Novel type vi crispr orthologs and systems

Abstract

To provide systems, methods, and compositions for targeting nucleic acids.SOLUTION: The invention provides non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA. More specifically, systems are provided that utilize a type VI-B CRISPR-Cas effector protein or a Cas13b effector protein, as well as a nucleic acid encoding such a protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications and Incorporation by Reference This application claims priority to U.S. Provisional Patent Application No. 62 / 484,791, filed April 12, 2017, U.S. Provisional Patent Application No. 62 / 561,662, filed September 21, 2017, and U.S. Provisional Patent Application No. 62 / 568,129, filed October 4, 2017, each of which is incorporated by reference herein in its entirety.

[0002] Reference is made to PCT applications, including, inter alia, PCT / US Patent Application Publication No. 2016 / 058302, filed October 21, 2016, specifically designating the United States. Reference is made to U.S. Provisional Patent Application No. 62 / 245,270, filed October 22, 2015, U.S. Provisional Patent Application No. 62 / 296,548, filed February 17, 2016, and U.S. Provisional Patent Applications Nos. 62 / 376,367 and 62 / 376,382, filed August 17, 2016. Reference is also made to U.S. Patent Application No. 62 / 471,792, filed March 15, 2017. Reference is also made to U.S. Provisional Patent Application No. 62 / 471,170, filed March 17, 2017. Further reference is made to U.S. Provisional Patent Application No. 62 / 484,791, filed April 12, 2017. Further reference is made to U.S. Provisional Patent Application No. 62 / 561,662, filed September 21, 2017. 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(Feb.16,2017)doi:10.1016 / j.molcel.2016.12.023.Epub Jan 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.Posted December 9,2017. Each of the foregoing applications and literature citations is hereby incorporated herein by reference.

[0003] All documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, manuals, 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 documents referenced are incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference.

[0004] Federally Sponsored Research Statement This invention was made with federal support under Grant Nos. MH100706 and MH110049 awarded by the National Institutes of Health. The federal government has certain rights in this invention.

[0005] The present invention relates generally to systems, methods, and compositions used to control gene expression, including gene transcript perturbation or nucleic acid editing, including sequence targeting, which may use vector systems related to clustered regularly interspaced short palindromic repeats (CRISPR) and its components. [Background technology]

[0006] Recent advances in genome sequencing technologies and analytical methods have rapidly improved our ability to catalog and map genetic factors associated with various biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by selectively perturbing individual genetic elements and advance synthetic biology, biotechnological, and medical applications. While genome editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available to generate targeted genome perturbations, there remains a need for novel genome and transcriptome engineering technologies that utilize novel strategies and molecular mechanisms and are inexpensive, easy to set up, scalable, and suitable for targeting multiple locations within eukaryotic genomes and transcriptomes. This will serve as a major resource for novel applications in genome engineering and biotechnology.

[0007] CRISPR-Cas systems in bacterial and archaeal adaptive immunity exhibit a high degree of diversity in terms of protein composition and genomic locus organization. CRISPR-Cas loci contain over 50 gene families, with no strictly universal genes, suggesting rapid evolution and a high degree of locus organization diversity. To date, a multi-pronged approach has comprehensively identified approximately 395 cas gene profiles for 93 Cas proteins. The classification includes signature gene profiles and locus organization signatures. A novel classification of CRISPR-Cas systems has been proposed, broadly dividing these systems into two classes: Class 1, which has multisubunit effector complexes, and Class 2, which has single-subunit effector modules, exemplified by the Cas9 protein. Novel effector proteins associated with Class 2 CRISPR-Cas systems can be developed as powerful genome engineering tools, and the prediction, engineering, and optimization of putative novel effector proteins are crucial.

[0008] The CRISPR-Cas adaptive immune system defends microorganisms against foreign genetic elements by DNA or RNA-DNA interference. Class 2 type VI single-component CRISPR-Cas effectors target RNA. One such system is Cas13a (also known as C2c2; see Shmakov et al. (2015) “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”; Molecular Cell 60:1-13; doi:http: / / dx.doi.org / 10.1016 / j.molcel.2015.10.008), which has been characterized as an RNA-guided RNase (Abudayyeh et al. (2016), Science, [Epub ahead of print], June 2; “C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector”; doi:10.1126 / science.aaf5573). In the current classification, Cas13a is a Class 2 Type VI-A CRISPR-Cas system. Another option is offered by Cas13b, a class 2 type VI-B effector protein. Class 2 type VI-B effector proteins include two subgroups, type VI-B1 and type VI-B2, also referred to as group 29 and group 30 proteins, which contain members that are RNA-programmable nucleases, RNA interference, and may be involved in bacterial adoptive immunity against RNA phages. (See Smargon A et al., "Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNAse differentially regulated by accessory proteins Csx27 and Csx28," Molecular Cell, online January 5, 2017. DOI: 10.1016 / j.molcel.2016.12.023.)

[0009] Group 29 and Group 30 systems contain a large single effector (approximately 1,100 amino acids) called Cas13b, and either one or neither of two small putative accessory proteins (approximately 200 amino acids long, termed Csx27 and Csx28) adjacent to the CRISPR array. Based on the adjacent small proteins, the systems are classified as Type VI-B1 (Csx27) or Type VI-B2 (Csx28). No additional proteins are conserved between species harboring each locus within 25 kilobase pairs upstream or downstream from the array. With few exceptions, CRISPR arrays contain a 36-nucleotide direct repeat sequence and a 30-nucleotide spacer sequence. The direct repeats are generally well conserved, particularly at the termini, where the 5'-terminal GTTG / GUUG sequence is reverse-complementary to the 3'-terminal CAAC sequence. This conservation suggests strong base pairing toward an RNA loop structure that may interact with proteins within the locus. A motif search complementary to the direct repeats did not reveal any candidate tracrRNAs flanking the array, indicating a single crRNA such as that found in the Cpf1 locus.

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

[0011] There is an urgent need for alternative and robust systems and techniques for targeting nucleic acids or polynucleotides (e.g., DNA or RNA, or any hybrid or derivative thereof) with a wide variety of applications, particularly in eukaryotic systems, and more particularly in mammalian systems. The present invention addresses this need and provides related advantages. The addition of the present novel RNA targeting system to the repertoire of genome, transcriptome, and epigenome targeting technologies can transform the study and perturbation or editing of specific target sites through direct detection, analysis, and manipulation, particularly in eukaryotic systems, and more particularly in mammalian systems (including cells, organs, tissues, or organisms). To effectively utilize the present RNA targeting system for RNA targeting without adverse effects, it is critical to understand the engineering and optimization aspects of these RNA targeting tools. [Means for solving the problem]

[0012] The Class 2 Type VI-B effector protein Cas13b is an RNA-guided RNase that can be efficiently programmed to degrade ssRNA. The inventors performed screens to identify several representative Cas13b orthologs from different species and determine their efficacy in eukaryotic cellular environments. In various embodiments, the present invention refers to, includes, or utilizes Type VI-B CRISPR-Cas effector proteins or Cas13b effector proteins, as well as nucleic acids encoding such proteins.

[0013] In some embodiments, the effector protein is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to a wild-type Cas13b effector protein of a prokaryote selected from the group consisting of Porphyromonas, Prevotella, Bacteroides, Riemerella, Bergeyella, Alistipes, Myroides, Capnocytophaga, and Flavobacterium.In some embodiments, the effector protein is selected from the group consisting of Porphyromonas gulae, Prevotella sp., Porphyromonas gingivalis, Bacteroides pyogenes, Riemerella anatipestifer, Bergeyella zoohelcum, Prevotella intermedia, Prevotella buccae, Alistipes sp., Prevotella aurantiaca, Myroides odoratimimus, Capnocytophaga canimorsus, and the like. The Cas13b effector protein is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to a wild-type Cas13b effector protein of a prokaryote selected from the group consisting of Flavobacterium canimorsus, Flavobacterium branchiophilum, and Flavobacterium columnare.In a preferred embodiment, the effector protein is selected from the group consisting of Porphyromonas gulae Cas13b (accession number WP_039434803), Prevotella sp. P5-125 Cas13b (accession number WP_044065294), Porphyromonas gingivalis Cas13b (accession number WP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession number WP_039428968), Bacteroides pyogenes Cas13b (accession number WP_034542281), Riemerella anatipestifera Cas13b (accession number WP_039428968), Porphyromonas gulae Cas13b (accession number WP_039434803), Prevotella sp. P5-125 Cas13b (accession number WP_044065294), Porphyromonas gingivalis Cas13b (accession number WP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession number WP_039428968), Bacteroides pyogenes Cas13b (accession number WP_034542281), Porphyromonas gingivalis Cas13b (accession number WP_039428968), Porphyromonas sp. COT-052 OH4946 Cas13b (accession number WP_039428968), Porphyromonas gingivalis ... or at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to a wild-type Cas13b effector protein selected from the group consisting of: (a) Cas13b (accession number WP_004919755);The most preferred effector proteins are Porphyromonas gulae Cas13b (accession number WP_039434803), Prevotella sp. P5-125 Cas13b (accession number WP_044065294), Porphyromonas gingivalis Cas13b (accession number WP_053444417), Porphyromonas sp. COT-052 OH4946 The Cas13b effector protein is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to a wild-type Cas13b effector protein selected from the group consisting of Cas13b (Accession No. WP_039428968), and most particularly preferred is Porphyromonas gulae Cas13b (Accession No. WP_039434803) or Prevotella sp. P5-125 Cas13b (Accession No. WP_044065294). The complete amino acid sequences of each of these Cas13b effector proteins and others are provided in Figure 1.

[0014] In some embodiments, the Cas13b effector protein (a) comprises 900-1800 amino acids and two HEPN domains, (b) is naturally present in the prokaryotic genome within 10 kb upstream or downstream of the CRISPR array, (c) is the only encoded protein containing more than 700 amino acids within 10 kb upstream or downstream of the CRISPR array, and / or (d) is free of Cas1 or Cas2 genes within 10 kb upstream or downstream of the CRISPR array. In some embodiments, at least one of Csx27 or Csx28 is also present within 10 kb upstream or downstream of the CRISPR array.

[0015] In certain embodiments, the Cas13b effector protein has a modified sequence compared to the wild-type protein. In certain embodiments, the effector protein is identical to the wild-type Cas13b effector protein in at least one or more common motifs shared by two or more Cas13b effector proteins. The common motif can be determined by standard sequence alignment tools to identify consensus sequences. In particular embodiments, the Cas13b effector protein is a protein comprising a sequence having at least 70% sequence identity with one or more of the sequences consisting of DKHXFGAFLNLARHN (SEQ ID NO: 1), GLLFFVSLFLDK (SEQ ID NO: 2), SKIXGFK (SEQ ID NO: 3), DMLNELXRCP (SEQ ID NO: 4), RXZDRFPYFALRYXD (SEQ ID NO: 5), and LRFQVBLGXY (SEQ ID NO: 6). In further detailed embodiments, the Cas13b effector protein comprises a sequence having at least 70% sequence identity with at least two, three, four, five, or all six of these sequences. In further detailed embodiments, the sequence identity with these sequences is at least 75%, 80%, 85%, 90%, 95%, or 100%. In further detailed embodiments, the Cas13b effector protein is a protein comprising a sequence having 100% sequence identity with GLLFFVSLFL (SEQ ID NO: 7) and RHQXRFPYF (SEQ ID NO: 8). In further detailed embodiments, the Cas13b effector is a Cas13b effector protein comprising a sequence having 100% sequence identity with RHQDRFPY (SEQ ID NO: 9).

[0016] The terms Cas enzyme, CRISPR enzyme, CRISPR protein, Cas protein and CRISPR Cas are generally used interchangeably and will be understood to refer by analogy to the CRISPR effector proteins described further herein unless otherwise clear, such as by specific reference to Cas9.

[0017] In embodiments of the invention, a Type VI-B system comprises a Cas13b effector protein and, optionally, a small accessory protein encoded upstream or downstream of the Cas13b effector protein. In certain embodiments, the small accessory protein enhances the ability of the Cas13b effector to target RNA.

[0018] In certain embodiments of the invention, a Type VI-B system comprises a Cas13b effector protein and, optionally, a small accessory protein encoded upstream or downstream of the Cas13b effector protein. In certain embodiments, the small accessory protein inhibits the ability of the Cas13b effector to target RNA.

[0019] The present invention provides a non-naturally occurring or engineered composition comprising i) a Type VI-B CRISPR-Cas effector protein and ii) a Type VI-B CRISPR-Cas crRNA, the crRNA comprising a) a guide sequence capable of hybridizing to a target RNA sequence, and b) a direct repeat sequence. The Type VI-B CRISPR-Cas effector protein forms a complex with the crRNA, and the guide sequence directs sequence-specific binding by the complex to the target RNA sequence, thereby forming a CRISPR complex comprising the Type VI-B CRISPR-Cas effector protein complexed with the guide sequence that hybridizes to the target RNA sequence. The complex formed upon specific hybridization of the guide sequence to the target RNA sequence involves interaction (recognition) of the protospacer flanking sequence (PFS).

[0020] In some embodiments, non-naturally occurring or engineered compositions of the invention may include a Type VI-B CRISPR-Cas accessory protein that enhances Type VI-B CRISPR-Cas effector protein activity. In certain such embodiments, the accessory protein that enhances Type VI-B CRISPR-Cas effector protein activity 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.

[0021] In some embodiments, the non-naturally occurring or engineered compositions of the invention comprise a Type VI-B CRISPR-Cas accessory protein that inhibits Type VI-B CRISPR-Cas effector protein activity. In certain such embodiments, the accessory protein that inhibits Type VI-B CRISPR-Cas effector protein activity is a csx27 protein. In such embodiments, the Type VI-B CRISPR-Cas effector protein and the Type VI-B CRISPR-Cas accessory protein can be from the same source or from different sources.

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

[0023] In some embodiments, the non-naturally occurring or engineered compositions of the present invention comprise a guide sequence that hybridizes to a target RNA sequence in a prokaryotic cell. In some embodiments, the non-naturally occurring or engineered compositions of the present invention comprise a guide sequence that hybridizes to a target RNA sequence in a eukaryotic cell. The CRISPR systems provided herein can utilize a crRNA or similar polynucleotide comprising a guide sequence, where the polynucleotide is RNA, DNA, or a mixture of RNA and DNA, and / or the polynucleotide comprises one or more nucleotide analogs. The sequence can comprise any structure, including, but not limited to, a structure of a natural crRNA, such as a bulge, hairpin, or stem-loop structure. In certain embodiments, the polynucleotide comprising the guide sequence forms a duplex with a second polynucleotide sequence, which can be an RNA or DNA sequence.

[0024] In certain embodiments, the method utilizes chemically modified guide RNAs. Examples of guide RNA chemical modifications include, without limitation, the incorporation of 2'-O-methyl (M), 2'-O-methyl 3' phosphorothioate (MS), or 2'-O-methyl 3' thio PACE (MSP) at one or more terminal nucleotides. Such chemically modified guide RNAs may exhibit increased stability and activity compared to unmodified guide RNAs, but on-target versus off-target specificity is unpredictable (see Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi:10.1038 / nbt.3290, published online 29 June 2015). Chemically modified guide RNAs further include, without limitation, RNAs containing phosphorothioate linkages and locked nucleic acid (LNA) nucleotides containing a methylene bridge between the 2' and 4' carbons of the ribose ring.

[0025] In some embodiments, the Type VI-B CRISPR-Cas effector protein comprises one or more nuclear localization signals (NLS).

[0026] In contrast to the catalytic mechanism of other known RNases found in CRISPR-Cas systems, Cas13b achieves RNA cleavage through conserved basic residues within its two HEPN domains. Mutation of the HEPN domain, such as alanine substitution of any of the four predicted HEPN domain catalytic residues, can convert Cas13b into an inactive programmable RNA-binding protein (dCas13b, similar to dCas9).

[0027] The ability of dCas13b to bind to designated sequences can be used in several embodiments of the present invention to (i) deliver effector modules to specific transcripts to modulate their function or translation (potentially useful for large-scale screening, building synthetic regulatory circuits, and other purposes); (ii) fluorescently tag specific RNAs to visualize their transport and / or localization; (iii) alter RNA localization with domains that have affinity for specific subcellular compartments; and (iv) capture specific transcripts (either by directly pulling down dCas13b or by using dCas13b to localize biotin ligase activity to specific transcripts) to enrich for nearby molecular partners, including RNAs and proteins.

[0028] Active Cas13b should also have many applications. Certain embodiments of the invention involve targeting specific transcripts for destruction. In addition, when primed by its cognate target, Cas13b can cleave other (non-complementary) RNA molecules in vitro and inhibit cell growth in vivo. Biologically, this promiscuous RNase activity may reflect a defense mechanism based on programmed cell death / dormancy (PCD / D) in the type VI-B CRISPR-Cas system. Thus, in certain embodiments of the invention, it could potentially be used to induce PCD or dormancy in specific cells—e.g., cancer cells expressing particular transcripts, certain classes of neurons, cells infected with specific pathogens, or other abnormal or otherwise undesirable cells.

[0029] The present invention provides a method for modifying a nucleic acid sequence associated with or at a target locus of interest, particularly a eukaryotic cell, tissue, organ, or organism, more particularly a mammalian cell, tissue, organ, or organism, comprising delivering to the locus a non-naturally occurring or engineered composition comprising a Type VI-B CRISPR-Cas locus 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 upon binding of the complex to the locus of interest, the effector protein induces modification of the 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, and the effector protein is encoded by a Type VI-B CRISPR-Cas locus. The complex can be formed in vitro or ex vivo and introduced into a cell, contacted with RNA, or formed in vivo.

[0030] The present invention provides a method for targeting (e.g., modifying) a sequence associated with or at a target locus of interest, comprising delivering a non-naturally occurring or engineered composition comprising a Cas13b locus effector protein (which may be catalytically active or alternatively catalytically inactive) and one or more nucleic acid components to the sequence associated with or at the locus, wherein the Cas13b effector protein forms a complex with the one or more nucleic acid components, and upon binding of the complex to the locus of interest, the effector protein induces modification of the 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 Cas13b effector protein forms a complex with one nucleic acid component, advantageously an engineered or non-naturally occurring nucleic acid component. The complex can be formed in vitro or ex vivo and introduced into a cell, contacted with RNA, or formed in vivo. Induction of modification of a sequence associated with or at a target locus of interest can be under the guidance of a Cas13b effector protein-nucleic acid. In a preferred embodiment, one nucleic acid component is a CRISPR RNA (crRNA). In a preferred embodiment, one 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 direct repeat sequence or a derivative thereof. In a preferred embodiment, the spacer sequence or a derivative thereof comprises a seed sequence, wherein the seed sequence is critical for recognition and / or hybridization with a sequence at the target locus.

[0031] Aspects of the present invention relate to Cas13b effector protein complexes having one or more non-naturally occurring, engineered, modified, or optimized nucleic acid components. In a preferred embodiment, the nucleic acid component of the complex can include a guide sequence linked to a direct repeat sequence, wherein the direct repeat sequence comprises one or more stem-loops or optimized secondary structures. In one embodiment, the direct repeat sequence can be approximately 36 nucleotides in length. In a specific embodiment, the direct repeat comprises GTTG / GUUG at the 5' end, reverse-complementary to CAAC at the 3' end. In certain embodiments, the direct repeat has a minimum length of 16 nt, e.g., at least 28 nt, and a single stem-loop. In further embodiments, the direct repeat is longer than 16 nt, preferably longer than 17 nt, e.g., at least 28 nt, in length, and has two or more stem-loops or optimized secondary structures. In a detailed embodiment, the direct repeat is 25 nt or longer, e.g., 26 nt, 27 nt, 28 nt or longer, and has one or more stem-loop structures. In a preferred embodiment, the direct repeats may be modified to include one or more protein-binding RNA aptamers. In a preferred embodiment, the direct repeats may be modified to include one or more protein-binding RNA aptamers. In a preferred embodiment, the one or more aptamers may be included as part of an optimized secondary structure. Such aptamers may be capable of binding to a bacteriophage coat protein. The bacteriophage coat protein may be selected from the group including Qβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, and PRR1. In a preferred embodiment, the bacteriophage coat protein is MS2. The invention also provides nucleic acid components of the complexes that are 30 or more, 40 or more, or 50 or more nucleotides in length.

[0032] The present invention provides cells comprising a Cas13b effector protein, and / or a guide, and / or a complex thereof with a target nucleic acid, including cells comprising a transiently expressed or introduced Cas13b effector protein, and / or a guide, and / or a complex thereof. In certain embodiments, the cell is a eukaryotic cell, including but not limited to a yeast cell, a plant cell, a mammalian cell, an animal cell, or a human cell.

[0033] The present invention also provides a method for modifying a target locus of interest, particularly a eukaryotic cell, tissue, organ, or organism, more particularly a mammalian cell, tissue, organ, or organism, comprising delivering to the locus a non-naturally occurring or engineered composition comprising a Cas13b locus effector protein and one or more nucleic acid components, wherein the Cas13b effector protein forms a complex with the one or more nucleic acid components, and upon binding of the complex to the locus of interest, the effector protein induces modification of the target locus of interest. In a preferred embodiment, the modification is the introduction of a strand break. The complex can be formed in vitro or ex vivo and introduced into a cell, contacted with RNA, or formed in vivo.

[0034] In such methods, the target locus of interest can be contained within an RNA molecule. Alternatively, the target locus of interest can be contained within a DNA molecule, and in certain embodiments, within a transcribed DNA molecule. In such methods, the target locus of interest can be contained within an in vitro nucleic acid molecule.

[0035] In such methods, a target locus of interest can be contained in a nucleic acid molecule within a cell, particularly a eukaryotic cell, such as a mammalian cell or a plant cell. The mammalian cell can be a non-human primate, bovine, porcine, rodent, or murine cell. The cell can be a non-mammalian eukaryotic cell, such as a poultry, fish, or shrimp cell. The plant cell can be a crop plant, such as cassava, corn, sorghum, wheat, or rice. The plant cell can also be algae, a tree, or a vegetable. The modification introduced into the cell by the present invention can be such that the cell and its progeny are altered for improved production of a biological product, such as an antibody, starch, alcohol, or other desired cellular product. The modification introduced into the cell by the present invention can be such that the cell and its progeny contain a change that alters the biological product produced.

[0036] The mammalian cell can be a non-human mammalian cell, such as a primate, bovine, ovine, porcine, canine, rodent, or Leporidae cell, such as a monkey, cow, sheep, pig, dog, rabbit, rat, or mouse cell. The cell can be a non-mammalian eukaryotic cell, such as a poultry avian (e.g., chicken), vertebrate fish (e.g., salmon), or crustacean (e.g., oyster, clam, lobster, shrimp) cell. The cell can also be a plant cell. The plant cell can be from a monocotyledonous or dicotyledonous plant or from a crop or cereal plant, such as cassava, corn, sorghum, soybean, wheat, oat, or rice. The plant cell can also be from an algae, a tree or productive plant, a fruit or vegetable (e.g., a citrus tree, such as an orange, grapefruit or lemon tree; a peach or nectarine tree; an apple or pear tree; a nut tree, such as an almond, walnut or pistachio tree; a Solanaceae plant; a Brassica plant; a Lactuca plant; a Spinacia plant; a Capsicum plant; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.).

[0037] The present invention provides a method of modifying a target locus of interest, the method comprising delivering to the locus a non-naturally occurring or engineered composition comprising a Type VI-B CRISPR-Cas locus 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 upon binding of the complex to the locus of interest, the effector protein induces modification of the target locus of interest. In a preferred embodiment, the modification is the introduction of a strand break.

[0038] The present invention also provides a method of modifying a target locus of interest, comprising delivering to the locus a non-naturally occurring or engineered composition comprising a Cas13b locus effector protein and one or more nucleic acid components, wherein the Cas13b effector protein forms a complex with the one or more nucleic acid components, and upon binding of the complex to the locus of interest, the effector protein induces modification of the target locus of interest. In a preferred embodiment, the modification is the introduction of a strand break.

[0039] In such methods, the target locus of interest can be comprised in a nucleic acid molecule in vitro. In such methods, the target locus of interest can be comprised in a nucleic acid molecule in a cell. Preferably, in such methods, the target locus of interest can be comprised in an RNA molecule in vitro. Also preferably, in such methods, the target locus of interest can be comprised in an RNA molecule in a cell. The cell can be a prokaryotic or eukaryotic cell. The cell can be a mammalian cell. The cell can be a rodent cell. The cell can be a mouse cell.

[0040] 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.

[0041] In a further embodiment of the present invention, the nucleic acid component may comprise a CRISPR RNA (crRNA) sequence. Without limitation, Applicants hypothesize that in such instances, the pre-crRNA may comprise a secondary structure sufficient for processing to yield the mature crRNA and loading of the crRNA onto an effector protein. By way of example and not limitation, such secondary structure may comprise, consist essentially of, or consist of a stem-loop within the pre-crRNA, more particularly within a direct repeat.

[0042] In any of the described methods, the effector protein and nucleic acid component may be provided by one or more polynucleotide molecules encoding the protein and / or nucleic acid component, where the one or more polynucleotide molecules are operably configured to express the protein and / or nucleic acid component. The one or more polynucleotide molecules may comprise one or more regulatory elements operably configured to express the protein and / or nucleic acid component. The one or more polynucleotide molecules may be contained within one or more vectors. In any of the described methods, the target locus of interest may be a genomic or epigenomic locus of interest. In any of the described methods, the complex may be delivered with multiple guides for multiplexed applications. In any of the described methods, two or more proteins may be used.

[0043] The regulatory element may comprise an inducible promoter. The polynucleotide and / or vector system may comprise an inducible system.

[0044] In any of the methods described, one or more polynucleotide molecules may be included in the delivery system, or one or more vectors may be included in the delivery system.

[0045] In any of the methods described, the non-naturally occurring or engineered composition may be delivered by liposomes, particles including nanoparticles, exosomes, microvesicles, gene guns, or one or more viral vectors.

[0046] The present invention also provides non-naturally occurring or engineered compositions that have the characteristics as discussed herein or are compositions defined in any of the methods described herein.

[0047] In certain embodiments, therefore, the invention provides non-naturally occurring or engineered compositions, such as compositions specifically capable of or configured to modify a target locus of interest, said compositions comprising a Type VI-B CRISPR-Cas locus 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 upon binding of the complex to the locus of interest, the effector protein induces modification of the target locus of interest. In certain embodiments, the effector protein may be a Cas13b locus effector protein.

[0048] In a further aspect, the present invention also provides non-naturally occurring or engineered compositions, such as compositions specifically capable of or configured to modify a target locus of interest, comprising: (a) a guide RNA molecule (or a combination of guide RNA molecules, such as for multiplexing, e.g., 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); and (b) a type VI-B CRISPR-Cas locus effector protein or a nucleic acid encoding a type VI-B CRISPR-Cas locus effector protein. In a specific embodiment, the effector protein may be a Cas13b locus effector protein.

[0049] In a further aspect, the present invention also provides a non-naturally occurring or engineered composition comprising: (a) a guide RNA molecule (or a combination of guide RNA molecules, e.g., 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); and (b) a Cas13b locus effector protein.

[0050] The present invention also provides a vector system comprising one or more vectors, wherein the one or more vectors comprise one or more polynucleotide molecules encoding components of a non-naturally occurring or engineered composition, the composition having the characteristics as defined in the methods described herein.

[0051] The present invention also provides a delivery system comprising one or more vectors or one or more polynucleotide molecules, wherein the one or more vectors or polynucleotide molecules comprise one or more polynucleotide molecules encoding components of a non-naturally occurring or engineered composition having the characteristics as discussed herein or being a composition defined by any of the methods described herein.

[0052] The present invention also provides non-naturally occurring or engineered compositions, or one or more polynucleotides encoding components of said compositions, or vectors or delivery systems comprising one or more polynucleotides encoding components of said compositions, for use in therapeutic treatment methods, which may include gene or transcriptome editing or gene therapy.

[0053] The present invention also provides methods and compositions in which one or more amino acid residues of an effector protein, such as an engineered or non-naturally occurring effector protein or Cas13b, can be modified. In some embodiments, the modification can include mutation of one or more amino acid residues of the effector protein. The one or more mutations can be in one or more catalytically active domains of the effector protein. The effector protein can have reduced or eliminated nuclease activity compared to an effector protein lacking the one or more mutations. The effector protein can fail to induce RNA strand cleavage at a target locus of interest. In a preferred embodiment, the one or more mutations can include two mutations. In a preferred embodiment, one or more amino acid residues are modified in a Cas13b effector protein, such as an engineered or non-naturally occurring effector protein or Cas13b. In certain embodiments, the effector protein contains one or more of the following mutations: R116A, H121A, R1177A, H1182A (wherein the amino acid positions are those of Bergeyella zoohelcum ATCC 154944). 43767), such as R116A, H121A, R1177A and H1182A, R116A, H121A and R1177A, R116A, H121A and H1182A, R116A, R1177A and H1182A, H121A, R1177A and H1182A, R116A and H121A, R116A and R1177A, R116A and H1182A, H121A and R1177A, H121A and H1182A, R1177A and H1182A, R116A, H121A, R1177A, H1182A, R116A, H121A, R1177A, H1182A. One skilled in the art will understand that corresponding amino acid positions in different Cas13b proteins can be mutated to the same effect.In certain embodiments, one or more of the mutations R116A, H121A, R1177A, H1182A, e.g., R116A, H121A, R1177A and H1182A, R116A, H121A and R1177A, R116A, H121A and H1182A, R116A, R1177A and H1182A, H121A, R1177A and H1182A , R116A and H121A, R116A and R1177A, R116A and H1182A, H121A and R1177A, H121A and H1182A, R1177A and H1182A, R116A, H121A, R1177A, H1182A completely or partially eliminate the catalytic activity of the protein (e.g., altered cleavage rate, altered specificity, etc.). In certain embodiments, wherein the amino acid positions correspond to amino acid positions of the Cas13b protein from Prevotella sp. P5-125, the effector protein comprises the H133A and H1058A mutations. In certain embodiments, the effector protein as described herein is a "dead" effector protein, such as a dead Cas13b effector protein (i.e., dCas13b). 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 comprise one or more nuclear localization signal (NLS) domains. The one or more heterologous functional domains may comprise at least two or more NLS domains. The one or more NLS domains may be located at, near, or in close proximity to the terminus of the effector protein (e.g., a Cas13b effector protein), and in the case of two or more NLSs, each of the two NLSs may be located at, near, or in close proximity to the terminus of the effector protein (e.g., a Cas13b effector protein). The one or more heterologous functional domains may comprise one or more transcription activation domains.In a preferred embodiment, the transcription activation domain may comprise VP64. The one or more heterologous functional domains may comprise one or more transcription repression domains. In a preferred embodiment, the transcription repression domain may comprise a KRAB domain or a SID domain (e.g., SID4X). The one or more heterologous functional domains may comprise one or more nuclease domains. In a preferred embodiment, the nuclease domain comprises Fok1.

