Novel type VI CRISPR orthologs and systems

Abstract

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

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 Apr. 12, 2017; U.S. Provisional Patent Application No. 62 / 561,662, filed Sep. 21, 2017; and U.S. Provisional Patent Application No. 62 / 568,129, filed Oct. 4, 2017, each of which is hereby incorporated by reference in its entirety.

[0002] Reference is made to PCT application filed on October 21, 2016, including the specification of PCT / US Patent Application Publication No. 2016 / 058302, which designates, inter alia, the United States. Reference is made to U.S. Provisional Patent Application No. 62 / 245,270 filed on October 22, 2015, U.S. Provisional Patent Application No. 62 / 296,548 filed on February 17, 2016, and U.S. Provisional Patent Application Nos. 62 / 376,367 and 62 / 376,382 filed on August 17, 2016. Further, reference is made to U.S. Patent Application No. 62 / 471,792 filed on March 15, 2017. Further, reference is made to U.S. Provisional Patent Application No. 62 / 471,170 filed on March 17, 2017. Further, reference is made to U.S. Provisional Patent Application No. 62 / 484,791 filed on April 12, 2017. Further, reference is made to U.S. Provisional Patent Application No. 62 / 561,662 filed on 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 are cited. Each of the foregoing applications and citations is hereby incorporated by reference herein.

[0003] All documents cited or referenced in the documents cited in this specification are hereby incorporated by reference into this specification and may be used in the practice of the present invention, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any product in any document mentioned in this specification or incorporated by reference herein. More specifically, all documents referenced are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0004] Description of federally sponsored research This invention was made with government support under grants No. MH100706 and No. MH110049 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] The present invention generally relates to systems, methods, and compositions for use in controlling gene expression that include sequence targeting using a vector system related to clustered regularly interspaced short palindromic repeats (CRISPR) and its components, such as perturbation of gene transcripts or nucleic acid editing. BACKGROUND OF THE INVENTION

[0006] Recent advances in genome sequencing techniques and analytical methods have led to a rapid improvement in the ability to catalog and map genetic factors associated with various biological functions and diseases. To enable systematic reverse engineering of causal genetic mutations by selectively perturbing individual genetic elements and to advance synthetic biology, biotechnology, and medical applications, accurate genome targeting technologies are required. Although genome editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available to generate targeted genome perturbations, there is still a need for new genome and transcriptome engineering technologies that utilize novel strategies and molecular mechanisms, are inexpensive, easy to set up, scalable, and suitable for targeting multiple positions within eukaryotic genomes and transcriptomes. This will be a major resource for new applications in genome engineering and biotechnology.

[0007] CRISPR-Cas systems of bacterial and archaeal adaptive immunity exhibit extremely high diversity with respect to protein composition and genomic locus architecture. The CRISPR-Cas locus has over 50 gene families and no strictly universal genes, suggesting rapid evolution and extremely high diversity of locus architecture. So far, by taking a multi-directional approach, approximately 395 profiles of cas genes for 93 Cas proteins have been comprehensively identified. Included in the classification are signature gene profiles + signatures of locus architecture. A new classification of CRISPR-Cas systems has been proposed, where these systems are roughly divided into two classes: class 1, which has a multi-subunit effector complex, and class 2, which has a single-subunit effector module 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 of putative novel effector proteins and their engineering and optimization are important.

[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 effector 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 RNase under an RNA guide (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 provided 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 proteins and group 30 proteins, and include members that are RNA-interfering and RNA-programmable nucleases that 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] Groups 29 and 30 systems contain a large single effector called Cas13b (about 1100 amino acids in length) and either or neither of two small putative accessory proteins (about 200 amino acids in length, called Csx27 and Csx28) adjacent to the CRISPR array. Based on this adjacent small protein, this system is classified as type VI-B1 (Csx27) or type VI-B2 (Csx28). There are no additional proteins conserved among species with each locus up to 25 kilobase pairs upstream or downstream from the array. Although there are slight exceptions, the CRISPR array contains direct repeat sequences 36 nucleotides in length and spacer sequences 30 nucleotides in length. The direct repeats are generally well conserved, especially at the ends, and the 5'-terminal GTTG / GUUG is reverse complementary to the 3'-terminal CAAC. This conservation suggests strong base pairing towards an RNA loop structure that may interact with proteins within the locus. From a search for motifs complementary to the direct repeats, no candidate tracrRNA adjacent to the array has been revealed, and it is a single crRNA as seen in the Cpf1 locus.

[0010] The citation or identification of any document in this application does not admit that such document is available as prior art for the present invention.

Summary of the Invention

Problems 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 hybrids or derivatives thereof) with broad applications, specifically in eukaryotic systems and more specifically in mammalian systems. The present invention addresses this need and provides related advantages. By adding the novel RNA targeting system of the present application to the repertoire of genome, transcriptome, and epigenome targeting technologies, research and perturbation or editing of specific target sites in eukaryotic systems, more specifically mammalian systems (including cells, organs, tissues, or organisms), can be altered through direct detection, analysis, and manipulation. To effectively utilize the RNA targeting system of the present application for RNA targeting without harmful effects, it is critically important to understand the aspects of engineering and optimization of these RNA targeting tools.

Means for Solving the Problems

[0012] Class 2 type VI-B effector protein Cas13b is an RNA-guided RNase that can be efficiently programmed to degrade ssRNA. The inventors have performed screening to identify several representative Cas13b orthologs from different species and determined the effectiveness of these orthologs in the eukaryotic cell environment. In various embodiments, the present invention refers to, includes, or utilizes a type VI-B CRISPR-Cas effector protein or a Cas13b effector protein and a nucleic acid encoding such a protein.

[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 identical or the same as 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 at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or the same as a wild-type Cas13b effector protein of a prokaryote 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, Flavobacterium branchiophilum and Flavobacterium columnare.In a preferred embodiment, the effector protein is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or the same as a wild-type Cas13b effector protein 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 anatipestifer Cas13b (accession number WP_004919755).The most preferred effector proteins are wild-type Cas13b effector proteins 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), and Porphyromonas sp. COT-052 OH4946 Cas13b (accession number WP_039428968), and are at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or the same as these, and most particularly preferred are Porphyromonas gulae Cas13b (accession number WP_039434803) or Prevotella sp. P5-125 Cas13b (accession number WP_044065294). Each of these Cas13b effector proteins and other complete amino acid sequences are provided in Figure 1.

[0014] In some embodiments, the Cas13b effector protein: (a) contains 900-1800 amino acids and two HEPN domains; (b) is naturally present within 10 kb upstream or downstream of the CRISPR array in a prokaryotic genome; (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) there is no Cas1 gene or Cas2 gene 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 when 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 for identifying consensus sequences. In a detailed embodiment, 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 a further detailed embodiment, the Cas13b effector protein comprises a sequence having at least 70% sequence identity with at least 2, 3, 4, 5, or all 6 of these sequences. In a further detailed embodiment, the sequence identity with these sequences is at least 75%, 80%, 85%, 90%, 95%, or 100%. In a further detailed embodiment, 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 a further detailed embodiment, 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 synonymously and will be understood to refer to the CRISPR effector proteins further described in this application by analogy whenever referred to herein, unless specifically indicated otherwise, such as by specific reference to Cas9.

[0017] In embodiments of the present invention, the 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 present invention, the 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 suppresses 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 comprising: a) a guide sequence having the ability to hybridize 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 of 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 when the guide sequence specifically hybridizes to the target RNA sequence involves interaction (recognition) of the protospacer flanking sequence (PFS).

[0020] In some embodiments, the non-naturally occurring or engineered compositions of the invention can 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 the csx28 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.

[0021] In some embodiments, the non-naturally occurring or engineered compositions of the invention include a type VI-B CRISPR-Cas accessory protein that suppresses type VI-B CRISPR-Cas effector protein activity. In certain such embodiments, the accessory protein that suppresses type VI-B CRISPR-Cas effector protein activity is the 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 include two or more type VI-B CRISPR-Cas crRNAs.

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

[0024] In certain embodiments, the method utilizes a chemically modified guide RNA. 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'-thioPACE (MSP) at one or more terminal nucleotides. Such chemically modified guide RNAs can include an increase in stability and an increase in activity when compared to unmodified guide RNAs, although 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 that include phosphorothioate linkages and locked nucleic acid (LNA) nucleotides that include 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 includes one or more nuclear localization signals (NLSs).

[0026] Unlike the catalytic mechanisms of other known RNases found in the CRISPR-Cas system, Cas13b achieves RNA cleavage by conserved basic residues within its two HEPN domains. Mutations in the HEPN domain, such as substitution of any of the four predicted HEPN domain catalytic residues (e.g., alanine), can convert Cas13b into an inactive programmable RNA-binding protein (dCas13b similar to dCas9).

[0027] If dCas13b can bind to the specified sequence, by using this ability in several aspects of the present invention, (i) an effector module can be brought to a specific transcript to regulate function or translation (which may be used for large-scale screening, construction of synthetic regulatory circuits, and other purposes), (ii) a fluorescent tag can be added to a specific RNA to visualize its transport and / or localization, (iii) RNA localization can be changed by a domain with affinity for a specific intracellular compartment, and (iv) a specific transcript can be captured (either by directly pulling down dCas13b or by localizing biotin ligase activity to the specific transcript using dCas13b) to enrich for proximity molecular partners including RNA and proteins.

[0028] There should also be many applications for active Cas13b. Some aspects of the present invention include targeting specific transcripts for destruction. In addition, Cas13b can cleave other (non-complementary) RNA molecules in vitro and inhibit cell growth in vivo when primed by its cognate target. Biologically, this promiscuous RNase activity may reflect the programmed cell death / dormancy (PCD / D)-based defense mechanism of the type VI-B CRISPR-Cas system. Thus, in some aspects of the present invention, it may be used to induce PCD or dormancy in specific cells - for example, cancer cells expressing a specific transcript, a given class of neurons, cells infected with a specific pathogen, or other abnormal cells or cells whose presence is otherwise undesirable.

[0029] The present invention provides a method for modifying a nucleic acid sequence associated with or present at a target locus of interest, particularly in a eukaryotic cell, tissue, organ or organism, more particularly in a mammalian cell, tissue, organ or organism, 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, when the complex binds to the target locus of interest, the effector protein induces modification of a sequence associated with or present 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 present at the target locus of interest comprises RNA and the effector protein is encoded by a type VI-B CRISPR-Cas locus. The complex may be formed in vitro or ex vivo and introduced into cells or contacted with RNA or may be formed in vivo.

[0030] The present invention provides a method for targeting (such as modifying) a sequence associated with or present at a target locus of interest, the method comprising delivering a non-naturally occurring or engineered composition comprising a Cas13b locus effector protein (which can be catalytically active or alternatively catalytically inactive) and one or more nucleic acid components to the sequence associated with or present at the locus, wherein the Cas13b effector protein forms a complex with the one or more nucleic acid components and, when the complex binds to the target locus of interest, the effector protein induces modification of the sequence associated with or present 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, preferably an engineered or non-naturally occurring nucleic acid component. The complex can be formed in vitro or ex vivo and introduced into cells, or contacted with RNA, or formed in vivo. Induction of modification of the sequence associated with or present at the target locus of interest can be under a Cas13b effector protein-nucleic acid guide. In a preferred embodiment, the one nucleic acid component is a CRISPR RNA (crRNA). In a preferred embodiment, the 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 derivatives thereof. In a preferred embodiment, the spacer sequence or a derivative thereof comprises a seed sequence, wherein the seed sequence is critically important for recognition and / or hybridization with the sequence at the target locus.

[0031] Aspects of the present invention relate to Cas13b effector protein complexes having one or more nucleic acid components that are not naturally occurring, or are engineered, or modified, or optimized. In preferred embodiments, the nucleic acid component of the complex can comprise a guide sequence linked to a direct repeat sequence, where the direct repeat sequence comprises one or more stem loops or an optimized secondary structure. In one embodiment, the direct repeat sequence can be about 36 nucleotides in length. In specific embodiments, the direct repeat comprises GTTG / GUUG at the 5' end that is reverse complementary to CAAC at the 3' end. In certain embodiments, the direct repeat has a minimum length of 16 nt, such as 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, such as at least 28 nt, and has two or more stem loops or an optimized secondary structure. In detailed embodiments, the direct repeat is 25 nt or more, such as 26 nt, 27 nt, 28 nt or more, and has one or more stem loop structures. In preferred embodiments, the direct repeat can be modified to include one or more protein-binding RNA aptamers. In preferred embodiments, the direct repeat can be modified to include one or more protein-binding RNA aptamers. In preferred embodiments, one or more aptamers can be included as part of an optimized secondary structure. Such aptamers can have the ability to bind to bacteriophage coat proteins. The bacteriophage coat proteins can be selected from the group consisting of Qβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s and PRR1. In preferred embodiments, the bacteriophage coat protein is MS2. The present invention also provides nucleic acid components of the complex that are 30 or more, 40 or more, or 50 or more nucleotides in length.

[0032] The present invention provides a cell comprising a Cas13b effector protein that is transiently expressed or introduced, and / or a guide, and / or a complex thereof, and a cell comprising a complex of the Cas13b effector protein, and / or a guide, and / or a target nucleic acid. 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 of modifying a target locus of interest, specifically in a eukaryotic cell, tissue, organ, or organism, more specifically in a mammalian cell, tissue, organ, or organism, the method 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 when the complex binds 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 cells, or contacted with RNA, or formed in vivo.

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

[0035] In such a method, the target locus of interest may be contained in a nucleic acid molecule within a cell, specifically within 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 cell (e.g., a chicken), a vertebrate fish cell (e.g., a salmon), or a crustacean cell (e.g., a oyster, clam, lobster, or shrimp). The plant cell can be a crop plant, such as a cassava, corn, sorghum, wheat, or rice. The plant cell can also be an algae, a tree, or a vegetable. The modification introduced into the cell according to the present invention can be such that it changes the cell and its progeny for the purpose of improving the production of biological products, such as antibodies, starch, alcohol, or other desired cell products. The modification introduced into the cell according to the present invention can be such that it includes changes in the cell and its progeny that alter the biological product produced.

[0036] The mammalian cell can be a non-human mammal, such as a primate, bovine, ovine, porcine, canine, rodent, leporidae (e.g., a monkey, a female bovine, an ovine, a porcine, a canine, a rabbit, a rat, or a murine cell). The cell can be a non-mammalian eukaryotic cell, such as a poultry cell (e.g., a chicken), a vertebrate fish cell (e.g., a salmon), or a crustacean cell (e.g., a oyster, clam, lobster, or shrimp). The cell can also be a plant cell. The plant cell can be a monocotyledonous or dicotyledonous plant cell, or a crop or cereal plant cell, such as a cassava, corn, sorghum, soybean, wheat, oat, or rice. The plant cell can also be an algae, a tree, or a production plant, a fruit, or a vegetable cell (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 solanaceous plant; a brassica plant; a lactuca plant; a spinach 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 for 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, when the complex binds to the target locus of interest, the effector protein induces modification of the target locus of interest. In a preferred embodiment, the modification is introduction of a strand break.

[0038] The present invention also provides a method for modifying a target locus of interest, the method 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, when the complex binds to the target locus of interest, the effector protein induces modification of the target locus of interest. In a preferred embodiment, the modification is introduction of a strand break.

[0039] In such methods, the target locus of interest can be contained in a nucleic acid molecule in vitro. In such methods, the target locus of interest can be contained in a nucleic acid molecule within a cell. Preferably, in such methods, the target locus of interest can be contained in an RNA molecule in vitro. Also preferably, in such methods, the target locus of interest can be contained in an RNA molecule within a cell. The cell can be a prokaryotic cell or a 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 methods described, the target locus of interest can be a target genomic or epigenomic locus. In any of the methods described, the complex can be delivered with multiple guides for multiplexed use. In any of the methods described, two or more proteins can be used.

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

[0042] In any of the methods described, the effector protein and the 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 include 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 methods described, the target locus of interest may be a genomic or epigenomic locus of interest. In any of the methods described, the complex may be delivered with multiple guides for multiplexed applications. In any of the methods described, two or more proteins may be used.

[0043] The regulatory element may include an inducible promoter. The polynucleotide and / or vector system may include 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, a 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 features as considered herein or are compositions defined by any of the methods described herein.

[0047] In certain embodiments, therefore, the present invention provides non-naturally occurring or engineered compositions, such as compositions that have the ability to modify or are configured to modify a particular 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, when the complex binds to the locus of interest, the effector protein induces modification of the target locus of interest. In certain embodiments, the effector protein can 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 that have the ability to modify or are configured to modify a particular target locus of interest, said compositions comprising (a) a guide RNA molecule (or a combination of guide RNA molecules, such as a first guide RNA molecule and a second guide RNA molecule, for example, for multiplexing) or a nucleic acid encoding a guide RNA molecule (or one or more nucleic acids encoding a combination of guide RNA molecules), (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 certain embodiments, the effector protein can 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 that has 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 that has the characteristics as contemplated herein or as defined in any of the methods described herein.

[0052] The present invention also provides a non - naturally occurring or engineered composition, or one or more polynucleotides encoding components of said composition, or a vector or delivery system comprising one or more polynucleotides encoding components of said composition, for use in a therapeutic treatment method. The therapeutic treatment method 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 can be modified, such as engineered or non-naturally occurring effector proteins or Cas13b. In certain embodiments, the modification can include a mutation of one or more amino acid residues of the effector protein. The one or more mutations can be in one or more catalytic domains of the effector protein. The effector protein can have reduced or abolished nuclease activity compared to an effector protein lacking the one or more mutations. The effector protein may not induce cleavage of the RNA strand at the target locus of interest. In preferred embodiments, the one or more mutations can include two mutations. In preferred embodiments, 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 has one or more of the following mutations: R116A, H121A, R1177A, H1182A (where the amino acid positions correspond to the amino acid positions of the Cas13b protein derived from Bergeyella zoohelcum ATCC 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. Those skilled in the art will understand that the corresponding amino acid positions in different Cas13b proteins can be mutated to have the same effect.In certain embodiments, one or more of the mutations R116A, H121A, R1177A, H1182A, 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 completely or partially abolish the catalytic activity of the protein (e.g., changes in cleavage rate, specificity, etc.). In certain embodiments, here, the amino acid positions correspond to the amino acid positions of the Cas13b protein derived from Prevotella sp. P5-125, and 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 both 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 adjacent to the end of the effector protein (e.g., Cas13b effector protein), and in the case of two or more NLSs, each of the two may be located at, near, or adjacent to the end of the effector protein (e.g., Cas13b effector protein). The one or more heterologous functional domains may comprise one or more transcriptional activation domains.In a preferred embodiment, the transcriptional activation domain may include VP64. The one or more heterologous functional domains may include one or more transcriptional repression domains. In a preferred embodiment, the transcriptional repression domain may include a KRAB domain or a SID domain (e.g., SID4X). The one or more heterologous functional domains may include one or more nuclease domains. In a preferred embodiment, the nuclease domain includes 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, translational activation activity, translational repression activity, transcriptional activation activity, transcriptional 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 of the heterologous functional domains may be at or near the amino terminus of the effector protein, and / or at least one or more of the heterologous functional domains may be at or near the carboxy terminus of the effector protein. The one or more heterologous functional domains may be fused to the effector protein. The one or more heterologous functional domains may be tethered to the effector protein. The one or more heterologous functional domains may 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 include an epitope tag or a reporter. Non-limiting examples of epitope tags include a histidine (His) tag, a V5 tag, a FLAG tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag, and a thioredoxin (Trx) tag. 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 an effector protein comprising an effector protein that is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical 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 further provides an effector protein comprising an effector protein of a wild-type Cas13b of a prokaryotic species 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, Flavobacterium branchiophilum, and Flavobacterium columnare, and an effector protein that is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or the same as the effector protein.The present invention further provides an effector protein comprising a wild-type Cas13b effector protein 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 anatipestifer Cas13b (accession number WP_004919755) and an effector protein that is at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or the same as said effector protein.The most preferred effector proteins are wild-type Cas13b effector proteins 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), and are at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or the same, and most particularly preferred are wild-type Porphyromonas gulae Cas13b (accession number WP_039434803) or Prevotella sp. P5-125 Cas13b (accession number WP_044065294) and are at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or the same. The effector protein may include 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 can be at least 700 amino acids in length. In preferred embodiments, the effector protein is from about 900 to about 1500 amino acids in length, such as from about 900 to about 1000 amino acids in length, from about 1000 to about 1100 amino acids in length, from about 1100 to about 1200 amino acids in length, or from about 1200 to about 1300 amino acids in length, or from about 1300 to about 1400 amino acids in length, or from about 1400 to about 1500 amino acids in length, such as about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700 or about 1800 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 within 10 kb upstream or downstream of the CRISPR array in a prokaryotic genome, (c) is the only protein containing more than 700 amino acids within 10 kb upstream or downstream of the CRISPR array, and / or (d) there is no Cas1 gene or Cas2 gene 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, contains at least one and preferably at least two, such as more preferably exactly two conserved RxxxxH motifs. The catalytic RxxxxH motif is specific to the HEPN (Higher Eukaryote and Prokaryote Nucleotide-binding) domain. Thus, in certain embodiments, the effector protein contains at least one and preferably at least two, such as more preferably exactly two HEPN domains. In certain embodiments, the HEPN domain can have RNase activity. In other embodiments, the HEPN domain can have DNase activity.

