the nucleic acid of claim 43 or 44 or the Cas13b effector protein of claim 42.

46. ​​A non-naturally occurring or engineered composition comprising: i) mRNA encoding the Cas13b effector protein shown in SEQ ID NO: 34, and ii) crRNA, wherein the crRNA comprises a) a guide sequence, which is capable of hybridizing to a target RNA sequence, and b) a direct repeat sequence.

47. A composition comprising: Cas13b effector protein shown by SEQ ID NO:34 Non-target polynucleotide comprising a detectable label At least one guide polynucleotide designed to form a complex with the Cas13b effector protein and comprising a guide sequence capable of hybridizing to a target sequence.

48. The composition of claim 47, wherein the Cas13b effector protein, once activated by the target sequence, exhibits bypass activity and cleaves the non-target polynucleotide sequence.

49. The composition of claim 47, further comprising an amplification reagent for amplifying the target sequence.

50. Use of the composition of any one of claims 47-49 in the preparation of a medicament for detecting one or more target polynucleotides in a sample, comprising: contacting one or more samples with the drug; and Signals from cleavage of the non-target polynucleotides are detected, thereby sequentially detecting the one or more target polynucleotides.

51. The use according to claim 50, further comprising amplifying the one or more target polynucleotides in the sample.

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