[0054] The present invention also provides that the one or more heterologous functional domains have one or more of the following activities: methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription termination 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 can be at or near the carboxy terminus of the effector protein. The one or more heterologous functional domains can be fused to the effector protein. The one or more heterologous functional domains can be tethered to the effector protein. The one or more heterologous functional domains can be linked to the effector protein by a linker moiety.

[0055] In certain embodiments of the present invention, one or more heterologous functional domains may comprise an epitope tag or reporter. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporters 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, such as blue fluorescent protein (BFP).

[0056] The present invention also provides effector proteins, including effector proteins that are at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to a wild-type Cas13b effector protein of a prokaryotic genus selected from the group consisting of Porphyromonas, Prevotella, Bacteroides, Riemerella, Bergeyella, Alistipes, Myroides, Capnocytophaga, and Flavobacterium.The present invention relates to Porphyromonas gulae, Prevotella sp., Porphyromonas gingivalis, Bacteroides pyogenes, Riemerella anatipestifer, Bergeyella zoohelcum, Prevotella intermedia, Prevotella buccae, Alistipes sp., Prevotella aurantiaca, Myroides odoratimimus, Capnocytophaga canimorsus, and the like. Further provided are effector proteins, including effector proteins that are at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to a wild-type Cas13b effector protein of a prokaryotic species selected from the group consisting of Flavobacterium canimorsus, Flavobacterium branchiophilum, and Flavobacterium columnare.The present invention additionally relates to Porphyromonas gulae Cas13b (Accession No. WP_039434803), Prevotella sp. P5-125 Cas13b (Accession No. WP_044065294), Porphyromonas gingivalis Cas13b (Accession No. WP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (Accession No. WP_039428968), Bacteroides pyogenes Cas13b (Accession No. WP_034542281), Riemerella anatipestifera Cas13b (Accession No. WP_039428968), and the like. The present invention provides effector proteins, including an effector protein that is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to a wild-type Cas13b effector protein selected from the group consisting of Cas13b (Cas13b anatipestifer) Cas13b (Accession No. WP_004919755).The most preferred effector proteins are Porphyromonas gulae Cas13b (accession number WP_039434803), Prevotella sp. P5-125 Cas13b (accession number WP_044065294), Porphyromonas gingivalis Cas13b (accession number WP_053444417), Porphyromonas sp. COT-052 OH4946 and at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to a wild-type Cas13b effector protein selected from the group consisting of Cas13b (Accession No. WP_039428968), and most particularly preferred is wild-type Porphyromonas gulae Cas13b (Accession No. WP_039434803) or Prevotella sp. P5-125. and at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to Cas13b (Accession No. WP_044065294). The effector protein may comprise a chimeric effector protein comprising a first fragment from a first effector protein ortholog and a second fragment from a second effector protein ortholog, wherein the first and second effector protein orthologs are different.

[0057] In certain embodiments, the effector protein may be at least 700 amino acids in length. In preferred embodiments, the effector protein may be about 900 to about 1500 amino acids in length, e.g., about 900 to about 1000 amino acids in length, about 1000 to about 1100 amino acids in length, about 1100 to about 1200 amino acids in length, about 1200 to about 1300 amino acids in length, about 1300 to about 1400 amino acids in length, or about 1400 to about 1500 amino acids in length, e.g., about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500 amino acids in length.

[0058] In some embodiments, the Cas13b effector protein (a) comprises 900-1800 amino acids and two HEPN domains, (b) is naturally present in the prokaryotic genome within 10 kb upstream or downstream of the CRISPR array, (c) is the only protein containing more than 700 amino acids within 10 kb upstream or downstream of the CRISPR array, and / or (d) is free of Cas1 or Cas2 genes within 10 kb upstream or downstream of the CRISPR array. In some embodiments, Csx27 or Csx28 is also present within 10 kb upstream or downstream of the CRISPR array.

[0059] In certain embodiments, the effector protein, particularly a type VI-B locus effector protein, more particularly Cas13b, comprises at least one and preferably at least two, e.g., more preferably, exactly two, conserved RxxxxH motifs. The catalytic RxxxxH motif is unique to the HEPN (higher eukaryotic and prokaryotic nucleotide-binding) domain. Thus, in certain embodiments, the effector protein comprises at least one and preferably at least two, e.g., more preferably, exactly two, HEPN domains. In certain embodiments, the HEPN domain may have RNase activity. In other embodiments, the HEPN domain may have DNase activity.

[0060] In certain embodiments, a Cas13b effector protein as contemplated herein may be associated with a locus that comprises a short CRISPR repeat that is 30-40 bp in length, more typically 34-38 bp in length, even more typically 36-37 bp in length, e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bp in length. In certain embodiments, the CRISPR repeat is a long or double repeat that is 80-350 bp in length, e.g., 80-200 bp in length, even more typically, 86-88 bp in length, e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 bp in length.

[0061] In certain embodiments, a protospacer adjacent motif (PAM) or PAM-like motif directs binding of an effector protein (e.g., a Cas13b effector protein) complex as disclosed herein to a target locus of interest. The PAM is sometimes referred to as a PFS or protospacer adjacent site. In some embodiments, the PAM can be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In other embodiments, the PAM can be a 3' PAM (i.e., located downstream of the 5' end of the protospacer). In other embodiments, both a 5' PAM and a 3' PAM are required. In certain embodiments of the invention, a PAM or PAM-like motif may not be required to direct binding of an effector protein (e.g., a Cas13b effector protein). In certain embodiments, the 5' PAM is D (i.e., A, G, or U). In certain embodiments, for type VI-B1 effectors, the 5' PAM is D. See Example 1, Table 2. Methods exist for determining consensus 5' and 3' PAMs for a given Cas13b system. In certain embodiments of the invention, cleavage in the repeat sequence can result in a crRNA (e.g., short or long crRNA) that contains a short nucleotide (e.g., 5, 6, 7, 8, 9, or 10 nt, or more if it is a double repeat) repeat sequence (which can be referred to as a crRNA "tag") at the 5' end and a complete spacer sequence flanked by the remainder of the repeat at the 3' end. In certain embodiments, targeting by the effector proteins described herein can require no homology between the crRNA tag and the target 5'-flanking sequence. This requirement may be similar to that 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 thought to distinguish between the true target on the invading nucleic acid and the CRISPR array itself, where the presence of the repeat sequence leads to perfect homology with the crRNA tag, preventing autoimmunity.

[0062] In certain embodiments, Cas13b effector proteins can be engineered to contain one or more mutations that reduce or eliminate nuclease activity, thereby reducing or eliminating RNA interference activity. Mutations can also be made in adjacent residues, such as amino acids near those involved in nuclease activity. In some embodiments, one or more putative catalytic nuclease domains are inactivated, resulting in an effector protein complex lacking cleavage activity and functioning as an RNA-binding complex. In preferred embodiments, the resulting RNA-binding complex can be linked to one or more functional domains as described herein.

[0063] In certain embodiments, the effector protein of the present invention (CRISPR enzyme, Cas13, effector protein) as described herein is a catalytically inactive or dead Cas13 effector protein (dCas13). In some embodiments, the dCas13 effector comprises a mutation in the nuclease domain. In some embodiments, the dCas13 effector protein is truncated. In some embodiments, to reduce the size of a fusion protein of a Cas13b effector with one or more functional domains, the C-terminus of the Cas13b effector can be truncated while still maintaining its RNA-binding function. For example, at least 20 amino acids, at least 50 amino acids, at least 80 amino acids, or at least 100 amino acids, or at least 150 amino acids, or at least 200 amino acids, or at least 250 amino acids, or at least 300 amino acids, or at least 350 amino acids, or up to 120 amino acids, or up to 140 amino acids, or up to 160 amino acids, or up to 180 amino acids, or up to 200 amino acids, or up to 250 amino acids, or up to 300 amino acids, or up to 350 amino acids, or up to 400 amino acids can be truncated at the C-terminus of the Cas13b effector. Specific examples of Cas13b truncations include C-terminal Δ984-1090, C-terminal Δ1026-1090, and C-terminal Δ1053-1090, C-terminal Δ934-1090, C-terminal Δ884-1090, C-terminal Δ834-1090, C-terminal Δ784-1090, and C-terminal Δ734-1090 (where the amino acid positions correspond to those of the Prevotella sp. P5-125 Cas13b protein). See Figure 15B.

[0064] In certain embodiments, one or more of the functional domains are regulatable, ie, inducible.

[0065] In certain embodiments of the present invention, the guide RNA or mature crRNA comprises, consists essentially of, or consists of a direct repeat sequence and a guide sequence or spacer sequence. In certain embodiments, the guide RNA or mature crRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or spacer sequence. In preferred embodiments of the present invention, the mature crRNA comprises a stem-loop, an optimized stem-loop structure, or an optimized secondary structure. In preferred embodiments, the mature crRNA comprises a stem-loop or an optimized stem-loop structure in the direct repeat sequence, where the stem-loop or 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 direct 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 stem-loop RNA duplex structure. In preferred embodiments, mutations that maintain the stem-loop RNA duplex can be introduced, thereby maintaining the cleavage activity of the effector protein complex. In other preferred embodiments, mutations can be introduced that disrupt the RNA duplex structure of the stem-loop, thereby completely abolishing the cleavage activity of the effector protein complex.

[0066] The present invention also provides nucleotide sequences encoding effector proteins that are codon-optimized for expression in eukaryotic organisms or cells in any of the methods or compositions described herein. In certain embodiments of the present invention, the nucleotide sequence encoding the codon-optimized effector protein encodes any Cas13b discussed herein and is codon-optimized for operation in a eukaryotic cell or organism, such as a cell or organism listed elsewhere herein, including, without limitation, a yeast cell or a mammalian cell or organism, such as a mouse cell, a rat cell, and a human cell, or a non-human eukaryotic organism, such as a plant.

[0067] In certain embodiments of the present invention, at least one nuclear localization signal (NLS) is added to a nucleic acid sequence encoding a Cas13b effector protein. In preferred embodiments, at least one or more C- or N-terminal NLSs are added (thus, a nucleic acid molecule encoding a Cas13b effector protein can contain coding for an NLS, and thus, an NLS will be attached or connected to the expressed product). In certain embodiments of the present invention, at least one nuclear export signal (NES) is added to a nucleic acid sequence encoding a Cas13b effector protein. In preferred embodiments, at least one or more C- or N-terminal NESs are added (thus, a nucleic acid molecule encoding a Cas13b effector protein can contain coding for an NES, and thus, an expression product will have an attached or connected NES). In preferred embodiments, a C- and / or N-terminal NLS or NES is added for optimal expression and nuclear targeting in eukaryotic cells, preferably human cells. In a preferred embodiment, the codon-optimized effector protein is Cas13b, and the spacer length of the guide RNA is 15 to 35 nt. In certain embodiments, the spacer length of the guide RNA is at least 16 nucleotides, such as at least 17 nucleotides, preferably at least 18 nt, such as preferably at least 19 nt, at least 20 nt, at least 21 nt, or at least 22 nt. In certain embodiments, the spacer length is 15-17 nt, 17-20 nt, 20-24 nt, such as 20, 21, 22, 23, or 24 nt, 23-25 ​​nt, such as 23, 24, or 25 nt, 24-27 nt, 27-30 nt, 30-35 nt, or 35 nt or more. In certain embodiments of the present invention, the codon-optimized effector protein is Cas13b, and the direct repeat length of the guide RNA is at least 16 nucleotides. In certain embodiments, the codon-optimized effector protein is Cas13b, and the direct repeat length of the guide RNA is 16-20 nt, such as 16, 17, 18, 19, or 20 nucleotides.In a particularly preferred embodiment, the direct repeat length of the guide RNA is 19 nucleotides.

[0068] The present invention also encompasses a method for delivering multiple nucleic acid components, each specific for a different target locus of interest, thereby modifying multiple target loci of interest. The nucleic acid components of the complex may include one or more protein-binding RNA aptamers. The one or more aptamers may be capable of binding to a bacteriophage coat protein. The bacteriophage coat protein may be selected from the group including Qβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, and PRR1. In a preferred embodiment, the bacteriophage coat protein is MS2. The invention also provides nucleic acid components of the complexes that are 30 or more, 40 or more, or 50 or more nucleotides in length.

[0069] In a further aspect, the present invention provides a eukaryotic cell comprising a modified target locus of interest, wherein the target locus of interest is modified by any of the methods described herein. A further aspect provides a cell line of said cell. Another aspect provides a multicellular organism comprising one or more said cells.

[0070] In certain embodiments, modification of a target locus of interest may 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.

[0071] In certain embodiments, the eukaryotic cell may be a mammalian cell or a human cell.

[0072] In further embodiments, the non-naturally occurring or engineered compositions, vector systems or delivery systems as described herein may be used for site-specific gene knockout, site-specific genome editing, RNA sequence-specific interference or multiplex genome engineering.

[0073] Also provided are gene products from cells, cell lines, or organisms as described herein. In certain embodiments, the amount of expressed gene product may be greater or less than the amount of the gene product from a cell that does not have an altered expression or edited genome. In certain embodiments, the gene product may be altered compared to the gene product from a cell that does not have an altered expression or edited genome.

[0074] Also provided are engineered and non-naturally occurring eukaryotic cells comprising at least one of (i) a Cas13b effector protein as described herein, or (ii) a guide RNA capable of forming a CRISPR-Cas complex with the Cas13b effector protein. In some embodiments, (i) and / or (ii) are transiently expressed or introduced into the cell. Also provided are organisms, cell lines, and progeny of the cell lines or organisms comprising such cells. The organism may be a vertebrate, such as a mammal. Alternatively, the organism may be a plant or a fungus.

[0075] In a further aspect, the present invention provides a eukaryotic cell comprising a nucleotide sequence encoding a CRISPR system described herein that ensures the generation of a modified target locus of interest, wherein the target locus of interest is modified by any of the methods described herein. A further aspect provides a cell line of said cell. Another aspect provides a multicellular organism comprising one or more of said cells.

[0076] In certain embodiments, modification of a target locus of interest may result in a eukaryotic cell comprising an altered (protein) expression of at least one gene product, a eukaryotic cell comprising an altered (protein) expression of at least one gene product wherein the (protein) expression of the at least one gene product is increased, a eukaryotic cell comprising an altered (protein) expression of at least one gene product wherein the (protein) expression of the at least one gene product is decreased, or a eukaryotic cell comprising an edited transcriptome.

[0077] In certain embodiments, the eukaryotic cell may be a mammalian cell or a human cell.

[0078] In further embodiments, non-naturally occurring or engineered compositions, vector systems or delivery systems as described herein may be used for RNA sequence-specific interference, RNA sequence-specific modification of expression (including isoform-specific expression), stability, localization, functionality (e.g., ribosomal RNA or miRNA), etc., or multiplexing of such processes.

[0079] In further embodiments, the non-naturally occurring or engineered compositions, vector systems, or delivery systems described herein can be used for RNA detection and / or quantification in a sample, such as a biological sample. In certain embodiments, RNA detection is in a cell. In some embodiments, the present invention provides a method for detecting a target RNA in a sample, the method comprising: (a) incubating the sample with i) a type VI-B CRISPR-Cas effector protein capable of cleaving RNA, ii) a guide RNA capable of hybridizing to the target RNA, and iii) an RNA-based cleavage-inducible reporter capable of being non-specifically and detectably cleaved by the effector protein; and (b) detecting the target RNA based on a signal generated by cleavage of the RNA-based cleavage-inducible reporter.

[0080] In some embodiments, the type VI-B CRISPR-Cas effector protein is a Cas13b effector protein, such as a Cas13b effector protein as described herein. In some embodiments, the RNA-based cleavage-inducible reporter construct comprises a fluorescent dye and a quencher. In certain embodiments, the sample comprises a cell-free biological sample. In other embodiments, the sample comprises a cellular sample, such as, without limitation, a plant cell or an animal cell. In some embodiments of the present invention, the target RNA comprises pathogen RNA, including, but not limited to, target RNA from a virus, bacterium, fungus, or parasite. In some embodiments, the guide RNA is designed to detect a target RNA or splice variant of an RNA transcript that contains a single nucleotide polymorphism. In some embodiments, the guide RNA comprises one or more mismatched nucleotides with the target RNA. In certain embodiments, the guide RNA hybridizes to a target molecule that is diagnostic for a disease state, such as, but not limited to, cancer or an immune disorder.

[0081] The present invention provides a ribonucleic acid (RNA) detection system comprising: a) a type VI-B CRISPR-Cas effector protein capable of cleaving RNA; b) a guide RNA capable of binding to a target RNA; and c) an RNA-based cleavage-inducible reporter capable of being non-specifically and detectably cleaved by the effector protein. The present invention also provides a kit for RNA detection comprising: a) a type VI-B CRISPR-Cas effector protein capable of cleaving RNA; and b) an RNA-based cleavage-inducible reporter capable of being non-specifically and detectably cleaved by the effector protein. In certain embodiments, the RNA-based cleavage-inducible reporter construct comprises a fluorescent dye and a quencher.

[0082] In further embodiments, the non-naturally occurring or engineered compositions, vector systems or delivery systems as described herein may be used in the generation of disease models and / or screening systems.

[0083] In further embodiments, the non-naturally occurring or engineered compositions, vector systems, or delivery systems as described herein may be used for site-specific transcriptome editing or perturbation, nucleic acid sequence-specific interference, or multiplex genome engineering.

[0084] In embodiments of the present invention, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or suppress viral pathogenesis, infection, or transmission in a mammalian subject. Embodiments of the present invention provide a Cas13b CRISPR system comprising: (a) a Cas13b CRISPR effector protein and / or a polynucleic acid encoding the Cas13b CRISPR effector protein; and (b) one or more guide RNAs designed to bind to one or more target molecules of a virus and / or one or more polynucleic acids encoding the one or more guide RNAs, for use in treating, protecting against, suppressing, and / or ameliorating viral pathogenesis, infection, and / or transmission in a subject. The Cas13b effector protein can be as defined herein, including with respect to preferred wild-type Ca13b proteins and preferred derivatives and variants thereof.

[0085] In some embodiments, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or inhibit Lassa virus pathogenesis, infection, or transmission in a mammalian subject. Lassa virus associates with DCs and vascular endothelial cells (see Kunz, S. et al. 2005. Journal of Virology).

[0086] In some embodiments, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or inhibit Ebola virus pathogenesis, infection, or transmission in mammalian subjects. Ebola virus is associated with many tissues and cell types, including DCs, macrophages, hepatocytes, etc. (See Martines, RB et al. 2015. Journal of Pathology).

[0087] In some embodiments, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or inhibit SARS-CoV pathogenesis, infection, or transmission in mammalian subjects. SARS-CoV is associated with lung tissue and cells (see To, KF. et al. 2004. Journal of Pathology).

[0088] In some embodiments, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or inhibit Zika virus pathogenesis, infection, or transmission in mammalian subjects. Zika virus is associated with many tissues and cell types, including body fluids, placenta, brain, etc. (See Miner, JJ & Diamond, MS 2017. Cell Host & Microbe).

[0089] In some embodiments, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or inhibit dengue virus pathogenesis, infection, or transmission in a mammalian subject. Dengue virus is associated with many tissues and cell types, including DCs, macrophages, liver, etc. (See Flipse, J. et al. 2016. Journal of General Virology).

[0090] In some embodiments, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or suppress Chikungunya virus pathogenesis, infection, or transmission in mammalian subjects. Chikungunya virus is associated with many tissues and cell types, including immune cells, the liver, the central nervous system, etc. (See Schwartz, O. & Albert, ML 2010. Nature Reviews).

[0091] In some embodiments, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or inhibit influenza virus pathogenesis, infection, or transmission in mammalian subjects. Influenza viruses are associated with lung epithelial cells and macrophages (see Medina, RA & Garcia-Sastre A. 2011 Nature Reviews).

[0092] In some embodiments, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or inhibit HIV viral pathogenesis, infection, or transmission in mammalian subjects. The HIV virus is associated with T cells and macrophages (see Weiss, RA 2002. IUBMB Life.).

[0093] In some embodiments, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or inhibit rotavirus viral pathogenesis, infection, or transmission in mammalian subjects. Rotavirus viruses are associated with intestinal tissues and cells (see Lopez, S & Arias, CF2006. CTMI).

[0094] In some embodiments, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or inhibit herpes simplex (HSV-1) pathogenesis, infection, or spread in a mammalian subject. HSV-1 is associated with epithelial and neuronal cells (see Schelhaas, M. et al. 2003. Journal of General Virology).

[0095] In some embodiments, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or inhibit HCV pathogenesis, infection, or transmission in a mammalian subject. HCV is associated with liver tissue and cells (see Ding, Q, et al. 2014. Cell Host & Microbe).

[0096] In some embodiments, the Cas13b effector proteins or systems described herein can be used to treat, protect against, prevent, or inhibit HBV pathogenesis, infection, or transmission in a mammalian subject. HBV is associated with liver tissue and cells (see Schieck, A. et al. 2013. Hepatology).

[0097] Also provided are gene products from cells, cell lines, or organisms as described herein. In certain embodiments, the amount of expressed gene product may be greater or less than the amount of the gene product from a cell that does not have an altered expression or edited genome. In certain embodiments, the gene product may be altered compared to the gene product from a cell that does not have an altered expression or edited genome.

[0098] These and other embodiments are disclosed or are apparent from and encompassed by the following detailed description.

[0099] Accordingly, it is an object of the present invention not to include within its scope any previously known product, process for making a product, or method for using a product, to which applicants reserve their rights and which hereby disclose any disclaimer of any previously known product, process, or method. It is further noted that the present invention is not intended to include within its scope any product, process for making a product, or method for using a product that does not meet the description and enablement requirements of the United States Patent and Trademark Office (USPTO) (35 U.S.C. § 112, first paragraph) or the European Patent Office (EPO) (Article 83 EPC), to which applicants reserve their rights and which hereby disclose any disclaimer of any previously described product, process for making a product, or method for using a product. Compliance with Article 53(c) EPC and Rule 28(b) and (c) EPC may be advantageous in the practice of the present invention. Nothing herein should be construed as prospective.

[0100] In this disclosure, and particularly in the claims and / or paragraphs, terms such as "comprise," "included," "comprising," and the like may have the meaning ascribed to them in U.S. patent law. For example, they may mean "include," "included," "comprising," and the like, and terms such as "consisting essentially of" and "consisting essentially of" have the meaning ascribed to them in U.S. patent law, for example, it is noted that these terms allow for elements not expressly recited, but exclude elements found in the prior art or that affect a basic or novel characteristic of the invention.

[0101] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0102] [Figure 1-1] A list of wild-type Cas13b orthologs and their amino acid sequences are provided, along with the source organism and protein accession number. [Figure 1-2] A list of wild-type Cas13b orthologs and their amino acid sequences are provided, along with the source organism and protein accession number. [Figure 1-3] A list of wild-type Cas13b orthologs and their amino acid sequences are provided, along with the source organism and protein accession number. [Figure 1-4] A list of wild-type Cas13b orthologs and their amino acid sequences are provided, along with the source organism and protein accession number. [Figure 1-5] A list of wild-type Cas13b orthologs and their amino acid sequences are provided, along with the source organism and protein accession number. [Figure 1-6] A list of wild-type Cas13b orthologs and their amino acid sequences are provided, along with the source organism and protein accession number. [Figure 1-7]A list of wild-type Cas13b orthologs and their amino acid sequences are provided, along with the source organism and protein accession number. [Figure 1-8] A list of wild-type Cas13b orthologs and their amino acid sequences are provided, along with the source organism and protein accession number. [Figure 1-9] A list of wild-type Cas13b orthologs and their amino acid sequences are provided, along with the source organism and protein accession number.

[0103] [Figure 2] Figure 1 shows the classification of each of the Cas13b orthologs along with their relative efficacy in knocking down luciferase expression in mammalian cell cultures.

[0104] [Figure 3] The activity of each of the active Cas13b orthologs is compared, controlling for the guide sequence.

[0105] [Figure 4] The activity of two Cas13b orthologs—Porphyromonas gulae WP_039434803 and Prevotella sp. P5-125 WP_044065294—among various guide sequences is compared to that of C2c2 / Cas13a.

[0106] [Figure 5]Characterization of highly active Cas13b orthologs for RNA knockdown. (A) Schematic of the stereotypical Cas13 locus and corresponding crRNA structure. (B) Characterization of 19 Cas13a, 15 Cas13b, and 7 Cas13c orthologs for luciferase knockdown using two different guides. The host organism name is indicated for orthologs with efficient knockdown using both guides. (C) Comparison of knockdown activity between PspCas13b and LwaCas13a by tiling guides against Gluc and measuring luciferase expression. (D) Comparison of knockdown activity between PspCas13b and LwaCas13a by tiling guides against Cluc and measuring luciferase expression. (E) Expression levels, expressed as log2 (transcripts per million (TPM)), of all genes detected in the RNA-seq library of the non-targeting control (x-axis) compared to the Gluc-targeting condition (y-axis) for LwaCas13a (red) and shRNA (black). Average values ​​for three biological replicates are shown. Gluc transcript data points are labeled. (F) Expression levels, expressed as log2 (transcripts per million (TPM)), of all genes detected in the RNA-seq library of the non-targeting control (x-axis) compared to the Gluc-targeting condition (y-axis) for PspCas13b (blue) and shRNA (black). Average values ​​for three biological replicates are shown. Gluc transcript data points are labeled. (G) Number of significant off-targets from Gluc knockdown for LwaCas13a, PspCas13b, and shRNA from the transcriptome-wide analysis in E and F.

[0107] [Figure 6]Engineering dCas13b-ADAR fusions for RNA editing. (A) Schematic of RNA editing by dCas13b-ADAR fusion proteins. (B) Schematic of Cypridina luciferase W85X target and targeting guide design. (C) Quantification of luciferase activity recovery for Cas13b-dADAR1 (left) and Cas13b-ADAR2-cd (right) tiled with guides of 30, 50, 70, or 84 nt in length. (D) Schematic of target sites for targeting Cypridina luciferase W85X. (E) Sequencing quantification of A→I editing for a 50 nt guide targeting Cypridina luciferase W85X.

[0108] [Figure 7] Measurement of sequence flexibility for RNA editing by REPAIRv1. (A) Schematic of the screen to determine the protospacer adjacent site (PFS) preference for RNA editing by REPAIRv1. (B) Distribution of RNA editing efficiency for all combinations of 4-N PFSs at two different editing sites. (C) Quantification of the percent editing rate of REPAIRv1 in Cluc W85 for all possible three-base motifs. (D) Heat map of 5' and 3' base preference for RNA editing in Cluc W85 for all possible three-base motifs.

[0109] [Figure 8]Correction of disease-associated mutations with REPAIRv1. (A) Schematic of the target and guide design for targeting AVPR2 878G>A. (B) The 878G>A mutation in AVPR2 is corrected to various percentages using REPAIRv1 with three different guide designs. (C) Schematic of the target and guide design for targeting FANCC 1517G>A. (D) The 1517G>A mutation in FANCC is corrected to various percentages using REPAIRv1 with three different guide designs. (E) Quantification of the percent editing rate 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) Distribution of editing motifs for all G>A mutations in ClinVar versus the editing efficiency by REPAIRv1 per motif as quantified on the Gluc transcript.

[0110] [Figure 9] Characterization of REPAIRv1 specificity. (A) Schematic of KRAS target site and guide design. (B) Quantification of percent editing rate for tiled KRAS-targeting guides. Editing rate percentages at on-target and adjacent adenosine sites are shown. For each guide, the region of duplex RNA is outlined in red. (C) Transcriptome-wide sites of significant RNA editing by REPAIRv1 with Cluc-targeting guides. The on-target Cluc site (254 A>G) is highlighted in orange. (D) Transcriptome-wide sites of significant RNA editing by REPAIRv1 with non-targeting guides.

[0111] [Figure 10]Rational mutagenesis of ADAR2 to improve the specificity of REPAIRv1. (A) Quantification of luciferase signal recovery by various dCas13-ADAR2 mutants and their specificity scores plotted along a schematic of contacts between key ADAR2 deaminase residues and the dsRNA target. The specificity score is defined as the ratio of luciferase signal between the targeting guide and non-targeting guide conditions. (B) Quantification of luciferase signal recovery by various dCas13-ADAR2 mutants against their specificity scores. (C) Measurement of on-target editing percentage and the number of significant off-targets for each dCas13-ADAR2 mutant by transcriptome-wide sequencing of mRNA. (D) Transcriptome-wide sites of significant RNA editing by REPAIRv1 and REPAIRv2 with guides targeting a premature termination site in Cluc. The on-target Cluc site (254 A>G) is highlighted in orange. (E) RNA sequencing reads surrounding the on-target Cluc editing site (254 A>G) highlighting the difference in 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 with guides targeting out-of-frame UAG sites in endogenous KRAS and PPIB transcripts. The percentage of on-target editing for each condition row is shown as a bar graph on the right. The duplex region formed by the guide RNA is indicated by a red box.