[0060] In certain embodiments, a Cas13b effector protein as contemplated herein can be associated with a locus containing short CRISPR repeats that are 30 to 40 bp in length, more typically 34 to 38 bp in length, even more typically 36 to 37 bp in length, such as 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bp in length. In certain embodiments, the CRISPR repeats are long or double repeats that are 80 to 350 bp in length, such as 80 to 200 bp in length, even more typically 86 to 88 bp in length, such as 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. A 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 a type VI-B1 effector, the 5’ PAM is D. See Example 1, Table 2. There are methods 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., a 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 full spacer sequence adjacent to the remainder of the repeat at the 3’ end. In certain embodiments, targeting by an effector protein as described herein may require that there be no homology between the crRNA tag and the target 5’ flanking sequence. This requirement can 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 the true target on the invading nucleic acid from the CRISPR array itself, where the presence of the repeat sequence leads to complete homology with the crRNA tag and prevents autoimmunity.

[0062] In certain embodiments, the Cas13b effector protein can include one or more mutations that engineer it to reduce or eliminate its RNase activity, thereby reducing or eliminating its RNA interference activity. Mutations can also be made to adjacent residues, such as amino acids in the vicinity of those involved in nuclease activity. In some embodiments, one or more putative catalytic nuclease domains are inactivated, and the effector protein complex lacks cleavage activity and functions as an RNA binding complex. In 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 (CRISPR enzyme, Cas13, effector protein) according to the invention as described herein is a catalytically inactive or dead Cas13 effector protein (dCas13). In some embodiments, the dCas13 effector contains a mutation in the nuclease domain. In some embodiments, the dCas13 effector protein is truncated. In some embodiments, to reduce the size of the fusion protein of the Cas13b effector and one or more functional domains while still maintaining its RNA binding function, the C-terminus of the Cas13b effector can be truncated. 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 the amino acid positions of the Prevotella sp. P5-125 Cas13b protein). See Figure 15B.

[0064] In certain embodiments, one or more functional domains are controllable, i.e., inducible.

[0065] In certain embodiments of the 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 invention, the mature crRNA comprises a stem loop, or 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 can be introduced that maintain the RNA duplex of the stem loop, 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 a nucleotide sequence encoding an effector protein codon-optimized for expression in a eukaryote or eukaryotic cell in any of the methods or compositions described herein. In certain embodiments of the invention, the nucleotide sequence encoding the codon-optimized effector protein encodes any of the Cas13b's discussed herein and is codon-optimized for operability in a eukaryotic cell or organism, such as a cell or organism as recited in other parts of this specification, such as, 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 eukaryote, such as a plant.

[0067] In certain embodiments of the invention, at least one nuclear localization signal (NLS) is added to the nucleic acid sequence encoding the Cas13b effector protein. In preferred embodiments, at least one or more C-terminal or N-terminal NLSs are added (thus, the nucleic acid molecule encoding the Cas13b effector protein can include the coding of the NLS, and thus the expressed product will have the NLS added or attached). In certain embodiments of the invention, at least one nuclear export signal (NES) is added to the nucleic acid sequence encoding the Cas13b effector protein. In preferred embodiments, at least one or more C-terminal or N-terminal NESs are added (thus, the nucleic acid molecule encoding the Cas13b effector protein can include the coding of the NES, and the expressed product will have the NES added or attached). In preferred embodiments, C-terminal and / or N-terminal NLSs or NESs are added for optimal expression and nuclear targeting in eukaryotic cells, preferably human cells. In preferred embodiments, the codon-optimized effector protein is Cas13b, and the spacer length of the guide RNA is 15-35 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 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 certain preferred embodiments, the guide RNA direct repeat length is 19 nucleotides.

[0068] The present invention also encompasses methods of delivering multiple nucleic acid components, where each nucleic acid component is specific for a different target locus of interest, thereby modifying multiple target loci of interest. The complex nucleic acid components can include one or more protein-binding RNA aptamers. The one or more aptamers can have the ability to bind to a bacteriophage coat protein. The bacteriophage coat protein can be selected from the group consisting of Qβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, and PRR1. In a preferred embodiment, the bacteriophage coat protein is MS2. The present invention also provides complex nucleic acid components 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, where 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.

[0070] In certain embodiments, modification of the target locus of interest can result in a eukaryotic cell comprising a change in the expression of at least one gene product, a eukaryotic cell comprising a change in the expression of at least one gene product, wherein the expression of at least one gene product is increased, a eukaryotic cell comprising a change in the expression of at least one gene product, wherein the expression of at least one gene product is decreased, or a eukaryotic cell comprising an edited genome.

[0071] In certain embodiments, the eukaryotic cell can 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 can 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 the expressed gene product may be more or less than the amount of the gene product from cells having an altered expression or an unedited genome. In certain embodiments, the gene product may be altered as compared to the gene product from cells having an altered expression or an unedited 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, progeny of said cell lines or organisms comprising such cells. The organism can be a vertebrate, such as a mammal. Alternatively, the organism can be a plant or a fungus.

[0075] In a further aspect, the invention provides a eukaryotic cell comprising a nucleotide sequence encoding a CRISPR system as described herein, which 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 cells. Another aspect provides a multicellular organism comprising one or more of said cells.

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

[0077] In certain embodiments, the eukaryotic cell can 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 can be used for RNA sequence-specific interference, RNA sequence-specific modification or multiplexing of such processes, including expression (including isoform-specific expression), stability, localization, functionality (e.g., ribosomal RNA or miRNA), etc.

[0079] In further embodiments, non-naturally occurring or engineered compositions, vector systems or delivery systems as described herein can be used for RNA detection and / or quantification in a sample, such as a biological sample. In certain embodiments, the RNA detection is in a cell. In one embodiment, 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 having the ability to cleave RNA, ii) a guide RNA having the ability to hybridize to the target RNA, and iii) an RNA-based cleavage-inducible reporter having the ability to be non-specifically and detectably cleaved by the effector protein; (b) detecting the target RNA based on a signal generated by cleavage of the RNA-based cleavage-inducible reporter.

[0080] In certain embodiments, the type VI-B CRISPR-Cas effector protein is a Cas13b effector protein, such as a Cas13b effector protein as described herein. In certain 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 cell sample, such as, without limitation, a plant cell or an animal cell. In certain embodiments of the invention, the target RNA comprises pathogen RNA, including but not limited to target RNA from viruses, bacteria, fungi, or parasites. In certain embodiments, the guide RNA is designed to detect a target RNA containing a single nucleotide polymorphism or a splice variant of an RNA transcript. In certain 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 serves as a diagnostic indicator of a disease state, such as, without limitation, cancer or an immune disease.

[0081] The present invention provides a ribonucleic acid (RNA) detection system comprising: a) a type VI-B CRISPR-Cas effector protein having the ability to cleave RNA; b) a guide RNA having the ability to bind to a target RNA; and c) an RNA-based cleavage-inducible reporter having the ability to be non-specifically and detectably cleaved by the effector protein. Further, the present invention provides a kit for RNA detection comprising: a) a type VI-B CRISPR-Cas effector protein having the ability to cleave RNA; and b) an RNA-based cleavage-inducible reporter having the ability to be 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, a non-naturally occurring or engineered composition, vector system, or delivery system as described herein can be used to create a disease model and / or screening system.

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

[0084] In aspects of the invention, the Cas13b effector proteins or systems as described herein can be used for the treatment, prophylaxis, prevention or inhibition of viral pathogenesis, infection or transmission in a mammalian subject. Aspects of the invention provide a Cas13b CRISPR system for use in the treatment, prophylaxis, inhibition and / or amelioration of viral pathogenesis, infection and / or transmission in a subject, comprising (a) a Cas13b CRISPR effector protein and / or a polynucleotide encoding a 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 polynucleotides encoding one or more guide RNAs. The Cas13b effector protein can be as defined herein, including those relating to the preferred wild-type Ca13b protein and its preferred derivatives and variants.

[0085] In some embodiments, the Cas13b effector proteins or systems as described herein can be used for the treatment, prophylaxis, prevention or inhibition of Lassa virus pathogenesis, infection or transmission in a mammalian subject. Lassa virus is associated 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 as described herein can be used for the treatment, prophylaxis, prevention or inhibition of Ebola virus pathogenesis, infection or transmission in a mammalian subject. Ebola virus is associated with many tissues and cell types including DCs, macrophages, hepatocytes, etc. (see Martines, R.B. et al. 2015. Journal of Pathology).

[0087] In some embodiments, the Cas13b effector protein or system described herein can be used for the treatment, defense, prevention, or suppression of SARS-CoV disease, infection, or transmission in mammalian subjects. SARS-CoV is associated with lung tissues and cells (see To, KF et al. 2004. Journal of Pathology).

[0088] In some embodiments, the Cas13b effector protein or system described herein can be used for the treatment, defense, prevention, or suppression of Zika virus disease, infection, or transmission in mammalian subjects. Zika virus is associated with many tissue 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 protein or system described herein can be used for the treatment, defense, prevention, or suppression of dengue virus disease, infection, or transmission in mammalian subjects. Dengue virus is associated with many tissue and cell types, including DC, macrophages, liver, etc. (see Flipse, J et al. 2016. Journal of General Virology).

[0090] In some embodiments, the Cas13b effector protein or system described herein can be used for the treatment, defense, prevention, or suppression of chikungunya virus disease, infection, or transmission in mammalian subjects. Chikungunya virus is associated with many tissue and cell types, including immune cells, liver, 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 for the treatment, prophylaxis, prevention or suppression of influenza virus pathogenesis, infection or transmission in a mammalian subject. Influenza virus is associated with lung epithelial cells and macrophages (see Medina, R.A. & Garcia-Sastre A. 2011 Nature Reviews).

[0092] In some embodiments, the Cas13b effector proteins or systems described herein can be used for the treatment, prophylaxis, prevention or suppression of HIV virus pathogenesis, infection or transmission in a mammalian subject. HIV virus is associated with T cells and macrophages (see Weiss, R.A. 2002. IUBMB Life.).

[0093] In some embodiments, the Cas13b effector proteins or systems described herein can be used for the treatment, prophylaxis, prevention or suppression of rotavirus virus pathogenesis, infection or transmission in a mammalian subject. Rotavirus virus is associated with intestinal tissue and cells (see Lopez, S&Arias, C.F. 2006. CTMI).

[0094] In some embodiments, the Cas13b effector proteins or systems described herein can be used for the treatment, prophylaxis, prevention or suppression of herpes simplex (HSV-1) pathogenesis, infection or transmission in a mammalian subject. HSV-1 is associated with epithelial cells and neurons (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 for the treatment, prophylaxis, prevention or suppression of 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 for the treatment, prophylaxis, prevention, or inhibition of HBV pathogenesis, infection, or transmission in mammalian subjects. 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 the expressed gene product may be more or less than the amount of the gene product from cells that do not have a change in expression or an edited genome. In certain embodiments, the gene product may be altered compared to the gene product from cells that do not have a change in expression or an edited genome.

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

[0099] Accordingly, it is noted that it is an object of the present invention not to include within the scope of the present invention any previously known product, process for making a product, or method of using a product such that the applicants reserve the right and disclaim any previously known product, process, or method by the disclosure herein. Further, the present invention is intended not to include within the scope of the present invention any product, process for making a product, or method of using a product that does not meet the written description and enablement requirements of the specification 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) such that the applicants reserve the right and disclaim any previously described product, process, or method by the disclosure herein. It may be advantageous to comply with Article 53(c) EPC and Rules 28(b) and (c) EPC in the practice of the present invention. Nothing herein is to be construed as a promise.

[0100] In this disclosure, and particularly in the claims and / or paragraphs, the terms "comprising," "comprised of," "including," etc. may have the meaning ascribed to them in U.S. patent law. For example, these may mean "including," "included," "containing," etc., and the terms "consisting essentially of" and "consisting essentially of" have the meaning ascribed to them in U.S. patent law, for example, these terms allow for elements not explicitly recited, but it is noted that elements found in the prior art or elements that affect the basic or novel characteristics of the invention are excluded.

[0101] The novel features of the invention are set forth in detail in the appended claims. A further understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description which illustrates exemplary embodiments in which the principles of the invention are utilized and the accompanying drawings thereof.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

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

[0125] Generally, a CRISPR-Cas or CRISPR system as used in the aforementioned documents such as International Publication No. WO 2014 / 093622 Pamphlet (Specification of PCT / U.S. Patent Application Publication No. 2013 / 074667) collectively refers to transcripts and other elements involved in the expression of CRISPR-associated ("Cas") genes or leading to their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeats" associated with 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 "RNA" as the term is used herein (e.g., RNA guiding Cas such as Cas9, e.g., CRISPR RNA and trans-activating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)), or other sequences and transcripts from the CRISPR locus. Generally, a CRISPR system is 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 an endogenous CRISPR system).

[0126] When the CRISPR protein is a class 2 type VI-B effector (e.g., a Cas13b effector protein), the tracrRNA is unnecessary. In the engineered systems of the invention, the direct repeat may include naturally occurring sequences or non-naturally occurring sequences. The direct repeats of the invention are not limited to the length and sequence of naturally occurring ones. The direct repeat can be 36 nt in length, however, longer or shorter direct repeats can vary. For example, the direct repeat can be 30 nt or more, such as 30 - 100 nt or more. For example, the direct repeat can be 30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt, 90 nt, 100 nt in length or more. In some embodiments, the direct repeats of the invention can include a synthetic nucleotide sequence inserted between the 5' and 3' ends of a naturally occurring direct repeat. In certain embodiments, the inserted sequence can be self-complementary, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% self-complementary. Further, the direct repeats of the invention can include nucleotide insertions (for association with functional domains), such as sequences that bind to an aptamer or an adapter 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 connection with the formation of the CRISPR complex, a "target sequence" refers to a sequence that is designed such that the guide sequence has complementarity thereto, where hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. The target sequence can include an RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the direct repeat can be identified in silico by searching for repetitive motifs that meet some or all of the following criteria: 1. Present in the genomic sequence of a 2 Kb window adjacent to the CRISPR locus, 2. Spanning 20 to 50 bp, and 3. Having a spacing of 20 to 50 bp. In some embodiments, two of these criteria, such as 1 and 2, 2 and 3, or 1 and 3, can be used. In some embodiments, all three criteria can be used.

[0128] In embodiments of the present invention, the terms guide sequence and guide RNA, i.e., an RNA having the ability to guide Cas13b to a target genomic locus, are used synonymously as in the aforementioned cited references such as WO 2014 / 093622 pamphlet (Specification of PCT / U.S. Patent Application Publication No. 2013 / 074667). Generally, a guide sequence is any polynucleotide sequence having complementarity with a target polynucleotide sequence sufficient to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or higher when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any algorithm suitable for sequence alignment, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., the Burrows-Wheeler aligner), ClustalW, ClustalX, BLAT, Novoalign (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 nucleotides in length or longer. 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 to 40 nucleotides in length, such as 20 to 30 or 20 to 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 the Cas13b effector is 10 to 30 nucleotides in length, such as 20 to 30 or 20 to 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 the Cas13b effector derived from Bergeyella zoohelcum (such as Bergeyella zoohelcum ATCC 43767) is 10 to 30 nucleotides in length, such as 20 to 30 nucleotides in length, such as 30 nucleotides in length or about 30 nucleotides in length. The ability of the guide sequence to direct sequence-specific binding of the CRISPR complex to the target sequence can be evaluated by any suitable assay. For example, the components of the CRISPR system sufficient to form the CRISPR complex, including the guide sequence to be tested, can be provided to a host cell having the corresponding target sequence, such as by transfection of a vector encoding the components of the CRISPR sequence, and subsequently the preferential cleavage within the target sequence can be evaluated, such as by the Surveyor assay as described herein. Similarly, cleavage of the target polynucleotide sequence can be determined in vitro by providing the components of the CRISPR complex, including the target sequence and the guide sequence to be tested, and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rates at the target sequence between the reaction of the test guide sequence and the control guide sequence. Other assays are possible and will be apparent to those skilled in the art.

[0129] In classical CRISPR-Cas systems, the degree of complementarity between the 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 nucleotides in length or longer, 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 present invention, off-target interactions can be reduced, for example, reducing the interaction of the guide with a target sequence of low complementarity. Indeed, certain natural mutations can distinguish between target sequences and off-target sequences having complementarity of 80% to greater than about 95%, such as 83% - 84%, or 88 - 89%, or 94 - 95% (e.g., distinguishing a target having 18 nucleotides from an 18-nucleotide off-target having 1, 2 or 3 mismatches), resulting in a CRISPR-Cas system. Thus, in the context of the present invention, the degree of complementarity between the 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%.The off-target has less than 100%, or 99.9%, or 99.5%, or 99%, or 99%, or 98.5%, or 98%, or 97.5%, or 97%, or 96.5%, or 96%, or 95.5%, or 95%, or 94.5%, or 94%, or 93%, or 92%, or 91%, or 90%, or 89%, or 88%, or 87%, or 86%, or 85%, or 84%, or 83%, or 82%, or 81%, or 80% complementarity between its sequence and the guide, and advantageously, the off-target has 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% complementarity between its sequence and the guide.

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

[0131] The method according to the present invention as described herein involves inducing one or more nucleotide modifications in eukaryotic cells (in vitro, i.e., in isolated eukaryotic cells) as considered herein, including delivering a vector as considered herein to the cells. Such mutations can include the introduction, deletion, or substitution of one or more nucleotides at each target sequence of the cell via a guide RNA or sgRNA. Such mutations can include the introduction, deletion, or substitution of 1 to 75 nucleotides at each target sequence of the cell via a guide RNA. Such mutations can 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 at each target sequence of the cell via a guide RNA. Such mutations can include the introduction, deletion, or substitution of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of the cell via a guide RNA. Such mutations can 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 at each target sequence of the cell via a guide RNA. Such mutations can include the introduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of the cell via a guide RNA. Such mutations can include the introduction, deletion, or substitution of 40, 45, 50, 75, 100, 200, 300, 400, or 500 nucleotides at each target sequence of the cell via a guide RNA.

[0132] To minimize toxicity and off-target effects, it may be important to control the concentrations of the delivered Cas mRNA or protein and guide RNA. The optimal concentrations 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 the endogenous CRISPR system, when a CRISPR complex (comprising a guide sequence that hybridizes to a target sequence and forms a complex with one or more Cas proteins) is formed, cleavage occurs at or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs or more from the target sequence), although in the case of RNA targets in particular, it may depend, for example, on secondary structure.

[0134] The nucleic acid molecule encoding Cas is preferably a codon-optimized Cas. Examples of codon-optimized sequences are, in this case, sequences optimized for expression in eukaryotes, such as humans (i.e., optimized for expression in humans) or other eukaryotes, animals or mammals as discussed herein. See, for example, the SaCas9 human codon-optimized sequence of WO 2014 / 093622 (PCT / US2013 / 074667). Although 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 particular cell, such as a eukaryotic cell. Eukaryotic cells can be of a particular organism, such as, but not limited to, mammals including humans or non-human eukaryotes or animals or mammals as discussed herein, such as mice, rats, rabbits, dogs, livestock or non-human mammals or primates, or can be derived therefrom. In some embodiments, methods of modifying the germline gene identity of humans or animals that cause no substantial medical benefit to humans or animals and may cause pain to them, and / or methods of modifying the gene identity of animals, and further animals obtained from such methods, can be excluded. Generally, codon optimization refers to a method of modifying a nucleic acid sequence to enhance expression in a target host cell by replacing at least one codon of a native sequence (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with a codon that is used more frequently or most frequently in the genes of that host cell while maintaining the native amino acid sequence. Different species exhibit a particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage among organisms) often correlates with the translation efficiency of messenger RNA (mRNA), which in turn is thought to depend, among other things, on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of a selected tRNA in a cell generally reflects the codons that are most frequently used in peptide synthesis.Accordingly, based on codon optimization, genes can be adjusted to suit optimal gene expression in a given organism. Codon usage tables are readily available, for example, in the "Codon Usage Database" available at www.kazusa.orjp / codon / , and these tables can be adapted in several ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms are also available for codon-optimizing a particular sequence for expression in a particular host cell, such as Gene Forge (Aptagen; Jacobus, PA). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all codons) in the sequence encoding Cas correspond to the codons most frequently used for a particular amino acid.

[0135] In certain embodiments, the method as described herein may comprise providing Cas transgenic cells in which one or more nucleic acids encoding one or more guide RNAs operably linked intracellularly to a regulatory element comprising a promoter of one or more target genes are provided or introduced. As used herein, the term “Cas transgenic cell” refers to a cell such as a eukaryotic cell in which the Cas gene is genomically integrated. The nature, type or origin of the cell is not particularly limited according to the present invention. Also, the method of introducing the Cas transgene into the cell can be various and can be any method as known in the art. In certain embodiments, the Cas transgenic cells are obtained by introducing the Cas transgene into isolated cells. In certain other embodiments, the Cas transgenic cells are obtained by isolating cells from a Cas transgenic organism. By way of example and without limitation, the Cas transgenic cells as referred to herein may be derived from a Cas transgenic eukaryote such as a Cas knock-in eukaryote. Reference is made to International Publication No. WO 2014 / 093622 pamphlet (PCT / US Patent Application Publication No. US 2013 / 74667) (incorporated herein by reference). The methods of US Patent Application Publication Nos. 20120017290 and 20110265198, assigned to Sangamo BioSciences, Inc., regarding targeting of the Rosa locus can be modified to utilize the CRISPR Cas system of the present invention. The method of US Patent Application Publication No. 20130236946, assigned to Cellectis, regarding targeting of the Rosa locus can also be modified to utilize the CRISPR Cas system of the present invention. As a further example, reference is made to Platt et.al. (Cell; 159(2):440-455(2014)) (incorporated herein by reference) which describes Cas9 knock-in mice. The Cas transgene may further comprise a Lox-Stop-polyA-Lox (LSL) cassette, thereby making Cas expression inducible by Cre recombinase.Alternatively, Cas transgenic cells can be obtained by introducing the Cas transgene into isolated cells. Delivery systems for transgenes are well known in the art. By way of example, the Cas transgene can be delivered, as described elsewhere in this specification, using, for example, vectors (e.g., AAV, adenovirus, lentivirus), and / or particles, and / or nanoparticle delivery in eukaryotic cells.

[0136] One of ordinary skill in the art will understand that cells such as Cas transgenic cells, as referred to herein, in addition to having an integrated Cas gene, can contain additional genomic changes, or mutations resulting from the sequence-specific action of Cas, such as one or more oncogenic mutations, when forming a complex 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 array 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, Cas contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs or NESs near or at the amino terminus, or contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs or NESs near or at the carboxy terminus, or a combination thereof (e.g., zero or at least 1 or more NLSs or NESs at the amino terminus and zero or 1 or more NLSs or NESs at the carboxy terminus). When two or more NLSs or NESs are present, each can be selected independently of the others, and thus a single NLS or NES can be present in two or more copies and / or can be present in combination with one or more other NLSs or NESs present in one or more copies. In a preferred embodiment of the invention, Cas contains up to 6 NLSs. In some embodiments, an NLS or NES is considered to be near the N terminus or C terminus when the nearest amino acid of the NLS or NES is within the range of about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more amino acids along the polypeptide chain from the N terminus or C terminus.Non-limiting examples of NLSs include the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: X), the bipartite NLS of nucleoplasmin 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 sequence of the IBB domain of importin-α, RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: X), the sequences of the myogenic T protein, VSRKRPRP (SEQ ID NO: X) and PPKKARED (SEQ ID NO: X), the sequence of human p53, POPKKKPL (SEQ ID NO: X), the sequence of mouse c-abl IV, SALIKKKKKMAP (SEQ ID NO: X), the sequences of influenza virus NS1, DRLRR (SEQ ID NO: X) and PKQKKRK (SEQ ID NO: X), the sequence of hepatitis delta antigen, RKLKKKIKKL (SEQ ID NO: X), the sequence of mouse Mx1 protein, REKKKFLKRR (SEQ ID NO: X), the sequence of human poly(ADP-ribose) polymerase, KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: X), and the sequence of the steroid hormone receptor (human) glucocorticoid, RKCLQAGMNLEARKTKK (SEQ ID NO: X). Non-limiting examples of NESs include the NES sequence LYPERLRRILT (ctgtaccctgagcggctgcggcggatcctgacc). In general, one or more NLSs or NESs are of sufficient strength to drive the accumulation of detectable amounts of Cas in the nucleus or cytoplasm, respectively, of eukaryotic cells. In general, the strength of the nuclear localization / nuclear export activity can be derived from the number of NLSs / NESs within Cas, the specific NLS or NES used, or a combination of these factors. Detection of accumulation in the nucleus / cytoplasm can be carried out by any suitable technique. For example, a detectable marker can be fused to Cas, thereby visualizing its location within the cell, such as in combination with means for detecting the location of the nucleus (e.g., staining specific for the nucleus, such as DAPI) or the cytoplasm.The cell nucleus can also be isolated from the cell and its contents can then be analyzed by any suitable protein detection method, such as immunohistochemistry, Western blot, or enzyme activity assay. The accumulation in the nucleus can be determined indirectly, such as by an assay for the effect of CRISPR complex formation (e.g., an assay for DNA cleavage or mutation at the target sequence or an assay for gene expression activity that has changed in response to the formation of the CRISPR complex and / or Cas enzyme activity), compared to a control not exposed to Cas or a control exposed to a Cas lacking one or more NLSs or NESs. In certain embodiments, without limitation, other localization tags can be fused to the Cas protein, such as for localizing Cas to specific sites within the cell, such as organelles, for example mitochondria, plastids, chloroplasts, vesicles, Golgi, (nuclear or cell) membranes, ribosomes, nucleosomes, ER, cytoskeleton, vacuoles, centrosomes, nucleosomes, granules, centrioles, etc.

[0138] In certain aspects, the invention relates to vectors for delivering or introducing into cells, for example, Cas and / or RNA having the ability to guide Cas to a target locus (i.e., guide RNA), 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. This is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted to effect replication of the inserted segment. Generally, a vector has the ability to replicate when associated with appropriate control elements. In general, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules, nucleic acid molecules with one or more free ends, nucleic acid molecules without free ends (e.g., circular), nucleic acid molecules containing DNA, RNA, or both, and various other polynucleotides known in the art. Certain vectors are "plasmids," which refer to circular double-stranded DNA loops into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, where the vector contains viral-derived DNA or RNA sequences for packaging into a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus, and adeno-associated virus (AAV)). Viral vectors also include the polynucleotides carried by the virus for transfection of host cells. Certain vectors have the ability to self-replicate in the 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) integrate into the genome of the host cell upon introduction into the host cell and are thereby replicated with the host genome. Further, certain vectors are capable of directing the expression of a gene 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] The recombinant expression vector can contain the nucleic acid of the present invention in a form suitable for the expression of the nucleic acid in a host cell, which means that, namely, the recombinant expression vector contains one or more regulatory elements operably linked to the nucleic acid sequence to be expressed (which can be selected based on the host cell used for expression). Within the scope of the recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element in such a way that expression (for example, in an in vitro transcription / translation system or in the host cell when the vector is introduced into the host cell) of the nucleotide sequence is possible. Regarding recombinant and cloning methods, reference is made to U.S. Patent Application No. 10 / 815,730, published as U.S. Patent Application Publication No. 2004-0171156 A1 on September 2, 2004 (the content of which is hereby incorporated by reference in its entirety herein).

[0140] The vector may contain regulatory elements, such as a promoter. The vector may contain a Cas coding sequence and / or a guide RNA (e.g., sgRNA) coding sequence that may contain a single but at least 3, or 8, or 16, or 32, or 48, or 50, for example, 1-2, 1-3, 1-4, 1-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNAs (e.g., sgRNAs). In a single vector, advantageously, when there are about 16 or fewer RNAs, each RNA (e.g., sgRNA) may have a promoter, and when a single vector provides more than 16 RNAs, one or more promoters may drive the expression of two or more of those RNAs. For example, when there are 32 RNAs, each promoter may drive the expression of 2 RNAs, and when there are 48 RNAs, each promoter may drive the expression of 3 RNAs. With simple arithmetic and well-established cloning protocols and the teachings of the present disclosure, one of ordinary skill in the art can readily implement the present invention with respect to suitable exemplary vectors such as AAV and suitable promoters such as the U6 promoter. For example, the packaging limit of AAV is about 4.7 kb. The length of a single U6-gRNA (+ restriction sites for cloning) is 361 bp. Thus, one of ordinary skill in the art can readily accommodate about 12-16, for example 13, U6-gRNA cassettes in a single vector. This can be assembled by any suitable means, such as the Golden Gate strategy (http: / / www.genome-engineering.org / taleffectors / ) used for TALE assembly. One of ordinary skill in the art can also use the tandem guide strategy to increase the number of U6-gRNAs by about 1.5-fold, for example, from about 12-16, such as 13, to about 18-24, such as about 19. Thus, one of ordinary skill in the art can readily arrive at about 18-24, for example about 19, promoter-RNAs, such as U6-gRNAs, in a single vector, such as an AAV vector.A further means of increasing 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 means of increasing the number of promoter-RNAs in a vector is to express an array of promoter-RNAs separated by cleavable sequences in the introns of the coding sequence or gene, in which case it is advantageous to use a polymerase II promoter, thereby increasing expression and enabling 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 a U6 tandem gRNA targeting up to about 50 genes. Thus, from the knowledge in the art and the teachings of the present disclosure, one of ordinary skill in the art can readily make and use a vector that expresses multiple RNAs or guides under the control of, or operably or functionally linked to, one or more promoters, such as a single vector (particularly with respect to the number of RNAs or guides contemplated herein) without any undue experimentation.

[0141] The coding sequence of the guide RNA and / or the Cas coding sequence can be operably or functionally linked to a regulatory element, such that the regulatory element drives expression. The promoter can be a constitutive promoter, and / or a conditional promoter, and / or 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 promoter and is U6.

[0142] Aspects of the invention relate to the identification and engineering of novel effector proteins related to class 2 CRISPR-Cas systems. In preferred embodiments, the effector protein comprises a single subunit effector module. In further embodiments, the effector protein functions in prokaryotic or eukaryotic cells in in vitro, in vivo or ex vivo applications.

[0143] The term "nucleic acid targeting system", where the nucleic acid is DNA or RNA and in some embodiments can also refer to DNA-RNA hybrids or derivatives thereof, collectively refers to transcripts and other elements involved in the expression of DNA or RNA targeting CRISPR associated ("Cas") genes or that induce their activity, which genes include sequences encoding DNA or RNA targeting Cas proteins and CRISPR RNA (crRNA) sequences and, in some but not all systems, trans-activating CRISPR-Cas system RNA (tracrRNA) sequences, and may include other sequences and transcripts from DNA or RNA targeting guide RNAs or DNA or RNA targeting CRISPR loci. Generally, RNA targeting systems are characterized by elements that promote the formation of DNA or RNA targeting complexes at the site of the target DNA or RNA sequence. In connection with the formation of DNA or RNA targeting complexes, a "target sequence" refers to a DNA or RNA sequence that is designed such that a DNA or RNA targeting guide RNA has complementarity thereto, where hybridization between the target sequence and the RNA targeting guide RNA promotes the formation of the RNA targeting complex. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell.

[0144] In certain embodiments of the invention, the novel RNA targeting system, also referred to herein as the RNA- or RNA targeting CRISPR / Cas or CRISPR-Cas system RNA targeting system, is based on an identified type VI-B Cas protein, which does not require the creation of customized proteins to target specific RNA sequences, but rather a single enzyme can be programmed by an RNA molecule to recognize a specific RNA target, in other words, the enzyme can be recruited to a specific RNA target using the RNA molecule.

[0145] In certain aspects of the invention, a novel DNA targeting system, also referred to herein as the DNA- or DNA-targeting CRISPR / Cas or CRISPR-Cas system RNA targeting system, is based on an identified type VI-B Cas protein, which does not require the creation of customized proteins to target specific RNA sequences, but rather a single enzyme can be programmed by an RNA molecule to recognize a specific DNA target, or in other words, the enzyme can be recruited to the specific DNA target using the RNA molecule.

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

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

[0148] Cas13b nuclease The Cas13b effector protein of the present invention is such a protein as shown in or from Figure 1, or includes it, or consists essentially of it, or consists of it, or is involved in or related to it. The preferred proteins of Figure 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), Bacteroides pyogenes Cas13b (Accession No. WP_034542281), Riemerella anatipestifer Cas13b (Accession No. WP_004919755). The most preferred proteins of Figure 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 particularly preferred is Porphyromonas gulae Cas13b (Accession No. WP_039434803) or Prevotella sp. P5-125 Cas13b (Accession No. WP_044065294).The present invention is intended to provide, relate to, concern with, include, consist essentially of, or consist of a protein as set forth in or shown from this specification, including its mutations or modifications as shown in this specification.

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

[0150] The RNase function in the CRISPR system is 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), bringing great advantages. Accordingly, a CRISPR-Cas system, composition or method for targeting RNA with the present effector protein is provided.

[0151] The target RNA, i.e., the RNA of interest, is the RNA to be targeted by the present invention that leads to the recruitment and binding of an effector protein to the target site of interest on the target RNA. The target RNA can be in any suitable form of RNA. This may include, in some embodiments, mRNA. In other embodiments, the target RNA may include tRNA or rRNA.

[0152] Cas13b guide 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 VI CRISPR-Cas locus effector protein includes any polynucleotide sequence having complementarity with a target nucleic acid sequence sufficient 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 similar polynucleotide containing a crRNA or guide sequence, where the polynucleotide is RNA, DNA, or a mixture of RNA and DNA, and / or the polynucleotide contains one or more nucleotide analogs. This sequence can include any structure, including but not limited to the structure of a native crRNA, such as a bulge, hairpin, or stem-loop structure. In certain embodiments, the polynucleotide containing the guide sequence forms a duplex with a second polynucleotide sequence that can be an RNA or DNA sequence.

[0154] In certain embodiments, the guides of the invention include non-naturally occurring nucleic acids, and / or non-naturally occurring nucleotides, and / or nucleotide analogs, and / or chemical modifications. Non-naturally occurring nucleic acids can include, for example, mixtures of naturally occurring nucleotides and non-naturally occurring nucleotides. Non-naturally occurring nucleotides and / or nucleotide analogs can have modified ribose, phosphate and / or base moieties. In certain embodiments of the invention, the guide nucleic acid includes ribonucleotides and non-ribonucleotides. In one such embodiment, the guide includes one or more ribonucleotides and one or more deoxyribonucleotides. In certain embodiments of the invention, the guide includes one or more non-naturally occurring nucleotides or nucleotide analogs, such as phosphorothioate linkages, nucleotides having boranophosphate linkages, locked nucleic acid (LNA) nucleotides or bridged nucleic acid (BNA) including a methylene bridge between the 2' and 4' carbons of the ribose ring. 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 (me1Ψ), 5-methoxyuridine (5moU), inosine, 7-methylguanosine. Examples of chemical modifications of guide RNA 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 high stability and high 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 by 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 contains 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 some embodiments of the invention, the deoxyribonucleotides and / or nucleotide analogs are incorporated into the engineered guide structure, such as, without limitation, the 5’ and / or 3’ ends, stem-loop regions, and seed regions. In certain embodiments, the modification is not in the 3’-handle of the stem-loop region. Chemical modifications at the 3’-handle of the guide’s stem-loop region may 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, such as 2’-F modifications, are introduced into the seed region. In some embodiments, the 2’-F modification is introduced at the 5’ and / or 3’ end of the guide. In certain embodiments, 3 to 5 nucleotides at the 5’ and / or 3’ end 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 the gene disruption level.In certain embodiments, more than 5 nucleotides at the 5’ and / or 3’ ends 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., 0215, PNAS, E7110-E7111). In some embodiments of the 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, protein or nanoparticle. Such chemically modified guides can be used for the identification or enrichment of cells genetically edited by the CRISPR system (see Lee et al., eLife, 2017, 6:e25312, DOI:10.7554).

[0156] In some embodiments, the modification of the guide is a chemical modification, insertion, deletion, or cleavage. In some embodiments, chemical modifications include, but are not limited to, 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 (me1Ψ), 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 at 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 a minor modification, such as 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, 5 or 10 nucleotides at the 5' end are chemically modified. Such chemical modification of the 5' end of Cas13b CrRNA can improve gene cleavage efficiency. In a specific embodiment, 5 nucleotides at the 5' end are replaced with 2'-fluoro analogs. In a specific embodiment, 10 nucleotides at the 5' end are replaced with 2'-fluoro analogs. In a specific embodiment, 5 nucleotides at the 5' end 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 a portion that is chemically linked or conjugated via a non-phosphodiester bond. In one aspect, the guide comprises, in non-limiting examples, direct repeats and targeting sequences that are chemically linked or conjugated via a non-nucleotide loop. In some embodiments, these portions are joined via a non-phosphodiester covalent linker. Examples of covalent linkers include, but are not limited to, carbamates, ethers, esters, amides, imines, amidines, aminotrizines, hydrozones, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides, ureas, thioureas, hydrazides, oximes, triazoles, photocleavable linkers, Diels-Alder cycloaddition pairs or ring-closing metathesis pairs, and chemical moieties selected from the group consisting of C-C bond-forming groups 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 include functional groups appropriate for ligation (Hermanson, G.T., 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 the two oligonucleotides. Examples of chemical bonds include, but are not limited to, carbamates, ethers, esters, amides, imines, amidines, aminotrizines, hydrozone, disulfides, thioethers, thioesters, phosphorothioates, phosphorodithioates, sulfonamides, sulfonates, fulfones, sulfoxides, ureas, thioureas, hydrazides, oximes, triazoles, photocleavable linkages, C-C bond forming groups such as Diels-Alder cycloaddition pairs or ring-closing metathesis pairs, and those based on Michael reaction pairs.

[0160] In some embodiments, one or more portions of the guide can be chemically synthesized. In some embodiments, chemical synthesis uses 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'-thiocarbamate (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, the guide portion can be covalently linked using various bioconjugation reactions, loops, crosslinks, and sugar modifications, internucleotide phosphate diester 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 a Huisgen 1,3-dipolar cycloaddition reaction involving an alkyne and an azide that results in 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 guide moiety and the 3'-azide guide moiety can be protected with a 2'-acetoxyethyl orthoester (2'-ACE) group, which can subsequently be 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 portion can be covalently linked via a linker (e.g., a non-nucleotide loop) that includes moieties such as a spacer, an attachment, a bioconjugate, a chromophore, a reporter group, a dye-labeled RNA, and unnatural nucleotide analogs. More specifically, suitable spacers for the purposes of the present invention include, but are not limited to, polyethers (e.g., polyethylene glycols, polyhydric alcohols, polypropylene glycols, or mixtures of ethylene and propylene glycols), polyamine groups (e.g., spermine, spermidine, and polymeric derivatives thereof), 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 the 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, polysaccharides. Suitable chromophores, reporter groups, 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 pamphlet.

[0164] The linker (e.g., a 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 pamphlet.