[0112] [Figure 11]Bacterial screening of Cas13b orthologs for in vivo efficiency and PFS determination. (A) Schematic of the bacterial assay to determine the PFS of Cas13b orthologs. Cas13b orthologs with a β-lactamase targeting spacer are co-transformed with a β-lactamase expression plasmid and subjected to double selection. (B) Quantification of the interference activity of β-lactamase-targeting Cas13b orthologs as measured by colony forming units (cfu). (C) PFS logos of Cas13b orthologs as determined by depletion sequencing from the bacterial assay.

[0113] [Figure 12] Optimization of Cas13b knockdown and further characterization of mismatch specificity. (A) Gluc knockdown is measured with two different guides using the top two Cas13a and top four Cas13b orthologs fused to various nuclear localization and export tags. (B) KRAS knockdown is measured with four different guides for LwaCas13a, RanCas13b, PguCas13b, and PspCas13b and compared to four position-matched shRNA controls. (C) Schematic of the single- and double-mismatch plasmid libraries used to assess the specificity of LwaCas13a and PspCas13b knockdown. All possible single and double mismatches are present at the target sequence and three positions immediately adjacent to the 5' and 3' ends of the target site. (D) Transcript depletion levels at the indicated single mismatches are plotted as a heatmap for both LwaCas13a and PspCas13b conditions. (E) Transcript depletion levels at the indicated double mismatches are plotted as a heatmap for both LwaCas13a and PspCas13b conditions.

[0114] [Figure 13]Characterization of design parameters for dCas13-ADAR2 RNA editing. (A) Knockdown efficiency of Gluc targeting for wild-type Cas13b and catalytically inactive H133A / H1058A Cas13b (dCas13b). (B) Quantification of luciferase activity restoration by dCas13b fused to either the wild-type ADAR2 catalytic domain or the highly active E488Q mutant ADAR2 catalytic domain, tested with tiling Cluc-targeting guides. (C) Guide design and sequencing quantification of A→I editing for a 30-nt guide targeting Cypridina luciferase W85X. (D) Guide design and sequencing quantification of A→I editing for a 50-nt guide targeting PPIB. (E) Effect of linker choice on luciferase activity restoration by REPAIRv1. (F) Effect of the opposite base identity of the target adenosine on luciferase activity restoration by REPAIRv1.

[0115] [Figure 14] ClinVar motif distribution for G>A mutations. Number of each possible triplet motif observed in the ClinVar database for all G>A mutations.

[0116] [Figure 15] (A) Cas13b truncations still have functional RNA editing. Various N- and C-terminal truncations of dCas13b enable RNA editing as measured by the restoration of luciferase signal. (B) Further examples of dCas13b-ADAR constructs with different C-terminal truncations of dCas13b.

[0117] [Figure 16]Comparison of dCas13-ADAR2 editing with other programmable ADAR systems. (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 (ADAR2DD(E488Q)) is fused to a small bacterial / viral protein called lambda N (λN), which specifically binds to a small RNA sequence called BoxB-λ. A guide RNA containing two BoxB-λ hairpins can then guide ADAR2DD(E488Q)-λN for site-specific editing. In the full-length ADAR2 scheme (bottom), the dsRNA-binding domain of ADAR2 binds to the guide RNA hairpins, enabling programmable ADAR2 editing. (B) Transcriptome-wide sites of significant RNA editing by BoxB-ADAR2DD(E488Q) in a Cluc-targeting guide and a non-targeting guide. The on-target Cluc site (254 A>G) is highlighted in orange. (C) Transcriptome-wide sites of significant RNA editing by ADAR2 with guides targeting Cluc and non-targeting guides. The on-target Cluc site (254 A>G) is highlighted in orange. (D) Transcriptome-wide sites of significant RNA editing by REPAIRv1 with guides targeting Cluc and non-targeting guides. The on-target Cluc site (254 A>G) is highlighted in orange. (E) Quantification of the on-target editing percentage of BoxB-ADAR2DD(E488Q), ADAR2, and REPAIRv1 for guides targeting Cluc. (F) Off-target site overlap between different targeting and non-targeting conditions for the programmable ADAR system.

[0118] [Figure 17]Efficiency and specificity of dCas13b-ADAR2 mutants. (A) Quantification of luciferase activity recovery by the dCas13b-ADAR2DD(E488Q) mutant for Cluc-targeting and non-targeting guides. (B) Relationship between the ratio of targeting and non-targeting guides and the number of RNA editing off-targets as quantified by transcriptome-wide sequencing. (C) Quantification of the number of transcriptome-wide off-target RNA editing sites relative to the on-target Cluc editing efficiency for the dCas13b-ADAR2DD(E488Q) mutant.

[0119] [Figure 18] Transcriptome-wide specificity of RNA editing by the dCas13b-ADAR2DD(E488Q) mutant. (A) Transcriptome-wide sites of significant RNA editing by the dCas13b-ADAR2DD(E488Q) mutant with a guide targeting Cluc. The on-target Cluc site (254 A>G) is highlighted in orange. (B) Transcriptome-wide sites of significant RNA editing by the dCas13b-ADAR2DD(E488Q) mutant with a non-targeting guide.

[0120] [Figure 19] Characterization of motif bias in off-target dCas13b-ADAR2DD(E488Q) editing. (A) For each dCas13b-ADAR2DD(E488Q) mutant, motifs present across all A>G off-target edits in the transcriptome are shown. (B) Distribution of off-target A>G edits by motif identity for REPAIRv1 with targeting and non-targeting guides is shown. (C) Distribution of off-target A>G edits by motif identity for REPAIRv2 with targeting and non-targeting guides is shown.

[0121] [Figure 20]Further characterization of REPAIRv1 and REPAIRv2 off-targets. (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) Predicted mutational effects of REPAIRv1 off-targets. (D) Distribution of potential oncogenic effects of REPAIRv1 off-targets. (E) Predicted mutational effects of REPAIRv2 off-targets. (F) Distribution of potential oncogenic effects of REPAIRv2 off-targets.

[0122] [Figure 21] RNA editing efficiency and specificity of REPAIRv1 and REPAIRv2. (A) Quantification of the percent editing rate of KRAS by KRAS targeting guide 1 at the target adenosine and adjacent sites for REPAIRv1 and REPAIRv2. (B) Quantification of the percent editing rate of KRAS by KRAS targeting guide 3 at the target adenosine and adjacent sites for REPAIRv1 and REPAIRv2. (C) Quantification of the percent editing rate of PPIB by PPIB targeting guide 2 at the target adenosine and adjacent sites for REPAIRv1 and REPAIRv2.

[0123] [Figure 22] Demonstration of all potential codon changes by A>G RNA editing. (A) Table of all potential codon conversions made possible by A>I editing. (B) Codon table demonstrating all potential codon conversions made possible by A>I editing. DETAILED DESCRIPTION OF THE INVENTION

[0124] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

[0125] Generally, CRISPR-Cas or CRISPR system, as used in the aforementioned documents, such as WO 2014 / 093622 (PCT / US 2013 / 074667), collectively refers to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeats" in the context of endogenous CRISPR systems and partial direct repeats processed by tracrRNA), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems) or "RNAs" as that term is used herein (e.g., RNAs that guide Cas, 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 a CRISPR locus. In general, CRISPR systems are characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of endogenous CRISPR systems).

[0126] When the CRISPR protein is a class 2 type VI-B effector (e.g., a Cas13b effector protein), tracrRNA is not required. In the engineered systems of the present invention, the direct repeats can comprise naturally occurring or non-naturally occurring sequences. The direct repeats of the present invention are not limited to naturally occurring lengths and sequences. The direct repeats can be 36 nt in length, but longer or shorter direct repeats can vary. For example, the direct repeats can be 30 nt or longer, e.g., 30-100 nt or longer. For example, the direct repeats can be 30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt or longer. In some embodiments, the direct repeats of the present invention can comprise a synthetic nucleotide sequence inserted between the 5' and 3' ends of the naturally occurring direct repeat. In certain embodiments, the inserted sequence may be self-complementary, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% self-complementary. Furthermore, the direct repeat of the present invention may contain an insertion of nucleotides (for association with a functional domain), such as a sequence that binds to an aptamer or an adaptor protein. In certain embodiments, one end of the direct repeat containing such an insertion is approximately the first half of the short DR, and the other end is approximately the second half of the short DR.

[0127] In the context of CRISPR complex formation, a "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between the target sequence and the guide sequence promotes CRISPR complex formation. The target sequence may comprise an RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, direct repeats may be identified in silico by searching for repetitive motifs that meet some or all of the following criteria: 1. Present in a 2 Kb window of genomic sequence adjacent to the CRISPR locus; 2. Spanning 20-50 bp and spaced 3. Between 20-50 bp. In some embodiments, two of these criteria may be used, e.g., 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all three criteria may be used.

[0128] In embodiments of the present invention, the terms guide sequence and guide RNA, i.e., RNA capable of guiding Cas13b to a target genomic locus, are used interchangeably, as in the aforementioned references, such as International Publication No. WO 2014 / 093622 (PCT / US Patent Application Publication No. 2013 / 074667). Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any algorithm suitable for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (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 guide sequence is 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. In some embodiments, the guide sequence is less than or equal to about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length. Preferably, the guide sequence is 10-40 nucleotides in length, such as 20-30 or 20-40 nucleotides in length or more, such as 30 nucleotides in length or about 30 nucleotides in length.In certain embodiments, the guide sequence for a Cas13b effector is 10-30 nucleotides in length, e.g., 20-30 or 20-40 nucleotides in length or more, e.g., 30 nucleotides in length or about 30 nucleotides in length. In certain embodiments, the guide sequence for a Cas13b effector derived from Bergeyella zoohelcum (such as Bergeyella zoohelcum ATCC 43767) is 10-30 nucleotides in length, e.g., 20-30 nucleotides in length, e.g., 30 nucleotides in length or about 30 nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence can be assessed by any suitable assay. For example, sufficient components of a CRISPR system to form a CRISPR complex, including the guide sequence to be tested, can be provided to a host cell having a corresponding target sequence, such as by transfection of a vector encoding the components of the CRISPR sequence, followed by assessing preferential cleavage within the target sequence, such as by a Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence can be determined in vitro by providing the target sequence, the components of the CRISPR complex, including the guide sequence to be tested, and a control guide sequence that differs from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between reactions with the test guide sequence and the control guide sequence. Other assays are possible and will occur to those skilled in the art.

[0129] In classical CRISPR-Cas systems, the degree of complementarity between a guide sequence and its corresponding target sequence can be about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100% or more, and the guide, or RNA, or sgRNA can be 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 sgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length or shorter. However, in certain embodiments of the invention, off-target interactions can be reduced, for example, by reducing guide interaction with less complementary target sequences. Indeed, certain natural mutations can result in CRISPR-Cas systems that can distinguish between target sequences and off-target sequences having 80% to greater than about 95% complementarity, e.g., 83% to 84%, or 88 to 89%, or 94 to 95% complementarity (e.g., distinguishing an 18-nucleotide target from an 18-nucleotide off-target with one, two, or three mismatches). Thus, in the context of the present invention, the degree of complementarity between a guide sequence and its corresponding target sequence can be 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 greater than 99.9%, or 100%.Off-target alignment 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%, or 94%, or 93%, or 92%, or 91%, or 90%, or 89%, or 88%, or 87%, or 86%, or is less than 85%, or 84%, or 83%, or 82%, or 81%, or 80% complementary, and 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% complementary between its sequence and the guide.

[0130] In certain embodiments, the introduction of one or more mismatches, such as one or two mismatches, between the spacer sequence and the target sequence can be used to adjust the cleavage efficiency, including the position of the mismatch along the spacer / target. For example, the closer to the center (i.e., not closer to the 3' or 5') the double mismatch is, the greater the impact on cleavage efficiency. Thus, the cleavage efficiency can be adjusted by selecting the mismatch position along the spacer. For example, if less than 100% cleavage of the target is desired (e.g., in a certain cell population), one or more, preferably two, mismatches between the spacer and the target sequence can be introduced into the spacer sequence. The closer to the center the mismatch position is along the spacer, the lower the cleavage rate.

[0131] The methods of the invention as described herein include inducing one or more nucleotide modifications in a eukaryotic cell as discussed herein (in vitro, i.e., in an isolated eukaryotic cell), comprising delivering a vector as discussed herein to the cell. The mutations may include the introduction, deletion, or substitution of one or more nucleotides in each target sequence of the cell via a guide RNA or sgRNA. The mutations may include the introduction, deletion, or substitution of 1 to 75 nucleotides in each target sequence of the cell via a guide RNA. The mutations may include the introduction, deletion, or substitution 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 in each target sequence of the cell via a guide RNA. 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 in each target sequence of the cell via the guide RNA.The mutation may include 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 in each target sequence of the cell via the guide RNA. 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 in each target sequence of the cell via the guide RNA.The mutation may include the introduction, deletion or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides in each target sequence of the cell via the guide RNA.

[0132] To minimize toxicity and off-target effects, it may be important to control the concentration of delivered Cas mRNA or protein and guide RNA. The optimal concentration of Cas mRNA or protein and guide RNA can be determined by testing various concentrations in cell models or non-human eukaryotic animal models and analyzing the extent of modification at potential off-target genomic loci using deep sequencing.

[0133] Typically, in the context of endogenous CRISPR systems, 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 at or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs of the target sequence), which can depend, for example, on secondary structure, particularly in the case of RNA targets.

[0134] The nucleic acid molecule encoding Cas is advantageously a codon-optimized Cas. An example of a codon-optimized sequence, in this case, is a sequence optimized for expression in a eukaryote, such as a human (i.e., optimized for human expression), or another eukaryote, animal, or mammal as discussed herein. See, for example, the SaCas9 human codon-optimized sequence in WO 2014 / 093622 (PCT / US 2013 / 074667). While this is preferred, it is understood that other examples are possible, and codon optimization for host species other than humans or for specific organs is known. In some embodiments, the enzyme coding sequence encoding Cas is codon-optimized for expression in a specific cell, e.g., a eukaryotic cell. The eukaryotic cell may be of or derived from a specific organism, e.g., a mammal, including, but not limited to, a human, or a non-human eukaryote or animal or mammal as discussed herein, e.g., a mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. In some embodiments, methods for modifying human germline genetic identity and / or methods for modifying animal genetic identity that may cause suffering to humans or animals without any substantial medical benefit to them, as well as animals resulting from such methods, may be excluded. Generally, codon optimization refers to a method of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the native sequence with a codon that is more frequently or most frequently used in the host cell's genes while maintaining the native amino acid sequence. Different species exhibit specific biases for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend, among other things, on the properties of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons most frequently used in peptide synthesis.Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. 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 a number of ways. See Nakamura, Y., et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000," Nucl. Acids Res. 28:292 (2000). Computer algorithms are also available for codon-optimizing a particular sequence for expression in a particular host cell, 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 a Cas-encoding sequence correspond to the most frequently used codon for a particular amino acid.

[0135] In certain embodiments, the methods described herein may include providing a Cas transgenic cell in which one or more nucleic acids encoding one or more guide RNAs operably linked within the cell to regulatory elements comprising promoters of one or more genes of interest are provided or introduced. As used herein, the term "Cas transgenic cell" refers to a cell, such as a eukaryotic cell, in which a Cas gene has been genomically integrated. The nature, type, or origin of the cell is not particularly limited according to the present invention. Also, the method of introducing a Cas transgene into a cell may vary and may be any method known in the art. In certain embodiments, the Cas transgenic cell is obtained by introducing a Cas transgene into an isolated cell. In certain other embodiments, the Cas transgenic cell is obtained by isolating cells from a Cas transgenic organism. By way of example and without limitation, the Cas transgenic cell as referred to herein may be derived from a Cas transgenic eukaryotic organism, such as a Cas knock-in eukaryotic organism. See International Publication No. WO 2014 / 093622 (PCT / US Patent Application Publication No. 13 / 74667), incorporated herein by reference. The methods of US Patent Application Publication Nos. 20120017290 and 20110265198, assigned to Sangamo BioSciences, Inc., relating to targeting the Rosa locus, can be modified to utilize the CRISPR Cas system of the present invention. The methods of US Patent Application Publication No. 20130236946, assigned to Cellectis, relating to targeting the Rosa locus can also be modified to utilize the CRISPR Cas system of the present invention. As a further example, see Platt et al. (Cell; 159(2):440-455(2014)), which describes Cas9 knock-in mice, incorporated herein by reference. The Cas transgene may further comprise a Lox-Stop-PolyA-Lox (LSL) cassette, making Cas expression inducible by Cre recombinase.Alternatively, Cas transgenic cells can be obtained by introducing a Cas transgene into isolated cells. Transgene delivery systems are well known in the art. For example, Cas transgenes can be delivered using vectors (e.g., AAV, adenovirus, lentivirus), and / or particle and / or nanoparticle delivery, for example, in eukaryotic cells, as also described elsewhere herein.

[0136] Those skilled in the art will understand that cells, such as Cas transgenic cells as referenced herein, in addition to having an integrated Cas gene, may contain additional genomic alterations or may contain mutations, such as one or more oncogenic mutations, that arise from the sequence-specific action of Cas when complexed with an RNA capable of guiding Cas to a target locus, as described, for example and without limitation, in Platt et al. (2014), Chen et al., (2014), or Kumar et al. (2009).

[0137] In some embodiments, the Cas sequence is fused to one or more nuclear localization sequences (NLSs) or nuclear export sequences (NESs), such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs or NESs. In some embodiments, the Cas comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs or NESs at or near the amino terminus, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs or NESs at or near the carboxy terminus, or a combination thereof (e.g., zero or at least one NLS or NES at the amino terminus and zero or one or more NLSs or NESs at the carboxy terminus). When more than one NLS or NES is present, each may be selected independently of the others, and thus a single NLS or NES may be present in two or more copies and / or in combination with one or more other NLSs or NESs present in one or more copies. In preferred embodiments of the invention, the Cas comprises up to six NLSs. In some embodiments, an NLS or NES is considered to be near the N- or C-terminus when the nearest amino acid of the NLS or NES 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- or C-terminus.Non-limiting examples of NLSs include the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: X), the nucleoplasmin bipartite NLS having the NLS of nucleoplasmin (e.g., the sequence KRPAATKKAGQAKKKK) (SEQ ID NO: X), the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: X) or RQRRNELKRSP (SEQ ID NO: X), the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: X), the IBB domain of importin-α having the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: X), the sequences VSRKRPRP (SEQ ID NO: X) and PPKKARED (SEQ ID NO: X) of the fibroid T protein, the sequence POPKKKPL (SEQ ID NO: X) of human p53, the mouse c-abl Examples of NLS sequences include those derived from the sequence SALIKKKKKMAP (SEQ ID NO: X), the sequences DRLRR (SEQ ID NO: X) and PKQKKRK (SEQ ID NO: X) of influenza virus NS1, the sequence RKLKKKIKKL (SEQ ID NO: X) of hepatitis virus delta antigen, the sequence REKKKFLKRR (SEQ ID NO: X) of mouse Mx1 protein, the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: X) of human poly(ADP-ribose) polymerase, and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: X) of steroid hormone receptor (human) glucocorticoid. Non-limiting examples of NESs include the NES sequence LYPERLRRILT (ctgtaccctgagcggctgcggcggatcctgacc). Generally, one or more NLSs or NESs are strong enough to drive the accumulation of detectable amounts of Cas in the nucleus or cytoplasm, respectively, of eukaryotic cells. In general, the strength of nuclear localization / export activity can be derived from the number of NLSs / NESs in the Cas, the specific NLSs or NESs used, or a combination of these factors. Detection of nuclear / cytoplasmic accumulation can be performed by any suitable technique. For example, a detectable marker can be fused to the Cas, thereby visualizing its location within the cell, for example, by combining it with a means to detect nuclear (e.g., a nuclear-specific stain such as DAPI) or cytoplasmic location.Cell nuclei can also be isolated from cells, and their contents can then be analyzed by any suitable protein detection method, such as immunohistochemistry, Western blot, or enzyme activity assays. Accumulation in the nucleus can also be determined indirectly, such as by assaying for the effect of CRISPR complex formation (e.g., assaying for DNA cleavage or mutation at the target sequence or assaying for altered gene expression activity affected by CRISPR complex formation and / or Cas enzymatic activity), compared to a control not exposed to the Cas or complex, or to a control exposed to Cas lacking one or more NLSs or NESs. In certain embodiments, other localization tags can be fused to the Cas protein, such as, without limitation, to localize Cas to specific sites within the cell, such as organelles, e.g., mitochondria, plastids, chloroplasts, vesicles, Golgi, (nuclear or cell) membranes, ribosomes, nucleoli, ER, cytoskeleton, vacuoles, centrosomes, nucleosomes, granules, centrioles, etc.

[0138] In certain aspects, the present invention relates to vectors for delivering or introducing, for example, Cas and / or RNA capable of guiding Cas to a target locus (i.e., guide RNA) into cells and for propagating these components (e.g., in prokaryotic cells). As used herein, a "vector" is a tool that enables or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted, resulting in replication of the inserted segment. Generally, a vector is capable of replication when associated with appropriate control elements. In general, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules, nucleic acid molecules containing one or more free ends, nucleic acid molecules without free ends (e.g., circular), nucleic acid molecules comprising DNA, RNA, or both, and various other polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which the vector contains virus-derived DNA or RNA sequences for packaging into a virus (e.g., retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, and adeno-associated virus (AAV)). Viral vectors also include virally carried polynucleotides for transfection into host cells. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." Common expression vectors useful in recombinant DNA techniques are often in the form of plasmids.

[0139] A recombinant expression vector can contain a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, meaning that the recombinant expression vector contains one or more regulatory elements (which can be selected based on the host cell used for expression) operably linked to the nucleic acid sequence to be expressed. Within the scope of a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory element in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). For recombination and cloning methods, see U.S. Patent Application No. 10 / 815,730, published September 2, 2004 as U.S. Patent Application Publication No. 2004-0171156 A1, the contents of which are incorporated herein by reference in their entirety.

[0140] The vector may include regulatory elements, such as a promoter. The vector may include a Cas coding sequence and / or a single guide RNA (e.g., sgRNA) coding sequence, but potentially may include at least 3, or 8, or 16, or 32, or 48, or 50 RNAs (e.g., sgRNAs), e.g., 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, or 3-50 RNAs (e.g., sgRNAs). A single vector advantageously may have a promoter for each RNA (e.g., sgRNA) if there are about 16 or fewer RNAs, and if a single vector provides more than 16 RNAs, one or more promoters may drive expression of two or more of those RNAs, e.g., if there are 32 RNAs, each promoter may drive expression of two RNAs, and if there are 48 RNAs, each promoter may drive expression of three RNAs. Using simple mathematical techniques, well-established cloning protocols, and the teachings of this disclosure, one skilled in the art can easily implement the present invention with RNA and a suitable promoter, such as the U6 promoter, for a suitable exemplary vector, such as AAV. For example, the packaging limit for AAV is approximately 4.7 kb. The length of a single U6-gRNA (plus restriction sites for cloning) is 361 bp. Therefore, one skilled in the art can easily fit approximately 12-16, e.g., 13, U6-gRNA cassettes into a single vector. These can be assembled by any suitable means, such as the Golden Gate strategy used for TALE assembly (http: / / www.genome-engineering.org / taleffectors / ). One skilled in the art can also use a tandem guide strategy to increase the number of U6-gRNAs by approximately 1.5-fold, e.g., from 12-16, e.g., 13, to approximately 18-24, e.g., approximately 19, U6-gRNAs. Thus, one skilled in the art can easily arrive at about 18 to 24, e.g., about 19 promoter-RNAs, e.g., U6-gRNAs, in a single vector, e.g., an AAV vector.Another way to increase the number of promoters and RNAs in a vector is to use a single promoter (e.g., U6) to express an array of RNAs separated by cleavable sequences. Yet another way to increase the number of promoter-RNAs in a vector is to express an array of promoter-RNAs separated by cleavable sequences in the coding sequence or intron of a gene, in which case it is advantageous to use a polymerase II promoter, which can increase expression and enable transcription of long RNAs in a tissue-specific manner (see, for example, http: / / nar.oxfordjournals.org / content / 34 / 7 / e53.short, http: / / www.nature.com / mt / journal / v16 / n9 / abs / mt2008144a.html). In an advantageous embodiment, AAV can package U6 tandem gRNAs targeting up to about 50 genes. Thus, from knowledge in the art and the teachings of this disclosure, one of ordinary skill in the art can readily make and use vectors, e.g., single vectors, that express multiple RNAs or guides under the control of, or operably or functionally linked to, one or more promoters (particularly with respect to the number of RNAs or guides discussed herein) without any undue experimentation.

[0141] The guide RNA coding sequence and / or the Cas coding sequence can be functionally or operably linked to a regulatory element, thereby driving expression. The promoter can be a constitutive promoter, a conditional promoter, an inducible promoter, and / or a tissue-specific promoter. The promoter can be selected from the group consisting of RNA polymerase, pol I, pol II, pol III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter. A preferred promoter is the U6 promoter.

[0142] Aspects of the present invention relate to the identification and engineering of novel effector proteins associated with Class 2 CRISPR-Cas systems. In preferred embodiments, the effector proteins comprise single-subunit effector modules. In further embodiments, the effector proteins function in prokaryotic or eukaryotic cells in in vitro, in vivo, or ex vivo applications.

[0143] The term "nucleic acid targeting system," in which the nucleic acid is DNA or RNA and in some embodiments can also refer to a DNA-RNA hybrid or derivative thereof, collectively refers to transcripts and other elements involved in the expression of or directing the activity of a DNA- or RNA-targeting CRISPR-associated ("Cas") gene, which may include sequences encoding the DNA- or RNA-targeting Cas protein and a CRISPR RNA (crRNA) sequence, and (in some, but not all systems) a DNA- or RNA-targeting guide RNA, including a trans-activating CRISPR-Cas system RNA (tracrRNA) sequence, or other sequences and transcripts from a DNA- or RNA-targeting CRISPR locus. Generally, RNA targeting systems are characterized by elements that promote the formation of a DNA- or RNA-targeting complex at the site of the target DNA or RNA sequence. In the context of forming a DNA or RNA targeting complex, "target sequence" refers to a DNA or RNA sequence to which a DNA or RNA targeting guide RNA is designed to have complementarity, where hybridization between the target sequence and the RNA targeting guide RNA promotes the formation of an RNA targeting complex. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell.

[0144] In one embodiment of the present invention, the novel RNA targeting system, also referred to as the RNA- or RNA-targeting CRISPR / Cas or CRISPR-Cas based RNA targeting system of the present application, is based on identified type VI-B Cas proteins, which do not require the creation of customized proteins to target specific RNA sequences, but rather allow a single enzyme to be programmed to recognize a specific RNA target by an RNA molecule, which in turn can be used to recruit an enzyme to a specific RNA target.

[0145] In one embodiment of the present invention, the present novel DNA targeting system, also referred to as DNA- or DNA-targeting CRISPR / Cas or CRISPR-Cas based RNA targeting system, is based on identified type VI-B Cas proteins, which do not require the creation of customized proteins to target specific RNA sequences, but rather allow a single enzyme to be programmed to recognize a specific DNA target by an RNA molecule, which in turn can be used to recruit the enzyme to a specific DNA target.

[0146] The nucleic acid targeting systems, vector systems, vectors and compositions described herein can be used in a variety of nucleic acid targeting applications, altering or modifying the synthesis of gene products such as proteins, nucleic acid cleavage, nucleic acid editing, nucleic acid splicing, target nucleic acid transport, target nucleic acid tracking, target nucleic acid isolation, target nucleic acid visualization, etc.

[0147] As used herein, Cas protein or CRISPR enzyme refers to any of the proteins represented in the novel class of CRISPR-Cas systems.

[0148] Cas13b nuclease A Cas13b effector protein of the present invention is, comprises, consists essentially of, consists of, involves or relates to such a protein from or as shown in Figure 1. Preferred proteins in FIG. 1 include Porphyromonas gulae Cas13b (accession number WP_039434803), Prevotella sp. P5-125 Cas13b (accession number WP_044065294), Porphyromonas gingivalis Cas13b (accession number WP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession number WP_039428968), Bacteroides pyogenes Cas13b (accession number WP_034542281), Riemerella anatipestifera Cas13b (accession number WP_039428968), and Porphyromonas gulae Cas13b (accession number WP_039434803). anatipestifer) Cas13b (accession number WP_004919755). The most preferred proteins of FIG. 1 are selected from the group consisting of Porphyromonas gulae Cas13b (Accession No. WP_039434803), Prevotella sp. P5-125 Cas13b (Accession No. WP_044065294), Porphyromonas gingivalis Cas13b (Accession No. WP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (Accession No. WP_039428968), and most especially preferred are Porphyromonas gulae Cas13b (Accession No. WP_039434803) or Prevotella sp. sp.) P5-125 Cas13b (accession number WP_044065294).The present invention is intended to provide or relate to or involve or comprise or consist essentially of or consist of proteins from or as set forth herein, including mutations or modifications thereof as set forth herein.

[0149] Thus, in some embodiments, the effector protein may be an RNA-binding protein, such as a dead Cas-type effector protein, which may optionally be functionalized with, for example, a transcription activator or repressor domain, an NLS, or other functional domain, as described herein. In some embodiments, the effector protein may be an RNA-binding protein that cleaves a single strand of 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 a double strand of RNA, for example, if it contains two RNase domains. If the bound RNA is dsRNA, the dsRNA is completely cleaved.

[0150] The RNase function in CRISPR systems is well known, and for example, mRNA targeting has been reported for certain type III CRISPR-Cas systems (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), providing significant advantages. Thus, a CRISPR-Cas system, composition, or method for targeting RNA by the effector protein is provided.