[0165] In some embodiments, the degree of complementarity is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or higher 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 transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (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 evaluated by any suitable assay. For example, the components of an RNA targeting CRISPR Cas13b system sufficient to form a nucleic acid targeting complex can be provided to a host cell having the corresponding target nucleic acid sequence, including the guide sequence to be tested, by transfection of a vector encoding the components of the nucleic acid targeting complex, and subsequent preferential targeting (e.g., cleavage) within the target nucleic acid sequence can be evaluated by, for example, the 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 different from the test guide sequence, and comparing the binding or cleavage rates at the target sequence between the reactions of the test guide sequence and the control guide sequence. Other assays are possible and will be apparent to those skilled in the art. A guide sequence, and thus an RNA targeting guide RNA or crRNA, can be selected to target any target nucleic acid sequence. The target sequence can be DNA.The target sequence can be any RNA sequence. In some embodiments, the target sequence is 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 such that the degree of secondary structure within the RNA targeting guide RNA or crRNA is reduced. In some embodiments, about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or less of the nucleotides of the RNA targeting guide RNA are involved 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 that uses the centroid structure prediction algorithm developed by the Institute for Theoretical Chemistry at the University of Vienna (see, for example, A.R. Gruber et al., 2008, Cell 106(1):23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12):1151-62).

[0167] In certain embodiments, the guide RNA or crRNA can comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence. In certain embodiments, the guide RNA or crRNA can 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 can be located upstream (i.e., 5' side) of the guide sequence or spacer sequence. In other embodiments, the direct repeat sequence can be located downstream (i.e., 3' side) of the guide sequence or spacer sequence. In other embodiments, multiple DRs (such as dual DRs) can 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 15-35 nt. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides. In certain embodiments, the spacer length is 15-17 nt, such as 15, 16 or 17 nt, 17-20 nt, such as 17, 18, 19 or 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, such as 24, 25, 26 or 27 nt, 27-30 nt, such as 27, 28, 29 or 30 nt, 30-35 nt, such as 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 can include interfering RNAs from both eukaryotes and prokaryotes, i.e., RNAs involved in the RNA interference pathway, such as shRNA, siRNA, etc. In other embodiments, the target RNA can include microRNA (miRNA). Controlling interfering RNA or miRNA can facilitate reducing off-target effects (OTE) seen in techniques by shortening the lifespan of interfering RNA or miRNA in vivo or in vitro.

[0171] In certain embodiments, the target is not the miRNA itself, but the miRNA binding site of the miRNA target.

[0172] In certain embodiments, the miRNA can be sequestered (such as including being relocated within the cell). In certain embodiments, the miRNA can be cleaved, such as by a hairpin without limitation.

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

[0174] When effector proteins and suitable guides are selectively expressed (e.g., under the control of spatially or temporally suitable promoters, such as tissue-specific or cell cycle-specific promoters and / or enhancers), this can be used to "protect" cells or systems (in vivo or in vitro) from RNAi in those cells. This may be useful in adjacent tissues or cells where RNAi is not required, or for purposes of comparing cells or tissues in which effector proteins and suitable guides are expressed and not expressed (i.e., where RNAi is not controlled and where it is controlled, respectively). Effector proteins can be used to control or bind to molecules that contain or consist of RNA, such as ribozymes, ribosomes or riboswitches. In embodiments of the invention, an RNA guide recruits an effector protein to such molecules, 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 therapies, assays and other applications, without undue experimentation, from this disclosure, to provide a basis for the present application to engineer this system based on information (see, for example, 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;doi:10.3390 / v7042099).

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

[0177] In some embodiments, the effector protein can be an optionally functionalized RNA-binding protein as described herein. In some embodiments, the effector protein can be an RNA-binding protein that cleaves a single strand of RNA. In either case, however, particularly when the RNA-binding protein cleaves a single strand of RNA, ribosome function can be regulated, specifically it can be reduced or destroyed. This can be applied to any ribosomal RNA and any ribosome subunit, and the sequences of rRNA are well known.

[0178] Therefore, control of ribosome activity by use of the present effector protein in combination with a suitable guide for a ribosome target is envisaged. This can be by cleavage of or binding to the ribosome. Specifically, reduction of ribosome activity is envisaged. This can also be useful in in vivo or in vitro assays of ribosome function and as a means of controlling in vivo or in vitro treatments based on ribosome activity. Furthermore, control (i.e., reduction) of protein synthesis in an in vivo or in vitro system is envisaged, and such control includes use as an antibiotic and use in research and diagnosis.

[0179] Riboswitch A riboswitch (also known as an aptazyme) is a regulatory segment of a messenger RNA molecule that binds a small molecule. This typically results in a change in the production of the protein encoded by the mRNA. Therefore, control of riboswitch activity by use of the present effector protein in combination with a suitable guide for a riboswitch target is thus envisaged. This can be by cleavage of or binding to the riboswitch. Specifically, reduction of riboswitch activity is envisaged. This can also be useful in in vivo or in vitro assays of riboswitch function and as a means of controlling in vivo or in vitro treatments based on riboswitch activity. Furthermore, control (i.e., reduction) of protein synthesis in an in vivo or in vitro system is envisaged. Such control can include use as an antibiotic and use in research and diagnosis insofar as it relates to rRNA.

[0180] Ribozyme A ribozyme is an RNA molecule with catalytic properties similar to an enzyme (which is of course a protein). Since ribozymes, both naturally occurring and engineered, contain or consist of RNA, they can similarly be targeted by this RNA-binding effector protein. In some embodiments, the effector protein can be an RNA-binding protein that cleaves the ribozyme, thereby inactivating it. Thus, control of ribozyme activity by use of this effector protein in combination with a suitable guide to a ribozyme target is envisioned. This can be by cleavage or binding to the ribozyme. Specifically, a reduction in ribozyme activity is envisioned. This can also be useful in in vivo or in vitro assays of ribozyme function and as a means to control in vivo or in vitro therapies based on ribozyme activity.

[0181] Gene expression including RNA processing The effector protein, together with a suitable guide, can also be used for targeting gene expression, including by control of RNA processing. Control of RNA processing can include RNA splicing, including alternative splicing by targeting of RNApol; viral replication, including that of plant viroids, satellite viruses, bacteriophages, and retroviruses such as HBV, HBC, and HIV, and other viruses exemplified herein; and RNA processing reactions such as tRNA biosynthesis. The effector protein and suitable guide can also be used for control of RNA activation (RNAa). RNAa leads to promotion of gene expression, and thus control of gene expression can be achieved in the form of disruption or reduction of RNAa, and thus reduced promotion of gene expression. This is considered in more detail below.

[0182] RNAi screen RNAi screening can identify gene products whose knockdown is associated with phenotypic changes, thereby examining biological pathways and identifying components. By using effector proteins and suitable guides to remove or reduce the activity of RNAi in a screen, and thus restore the activity of the gene product (by removing or reducing interference / suppression), control can be exerted over such screens or between screens.

[0183] Satellite RNAs (satRNAs) and satellite viruses can also be processed.

[0184] The control described herein related to RNase activity generally means reduction, negative disruption or knockdown or knockout.

[0185] In vivo RNA application Inhibition of gene expression The target-specific RNases provided herein enable extremely specific cleavage of target RNAs. Interference at the RNA level allows for regulation in a spatial and temporal, and non-invasive manner since the genome is not modified.

[0186] It has been demonstrated that several diseases are treatable by mRNA targeting. Most of those studies relate to the administration of siRNAs, but 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 Naion), 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 for the treatment of RNA virus-mediated diseases such as HIV (targeting of HIV Tet and Rev), RSV (targeting of RSV nucleocapsid) and HCV (targeting of miR-122) (Burnett and Rossi Chem Biol. 2012, 19(1):60-71).

[0188] Furthermore, it is contemplated 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 the transcribed mRNA containing the mutation or the allele-specific sequence. Such specific knockdown is particularly suitable for therapeutic applications related to 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 the apolipoprotein B protein: a short version (ApoB-48) and a longer version (ApoB-100). Some ApoB gene mutations leading to FHBL abnormally shorten both versions of ApoB. Specifically targeting and knocking down the mutated ApoB mRNA transcript with the RNA targeting effector proteins of the present invention may be beneficial in the treatment of FHBL. As another example, Huntington's disease (HD) is caused by the production of an abnormal protein due to an expansion of the CAG triplet repeat in the gene encoding the huntingtin protein. Specifically targeting and knocking down the mutated or allele-specific mRNA transcript encoding the huntingtin protein with the RNA targeting effector proteins of the present invention may be beneficial in the treatment of HD.

[0189] In connection with this and more generally for the 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 two parts of the Cas13b enzyme substantially make up functional Cas13b. Ideally, the split should always be such that the catalytic domain is not affected. 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 mutations in its catalytic domain.

[0190] Each half of split Cas13b can be fused to a dimerization partner. By way of example and not limitation, the use of a rapamycin-sensitive dimerization domain enables the creation of 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 this rapamycin-sensitive dimerization domain can be used to reassemble Cas13b in a controlled form. The two parts of split Cas13b can be considered the N'-terminal part and the C'-terminal part of split Cas13b. This fusion is typically at the split point of Cas13b. In other words, the C'-terminal of the N'-terminal part of split Cas13b fuses to one of the dimer halves while the N'-terminal of the C'-terminal part fuses to the other dimer half.

[0191] Cas13b need not be split in the sense that a cleavage point is newly created. The split points are typically designed in silico and cloned into constructs. Together, the two parts of split Cas13b, the N'-terminal part and the C'-terminal part, preferably comprise at least 70% or more, 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) to form a complete Cas13b. There may be some trimming and mutants are envisioned. Non-functional domains can be completely removed. The important point is that the two parts can be joined together and the desired Cas13b function is restored or returned. The dimer can be a homodimer or a heterodimer.

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

[0193] The RNA targeting effector protein can be further fused to another functional RNase domain, such as a non-specific RNase or Argonaute 2, that synergistically increases RNase activity or ensures further degradation of the message.

[0194] Regulation of gene expression by regulation of RNA function

[0195] Apart from the direct effect on gene expression by cleavage of mRNA, RNA targeting can also be used to affect specific aspects of RNA processing in the cell, which may enable more delicate regulation of gene expression. Generally, the regulation can be mediated, for example, by interfering with the binding of proteins to RNA, such as by blocking protein binding or recruiting RNA-binding proteins. In fact, the regulation can be ensured at various levels, such as mRNA splicing, transport, localization, translation, and metabolic turnover. Similarly, in the context of therapy, it is envisioned that RNA-specific targeting molecules can be used to address (pathogenic) dysfunction at each of these levels. In these embodiments, often, the RNA targeting protein is preferably a "dead" Cas13b that has lost its ability to cleave the RNA target, such as the mutant Cas13b described herein, but maintains its ability to bind to it.

[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 abnormal splicing that leads to loss or gain of function of the expressed gene. Some of these diseases are caused by mutations that cause splicing defects, while many 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, affect exon inclusion or exclusion, and influence the expression of specific isoforms and / or stimulate the expression of alternative protein products. Such applications are described in more 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 targeting a splice site can block splicing at that site and, optionally, redirect splicing to an adjacent site. For example, the 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, which is advantageous for exon skipping of that exon. Alternatively, an RNA-targeting effector protein targeting a splicing enhancer or silencer can interfere with the binding of a trans-acting regulatory splicing factor at the target site and can effectively block or promote splicing. Furthermore, exon exclusion can be achieved by recruiting ILF2 / 3 near the exon of the pre-mRNA by an RNA-targeting effector protein as described herein. As yet another example, a glycine-rich domain can be added for the recruitment of hnRNP A1 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 targeting of a specific splice variant while other splice variants are not targeted.

[0200] In some cases, an RNA-targeting effector protein can be used to promote splicing (e.g., when splicing is defective). For example, for additional splicing, the RNA-targeting effector protein can be associated with an effector capable of stabilizing a splicing regulatory stem-loop. Linking an RNA-targeting effector protein to the consensus binding site sequence of a specific splicing factor can recruit the protein to the target DNA or RNA.

[0201] Examples of diseases associated with abnormal splicing include, but are not limited to, paraneoplastic opsoclonus-myoclonus ataxia (or POMA) caused by loss of the Nova protein that regulates splicing of proteins functioning at synapses and cystic fibrosis caused by splicing defects of the cystic fibrosis transmembrane conductance regulator that result in production of non-functional chloride channels. In other diseases, abnormal RNA splicing results in a gain of function. This includes, for example, myotonic dystrophy caused by CUG triplet repeat expansions (50 to >1500 repeats) in the 3’ UTR of mRNA that cause splicing defects.

[0202] By using an RNA targeting effector protein to recruit a splicing factor (such as U1) to the 5’ splicing site to promote excision of the intron around the desired exon, the exon can be excluded. Such recruitment may 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 an RNA targeting effector protein can be used to block the splicing machinery at a desired locus, thereby preventing exon recognition and expression of another protein product. An example of a disorder that can be treated is Duchenne muscular dystrophy (DMD) caused by mutations in the gene encoding the dystrophin protein. Almost all DMD mutations lead to frameshifts and disrupt dystrophin translation. By combining an RNA targeting effector protein with a splice junction or exon splicing enhancer (ESE), exon recognition can be prevented, thereby resulting in translation of a partially functional protein. This converts the lethal Duchenne phenotype to the less severe Becker phenotype.

[0204] B) RNA modification

[0205] RNA editing is a natural process in which small modifications of RNA increase the diversity of gene products of a given sequence. Typically, this modification involves the conversion of adenosine (A) to inosine (I), which results in an RNA sequence different from that encoded by its genome. RNA modification is generally ensured by ADAR enzymes, according to which the pre-RNA target forms an imperfect double-stranded RNA by base pairing between the exon containing the adenosine to be edited and the intron non-coding element. A classical example of A-I editing is the glutamate receptor GluR-B mRNA, according to which the conductance properties of the channel will be modified by this change (Higuchi M, et al. Cell. 1993;75:1361-70).

[0206] According to the present invention, enzymatic techniques are used to induce a transition (change in A⇔G or C⇔U) or a transversion (any purine to any pyrimidine or vice versa) in the RNA bases of a given transcript. The transition can be directly induced using adenosine (ADAR1 / 2)) or cytosine deaminase (APOBEC, AID), which convert A to I or C to U, respectively. The transversion can be indirectly induced by localizing damage by reactive oxygen species to the base of interest, thereby adding a chemical modification to the affected base, such as the conversion of guanine to oxoguanine. Oxoguanine is recognized as T and thus will base pair with adenine and affect translation. Proteins that can be mobilized for ROS-mediated base damage include APEX and mini-SOG. Both techniques can fuse these effectors to catalytically inactive Cas13b and mobilize them to sites on the transcript where that type of mutation is desired.

[0207] In humans, heterozygous loss-of-function mutations in the ADAR1 gene lead to skin diseases, human hereditary dermatoses (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 an RNA adenosine methyltransferase (N(6)-methyladenosine) can be fused to the RNA targeting effector protein of the present invention and targeted to a transcript of interest. This methyltransferase causes reversible methylation, has a regulatory role, and can affect gene expression and cell fate determination by regulating multiple RNA-related cellular pathways (Fu et al Nat Rev Genet. 2014;15(5):293-306).

[0209] C) Polyadenylation

[0210] Polyadenylation of mRNA is important for nuclear export, translation efficiency, and stability of mRNA, and all of these as well as the polyadenylation process are dependent on specific RBPs. Many eukaryotic mRNAs receive a 3’ poly(A) tail of approximately 200 nucleotides post-transcriptionally. 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 protein provided herein can be used to interfere with or promote the interaction between an RNA-binding protein and RNA.

[0211] An example of a disease associated with a defective protein involved in polyadenylation is oculopharyngeal muscular dystrophy (OPMD) (Brais B, et al. Nat Genet. 1998;18:164-7).

[0212] D) RNA nuclear export After pre-mRNA processing, the mRNA is transported from the nucleus to the cytoplasm. This is ensured by cellular machinery that includes the formation of a carrier complex, which then moves through nuclear pore complexes, releases the mRNA in the cytoplasm, and subsequently the carrier is recycled.

[0213] Overexpression of proteins (such as TAP) that play a role in nuclear export of RNA has been shown to increase the nuclear export of transcripts that are 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. 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 localization elements to the RNA of interest. Effector proteins can be designed to bind to the target transcript and shuttle it to an intracellular location determined by its peptide signal tag. For example, more specifically, the localization of RNA 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 zip code binding protein 1 (ZBP1) that ensures the localization of β-actin 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 the mitochondria), the peroxisome targeting signal (localization to the peroxisome), and the m6A modification / YTHDF2 (localization to the p body). Other envisioned approaches are fusions of RNA-targeting effector proteins with proteins of known localization (such as membranes, synapses).

[0217] Instead, the effector protein according to the present invention can be used, for example, for locus-dependent knockdown. By fusing the effector protein with an appropriate localization signal, the effector is 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, thus enabling locus-dependent knockdown.

[0218] F) Translation

[0219] Translation can be enhanced or suppressed using the RNA targeting effector proteins described herein. Upregulation of translation is envisioned as a very robust way to control cell circuitry. Additionally, for functional studies, protein translation screens may be advantageous compared to transcriptional upregulation screens, which have the drawback that upregulation of transcripts does not lead to an increase in protein production.

[0220] It is envisioned that the translation initiation factor such as EIF4G can be brought in the vicinity of the 5' untranslated repeat (5'UTR) of the target messenger RNA using the RNA targeting effector proteins described herein 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, GLD2 of cytoplasmic poly(A) polymerase can be recruited to the target mRNA by an RNA targeting effector protein. According to this, directed polyadenylation of the target mRNA and thereby stimulation of translation may be possible.

[0221] Similarly, using RNA-targeting effector proteins envisioned herein, translational repressors of mRNA, such as ZBP1, can be blocked (Huttelmaier S, et al. Nature. 2005;438:512-5). Translation can be directly affected by binding to the translation initiation site of the target RNA.

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

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

[0224] Furthermore, using an RNA-targeting effector protein to recruit Caf1, which is a component of the CCR4-NOT deadenylase complex, to the target mRNA can result 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 where translation can be downregulated or upregulated using RNA targeting effector proteins are amyotrophic lateral sclerosis (ALS) and cardiovascular disorders. In ALS motor cortex and spinal cord, reduced levels of the glial glutamate transporter EAAT2 have been reported, and multiple abnormal EAAT2 mRNA transcripts have also been reported in ALS brain tissue. Loss of EAAT2 protein and function is thought to be a major cause of excitotoxicity in ALS. Restoration of EAAT2 protein levels and function could provide a therapeutic benefit. Thus, RNA targeting effector proteins can be beneficially used, for example, to upregulate the expression of EAAT2 protein 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 thought to be anti-atherogenic. It is envisioned that RNA targeting effector proteins can be beneficially used to upregulate the expression of ApoA1, for example, by blocking translational repressors or stabilizing mRNA as described above.

[0226] G) mRNA metabolism turnover

[0227] Translation is closely linked to mRNA metabolism turnover and regulated mRNA stability. Specific proteins have been described as being involved in transcript stability (ELAV / Hu proteins in neurons, Keene JD, 1999, Proc Natl Acad Sci U S A. 96:5-7) and tristetraprolin (TTP) etc. 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 contemplated that the RNA targeting effector proteins of the present invention can interfere with or promote the activity of proteins that play a role in stabilizing mRNA transcripts such that mRNA metabolism turnover is affected. For example, using an RNA targeting effector protein to recruit human TTP to a target RNA may enable 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 proto-oncogenes, nuclear transcription factors, and cytokines, and promote RNA stability. As another example, fusing an RNA targeting effector protein to HuR (Hinman MN and Lou H, Cell Mol Life Sci 2008;65:3168-81), another mRNA stabilizing protein, and recruiting it to a target transcript can extend its lifespan or stabilize short-lived mRNAs.

[0229] Furthermore, it is contemplated that the RNA targeting effector proteins described herein can be used to promote the degradation of target transcripts. For example, recruiting an m6A methyltransferase to a target transcript and localizing the transcript to P bodies can result in target degradation.

[0230] As yet another example, fusing an RNA targeting effector protein as described herein to the non-specific endonuclease domain PilT N-terminal (PIN) can recruit it to a target transcript and enable its degradation.

[0231] Patients with paraneoplastic neurological disorders (PND)-associated encephalomyelitis and neuropathy generate autoantibodies against Hu proteins in tumors outside the central nervous system (Szabo A et al. 1991, Cell.;67:325-33), which then pass through the blood-brain barrier in patients. It is contemplated that the RNA targeting effector proteins of the present invention can interfere with the binding of autoantibodies to mRNA transcripts.

[0232] Patients with myotonic dystrophy type 1 (DM1) caused by the expansion of (CUG)n 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 envisioned that the RNA targeting effector proteins of the present invention, which are fused to an endonuclease targeting (CUG)n repeats, may inhibit the accumulation of such abnormal transcripts.