[0151] The target RNA, i.e., the RNA of interest, is the RNA to be targeted by the present invention, which leads to the recruitment and binding of an effector protein to a desired target site 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.

[0152] Cas13b Guide As used herein, the terms "crRNA," or "guide RNA," or "single guide RNA," or "sgRNA," or "one or more nucleic acid components" of a Type VI CRISPR-Cas locus effector protein include any polynucleotide sequence that has sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of an RNA targeting complex to the target RNA sequence.

[0153] In certain embodiments, the CRISPR system as provided herein can utilize a crRNA or similar polynucleotide comprising a guide sequence, where the polynucleotide is RNA, DNA, or a mixture of RNA and DNA, and / or the polynucleotide comprises one or more nucleotide analogs. This sequence can comprise any structure, including, but not limited to, the structure of a naturally occurring crRNA, such as a bulge, hairpin, or stem-loop structure. In certain embodiments, the polynucleotide comprising the guide sequence forms a duplex with a second polynucleotide sequence, which can be an RNA or DNA sequence.

[0154] In certain embodiments, the guide of the present invention comprises a non-naturally occurring nucleic acid, and / or a non-naturally occurring nucleotide, and / or a nucleotide analog, and / or a chemical modification. Non-naturally occurring nucleic acids can include, for example, a mixture of naturally occurring and non-naturally occurring nucleotides. The non-naturally occurring nucleotides and / or nucleotide analogs can be modified at the ribose, phosphate, and / or base moieties. In certain embodiments of the present invention, the guide nucleic acid comprises ribonucleotides and non-ribonucleotides. In one such embodiment, the guide comprises one or more ribonucleotides and one or more deoxyribonucleotides. In certain embodiments of the present invention, the guide comprises one or more non-naturally occurring nucleotides or nucleotide analogs, such as a nucleotide having a phosphorothioate linkage, a boranophosphate linkage, a locked nucleic acid (LNA) nucleotide containing a methylene bridge between the 2' and 4' carbons of the ribose ring, or a bridged nucleic acid (BNA). Other examples of modified nucleotides include 2'-O-methyl analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, or 2'-fluoro analogs. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine (Ψ), N1-methylpseudouridine (melΨ), 5-methoxyuridine (5moU), inosine, 7-methylguanosine. Examples of chemical modifications of guide RNAs include, without limitation, incorporation of 2'-O-methyl (M), 2'-O-methyl-3'-phosphorothioate (MS), S-constrained ethyl (cEt), or 2'-O-methyl-3'-thioPACE (MSP) at one or more terminal nucleotides.Such chemically modified guides can include increased stability and activity when compared to unmodified guide RNAs; however, on-target versus off-target specificity is unpredictable (Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi:10.1038 / nbt.3290, published online 29 June 2015; Allerson et al., J. Med. Chem. 2005, 48:901-904; Bramsen et al., Front. Genet., 2012, 3:154; Deng et al., PNAS, 2015, 112:11870-11875; Sharma et al., MedChemComm., 2014, 5:1454-1471; Li et al., Nature Biomedical Engineering, 2017, 1,0066 DOI:10.1038 / s41551-017-0066).

[0155] In some embodiments, the 5' and / or 3' ends of the guide RNA are modified with various functional moieties, including fluorescent dyes, polyethylene glycol, cholesterol, proteins, or detection tags (see Kelly et al., 2016, J. Biotech. 233:74-83). In certain embodiments, the guide comprises ribonucleotides in the region that binds to the target RNA and one or more deoxyribonucleotides and / or nucleotide analogs in the region that binds to Cas13b. In certain embodiments of the invention, deoxyribonucleotides and / or nucleotide analogs are incorporated into the engineered guide structure, including, without limitation, the 5' and / or 3' ends, the stem-loop region, and the seed region. In certain embodiments, the modification is not in the 3'-handle of the stem-loop region. Chemical modification of the 3'-handle of the stem-loop region of the guide can abolish its function. In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides of the guide are chemically modified. In some embodiments, 3 to 5 nucleotides at either the 3' or 5' end of the guide are chemically modified. In some embodiments, only minor modifications are introduced in the seed region, such as 2'-F modifications. In some embodiments, 2'-F modifications are introduced at the 5' and / or 3' end of the guide. In certain embodiments, 3 to 5 nucleotides at the 5' and / or 3' ends of the guide are chemically modified with 2'-O-methyl (M), 2'-O-methyl-3'-phosphorothioate (MS), S-constrained ethyl (cEt), or 2'-O-methyl-3'-thioPACE (MSP). Such modifications can enhance genome editing efficiency (see Hendel et al., Nat. Biotechnol. (2015) 33(9):985-989). In certain embodiments, all phosphodiester bonds of the guide are replaced with phosphorothioate (PS) to enhance gene disruption levels.In certain embodiments, more than five nucleotides at the 5' and / or 3' end of the guide are chemically modified with 2'-O-Me, 2'-F, or S-constrained ethyl (cEt). Such chemically modified guides can mediate enhanced levels of gene disruption (see Ragdarm et al., 20215, PNAS, E7110-E7111). In certain embodiments of the present invention, the guide is modified to include a chemical moiety at its 3' and / or 5' end. Such moieties include, but are not limited to, amine, azide, alkyne, thio, dibenzocyclooctyne (DBCO), or rhodamine. In certain embodiments, the chemical moiety is conjugated to the guide by a linker such as an alkyl chain. In certain embodiments, the chemical moiety of the modified guide can be used to attach the guide to another molecule, such as DNA, RNA, a protein, or a nanoparticle. Such chemically modified guides can be used to identify or enrich for cells that have been gene-edited by the CRISPR system (see Lee et al., eLife, 2017, 6:e25312, DOI:10.7554).

[0156] In some embodiments, the guide modification is a chemical modification, insertion, deletion, or split. In some embodiments, the chemical modification includes, but is not limited to, the incorporation of 2'-O-methyl (M) analogs, 2'-deoxy analogs, 2-thiouridine analogs, N6-methyladenosine analogs, 2'-fluoro analogs, 2-aminopurine, 5-bromo-uridine, pseudouridine (Ψ), N1-methylpseudouridine (melΨ), 5-methoxyuridine (5moU), inosine, 7-methylguanosine, 2'-O-methyl-3'-phosphorothioate (MS), S-constrained ethyl (cEt), phosphorothioate (PS), or 2'-O-methyl-3'-thioPACE (MSP). In some embodiments, the guide comprises one or more phosphorothioate modifications. In certain embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 nucleotides of the guide are chemically modified. In certain embodiments, one or more nucleotides of the seed region are chemically modified. In certain embodiments, one or more nucleotides of the 5'-end are chemically modified. In certain embodiments, no nucleotides of the 3'-handle are chemically modified. In some embodiments, the chemical modification of the seed region is minor, such as the incorporation of a 2'-fluoro analog. In a specific embodiment, one nucleotide of the seed region is replaced with a 2'-fluoro analog. In some embodiments, five or ten nucleotides of the 5'-end are chemically modified. Such chemical modification of the 5'-end of the Cas13b CrRNA can improve gene cleavage efficiency. In a specific embodiment, five nucleotides of the 5'-end are replaced with a 2'-fluoro analog. In a specific embodiment, ten nucleotides of the 5'-end are replaced with a 2'-fluoro analog. In a specific embodiment, the 5' terminal five nucleotides are replaced with 2'-O-methyl (M) analogs.

[0157] In some embodiments, the loop of the 3'-handle of the guide is modified. In some embodiments, the loop of the 3'-handle of the guide is modified to have a deletion, insertion, split, or chemical modification. In certain embodiments, the loop comprises 3, 4, or 5 nucleotides. In certain embodiments, the loop comprises the sequence UCUU, UUUU, UAUU, or UGUU.

[0158] In one aspect, the guide comprises moieties that are chemically linked or conjugated via non-phosphodiester bonds. In one aspect, the guide comprises, in a non-limiting example, a direct repeat and a targeting sequence that are chemically linked or conjugated via a non-nucleotide loop. In some embodiments, these moieties are joined via a non-phosphodiester covalent linker. Examples of covalent linkers include, but are not limited to, chemical moieties selected from the group consisting of carbamates, ethers, esters, amides, imines, amidines, aminotrizines, hydrozones, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, sulphonates, fulfones, sulfoxides, ureas, thioureas, hydrazides, oximes, triazoles, photolabile linkages, C-C bond forming groups such as Diels-Alder cycloaddition pairs or ring-closing metathesis pairs, and Michael reaction pairs.

[0159] In some embodiments, the guide portion is first synthesized using standard phosphoramidite synthesis protocols (Herdewijn, P., ed., Methods in Molecular Biology Col 288, "Oligonucleotide Synthesis: Methods and Applications", Humana Press, New Jersey (2012)). In some embodiments, the non-targeting guide portion can be functionalized using standard protocols known in the art to contain functional groups suitable for ligation (Hermanson, GT, Bioconjugate Techniques, Academic Press (2013)). Examples of functional groups include, but are not limited to, hydroxyl, amine, carboxylic acid, carboxylic acid halide, carboxylic acid active ester, aldehyde, carbonyl, chlorocarbonyl, imidazolylcarbonyl, hydrozide, semicarbazide, thiosemicarbazide, thiol, maleimide, haloalkyl, sufonyl, ally, propargyl, diene, alkyne, and azide. When the non-targeting portion of the guide is functionalized, a covalent chemical bond or linkage can be formed between two oligonucleotides. Examples of chemical bonds include, but are not limited to, those based on carbamates, ethers, esters, amides, imines, amidines, aminotrizines, hydrozones, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides, ureas, thioureas, hydrazides, oximes, triazoles, photolabile linkages, C-C bond forming groups such as Diels-Alder cycloaddition pairs or ring-closing metathesis pairs, and Michael reaction pairs.

[0160] In some embodiments, one or more portions of the guide can be chemically synthesized using an automated solid-phase oligonucleotide synthesizer with 2'-acetoxyethyl orthoester (2'-ACE) (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317:3-18) or 2'-thionocarbamate (2'-TC) chemistry (Dellinger et al., J. Am. Chem. Soc. (2011) 133:11540-11546; Hendel et al., Nat. Biotechnol. (2015) 33:985-989).

[0161] In some embodiments, guide moieties can be covalently linked using various bioconjugation reactions, loops, cross-linking and sugar modifications, internucleotide phosphodiester bonds, and non-nucleotide linkages via purine and pyrimidine residues (Sletten et al., Angew. Chem. Int. Ed. (2009) 48:6974-6998; Manoharan, M. Curr. Opin. Chem. Biol. (2004) 8:570-9; Behlke et al., Oligonucleotides (2008) 18:305-19; Watts, et al., Drug. Discov. Today (2008) 13:842-55; Shukla, et al., ChemMedChem (2010) 5:328-49).

[0162] In some embodiments, the guide moiety can be covalently linked using click chemistry. In some embodiments, the guide moiety can be covalently linked using a triazole linker. In some embodiments, the guide moiety can be covalently linked using the Huisgen 1,3-dipolar cycloaddition reaction involving an alkyne and an azide, which generates a highly stable triazole linker (He et al., ChemBioChem (2015) 17:1809-1812; WO 2016 / 186745). In some embodiments, the guide moiety can be covalently linked by ligation of a 5'-hexyne moiety and a 3'-azide moiety. In some embodiments, either or both of the 5'-hexyne and 3'-azide guide moieties can be protected with 2'-acetoxyethyl orthoester (2'-ACE) groups, which can be subsequently removed using the Dharmacon protocol (Scaringe et al., J. Am. Chem. Soc. (1998) 120:11820-11821; Scaringe, Methods Enzymol. (2000) 317:3-18).

[0163] In some embodiments, the guide moiety can be covalently linked via a linker (e.g., a non-nucleotide loop) including moieties such as spacers, attachments, bioconjugates, chromophores, reporter groups, dye-labeled RNA, and non-naturally occurring nucleotide analogs. More specifically, spacers suitable for purposes of the present invention include, but are not limited to, polyethers (e.g., polyethylene glycols, polyhydric alcohols, polypropylene glycol, or mixtures of ethylene and propylene glycols), polyamine groups (e.g., spennine, spermidine, and its polymeric derivatives), polyesters (e.g., poly(ethyl acrylate)), polyphosphodiesters, alkylenes, and combinations thereof. Suitable attachments include, but are not limited to, any moiety that can be added to a linker to impart additional properties to the linker, such as a fluorescent label. Suitable bioconjugates include, but are not limited to, peptides, glycosides, lipids, cholesterol, phospholipids, diacylglycerols and dialkylglycerols, fatty acids, hydrocarbons, enzyme substrates, steroids, biotin, digoxigenin, carbohydrates, and polysaccharides. Suitable chromophore-, reporter-, and dye-labeled RNAs include, but are not limited to, fluorescent dyes such as fluorescein and rhodamine, chemiluminescent, electrochemiluminescent, and bioluminescent marker compounds. Exemplary linker designs for conjugating two RNA components are also described in WO 2004 / 015075.

[0164] The linker (e.g., non-nucleotide loop) can be of any length. In some embodiments, the linker has a length equal to about 0 to 16 nucleotides. In some embodiments, the linker has a length equal to about 0 to 8 nucleotides. In some embodiments, the linker has a length equal to about 0 to 4 nucleotides. In some embodiments, the linker has a length equal to about 2 nucleotides. Exemplary linker designs are also described in WO 2011 / 008730.

[0165] In some embodiments, the degree of complementarity is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any algorithm suitable for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (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). The ability of a guide sequence (within an RNA-targeting guide RNA or crRNA) to direct sequence-specific binding of a nucleic acid targeting complex to a target nucleic acid sequence can be assessed by any suitable assay. For example, sufficient components of an RNA-targeting CRISPR Cas13b system to form a nucleic acid targeting complex can be provided to a host cell containing the corresponding target nucleic acid sequence, including the guide sequence to be tested, such as by transfection of a vector encoding the components of the nucleic acid targeting complex, followed by assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by a Surveyor assay as described herein. Similarly, cleavage of a target nucleic acid sequence can be determined in vitro by providing the target nucleic acid sequence, the components of the nucleic acid targeting complex, including the guide sequence to be tested, and a control guide sequence that differs from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between reactions with the test and control guide sequences. Other assays are possible and will occur to those skilled in the art. The guide sequence, and thus the RNA-targeting guide RNA or crRNA, can be selected to target any target nucleic acid sequence. The target sequence may be DNA.The target sequence can be any RNA sequence. In some embodiments, the target sequence can be a sequence within an RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double-stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and small cytoplasmic RNA (scRNA). In some preferred embodiments, the target sequence can be a sequence within an RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments, the target sequence can be a sequence within an RNA molecule selected from the group consisting of ncRNA and lncRNA. In some more preferred embodiments, the target sequence can be a sequence within an mRNA molecule or a pre-mRNA molecule.

[0166] In some embodiments, the RNA targeting guide RNA or crRNA is selected to reduce the degree of secondary structure within the RNA targeting guide RNA or crRNA. In some embodiments, no more than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or fewer of the nucleotides of the RNA targeting guide RNA participate in self-complementary base pairing when optimally folded. Optimal folding can be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another exemplary folding algorithm is the online web server RNAfold, developed at the Institute for Theoretical Chemistry at the University of Vienna, which uses a centroid structure prediction algorithm (see, e.g., A.R. Gruber et al., 2008, Cell 106(1):23-24; and P.A. Carr and G.M. Church, 2009, Nature Biotechnology 27(12):1151-62).

[0167] In certain embodiments, a guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence. In certain embodiments, a guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence. In certain embodiments, the direct repeat sequence may be located upstream (i.e., 5') of the guide sequence or spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (i.e., 3') of the guide sequence or spacer sequence. In other embodiments, multiple DRs (e.g., dual DRs) may be present.

[0168] In certain embodiments, the crRNA comprises a stem-loop, preferably a single stem-loop. In certain embodiments, the direct repeat sequence forms a stem-loop, preferably a single stem-loop.

[0169] In certain embodiments, the spacer length of the guide RNA is 15 to 35 nt. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides. In certain embodiments, the spacer length is 15 to 17 nt, for example, 15, 16, or 17 nt, 17 to 20 nt, for example, 17, 18, 19, or 20 nt, 20 to 24 nt, for example, 20, 21, 22, 23, or 24 nt, 23 to 25 nt, for example, 23, 24, or 25 nt, 24 to 27 nt, for example, 24, 25, 26, or 27 nt, 27 to 30 nt, for example, 27, 28, 29, or 30 nt, 30 to 35 nt, for example, 30, 31, 32, 33, 34, or 35 nt, or 35 nt or more.

[0170] Interfering RNA (RNAi) and microRNA (miRNA) In other embodiments, the target RNA may include both eukaryotic and prokaryotic interfering RNAs, i.e., RNAs involved in the RNA interference pathway, such as shRNA, siRNA, etc. In other embodiments, the target RNA may include microRNA (miRNA). Controlling interfering RNA or miRNA can help reduce the off-target effects (OTE) seen in methods by shortening the lifespan of interfering RNA or miRNA in vivo or in vitro.

[0171] In certain embodiments, the target is the miRNA binding site of the miRNA target, rather than the miRNA itself.

[0172] In certain embodiments, the miRNA may be sequestered (including, for example, being relocated within the cell). In certain embodiments, the miRNA may be truncated, such as, without limitation, a hairpin.

[0173] In certain embodiments, miRNA processing (including, for example, turnover) is increased or decreased.

[0174] If the effector protein and suitable guide are selectively expressed (e.g., under the control of a spatially or temporally suitable promoter, such as a tissue- or cell cycle-specific promoter and / or enhancer), this could be used to "protect" a cell or system (in vivo or in vitro) from RNAi in that cell. This could be useful in adjacent tissues or cells where RNAi is not required, or for purposes of comparing cells or tissues in which the effector protein and suitable guide are expressed and not expressed (i.e., RNAi is not and is controlled, respectively). The effector protein may be used to regulate or bind to molecules that comprise or consist of RNA, such as ribozymes, ribosomes, or riboswitches. In embodiments of the invention, the RNA guide recruits the effector protein to such molecules, thereby enabling the effector protein to bind to them.

[0175] The protein system of the present invention can be applied in the area of ​​RNAi technology, including therapeutics, assays, and other applications, without undue experimentation from this disclosure, as this application provides a basis for informed engineering of this system (see, e.g., Guidi et al., PLoS Negl Trop Dis 9(5):e0003801.doi:10.1371 / journal.pntd; Crotty et al., "In vivo RNAi screens: concepts and applications." Shane Crotty...2015 Elsevier Ltd. Published by Elsevier Inc., Pesticide Biochemistry and Physiology (Impact Factor:2.01).01 / 2015;120.DOI:10.1016 / j.pestbp.2015.01.002 and Makkonen et al., Viruses 2015,7(4),2099-2125; see doi:10.3390 / v7042099).

[0176] ribosomal RNA (rRNA) For example, azalide antibiotics such as azithromycin are well known. They target and destroy the 50S ribosomal subunit. In some embodiments, the effector protein, together with a suitable guide RNA targeting the 50S ribosomal subunit, can be recruited to and bind to the 50S ribosomal subunit. Thus, the effector protein is provided in combination with a suitable guide directed to the ribosome (particularly the 50S ribosomal subunit). The use of this effector protein in combination with a suitable guide directed to the ribosome (particularly the 50S ribosomal subunit) can include use as an antibiotic. In particular, the use as an antibiotic is similar to the function of azalide antibiotics such as azithromycin. In some embodiments, prokaryotic ribosomal subunits, such as the prokaryotic 70S subunit, the above-mentioned 50S subunit, 30S subunit, and 16S and 5S subunits, can be targeted. In other embodiments, eukaryotic ribosomal subunits may be targeted, such as the eukaryotic 80S subunit, 60S subunit, 40S subunit, and 28S, 18S, 5.8S, and 5S subunits.

[0177] In some embodiments, 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 a single strand of RNA. In either case, but particularly when the RNA binding protein cleaves a single strand of RNA, ribosome function may be regulated, and in particular, reduced or disrupted. This may apply to any ribosomal RNA and any ribosomal subunit, and the sequence of rRNA is well known.

[0178] Thus, control of ribosomal activity is contemplated through the use of the present effector proteins in conjunction with a suitable guide to a ribosomal target. This may be by cleaving or binding to the ribosome. In particular, reduction of ribosomal activity is contemplated. This may be useful in in vivo or in vitro assays of ribosomal function and also as a means of controlling ribosomal activity-based therapies in vivo or in vitro. Furthermore, control (i.e., reduction) of protein synthesis in in vivo or in vitro systems is contemplated, including use as an antibiotic and in research and diagnostic uses.

[0179] Riboswitches Riboswitches (also known as aptazymes) are regulatory segments of messenger RNA molecules that bind to small molecules. This typically results in altered production of the protein encoded by the mRNA. Therefore, control of riboswitch activity through the use of the present effector proteins in conjunction with suitable guides to the riboswitch target is therefore contemplated. This may be by cleaving the riboswitch or by binding to it. In particular, reduction of riboswitch activity is contemplated. This may be useful in in vivo or in vitro assays of riboswitch function and as a means of controlling therapeutics based on riboswitch activity in vivo or in vitro. Furthermore, control (i.e., reduction) of protein synthesis in in vivo or in vitro systems is contemplated. This control, insofar as rRNA is concerned, may include use as antibiotics and in research and diagnostic uses.

[0180] Ribozymes Ribozymes are RNA molecules with catalytic properties similar to enzymes (which, of course, are proteins). Because ribozymes, both naturally occurring and engineered, contain or consist of RNA, they can similarly be targets for the present RNA-binding effector proteins. In some embodiments, the effector protein can be an RNA-binding protein that cleaves the ribozyme, thereby disabling it. Thus, control of ribozyme activity is envisioned by using the present effector proteins in conjunction with a suitable guide to the ribozyme target. This can be by cleaving or binding to the ribozyme. In particular, reduction of ribozyme activity is envisioned. This can be useful in in vivo or in vitro assays of ribozyme function, and also as a means of controlling ribozyme activity-based therapies in vivo or in vitro.

[0181] Gene expression including RNA processing Effector proteins, together with a suitable guide, can also be used to target gene expression, including by controlling RNA processing. Control of RNA processing can include RNA splicing, including alternative splicing by targeting RNApol; viral replication, including plant viroids (particularly those of satellite viruses, bacteriophages, and retroviruses, such as HBV, HBV, and HIV, as well as other viruses listed herein); and RNA processing reactions such as tRNA biosynthesis. Effector proteins and a suitable guide can also be used to control RNA activation (RNAa). RNAa leads to enhanced gene expression, so gene expression can be controlled by disrupting or reducing RNAa, thereby reducing the enhanced gene expression. This will be discussed in more detail below.

[0182] RNAi screen RNAi screens allow for the investigation of biological pathways and the identification of components by identifying gene products whose knockdown is associated with phenotypic changes. Control can also be exerted during such screens by using effector proteins and suitable guides to remove or reduce the activity of RNAi in the screen, thus restoring the activity of the (previously interfered) gene product (by removing or reducing interference / repression).

[0183] Satellite RNA (satRNA) and satellite viruses may also be treated.

[0184] Regulation herein in relation to RNase activity generally means reduction, negative disruption or knockdown or knockout.

[0185] In vivo RNA applications Inhibition of gene expression The target-specific RNases provided herein are capable of highly specific cleavage of target RNAs. Interference at the RNA level can be regulated both spatially and temporally and in a non-invasive manner since the genome is not altered.

[0186] It has been demonstrated that several diseases can be treated 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 the same way.

[0187] Examples of mRNA targets (and corresponding disease treatments) are VEGF, VEGF-R1 and RTP801 (for the treatment of AMD and / or DME), caspase 2 (for the treatment of Na+), ADRB2 (for the treatment of intraocular pressure), TRPVI (for the treatment of dry eye syndrome, Syk kinase (for the treatment of asthma), Apo B (for the treatment of hypercholesterolemia or hypobetalipoproteinemia), PLK1, KSP and VEGF (for the treatment of solid tumors), Ber-Abl (for the treatment of CML) (Burnett and Rossi Chem Biol. 2012, 19(1):60-71)). Similarly, RNA targeting has been demonstrated 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).

[0188] It is further envisioned that the RNA targeting effector proteins of the present invention can be used for mutation-specific or allele-specific knockdown. Guide RNAs can be designed to specifically target the sequence of transcribed mRNA containing a mutation or an allele-specific sequence. Such specific knockdown is particularly suitable for therapeutic applications involving disorders associated with mutated or allele-specific gene products. For example, most cases of familial hypobetalipoproteinemia (FHBL) are caused by mutations in the ApoB gene. This gene encodes two versions of apolipoprotein B protein: a short version (ApoB-48) and a longer version (ApoB-100). Some ApoB gene mutations that lead to FHBL cause both versions of ApoB to be abnormally short. Specific targeting and knockdown of mutated ApoB mRNA transcripts with the RNA targeting effector proteins of the present invention may be beneficial in treating FHBL. As another example, Huntington's disease (HD) is caused by an expansion of a CAG triplet repeat in the gene encoding huntingtin, resulting in an abnormal protein. Specific targeting and knockdown of mutated or allele-specific mRNA transcripts encoding huntingtin protein with the RNA targeting effector proteins of the present invention may be beneficial in the treatment of HD.

[0189] In this regard, and more generally, for various applications as described herein, it is noted that the use of split versions of RNA targeting effector proteins can be envisioned. Indeed, this can not only allow for increased specificity, but can also be advantageous for delivery. Cas13b is split in the sense that the two parts of the Cas13b enzyme essentially form a functional Cas13b. Ideally, the split should always be such that the catalytic domain remains unaffected. This Cas13b can function as a nuclease, or it can be a dead Cas13b, an RNA-binding protein that essentially has little or no catalytic activity, typically due to a mutation in its catalytic domain.

[0190] Each half of the split Cas13b can be fused to a dimerization partner. By way of example and not limitation, a rapamycin-sensitive dimerization domain can be used to create a chemically inducible split Cas13b for temporal control of Cas13b activity. Thus, Cas13b can be made chemically inducible by splitting it into two fragments, and the rapamycin-sensitive dimerization domain can be used to reassemble Cas13b in a controlled manner. The two parts of the split Cas13b can be considered the N'-terminal and C'-terminal parts of the split Cas13b. This fusion is typically the split point of Cas13b. In other words, the C'-terminus of the N'-terminal part of the split Cas13b is fused to one dimer half, while the N'-terminus of the C'-terminal part is fused to the other dimer half.

[0191] Cas13b does not need to be split in the sense that the breakpoint is created de novo. The splitpoint is typically designed in silico and cloned into the construct. Together, the two parts of the split Cas13b, the N'-terminal part and the C'-terminal part, preferably form a complete Cas13b containing at least 70% or more of the wild-type amino acids (or the 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 of the wild-type amino acids (or the nucleotides encoding them). Some trimming may be possible, and mutants are envisioned. Non-functional domains may be completely removed. The important point is that the two parts can be brought together and the desired Cas13b function is restored or reversed. The dimer can be a homodimer or a heterodimer.

[0192] In certain embodiments, Cas13b effectors as described herein can be used for mutation- or allele-specific targeting, such as mutation- or allele-specific knockdown.

[0193] The RNA targeting effector protein can further be fused to another functional RNase domain, such as nonspecific RNase or Argonaute 2, which act synergistically to increase RNase activity or ensure further degradation of the message.

[0194] Regulation of gene expression by modulating RNA function

[0195] In addition to its direct effect on gene expression through mRNA cleavage, RNA targeting can also be used to affect specific aspects of RNA processing in cells, potentially enabling more delicate regulation of gene expression. Generally, regulation can be mediated by, for example, interfering with protein binding to RNA, for example, by blocking protein binding or recruiting RNA-binding proteins. Regulation can be achieved at various levels, including mRNA splicing, transport, localization, translation, and turnover. Similarly, in therapeutic contexts, it is conceivable to address (pathogenic) dysfunctions at each of these levels by using RNA-specific targeting molecules. In these embodiments, it is often preferred that the RNA targeting protein be a "dead" Cas13b, such as the mutant Cas13b described herein, that has lost the ability to cleave RNA targets but still retains its ability to bind to them.

[0196] A) Alternative splicing

[0197] Many human genes express multiple mRNAs as a result of alternative splicing. Various diseases have been shown to be associated with aberrant splicing, leading to loss-of-function or gain-of-function of the expressed gene. Some of these diseases are caused by mutations that result in splicing defects, but many others are not. One therapeutic option is to directly target the splicing machinery. The RNA targeting effector proteins described herein can be used, for example, to block or promote splicing, to include or exclude exons, and to influence the expression of specific isoforms and / or to stimulate the expression of alternative protein products. Such applications are described in further detail below.

[0198] When an RNA targeting effector protein binds to a target RNA, it can sterically block the access of splicing factors to the RNA sequence. An RNA targeting effector protein that targets a splice site can block splicing at that site and optionally redirect splicing to an adjacent site. For example, binding of an RNA targeting effector protein that binds to a 5' splice site can block the recruitment of the U1 component of the spliceosome, favoring the skipping of that exon. Alternatively, an RNA targeting effector protein that targets a splicing enhancer or silencer can prevent the binding of trans-acting regulatory splicing factors at the target site, effectively blocking or promoting splicing. Furthermore, exon exclusion can be achieved by recruiting ILF2 / 3 to the vicinity of an exon in a precursor mRNA using an RNA targeting effector protein as described herein. As yet another example, a glycine-rich domain can be added for hnRNP A1 recruitment and exon exclusion (Del Gatto-Konczak et al. Mol Cell Biol. 1999 Jan;19(1):251-60).

[0199] In certain embodiments, appropriate selection of the gRNA can result in specific splice variants being targeted while other splice variants are not targeted.

[0200] In some cases, RNA targeting effector proteins can be used to promote splicing (e.g., in cases where splicing is deficient). For example, RNA targeting effector proteins can be associated with effectors capable of stabilizing splicing-regulatory stem-loops for further splicing. Linking RNA targeting effector proteins to consensus binding site sequences for specific splicing factors can recruit proteins to target DNA or RNA.

[0201] Examples of diseases associated with aberrant splicing include, but are not limited to, paraneoplastic opsoclonus-myoclonus ataxia (POMA), which is caused by loss of the Nova protein, which regulates the splicing of proteins that function at synapses, and cystic fibrosis, which is caused by a splicing defect in the cystic fibrosis transmembrane conductance regulator, resulting in the production of nonfunctional chloride channels. In other diseases, aberrant RNA splicing results in a gain of function, such as myotonic dystrophy, which is caused by a CUG triplet repeat expansion (50 to >1500 repeats) in the 3' UTR of mRNA, resulting in a splicing defect.

[0202] RNA-targeting effector proteins can be used to exclude exons by recruiting splicing factors (such as U1) to the 5' splice site to promote excision of the intron surrounding the desired exon. Such recruitment can be mediated by fusion with an arginine / serine-rich domain that functions as a splicing activator (Gravely BR and Maniatis T, Mol Cell. 1998(5):765-71).

[0203] It is envisioned that RNA targeting effector proteins can be used to block the splicing machinery at a desired gene locus, thereby preventing exon recognition and the expression of an alternative protein product. An example of a treatable disorder is Duchenne muscular dystrophy (DMD), which is caused by mutations in the gene encoding the dystrophin protein. Nearly all DMD mutations lead to frameshifts, resulting in impaired dystrophin translation. By combining RNA targeting effector proteins with splice junctions or exon splicing enhancers (ESEs), thereby preventing exon recognition, it is possible to result in the translation of a partially functional protein. This converts the lethal Duchenne phenotype into the less severe Becker phenotype.

[0204] B) RNA modification

[0205] RNA editing is a natural process in which small modifications to RNA increase the diversity of gene products of a given sequence. Typically, this modification involves the conversion of adenosine (A) to inosine (I), resulting in an RNA sequence that differs from that encoded by the genome. RNA modification is generally achieved by ADAR enzymes, whereby the pre-RNA target forms an incomplete double-stranded RNA through base pairing between the exon containing the edited adenosine and an intronic non-coding element. A classic example of AI editing is the glutamate receptor GluR-B mRNA, where this change results in an alteration of the channel's conductance properties (Higuchi M, et al. Cell. 1993; 75: 1361-70).

[0206] According to the present invention, enzymatic approaches are used to induce transitions (A⇔G or C⇔U changes) or transversions (any purine to any pyrimidine or vice versa) in RNA bases of a given transcript. Transitions can be induced directly using adenosine (ADAR1 / 2) or cytosine deaminases (APOBEC, AID), which convert A to I or C to U, respectively. Transitions can be induced indirectly by localizing reactive oxygen species damage to the target base, resulting in a chemical modification of the affected base, such as the conversion of guanine to oxoguanine. Oxoguanine is recognized as T and therefore base pairs with adenine, affecting translation. Proteins that can be recruited for ROS-mediated base damage include APEX and mini-SOG. In both approaches, these effectors can be fused to catalytically inactive Cas13b and recruited to sites on the transcript where these types of mutations are desired.

[0207] In humans, heterozygous functional null mutations in the ADAR1 gene lead to the skin disease human pigmented genodermatosis (Miyamura Y, et al. Am J Hum Genet. 2003; 73: 693-9). It is envisioned that the RNA targeting effector protein of the present invention can be used to correct dysfunctional RNA modifications.

[0208] Furthermore, it is envisioned that RNA adenosine methylase (N(6)-methyladenosine) can be fused to the RNA targeting effector proteins of the invention to target transcripts of interest. This methylase causes reversible methylation, has a regulatory role, and can affect gene expression and cell fate decisions by modulating multiple RNA-related cellular pathways (Fu et al Nat Rev Genet. 2014;15(5):293-306).

[0209] C) Polyadenylation

[0210] Polyadenylation of mRNA is important for the nuclear transport, translation efficiency, and stability of mRNA, all of which, as well as the polyadenylation process, depend on specific RBPs. Many eukaryotic mRNAs receive a 3' poly(A) tail of about 200 nucleotides after transcription. Polyadenylation involves various RNA-binding protein complexes that 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 proteins provided herein can be used to interfere with or promote the interaction between RNA-binding proteins and RNA.

[0211] An example of a disease that has been linked to defective proteins involved in polyadenylation is oculopharyngeal muscular dystrophy (OPMD) (Brais B, et al. Nat Genet. 1998;18:164-7).

[0212] D) RNA nuclear export After pre-mRNA processing, the mRNA is transported from the nucleus to the cytoplasm, which is ensured by cellular mechanisms that involve the generation of a carrier complex, which then translocates through nuclear pores and releases the mRNA in the cytoplasm, where the carrier is subsequently recycled.

[0213] Overexpression of proteins that play a role in the nuclear export of RNA (such as TAP) has been shown to increase the nuclear export of transcripts that are normally inefficiently exported in Xenopus laevis (Katahira J, et al. EMBO J. 1999;18:2593-609).

[0214] E)mRNA localization

[0215] mRNA localization ensures spatially regulated protein production. The localization of transcripts to specific regions of the cell can be ensured by localization elements. In a detailed embodiment, it is envisioned that the effector proteins described herein can be used to target the localization elements to the RNA of interest. The effector protein can be designed to bind to the target transcript and shuttle it to a location within the cell determined by its peptide signal tag. For example, more specifically, the RNA localization can be altered using an RNA targeting effector protein fused to one or more nuclear localization signals (NLS) and / or one or more nuclear export signals (NES).

[0216] Further examples of localization signals include the zipcode binding protein (ZBP1), which ensures β-actin localization to the cytoplasm in some asymmetric cell types, the KDEL retention sequence (localization to the endoplasmic reticulum), the nuclear export signal (localization to the cytoplasm), the mitochondrial targeting signal (localization to mitochondria), the peroxisomal targeting signal (localization to peroxisomes), and the m6A tag / YTHDF2 (localization to p-bodies). Another envisioned approach is the fusion of an RNA-targeting effector protein with a protein of known localization (e.g., membrane, synapse).

[0217] Alternatively, the effector protein of the present invention can be used, for example, for localization-dependent knockdown. By fusing the effector protein with an appropriate localization signal, the effector can be targeted to a specific intracellular compartment. Only the target RNA in this compartment will be effectively targeted, while targets in other identical but different intracellular compartments will not be targeted, thereby achieving localization-dependent knockdown.

[0218] F) Translation

[0219] The RNA targeting effector protein described herein can be used to enhance or suppress translation.It is expected that translation upregulation is a very robust method to control cellular circuits.In addition, for functional studies, protein translation screens can be advantageous compared to transcription upregulation screens, which have the drawback that transcript upregulation does not lead to increased protein production.

[0220] It is envisioned that the RNA targeting effector proteins described herein can be used to bring translation initiation factors such as EIF4G into proximity with the 5' untranslated repeat (5'UTR) of a target messenger RNA to drive translation (as described for non-reprogrammable RNA-binding proteins in De Gregorio et al. EMBO J. 1999;18(17):4865-74). As another example, the cytoplasmic poly(A) polymerase GLD2 can be recruited to a target mRNA by an RNA targeting effector protein. This can allow for directed polyadenylation of the target mRNA and thereby stimulate translation.

[0221] Similarly, the RNA targeting effector proteins envisioned herein can be used to block translation repressors of mRNA, such as ZBP1 (Huttelmaier S, et al. Nature. 2005; 438: 512-5). Binding of target RNA to the translation start site can directly affect translation.

[0222] Additionally, fusing an RNA targeting effector protein to a protein that stabilizes the mRNA, for example by preventing its degradation, such as an RNase inhibitor, can increase protein production from the transcript of interest.

[0223] It is envisioned that the RNA targeting effector proteins described herein may be used to bind to the 5UTR region of an RNA transcript and inhibit translation by preventing ribosome formation and translation initiation.

[0224] Furthermore, RNA-targeting effector proteins can be used to recruit Caf1, a component of the CCR4-NOT deadenylase complex, to target mRNAs, resulting in deadenylation of the target transcript and inhibition of protein translation.

[0225] For example, the RNA-targeting effector proteins 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 down- or up-regulate translation are amyotrophic lateral sclerosis (ALS) and cardiovascular disorders. Reduced levels of the glial glutamate transporter EAAT2 have been reported in the ALS motor cortex and spinal cord, and multiple abnormal EAAT2 mRNA transcripts have also been reported in ALS brain tissue. Loss of EAAT2 protein and function is believed to be the primary cause of excitotoxicity in ALS. Restoring EAAT2 protein levels and function can provide therapeutic benefits. Therefore, RNA-targeting effector proteins can be beneficially used to up-regulate EAAT2 protein expression, for example, by blocking translational repressors or stabilizing mRNA, as described above. Apolipoprotein A1 is a major protein component of high-density lipoprotein (HDL), and ApoA1 and HDL are generally considered to be anti-atherogenic. It is envisioned that RNA targeting effector proteins may be beneficially used to upregulate ApoA1 expression, for example, by blocking translational repressors or stabilizing mRNA as described above.

[0226] G) mRNA turnover

[0227] Translation is closely linked to mRNA turnover and regulated mRNA stability. Specific proteins have been described to be involved in transcript stability (e.g., ELAV / Hu proteins in neurons, Keene JD, 1999, Proc Natl Acad Sci U S A. 96:5-7) and tristetraprolin (TTP). These proteins stabilize target mRNAs by protecting the message from degradation in the cytoplasm (Peng SS et al., 1988, EMBO J. 17:3461-70).

[0228] It is conceivable that the RNA targeting effector proteins of the present invention can be used to interfere with or promote the activity of proteins that stabilize mRNA transcripts, such that mRNA turnover is affected. For example, the RNA targeting effector proteins can be used to recruit human TTP to target RNAs, enabling adenylate-uridylate-rich element (AU-rich element)-mediated translational repression and target degradation. AU-rich elements are found in the 3'UTRs of many mRNAs encoding protooncogenes, nuclear transcription factors, and cytokines, and promote RNA stability. As another example, the RNA targeting effector protein can be fused to another mRNA stabilizing protein, HuR (Hinman MN and Lou H, Cell Mol Life Sci 2008;65:3168-81), and recruited to target transcripts to extend their lifespan or stabilize short-lived mRNAs.

[0229] It is further contemplated that the RNA targeting effector proteins described herein can be used to promote degradation of target transcripts, for example, by recruiting m6A methyltransferase to target transcripts and localizing the transcripts to P-bodies, thereby resulting in target degradation.

[0230] 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 target transcripts and enable their degradation.

[0231] Patients with paraneoplastic neuropathy (PND)-associated encephalomyelitis and neuropathy 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 may be used to interfere with the binding of autoantibodies to mRNA transcripts.

[0232] Patients with dystrophy type 1 (DM1), caused by a (CUG)n expansion in the 3'UTR of the myotonic dystrophy protein kinase (DMPK) gene, are characterized by the accumulation of such transcripts in the nucleus. It is anticipated that the RNA-targeting effector protein of the present invention fused to an endonuclease that targets the (CUG)n repeat can inhibit the accumulation of such abnormal transcripts.

[0233] H) Interaction with multifunctional proteins

[0234] Some RNA binding proteins bind to multiple sites on many RNAs and function in various processes.For example, it has been found that hnRNP A1 protein binds to exon splicing silencer sequences to antagonize splicing factors, associates with telomere ends (thereby stimulating telomere activity), and binds to miRNA to promote Drosha-mediated processing, thereby affecting maturation.It is assumed that the RNA binding effector protein of the present invention can interfere with the binding of RNA binding proteins at one or more positions.

[0235] I) RNA folding

[0236] RNA adopts a defined structure to exert its biological activity. Conformational transitions between alternative tertiary structures are crucial for many RNA-mediated processes. However, RNA folding can be associated with several problems. For example, RNA may tend to fold into and maintain inappropriate alternative conformations, and / or the correct tertiary structure may not be sufficiently thermodynamically favorable compared to alternative structures. The RNA targeting effector proteins of the present invention, particularly cleavage-deficient or dead RNA targeting proteins, can be used to direct (m)RNA folding and / or ensure its correct tertiary structure.

[0237] In certain embodiments, Cas13b complexed with crRNA is activated upon binding to the target RNA and subsequently cleaves any nearby ssRNA targets (i.e., the "collateral" or "bystander" effect). When primed by its cognate target, Cas13b can cleave other (non-complementary) RNA molecules. Such indiscriminate RNA cleavage can potentially cause cytotoxicity or otherwise affect cellular physiology or state.

[0238] Thus, in certain embodiments, a non-naturally occurring or engineered composition, vector system, or delivery system as described herein is used or for use in inducing cellular dormancy. In certain embodiments, a non-naturally occurring or engineered composition, vector system, or delivery system as described herein is used or for use in inducing cell cycle arrest. In certain embodiments, a non-naturally occurring or engineered composition, vector system, or delivery system as described herein is used or for use in reducing cell growth and / or cell proliferation. In certain embodiments, a non-naturally occurring or engineered composition, vector system, or delivery system as described herein is used or for use in inducing cellular anergy. In certain embodiments, a non-naturally occurring or engineered composition, vector system, or delivery system as described herein is used or for use in inducing cellular apoptosis. In certain embodiments, a non-naturally occurring or engineered composition, vector system, or delivery system as described herein is used or for use in inducing cellular necrosis. In certain embodiments, a non-naturally occurring or engineered composition, vector system, or delivery system as described herein is used or for use in inducing cell death. In certain embodiments, a non-naturally occurring or engineered composition, vector system, or delivery system as described herein is used or for use in inducing programmed cell death.

[0239] In certain embodiments, the present invention relates to a method for inducing cellular dormancy, 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 method for inducing cell cycle arrest, 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 method for reducing cell growth and / or cell proliferation, 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 method for inducing cellular anergy, 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 method for inducing cellular apoptosis, 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 method for inducing cellular necrosis, 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 methods of inducing cell death 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 methods of inducing programmed cell death comprising introducing or inducing a non-naturally occurring or engineered composition, vector system, or delivery system as described herein.

[0240] The methods and uses as described herein may be therapeutic or prophylactic and may target specific cells, cell (sub)populations, or cell / tissue types. In particular, the methods and uses as described herein may be therapeutic or prophylactic and may target specific cells, cell (sub)populations, or cell / tissue types that express one or more target sequences, such as one or more specific target RNAs (e.g., ssRNAs). Without limitation, target cells may be, for example, cancer cells that express a specific transcript, e.g., neurons of a given class, e.g., (immune) cells that cause autoimmunity, or cells infected with a specific (e.g., viral) pathogen, etc.

[0241] Thus, in certain embodiments, the present invention relates to a method of treating a pathological condition characterized by the presence of unwanted 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 the use of a non-naturally occurring or engineered composition, vector system, or delivery system as described herein to treat a pathological condition characterized by the presence of unwanted 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 for use in treating a pathological condition characterized by the presence of unwanted cells (host cells). It should be understood that preferably, the CRISPR-Cas system targets a target specific to the unwanted 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 to treat, prevent, or alleviate cancer. In certain embodiments, the present invention relates to a non-naturally occurring or engineered composition, vector system, or delivery system as described herein for use in treating, preventing, or alleviating cancer. In certain embodiments, the present invention relates to a method of treating, preventing, or alleviating 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 alleviating infection of cells 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 for use in treating, preventing, or alleviating infection of cells by a pathogen.In certain embodiments, the present invention relates to a method of treating, preventing, or alleviating 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. Preferably, it should be understood that the CRISPR-Cas system targets a target specific to a cell infected by the pathogen (e.g., a target derived from the 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 alleviating an autoimmune disorder. In certain embodiments, the present invention relates to a non-naturally occurring or engineered composition, vector system, or delivery system as described herein for use in treating, preventing, or alleviating an autoimmune disorder. In certain embodiments, the present invention relates to a method of treating, preventing, or alleviating an autoimmune disorder, comprising introducing or inducing a non-naturally occurring or engineered composition, vector system, or delivery system as described herein. Preferably, it should be understood that the CRISPR-Cas system targets a target specific to a cell involved in the autoimmune disorder (e.g., a specific immune cell).

[0242] Use of RNA-targeting effector proteins in RNA or protein detection It is further envisioned that the RNA targeting effector proteins may be used for the detection of nucleic acids or proteins in biological samples. The samples may be cellular or cell-free.

[0243] Furthermore, it is envisioned that RNA targeting effector proteins can be used in Northern blot assays. Northern blotting involves the size separation of RNA samples using electrophoresis. RNA targeting effector proteins can be used to specifically bind and detect target RNA sequences.

[0244] RNA targeting effector proteins can also be fused to fluorescent proteins (such as GFP) and used to track RNA localization in live cells. More specifically, the RNA targeting effector protein can be inactivated at the point where it no longer cleaves RNA. In detailed embodiments, to ensure more precise visualization, it is envisioned that split RNA targeting effector proteins can be used, whereby the signal depends on the binding of both subproteins. Alternatively, split fluorescent proteins can be used that are reconstituted upon binding of multiple RNA targeting effector protein complexes to the target transcript. Furthermore, it is envisioned that transcripts can be targeted at multiple binding sites along the mRNA, thereby amplifying the true signal and enabling localized discrimination. As yet another alternative, fluorescent proteins can be reconstituted from split inteins.

[0245] RNA targeting effector proteins are suitable for use in, for example, determining the localization of RNA or specific splice variants, mRNA transcript levels, transcript up- or down-regulation, and disease-specific diagnosis. RNA targeting effector proteins can be used, for example, to visualize RNA in (live) cells using fluorescence microscopy or flow cytometry, such as fluorescence-activated cell sorting (FACS), which allows high-throughput screening of cells and the recovery of live cells after cell sorting. Furthermore, the expression levels of various transcripts can be simultaneously assessed under stress, such as the inhibition of cancer growth using molecular inhibitors or hypoxic conditions for cells. Another application could be tracking the localization of transcripts to synaptic junctions during neuronal stimulation using two-photon microscopy.

[0246] In certain embodiments, the components or complexes of the invention as described herein can be used in multiplexed error-robust fluorescence in situ hybridization (MERFISH; Chen et al. Science; 2015; 348(6233)), e.g., with (fluorescently) labeled Cas13b effectors.

[0247] In vitro APEX labeling Cellular processes depend on a network of molecular interactions between proteins, RNA, and DNA. Accurate detection of protein-DNA and protein-RNA interactions is important for understanding such processes. In vitro proximity labeling techniques utilize affinity tags in combination with, for example, photoactivatable probes to label polypeptides and RNAs in vitro near a protein or RNA of interest. After UV irradiation, the photoactivatable groups react with proteins and other molecules in close proximity to the tagged molecule, thereby labeling them. The labeled interacting molecules can then be recovered and identified. The RNA targeting effector proteins of the present invention can be used, for example, to target probes to selected RNA sequences.

[0248] These applications may also be applicable in animal models for disease-related applications or in vivo imaging of difficult-to-culture cell types.

[0249] The present invention provides agents and methods for diagnosing and monitoring health conditions through noninvasive sampling of cell-free RNA, including risk assessment and guidance for RNA-targeted therapy, useful in situations where rapid administration of treatment is critical to treatment outcome. In one embodiment, the present invention provides cancer detection methods and agents related to circulating tumor RNA, including monitoring for recurrence and / or the occurrence of common drug resistance mutations. In another embodiment, the present invention provides detection methods and agents for detecting and / or identifying bacterial species directly from blood or serum, thereby monitoring, for example, disease progression and sepsis. In one embodiment of the present invention, Cas13b proteins and derivatives are used to distinguish and diagnose common illnesses, such as rhinovirus infections or upper respiratory tract infections, from more serious infections, such as bronchitis.

[0250] The present invention provides methods and agents for rapid genotyping towards emergency pharmacogenomics, including guiding the administration of anticoagulants based on, for example, VKORC1, CYP2C9 and CYP2C19 genotyping in the treatment of myocardial infarction or stroke.

[0251] The present invention provides agents and methods for monitoring bacterial contamination of food products at any point along the food production and distribution chain. In another embodiment, the present invention provides quality control and monitoring, for example, by determining the identity and purity of food ingredients. In one non-limiting example, the present invention may be used to identify or verify food ingredients, such as animal meats and seafood species.

[0252] In another embodiment, the present invention is used in forensic determination, for example, crime scene samples containing blood or other bodily fluids. In one embodiment of the present invention, the present invention is used to identify nucleic acid samples from fingerprints.

[0253] Use of RNA-targeting effector proteins in RNA origami / in vitro assembly lines - Combinatrix RNA origami refers to nanoscale folding of structures using RNA as an integrated template to create two-dimensional or three-dimensional structures. The folding structure is encoded by RNA, and therefore the shape of the resulting RNA is determined by the synthesized RNA sequence (Geary, et al. 2014. Science, 345 (6198), pp. 799-804). RNA origami can serve as a scaffold for arranging other components, such as proteins, into a complex. Using the RNA targeting effector protein of the present invention, a protein of interest can be targeted to the RNA origami using, for example, a suitable guide RNA.

[0254] Use of RNA targeting effector proteins in RNA isolation or purification, enrichment or depletion It is further envisioned that RNA-targeting effector proteins, when complexed with RNA, can be used to isolate and / or purify RNA. For example, the RNA-targeting effector protein can be fused to an affinity tag that can be used to isolate and / or purify the RNA-RNA-targeting effector protein complex. Such applications are useful, for example, in analyzing gene expression profiles in cells. In particular embodiments, it is envisioned that RNA-targeting effector proteins can be used to target specific non-coding RNAs (ncRNAs), thereby blocking their activity and providing useful functional probes. In certain embodiments, effector proteins as described herein can be used to specifically enrich specific RNAs (including, but not limited to, increasing stability) or alternatively, specifically deplete specific RNAs (e.g., without limitation, specific splice variants, isoforms, etc.).

[0255] Examination of lincRNA function and other nuclear RNAs Current RNA knockdown strategies, such as siRNA, have the disadvantage that the protein machinery is cytoplasmic, so they are mostly limited to targeting cytoplasmic transcripts. The advantage of the RNA-targeting effector protein of the present invention, an exogenous system that is not essential for cellular function, is that it can be used in any compartment of the cell. By fusing an NLS signal to the RNA-targeting effector protein, it can be directed to the nucleus, enabling targeting of nuclear RNA. For example, probing the function of lincRNAs is envisioned. Long intergenic non-coding RNAs (lincRNAs) are a highly extensively investigated research area. Many lincRNAs have as yet unexplored functions, which could potentially be studied using the RNA-targeting effector proteins of the present invention.

[0256] Identification of RNA-binding proteins Identifying proteins that bind to specific RNAs can be useful for understanding the role of many RNAs. For example, many lincRNAs are involved in transcriptional and epigenetic regulation of transcription. Understanding which proteins bind to a given lincRNA can facilitate elucidating the components of a given regulatory pathway. The RNA targeting effector proteins of the present invention can be designed to recruit biotin ligase to specific transcripts, thereby locally labeling the bound protein with biotin. The protein can then be pulled down and analyzed by mass spectrometry to identify it.

[0257] Assembly of the complex onto RNA and substrate shuttling Furthermore, the RNA targeting effector proteins of the present invention can be used to assemble complexes on RNA. This can be achieved by functionalizing the RNA targeting effector protein with multiple related proteins (e.g., components of a specific synthetic pathway). Alternatively, multiple RNA targeting effector proteins can be functionalized with such various related proteins to target the same or adjacent target RNAs. A useful application of assembling complexes on RNA is, for example, promoting substrate shuttling between proteins.

[0258] synthetic biology The development of biological systems has broad utility, including clinical applications. It is envisioned that the programmable RNA-targeting effector proteins of the present invention can be used to fuse toxic domains to split proteins for targeted cell death using, for example, cancer-associated RNAs as target transcripts. Furthermore, in synthetic biology systems, pathways involving protein-protein interactions can be influenced by fusion complexes with appropriate effectors, such as kinases or other enzymes.

[0259] Protein splicing: inteins Protein splicing is a post-translational process in which an intervening polypeptide, called an intein, catalyzes its own excision from adjacent polypeptides, called exteins, and subsequent ligation of the exteins. The assembly of two or more RNA-targeting effector proteins as described herein onto a target transcript can be used to direct the release of a split intein (Topilina and Mills Mob DNA. 2014 Feb 4;5(1):5), thereby enabling direct accounting of the presence of mRNA transcripts and subsequent release of a protein product, such as a metabolic enzyme or transcription factor (for downstream operation of a transcription pathway). This application may have significant implications in synthetic biology (see above) or large-scale bioproduction (product production only under certain conditions).

[0260] Inducible, Administered, and Self-Inactivating Systems In one embodiment, a fusion complex comprising an RNA targeting effector protein of the invention and an effector component is designed to be inducible, e.g., light- or chemically inducible, allowing the effector component to be activated at a desired time.

[0261] Light-inducibility can be achieved, for example, by designing fusion complexes that utilize CRY2PHR / CIBN pairing for fusion, a system particularly useful for light-induction of protein interactions in living cells (Konermann S, et al. Nature. 2013;500:472-476).

[0262] Chemical inducibility can be provided, for example, by designing fusion complexes that use FKBP / FRB (FK506 binding protein / FKBP rapamycin binding) pairing for fusion. Using this system, rapamycin is required for protein binding (Zetsche et al. Nat Biotechnol. 2015;33(2):139-42 describes the use of this system with Cas9).

[0263] Furthermore, when introduced into cells as DNA, the RNA targeting effector proteins of the invention can be regulated by inducible promoters, such as tetracycline- or doxycycline-controlled transcriptional activation (Tet-On and Tet-Off expression systems), hormone-inducible gene expression systems, such as the ecdysone-inducible gene expression system, and arabinose-inducible gene expression systems. When delivered as RNA, expression of the RNA targeting effector proteins can be regulated by riboswitches, which can sense small molecules such as tetracycline (as described in Goldfless et al. Nucleic Acids Res. 2012;40(9):e64).

[0264] In one embodiment, delivery of the RNA targeting effector proteins of the present invention can be modulated to alter the amount of protein or crRNA in the cell, thereby altering the magnitude of the desired effect or any undesired off-target effects.

[0265] In one embodiment, the RNA targeting effector proteins described herein can be designed to self-inactivate, which when delivered to a cell as RNA, either mRNA or a replication RNA therapeutic (Wrobleska et al Nat Biotechnol. 2015 Aug;33(8):839-841), can self-inactivate expression and subsequent effects by destroying self-RNA, thereby reducing resident and potentially undesirable effects.

[0266] For further in vivo applications of RNA targeting effector proteins as described herein, see 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 invention, preferably by using catalytically inactive Cas13b, the following applications are envisioned: translation enhancement (e.g., Cas13b-translation enhancer fusions (e.g., eIF4 fusions)); translation repression (e.g., gRNAs targeting ribosome binding sites); exon skipping (e.g., gRNAs targeting splice donor and / or acceptor sites); exon inclusion (e.g., gRNAs or spliceosome components (e.g., U1 fusions) that target specific exon splice donor and / or acceptor sites for inclusion). Cas13b fused to or recruiting snRNA); access to RNA localization (e.g., Cas13b-marker fusions (e.g., EGFP fusions)); alteration of RNA localization (e.g., Cas13b-localization signal fusions (e.g., NLS or NES fusions)); RNA degradation (in this case, if dependent on Cas13b activity, catalytically inactive Cas13b should not be used; instead, and for increased specificity, split Cas13b may be used); inhibition of non-coding RNA function (e.g., miRNA), such as by degradation of gRNA or binding to functional sites (potentially titrated out at specific sites by relocalization with Cas13b-signal sequence fusions).

[0267] Cas13b function is robust against 5' or 3' extension of the crRNA and extension of the crRNA loop. Therefore, it is conceivable that MS2 loops and other recruitment domains can be added to the crRNA without affecting complex formation and binding to target transcripts. Such modifications to the crRNA to recruit various effector domains are applicable in the use of the RNA-targeting effector proteins described above.

[0268] Cas13b has the ability to mediate RNA phage resistance, and therefore it is envisioned that Cas13b can be used to immunize, for example, animals, humans, and plants against RNA-only pathogens, including but not limited to, retroviruses (e.g., lentiviruses such as HIV), HCV, Ebola virus, and Zika virus.

[0269] In certain embodiments, Cas13b can process (cleave) its own array. This applies to both wild-type Cas13b proteins and mutant Cas13b proteins containing one or more mutated amino acid residues described herein. It is therefore envisioned 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 one promoter. Such a delivery method has the advantages of being substantially more compact, easier to synthesize, and easier to deliver in viral systems. It will be understood that for orthologs of Cas13b herein, the exact amino acid positions may differ, which can be appropriately determined by protein alignment, as known in the art and described elsewhere herein. Aspects of the present invention also encompass methods and uses of the compositions and systems described herein for, for example, altering or manipulating the expression of one or more genes or one or more gene products in genome engineering in vitro, in vivo, or ex vivo in prokaryotic or eukaryotic cells.

[0270] Aspects of the invention also include methods and uses of the compositions and systems described herein for, for example, altering or manipulating (protein) expression of one or more genes or one or more gene products in genome or transcriptome engineering in vitro, in vivo or ex vivo in prokaryotic or eukaryotic cells.

[0271] In certain aspects, the present invention provides methods and compositions for modulating, e.g., reducing, target RNA (protein) expression in a cell, wherein the Cas13b system of the present invention interferes with RNA transcription, stability, and / or translation.

[0272] In certain embodiments, an effective amount of Cas13b system is used to cleave RNA or otherwise inhibit RNA expression.In this regard, this system has the same use as siRNA and shRNA, and therefore can also be substituted for such methods.This method includes, but is not limited to, for example, the use of Cas13b system instead of interfering ribonucleic acid (such as siRNA or shRNA) or its transcription template, for example, the DNA encoding shRNA.The Cas13b system is introduced into target cells, for example, by administering to a mammal that contains target cells.