[0233] H) Interaction with multifunctional proteins

[0234] Some RNA-binding proteins bind to multiple sites on multiple RNAs and function in diverse processes. For example, the hnRNP A1 protein has been shown to bind to exon splicing silencer sequences to antagonize splicing factors, associate with telomere ends (thereby stimulating telomere activity), and bind to miRNAs to promote Drosha-mediated processing, thereby affecting maturation. It is envisioned that the RNA-binding effector proteins of the present invention may 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. The conformational transition between alternative tertiary structures is critically important for many RNA-mediated processes. However, RNA folding can be associated with several problems. For example, RNA may tend to fold into inappropriate alternative conformations and be maintained therein, 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 the folding of (m)RNA 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 proximal ssRNA targets (i.e., the “collateral” or “bystander” effect). Cas13b can cleave other (non-complementary) RNA molecules when primed by cognate targets. Such indiscriminate RNA cleavage can potentially cause cytotoxicity or otherwise affect cellular physiology or cell state.

[0238] Thus, in certain embodiments, the non-naturally occurring or engineered compositions, vector systems or delivery systems as described herein are for use in or for the induction of cell quiescence. In certain embodiments, the non-naturally occurring or engineered compositions, vector systems or delivery systems as described herein are for use in or for the induction of cell cycle arrest. In certain embodiments, the non-naturally occurring or engineered compositions, vector systems or delivery systems as described herein are for use in or for the reduction of cell growth and / or cell proliferation. In certain embodiments, the non-naturally occurring or engineered compositions, vector systems or delivery systems as described herein are for use in or for the induction of cell anergy. In certain embodiments, the non-naturally occurring or engineered compositions, vector systems or delivery systems as described herein are for use in or for the induction of cell apoptosis. In certain embodiments, the non-naturally occurring or engineered compositions, vector systems or delivery systems as described herein are for use in or for the induction of cell necrosis. In certain embodiments, the non-naturally occurring or engineered compositions, vector systems or delivery systems as described herein are for use in or for the induction of cell death. In certain embodiments, the non-naturally occurring or engineered compositions, vector systems or delivery systems as described herein are for use in or for the induction of programmed cell death.

[0239] In certain embodiments, the invention relates to a method of inducing cell dormancy, comprising introducing or inducing a non-naturally occurring or engineered composition, vector system or delivery system as described herein. In certain embodiments, the invention relates to a method of 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 invention relates to a method of 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 invention relates to a method of inducing cell anergy, comprising introducing or inducing a non-naturally occurring or engineered composition, vector system or delivery system as described herein. In certain embodiments, the invention relates to a method of inducing apoptosis, comprising introducing or inducing a non-naturally occurring or engineered composition, vector system or delivery system as described herein. In certain embodiments, the invention relates to a method of inducing necrosis, comprising introducing or inducing a non-naturally occurring or engineered composition, vector system or delivery system as described herein. In certain embodiments, the invention relates to a method 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 invention relates to a method 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 can be therapeutic or prophylactic and can target specific cells, cell (sub)populations or cell types / tissue types. In particular, the methods and uses as described herein can be therapeutic or prophylactic and can target one or more target sequences, such as specific cells, cell (sub)populations or cell types / tissue types that express one or more specific target RNAs (e.g., ssRNA). Without limitation, the target cells can be, for example, cancer cells that express a particular transcript, such as a given class of neurons, such as (immune) cells that cause autoimmunity or cells infected with a specific (e.g., viral) pathogen, etc.

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

[0242] Use of an RNA-targeting effector protein in RNA detection or protein detection Furthermore, it is contemplated that an RNA-targeting effector protein can be used for the detection of nucleic acids or proteins in a biological sample. The sample can be cellular or cell-free.

[0243] Furthermore, it is contemplated that an RNA-targeting effector protein can be used in a Northern blot assay. Northern blotting involves separation of RNA samples by size using electrophoresis. The RNA-targeting effector protein can be used to specifically bind and detect the target RNA sequence.

[0244] RNA targeting effector proteins can also be fused to a fluorescent protein (such as GFP) and used to track RNA localization in living cells. More specifically, the RNA targeting effector protein can be inactivated in that it no longer cleaves RNA. In a detailed embodiment, it is envisioned that split RNA targeting effector proteins may be used to ensure more precise visualization, according to which the signal depends on the binding of both sub-proteins. Alternatively, a split fluorescent protein that is reconstituted when multiple RNA targeting effector protein complexes bind to the target transcript can be used. Furthermore, it is envisioned that the transcript is targeted at multiple binding sites along the mRNA, such that the fluorescent signal can amplify the true signal to enable localized discrimination. As yet another alternative, the fluorescent protein can be reconstituted from a split intein.

[0245] RNA targeting effector proteins are preferably used, for example, for the localization of RNA or specific splice variants, the level of mRNA transcripts, the up- or down-regulation of transcripts, and the determination of disease-specific diagnoses. RNA targeting effector proteins can be used, for example, for the visualization of RNA in (living) cells using fluorescence microscopy or flow cytometry, such as fluorescence-activated cell sorting (FACS) which enables high-throughput screening of cells and the recovery of living cells after cell sorting. Furthermore, the expression levels of various transcripts can be simultaneously evaluated under stress, for example, under inhibition of cancer growth using molecular inhibitors or hypoxic conditions for the cells. Another application can be to track the localization of transcripts to synaptic junctions during nerve stimulation using two-photon microscopy.

[0246] In certain embodiments, the components or complexes according to the invention as described herein can be used, for example, in multiplexed error-robust fluorescence in situ hybridization (MERFISH; Chen et al. Science; 2015; 348(6233)), such as by means of a (fluorescently) labeled Cas13b effector.

[0247] In vitro APEX labeling Cellular processes rely on a network of molecular interactions among proteins, RNAs, and DNAs. Accurate detection of protein-DNA and protein-RNA interactions is important for understanding such processes. In vitro proximity labeling techniques utilize an affinity tag in combination with, for example, a photoactivatable probe to label polypeptides and RNAs near a protein or RNA of interest in vitro. After UV irradiation, the photoactivatable group reacts with proteins and other molecules in close proximity to the tagged molecule, thereby labeling them. The labeled interacting molecules can subsequently be recovered and identified. Using the RNA targeting effector protein of the present invention, for example, a probe can be targeted to a selected RNA sequence.

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

[0249] The present invention provides agents and methods for diagnosing and monitoring health status by non-invasive sampling of cell-free RNA, including risk assays and guidance for RNA-targeted therapies, which are useful in situations where rapid administration of treatment is critical for 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 directly detecting and / or identifying bacterial species from blood or serum, thereby monitoring, for example, disease progression and sepsis. In certain embodiments of the present invention, Cas13b proteins and derivatives are used to distinguish and diagnose common diseases such as rhinovirus infection or upper respiratory tract infection from more severe infections such as bronchitis.

[0250] The present invention provides a method and an agent for rapid gene typing for emergency pharmacogenomics, including a guide for the administration of anticoagulants based on, for example, VKORC1, CYP2C9, and CYP2C19 gene typing during myocardial infarction or stroke treatment.

[0251] The present invention provides an agent and a method for monitoring bacterial contamination of food at any point along the food production and distribution chain. In another embodiment, the present invention provides quality control and monitoring, for example, by identifying food ingredients and determining their purity. In one non-limiting example, the present invention can be used to identify or confirm food ingredients such as animal meat and seafood species.

[0252] In another embodiment, the present invention is used in forensic determinations. For example, crime scene samples containing blood or other body fluids. In certain embodiments of the present invention, nucleic acid samples are identified from fingerprints using the present invention.

[0253] Use of RNA targeting effector proteins in RNA origami / in vitro assembly lines - Combinatorics RNA origami refers to a nanoscale folded structure for creating two-dimensional or three-dimensional structures using RNA as an incorporated template. The folded structure is encoded in the RNA, and thus 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 complexes. Using the RNA targeting effector proteins of the present invention, for example, a target protein can be targeted to RNA origami using a suitable guide RNA.

[0254] Use of RNA targeting effector proteins in RNA isolation or purification, enrichment or depletion Furthermore, it is envisioned that RNA can be isolated and / or purified using an RNA targeting effector protein when complexed with RNA. For example, an affinity tag that can be used for the isolation and / or purification of an RNA-RNA targeting effector protein complex can be fused to the RNA targeting effector protein. Such applications are useful, for example, in the analysis of gene expression profiles in cells. In a detailed embodiment, it can be envisioned that an RNA targeting effector protein can be used to target a specific non-coding RNA (ncRNA), thereby blocking its activity and providing a useful functional probe. In certain embodiments, a specific RNA can be specifically enriched (including, but not limited to, increasing stability, etc.) using an effector protein as described herein, or alternatively, a specific RNA (without limitation, such as a specific splice variant, isoform, etc.) can be specifically depleted.

[0255] Examination of lincRNA function and other nuclear RNAs Current RNA knockdown strategies such as siRNA have the disadvantage that most are limited to targeting cytoplasmic transcripts because the protein machinery is cytoplasmic. The advantage of the RNA targeting effector protein of the present invention, which is an exogenous system not essential for cell 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, it is envisioned that the function of lincRNA can be probed. Long intergenic non-coding RNAs (lincRNAs) are a field of research that has been investigated very extensively. Many lincRNAs have functions that remain unexplained to date, which could be the subject of research using the RNA targeting effector protein of the present invention.

[0256] Identification of RNA-binding proteins The identification of proteins that bind to specific RNAs can be useful for understanding the roles of many RNAs. For example, many lincRNAs are associated with transcriptional and epigenetic regulators in the control of transcription. Understanding which proteins bind to a given lincRNA can facilitate elucidating the components of a given regulatory pathway. To locally label the bound proteins with biotin, the RNA targeting effector proteins of the present invention can be designed to recruit biotin ligase to specific transcripts. Next, the proteins can be pulled down and analyzed by mass spectrometry to identify them.

[0257] Assembly of complexes to RNA and substrate shuttling Furthermore, the RNA targeting effector proteins of the present invention can be used to assemble complexes to RNA. This can be achieved by functionalizing the RNA targeting effector proteins with a plurality of related proteins (e.g., components of a specific synthetic pathway). Alternatively, a plurality of RNA targeting effector proteins can be functionalized with such various related proteins to target the same or adjacent target RNAs. A useful application of the assembly of complexes to RNA is, for example, the promotion of 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, for example, to fuse to split proteins of toxic domains for targeted cell death using cancer-related RNAs as target transcripts. Furthermore, in synthetic biological systems, it is possible to affect pathways involving protein-protein interactions by fusion complexes with appropriate effectors such as kinases or other enzymes.

[0259] Protein splicing: intein 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. Steering the release of split inteins by assembling two or more RNA targeting effector proteins as described herein to a target transcript (Topilina and Mills Mob DNA. 2014 Feb 4;5(1):5), thereby enabling direct calculation regarding the presence of the mRNA transcript and subsequent release of protein products (for downstream action of the transcription pathway) such as metabolic enzymes or transcription factors. This application may have great significance in synthetic biology (see above) or large-scale bioproduction (producing products only under certain conditions).

[0260] Inducible system, administration system, and self-inactivating system In one embodiment, the fusion complex comprising the RNA targeting effector protein and effector component of the present invention is designed to be inducible, for example, photoinducible or chemically inducible. Such inducibility enables activation of the effector component at a desired time point.

[0261] Photoinducibility is achieved, for example, by designing a fusion complex in which the formation of the CRY2PHR / CIBN pair is used for fusion. This system is particularly useful for photoinduction of protein interactions in living cells (Konermann S, et al. Nature. 2013;500:472-476).

[0262] Chemical inducibility is provided, for example, by designing a fusion complex in which the formation of the FKBP / FRB (FK506 binding protein / FKBP rapamycin binding) pair is used for fusion. Use of this system requires rapamycin for protein binding (Zetsche et al. Nat Biotechnol. 2015;33(2):139-42 describes the use of this system for Cas9).

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

[0264] In one embodiment, the delivery of the RNA targeting effector protein of the present invention can be regulated to vary the amount of protein or crRNA in the cell, thereby varying the magnitude of the desired effect or any unwanted off-target effect.

[0265] In one embodiment, the RNA targeting effector proteins described herein can be designed to be self-inactivating. This can inactivate expression and subsequent effects by destroying self-RNA when delivered to cells as RNA, either as mRNA or a replication RNA therapeutic (Wrobleska et al Nat Biotechnol. 2015 Aug;33(8):839-841), thereby reducing persistence and potentially unwanted 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 hereby incorporated by reference. Specifically, in certain embodiments of the invention, the following applications are contemplated in certain embodiments, preferably by using catalytically inactive Cas13b: enhancement of translation (e.g., Cas13b-translation promoting factor fusions (e.g., eIF4 fusions)); suppression of translation (e.g., gRNAs targeting ribosome binding sites); exon skipping (e.g., gRNAs targeting splice donor and / or acceptor sites); exon inclusion (e.g., gRNAs targeted to include specific exon splice donors and / or acceptor sites or Cas13b fused to or recruiting spliceosome components (e.g., U1 snRNA)); access to RNA localization (e.g., Cas13b-marker fusions (e.g., EGFP fusions)); changes in RNA localization (e.g., Cas13b-localization signal fusions (e.g., NLS or NES fusions)); RNA degradation (in this case, if dependent on the activity of Cas13b, catalytically inactive Cas13b is not used and instead split Cas13b may be used for increased specificity); inhibition of non-coding RNA function (e.g., miRNA), such as by degradation of gRNA or binding to functional sites (possibly titrated out at specific sites by relocalization by a Cas13b-signal sequence fusion).

[0267] The Cas13b function is robust to 5’ or 3’ extensions of crRNA and extensions of the crRNA loop. Thus, it is envisioned that MS2 loops and other recruitment domains can be added to crRNA without affecting complex formation and binding to target transcripts. Such modifications to crRNA for recruiting 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. Thus, 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 protein and mutant Cas13b proteins containing one or more of the mutant amino acid residues described herein. Thus, it is envisioned that multiple crRNAs designed for various target transcripts and / or applications can be delivered as a single pre-crRNA or as a single transcript driven by one promoter. Such delivery methods have the advantages of being substantially more compact, easier to synthesize, and easier to deliver in a viral system. For the orthologs of Cas13b herein, the exact amino acid positions may vary and can be appropriately determined by protein alignment as known in the art and as described elsewhere herein. Aspects of the invention also include methods and uses of the compositions and systems described herein for, e.g., changing or manipulating the expression of one or more genes or one or more gene products in in vitro, in vivo, or ex vivo genomic engineering in prokaryotic or eukaryotic cells.

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

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

[0272] In certain embodiments, an effective amount of the Cas13b system is used to cleave the RNA or otherwise inhibit RNA expression. In this regard, this system has a similar use as siRNA and shRNA and thus can also replace such methods. The methods include, without limitation, the use of the Cas13b system as an alternative to, e.g., interfering ribonucleic acids (such as siRNA or shRNA) or their transcription templates, such as DNA encoding shRNA. The Cas13b system is introduced into the target cells, for example, by administration to a mammal containing the target cells.

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

[0274] Destabilizing Cas13b In certain embodiments, an effector protein (CRISPR enzyme; Cas13b) according to the invention as described herein is associated with or fused to a destabilizing domain (DD). In some embodiments, the DD is ER50. The corresponding stabilizing ligand for this DD is 4HT in some embodiments. Thus, in some embodiments, one of 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 for this DD is TMP in some embodiments. Thus, in some embodiments, one of at least one DD is DHFR50, and thus the stabilizing ligand is TMP. In some embodiments, the DD is ER50. The corresponding stabilizing ligand for this DD is CMP8 in some embodiments. Thus, CMP8 can be an alternative stabilizing ligand to 4HT in the ER50 system. It is possible that CMP8 and 4HT can be used competitively / should be used, but some cell types may be more susceptible to the effects of either of these two ligands, and from the present disclosure and knowledge in the art, one of ordinary skill 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 side of the CRISPR enzyme. In some embodiments, at least two DDs associate with the CRISPR enzyme and those DDs are the same DD, i.e., the DDs are homologous. Thus, both (or more than two) of the DDs can be ER50 DDs. This is preferred in some embodiments. Alternatively, both (or more than two) of the DDs can be DHFR50 DDs. This is also preferred in some embodiments. In some embodiments, at least two DDs associate with the CRISPR enzyme and those DDs are different DDs, i.e., the DDs are heterologous. Thus, one of the DDs can be ER50 while one or more or any other of the DDs can be DHFR50. Having two or more heterologous DDs can be advantageous as it can result in a higher level of degradation control. Tandem fusions of two or more DDs at the N-terminus or C-terminus can enhance degradation and such tandem fusions can be, for example, ER50-ER50-Cas13b or DHFR-DHFR-Cas13b. High levels of degradation occur in the absence of any stabilizing ligands, intermediate levels of degradation can occur when one stabilizing ligand is absent and the other (or another) stabilizing ligand is present, while low levels of degradation can be expected when both (or more than two) of the stabilizing ligands are present. 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 DD includes a linker between 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). This can be included in addition to the NES. In some embodiments, the CRISPR enzyme includes, consists essentially of, or consists of a localization (nuclear import or nuclear export) signal as or as part of the linker between the CRISPR enzyme and DD. HA or Flag tags are also within the scope of the present invention as linkers. The applicants use NLS and / or NES as linkers and also use glycine-serine linkers ranging from as short as GS to up to (GGGGS) 3 and up to.

[0277] Destabilizing domains have general utility in conferring instability on 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, the temperature-sensitive mutant of mammalian DHFR (DHFRts), which is an N-terminal rule destabilizing residue, has been found to be stable at the permissive temperature but unstable at 37°C. When methotrexate, a high-affinity ligand for mammalian DHFR, was added to cells expressing DHFRts, protein degradation was partially inhibited. This was an important demonstration that small molecule ligands can stabilize proteins that are otherwise targets of degradation in cells. Using a rapamycin derivative, the unstable mutant (FRB*) of the FRB domain of mTOR was stabilized, and the function of the fused kinase GSK-3β was restored6,7. This system demonstrated that ligand-dependent stability corresponds to an attractive strategy for regulating the function of specific proteins in a complex biological environment. The protein activity control system may involve DD, which becomes functional when ubiquitin complementarity occurs by dimerization of FK506-binding protein induced by rapamycin and 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 part of possible destabilizing domains (DDs) useful in the practice of the present invention, and the instability of the DD as a fusion with a CRISPR enzyme results in proteasome-mediated degradation of the entire fusion protein, the CRISPR protein. Shield-1 and TMP bind to the DD in a dose-dependent manner and stabilize it. The estrogen receptor ligand-binding domain (ERLBD, residues 305-549 of ERS1) can also be engineered as a destabilizing domain. Since the estrogen receptor signaling pathway is involved in various diseases such as breast cancer, this pathway has been widely studied, and many estrogen receptor agonists and antagonists have been developed.Therefore, pairs of ERLBDs and drugs with compatibility are known. There are ligands that bind to the mutant form of ERLBD but not to the wild-type form of ERLBD. By using one of these mutant domains encoding three mutations (L384M, M421G, G521R), it is possible to regulate the stability of 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 and configure it as 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, whereby the CRISPR enzyme has a DD. Another DD can 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, DD can be a modified FK506-binding protein 12 (FKBP12) that binds to and is thereby reversibly stabilized by the synthetically biologically inert small molecule, Shield-1. See, for example, 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 silent small molecules”. The Journal of biological chemistry. 2007;282:24866-24872; and Rodriguez, Chem Biol. Mar 23, 2012;19(3):391-398 (all of which are incorporated herein by reference), and can be used in the practice of the present invention in the selection of DD to associate with the CRISPR enzyme in the practice of the present invention.As can be appreciated, the knowledge in the art includes several DDs, and the DDs can be associated, advantageously with a linker, e.g., fused, with a CRISPR enzyme, thereby stabilizing the DD in the presence of a ligand and destabilizing the DD when it is not present, thereby destabilizing the CRISPR enzyme as a whole, or the DD can be stabilized when the ligand is absent and destabilized when the ligand is present, and the DD can regulate or control the CRISPR enzyme, and thus the CRISPR-Cas complex or system - so to speak, it is possible to switch on and off, thereby providing a means to regulate or control the system, for example, in an in vivo or in vitro environment. For example, when expressing a protein of interest as a fusion with a DD tag, it is destabilized in the cell and rapidly degraded, for example, by the proteasome. Thus, in the absence of a stabilizing ligand, it leads to the degradation of Cas associated with D. Fusing a novel DD to a protein of interest confers instability on the protein of interest and results in rapid degradation of the entire fusion protein. The peak activity of Cas is sometimes beneficial for reducing off-target effects. Thus, a short burst of high activity is preferred. The present invention is capable of providing such a peak. In a sense, this system is inducible. In another sense, this system is repressed in the absence of a stabilizing ligand and the repression is released in the presence of a stabilizing ligand.