[0273] Advantageously, the Cas13b system of the present invention is specific: for example, while interfering ribonucleic acid (such as siRNA or shRNA) polynucleotide systems suffer from design and stability issues as well as off-target binding, the Cas13b system of the present invention can be designed with high specificity.

[0274] Destabilized Cas13b In certain embodiments, the effector protein of the present invention (CRISPR enzyme; Cas13b) as described herein is associated with or fused to a destabilization domain (DD). In some embodiments, the DD is ER50. The corresponding stabilizing ligand of this DD is, in some embodiments, 4HT. Therefore, in some embodiments, one of the at least one DD is ER50, and thus the stabilizing ligand is 4HT or CMP8. In some embodiments, the DD is DHFR50. The corresponding stabilizing ligand of this DD is, in some embodiments, TMP. Therefore, in some embodiments, one of the at least one DD is DHFR50, and thus the stabilizing ligand is TMP. In some embodiments, the DD is ER50. The corresponding stabilizing ligand of this DD is, in some embodiments, CMP8. Therefore, CMP8 can be an alternative stabilizing ligand to 4HT in the ER50 system. Although CMP8 and 4HT may / should be used competitively, some cell types may be more susceptible to either of these two ligands, and based on this disclosure and knowledge in the art, one skilled in the art can use CMP8 and / or 4HT.

[0275] In some embodiments, one or two DDs may be fused to the N-terminal end of the CRISPR enzyme, and one or two DDs may be fused to the C-terminal end of the CRISPR enzyme. In some embodiments, at least two DDs are associated with the CRISPR enzyme, and the DDs are the same DD, i.e., the DDs are homologous. Thus, both (or two or more) of the DDs may be ER50 DDs. This is preferred in some embodiments. Alternatively, both (or two or more) of the DDs may be DHFR50 DDs. This is also preferred in some embodiments. In some embodiments, at least two DDs are associated with the CRISPR enzyme, and the DDs are different DDs, i.e., the DDs are heterologous. Thus, one of the DDs may be ER50, while one or more of the DDs or any other DD may be DHFR50. Having two or more heterologous DDs may be advantageous, as it may result in a higher level of degradation control. Tandem fusion of two or more DDs at the N- or C-terminus can enhance degradation, and such tandem fusions can be, for example, ER50-ER50-Ca13b or DHFR-DHFR-Ca13b. It is envisioned that high levels of degradation occur in the absence of either stabilizing ligand, intermediate levels of degradation occur in the absence of one stabilizing ligand and the presence of the other (or another) stabilizing ligand, while low levels of degradation occur in the presence of both (or more) stabilizing ligands. Control can also be provided by having an N-terminal ER50 DD and a C-terminal DHFR50 DD.

[0276] In some embodiments, the fusion of the CRISPR enzyme and the DD includes a linker between the DD and the CRISPR enzyme. In some embodiments, the linker is a GlySer linker. In some embodiments, the DD-CRISPR enzyme further includes at least one nuclear export signal (NES). In some embodiments, the DD-CRISPR enzyme includes two or more NESs. In some embodiments, the DD-CRISPR enzyme includes at least one nuclear localization signal (NLS), which may be in addition to an NES. In some embodiments, the CRISPR enzyme includes, consists essentially of, or consists of a localization (nuclear import or nuclear export) signal as or part of the linker between the CRISPR enzyme and the DD. HA or Flag tags are also within the scope of the present invention as linkers. Applicants use NLSs and / or NESs as linkers, as well as glycine-serine linkers as short as GS up to (GGGGS)3.

[0277] Destabilizing domains have general utility in conferring instability to a wide range of proteins. See, for example, Miyazaki, J Am Chem Soc. Mar 7, 2012;134(9):3942-3945 (incorporated herein by reference). CMP8 or 4-hydroxytamoxifen can be destabilizing domains. More generally, a temperature-sensitive mutant of mammalian DHFR (DHFRts), which is destabilized by the N-end rule, was 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 was an important demonstration that small molecule ligands can stabilize proteins that are normally targeted for degradation in cells. A rapamycin derivative stabilized a destabilizing mutant of the FRB domain of mTOR (FRB*) and restored function of the fused kinase GSK-3β. 6,7 This system demonstrated that ligand-dependent stability represents an attractive strategy for modulating the function of specific proteins in complex biological environments. Regulation of protein activity may involve a DD that becomes functional upon ubiquitin complementation via rapamycin-induced dimerization of FK506-binding protein with FKBP12. Mutants of human FKBP12 or ecDHFR proteins 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 of the potential destabilization domains (DDs) useful in the practice of the present invention, and the instability of the DD as a fusion with a CRISPR enzyme leads to CRISPR proteolysis of the entire fusion protein by the proteasome. Shield-1 and TMP bind to and stabilize the DD in a dose-dependent manner. The estrogen receptor ligand-binding domain (ERLBD, residues 305–549 of ERS1) can also be engineered as a destabilization domain. Because the estrogen receptor signaling pathway is involved in various diseases, such as breast cancer, this pathway has been extensively studied and many estrogen receptor agonists and antagonists have been developed.Therefore, compatible pairs of ERLBD and drugs are known. There are ligands that bind to mutant forms of ERLBD but not to wild-type forms of ERLBD. By using one of these mutant domains, encoding three mutations (L384M, M421G, G521R), it is possible to modulate the stability of the DD derived from ERLBD using a ligand that does not disrupt the endogenous estrogen-sensitive network. An additional mutation (Y537S) can be introduced to further destabilize the ERLBD, making it a potential DD candidate. This quadruple mutant is an advantageous DD development. This mutant ERLBD can be fused to a CRISPR enzyme, and its stability can be regulated or disrupted using a ligand, resulting in the CRISPR enzyme having a DD. Another DD could be a 12 kDa (107 amino acids) tag based on a mutant FKBP protein that is stabilized by the Shield1 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 a synthetic, biologically inert small molecule, Shield-1, 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 directed approach for engineering conditional protein stability using biologically See, "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 may be used in the practice of the invention in selecting a DD to associate with a CRISPR enzyme.As can be appreciated, the art includes numerous DDs, which can be associated with, e.g., fused to, a CRISPR enzyme, preferably with a linker, such that the DD can be stabilized in the presence of a ligand and destabilized in its absence, thereby destabilizing the CRISPR enzyme as a whole, or the DD can be stabilized in the absence of a ligand and destabilized in the presence of a ligand, thereby regulating or controlling the CRISPR enzyme and thus the CRISPR-Cas complex or system—essentially turning it on and off, so to speak—and thereby providing a means of regulating or controlling the system, for example, in vivo or in vitro. For example, expressing a protein of interest as a fusion with a DD tag destabilizes it in cells and causes rapid degradation, e.g., by the proteasome. Thus, the absence of a stabilizing ligand leads to degradation of the D-associated Cas. Fusing a novel DD to a protein of interest confers instability to the protein of interest, resulting in rapid degradation of the entire fusion protein. Peak Cas activity is sometimes beneficial for reducing off-target effects. Therefore, a short burst of high activity is desirable. The present invention can provide such a peak. In one sense, the system is inducible. In another sense, the system is repressed in the absence of a stabilizing ligand and derepressed in the presence of a stabilizing ligand.

[0278] Application of RNA targeting CRISPR systems to plants and yeast Definition: In general, the term "plant" refers to any of a variety of photosynthetic, eukaryotic, unicellular or multicellular organisms of the kingdom Plantae that grow characteristically by cell division, contain chloroplasts, and have cell walls composed of cellulose. The term plant includes monocotyledonous and dicotyledonous plants. Specific plants include, without limitation, acacia, alfalfa, amaranth, apple, apricot, artichoke, ash tree, asparagus, avocado, banana, barley, beans, sugar beet, birch, beech, blackberry, blueberry, broccoli, Brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, cedar, grains, celery, chestnut, cherry, Chinese cabbage, citrus fruits, clementine, clover, coffee, corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus, fennel, fig, fir, geranium, grape, grapefruit, peanut, ground cherry, gum hemlock, and the like. hemlock), hickory, kale, kiwi fruit, kohlrabi, larch, lettuce, chives, lemon, lime, black locust, pine, maidenhair, corn, mango, maple, melon, millet, mushrooms, mustard, nuts, oak, oats, oil palm, okra, onion, orange, ornamental plants or decorative flowers or trees, papaya, palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper, persimmon, pigeon pea, pine, pineapple, plantain, It is intended to include angiosperms and gymnosperms such as plum, pomegranate, potato, pumpkin, radish, rapeseed, raspberry, rice, rye, sorghum, safflower, wild willow, soybean, spinach, spruce, pumpkin, strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn, tangerine, tea, tobacco, tomato, trees, triticale, turfgrass, turnip, vines, walnut, watercress, watermelon, wheat, yam, yew, and zucchini. The term plant also encompasses algae, which are mostly photoautotrophs, united primarily by their lack of roots, leaves, and other organs that characterize higher plants.

[0279] The method of regulating gene expression using the RNA targeting system as described herein can be used to confer desired traits to essentially any plant. A wide variety of plants and plant cell lines can be engineered for the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transformation methods described above. In preferred embodiments, plants and plant cells targeted for engineering include monocotyledonous and dicotyledonous plants, such as crops including, but not limited to, cereal crops (e.g., wheat, corn, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root crops (e.g., carrot, potato, sugar beet, yam), leafy vegetable crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), coniferous and pine trees (e.g., fir, spruce); plants used in phytoremediation (e.g., heavy metal accumulating plants); oil crops (e.g., sunflower, rapeseed) and plants used for experimental purposes (e.g., Arabidopsis). Thus, the present methods and CRISPR-Cas systems can be used across a wide range of plants, including, for example, Magniolales, Illiciales, Laurales, Piperales, Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelids, and the like. melidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales,Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulaceae, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales, Santalales It can be used for dicotyledonous plants belonging to the orders of Asterales, Rafflesiales, Celastraceae, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales and Asterales. The methods and CRISPR-Cas systems are applicable to plants from the following orders: Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales,Plants belonging to the monocotyledonous or gymnospermae order, such as those belonging to the Liliales and Orchids orders, can be used, for example those belonging to the Pinales, Ginkgoales, Cycadales, Araucariales, Cupressales and Gnetales orders.

[0280] The RNA targeting CRISPR systems and methods of use described herein can be used in a wide range of plant species within the following non-limiting list of dicotyledonous, monocotyledonous, or gymnosperm genera: Atropa, Alseodaphne, Anacardium, Arachis, Beilschmiedia, Brassica, Carthamus, Cocculus, Chlorella, and the like. Croton, Cucumis, Citrus, Citrullus, Capsicum, Catharanthus, Cocos, Coffea, Cucurbita, Daucus, Duguetia, Eschscholzia, Ficus, Fragaria, Glaucium, Glycine, Gossypium, Helianthus, Hevea, Hyoscyamus, Lactuca, Landolphia, Linum, Litsea, Lycopersicon, Lupin, Manihot, Majorana, Malus, Medicago, Nicotiana na), Olive (Olea), Parthenium (Parthenium), Papaver (Papaver), Persea (Persea), Phaseolus (Phaseolus), Pistacia (Pistacia), Pisum (Pisum), Pyrus (Pyrus), Prunus (Prunus), Raphanus (Raphanus), Ricinus (Ricinus), Senecio (Senecio), Sinomenium (Sinomenium), Stephania (Stephania), Sinapis (Sinapis), Solanum (Solanum),Theobroma, Trifolium, Trigonella, Vicia, Vinca, Vilis, and Vigna; and Allium, Andropogon, Aragrostis, Asparagus, Avena, Cynodon, Elaeis, Festuca, Festuca, Heterococcus, and Heterococcus. allis, barley (Hordeum), duckweed (Lemna), rye (Lolium), musa (Musa), rice (Oryza), millet (Panicum), pennesetum (Pannesetum), timothy grass (Phleum), rye (Poa), rye (Secale), sorghum (Sorghum), wheat (Triticum), corn (Zea), fir (Abies), Chinese fir (Cunninghamia), ephedra (Ephedra), spruce (Picea), pine (Pinus) and Pseudotsuga.

[0281] The RNA targeting CRISPR systems and methods of use can also be used with a wide range of "algae" or "algal cells," including, for example, algae selected from several eukaryotic phyla, including Rhodophyta (red algae), Chlorophyta (green algae), Phaeophyta (brown algae), Bacillariophyta (diatoms), Eustigmatophyta, and Dinoflagellates, as well as the prokaryotic phylum Cyanobacteria (blue-green algae).The term "algae" includes, for example, species of the genera Amphora, Anabaena, Ankistrodesmis, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella, Chlorococcum, Cyclotella, Cylindrotheca, The genera Dunaliella, Emiliana, Euglena, Hematococcus, Isochrysis, Monochrysis, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Nephrochloris phrochloris, Nephroselmis, Nitzschia, Nodularia, Nostoc, Oochromonas, Oocystis, Oscillartoria, Pavlova, Phaeodactylum, Playtmonas, Pleuro chrysis, Porhyra, Pseudoanabaena, Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetraselmis, Thalassiosira, and Trichodesmium.

[0282] Plant parts, i.e., "plant tissue," can be treated according to the methods of the present invention to produce improved plants. Plant tissue also includes plant cells. The term "plant cell," as used herein, refers to an individual unit of a living plant, either an intact whole plant, or alternatively, in isolated form grown in in vitro tissue culture on media or agar, in suspension in a growth medium or buffer, or as part of a more highly organized unit, such as a plant tissue, plant organ, or whole plant.

[0283] "Protoplast" refers to a plant cell whose protective cell wall has been completely or partially removed, e.g., using mechanical or enzymatic means, resulting in an intact, biochemically competent unit of a living plant that can reform its cell wall, grow, and regenerate to develop into a whole plant under appropriate growth conditions.

[0284] The term "transformation" broadly refers to a method in which a plant host is genetically modified by the introduction of DNA using Agrobacteria or one of a variety of chemical or physical methods. As used herein, the term "plant host" refers to a plant, including any cell, tissue, organ, or progeny of the plant. Many suitable plant tissues or plant cells can be transformed, including, but not limited to, protoplasts, somatic embryos, pollen, leaves, seedlings, stems, callus, stolon, microtubules, and shoots. Plant tissue also refers to any clones of such plants, seeds, progeny, propagules, and progeny of any of these, such as cuttings or seeds, whether produced sexually or asexually.

[0285] The term "transformed," as used herein, refers to a cell, tissue, organ, or organism into which an exogenous DNA molecule, such as a construct, has been introduced. The introduced DNA molecule may be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism such that the introduced DNA molecule is passed on to subsequent progeny. In these embodiments, a "transformed" or "transgenic" cell or plant can also include the progeny of that cell or plant and progeny produced from breeding programs using such transformed plants as parents in crosses, and which exhibit a phenotypic change resulting from the presence of the introduced DNA molecule. Preferably, the transgenic plant is fertile, capable of passing on the introduced DNA to progeny through sexual reproduction.

[0286] The term "progeny," such as the offspring of a transgenic plant, is one that is born from, produced from, or derived from a plant or transgenic plant. The introduced DNA molecule may also be transiently introduced into a recipient cell, such that the introduced DNA molecule will not be inherited by subsequent progeny and is therefore not considered "transgenic." Thus, as used herein, a "non-transgenic" plant or plant cell is one that does not contain foreign DNA stably integrated into its genome.

[0287] The term "plant promoter," as used herein, is a promoter capable of initiating transcription in plant cells, regardless of whether its origin is a plant cell. Exemplary suitable plant promoters include, but are not limited to, those obtained from bacteria such as Agrobacterium or Rhizobium, which contain genes expressed in plants, plant viruses, and plant cells.

[0288] As used herein, "fungal cell" refers to any type of eukaryotic cell within the kingdom Fungi. Phylums within the kingdom Fungi include Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cells can include yeast, mold, and filamentous fungi. In some embodiments, the fungal cell is a yeast cell.

[0289] As used herein, the term "yeast cell" refers to any fungal cell within the phyla Ascomycota and Basidiomycota. Yeast cells can include budding yeast cells, fission yeast cells, and mold cells. Many types of yeast used in laboratory and industrial settings are part of the phylum Ascomycota, although not limited to these organisms. In some embodiments, the yeast cell is a S. cerevisiae, Kluyveromyces marxianus, or Issatchenkia orientalis cell. Other yeast cells include, without limitation, Candida spp. (e.g., Candida albicans), Yarrowia spp. (e.g., Yarrowia lipolytica), Pichia spp. (e.g., Pichia pastoris), Kluyveromyces spp. (e.g., Kluyveromyces lactis and Kluyveromyces marxianus), Neurospora spp. (e.g., Neurospora crassa), Fusarium spp. spp. (e.g., Fusarium oxysporum) and Issatchenkia spp. (e.g., Issatchenkia orientalis, also known as Pichia kudriavzevii and Candida acidothermophilum). In some embodiments, the fungal cell is a filamentous fungal cell. As used herein, the term "filamentous fungal cell" refers to any type of fungal cell that grows in a filamentous manner, i.e., as a hypha or mycelium.Examples of filamentous fungal cells include, without limitation, Aspergillus spp. (e.g., Aspergillus niger), Trichoderma spp. (e.g., Trichoderma reesei), Rhizopus spp. (e.g., Rhizopus oryzae), and Mortierella spp. (e.g., Mortierella isabellina).

[0290] In some embodiments, the fungal cell is an industrial strain. As used herein, "industrial strain" refers to any strain of fungal cell used in or isolated from an industrial process, e.g., the production of a product on a commercial or industrial scale. An industrial strain may refer to a fungal species typically used in industrial processes, or it may refer to an isolate of a fungal species that may also be used for non-industrial purposes (e.g., laboratory research). Examples of industrial processes can include fermentation (e.g., in the production of food or beverage products), distillation, biofuel production, chemical compound production, and polypeptide production. Examples of industrial strains can include, without limitation, JAY270 and ATCC4124.

[0291] In some embodiments, the fungal cell is a polyploid cell. As used herein, a "polyploid" cell can refer to any cell in which the genome exists in two or more copies. A polyploid cell can refer to a type of cell that is naturally found in a polyploid state, or it can refer to a cell that has been induced to exist in a polyploid state (e.g., by specifically regulating, altering, inactivating, activating, or modifying meiosis, cytokinesis, or DNA replication). A polyploid cell can refer to a cell whose entire genome is polyploid, or it can refer to a cell that is polyploid at a particular genomic locus of interest. Without wishing to be bound by theory, it is believed that the methods using the Cas13b CRISPR system described herein can take advantage of the use of certain fungal cell types, as guide RNA abundance can often be a rate-limiting component in genome engineering of polyploid cells compared to haploid cells.

[0292] In some embodiments, the fungal cell is a diploid cell. As used herein, a "diploid" cell can refer to any cell in which the genome exists in two copies. A diploid cell can refer to a type of cell that is naturally found in a diploid state, or it can refer to a cell that has been induced to exist in a diploid state (e.g., by specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, S. cerevisiae strain S228C can be maintained in a haploid or diploid state. A diploid cell can refer to a cell in which the entire genome is diploid, or it can refer to a cell that is diploid at a specific genomic locus of interest. In some embodiments, the fungal cell is a haploid cell. As used herein, a "haploid" cell can refer to any cell in which the genome exists in one copy. A haploid cell can refer to a type of cell that is naturally found in a haploid state, or it can refer to a cell that has been induced to exist in a haploid state (e.g., by specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, S. cerevisiae strain S228C can be maintained in a haploid or diploid state. A haploid cell can refer to a cell whose entire genome is haploid, or it can refer to a cell that is haploid at a particular genomic locus of interest.

[0293] As used herein, a "yeast expression vector" refers to a nucleic acid containing one or more sequences encoding an RNA and / or a polypeptide, and may further contain any desired elements that control expression of the nucleic acid and any elements that allow for replication and maintenance of the expression vector within a yeast cell. Many suitable yeast expression vectors and their characteristics are known in the art. For example, various vectors and techniques are exemplified in "Yeast Protocols, 2nd edition," Xiao, W., ed. (Humana Press, New York, 2007) and Buckholz, RG and Gleeson, MA (1991) Biotechnology (NY) 9(11):1067-72. Yeast vectors may contain, without limitation, a centromere (CEN) sequence, an autonomously replicating sequence (ARS), a promoter such as an RNA polymerase III promoter operably linked to a sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., an auxotroph, antibiotic, or other selectable marker). Examples of expression vectors used in yeast include plasmids, yeast artificial chromosomes, 2μ plasmids, yeast integrating plasmids, yeast replicating plasmids, shuttle vectors, and episomal plasmids.

[0294] Stable integration of RNA-targeting CRISPR system components in the genomes of plants and plant cells In particular embodiments, it is envisioned that polynucleotides encoding components of an RNA-targeting CRISPR system are introduced for stable integration into the genome of a plant cell. In these embodiments, the design of the transformation vector or expression system can be tailored depending on when, where, and under what conditions the guide RNA and / or RNA-targeting gene is to be expressed.

[0295] In particular embodiments, it is contemplated that components of an RNA-targeting CRISPR system are stably introduced into the genomic DNA of a plant cell. Additionally or alternatively, it is contemplated that components of an RNA-targeting CRISPR system are introduced for stable integration into the DNA of a plant organelle, such as, but not limited to, a plastid, mitochondria, or chloroplast.

[0296] Expression systems for stable integration into the genome of plant cells may contain one or more of the following elements: a promoter element that can be used to express the guide RNA and / or RNA targeting enzyme in plant cells; a 5' untranslated region that enhances expression; an intron element that further enhances expression in certain cells, such as monocotyledonous plant cells; a multiple cloning site that provides convenient restriction sites for inserting one or more guide RNA and / or RNA targeting gene sequences and other desired elements; and a 3' untranslated region that provides efficient termination of the expressed transcript.

[0297] The elements of the expression system may be on one or more expression constructs that are either circular, such as a plasmid or transformation vector, or non-circular, such as linear double-stranded DNA. In particular embodiments, the RNA targeting CRISPR expression system comprises at least: (a) a nucleotide sequence encoding a guide RNA (gRNA) that hybridizes with a target sequence in a plant, the guide RNA comprising a guide sequence and a direct repeat sequence; and (b) a nucleotide sequence encoding an RNA targeting protein; wherein components (a) or (b) are located on the same or different constructs, whereby the different nucleotide sequences can be under the control of the same or different regulatory elements operable in a plant cell.

[0298] DNA constructs containing the components of an RNA-targeting CRISPR system and, if applicable, a template sequence can be introduced into the genome of a plant, plant part, or plant cell by a variety of conventional techniques. This process generally involves selecting a suitable host cell or tissue, introducing the construct into the host cell or tissue, and regenerating a plant cell or plant therefrom. In particular embodiments, DNA constructs can be introduced into plant cells using techniques such as, but not limited to, electroporation, microinjection, aerosol beam injection of plant cell protoplasts, or DNA constructs can be directly introduced into plant tissues using biolistic methods such as DNA particle bombardment (see also Fu et al., Transgenic Res. 2000 Feb;9(1):11-9). The basis of particle bombardment is to accelerate particles coated with a gene of interest toward the cell, allowing the particles to penetrate the cytoplasm and typically result in stable integration into the genome (see, e.g., Klein et al., Nature (1987); Klein et al., Bio / Technology (1992); Casas et al., Proc. Natl. Acad. Sci. USA (1993)).

[0299] In a detailed embodiment, a DNA construct containing components of an RNA-targeting CRISPR system can be introduced into a plant via Agrobacterium-mediated transformation. The DNA construct can be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. Foreign DNA can be incorporated into the plant genome by infecting the plant or by incubating plant protoplasts with Agrobacterium bacteria containing one or more Ti (tumor-inducing) plasmids (see, e.g., Fraley et al., (1985), Rogers et al., (1987), and U.S. Patent No. 5,563,055).

[0300] Plant promoters To ensure proper expression in plant cells, the components of the Cas13b CRISPR system described herein are typically placed under the control of a plant promoter, i.e., a promoter that is operable in plant cells. The use of various types of promoters is contemplated.

[0301] A constitutive plant promoter is a promoter that is capable of causing the open reading frame (ORF) it controls to be expressed in all or nearly all plant tissues at all or nearly all developmental stages of the plant (referred to as "constitutive expression"). One non-limiting example of a constitutive promoter is the cauliflower mosaic virus 35S promoter. The present invention also contemplates methods for modifying RNA sequences and thus regulating the expression of plant biomolecules. Thus, in specific embodiments of the present invention, it is advantageous to place one or more elements of an RNA targeting CRISPR system under the control of a promoter that may be regulated. A "regulated promoter" refers to a promoter that directs gene expression in a non-constitutive but temporally and / or spatially regulated manner, including tissue-specific, tissue-preferred, and inducible promoters. Different promoters may direct the expression of genes in different tissues or cell types, at different developmental stages, or in response to different environmental conditions. In particular embodiments, one or more of the RNA targeting CRISPR components are expressed under the control of a constitutive promoter such as the cauliflower mosaic virus 35S promoter, and by utilizing tissue-preferred promoter, can target the enhanced expression in certain cell types in specific plant tissue, for example, the vascular cells of leaves or roots or the specific cells of seeds.For the detailed promoter examples used in the RNA targeting CRISPR system, refer to Kawamata et al., (1997) Plant Cell Physiol 38:792-803; Yamamoto et al., (1997) Plant J 12:255-65; Hire et al., (1992) Plant Mol Biol 20:207-18; Kuster et al., (1995) Plant Mol Biol 29:759-72 and Capana et al., (1994) Plant Mol Biol 25:681-91. Examples of promoters that are inducible and allow for gene editing or spatiotemporal control of gene expression may use some form of energy.The form of energy may include, but is not limited to, acoustic energy, electromagnetic radiation, chemical energy, and / or thermal energy. Examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activation systems (FKBP, ABA, etc.), or light-inducible systems (phytochromes, LOV domains, or cryptochromes), such as light-inducible transcriptional effectors (LITEs) that induce sequence-specific changes in transcriptional activity. Components of light-inducible systems may include RNA-targeting CRISPR enzymes, light-responsive cytochrome heterodimers (e.g., from Arabidopsis thaliana), and transcriptional activation / repression domains. Further examples of inducible DNA-binding proteins and methods of use thereof are provided in U.S. Provisional Patent Application Nos. 61 / 736465 and 61 / 721,283, which are hereby incorporated by reference in their entireties.

[0302] In particular embodiments, transient or inducible expression can be achieved using, for example, chemically regulated promoters, whereby gene expression is induced upon addition of an exogenous chemical. Regulation of gene expression can also be achieved through chemically repressible promoters, whereby gene expression is repressed upon addition of a chemical. Chemically inducible promoters include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide antidotes (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-II-27, WO 93 / 01294), which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7). Antibiotic-regulated promoters, such as tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156), can also be used herein.

[0303] Transport to and / or expression in specific plant organelles The expression system may contain elements for translocation to and / or expression in specific plant organelles.

[0304] Chloroplast targeting In particular embodiments, it is envisioned that RNA targeting CRISPR system is used to specifically modify the expression and / or translation of chloroplast gene, or ensure the expression in chloroplast.For this purpose, chloroplast transformation method or RNA targeting CRISPR components are used to compartmentalize in chloroplast.For example, introducing genetic modification into plastid genome can reduce biosafety issues such as gene flow through pollen.

[0305] Chloroplast transformation methods are known in the art and include particle bombardment, PEG treatment, and microinjection. In addition, methods involving the transfer of a transformation cassette from the nuclear genome to the plastid can be used, as described in WO2010061186.

[0306] Alternatively, it is envisioned that one or more RNA-targeting CRISPR components are targeted to plant chloroplasts. This is achieved by incorporating into the expression construct a sequence encoding a chloroplast transit peptide (CTP) or plastid transit peptide operably linked to the 5' region of the sequence encoding the RNA-targeting protein. The CTP is removed in a processing step during translocation to the chloroplasts. Chloroplast targeting of expressed proteins is well known to those skilled in the art (see, for example, Protein Transport into Chloroplasts, 2010, Annual Review of Plant Biology, Vol. 61:157-180). In such embodiments, it is also desirable to target one or more guide RNAs to plant chloroplasts. Methods and constructs that can be used to translocate guide RNAs to chloroplasts using chloroplast-localizing sequences are described, for example, in U.S. Patent Application Publication No. 20040142476 (incorporated herein by reference). By incorporating such various constructs into the expression system of the present invention, RNA-targeting guide RNAs can be efficiently translocated.

[0307] Introduction of polynucleotides encoding the CRISPR-RNA targeting system into algal cells Transgenic algae (or other plants, such as rapeseed) may be particularly useful for the production of vegetable oils or biofuels, such as alcohols (especially methanol and ethanol), or other products. They can be engineered to express or overexpress high levels of oils or alcohols for use in the oil or biofuel industry.

[0308] U.S. Patent No. 8,945,839 describes a method for engineering microalgae (Chlamydomonas reinhardtii cells) using Cas9. Using similar tools, the RNA-targeting CRISPR-based method described herein can be applied to Chlamydomonas species and other algae. In a detailed embodiment, an RNA-targeting protein and guide RNA are introduced and expressed in algae using a vector expressing the RNA-targeting protein under the control of a constitutive promoter, such as Hsp70A-Rbc S2 or β2-tubulin. The guide RNA is optionally delivered using a vector containing a T7 promoter. Alternatively, RNA-targeting mRNA and in vitro transcribed guide RNA can be delivered to algae cells. Electroporation protocols are available to those skilled in the art, such as the standard recommended protocol from the GeneArt Chlamydomonas Engineering Kit.

[0309] Introduction of polynucleotides encoding RNA targeting components into yeast cells In a specific embodiment, the present invention relates to the use of RNA targeting CRISPR system for RNA editing in yeast cells. The transformation method of yeast cells that can be used to introduce polynucleotides encoding RNA targeting CRISPR system components is well known to those skilled in the art and is reviewed by Kawai et al., 2010, Bioeng Bugs. 2010 Nov-Dec; 1 (6): 395-403). Non-limiting examples include transformation of yeast cells by lithium acetate treatment (which may further include carrier DNA and PEG treatment), bombardment or electroporation.

[0310] Transient expression of RNA-targeting CRISPR system components in plants and plant cells In particular embodiments, it is envisaged that the guide RNA and / or the RNA targeting gene are transiently expressed in plant cells. In these embodiments, the RNA targeting CRISPR system will only modify the RNA target molecule when both the guide RNA and the RNA targeting protein are present in the cell, thus further controlling gene expression. Because the expression of the RNA targeting enzyme is transient, the plant regenerated from such plant cells typically does not contain foreign DNA. In particular embodiments, the RNA targeting enzyme is stably expressed by the plant cell, and the guide sequence is transiently expressed.

[0311] In a particularly preferred embodiment, RNA-targeting CRISPR system components can be introduced into plant cells using plant viral vectors (Scholthof et al. 1996, Annu Rev Phytopathol. 1996;34:299-323). In further detailed embodiments, the viral vector is a vector derived from a DNA virus. For example, a geminivirus (e.g., cabbage leaf curl virus, bean yellows virus, wheat dwarf virus, tomato leaf curl virus, corn streak virus, tobacco leaf curl virus, or tomato golden mosaic virus) or a nanovirus (e.g., broad bean spotted wilt virus). In other detailed embodiments, the viral vector is a vector derived from an RNA virus. For example, a tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus), a potexvirus (e.g., potato virus X), or a hordeivirus (e.g., wheat stripe mosaic virus). Replicating genomes of plant viruses are non-integrating vectors, which is advantageous in the context of avoiding the creation of GMO plants.

[0312] In a detailed embodiment, the vector used for transient expression of RNA targeting CRISPR constructs is, for example, a pEAQ vector, which has been adapted for Agrobacterium-mediated transient expression in protoplasts (Sainsbury F. et al., Plant Biotechnol J. 2009 Sep; 7(7): 682-93). Precise targeting of genomic locations has been demonstrated using a modified cabbage leaf curl virus (CaLCuV) vector to express gRNA in stable transgenic plants expressing CRISPR enzymes (Scientific Reports 5, Article number: 14926 (2015), doi: 10.1038 / srep14926).

[0313] In a detailed embodiment, double-stranded DNA fragments encoding guide RNAs and / or RNA targeting genes can be transiently introduced into plant cells. In such embodiments, the introduced double-stranded DNA fragments are provided in an amount sufficient to modify RNA molecules in the cells, but not to remain after a desired time or one or more cell divisions. Methods for directing DNA transfer in plants are known to those skilled in the art (see, for example, Davey et al. Plant Mol Biol. 1989 Sep;13(3):273-85).

[0314] In another embodiment, an RNA polynucleotide encoding an RNA targeting protein is introduced into a plant cell in an amount sufficient to modify the RNA molecule cell (in the presence of at least one guide RNA), but not remaining after a predetermined time or one or more cell divisions, which is then translated and processed by the host cell to produce the protein. Methods for introducing mRNA into plant protoplasts for transient expression are known to those skilled in the art (see, for example, Gallie, Plant Cell Reports (1993), 13; 119-122). Combinations of the different methods described above are also envisioned.

[0315] Delivery of RNA-targeting CRISPR components into plant cells In detailed embodiments, it is beneficial to deliver one or more components of the RNA-targeting CRISPR system directly into plant cells. This is particularly beneficial for generating non-transgenic plants (see below). In detailed embodiments, one or more of the RNA-targeting components are prepared outside of a plant or plant cell and delivered to the cell. For example, in detailed embodiments, the RNA-targeting protein is prepared in vitro and then introduced into a plant cell. The RNA-targeting protein can be prepared by various methods known to those skilled in the art, including recombinant production. After expression, the RNA-targeting protein is isolated, refolded as needed, purified, and optionally treated to remove any purification tag, such as a His-tag. Once crude, partially purified, or more completely purified RNA-targeting protein is obtained, the protein can be introduced into a plant cell.

[0316] In particular embodiments, an RNA targeting protein is mixed with a guide RNA that targets an RNA of interest to form a pre-assembled ribonucleoprotein.

[0317] Individual components or pre-assembled ribonucleoproteins can be introduced into plant cells by electroporation, by bombardment of particles coated with RNA targeting-related gene products, by chemical transfection, or by any other means of transport across cell membranes.For example, the transfection of plant protoplasts with pre-assembled CRISPR ribonucleoproteins has been demonstrated to ensure targeted modification of plant genomes (as described by Woo et al. Nature Biotechnology, 2015; DOI:10.1038 / nbt.3389).These methods can be modified to achieve targeted modification of RNA molecules in plants.

[0318] In particular embodiments, RNA-targeting CRISPR system components are introduced into plant cells using nanoparticles. The components can be uploaded onto or packaged in nanoparticles and applied to plants as either proteins or nucleic acids, or alternatively, a combination thereof (e.g., as described in WO2008042156 and U.S. Patent Application Publication No. 20130185823). Specifically, embodiments of the present invention include nanoparticles uploaded or packaged with a DNA molecule encoding an RNA-targeting protein, a DNA molecule encoding a guide RNA, and / or an isolated guide RNA, as described in WO2015089419.

[0319] A further means of introducing one or more components of an RNA-targeting CRISPR system into a plant cell is by using a cell-penetrating peptide (CPP). Accordingly, in particular, embodiments of the present invention include compositions comprising a cell-penetrating peptide linked to an RNA-targeting protein. In particular embodiments of the invention, the RNA targeting protein and / or guide RNA are coupled to one or more CPPs for efficient transport inside the plant protoplast (see Ramakrishna (2014 Genome Res. 2014 Jun;24(6):1020-7) for Cas9 in human cells). In other embodiments, the RNA targeting gene and / or guide RNA are encoded by one or more circular or acyclic DNA molecules coupled to one or more CPPs for plant protoplast delivery. The plant protoplasts are then regenerated into plant cells and further plants. CPPs are generally described as short peptides of less than 35 amino acids derived from either proteins or chimeric sequences that have the ability to transport biomolecules across cell membranes in a receptor-independent manner. CPPs can be cationic peptides, peptides with hydrophobic sequences, amphipathic peptides, peptides with proline-rich antimicrobial sequences, and chimeric or bipartite peptides (Pooga and Langel CPPs are capable of penetrating biological membranes, thereby causing the movement of various biomolecules across the cell membrane and into the cytoplasm, and improving their intracellular transport, thereby facilitating the interaction of biomolecules with their targets. Examples of CPPs include Tat, a nuclear transcriptional activator protein required for HIV type 1 viral replication; penetratin; Kaposi's fibroblast growth factor (FGF) signal peptide sequence; integrin β3 signal peptide sequence; polyarginine peptide Arg sequence; guanine-rich molecular transporter; and sweet arrow peptide, among others.

[0320] Target RNA envisaged for plant, algae or fungal applications A target RNA, i.e., an RNA of interest, is an RNA that is targeted by the present invention, leading to the recruitment of an RNA targeting protein to a desired target site on the target RNA and binding thereto. The target RNA can be any suitable form of RNA, which in some embodiments may include mRNA. In other embodiments, the target RNA may include transfer RNA (tRNA) or ribosomal RNA (rRNA). In other embodiments, the target RNA may include interfering RNA (RNAi), microRNA (miRNA), microswitches, microzymes, satellite RNA, and RNA viruses. The target RNA may be located in the cytoplasm of a plant cell, or in the nucleus, or in a plant organelle such as a mitochondria, chloroplast, or plastid.

[0321] In particular embodiments, RNA-targeting CRISPR systems are used to cleave RNA or otherwise inhibit RNA expression.

[0322] Use of RNA-targeting CRISPR systems to regulate plant gene expression by RNA regulation RNA targeting proteins, in combination with suitable guide RNAs, can also be used to target gene expression by controlling RNA processing. Control of RNA processing can include RNA splicing, including alternative splicing or specific targeting of particular splice variants or isoforms; RNA processing reactions such as viral replication (particularly that of plant viruses, including plant viroids); and tRNA biosynthesis. RNA targeting proteins in combination with suitable guide RNAs can also be used to control RNA activation (RNAa). RNAa leads to enhanced gene expression, so control of gene expression can be achieved by disrupting or reducing RNAa, thereby reducing the enhanced gene expression.

[0323] The RNA targeting effector proteins of the present invention can also be used for antiviral activity in plants, particularly against RNA viruses. The effector protein can be targeted to viral RNA using a suitable guide RNA selective for the selected viral RNA sequence. In particular, the effector protein can be an active nuclease that cleaves RNA, such as single-stranded RNA. Thus, the use of the RNA targeting effector proteins of the present invention as antiviral agents is provided. Examples of viruses that can be antagonized in this way include, but are not limited to, tobacco mosaic virus (TMV), tomato spotted wilt virus (TSWV), cucumber mosaic virus (CMV), potato virus Y (PVY), cauliflower mosaic virus (CaMV) (RT virus), plum pox virus (PPV), brome mosaic virus (BMV), and potato virus X (PVX).

[0324] Examples of modulation of RNA expression in plants, algae or fungi as an alternative to targeted gene modification are further described herein.

[0325] Of particular interest is regulated gene expression control through regulated mRNA cleavage, which can be achieved by placing RNA targeting elements under the control of a regulated promoter as described herein.

[0326] Using RNA-targeting CRISPR systems to restore function to tRNA molecules Pring et al. describe RNA editing in plant mitochondria and chloroplasts, which alters mRNA sequences to encode proteins different from those in DNA (Plant Mol. Biol. (1993) 21(6):1163-1170. doi:10.1007 / BF00023611). In particular embodiments of the present invention, elements of an RNA-targeting CRISPR system that specifically target mitochondrial and chloroplast mRNAs can be introduced into plants or plant cells to express different proteins in such plant organelles, mimicking the process that occurs in vivo.

[0327] Using RNA-targeting CRISPR systems as an alternative to RNA interference to inhibit RNA expression RNA targeting CRISPR system has the same application as RNA inhibition or RNA interference, and therefore can be substituted for such method.In particular embodiment, the method of the present invention comprises, for example, using RNA targeting CRISPR instead of interfering ribonucleic acid (such as siRNA or shRNA or dsRNA).The example of inhibiting RNA expression in plants, algae or fungi as an alternative to targeted gene modification is further described herein.

[0328] Using RNA-targeting CRISPR systems to control RNA interference The regulation of interfering RNA or miRNA can help reduce the off-target effects (OTE) seen in methods by shortening the lifespan of interfering RNA or miRNA in vivo or in vitro. In particular embodiments, the target RNA can include interfering RNA, i.e., RNA involved in the RNA interference pathway, such as shRNA, siRNA, etc. In other embodiments, the target RNA can include microRNA (miRNA) or double-stranded RNA (dsRNA).

[0329] In other detailed embodiments, when an RNA targeting protein and a suitable guide RNA are selectively expressed (e.g., under the control of a spatially or temporally regulated promoter, such as a tissue-specific or cell cycle-specific promoter and / or enhancer), they can be used to "protect" a cell or system (in vivo or in vitro) from RNAi in that cell. This can be useful in adjacent tissues or cells where RNAi is not required, or for purposes of comparing cells or tissues where the effector protein and a suitable guide are expressed and not expressed (i.e., where RNAi is not and is controlled, respectively). The RNA targeting protein can be used to regulate or bind to molecules that contain or consist of RNA, such as ribozymes, ribosomes, or riboswitches. In embodiments of the invention, the guide RNA recruits the RNA targeting protein to such molecules, thereby enabling the RNA targeting protein to bind to them.

[0330] The RNA targeting CRISPR system of the present invention can be applied in the area of ​​in planta RNAi technology, including pest management, plant disease management and herbicide resistance management, and in plant assays and for other applications without undue experimentation from this disclosure, as the present application provides a basis for informed engineering of this system (see, for example, Kim et al., Pesticide Biochemistry and Physiology (Impact Factor: 2.01). 01 / 2015; 120. DOI: 10.1016 / j.pestbp.2015.01.002; Sharma et al. in Academic Journals (2015), Vol. 12(18) pp2303-2312; Green JM, in Pest Management Science, Vol 70(9), pp 1351-1357).

[0331] Using RNA-targeting CRISPR systems to engineer riboswitches and control metabolic regulation in plants, algae, and fungi Riboswitches (also known as aptazymes) are regulatory segments of messenger RNA that bind small molecules and thus regulate gene expression. This mechanism allows cells to sense the intracellular concentration of these small molecules. A particular riboswitch typically regulates its neighboring gene by altering the transcription, translation, or splicing of that gene. Therefore, specific embodiments of the present invention contemplate controlling riboswitch activity by using an RNA targeting protein in combination with a suitable guide RNA that targets the riboswitch. This can be by cleaving or binding to the riboswitch. In specific embodiments, reducing riboswitch activity is contemplated. Recently, riboswitches that bind to thiamine pyrophosphate (TPP) have been characterized and found to regulate thiamine biosynthesis in plants and algae. Furthermore, this element appears to be an essential regulator of primary metabolism in plants (Bocobza and Aharoni, Plant J. 2014 Aug;79(4):693-703. doi:10.1111 / tpj.12540. Epub 2014 Jun 17). TPP riboswitches are also found in certain fungi, such as Neurospora crassa, where they control alternative splicing to conditionally produce upstream open reading frames (uORFs), thereby affecting downstream gene expression (Cheah MT et al., (2007) Nature 447(7143):497-500. doi:10.1038 / nature05769). The RNA targeting CRISPR system described herein can be used to manipulate the endogenous riboswitch activity of plants, algae or fungi, thereby changing the expression of downstream genes controlled by it.In particular embodiments, the RNA targeting CRISPR system can be used to assay the function of riboswitch in vivo or in vitro and study its relationship to metabolic networks.In particular embodiments, the RNA targeting CRISPR system can potentially be used to engineer riboswitches as metabolite sensors in plants and gene regulation platforms.

[0332] Use of RNA-targeting CRISPR systems in RNAi screens in plants, algae, or fungi RNAi screens allow for the identification of gene products whose knockdown is associated with phenotypic changes, allowing for biological pathways to be explored and components to be identified. In particular embodiments of the invention, control can be exerted over or during such screens by using the Cas13b protein and suitable guide RNAs described herein to remove or reduce the activity of RNAi in the screen, thereby restoring (by removing or reducing interference / repression) the activity of the (previously interfered) gene product.

[0333] Use of RNA targeting proteins to visualize RNA molecules in vivo and in vitro In a specific embodiment, the present invention provides a nucleic acid binding system.The in situ hybridization of RNA and complementary probe is a powerful technique.Typically, fluorescent DNA oligonucleotides are used to detect nucleic acids by hybridization.Although some modifications, such as locked nucleic acid (LNA), have achieved increased efficiency, there is still a need for efficient and versatile alternatives.In this way, the labeling element of RNA targeting system can be used as an alternative to the efficient and adaptable system for in situ hybridization.

[0334] Further applications of RNA-targeting CRISPR systems in plants and yeast Use of RNA-targeting CRISPR systems in biofuel production The term "biofuel," as used herein, refers to alternative fuels made from plants and plant-derived resources. Renewable biofuels can be extracted from organic matter that has yielded energy through the process of carbon fixation, or are made by the use or conversion of biomass. This biomass can be used directly for biofuel or converted into convenient energy-containing substances through thermal, chemical, and biochemical conversion. This biomass conversion can result in fuel in solid, liquid, or gaseous form. There are two types of biofuels: bioethanol and biodiesel. Bioethanol is primarily produced by the sugar fermentation process of cellulose (starch), which is mostly derived from corn and sugarcane. Biodiesel, on the other hand, is primarily produced from oil crops such as rapeseed, palm, and soybeans. Biofuels are primarily used for transportation.

[0335] Enhancing plant traits for biofuel production In particular embodiments, the method of using RNA-targeting CRISPR as described herein is used to change the properties of the cell wall to make it more accessible to key hydrolyzing agents so that sugars are more efficiently released during fermentation. In particular embodiments, the biosynthesis of cellulose and / or lignin is modified. Cellulose is the main component of the cell wall. The biosynthesis of cellulose and lignin is co-regulated. By reducing the proportion of lignin in the plant, the proportion of cellulose can be increased. In particular embodiments, the method described herein is used to downregulate lignin biosynthesis in plants, thereby increasing fermentable sugars. More specifically, as disclosed in WO2008064289 A2, the methods described herein downregulate at least a first lignin biosynthetic gene selected from the group consisting of 4-coumarate 3-hydroxylase (C3H), phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyltransferase (HCT), caffeic acid O-methyltransferase (COMT), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl-CoA-reductase (CCR), 4-coumarate-CoA ligase (4CL), monolignol-lignin-specific glycosyltransferase, and aldehyde dehydrogenase (ALDH).

[0336] In particular embodiments, the methods described herein are used to produce plant mass that generates lower levels of acetic acid during fermentation (see also WO2010096488).

[0337] Yeast engineering for biofuel production In detailed embodiments, the RNA-targeting enzymes provided herein are used in the production of bioethanol by recombinant microorganisms. For example, RNA-targeting enzymes can be used to engineer microorganisms, such as yeast, to create biofuels or biopolymers from fermentable sugars and, optionally, to degrade plant-derived lignocellulose obtained from agricultural waste as a source of fermentable sugars. More specifically, the present invention provides methods for using RNA-targeting CRISPR complexes to modify the expression of endogenous genes required for biofuel production and / or to modify endogenous genes that may interfere with biofuel synthesis. More specifically, the methods involve stimulating the expression in a microorganism, such as yeast, of one or more nucleotide sequences encoding enzymes involved in the conversion of pyruvate to ethanol or another product of interest. In detailed embodiments, the methods ensure the stimulation of expression of one or more enzymes, such as cellulases, that enable the microorganism to degrade cellulose. In yet another embodiment, RNA-targeting CRISPR complexes are used to suppress endogenous metabolic pathways that compete with biofuel production pathways.

[0338] Modification of algae and plants for the production of vegetable oils or biofuels Transgenic algae or other plants, such as rapeseed, may be particularly useful for the production of vegetable oils or biofuels, such as alcohols (especially methanol and ethanol). They can be engineered to express or overexpress high levels of oils or alcohols for use in the oil or biofuel industry.

[0339] U.S. Patent No. 8,945,839 describes a method for engineering microalgae (Chlamydomonas reinhardtii cells) using Cas9. Using similar tools, the RNA-targeting CRISPR-based method described herein can be applied to Chlamydomonas species and other algae. In a detailed embodiment, an RNA-targeting effector protein and guide RNA are introduced and expressed in algae using a vector expressing the RNA-targeting effector protein under the control of a constitutive promoter, such as Hsp70A-Rbc S2 or β2-tubulin. The guide RNA will be delivered using a vector containing a T7 promoter. Alternatively, in vitro transcribed guide RNA can be delivered to algae cells. The electroporation protocol follows the standard recommended protocol for the GeneArt Chlamydomonas Engineering Kit.

[0340] Detailed application of RNA targeting enzymes in plants In a detailed embodiment, the present invention can be used as a therapeutic agent for virus elimination in plant systems because it can cleave viral RNA.Previous studies in human systems have demonstrated the successful use of CRISPR in targeting single-stranded RNA viruses, such as hepatitis C (A. Price, et al., Proc. Natl. Acad. Sci, 2015).These methods can also be adapted to use RNA targeting CRISPR systems in plants.

[0341] improved plants The present invention also provides plants and yeast cells obtainable by and obtained by the methods provided herein. Improved plants obtained by the methods described herein may be useful in food or feed production, for example, by modifying the expression of genes that ensure tolerance to plant pests, herbicides, drought, low or high temperatures, excess water, etc.

[0342] Improved plants, particularly crops and algae, obtained by the methods described herein may be useful in food or feed production, for example, due to the expression of higher protein, carbohydrate, nutrient or vitamin levels than would normally be found in the wild-type. Improved plants, particularly pulses and tubers, are preferred in this regard.

[0343] Improved algae or other plants, such as rapeseed, may be particularly useful for the production of vegetable oils or biofuels, such as alcohols (especially methanol and ethanol). They can be engineered to express or overexpress high levels of oils or alcohols for use in the oil or biofuel industry.

[0344] The present invention also provides improved plant parts. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. Plant parts as contemplated herein can be viable, non-viable, regenerable, and / or non-regenerable.

[0345] Also included herein are plant cells and plants produced by the methods of the present invention. Also included within the scope of the present invention are gametes, seeds, embryos (whether zygotic or somatic), progeny, or hybrids of plants containing genetic modifications produced by conventional breeding methods. Such plants may contain heterologous or foreign DNA sequences inserted into or in place of a target sequence. Alternatively, such plants may contain only certain changes (mutations, deletions, insertions, substitutions) in one or more nucleotides. Thus, such plants differ from their progenitor plants only by the presence of a particular modification.

[0346] In some embodiments of the present invention, the Cas13b system is used to engineer pathogen-resistant plants by creating resistance to diseases caused by, for example, bacteria, fungi, or viruses. In certain embodiments, pathogen resistance can be achieved by engineering crops to create a Cas13b system that will be ingested by pests, leading to mortality. In some embodiments of the present invention, the Cas13b system is used to engineer abiotic stress tolerance. In other embodiments, the Cas13b system is used to engineer drought stress tolerance, salt stress tolerance, or cold or heat stress tolerance. Younis et al. 2014, Int. J. Biol. Sci. 10;1150 reviews potential targets for plant breeding methods, all of which are suitable for modification or improvement using the Cas13b system described herein. Some non-limiting target crops include rice (Oryza sativa L.), plum (Prunus domestica L.), cotton (Gossypium hirsutum), Nicotiana rustica, maize (Zea mays), alfalfa (Medicago sativa), tobacco (Nicotiana benthamiana), and Arabidopsis thaliana.

[0347] In one embodiment of the invention, the Cas13b system is used to manage crop pests. For example, a Cas13b system operable in a crop pest can be expressed from a plant host or directly transferred to the target, for example, using a viral vector.

[0348] In one embodiment, the present invention provides a method for efficiently producing homozygous organisms from heterozygous non-human starting organisms. In one embodiment, the present invention is used in plant breeding. In another embodiment, the present invention is used in animal breeding. In such embodiments, homozygous organisms, such as plants or animals, are produced by preventing or suppressing recombination by interfering with at least one target gene involved in double-strand breaks, chromosome pairing, and / or strand exchange.

[0349] Application of Cas13b protein in an optimized functional RNA targeting system In some embodiments, the present invention provides a system for specifically delivering functional components to RNA environments.This can be ensured by using a CRISPR system that includes the RNA targeting effector protein of the present invention, which allows specific targeting of various components to RNA.More specifically, such components include activators or repressors, such as activators or repressors of RNA translation, degradation, etc.The application of this system will be described elsewhere herein.

[0350] In one aspect, the present invention provides a non-naturally occurring or engineered composition comprising a guide RNA comprising a guide sequence capable of hybridizing to a target sequence within a genomic locus of interest in a cell, wherein the guide RNA is modified by the insertion of one or more distinct RNA sequences that bind to an adaptor protein. In a detailed embodiment, the RNA sequence can bind to two or more adaptor proteins (e.g., aptamers), and each adaptor protein is associated with one or more functional domains. The guide RNA of the Cas13b enzyme described herein is shown to be suitable for guide sequence modification. In a detailed embodiment, the guide RNA is modified by the insertion of distinct RNA sequences 5' to the direct repeat, within the direct repeat, or 3' to the guide sequence. When there are two or more functional domains, the functional domains can be the same or different, for example, two identical or two different activators or repressors. In one aspect, the present invention provides a composition as discussed herein, wherein one or more functional domains are added to the RNA-targeting enzyme such that, upon binding to the target RNA, the functional domains are spatially arranged to enable the functional domains to function in their assigned function. In certain aspects, the present invention provides a composition as discussed herein, wherein the composition comprises a CRISPR-Cas complex having at least three functional domains, at least one of which is associated with an RNA targeting enzyme and at least two of which are associated with a gRNA.

[0351] Thus, in certain aspects, the invention provides non-naturally occurring or engineered CRISPR-Cas13b complex compositions comprising a guide RNA as discussed herein and an RNA-targeting enzyme, Cas13b, wherein optionally the RNA-targeting enzyme comprises at least one mutation that results in the RNA-targeting enzyme having 5% or less of the nuclease activity of an enzyme lacking that at least one mutation, and optionally one or more nuclear localization sequences. In particular embodiments, the guide RNA is additionally or alternatively modified (as detailed elsewhere herein) to still ensure binding of the RNA-targeting enzyme, but to prevent cleavage by the RNA-targeting enzyme.

[0352] In particular embodiments, the RNA targeting enzyme is a Cas13b enzyme that has at least 97% or 100% reduced nuclease activity compared to a Cas13b enzyme that does not have the at least one mutation. In certain aspects, the invention provides a composition as discussed herein, wherein the Cas13b enzyme comprises two or more mutations as described elsewhere herein.

[0353] In a specific embodiment, there is provided an RNA targeting system as described herein above, comprising two or more functional domains. In a specific embodiment, the two or more functional domains are heterologous functional domains. In a specific embodiment, the system comprises an adaptor protein that is a fusion protein comprising a functional domain, and the fusion protein optionally comprises a linker between the adaptor protein and the functional domain. In a specific embodiment, the linker comprises a GlySer linker. Additionally or alternatively, one or more functional domains are attached to the RNA effector protein by a linker, optionally a GlySer linker. In a specific embodiment, one or more functional domains are attached to the RNA targeting enzyme at one or both of the HEPN domains.

[0354] In certain embodiments, the present invention provides a composition as discussed herein, wherein one or more functional domains associated with an adaptor protein or RNA targeting enzyme are domains capable of activating or repressing RNA translation. In certain embodiments, the present invention provides a composition as discussed herein, wherein at least one of the one or more functional domains associated with the adaptor protein has one or more activities including methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcription termination factor activity, histone modification activity, DNA integration activity, RNA cleavage activity, DNA cleavage activity, or nucleic acid binding activity, or molecular switch activity, or chemical or light inducibility.

[0355] In some embodiments, the present invention provides a composition as discussed herein, comprising an aptamer sequence. In particular embodiments, the aptamer sequences are two or more aptamer sequences specific to the same adaptor protein. In some embodiments, the present invention provides a composition as discussed herein, wherein the aptamer sequences are two or more aptamer sequences specific to different adaptor proteins. In some embodiments, the present invention provides a composition as discussed herein, wherein the adaptor proteins include MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, and PRR1. Thus, in particular embodiments, the aptamer is selected from binding proteins that specifically bind any one of the adaptor proteins listed above. In some aspects, the invention provides a composition as discussed herein, wherein the cell is a eukaryotic cell. In some aspects, the invention provides a composition as discussed herein, wherein the eukaryotic cell is a mammalian cell, a plant cell, or a yeast cell, whereby the mammalian cell is optionally a mouse cell. In some aspects, the invention provides a composition as discussed herein, wherein the mammalian cell is a human cell.

[0356] In some embodiments, the present invention provides compositions as discussed hereinabove, wherein there are two or more gRNAs, and these gRNAs target different sequences, thereby providing multiplexing when the compositions are used. In some embodiments, the present invention provides compositions wherein there are two or more gRNAs modified by the insertion of distinct RNA sequences that bind to one or more adaptor proteins.

[0357] In some embodiments, the present invention provides a composition as discussed herein, wherein one or more adaptor proteins associated with one or more functional domains are present to bind to distinct RNA sequences inserted into the guide RNA.

[0358] In certain embodiments, the present invention provides a composition as discussed herein, wherein the guide RNA is modified to have at least one non-coding functional loop, e.g., at least one non-coding functional loop is inhibitory, e.g., at least one non-coding functional loop comprises an Alu.

[0359] In certain aspects, the present invention provides methods for modifying gene expression comprising administration to a host or in vivo expression in a host of one or more of the compositions as discussed herein.

[0360] In some embodiments, the invention provides methods as discussed herein, comprising delivery of a composition or a nucleic acid molecule encoding same, wherein said nucleic acid molecule is operably linked to a regulatory sequence and expressed in vivo. In some embodiments, the invention provides methods as discussed herein, wherein in vivo expression is via lentivirus, adenovirus, or AAV.

[0361] In certain aspects, the invention provides a mammalian cell line of a cell as discussed herein, wherein the cell line is optionally a human cell line or a mouse cell line. In certain aspects, the invention provides a transgenic mammalian model, optionally a mouse, wherein the model has been transformed with a composition as discussed herein or is the progeny of said transformant.

[0362] In some embodiments, the present invention provides a nucleic acid molecule encoding a guide RNA or RNA-targeting CRISPR-Cas complex or composition as discussed herein. In some embodiments, the present invention provides a vector comprising a nucleic acid molecule encoding a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, wherein the direct repeats of the gRNA are modified by the insertion of distinct RNA sequences that bind to two or more adaptor proteins, and each adaptor protein is associated with one or more functional domains, or the gRNA is modified to have at least one non-coding functional loop. In some embodiments, the present invention provides a vector comprising a nucleic acid molecule encoding a non-naturally occurring or engineered CRISPR-Cas complex composition comprising a gRNA as discussed herein and an RNA-targeting enzyme, optionally comprising at least one mutation such that the RNA-targeting enzyme has 5% or less of the nuclease activity of the RNA-targeting enzyme without the at least one mutation, and optionally comprising at least one or more nuclear localization sequences. In certain embodiments, the vector may further comprise regulatory elements operable in a eukaryotic cell operably linked to a nucleic acid molecule encoding a guide RNA (gRNA) and / or a nucleic acid molecule encoding an RNA targeting enzyme and / or an optional nuclear localization sequence.

[0363] In one aspect, the present invention provides a kit comprising one or more of the components described above. In some embodiments, the kit comprises a vector system as described above and instructions for use of the kit.