[0278] Application of RNA-targeting CRISPR systems to plants and yeast Definition: Generally, the term "plant" refers to any of various photosynthetic organisms, eukaryotes, unicellular or multicellular organisms of the kingdom Plantae that characteristically grow by cell division, contain chloroplasts, and have cell walls composed of cellulose. The term "plant" includes monocotyledonous and dicotyledonous plants. Specifically, plants include, without limitation, acacia, alfalfa, amaranth, apple, apricot, artichoke, basswood, asparagus, avocado, banana, barley, beans, beet, beech, birch, blackberry, blueberry, broccoli, Brussels sprout, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, Douglas fir, grains, celery, clover, coffee, corn, cotton, cowpea, cucumber, elm, eggplant, eucalyptus, fennel, fig, fir, geranium, grape, grapefruit, legumes, ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch, lettuce, leek, lemon, lime, mimosa, pine, maidenhair, corn, mango, maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm, okra, onion, orange, ornamental plants or decorative flowers or ornamental trees, papaya, palm, parsley, parsnip, pea, peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sedge, turnip, vine plants, walnut, winter squash, wheat, yam, yew, and zucchini, and other angiosperms and gymnosperms. The term "plant" also includes algae, which are mostly photoautotrophic organisms that are mainly unified by the absence of roots, leaves, and other organs that characterize higher plants.

[0279] Using the method for regulating gene expression using an RNA targeting system as described herein, essentially any plant can be imparted with a desired trait. For the desired physiological and agronomic characteristics described herein, various plant and plant cell lines can be engineered using the nucleic acid constructs of the present disclosure and the various transformation methods described above. In preferred embodiments, the plants and plant cells targeted for engineering include, but are 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 crops (e.g., lettuce, spinach); flowering plants (e.g., petunia, rose, chrysanthemum), conifers 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), including crops such as monocotyledonous and dicotyledonous plants. Thus, the present method and the CRISPR-Cas system extend to a wide range of plants, for example, Magnoliales, Illiciales, Laurales, Piperales, Aristochiales, Nymphaeales, Ranunculales, Papaverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales,It can be used in dicotyledonous plants belonging to the orders Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales, Santalales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales. This method and the CRISPR-Cas system are applicable to the orders Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales,It can be used for monocotyledonous plants belonging to Lilliales and Orchidales, etc., or plants belonging to Gymnospermae, for example, those belonging to Pinales, Ginkgoales, Cycadales, Araucariales, Cupressales and Gnetales.

[0280] The RNA-targeting CRISPR systems and methods of use described herein can be used in a wide range of plant species included in the following non-limiting lists of dicotyledonous, monocotyledonous, or gymnosperm genera: Atropa, Alseodaphne, Anacardium, Arachis, Beilschmiedia, Brassica, Carthamus, Cocculus, 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, Lupinus, Manihot, Majorana, Malus, Medicago, Nicotiana, Olea, Parthenium, Papaver, Persea, Phaseolus, Pistacia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Senecio, Sinomenium, Stephania, Sinapis, Solanum,Genus Theobroma, Trifolium, Trigonella, Vicia, Vinca, Vitis and Vigna; and Allium, Andropogon, Aragrostis, Asparagus, Avena, Cynodon, Elaeis, Festuca, Festulolium, Heterocallis, Hordeum, Lemna, Lolium, Musa, Oryza, Panicum, Pannesetum, Phleum, Poa, Secale, Sorghum, Triticum, Zea, Abies, Cunninghamia, Ephedra, Picea, Pinus and Pseudotsuga.,

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

[0282] A part of a plant, i.e., "plant tissue", can be treated according to the method of the present invention to create an improved plant. Plant tissue also includes plant cells. The term "plant cell", as used herein, refers to an individual unit of a living plant, whether it is an intact whole plant or, alternatively, in an isolated form that has grown as part of a highly organized unit, such as a plant tissue, plant organ, or whole plant, in a medium or on agar under in vitro tissue culture, in a suspension in a growth medium or buffer, or otherwise.

[0283] "Protoplast" refers to a plant cell whose protective cell wall has been completely or partially removed, for example, by using mechanical or enzymatic means that result in a viable, biochemically competent unit of a living plant that can reform its cell wall, proliferate, and regenerate into a whole plant under appropriate growth conditions.

[0284] The term "transformation", in a broad sense, refers to a method by which a plant host is genetically modified by the introduction of DNA using Agrobacteria or one of various 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, stolons, microtubules, and shoots. Plant tissue refers to any of these, as well as any progeny thereof, whether produced sexually or asexually, such as plants, seeds, progeny, any clone of a scion, and cuttings or seeds.

[0285] As used herein, the term "transformed" 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 can 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, "transformed" or "transgenic" cells or plants can include the progeny of such cells or plants and progeny produced from breeding programs using such transformed plants as parents and exhibiting phenotypic changes resulting from the presence of the introduced DNA molecule. Preferably, the transgenic plant is fertile and capable of transmitting the introduced DNA to its progeny through sexual reproduction.

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

[0287] As used herein, the term "plant promoter" is a promoter that has the ability to initiate transcription in a plant cell, regardless of whether its origin is in a plant cell. Exemplary suitable plant promoters include, but are not limited to, those obtained from bacteria such as Agrobacterium or Rhizobium that 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 of fungi. Phyla within the kingdom of fungi include Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cells can include yeasts, molds, 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. Without limitation to these organisms, many types of yeasts used in laboratory and industrial settings are part of the phylum Ascomycota. In some embodiments, the yeast cell is an 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. (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 filamentously, i.e., as hyphae or mycelia.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 a fungal cell that is used in an industrial process, such as the production of a product on a commercial or industrial scale, or isolated therefrom. An industrial strain can typically refer to a fungal species used in an industrial process, or it can refer to an isolate of a fungal species that can also be used for non-industrial purposes (e.g., experimental research). Examples of industrial processes include fermentation (e.g., in the production of food or beverage products), distillation, biofuel production, compound production, and polypeptide production. Examples of industrial strains 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 is present 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 be in a polyploid state (e.g., by specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). A polyploid cell can refer to a cell in which the 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 thought that the methods using the Cas13b CRISPR system described herein may take advantage of the use of specific fungal cell types because the abundance of guide RNA is often the 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 is present 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 be in a diploid state (e.g., by specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the 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 particular 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 is present 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 be in a haploid state (e.g., by specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the S. cerevisiae strain S228C can be maintained in a haploid or diploid state. A haploid cell can refer to a cell in which the 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, "yeast expression vector" refers to a nucleic acid containing one or more sequences encoding RNA and / or polypeptide, and which may further contain any desired elements that control the expression of the nucleic acid and any elements that enable the replication and maintenance of the expression vector within yeast cells. 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, R.G. and Gleeson, M.A. (1991) Biotechnology (NY) 9(11):1067-72. Yeast vectors can contain, without limitation, a centromere (CEN) sequence, an autonomous replication sequence (ARS), a promoter such as an RNA polymerase III promoter operably linked to the sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., 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 certain embodiments, it is contemplated that polynucleotides encoding the 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 adjusted according to when, where, and under what conditions the guide RNA and / or RNA targeting gene are expressed.

[0295] In a detailed embodiment, it is contemplated that the components of the RNA-targeting CRISPR system are stably introduced into the genomic DNA of plant cells. Additionally or alternatively, it is contemplated that the components of the RNA-targeting CRISPR system are introduced for stable integration into the DNA of plant cell organelles such as, but not limited to, plastids, mitochondria or chloroplasts.

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

[0297] The elements of the expression system can be on one or more expression constructs that are either circular, such as a plasmid or a transformation vector, or linear double-stranded DNA, such as an uncircularized one. In a detailed embodiment, the RNA-targeting CRISPR expression system comprises at least (a) a guide RNA (gRNA) that hybridizes to a target sequence of a plant, the nucleotide sequence encoding a 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] The components of the RNA-targeting CRISPR system and, where applicable, the DNA construct containing the template sequence can be introduced into the genome of plants, plant parts or plant cells by various prior art methods. This process generally includes the steps of selecting a suitable host cell or host tissue, introducing the construct into the host cell or host tissue, and then regenerating plant cells or plants therefrom. In a detailed embodiment, the DNA construct can be introduced into plant cells using techniques such as, but not limited to, electroporation, microinjection, aerosol beam injection of plant cell protoplasts, or the DNA construct can also be directly introduced into plant tissues using a particle bombardment method 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 the gene of interest towards the cells to allow the particles to penetrate the protoplasm and typically obtain stable integration into the genome (see, for example, 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, the DNA construct containing the components of the RNA-targeting CRISPR system can be introduced into plants by Agrobacterium-mediated transformation. The DNA construct can be combined with a suitable T-DNA flanking region and introduced into a conventional Agrobacterium tumefaciens host vector. The foreign DNA can be incorporated into the genome of the plant by infecting the plant or by incubating plant protoplasts with Agrobacterium bacteria containing one or more Ti (tumor-inducing) plasmids (see, for example, Fraley et al., (1985), Rogers et al., (1987) and U.S. Patent No. 5,563,055).

[0300] Plant promoter 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 envisioned.

[0301] A constitutive plant promoter is a promoter capable of causing the open reading frame (ORF) it controls to be expressed (referred to as "constitutive expression") in all or substantially all plant tissues at all or substantially all stages of plant development. One non-limiting example of a constitutive promoter is the cauliflower mosaic virus 35S promoter. The present invention contemplates methods of modifying RNA sequences and, therefore, also contemplates regulating the expression of plant biomolecules. Thus, in a detailed embodiment of the present invention, it is advantageous to place one or more elements of the RNA-targeting CRISPR system under the control of a promoter that can be regulatable. A "regulatory promoter" refers to a promoter that directs gene expression in a non-constitutive, temporally and / or spatially regulated manner and includes tissue-specific, tissue-preferred, and inducible promoters. Different promoters can direct the expression of a gene in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. In a detailed embodiment, 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 a tissue-preferred promoter, it is possible to target the enhancement of expression in certain cell types within a particular plant tissue, such as vascular cells of leaves or roots or specific cells of seeds. For examples of specific promoters used in the RNA-targeting CRISPR system, reference is made 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 enable spatiotemporal control of gene editing or gene expression can utilize a certain form of energy.The forms of energy include, but are not limited to, acoustic energy, electromagnetic radiation, chemical energy, and / or thermal energy. Examples of inducible systems include the tetracycline-inducible promoter (Tet-On or Tet-Off), small molecule two-hybrid transcriptional activation systems (FKBP, ABA, etc.) or light-inducible systems (phytochrome, LOV domain or cryptochrome), such as the light-inducible transcriptional effector (LITE) that leads to sequence-specific changes in transcriptional activity. The components of the light-inducible system can include an RNA-targeting CRISPR enzyme, a light-responsive cytochrome heterodimer (e.g., from Arabidopsis thaliana), and a transcriptional activation / suppression domain. Further examples of inducible DNA-binding proteins and methods of using them are provided in U.S. Provisional Patent Application No. 61 / 736,465 and U.S. Provisional Patent Application No. 61 / 721,283, which are hereby incorporated by reference in their entirety.

[0302] In a detailed embodiment, for example, a chemically regulated promoter can be used to achieve transient or inducible expression, i.e., according to this, when an exogenous chemical substance is added, gene expression is induced. Regulation of gene expression can also be achieved by a chemically repressive promoter, where gene expression is repressed when a chemical substance is added. Examples of chemically inducible promoters include, but are not limited to, the maize In2-2 promoter activated by a benzenesulfonamide herbicide safener (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-II-27, WO 93 / 01294) activated by a hydrophobic electrophilic compound used as a pre-emergence herbicide, and the tobacco PR-1a promoter activated by salicylic acid (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7). Promoters regulated by antibiotics, such as tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; US Pat. Nos. 5,814,618 and 5,789,156) can also be used herein.

[0303] Translocation to and / or expression in a specific plant cell organelle The expression system may include elements for translocation to and / or expression in a specific plant cell organelle.

[0304] Chloroplast targeting In a detailed embodiment, it is contemplated that an RNA-targeting CRISPR system is used to specifically modify the expression and / or translation of chloroplast genes or to ensure expression in chloroplasts. For this purpose, a chloroplast transformation method or compartmentalization of RNA-targeting CRISPR components into chloroplasts is used. For example, introducing genetic modifications into the plastid genome can reduce biosafety problems such as gene flow through pollen.

[0305] Methods for chloroplast transformation are known in the art and include particle bombardment, PEG treatment, and microinjection. Additionally, as described in WO 2010 / 061186, methods involving the transfer of a transformation cassette from the nuclear genome to the plastid can be used.

[0306] Alternatively, it is contemplated to target one or more of the RNA targeting CRISPR components to the plant chloroplast. This is achieved by incorporating into the expression construct a sequence encoding a chloroplast transit peptide (CTP) or a plastid transit peptide operably linked to the 5' region of the sequence encoding the RNA targeting protein. The CTP is removed during the processing step during import into the chloroplast. Chloroplast targeting of the expressed protein is well known to those skilled in the art (see, e.g., 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 the plant chloroplast. Methods and constructs that can be used to translocate guide RNAs to the chloroplast using a chloroplast localization sequence are described, for example, in US Patent Application Publication No. 20040142476, which is incorporated herein by reference. By incorporating such various constructs into the expression system of the present invention, the RNA targeting guide RNA can be efficiently translocated.

[0307] Introduction of a polynucleotide encoding a CRISPR - RNA targeting system in algal cells Transgenic algae (or other plants such as Brassica napus) can be particularly useful for the production of vegetable oils or biofuels, such as alcohols (in particular methanol and ethanol) or other products. They can be engineered to express or over - express high levels of oil or alcohol used in the oil or biofuel industries.

[0308] U.S. Patent No. 8,945,839 describes a method for engineering microalgae (Chlamydomonas reinhardtii cells) using Cas9. Similar tools can be used to apply the RNA targeting CRISPR system methods described herein to Chlamydomonas species and other algae. In a detailed embodiment, an RNA targeting protein and a guide RNA are introduced and expressed in algae using a vector that expresses 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 algal cells. Those skilled in the art have available electroporation protocols such as standard recommended protocols from the GeneArt Chlamydomonas engineering kit.

[0309] Introduction of a polynucleotide encoding an RNA targeting component in yeast cells In a detailed embodiment, the invention relates to the use of an RNA targeting CRISPR system for RNA editing in yeast cells. Methods for transforming yeast cells that can be used for the introduction of polynucleotides encoding RNA targeting CRISPR system components are well known to those skilled in the art and have been 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 a detailed embodiment, it is envisioned that the guide RNA and / or the RNA targeting gene are transiently expressed in a plant cell. In these embodiments, the RNA targeting CRISPR system modifies the RNA target molecule only when both the guide RNA and the RNA targeting protein are present within the cell, and thus gene expression can be further controlled. Since the expression of the RNA targeting enzyme is transient, plants regenerated from such plant cells typically do not contain foreign DNA. In a detailed embodiment, the RNA targeting enzyme is stably expressed by the plant cell and the guide sequence is transiently expressed.

[0311] In a particularly preferred embodiment, the RNA targeting CRISPR system components can be introduced into plant cells using a plant virus vector (Scholthof et al. 1996, Annu Rev Phytopathol. 1996;34:299-323). In a more detailed embodiment, the virus vector is a vector derived from a DNA virus. For example, geminivirus (e.g., cabbage leaf curl virus, bean yellow dwarf virus, wheat dwarf virus, tomato leaf curl virus, maize streak virus, tobacco leaf curl virus or tomato golden mosaic virus) or nanovirus (e.g., faba bean necrotic yellows virus). In another detailed embodiment, the virus vector is a vector derived from an RNA virus. For example, tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus), potexvirus (e.g., potato virus X) or hordeivirus (e.g., barley stripe mosaic virus). The replication genome of a plant virus is a non-integrating vector, which is advantageous in relation to the avoidance of creating GMO plants.

[0312] In a detailed embodiment, the vector used for transient expression of an RNA targeting CRISPR construct 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 loci has been demonstrated using a modified cabbage leaf curl virus (CaLCuV) vector to express gRNA in stable transgenic plants expressing a CRISPR enzyme (Scientific Reports 5, Article number: 14926 (2015), doi:10.1038 / srep14926).

[0313] In a detailed embodiment, a double-stranded DNA fragment encoding a guide RNA and / or an RNA targeting gene can be transiently introduced into a plant cell. In such an embodiment, the double-stranded DNA fragment introduced is provided in an amount sufficient to modify the RNA molecule in the cell but that does not remain after the intended time has elapsed or after one or more cell divisions. Methods for effecting 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 other embodiments, 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 that does not remain after the intended time has elapsed or after one or more cell divisions, and 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 to Plant Cells In certain embodiments, it is beneficial to directly deliver one or more components of an RNA-targeting CRISPR system to plant cells. This is particularly useful for generating non-transgenic plants (see below). In certain embodiments, one or more of the RNA-targeting components are prepared outside of the plant or plant cell and delivered to the cell. For example, in certain embodiments, an RNA-targeting protein is prepared in vitro and then introduced into a plant cell. The RNA-targeting protein can be prepared by a variety of methods known to those of skill in the art, including recombinant production. After expression, the RNA-targeting protein is isolated, optionally refolded, purified, and optionally processed to remove any purification tags, such as a His tag. Once a crude, partially purified, or more fully purified RNA-targeting protein is obtained, the protein can be introduced into a plant cell.

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

[0317] The individual components or pre-assembled ribonucleoproteins can be introduced into plant cells by electroporation, by bombardment with particles coated with RNA-targeting related gene products, by chemical transfection, or by any other means of transporting across the cell membrane. For example, transfection of plant protoplasts with pre-assembled CRISPR ribonucleoproteins has been demonstrated to ensure targeted modification of the plant genome (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 a detailed embodiment, the RNA targeting CRISPR system components are introduced into plant cells using nanoparticles. The components can be uploaded or packaged onto the nanoparticles and applied to plants as either proteins or nucleic acids, or alternatively as combinations thereof (for example, as described in WO 2008 / 042156 pamphlet and US Patent Application Publication No. 2013 / 0185823). Specifically, embodiments of the present invention include nanoparticles onto which a DNA molecule encoding an RNA targeting protein, a DNA molecule encoding a guide RNA, and / or an isolated guide RNA are uploaded or packaged, as described in WO 2015 / 089419 pamphlet.

[0319] A further means of introducing one or more components of the RNA-targeting CRISPR system into plant cells is by using a cell-penetrating peptide (CPP). Thus, in particular, embodiments of the invention include compositions comprising a cell-penetrating peptide linked to an RNA-targeting protein. In a detailed embodiment of the invention, the RNA-targeting protein and / or guide RNA are coupled to one or more CPPs for their efficient transport inside the plant protoplasts (for Cas9 in human cells, see Ramakrishna (2014 Genome Res. 2014 Jun;24(6):1020-7)). In other embodiments, the RNA-targeting gene and / or guide RNA are encoded by one or more circular or non-circular DNA molecules coupled to one or more CPPs for plant protoplast delivery. The plant protoplasts are then regenerated into plant cells and further into plants. CPPs are generally described as short peptides of less than 35 amino acids, either proteins or chimeric sequences, having the ability to transport biomolecules across the cell membrane in a receptor-independent manner. CPPs can be cationic peptides, peptides having hydrophobic sequences, amphiphilic peptides, peptides having proline-rich antimicrobial sequences, and chimeric or bipartite peptides (Pooga and Langel 2005). CPPs are able to penetrate biological membranes, thus causing the movement of various biomolecules across the cell membrane into the cytoplasm and enabling their intracellular transport, thus promoting the interaction between the biomolecule and the target. Examples of CPPs include, among others, Tat, an endonuclear transcriptional activation protein required for viral replication by human immunodeficiency virus type 1, penetratin, the Kaposi fibroblast growth factor (FGF) signal peptide sequence, the integrin β3 signal peptide sequence; the polyarginine peptide Arg sequence, the guanine-rich molecular transporter, the sweet arrow peptide, etc.