[0364] In certain aspects, the invention provides a method of screening for gain of function (GOF) or loss of function (LOF) or a method of screening for non-coding RNAs or potential regulatory regions (e.g., enhancers, repressors), comprising introducing cells of a cell line as discussed herein or a model as discussed herein that contain or express an RNA targeting enzyme and a composition as discussed herein into cells of the cell line or model, whereby the gRNA comprises either an activator or a repressor, and monitoring the GOF or LOF, respectively, for those cells in which the introduced gRNA comprises an activator or for those cells in which the introduced gRNA comprises a repressor.

[0365] In some embodiments, the present invention provides a library of non-naturally occurring or engineered compositions, each comprising an RNA-targeting CRISPR guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target RNA sequence of interest in a cell, an RNA-targeting enzyme, wherein the RNA-targeting enzyme comprises at least one mutation such that the RNA-targeting enzyme has 5% or less of the nuclease activity of the RNA-targeting enzyme without the at least one mutation, the gRNA being modified by the insertion of a distinct RNA sequence that binds to one or more adaptor proteins, and the adaptor proteins being associated with one or more functional domains, the composition comprising one or more or two or more adaptor proteins, each protein being associated with one or more functional domains, and the gRNA comprising a genome-wide library comprising a plurality of RNA-targeting guide RNAs (gRNAs). In some embodiments, the present invention provides a library as discussed herein, wherein the RNA-targeting RNA-targeting enzyme has a reduced nuclease activity of at least 97% or 100% compared to the RNA-targeting enzyme without the at least one mutation. In some embodiments, the present invention provides a library as discussed herein, wherein the adaptor protein is a fusion protein comprising a functional domain. In some embodiments, the present invention provides a library as discussed herein, wherein the gRNA is not modified by insertion of a distinct RNA sequence that binds to one or more adaptor proteins. In some embodiments, the present invention provides a library as discussed herein, wherein the one or more functional domains are associated with an RNA targeting enzyme. In some embodiments, the present invention provides a library as discussed herein, wherein the cell population of cells is a population of eukaryotic cells. In some embodiments, the present invention provides a library as discussed herein, wherein the eukaryotic cells are mammalian cells, plant cells, or yeast cells. In some embodiments, the present invention provides a library as discussed herein, wherein the mammalian cells are human cells.In an embodiment, the invention provides a library as discussed herein, wherein the population of cells is a population of embryonic stem (ES) cells.

[0366] In some embodiments, the present invention provides a library as discussed herein, wherein the targeting is about 100 or more RNA sequences. In some embodiments, the present invention provides a library as discussed herein, wherein the targeting is about 1000 or more RNA sequences. In some embodiments, the present invention provides a library as discussed herein, wherein the targeting is about 20,000 or more sequences. In some embodiments, the present invention provides a library as discussed herein, wherein the targeting is transcriptome-wide. In some embodiments, the present invention provides a library as discussed herein, wherein the targeting is a panel of target sequences focused on a relevant or desirable pathway. In some embodiments, the present invention provides a library as discussed herein, wherein the pathway is an immune pathway. In some embodiments, the present invention provides a library as discussed herein, wherein the pathway is a cell division pathway.

[0367] In one aspect, the present invention provides a method for generating a model eukaryotic cell comprising a gene whose expression has been altered. In some embodiments, the disease gene is any gene associated with an increased risk of suffering from or developing a disease. In some embodiments, the method comprises: (a) introducing one or more vectors encoding components of the system described hereinabove into a eukaryotic cell; and (b) allowing a CRISPR complex to bind to a target polynucleotide to alter the expression of the gene, thereby generating a model eukaryotic cell comprising an altered gene expression.

[0368] The structural information provided herein allows for investigation of guide RNA interactions with target RNAs and RNA-targeting enzymes, allowing for engineering or altering guide RNA structures to optimize the functionality of the overall RNA-targeting CRISPR-Cas system. For example, the insertion of adaptor proteins capable of binding to RNA allows for the elongation of guide RNAs without conflict with the RNA-targeting protein. These adaptor proteins can further recruit effector proteins or fusions containing one or more functional domains.

[0369] One aspect of the invention is that the above elements are contained in a single composition or in separate compositions that can be advantageously applied to a host to induce a functional effect at the genomic level.

[0370] Those skilled in the art will understand that modifications to the guide RNA that allow for the binding of the adaptor+functional domain but do not allow the adaptor+functional domain to be properly positioned (e.g., due to steric hindrance within the three dimensional structure of the CRISPR complex) are unintended modifications. One or more modified guide RNAs can be modified by the introduction of a separate RNA sequence 5' to the direct repeat, within the direct repeat, or 3' to the guide sequence.

[0371] The modified guide RNA, the inactivating RNA targeting enzyme (with or without functional domains), and the binding protein having one or more functional domains can each be included in a separate composition and administered to the host individually or together. Alternatively, these components can be provided in a single composition for administration to the host. Administration to the host can be carried out using a viral vector (e.g., a lentiviral vector, an adenoviral vector, an AAV vector) known to those skilled in the art or described herein for delivery to the host. As described herein, using different selection markers (e.g., for lentiviral gRNA selection) and concentrations of gRNA (e.g., depending on whether multiple gRNAs are used) can be advantageous to produce improved effects.

[0372] Using the provided compositions, one skilled in the art can advantageously and specifically target single or multiple loci with the same or different functional domains to induce one or more genomic events. The compositions can be applied to a wide variety of methods for screening libraries of cells and in vivo functional modeling (e.g., gene activation and functional identification of lincRNAs; gain-of-function modeling; loss-of-function modeling; use of the compositions of the invention to establish cell lines and transgenic animals for optimization and screening purposes).

[0373] The present invention encompasses the use of the compositions of the invention to establish and utilize conditional or inducible CRISPR RNA targeting events. (See, e.g., Platt et al., Cell (2014), http: / / dx.doi.org / 10.1016 / j.cell.2014.09.014, or the PCT patent publications cited herein, such as WO 2014 / 093622 (PCT / US 2013 / 074667), which are not believed to prior art to the present invention or application.) For example, a target cell may contain an RNA-targeting CRISPR enzyme, conditionally or inducibly (e.g., in the form of a Cre-dependent construct), and / or contain an adapter protein, conditionally or inducibly, and upon expression of the vector introduced into the target cell, the vector is expressed in a manner that induces or creates a condition for RNA-targeting enzyme expression and / or adapter expression in the target cell. By applying the teachings and compositions of the present invention together with known methods for generating CRISPR complexes, inducible gene expression influenced by functional domains is also an aspect of the present invention. Alternatively, an adaptor protein can be provided as a conditional or inducible element along with a conditional or inducible RNA targeting enzyme, providing a useful model for screening purposes, which advantageously requires only minimal design and administration of specific gRNAs for a wide variety of applications.

[0374] Guide RNAs of the present invention containing dead guide sequences In one aspect, the present invention provides guide sequences that are modified to allow successful CRISPR complex formation and target binding while not allowing successful nuclease activity (i.e., no nuclease activity / no indel activity). For purposes of explanation, such modified guide sequences are referred to as "dead guides" or "dead guide sequences." These dead guides or dead guide sequences can be considered catalytically inactive or conformationally inactive with respect to nuclease activity. In fact, dead guide sequences may not sufficiently participate in productive base pairing in terms of their ability to promote catalytic activity or distinguish between on-target and off-target binding activity. Briefly, the assay involves synthesizing a CRISPR target RNA and a guide RNA containing a mismatch with the target RNA, combining them with an RNA-targeting enzyme, analyzing cleavage on a gel based on the presence of bands generated by the cleavage products, and quantifying cleavage based on relative band intensity.

[0375] Accordingly, in a related aspect, the present invention provides a non-naturally occurring or engineered compositional RNA-targeting CRISPR-Cas system comprising a functional RNA-targeting and guide RNA (gRNA) as described herein, wherein the gRNA comprises a dead guide sequence, such that the gRNA is capable of hybridizing to a target sequence such that the RNA-targeting CRISPR-Cas system is guided to a genomic locus of interest within a cell without detectable RNA-cleavage activity of the non-mutant RNA-targeting enzyme of the system. It should be understood that any of the gRNAs of the present invention as described elsewhere herein may be used as gRNAs comprising a dead gRNA / dead guide sequence as described herein below. Any of the methods, products, compositions, and uses as described elsewhere herein are equally applicable with gRNAs comprising a dead gRNA / dead guide sequence as further detailed below. The following detailed aspects and embodiments are provided for further guidance.

[0376] The ability of a dead guide sequence to direct sequence-specific binding of a CRISPR complex to an RNA target sequence can be evaluated by any suitable assay. For example, sufficient components of a CRISPR system to form a CRISPR complex can be provided to a host cell having a corresponding target sequence, including the dead guide sequence to be tested, such as by transfecting a vector encoding the components of the CRISPR sequence, and then the preferential cleavage within the target sequence can be evaluated. For example, the cleavage of a target RNA polynucleotide sequence can be determined in vitro by providing the target sequence, the components of the CRISPR complex, including the dead guide sequence to be tested, and a control guide sequence that is different from the dead guide sequence to be tested, and comparing the binding or cleavage rate at the target sequence between the reaction with the test guide sequence and the control guide sequence. Other assays are also possible and will occur to those skilled in the art. A dead guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of a cell.

[0377] As further described herein, several structural parameters can lead to suitable frameworks for such dead guides. Dead guide sequences are typically shorter than the respective guide sequences that result in active RNA cleavage. In specific embodiments, the dead guides are 5%, 10%, 20%, 30%, 40%, or 50% shorter than the respective guide sequences for the same.

[0378] As described below and known in the art, one aspect of gRNA—RNA targeting specificity—is a direct repeat sequence, which must be appropriately linked to such a guide. Specifically, this implies that the direct repeat sequence is designed according to the origin of the RNA targeting enzyme. Therefore, structural data available for validated dead guide sequences can be used to design Cas13b-specific equivalents. For example, the structural similarity between the orthologous nuclease domains HEPN of two or more Cas13b effector proteins can be used to transfer equivalently designed dead guides. Therefore, the dead guides herein can be appropriately modified in length and sequence to reflect such Cas13b-specific equivalents, thereby enabling successful CRISPR complex formation and binding to the target RNA while simultaneously disallowing successful nuclease activity.

[0379] In the context of this specification and the state of the art, the use of dead guide provides an unexpected and unexpected platform for network biology and / or systems biology in both in vitro, ex vivo and in vivo applications, allowing multiple gene targeting, particularly bidirectional multiple gene targeting.Before using dead guide, addressing multiple targets has been challenging and sometimes impossible.Using dead guide, for example, multiple targets and therefore multiple activities can be addressed in the same cell, the same animal or the same patient.Such multiplexing can occur simultaneously or can occur staggered in a desired time frame.

[0380] For example, dead guides allow the use of gRNAs as a means of gene targeting without nuclease activity, while also providing a means for inducible activation or repression. Guide RNAs containing dead guides can be further modified to include protein adaptors (e.g., aptamers) as described elsewhere herein, which allow for functional placement of elements, particularly gene effectors (e.g., activators or repressors of gene activity), in a manner that allows for activation or repression of gene activity. One example is the incorporation of aptamers, as described herein and in the state of the art. By engineering gRNAs containing dead guides to incorporate protein-interacting aptamers (Konermann et al., "Genome-scale transcription activation by an engineered CRISPR-Cas9 complex," doi:10.1038 / nature14136, incorporated herein by reference), multiple distinct effector domains can be assembled. This can be modeled after natural processes.

[0381] Offer overview In one aspect, the present invention provides a nucleic acid binding system. In situ hybridization between RNA and a complementary probe is a powerful technique. Typically, fluorescent DNA oligonucleotides are used to detect nucleic acids by hybridization. Although increased efficiency has been achieved through certain modifications, such as locked nucleic acids (LNA), there remains a need for efficient and versatile alternatives. The present invention provides an efficient and adaptable system for in situ hybridization.

[0382] In embodiments of the present invention, the terms guide sequence and guide RNA are used interchangeably as in the aforementioned references, such as WO 2014 / 093622 (PCT / US 2013 / 074667). Generally, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any algorithm suitable for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (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 guide sequence is 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. In some embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides in length, or shorter. Preferably, the guide sequence is 10-30 nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence can be assessed by any suitable assay.For example, sufficient components of a CRISPR system to form a CRISPR complex, including the guide sequence to be tested, can be provided to a host cell having a corresponding target sequence, such as by transfection of a vector encoding the components of the CRISPR sequence, followed by evaluation of preferential cleavage within the target sequence, such as by a Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence can be determined in vitro by providing the target sequence, the components of the CRISPR complex, including the guide sequence to be tested, and a control guide sequence that is different from the test guide sequence, and comparing the binding or cleavage rate at the target sequence between reactions with the test guide sequence and the control guide sequence. Other assays are possible and will occur to those skilled in the art. The guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of a cell. Exemplary target sequences include those that are unique to the target genome.

[0383] Generally, and throughout this specification, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends or no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, in which the vector contains virus-derived DNA or RNA sequences for packaging into a virus (e.g., retrovirus, replication-deficient retrovirus, adenovirus, replication-deficient adenovirus, and adeno-associated virus). Viral vectors also include polynucleotides carried by viruses for transfection into host cells. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Vectors that are intended for and result in expression in eukaryotic cells may be referred to herein as "eukaryotic expression vectors." Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0384] A recombinant expression vector can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, meaning that the recombinant expression vector comprises one or more regulatory elements (which can be selected based on the host cell used for expression) operably linked to the nucleic acid sequence to be expressed. Within the scope of a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory element in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0385] The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and polyU sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in specific host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired target tissue, such as muscle, neuron, bone, skin, blood, a specific organ (e.g., liver, pancreas), or a specific cell type (e.g., lymphocyte). Regulatory elements can also direct expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, and this expression may or may not be tissue- or cell-type-specific. In some embodiments, the vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1α promoter.The term "regulatory element" also encompasses enhancer elements such as the WPRE; the CMV enhancer; the R-U5' segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). Those skilled in the art will understand that the design of the expression vector may depend on factors such as the choice of host cell to be transformed and the desired expression level. The vector can be introduced into a host cell, thereby producing transcripts, proteins, or peptides encoded by nucleic acids as described herein, including fusion proteins or peptides (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutants thereof, fusion proteins thereof, etc.).

[0386] Advantageous vectors include lentiviruses and adeno-associated viruses, and the type of vector can also be selected to target specific cell types.

[0387] As used herein, the term "crRNA" or "guide RNA" or "single guide RNA" or "sgRNA" or "one or more nucleic acid components" of a Type V or Type VI CRISPR-Cas locus effector protein includes any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with and direct sequence-specific binding of a nucleic acid targeting complex to the target nucleic acid sequence.

[0388] In certain embodiments, the CRISPR system as provided herein can utilize a crRNA or similar polynucleotide comprising a guide sequence, where the polynucleotide is RNA, DNA, or a mixture of RNA and DNA, and / or the polynucleotide comprises one or more nucleotide analogs. This sequence can comprise any structure, including, but not limited to, the structure of a naturally occurring crRNA, such as a bulge, hairpin, or stem-loop structure. In certain embodiments, the polynucleotide comprising the guide sequence forms a duplex with a second polynucleotide sequence, which can be an RNA or DNA sequence.

[0389] In certain embodiments, chemically modified guide RNAs are utilized. Examples of chemical modifications of guide RNAs include, but are not limited to, the incorporation of 2'-O-methyl (M), 2'-O-methyl 3' phosphorothioate (MS), or 2'-O-methyl 3' thio PACE (MSP) at one or more terminal nucleotides. Such chemically modified guide RNAs can have higher stability and activity compared to unmodified guide RNAs; however, on-target versus off-target specificity is unpredictable (see Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi:10.1038 / nbt.3290, published online 29 June 2015). Chemically modified guide RNAs further include, but are not limited to, RNAs with phosphorothioate bonds and locked nucleic acid (LNA) nucleotides containing a methylene bridge between the 2' and 4' carbons of the ribose ring.

[0390] In some embodiments, the degree of complementarity is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any algorithm suitable for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, al...

Claims

1. 1. A method of modifying a target locus of interest in a eukaryotic cell, preferably a mammalian cell, comprising delivering to said locus a non-naturally occurring or engineered composition comprising a Cas13b effector protein, optionally fused to one or more localization signals, and one or more nucleic acid components, wherein at least said one or more nucleic acid components are engineered, said one or more nucleic acid components directing a complex to said target of interest, and said effector protein forming a complex with said one or more nucleic acid components, and said complex binding to said target locus of interest.

2. 1. A non-naturally occurring or engineered composition comprising a Cas13b effector protein, optionally fused to one or more localization signals, and one or more nucleic acid components, wherein at least said one or more nucleic acid components are engineered for use in modifying a target locus of interest in a eukaryotic cell, said one or more nucleic acid components directing a complex to said target, and said effector protein forming a complex with said one or more nucleic acid components, and said complex binding to said target locus of interest.

3. 1. Use of a non-naturally occurring or engineered composition comprising a Cas13b effector protein, optionally fused to one or more localization signals, and one or more nucleic acid components, wherein at least said one or more nucleic acid components are engineered to modify a target locus of interest in a eukaryotic cell, said one or more nucleic acid components directing a complex to said target of interest, and said effector protein forming a complex with said one or more nucleic acid components, and said complex binding to said target locus of interest.

4. 4. The method, composition or use of any one of claims 1 to 3, wherein the Cas13b effector protein is at least 80% homologous or identical to a wild-type Cas13b effector protein of a prokaryote selected from the group consisting of Porphyromonas, Prevotella, Bacteroides, Riemerella, Bergeyella, Alistipes, Myroides, Capnocytophaga and Flavobacterium.

5. The Cas13b effector protein is effective against Porphyromonas gulae, Prevotella sp., Porphyromonas gingivalis, Bacteroides pyogenes, Riemerella anatipestifer, Bergeyella zoohelcum, Prevotella intermedia, Prevotella buccae, Alistipes sp. sp.), Prevotella aurantiaca, Myroides odoratimimus, Capnocytophaga canimorsus, Flavobacterium branchiophilum and Flavobacterium columnare.

6. The Cas13b effector protein may be selected from Porphyromonas gulae Cas13b (accession number WP_039434803), Prevotella sp. P5-125 Cas13b (accession number WP_044065294), Porphyromonas gingivalis Cas13b (accession number WP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession number WP_039428968), Bacteroides pyogenes ... pyogenes Cas13b (accession number WP_034542281), Riemerella anatipestifer Cas13b (accession number WP_004919755), Bergeyella zoohelcum Cas13b (accession number WP_002664492), Prevotella intermedia Cas13b (accession number WP_036860899), Prevotella buccae Cas13b (accession number WP_004343973), Porphyromonas gingivalis Cas13b (accession number WP_012458151), Alistipes sp. ZOR0009 Cas13b (accession number WP_047447901), Prevotella sp. MA2016 Cas13b (accession number WP_036929175), Prevotella aurantiaca aurantiaca Cas13b (accession number WP_025000926), Myroides odoratimimus CCUG 10230 Cas13b (accession number EHO06562), Prevotella intermedia Cas13b (accession number WP_061868553), Capnocytophaga canimorsuscanimorsus Cas13b (accession number WP_013997271), Flavobacterium branchiophilum Cas13b (accession number WP_014084666), Myroides odoratimimus Cas13b (accession number WP_058700060), Flavobacterium columnare Cas13b (accession number WP_065213424), and Prevotella intermedia 6. The method, composition or use of any one of claims 1 to 5, wherein the effector protein is at least 80% homologous or identical to a wild-type Cas13b effector protein selected from the group consisting of: (i) Cas13b (accession number WP_050955369);

7. The Cas13b effector protein may be selected from Porphyromonas gulae Cas13b (accession number WP_039434803), Prevotella sp. P5-125 Cas13b (accession number WP_044065294), Porphyromonas gingivalis Cas13b (accession number WP_053444417), Porphyromonas sp. COT-052 OH4946 Cas13b (accession number WP_039428968), Bacteroides pyogenes ...

7. The method, composition or use of any one of claims 1 to 6, wherein the Cas13b effector protein is at least 80% homologous to a wild-type Cas13b effector protein selected from the group consisting of: S. pyogenes Cas13b (Accession No. WP_034542281) and Riemerella anatipestifer Cas13b (Accession No. WP_004919755).

8. 8. The method, composition or use of any one of claims 1 to 7, wherein the Cas13b effector protein is at least 80% homologous to a wild-type Cas13b effector protein selected from the group consisting of Porphyromonas gulae Cas13b (Accession No. WP_039434803) and Prevotella sp. P5-125 Cas13b (Accession No. WP_044065294).

9. The method, composition or use of any one of claims 1 to 8, wherein the Cas13b effector protein is a protein comprising a sequence having at least 70% sequence identity with one or more of the sequences consisting of DKHXFGAFLNLARHN (SEQ ID NO: 1), GLLFFVSLFLDK (SEQ ID NO: 2), SKIXGFK (SEQ ID NO: 3), DMLNELXRCP (SEQ ID NO: 4), RXZDRFPYFALRYXD (SEQ ID NO: 5) and LRFQVBLGXY (SEQ ID NO: 6).

10. 10. The method, composition or use of any one of claims 1 to 9, wherein said target locus of interest comprises RNA.

11. 11. The method, composition or use of any one of claims 1 to 10, wherein said Cas13b effector protein is fused to at least one localization signal, said localization signal being a nuclear localization signal (NLS) or a nuclear export signal (NES), preferably a NES.

12. 12. The method, composition or use of any one of claims 1 to 11, wherein said modification of said target locus of interest comprises a strand break.

13. 13. The method, composition or use of any one of claims 1 to 12, wherein said Cas13b effector protein is codon-optimized for expression in mammalian cells.

14. 14. The method, composition or use of any one of claims 1 to 13, wherein the Cas13b effector protein is associated with one or more functional domains, and optionally the effector protein contains one or more mutations, optionally within the HEPN domain, such as R116A, H121A, R1177A, H1182A (wherein the amino acid positions correspond to the amino acid positions of the Cas13b protein from Bergeyella zoohelicum ATCC 43767), whereby the complex is capable of delivering an epigenetic modifier or a transcriptional or translational activation or repression signal.

15. The method, composition or use of any one of claims 1 to 14, wherein said functional domain modifies transcription or translation of said target locus.

16. 16. The method, composition or use of any one of claims 1 to 15, wherein said target locus of interest is comprised in a nucleic acid molecule within a cell.

17. The method, composition or use of any one of claims 1 to 16, wherein the modification is in vivo or ex vivo.

18. 18. The method, composition or use of any one of claims 1 to 17, wherein said nucleic acid component, when complexed with said effector protein, is capable of causing sequence-specific binding of said complex to a target sequence in said target locus of interest.

19. 19. The method, composition or use of any one of claims 1 to 18, wherein the nucleic acid component comprises a dual direct repeat sequence.

20. 20. The method, composition or use of any one of claims 1 to 19, wherein the effector protein and the nucleic acid component are provided by one or more polynucleotide molecules encoding the polypeptide and / or the nucleic acid component, the one or more polynucleotide molecules being operably configured to express the polypeptide and / or the nucleic acid component.

21. 21. The method, composition or use of claim 20, wherein the one or more polynucleotide molecules comprise one or more regulatory elements operably configured to express the polypeptide and / or the nucleic acid component, and optionally the one or more regulatory elements comprise a promoter or an inducible promoter.

22. 22. The method, composition or use of claim 20 or 21, wherein said one or more polynucleotide molecules are comprised within one or more vectors.

23. 22. The method, composition or use of claim 20 or 21, wherein the one or more polynucleotide molecules are contained within a vector.

24. 24. The method, composition or use of claim 22 or 23, wherein the one or more vectors comprise a viral vector.

25. 25. The method, composition or use of claim 24, wherein said one or more viral vectors comprise one or more retroviral, lentiviral, adenoviral, adeno-associated or herpes simplex viral vectors.

26. 26. The method, composition or use of claim 20 or 21, wherein the one or more polynucleotide molecules are comprised in a delivery system; or the method, composition or use of claim 22 or 23, wherein the one or more vectors are comprised in a delivery system; or the method, composition or use of any one of claims 1 to 25, wherein the assembled complex is comprised in a delivery system.

27. 27. The method, composition or use of any one of claims 1 to 26, wherein the non-naturally occurring or engineered composition is delivered by a delivery vehicle comprising a liposome, a particle, an exosome, a microvesicle, a gene gun or one or more viral vectors.

28. 28. The method, composition or use of any one of claims 1 to 27, wherein said composition comprises an accessory protein that enhances Cas13b effector protein activity, preferably a csx28 protein.

29. 29. The method, composition or use of any one of claims 1 to 28, wherein said composition comprises an accessory protein, preferably a csx27 protein, that suppresses Cas13b effector protein activity.

30. 30. The method, composition or use of any one of claims 1 to 29, wherein the Cas13b effector protein is linked to at least one functional domain, wherein the Cas13b effector protein comprises one or more mutations in the HEPN domain, and wherein the Cas13b effector protein is truncated at its C-terminus, wherein the truncation is optionally selected from: Δ1053-1090, Δ1026-1090, Δ984-1090, Δ934-1090, Δ884-1090, Δ834-1090, Δ784-1090 and Δ734-1090, which correspond to amino acid positions of a Prevotella sp. P5-125 Cas13b protein.

31. A mammalian cell that has been modified by a method according to any one of the preceding or following claims, or that has been engineered to contain or express, optionally inducibly or constitutively, a composition or component thereof according to any one of the preceding or following claims.

32. The modification is - said cell comprising an alteration in the transcription or translation of at least one RNA product, - the cell comprising an alteration in the transcription or translation of at least one RNA product, wherein the expression of said at least one product is increased, or - the cell comprising an alteration in the transcription or translation of at least one RNA product, wherein the expression of said at least one product is reduced.

32. The mammalian cell of claim 31 ,

33. In vivo or ex vivo use in eukaryotic cells, preferably mammalian cells: - RNA sequence-specific interference, - RNA sequence-specific gene regulation, - screening of RNA or RNA products, or lincRNA or non-coding RNA, or nuclear RNA, or mRNA, - mutagenesis, - Fluorescence in situ hybridization, - Breeding, - in vitro or in vivo induction of cell dormancy, - in vitro or in vivo induction of cell cycle arrest, - reduction of cell growth and / or cell proliferation in vitro or in vivo, - in vitro or in vivo induction of cellular anergy, - in vitro or in vivo induction of cell apoptosis, - in vitro or in vivo induction of cell necrosis, - in vitro or in vivo induction of cell death, or - In vitro or in vivo induction of programmed cell death 31. A non-naturally occurring or engineered composition according to or referred to in any one of claims 1 to 30 for:

34. 33. A cell according to claim 31 or 32, or a cell line comprising the same, or a progeny thereof.

35. 33. A eukaryotic organism, preferably a mammal, comprising one or more cells according to claim 31 or 32.

36. 36. A eukaryotic model, preferably a mammalian model, comprising one or more cells according to claim 31 or 32, said cells optionally inducibly or constitutively expressing a composition or components thereof according to any one of claims 1 to 35.

37. 37. A product from a cell of claim 31 or 32, or a cell line or organism of claim 34 or 35, or a mammalian model of claim 36, wherein said one or more cells of a mammal of said cell line or mammalian model optionally inducibly or constitutively express a composition or components thereof according to any one of claims 1 to 36.

38. An assay, screening method or mutagenesis method comprising the method, composition or use of any one of claims 1 to 30.

39. 31. An RNA based assay, screening or mutagenesis method, wherein the improvement comprises, instead of using RNA, said method comprising using a non-naturally occurring or engineered composition according to any one of claims 1 to 30 or referred to in any one of claims 1 to 30, and optionally said RNA based assay, screening or mutagenesis method is an RNAi or fluorescence in situ hybridization method.

40. 31. A method for modulating translation of a eukaryotic target locus of interest, comprising delivering to said locus a non-naturally occurring or engineered composition according to or mentioned in any one of claims 1 to 30, wherein said Cas13b effector protein is fused to a (heterologous) translational regulator such as a translation activator or a translation repressor and optionally one or more localization signals, at least said one or more nucleic acid components are engineered, said one or more nucleic acid components direct a complex to said target of interest, and said effector protein forms a complex with said one or more nucleic acid components, and said complex binds to said target locus of interest, preferably said heterologous domain is EIF4, such as EIF4E.

41. 1. A method for detecting a target RNA in a sample, comprising: (a) incubating said sample with a non-naturally occurring or engineered composition of matter according to any one of claims 1 to 30 or mentioned in any one of claims 1 to 30 and an RNA-based cleavage-inducible reporter capable of being non-specifically and detectably cleaved by said effector protein; (b) detecting the target RNA based on a signal generated by cleavage of the RNA-based cleavage-inducible reporter. A method comprising:

42. 42. The method of claim 41 , wherein the RNA-based cleavage-inducible reporter construct comprises a fluorescent dye and a quencher.

43. 43. The method of claim 41 or 42, wherein the target RNA comprises pathogen RNA.

44. 44. The method of claim 43, wherein the pathogen comprises a virus, bacterium, fungus, or parasite.

45. 45. The method of any one of claims 41 to 44, comprising a guide RNA designed to detect a single nucleotide polymorphism of a target RNA or a splice variant of an RNA transcript.

46. 46. ​​The method of any one of claims 41 to 45, wherein the guide RNA comprises one or more mismatched nucleotides with the target RNA.

47. 47. The method of any one of claims 41 to 46, wherein the guide RNA binds to a target molecule that is diagnostic for a disease state.

48. 48. The method of claim 47, wherein the disease state comprises cancer.

49. 48. The method of claim 47, wherein the disease state comprises an autoimmune disease.

50. 1. A ribonucleic acid (RNA) detection system comprising: a) a non-naturally occurring or engineered composition according to or referred to in any one of claims 1 to 30, and c) an RNA-based cleavage-inducible reporter capable of being non-specifically and detectably cleaved by said effector protein; A ribonucleic acid (RNA) detection system comprising:

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