[0320] Target RNA for which plant, algal or fungal applications are envisaged The target RNA, i.e., the RNA of interest, is the RNA targeted by the present invention, which leads to the recruitment and binding of the RNA targeting protein to the target site of interest on the target RNA. The target RNA can be any suitable form of RNA. In some embodiments, this 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), microswitch, ribozyme, satellite RNA, and RNA virus. The target RNA can be located in the cytoplasm of plant cells, or in the cell nucleus, or in plant cell organelles such as mitochondria, chloroplasts, or plastids.

[0321] In a specific embodiment, the RNA is cleaved or otherwise the RNA expression is inhibited using an RNA targeting CRISPR system.

[0322] Use of an RNA targeting CRISPR system for the regulation of plant gene expression by RNA regulation The RNA targeting protein, together with a suitable guide RNA, can also be used for the targeting of gene expression by the control of RNA processing. The control of RNA processing includes RNA splicing, including alternative splicing or the specific targeting of specific splice variants or isoforms; viral replication (specifically, those of plant viruses including viroids in plants and RNA processing reactions such as tRNA biosynthesis). The RNA targeting protein in combination with a suitable guide RNA can also be used for the control of RNA activation (RNAa). RNAa leads to the promotion of gene expression, and thus the control of gene expression can be achieved in the form of the disruption or reduction of RNAa, and thus the reduction of the promotion of gene expression.

[0323] The RNA-targeting effector protein of the present invention can further 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 a selected viral RNA sequence. Specifically, the effector protein can be an active nuclease that cleaves RNA, such as single-stranded RNA. Accordingly, the use of the RNA-targeting effector protein of the present invention as an antiviral agent 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), Bromo mosaic virus (BMV), and Potato virus X (PVX).

[0324] Examples of the regulation of RNA expression in plants, algae, or fungi as an alternative means of target gene modification are further described herein.

[0325] In particular, regulated gene expression control by regulated mRNA cleavage is of interest. This can be achieved by placing the RNA-targeting element under the control of a regulatable promoter as described herein.

[0326] Use of an RNA-targeting CRISPR system to restore the function of tRNA molecules Pring et al. have described RNA editing in plant mitochondria and chloroplasts that changes the mRNA sequence to encode a protein different from DNA (Plant Mol. Biol. (1993) 21(6): 1163-1170. doi: 10.1007 / BF00023611). In a detailed embodiment of the present invention, by introducing elements of an RNA targeting CRISPR system that specifically targets mitochondrial and chloroplast mRNAs into a plant or a plant cell, different proteins can be expressed in such plant cell organelles to mimic the processes that occur in vivo.

[0327] Use of an RNA targeting CRISPR system as an alternative to RNA interference for inhibiting RNA expression The RNA targeting CRISPR system has uses similar to RNA inhibition or RNA interference and can thus replace such methods. In a detailed embodiment, the method of the present invention includes, for example, the use of RNA targeting CRISPR in place of interfering ribonucleic acids (such as siRNA or shRNA or dsRNA). Examples of inhibition of RNA expression in plants, algae or fungi as an alternative means of targeted gene modification are further described herein.

[0328] Use of an RNA targeting CRISPR system for controlling RNA interference Control of interfering RNA or miRNA can facilitate reducing off-target effects (OTE) seen in techniques by shortening the lifespan of interfering RNA or miRNA in vivo or in vitro. In a detailed embodiment, 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 specific embodiments, when the RNA targeting protein and the 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), this can be used to "protect" cells or systems (in vivo or in vitro) from RNAi in those cells. This can be useful for comparing adjacent tissues or cells where RNAi is not required, or for comparing cells or tissues in which the effector protein and suitable guide are expressed and not expressed (i.e., where RNAi is not controlled and where it is controlled, respectively). RNA targeting proteins can be used to control or bind to molecules containing or consisting of RNA, such as ribozymes, ribosomes or riboswitches. In embodiments of the invention, by the guide RNA recruiting the RNA targeting protein to such molecules, the RNA targeting protein becomes capable of binding to them.

[0330] The RNA targeting CRISPR system of the present invention can be applied from this disclosure, without undue experimentation, in the areas of pest management, plant disease management and herbicide tolerance management, and in the area of implant RNAi technology for plant assays and other applications, including providing a basis for engineering this system based on information (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 J.M, in Pest Management Science, Vol 70(9), pp 1351 - 1357).

[0331] Use of an RNA targeting CRISPR system for modifying riboswitches and controlling 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 concentrations of those small molecules. Certain riboswitches typically regulate their adjacent genes by changing the transcription, translation, or splicing of that gene. Thus, in detailed embodiments of the present invention, control of riboswitch activity by using an RNA targeting protein in combination with a suitable guide RNA that targets the riboswitch is envisioned. This can be by cleavage of or binding to the riboswitch. In detailed embodiments, reduction of riboswitch activity is envisioned. Recently, riboswitches that bind thiamine pyrophosphate (TPP) have been characterized and found to regulate thiamine biosynthesis in plants and algae. Furthermore, this element is considered 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 it controls alternative splicing to conditionally produce an upstream open reading frame (uORF), thereby affecting the expression of downstream genes (Cheah MT et al., (2007) Nature 447(7143):497-500.doi:10.1038 / nature05769). The RNA targeting CRISPR systems described herein can be used to manipulate the endogenous riboswitch activity in plants, algae, or fungi and thus change the expression of downstream genes that are thereby controlled. In detailed embodiments, the RNA targeting CRISPR systems can be used in assays of riboswitch function in vivo or in vitro and in studies of its relevance to metabolic networks. In detailed embodiments, the RNA targeting CRISPR systems can potentially be used in the engineering of riboswitches as metabolite sensors in plants and gene control platforms.

[0332] Use of RNA targeting CRISPR systems in RNAi screens of plants, algae or fungi RNAi screens can identify gene products whose knockdown is associated with a phenotypic change, to investigate biological pathways and identify components. In a particular embodiment of the invention, the Cas13b protein and suitable guide RNAs described herein are used to remove or reduce the activity of RNAi in a screen, and thus (by removing or reducing interference / suppression) restore the activity of the gene product (which was previously subject to interference), thereby allowing control both within and between such screens.

[0333] Use of RNA targeting proteins for visualizing RNA molecules in vivo and in vitro In a particular embodiment, the invention provides a nucleic acid binding system. In situ hybridization of RNA with a complementary probe is a powerful technique. Typically, fluorescent DNA oligonucleotides are used for the detection of nucleic acids by hybridization. Although certain modifications, such as locked nucleic acids (LNAs), have achieved increased efficiency, an efficient and versatile alternative is still needed. Thus, the labeling element of an RNA targeting system can be used as an alternative to an 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 As used herein, the term "biofuel" is an alternative fuel made from plants and plant-derived resources. Renewable biofuels can be extracted from organic matter that has obtained energy through the process of carbon fixation, or can be produced by the use or conversion of biomass. This biomass can be used directly as biofuel or can be converted into a convenient energy-containing substance by thermal conversion, chemical conversion, and biochemical conversion. Through this biomass conversion, fuels in solid, liquid, or gaseous form can be obtained. There are two types of biofuels: bioethanol and biodiesel. Bioethanol is mainly produced by the sugar fermentation process of cellulose (starch), most of which is derived from corn and sugarcane. On the other hand, biodiesel is mainly produced from oil crops such as rapeseed, palm, and soybean. Biofuels are mainly used in transportation vehicles.

[0335] Enhancement of Plant Characteristics for Biofuel Production In a detailed embodiment, to facilitate access of the primary hydrolyzing agent so that sugars are released more efficiently during fermentation, the cell wall properties are altered using the method of RNA targeting CRISPR as described herein. In a detailed embodiment, the biosynthesis of cellulose and / or lignin is modified. Cellulose is a major 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 increase. In a detailed embodiment, lignin biosynthesis in the plant is downregulated using the method described herein, thereby increasing the fermentable carbohydrates. More specifically, using the method described herein, as disclosed in WO 2008 / 064289 A2, at least a first lignin biosynthesis gene selected from the group consisting of 4-coumarate 3-hydroxylase (C3H), phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), hydroxycinnamoyl transferase (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) is downregulated.

[0336] In a detailed embodiment, a plant mass with lower acetic acid levels generated during fermentation is produced using the method described herein (see also WO 2010 / 096488).

[0337] Modification of Yeast for Biofuel Production In a detailed embodiment, the RNA targeting enzymes provided herein are used in the production of bioethanol by recombinant microorganisms. For example, by engineering microorganisms such as yeast using RNA targeting enzymes, biofuels or biopolymers can be created from fermentable sugars and, optionally, plant-derived lignocellulose obtained from agricultural waste as a source of fermentable sugars can be degraded. More specifically, the present invention provides a method of using an RNA targeting CRISPR complex 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 method includes stimulating the expression in a microorganism such as yeast of one or more nucleotide sequences encoding an enzyme involved in the conversion of pyruvate to ethanol or another desired product. In a detailed embodiment, the method ensures the stimulation of the expression of one or more enzymes that enable the degradation of cellulose by microorganisms, such as cellulases. In yet another embodiment, an RNA targeting CRISPR complex is used to suppress endogenous metabolic pathways that compete with the biofuel production pathway.

[0338] Modification of algae and plants for the production of vegetable oils or biofuels Transgenic algae or other plants such as Brassica napus can 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 oil or alcohol used in the oil or biofuel industries.

[0339] U.S. Patent No. 8,945,839 describes a method for engineering microalgae (Chlamydomonas reinhardtii cells) using Cas9. Using similar tools, the methods of the RNA-targeting CRISPR systems described herein can be applied to Chlamydomonas species and other algae. In a detailed embodiment, an RNA-targeting effector protein and a guide RNA are introduced and expressed in algae using a vector that expresses 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 algal cells. The electroporation protocol follows the standard recommended protocol of the GeneArt Chlamydomonas engineering kit.

[0340] Detailed applications of RNA-targeting enzymes in plants In a detailed embodiment, since the present invention is capable of cleaving viral RNA, it can be used as a therapeutic agent for virus removal in plant systems. Prior studies in human systems have demonstrated the success of using CRISPR for targeting single-stranded RNA viruses, hepatitis C (A. Price, et al., Proc. Natl. Acad. Sci, 2015). These methods can also be adapted for use of the RNA-targeting CRISPR system in plants.

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

[0342] Improved plants obtained by the methods described herein, particularly crops and algae, can be useful in food or feed production, for example, by expressing higher levels of proteins, carbohydrates, nutrients or vitamins than would typically be found in the wild type. In this regard, improved plants, particularly legumes and tubers, are preferred.

[0343] Improved algae or other plants such as Brassica napus can be particularly useful for the production of vegetable oils or biofuels, such as alcohols (particularly methanol and ethanol). These can be engineered to express or overexpress high levels of oil or alcohol for use in the oil or biofuel industries.

[0344] The present invention also provides improved parts of plants. 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 is the provision of plant cells and plants created by the methods of the present invention. Gametes, seeds, embryos (whether zygotic or somatic), progeny or hybrids of plants containing genetic modifications made by conventional breeding methods are also within the scope of the present invention. Such plants can contain heterologous or foreign DNA sequences inserted into or instead of the target sequence. Alternatively, such plants can contain only one or more nucleotides with a particular change (mutation, deletion, insertion, substitution). As such, such plants will differ from their ancestral plants only by the presence of the specific modification.

[0346] In certain embodiments of the invention, pathogen-resistant plants are engineered using a Cas13b system, for example, by creating resistance to diseases caused by bacteria, fungi, or viruses. In certain embodiments, pathogen resistance can be achieved by engineering a crop such that a Cas13b system is created that will be ingested by a pest and lead to its death. In certain embodiments of the invention, abiotic stress tolerance is engineered using a Cas13b system. In another embodiment, drought stress tolerance or salt stress tolerance or low temperature or heat stress tolerance is engineered using a Cas13b system. 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 systems described herein. Some non-limiting target crops include rice (Oryza sativa L), European plum (Prunus domestica L.), cotton (Gossypium hirsutum), Nicotiana rustica, maize (Zea mays), alfalfa (Medicago sativa), Nicotiana benthamiana, and Arabidopsis thaliana.

[0347] In certain embodiments of the invention, the Cas13b system is used for the management of crop pests. For example, a Cas13b system that is 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 certain embodiments, the present invention provides a method for efficiently producing a homozygous organism from a heterozygous non-human starting organism. In certain embodiments, the present invention is used in plant breeding. In another embodiment, the present invention is used in animal breeding. In such embodiments, a homozygous organism such as a plant or an animal is created by preventing or suppressing recombination by interfering with at least one target gene involved in double-strand break, chromosome pairing and / or strand exchange.

[0349] Application of Cas13b protein in an optimized functional RNA targeting system In one aspect, the present invention provides a system for specifically delivering a functional component to an RNA environment. This can be ensured using a CRISPR system comprising an RNA targeting effector protein of the present invention that enables 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 is described in other parts of this specification.

[0350] According to 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 target genomic locus of a cell, wherein the guide RNA is modified by the insertion of one or more individual RNA sequences that bind to an adapter protein. In a detailed embodiment, the RNA sequence can bind to two or more adapter proteins (e.g., aptamers), and each adapter protein is associated with one or more functional domains. The guide RNA of the Cas13b enzyme described herein is shown to be suitable for modification of the guide sequence. In a detailed embodiment, the guide RNA is modified by the insertion of an individual RNA sequence 5' to the direct repeat, within the direct repeat, or 3' to the guide sequence. When there are two or more functional domains, they can be the same or different, for example, they can be the same two or two different activators or repressors. In one aspect, the present invention provides a composition as contemplated herein, wherein, because one or more functional domains are added to an RNA targeting enzyme, when the functional domain binds to the target RNA, it results in a spatial arrangement that allows the functional domain to function in its attributed function. In one aspect, the present invention provides a composition as contemplated 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 the gRNA.

[0351] Accordingly, in one aspect, the present invention provides a non-naturally occurring or engineered CRISPR-Cas13b complex composition comprising a guide RNA and an RNA targeting enzyme, Cas13b, as contemplated herein, where optionally, the RNA targeting enzyme comprises at least one mutation and optionally one or more nuclear localization sequences, such that the RNA targeting enzyme has nuclease activity that is 5% or less of the enzyme that has no mutations in at least one of its mutations. In a detailed embodiment, the guide RNA is modified (as detailed elsewhere herein) such that, in addition to or instead of, binding of the RNA targeting enzyme is still ensured, cleavage by the RNA targeting enzyme is prevented.

[0352] In a detailed embodiment, the RNA targeting enzyme is a Cas13b enzyme having at least a 97% or 100% reduced nuclease activity when compared to a Cas13b enzyme having no mutations in at least one mutation. In one aspect, the present invention provides a composition as contemplated herein, where the Cas13b enzyme comprises two or more mutations as described elsewhere herein.

[0353] In a detailed embodiment, an RNA targeting system as described above herein comprising two or more functional domains is provided. In a detailed embodiment, the two or more functional domains are heterologous functional domains. In a detailed embodiment, the system comprises an adapter protein that is a fusion protein comprising a functional domain, where the fusion protein optionally comprises a linker between the adapter protein and the functional domain. In a detailed embodiment, the linker comprises a GlySer linker. In addition to or instead of, one or more functional domains are added to the RNA effector protein by a linker, optionally a GlySer linker. In a detailed embodiment, one or more functional domains are added to the RNA targeting enzyme by one or both of the HEPN domains.

[0354] In certain aspects, the invention provides a composition as contemplated herein, where one or more functional domains associated with an adapter protein or an RNA targeting enzyme are domains having the ability to activate or suppress RNA translation. In certain aspects, the invention provides a composition as contemplated herein, where at least one of the one or more functional domains associated with an adapter 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 inducibility or photoinducibility.

[0355] In certain aspects, the invention provides a composition as contemplated herein that includes an aptamer sequence. In a detailed embodiment, the aptamer sequence is two or more aptamer sequences specific for the same adapter protein. In certain aspects, the invention provides a composition as contemplated herein, where the aptamer sequence is two or more aptamer sequences specific for different adapter proteins. In certain aspects, the invention provides a composition as contemplated herein, where the adapter protein includes 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, PRR1. Thus, in a detailed embodiment, the aptamer is selected from binding proteins that specifically bind any one of the adapter proteins listed above. In certain aspects, the invention provides a composition as contemplated herein, where the cell is a eukaryotic cell. In certain aspects, the invention provides a composition as contemplated herein, where the eukaryotic cell is a mammalian cell, a plant cell, or a yeast cell, whereby the mammalian cell is optionally a mouse cell. In certain aspects, the invention provides a composition as contemplated herein, where the mammalian cell is a human cell.

[0356] In certain aspects, the present invention provides the compositions discussed above herein, where there are two or more gRNAs, and these gRNAs target different sequences, such that when the present composition is used, there is multiplexing. In certain aspects, the present invention provides a composition having two or more gRNAs modified by the insertion of individual RNA sequences that bind to one or more adapter proteins.

[0357] In certain aspects, the present invention provides the compositions discussed herein, where there are one or more adapter proteins associated with one or more functional domains that bind to the individual RNA sequences inserted into the guide RNA.

[0358] In certain aspects, the present invention provides the compositions discussed herein, where the guide RNA is modified to have at least one non-coding functional loop, for example, at least one non-coding functional loop is inhibitory, for example, at least one non-coding functional loop contains an Alu.

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

[0360] In certain aspects, the present invention provides the methods discussed herein, where the methods include delivery of the composition or a nucleic acid molecule encoding the same, where the nucleic acid molecule is operably linked to regulatory sequences and is expressed in vivo. In certain aspects, the present invention provides the methods discussed herein, where the in vivo expression is via lentivirus, adenovirus, or AAV.

[0361] In certain aspects, the present invention provides mammalian cell lines of cells as contemplated herein, where the cell line is optionally a human cell line or a mouse cell line. In certain aspects, the present invention provides a transgenic mammalian model, optionally a mouse, where the model is transformed with a composition as contemplated herein or is a progeny of said transformant.

[0362] In certain aspects, the present invention provides a nucleic acid molecule encoding a guide RNA or an RNA-targeting CRISPR-Cas complex or composition as contemplated herein. In certain aspects, the present invention provides a vector comprising a nucleic acid molecule encoding a guide RNA (gRNA) comprising a guide sequence hybridizable to a target sequence at a target genomic locus of a cell, where the direct repeat of the gRNA is modified by insertion of individual RNA sequences that bind to two or more adapter proteins, and each adapter protein is associated with one or more functional domains, or the gRNA is modified to have at least one non-coding functional loop. In certain aspects, 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 contemplated herein and an RNA-targeting enzyme [optionally the RNA-targeting enzyme comprises at least one mutation such that the RNA-targeting enzyme has nuclease activity of no more than 5% of an RNA-targeting enzyme having no such at least one mutation and optionally comprises one or more nuclear localization sequences]. In certain aspects, the vector may further comprise regulatory elements operable in eukaryotic cells 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 one aspect, the present invention provides a method for screening for gain-of-function (GOF) or loss-of-function (LOF), or for screening non-coding RNAs or potential regulatory regions (e.g., enhancers, repressors), the method comprising introducing into a cell line or a cell of a model as contemplated herein that contains or expresses an RNA-targeting enzyme, and a composition as contemplated herein, such that the gRNA contains either an activator or a repressor, and monitoring for GOF or LOF, respectively, with respect to whether the gRNA introduced into those cells contains an activator or with respect to whether the gRNA introduced into those cells contains a repressor.

[0365] In certain aspects, 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 nuclease activity that is 5% or less of an RNA targeting enzyme that does not have the at least one mutation, the gRNA is modified by the insertion of an individual RNA sequence that binds to one or more adapter proteins, and the adapter protein associates with one or more functional domains, the composition comprising one or more or two or more adapter proteins, each protein associating with one or more functional domains, and the gRNA comprising a genome-wide library of multiple RNA targeting guide RNAs (gRNAs). In certain aspects, the present invention provides a library as contemplated herein, wherein the RNA targeting RNA targeting enzyme has a nuclease activity that is at least 97% or 100% decreased when compared to an RNA targeting enzyme that does not have the at least one mutation. In certain aspects, the present invention provides a library as contemplated herein, wherein the adapter protein is a fusion protein comprising a functional domain. In certain aspects, the present invention provides a library as contemplated herein, wherein the gRNA is not modified by the insertion of an individual RNA sequence that binds to one or two or more adapter proteins. In certain aspects, the present invention provides a library as contemplated herein, wherein one or two or more functional domains are associated with the RNA targeting enzyme. In certain aspects, the present invention provides a library as contemplated herein, wherein the cell population of cells is a population of eukaryotic cells. In certain aspects, the present invention provides a library as contemplated herein, wherein the eukaryotic cells are mammalian cells, plant cells, or yeast cells. In certain aspects, the present invention provides a library as contemplated herein, wherein the mammalian cells are human cells.In one aspect, the present invention provides a library as contemplated herein, where the population of cells is a population of embryonic stem (ES) cells.

[0366] In one aspect, the present invention provides a library as contemplated herein, where the targeting is at least about 100 RNA sequences. In one aspect, the present invention provides a library as contemplated herein, where the targeting is at least about 1000 RNA sequences. In one aspect, the present invention provides a library as contemplated herein, where the targeting is at least about 20,000 sequences. In one aspect, the present invention provides a library as contemplated herein, where the targeting is the entire transcriptome. In one aspect, the present invention provides a library as contemplated herein, where the targeting is a panel of target sequences focused on a relevant or desirable pathway. In one aspect, the present invention provides a library as contemplated herein, where the pathway is an immune pathway. In one aspect, the present invention provides a library as contemplated herein, where the pathway is a cell division pathway.

[0367] In one aspect, the present invention provides a method of creating a model eukaryotic cell comprising a gene with modified expression. In some embodiments, the disease gene is any gene associated with an increased risk of developing or having a disease. In some embodiments, the method comprises (a) introducing into a eukaryotic cell one or more vectors encoding the components of the system described above herein, and (b) binding a CRISPR complex to a target polynucleotide to modify gene expression, thereby creating a model eukaryotic cell comprising a modification of gene expression.

[0368] The provided structural information in this specification enables investigation of the guide RNA interaction with the target RNA and the RNA-targeting enzyme, and enables engineering or alteration of the guide RNA structure so that the functionality of the entire RNA-targeting CRISPR-Cas system is optimized. For example, insertion of an adapter protein capable of binding to RNA can elongate the guide RNA without collision with the RNA-targeting protein. These adapter 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 included in a single composition or in individual compositions. These compositions can advantageously be 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 enable the binding of the adapter + functional domain but cannot properly position the adapter + functional domain (e.g., due to steric hindrance within the three-dimensional structure of the CRISPR complex) are not intended modifications. One or more modified guide RNAs can be modified by introduction of individual RNA sequences to the 5' side of the direct repeat, within the direct repeat, or to the 3' side of the guide sequence.

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

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

[0373] The present invention encompasses the use of the compositions of the present invention for establishing and utilizing 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 PCT patent publications cited herein, such as WO 2014 / 093622 pamphlet (PCT / US patent application publication No. 2013 / 074667), which are not considered to be prior to the present invention or this application). For example, the target cell conditionally or inducibly (e.g., in the form of a Cre-dependent construct) contains an RNA targeting CRISPR enzyme and / or conditionally or inducibly contains an adapter protein, and upon expression of a vector introduced into the target cell, the vector induces or gives rise to the conditions for RNA targeting enzyme expression and / or adapter expression in the target cell. Inducible gene expression affected by a functional domain is also an aspect of the present invention by applying the teachings and compositions of the present invention together with known methods of creating CRISPR complexes. Alternatively, by providing an adapter protein together with a conditional or inducible RNA targeting enzyme as a conditional or inducible element, a model effective for screening purposes can be provided, which advantageously requires only minimal design and administration of specific gRNAs for a wide variety of applications.

[0374] The guide RNA according to the present invention containing a dead guide sequence In one aspect, the present invention provides a guide sequence that allows for the formation of a CRISPR complex and successful binding to a target while not allowing for successful nuclease activity (i.e., no nuclease activity / no indel activity). For illustrative purposes, such a modified guide sequence is referred to as a "dead guide" or "dead guide sequence". These dead guides or dead guide sequences can be considered catalytically inactive or structurally inactive in terms of nuclease activity. In fact, a dead guide sequence may not be sufficiently involved in productive base pairing in terms of the ability to promote catalytic activity or distinguish between on-target and off-target binding activity. Briefly, this assay involves synthesizing a CRISPR target RNA and a guide RNA that includes mismatches to the target RNA, combining them with an RNA-targeting enzyme, analyzing cleavage based 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 composition RNA-targeting CRISPR-Cas system that includes functional RNA targeting and a guide RNA (gRNA) as described herein, where the gRNA includes a dead guide sequence, whereby the gRNA has the ability to hybridize to a target sequence such that the RNA-targeting CRISPR-Cas system is directed to a genomic locus of interest within a cell without detectable RNA cleavage activity of the non-mutated RNA-targeting enzyme of the system. It should be understood that any of the gRNAs according to the present invention as described in other parts of this specification can be used as a gRNA that includes a dead gRNA / dead guide sequence as described below in this specification. Any of the methods, products, compositions, and uses as described in other parts of this specification are equally applicable with a gRNA that includes a dead gRNA / dead guide sequence as described in further detail below. Further guidance is provided in the detailed aspects and embodiments below.

[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, the components of a CRISPR system sufficient 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 transfection of vectors encoding the components of the CRISPR sequence, and subsequently preferential cleavage within the target sequence can be evaluated. For example, cleavage of a target RNA polynucleotide sequence can be determined in vitro by providing the components of the CRISPR complex including the target sequence, the dead guide sequence to be tested, and a control guide sequence different from the test dead guide sequence, and comparing the binding or cleavage rates at the target sequence between the reaction of the test guide sequence and the control guide sequence. Other assays are possible and will be apparent to those skilled in the art. The 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, it is possible to arrive at such a dead guide with an appropriate framework by several structural parameters. The dead guide sequence is typically shorter than each guide sequence that results in active RNA cleavage. In a detailed embodiment, the dead guide is 5%, 10%, 20%, 30%, 40%, 50% shorter than each guide for the same.

[0378] As described below and as is known in the art, one aspect of gRNA - RNA targeting specificity is the direct repeat sequence, which should 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. Thus, in the design of Cas13b - specific equivalents, available structural data of the validated dead guide sequences can be used. For example, by using the structural similarity between the orthologous nuclease domains HEPN of two or more Cas13b effector proteins, dead guides of equivalent design can be transferred. Thus, the dead guides herein can be appropriately modified in length and sequence to reflect such Cas13b - specific equivalents, thereby achieving the formation of the CRISPR complex and successful binding to the target RNA, while at the same time not allowing the success of nuclease activity.

[0379] In connection with the present specification and the state of the art in the field, the use of dead guides provides an unexpected and unforeseen platform for network biology and / or systems biology in both in vitro, ex vivo and in vivo applications, enabling multiplex gene targeting, specifically bidirectional multiplex gene targeting. Prior to the use of dead guides, dealing with multiple targets was a challenge and in some cases impossible. With the use of dead guides, it is possible to address multiple targets, and thus multiple activities, in, for example, the same cell, the same animal or the same patient. Such multiplexing can occur simultaneously or can occur at different times within a desired time frame.

[0380] For example, a dead guide enables the use of gRNA as a means of gene targeting without nuclease activity, while at the same time providing an inducible activation or repression means. Guide RNAs containing dead guides can be modified to further include protein adapters (e.g., aptamers) as described elsewhere in this specification that enable the functional placement of elements, specifically gene effectors (e.g., activators or repressors of gene activity), in a form that enables the activation or repression of gene activity. One example is the incorporation of aptamers as described in this specification and the state of the art. By engineering guide RNAs containing dead guides to incorporate protein-interactive aptamers (Konermann et al., “Genome-scale transcription activation by an engineered CRISPR-Cas9 complex”, doi:10.1038 / nature14136, incorporated herein by reference), multiple individual effector domains can be assembled. This can be modeled after natural processes.

[0381] Summary of the Offerings In one aspect, the present invention provides a nucleic acid binding system. In situ hybridization of RNA with a complementary probe is a powerful technique. Typically, fluorescent DNA oligonucleotides are used for the detection of nucleic acids by hybridization. Although certain modifications, such as locked nucleic acids (LNAs), have achieved increased efficiency, an efficient and versatile alternative is still needed. 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 synonymously as described in the foregoing cited references such as WO 2014 / 093622 pamphlet (US Patent Application Publication No. 2013 / 074667). Generally, a guide sequence is any polynucleotide sequence having complementarity with a target polynucleotide sequence sufficient to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or higher when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any algorithm suitable for sequence alignment, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., the Burrows-Wheeler Aligner), ClustalW, ClustalX, BLAT, Novoalign (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 nucleotides in length or longer. 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 the guide sequence to direct sequence-specific binding of the CRISPR complex to the target sequence can be evaluated by any suitable assay.For example, the components of the CRISPR system sufficient to form a CRISPR complex 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 array, including the guide sequence to be tested, and subsequently, the preferential cleavage within the target sequence can be evaluated, such as by the Surveyor assay as described herein. Similarly, cleavage of the target polynucleotide sequence can be determined in vitro by providing the components of the CRISPR complex, including the target sequence and the guide sequence to be tested, and a control guide sequence different from the test guide sequence, and comparing the binding or cleavage rates in the target sequence between the reactions of those test guide sequences and the control guide sequence. Other assays are possible and will be apparent 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 the cell. Exemplary target sequences include those that are unique in the target genome.

[0383] Generally and throughout this specification, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. Vectors include, but are not limited to, single-stranded, double-stranded or partially double-stranded nucleic acid molecules; nucleic acid molecules without free ends (e.g., circular) containing one or more free ends; nucleic acid molecules containing DNA, RNA or both; and other types of polynucleotides known in the art. Certain vectors are "plasmids", which refer to circular double-stranded DNA loops into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, where the vector contains viral-derived DNA or RNA sequences for packaging into a virus (e.g., retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus and adeno-associated virus). Viral vectors also include polynucleotides carried by a virus for transfection of a host cell. Certain vectors have the ability to self-replicate in the 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 are thereby replicated with the host genome. Further, certain vectors are capable of directing the expression of a gene to which they are operably linked. Such vectors are referred to herein as "expression vectors". Vectors for expression in and effecting expression in eukaryotic cells can be referred to herein as "eukaryotic cell expression vectors". General expression vectors useful in recombinant DNA techniques are often in the form of plasmids.

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

[0385] The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRESs), 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 certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can primarily direct expression in a desired target tissue, such as muscle, neurons, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). 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 can also be tissue- or cell-type specific or not. In some embodiments, the vector includes 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, the 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 phosphoglycerate kinase (PGK) promoter, and the EF1α promoter.In addition, the term "regulatory element" includes enhancer elements such as WPRE; CMV enhancer; the R-U5' segment in the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); 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). It will be understood by those skilled in the art 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 the host cell, whereby transcripts, proteins or peptides encoded by the nucleic acids as described herein, including fusion proteins or peptides (e.g., clustered regular...

Claims

1. 1. A method of modifying a target locus of interest in a eukaryotic cell, comprising delivering to said locus a non-naturally occurring or engineered composition comprising a Cas13b effector protein and one or more nucleic acid components, at least said one or more nucleic acid components being 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, said Cas13b effector protein being selected from the group consisting of Bacteroides pyogenes Cas13b (Accession No. WP_034542281), Riemerella anatipestifer Cas13b (Accession No. WP_004919755), Porphyromonas gulae Cas13b (Accession No. WP_004919756), Porphyromonas pyogenes Cas13b (Accession No. WP_004919757), Porphyromonas pyogenes Cas13b (Accession No. WP_004919758), Porphyromonas pyogenes Cas13b (Accession No. WP_004919759 ... gulae Cas13b (Accession No. WP_039434803), Prevotella sp. P5-125 Cas13b (Accession No. WP_044065294), or Porphyromonas sp. COT-052 OH4946 Cas13b (Accession No. WP_039428968), except for methods of treating humans.

2. The method of claim 1 , wherein the eukaryotic cell is a mammalian cell.

3. The method of claim 1 or 2, wherein the Cas13b effector protein is fused to one or more localization signals.

4. The method of any one of claims 1 to 3, wherein the target locus of interest comprises RNA.

5. The method of any one of claims 1 to 4, wherein the Cas13b effector protein is fused to at least one localization signal, the localization signal being a nuclear localization signal (NLS) or a nuclear export signal (NES).

6. 6. The method of any one of claims 1 to 5, wherein the modification of the target locus of interest comprises a strand break.

7. 7. The method of any one of claims 1 to 6, wherein the Cas13b effector protein is associated with one or more functional domains, whereby the complex is capable of delivering an epigenetic modifier or a transcriptional or translational activation or repression signal.

8. The effector protein contains one or more mutations or The effector protein contains one or more mutations in the HEPN domain, or The effector protein contains one or more of the following mutations: R116A, H121A, R1177A, H1182A (wherein the amino acid positions correspond to the amino acid positions of the Cas13b protein from Bergeyella zoohelcum ATCC 43767 (Accession No. WP_002664492)); The method according to claim 7.

9. The method of claim 7 or 8, wherein the functional domain alters transcription or translation of the target locus.

10. The method of any one of claims 1 to 9, wherein the target locus of interest is comprised in a nucleic acid molecule within a cell.

11. The method according to any one of claims 1 to 10, wherein the modification is in vivo or ex vivo.

12. 12. The method of any one of claims 1 to 11, wherein the nucleic acid component, when complexed with the effector protein, is capable of effecting sequence-specific binding of the complex to a target sequence of the target locus of interest.

13. The method of any one of claims 1 to 12, wherein the nucleic acid moiety comprises a dual direct repeat sequence.

14. 14. The method of any one of claims 1 to 13, wherein the effector protein and the nucleic acid component are provided by one or more polynucleotide molecules encoding one or more polypeptides and / or the nucleic acid component, the one or more polynucleotide molecules being operatively configured to express the polypeptides and / or the nucleic acid component.

15. The method of claim 14 , 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.

16. 16. The method of claim 15, wherein the one or more regulatory elements comprise a promoter or an inducible promoter.

17. 17. The method of any one of claims 14 to 16, wherein the one or more polynucleotide molecules are contained within one or more vectors, or the one or more polynucleotide molecules are contained within a single vector.

18. 20. The method of claim 17, wherein the one or more vectors comprise a viral vector.

19. 20. The method of claim 18, wherein the one or more viral vectors comprise one or more retroviral, lentiviral, adenoviral, adeno-associated, or herpes simplex viral vectors.

20. The method according to any one of claims 1 to 19, wherein said one or more polynucleotide molecules are comprised in a delivery system or said one or more vectors are comprised in a delivery system.

21. The method of claim 20, wherein the assembled complex is included in a delivery system.

22. 22. The method of any one of claims 1 to 21, 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.

23. The composition comprises an accessory protein that enhances Cas13b effector protein activity, or The composition comprises a csx28 protein, or The composition comprises an accessory protein that inhibits Cas13b effector protein activity; or The composition comprises a csx27 protein.

23. The method according to any one of claims 1 to 22.

24. The Cas13b effector protein is linked to at least one functional domain, the Cas13b effector protein comprises one or more mutations in the HEPN domain, or the Cas13b effector protein is truncated at its C-terminus.

24. The method according to any one of claims 1 to 23.

25. 1. A non-naturally occurring or engineered composition for use in modifying a target locus of interest in a eukaryotic cell, comprising a Cas13b effector protein and one or more nucleic acid components, at least said one or more nucleic acid components being 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, said Cas13b effector protein being selected from the group consisting of Bacteroides pyogenes Cas13b (Accession No. WP_034542281), Riemerella anatipestifer, anatipestifer Cas13b (accession number WP_004919755), Porphyromonas gulae Cas13b (accession number WP_039434803), Prevotella sp. P5-125 Cas13b (accession number WP_044065294), or Porphyromonas sp. COT-052 OH4946 Cas13b (accession number WP_039428968).

26. The composition of claim 25, wherein the Cas13b effector protein is fused to one or more localization signals.

27. 27. The composition of claim 25 or 26, wherein the target locus of interest comprises RNA.

28. The composition of any one of claims 26 to 27, wherein the Cas13b effector protein is fused to at least one localization signal, the localization signal being a nuclear localization signal (NLS) or a nuclear export signal (NES).

29. The composition of any one of claims 25 to 28, wherein the modification of the target locus of interest comprises a strand break.

30. 30. The composition of any one of claims 25 to 29, wherein the Cas13b effector protein is associated with one or more functional domains, whereby the complex is capable of delivering an epigenetic modifier or a transcriptional or translational activation or repression signal.

31. The effector protein contains one or more mutations or The effector protein contains one or more mutations in the HEPN domain, or The effector protein contains one or more of the following mutations: R116A, H121A, R1177A, H1182A (wherein the amino acid positions correspond to the amino acid positions of the Cas13b protein from Bergeyella zoohelcum ATCC 43767 (Accession No. WP_002664492)); 31. The composition of claim 30.

32. 32. The composition of claim 30 or 31, wherein the functional domain alters transcription or translation of the target locus.

33. The composition of any one of claims 25 to 32, wherein the target locus of interest is comprised in a nucleic acid molecule within a cell.

34. The composition of any one of claims 25 to 33, wherein the modification is in vivo or ex vivo.

35. 35. The composition of any one of claims 25 to 34, wherein the nucleic acid component, when complexed with the effector protein, is capable of effecting sequence-specific binding of the complex to a target sequence at the target locus of interest.

36. The composition of any one of claims 25 to 35, wherein the nucleic acid component comprises a dual direct repeat sequence.

37. 37. The composition of any one of claims 25 to 36, wherein the Cas13b effector protein and the nucleic acid components are obtained by expression of one or more polynucleotide molecules encoding one or more polypeptides and / or nucleic acid components, the one or more polynucleotide molecules being operatively configured to express the polypeptides and / or nucleic acid components.

38. The composition of claim 37 , wherein the one or more polynucleotide molecules comprise one or more regulatory elements operably configured to express the polypeptide and / or the nucleic acid components.

39. The composition of claim 38 , wherein the one or more regulatory elements comprises a promoter or an inducible promoter.

40. 40. The composition of any one of claims 37 to 39, wherein the one or more polynucleotide molecules are contained within one or more vectors, or the one or more polynucleotide molecules are contained within a single vector.

41. 41. The composition of claim 40, wherein the one or more vectors comprise a viral vector.

42. 42. The composition of claim 41, wherein the one or more viral vectors comprise one or more retroviral, lentiviral, adenoviral, adeno-associated, or herpes simplex viral vectors.

43. The composition of any one of claims 25 to 42, wherein the one or more polynucleotide molecules are comprised in a delivery system or the one or more vectors are comprised in a delivery system.

44. The composition of claim 43, wherein the assembled complex is included in a delivery system.

45. 45. The composition of any one of claims 25 to 44, 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.

46. The composition comprises an accessory protein that enhances Cas13b effector protein activity, or The composition comprises a csx28 protein, or The composition comprises an accessory protein that inhibits Cas13b effector protein activity; or The composition comprises a csx27 protein. The composition according to any one of claims 25 to 45.

47. The Cas13b effector protein is linked to at least one functional domain, the Cas13b effector protein comprises one or more mutations in the HEPN domain, or the Cas13b effector protein is truncated at its C-terminus. The composition according to any one of claims 25 to 46.

48. In vivo or ex vivo uses in eukaryotic or 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, - in vitro or in vivo reduction of cell growth and / or cell proliferation, - 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 The composition according to any one of claims 25 to 47 for:

49. A method for assaying, screening or mutagenesis comprising the method of any one of claims 1 to 24, excluding methods for treating humans.

50. 50. A method of modulating translation of a eukaryotic target locus of interest, comprising delivering to said locus a non-naturally occurring or engineered composition of any one of claims 25-49, wherein said Cas13b effector protein is fused to a heterologous translational regulator, or is fused to a heterologous translational regulator and one or more localization signals, and 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, except for methods of treating humans.

51. The method described in claim 50, wherein the heterologous translation regulatory factor is a translation activator or a translation repressor.

52. 52. The method of claim 50 or 51, wherein the heterologous translational regulatory factor is EIF4 or EIF4E.

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